More than 400 publications on HepaRG!

2018

• Co-exposure to benzo[a]pyrene and ethanol induces a pathological progression of liver steatosis in vitro and in vivo.Bucher S,  Tête A,  Podechard N,  Liamin M,  Le Guillou D,  Chevanne M,  Coulouarn C,  Imran M,  Gallais I2,  Fernier M,  Hamdaoui Q,  Robin MA,  Sergent O
  Sci Rep, Apr 2018
  Abstract : Hepatic steatosis (i.e. lipid accumulation) and steatohepatitis have been related to diverse etiologic factors, including alcohol, obesity, environmental pollutants. However, no study has so far analyzed how these different factors might interplay regarding the progression of liver diseases. The impact of the co-exposure to the environmental carcinogen benzo[a]pyrene (B[a]P) and the lifestyle-related hepatotoxicant ethanol, was thus tested on in vitro models of steatosis (human HepaRG cell line; hybrid human/rat WIF-B9 cell line), and on an in vivo model (obese zebrafish larvae). Steatosis was induced prior to chronic treatments (14, 5 or 7 days for HepaRG, WIF-B9 or zebrafish, respectively). Toxicity and inflammation were analyzed in all models; the impact of steatosis and ethanol towards B[a]P metabolism was studied in HepaRGcells. Cytotoxicity and expression of inflammation markers upon co-exposure were increased in all steatotic models, compared to non steatotic counterparts. A change of B[a]P metabolism with a decrease in detoxification was detected in HepaRG cells under these conditions. A prior steatosis therefore enhanced the toxicity of B[a]P/ethanol co-exposure in vitro and in vivo; such a co-exposure might favor the appearance of a steatohepatitis-like state, with the development of inflammation. These deleterious effects could be partly explained by B[a]P metabolism alterations.
• Bavachinin Induces Oxidative Damage in HepaRG Cells through p38/JNK MAPK Pathways.Wang S,  Wang M,  Wang M,  Tian Y,  Sun X,  Sun G,  Sun X
  Toxins, Apr 2018
  Abstract : Drug-induced liver injury is one of the main causes of drug non-approval and drug withdrawal by the Food and Drug Administration (FDA). Bavachinin (BVC) is a natural product derived from the fruit of the traditional Chinese herb Fructus Psoraleae (FP). There have been reports of acute liver injury following the administration of FP and its related proprietary medicines. To explore BVC hepatotoxicity and its mechanisms, we used the HepaRG cell line. In our recent research, we showed that BVC induces HepaRG cell death, mainly via BVC-induced oxidative damage. The formation of ROS is closely related to the activation of the stress-activated kinases, JNK and p38, while SP600125 (SP, JNK inhibitor) and SB203580 (SB, p38 inhibitor) pretreatment inhibited the generation of ROS. On the other hand, N-acetylcysteine (NAC) pretreatment prevented the phosphorylation of p38 but not that of JNK. Taken together, these data reveal that BVC induces HepaRG cell death via ROS and the JNK/p38 signaling pathways.
• Cytochrome P450-dependent drug oxidation activities in commercially available hepatocytes derived from human induced pluripotent stem cells cultured for 3 weeks.Murayama N,  Yamazaki H
  J Toxicol Sci, Apr 2018
  Abstract : Hepatocyte-like cells differentiated from human induced pluripotent stem (iPS) cells are of great interest for applications in pharmacological research. For drug metabolism testing, commercially available hepatocytes derived from human iPS cells are generally recommended to be used 1 week after seeding on plates. In this study, however, after 3-4 weeks of culture according to the manufacturer's instructions, human cytochrome P450 (P450) 2C9- and 2C19-dependent diclofenac 4'-hydroxylation and omeprazole 5-hydroxylation activities of the iPS-derived hepatocytes had significantly increased above the activities at 1 week and had reached levels similar to those in HepaRG cells, a human hepatocyte-like cell line. This increase in activities was associated with increasing P450 2C9 and 2C19 mRNA levels. Human P450 3A4-dependent midazolam 1'/4-hydroxylation activities in the iPS-derived hepatocytes were also enhanced after 3 weeks of culture, but the levels were low compared with those of HepaRG cells. These results indicate that the induction of mRNA of typical P450s in human iPS-derived hepatocyte-like cells occurred after 3 weeks of normal culture conditions. However, the induction levels varied considerably depending on the pregnane X receptor pathway and/or the P450 isoform. Our findings that the hepatic functions of human iPS-derived hepatocytes were enhanced by 3 weeks of simple culture could facilitate the use of these cells for drug metabolism and toxicity testing.
• Rhein Induces Cell Death in HepaRG Cells through Cell Cycle Arrest and Apoptotic Pathway.You L,  Dong X,  Yin X,  Yang C,  Leng X,  Wang W,  Ni J
  Int J Mol Sci., Apr 2018
  Abstract : Rhein, a naturally occurring active anthraquinone found abundantly in various medicinal and nutritional herbs, possesses a wide spectrum of pharmacological effects. Furthermore, previous studies have reported that rhein could induce hepatotoxicity in rats. However, its cytotoxicity and potential molecular mechanisms remain unclear. Therefore, the present study aimed to investigate the cytotoxicity of rhein on HepaRGcells and the underlying mechanisms of its cytotoxicity. Our results demonstrate, by 3-(4,5-dimethyl thiazol-2-yl-)-2,5-diphenyl tetrazolium bromide (MTT) and Annexin V-fluoresce isothiocyanate (FITC)/propidium iodide (PI) double-staining assays, that rhein significantly inhibited cell viability and induced apoptosis in HepaRG cells. Moreover, rhein treatment resulted in the generation of reactive oxygen species (ROS), loss of mitochondrial membrane potential (MMP), and S phase cell cycle arrest. The results of Western blotting showed that rhein treatment resulted in a significant increase in the protein levels of Fas, p53, p21, Bax, cleaved caspases-3, -8, -9, and poly(ADP-ribose)polymerase (PARP). The protein expression of Bcl-2, cyclin A, and cyclin-dependent kinase 2 (CDK 2) was decreased. In conclusion, these results suggest that rhein treatment could inhibit cell viability of HepaRG cells and induce cell death through cell cycle arrest in the S phase and activation of Fas- and mitochondrial-mediated pathways of apoptosis. These findings emphasize the need to assess the risk of exposure for humans to rhein.
• A threshold dose distribution approach for the study of PDT resistance development: A threshold distribution approach for the study of PDT resistance.de Faria CMG,  Inada NM,  Vollet-Filho JD,  Bagnato VS
  J Photochem Photobiol B, Mar 2018
  Abstract : Photodynamic therapy (PDT) is a technique with well-established principles that often demands repeated applications for sequential elimination of tumor cells. An important question concerns the way surviving cells from a treatment behave in the subsequent one. Threshold dose is a core concept in PDT dosimetry, as the minimum amount of energy to be delivered for cell destruction via PDT. Concepts of threshold distribution have shown to be an important tool for PDT results analysis in vitro. In this study, we used some of these concepts for demonstrating subsequent treatments with partial elimination of cells modify the distribution, which represents an increased resistance of the cells to the photodynamic action. HepG2 and HepaRG were used as models of tumor and normal liver cells and a protocol to induce resistance, consisted of repeated PDT sessions using Photogem® as a photosensitizer, was applied to the tumor ones. The response of these cells to PDT was assessed using a standard viability assay and the dose response curves were used for deriving the threshold distributions. The changes in the distribution revealed that the resistance protocol effectively eliminated the most sensitive cells. Nevertheless, HepaRG cell line was the most resistant one among the cells analyzed, which indicates a specificity in clinical applications that enables the use of high doses and drug concentrations with minimal damage to the surrounding normal tissue.
• Direct antiviral properties of TLR ligands against HBV replication in immune-competent hepatocytes.Lucifora J,  Bonnin M,  Aillot L,  Fusil F,  Maadadi S,  Dimier L,  Michelet M,  Floriot O,  Ollivier A,  Rivoire M,  Ait-Goughoulte M,  Daffis S,  Fletcher SP
  Sci rep, Mar 2018
  Abstract : Current therapies for chronic hepatitis B virus (HBV) infections are effective at decreasing the viral load in serum, but do not lead to viral eradication. Recent studies highlighted the therapeutic or "adjuvant" potential of immune-modulators. Our aim was to explore the direct anti-HBV effect of Toll-Like-Receptors (TLR) agonists in hepatocytes. HBV-infected primary human hepatocytes (PHH) or differentiated HepaRGcells (dHepaRG) were treated with various TLR agonists. Amongst all TLR ligands tested, Pam3CSK4 (TLR1/2-ligand) and poly(I:C)-(HMW) (TLR3/MDA5-ligand) were the best at reducing all HBV parameters. No or little viral rebound was observed after treatment arrest, implying a long-lasting effect on cccDNA. We also tested Riboxxol that features improved TLR3 specificity compared to poly(I:C)-(HMW). This agonist demonstrated a potent antiviral effect in HBV-infected PHH. Whereas, poly(I:C)-(HMW) and Pam3CSK4 mainly induced the expression of classical genes from the interferon or NF-κB pathway respectively, Riboxxol had a mixed phenotype. Moreover, TLR2 and TLR3 ligands can activate hepatocytes and immune cells, as demonstrated by antiviral cytokines produced by stimulated hepatocytes and peripheral blood mononuclear cells. In conclusion, our data highlight the potential of innate immunity activation in the direct control of HBV replication in hepatocytes, and support the development of TLR-based antiviral strategies.
• Bioprinting Perfusion-Enabled Liver Equivalents for Advanced Organ-on-a-Chip Applications.Grix T,  Ruppelt A,  Thomas A,  Amler AK,  Noichl BP,  Lauster R,  Kloke L
  Genes (Basel), Mar 2018
  Abstract : Many tissue models have been developed to mimic liver-specific functions for metabolic and toxin conversion in in vitro assays. Most models represent a 2D environment rather than a complex 3D structure similar to native tissue. To overcome this issue, spheroid cultures have become the gold standard in tissue engineering. Unfortunately, spheroids are limited in size due to diffusion barriers in their dense structures, limiting nutrient and oxygen supply. Recent developments in bioprinting techniques have enabled us to engineer complex 3D structures with perfusion-enabled channel systems to ensure nutritional supply within larger, densely-populated tissue models. In this study, we present a proof-of-concept for the feasibility of bioprinting a liver organoid by combining HepaRG and human stellate cells in a stereolithographic printing approach, and show basic characterization under static cultivation conditions. Using standard tissue engineering analytics, such as immunohistology and qPCR, we found higher albumin and cytochrome P450 3A4 (CYP3A4) expression in bioprinted liver tissues compared to monolayer controls over a two-week cultivation period. In addition, the expression of tight junctions, liver-specific bile transporter multidrug resistance-associated protein 2 (MRP2), and overall metabolism (glucose, lactate, lactate dehydrogenase (LDH)) were found to be stable. Furthermore, we provide evidence for the perfusability of the organoids' intrinsic channel system. These results motivate new approaches and further development in liver tissue engineering for advanced organ-on-a-chip applications and pharmaceutical developments.
• Hepatitis D Virus replication is sensed by MDA5 and induces IFN-?/? responses in hepatocytes.Zhang Z,  Filzmayer C,  Ni Y,  Sültmann H,  Mutz P,  Hiet MS,  Vondran FWR,  Bartenschlager R,  Urban S, , , ,
  J Hepatol, Mar 2018
  Abstract : Microarray analysis revealed that HDV but not HBV infection activated a broad range of interferon stimulated genes (ISGs) in HepG2NTCP cells. HDV strongly activated IFN-β and IFN-λ in cell lines and PHH. HDV induced IFN levels remained unaltered upon RIG-I or TLR3 knock-down, but were almost completely abolished upon MDA5 depletion. Conversely, overexpression of MDA5 but not RIG-I and TLR3 in Huh7.5NTCP cells partially restored ISG induction. During long-term infection, IFN levels gradually diminished in both HepG2NTCPand HepaRGNTCP cell lines. MDA5 depletion had little effect on HDV replication despite dampening HDV-induced IFN response. Moreover, treatment with type I or type III IFNs did not abolish HDV replication.
• Structure-dependent induction of apoptosis by hepatotoxic pyrrolizidine alkaloids in the human hepatoma cell line HepaRG: Single versus repeated exposure.Waizenegger J,  Braeuning A,  Templin M,  Lampen A,  Hessel-Pras S
  Food Chem Toxicol, Feb 2018
  Abstract : Pyrrolizidine alkaloids (PA) are secondary plant compounds. PA intoxication in humans causes severe acute and chronic hepatotoxicity. However, the molecular mechanisms of PA hepatotoxicity in humans are not well understood yet. Therefore, we investigated cell death parameters in human HepaRG cells following either single (24?h) or repeated dose treatment (14?d) with structurally different PA of the retronecine (echimidine, senecionine), heliotridine (heliotrine), and otonecine type (senkirkine). After 24?h of exposure only retronecine-type PA were cytotoxic in HepaRG cells and induced apoptosis indicated by a loss of membrane asymmetry, disruption of the mitochondrial membrane potential, and increased pro-caspase and PARP cleavage. In contrast, after 14?d all four PA exerted the aforementioned effects. Furthermore, the apoptotic events caspase 3, 8 and 9 activation as well as nuclear condensation and DNA fragmentation were only detected for the retronecine-type PA after single exposure (6?h). Overall, our studies revealed a time- and structure-dependent apoptosis after PA exposure, suggesting that retronecine-type PA seem to be more potent apoptosis inducers than heliotridine- or otonecine-type PA. Furthermore, our results suggest that PA-induced apoptosis in HepaRG cells occur most probably by involving both, the extrinsic death receptor pathway as well as the intrinsic mitochondrial pathway.
• Comparison of drug metabolism and its related hepatotoxic effects in HepaRG, cryopreserved human hepatocytes, and HepG2 cell cultures.Yokoyama Y,  Sasaki Y,  Terasaki N,  Kawataki T,  Takekawa K,  Iwase Y,  Shimizu T,  Sanoh S,  Ohta S
  Biol Pharm Bull, Feb 2018
  Abstract : Differentiated HepaRG cells maintain liver-specific functions such as drug-metabolizing enzymes. In this study, the feasibility of HepaRG cells as a human hepatocyte model for in vitro toxicity assessment was examined using selected hepatotoxic compounds. First, basal drug-metabolizing enzyme activities (CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4, uridine 5'-diphospho-glucuronosyltransferase [UGT], and sulfotransferases [SULT]) were measured in HepaRG, human hepatocytes, and HepG2 cells. Enzyme activities in differentiated HepaRG cells were comparable to those in human hepatocytes and much higher than those in HepG2 cells, except for SULT activity. Second, we examined the cytotoxicity of hepatotoxic compounds, acetaminophen (APAP), aflatoxin B1 (AFB1), cyclophosphamide (CPA), tamoxifen (TAM), and troglitazone (TGZ) in HepaRG cells and human hepatocytes. AFB1- and CPA-induced cytotoxicities against HepaRGcells were comparable to those against human hepatocytes. Furthermore, the cytotoxicities of these compounds were inhibited by 1-aminobenzotriazole (ABT), a broad CYP inhibitor, in both cells and were likely mediated by metabolic activation by CYP. Finally, toxicogenomics analysis of HepG2 and HepaRG cells after exposure to AFB1 and CPA revealed that numerous p53-related genes were upregulated and the expression of these genes was greater in HepaRG than in HepG2 cells. These results suggest that gene expression profiles of HepaRG cells were affected more considerably by the toxic mechanisms of AFB1 and CPA than the profiles of HepG2 cells were. Therefore, our investigation shows that HepaRG cells could be useful human hepatic cellular models for toxicity studies.
• Interaction between TLR9-CpG-ODNs and HBV virions leads to entry inhibition in hepatocytes and reduction of IFN? production by pDC.Aillot L,  Bonnin M,  Ait-Goughoulte M,  Bendriss-Vermare N,  Maadadi S,  Dimier L,  Subic M,  Scholtes C,  Najera I,  Zoulim F,  Lucifora J,  Durantel D
  Antimicrob Agents Chemother, Feb 2018
  Abstract : We previously reported that TLR9 CpG-oligonucleotides could inhibit the establishment of HBV infections in hepatocytes. Our aim was to uncover the underlying mechanisms of this inhibition. HepaRG, RPMI-B lymphoblastoma and primary plasmacytoid dendritic cells exposed to HBV and TLR9 ligands/agonists in various configurations were used. We observed an inhibition of HBV infection upon TLR9 stimulations only when agonist was applied during inoculation. This inhibition was independent of IL6/IP10 production as well as of TLR9 expression in hepatocytes. We further demonstrated an entry inhibition mechanism by evidencing a non-covalent binding of TLR9 agonist to HBV particles. Besides inhibiting HBV entry into hepatocytes, this biophysical interaction between HBV virions and TLR9 agonist was responsible for a reduction of IFNα expression by pDCs. Interestingly subviral-particles composed of only HBsAg were capable to genuinely inhibit the TLR9 pathway, without "titering out" TLR9 ligands. To conclude, our data suggest that synthetic TLR9-CpG-oligonucleotides can strongly inhibit HBV entry by "coating" HBV virions and thereby preventing their interaction with cellular receptor. This titration effect of TLR9 agonist is also artefactually responsible for the inhibition of TLR9 engagement in pDC, whereas a genuine inhibition of this innate pathway was confirmed with HBsAg subviral-particles.
• Hepatitis B virus DNA integration occurs early in the viral life cycle in an in vitro infection model via NTCP-dependent uptake of enveloped virus particles.Tu T,  Budzinska MA,  Vondran FWR,  Shackel NA,  Urban S
  J Virol, Feb 2018
  Abstract : Chronic infection by the Hepatitis B Virus (HBV) is the major contributor to liver disease worldwide. Though HBV replicates via a nuclear episomal DNA (cccDNA), integration of HBV DNA into the host cell genome is regularly observed in the liver of infected patients. While reported as a pro-oncogenic alteration, the mechanism(s) and timing of HBV DNA integration are not well-understood, chiefly due to the lack of in vitro infection models that have detectable integration events. Here, we have established an in vitro system in which integration can be reliably detected following HBV infection. We measured HBV DNA integration using inverse nested PCR in primary human hepatocytes, HepaRG-NTCP, HepG2-NTCP, and Huh7-NTCP cells after HBV infection. Integration was detected in all cell types at a rate of >1 per 10000 cells, with the most consistent detection in Huh7-NTCP cells. Integration rate remained stable between 3 and 9 days post-infection. HBV DNA integration was efficiently blocked by treatment with 200nM of the HBV entry inhibitor Myrcludex B, but not with 10μM Tenofovir, 100U Interferon alpha, or 1μM of the capsid assembly inhibitor GLS4. This suggests integration of HBV DNA occurs immediately after infection of hepatocytes and is likely independent of de novo HBV replication in this model. Site analysis revealed that HBV DNA integrations were distributed over the entire human genome. Further, integrated HBV DNA sequences were consistent with double-stranded linear HBV DNA being the major precursor. Thus, we have established an in vitro system to interrogate the mechanisms of HBV DNA integration.ImportanceHepatitis B Virus (HBV) is a common blood-borne pathogen and, following a chronic infection, can cause liver cancer and liver cirrhosis. Integration of HBV DNA into the host genome occurs in all known members of the hepadnaviridae family, despite this form not being necessary for viral replication. HBV DNA integration has been reported to drive liver cancer formation and persistence of virus infection. However, when and the mechanism(s) by which HBV DNA integration occurs is not clear. Here, we have developed and characterized an in vitro system to reliably detect HBV DNA integrations that result from a true HBV infection event and that closely resemble those found in patient tissues. Using this model, we show that integration already occurs when the infection is first established. Importantly, we provide here a system to analyze molecular factors involved in HBV integration, which can be used to develop strategies to halt its formation.
• Posttranscriptional regulation of UGT2B10 hepatic expression and activity by alternative splicing.Labriet A,  Allain EP,  Rouleau M,  Audet-Delage Y,  Villeneuve L,  Guillemette C
  Drug Metab Dispos, Feb 2018
  Abstract : The detoxification enzyme UDP-glucuronosyltransferase UGT2B10 is specialized in the N-linked glucuronidation of many drugs and xenobiotics. Preferred substrates possess tertiary aliphatic amines and heterocyclic amines such as tobacco carcinogens and several anti-depressants and anti-psychotics. We hypothesized that alternative splicing (AS) constitutes a mean to regulate steady state levels of UGT2B10 and enzyme activity. We established the transcriptome of UGT2B10 in normal and tumoral tissues of multiple individuals. Highest expression was in the liver, where ten AS transcripts represented 50% of the UGT2B10 transcriptome in 50 normal livers and 44 hepatocellular carcinomas. One abundant class of transcripts involves a novel exonic sequence and leads to two alternative (alt.) variants with novel in-frame C-termini of 10 or 65 amino acids. Their hepatic expression was highly variable among individuals, correlated with canonical transcript levels, and was 3.5 fold higher in tumors. Evidence for their translation in liver tissues was acquired by mass spectrometry. In cell models, they co-localized with the enzyme and influenced the conjugation of amitriptyline and levomedetomidine by repressing or activating the enzyme (40-70%; PHepaRG hepatic cell model, which favored alt. variants expression over the canonical transcript. Our findings support a significant contribution of AS in the regulation of UGT2B10 expression in the liver with an impact on enzyme activity.
• Silencing Retinoid X Receptor Alpha Expression Enhances Early-stage Hepatitis B Virus Infection In Cell Cultures.Song M,  Sun Y,  Tian J,  He W,  Xu G,  Jing Z,  Li W
  J Virol, Feb 2018
  Abstract : Multiple steps of the life cycle of Hepatitis B virus (HBV) are known to be coupled to hepatic metabolism. However the details of involvement of hepatic metabolic milieu in HBV infection remain incompletely understood. Hepatic lipid metabolism is controlled by a complicated transcription factors network centered by Retinoid X receptor alpha (RXRα). Here, we report that RXRα negatively regulates HBV infection at early stage in cell cultures. RXR specific agonist Bexarotene inhibits HBV in HepG2 cells expressing the sodium taurocholate cotransporting polypeptide (NTCP) (HepG2-NTCP), HepaRG cells and primary Tupaia hepatocytes (PTHs); Reducing RXRα expression significantly enhanced HBV infection in the cells. RNAseq analysis of HepG2-NTCP cells with disrupted RXRα gene revealed that, reduced gene expression in arachidonic acid (AA)/eicosanoids biosynthesis pathways, including the AA synthases Phospholipase A2 Group IIA (PLA2G2A), is associated with increased HBV infection. Moreover, exogenous treatment of AA inhibits HBV infection in HepG2-NTCP cells. These data demonstrate that RXRα is an important cellular factor in modulating HBV infection and implicate the participation of AA/eicosanoids biosynthesis pathways in the regulation of HBV infection.IMPORTANCEUnderstanding how HBV infection is connected with hepatic lipid metabolism may provide new insights into the viral infection and its pathogenesis. By a series of genetic studies in combination with transcriptome analysis and pharmacological assays, we here investigated the role of cellular Retinoid X receptor alpha (RXRα), a crucial transcription factor for controlling hepatic lipid metabolism, in de novo HBV infection in cell cultures. We found that silencing of RXRα resulted in enhanced HBV cccDNA formation and viral antigens production, while activation of RXRα reduced HBV infection efficiency. Our results also showed that silencing Phospholipase A2 Group IIA (PLA2G2A), a key enzyme of arachidonicacid (AA) synthases enhanced HBV infection efficiency in HepG2-NTCP cells and exogenous AA treatment reduced de novo HBV infection in the cells. These findings unveil RXRα as an important cellular factor in modulating HBV infection and may point to a new strategy for host-targeted therapies against HBV.
• A novel genotoxin-specific qPCR array based on the metabolically competent human HepaRG? cell line as a rapid and reliable tool for improved in vitro hazard assessment.Ates G,  Mertens B,  Heymans A,  Verschaeve L,  Milushev D,  Vanparys P,  Roosens NHC6,  De Keersmaecker SCJ,  Rogiers V3,  Doktorova TY, , ,
  Arch Toxicol, Feb 2018
  Abstract : Although the value of the regulatory accepted batteries for in vitro genotoxicity testing is recognized, they result in a high number of false positives. This has a major impact on society and industries developing novel compounds for pharmaceutical, chemical, and consumer products, as afflicted compounds have to be (prematurely) abandoned or further tested on animals. Using the metabolically competent human HepaRG™ cell line and toxicogenomics approaches, we have developed an upgraded, innovative, and proprietary gene classifier. This gene classifier is based on transcriptomic changes induced by 12 genotoxic and 12 non-genotoxic reference compounds tested at sub-cytotoxic concentrations, i.e., IC10 concentrations as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The resulting gene classifier was translated into an easy-to-handle qPCR array that, as shown by pathway analysis, covers several different cellular processes related to genotoxicity. To further assess the predictivity of the tool, a set of 5 known positive and 5 known negative test compounds for genotoxicity was evaluated. In addition, 2 compounds with debatable genotoxicity data were tested to explore how the qPCR array would classify these. With an accuracy of 100%, when equivocal results were considered positive, the results showed that combining HepaRG™ cells with a genotoxin-specific qPCR array can improve (geno)toxicological hazard assessment. In addition, the developed qPCR array was able to provide additional information on compounds for which so far debatable genotoxicity data are available. The results indicate that the new in vitro tool can improve human safety assessment of chemicals in general by basing predictions on mechanistic toxicogenomics information.
• Oxygen drives hepatocyte differentiation and phenotype stability in liver cell lines.van Wenum M,  Adam AAA,  van der Mark VA,  Chang JC,  Wildenberg ME,  Hendriks EJ,  Jongejan A,  Moerland PD,  van Gulik TM,  Oude Elferink RP,  Chamuleau RAFM,  Hoekstra R
  J Cell Commun Signal., Feb 2018
  Abstract : The in vitro generation of terminally differentiated hepatocytes is an unmet need. We investigated the contribution of oxygen concentration to differentiation in human liver cell lines HepaRG and C3A. HepaRG cells were cultured under hypoxia (5%O2), normoxia (21%O2) or hyperoxia (40%O2). Cultures were analysed for hepatic functions, gene transcript levels, and protein expression of albumin, hepatic transcription factor CEBPα, hepatic progenitor marker SOX9, and hypoxia inducible factor (HIF)1α. C3A cells were analysed after exposure to normoxia or hyperoxia. In hyperoxic HepaRG cultures, urea cycle activity, bile acid synthesis, CytochromeP450 3A4 (CYP3A4) activity and ammonia elimination were 165-266% increased. These effects were reproduced in C3A cells. Whole transcriptome analysis of HepaRG cells revealed that 240 (of 23.223) probes were differentially expressed under hyperoxia, with an overrepresentation of genes involved in hepatic differentiation, metabolism and extracellular signalling. Under hypoxia, CYP3A4 activity and ammonia elimination were inhibited almost completely and 5/5 tested hepatic genes and 2/3 tested hepatic transcription factor genes were downregulated. Protein expression of SOX9 and HIF1α was strongly positive in hypoxic cultures, variable in normoxic cultures and predominantly negative in hyperoxic cultures. Conversely, albumin and CEBPα expression were highest in hyperoxic cultures. HepaRG cells that were serially passaged under hypoxia maintained their capacity to differentiate under normoxia, in contrast to cells passaged under normoxia. Hyperoxia increases hepatocyte differentiation in HepaRG and C3A cells. In contrast, hypoxia maintains stem cell characteristics and inhibits hepatic differentiation of HepaRG cells, possibly through the activity of HIF1α.
• Cell-based assay using glutathione-depleted HepaRG and HepG2 human liver cells for predicting drug-induced liver injury.Xu J,  Oda S,  Yokoi T
  Toxicol In Vitro, Jan 2018
  Abstract : Immortalized liver cells have been used for evaluating the toxicity of compounds; however, excessive glutathione is considered to lessen cytotoxicity. In this study, we compared the effects of glutathione depletion on cytotoxicities of drugs using HepaRG and HepG2 cells, which express and lack drug-metabolizing enzymes, respectively, for predicting drug-induced liver injury (DILI) risks. These cells were pre-incubated with L-buthionine-S,R-sulfoximine (BSO) and then exposed to 34 test compounds with various DILI risks for 24?h. ATP level exhibited the highest predictability of DILI among tested parameters. BSO treatment rendered cells susceptible to drug-induced cytotoxicity when evaluated by cell viability and caspase 3/7 activity with the sensitivity of cell viability from 50% in non-treated HepaRG cells to 71% in BSO-treated HepaRG cells. These results indicate that cytotoxicity assays using GSH-depleted HepaRG cells improve the predictability of DILI risks. However, HepaRG cells were not always superior to HepG2 cells when assessed by ATP level. The combination of HepG2 and HepaRG cells index produced the best prediction in the cases of caspase 3/7 acitivity and ATP level. In conclusions, the developed highly sensitive cell-based assay using GSH-reduced cells would be useful for predicting potential DILI risks at an early stage of drug development.
• HBV Bypasses the Innate Immune Response and Does not Protect HCV From Antiviral Activity of Interferon.Mutz P,  Metz P,  Lempp FA,  Bender S,  Qu B,  Schöneweis K,  Seitz S,  Tu T,  Restuccia A,  Frankish J,  Dächert C,  Schusser B,  Koschny R
  Gastroenterology, Jan 2018
  Abstract : HBV infection of dHepaRGNTCP cells and PHHs neither activated nor inhibited signaling via pattern recognition receptors. Incubation of dHepaRGNTCP cells and PHHs with IFN had little effect on HBV replication or levels of cccDNA. HBV infection of these cells did not inhibit JAK-STAT signaling or upregulation of IFN-stimulated genes. In co-infected cells, HBV did not prevent IFN-induced suppression of HCV replication.
• Optimized Methods to Explore the Mechanistic and Biomarker Potential of Hepatocyte-Derived Exosomes in Drug-Induced Liver Injury.Thacker SE,  Nautiyal M,  Otieno MA,  Watkins PB,  Mosedale M
  Toxicol Sci., Jan 2018
  Abstract : Recent evidence supports that alterations in hepatocyte-derived exosomes (HDE) may play a role in the pathogenesis of drug-induced liver injury (DILI). HDE-based biomarkers also hold promise to improve the sensitivity of existing in vitro assays for predicting DILI liability. Primary human hepatocytes (PHH) provide a physiologically relevant in vitro model to explore the mechanistic and biomarker potential of HDE in DILI. However, optimal methods to study exosomes in this culture system have not been defined. Here we use HepG2 and HepaRG cells along with PHH to optimize methods for in vitro HDE research. We compared the quantity and purity of HDE enriched from HepG2 cell culture medium by three widely used methods: ultracentrifugation (UC), OptiPrep density gradient ultracentrifugation (ODG), and ExoQuick (EQ) - a commercially available exosome precipitation reagent. While EQ resulted in the highest number of particles, UC resulted in more exosomes as indicated by the relative abundance of exosomal CD63 to cellular prohibitin-1 as well as the comparative absence of contaminating extravesicular material. To determine culture conditions that best supported exosome release, we also assessed the effect of Matrigel matrix overlay at concentrations ranging from 0-0.25 mg/ml in HepaRG cells and compared exosome release from fresh and cryopreserved PHH from same donor. Sandwich culture did not impair exosome release, and freshly prepared PHH yielded a higher number of HDE overall. Taken together, our data support the use of UC-based enrichment from fresh preparations of sandwich-cultured PHH for future studies of HDE in DILI.
• Effect of Oridonin on Cytochrome P450 Expression and Activities in HepaRG Cell.Zhang YW,  Zheng XW,  Liu YJ,  Fang L,  Pan ZF,  Bao MH,  Huang P
  Pharmacol, Jan 2018
  Abstract : Oridonin, the major terpene found in Rabdosia rubescens, is widely used as a dietary supplement or therapeutic drug. However, the effects of oridonin on major CYP450s are still unclear. As oridonin can enhance the effect of other clinical drugs, in this study, we investigated the influence of oridonin on CYP450s mRNA expression and its impact on activities in human HepaRG cell to evaluate the safety by studying its potential drug interaction. HepaRG cells were cultured with series concentrations of oridonin (1, 5, 10, and 20 μmol/L), and the major CYP450s mRNA and protein expression, as well as enzyme activities were analyzed by real-time polymerase chain reaction, Western blot analysis and UPLC-MS/MS-based metabolite assay. In general, ordonin has induced effects on the major member of CYP450s mRNA and protein expression, as well as on the enzyme activity in human HepaRG cells, especially on CYP3A4 and CYP2C9. To our knowledge, this is the first systematic research about the inductive effects of oridonin on the major member of CYP450s in human cell line. These results may provide at least partly of the basis for potential drug-drug interactions and oridonin should be used with caution to avoid potential risk.
• Metabolomics profiling of steatosis progression in HepaRG® cells using sodium valproate.Cuykx M,  Claes L,  Rodrigues RM,  Vanhaecke T,  Covaci A
  Toxicol Lett, Jan 2018
  Abstract : Non-alcoholic Fatty Liver Disease (NAFLD) is a frequently encountered Drug-Induced Liver Injury (DILI). Although this stage of the disease is reversible, it can lead to irreversible damage provoked by non-alcoholic steatohepatitis (NASH), fibrosis and cirrhosis. Therefore, the assessment of NAFLD is a paramount objective in toxicological screenings of new drug candidates. In this study, a metabolomic fingerprint of NAFLD induced in HepaRG® cells at four dosing schemes by a reference toxicant, sodium valproate (NaVPA), was obtained using liquid-liquid extraction followed by liquid chromatography and accurate mass-mass spectrometry (LC-AM/MS). The combination of a strict design of experiment with a robust detection method, applied on sodium valproate, validated the possibilities of untargeted metabolomics in hepatic toxicological research. Distinctive patterns between exposed and control cells were consistently observed, multivariate analyses selected up to 200 features of interest, revealing hallmark NAFLD-biomarkers, such as diacylglycerol and triglyceride accumulation and carnitine deficiency. Initial toxic responses show increased levels of S-adenosylmethionine and mono-acetylspermidine in combination with only a moderate increase in triglycerides. New specific markers of toxicity have been observed, such as spermidines, creatine, and acetylcholine. The described design of experiment provides a valuable metabolomics platform for mechanistic research of toxicological hazards and identified new markers for steatotic progression.
• Cytochrome P450 1A2 Messenger RNA is a More Reliable Marker than Cytochrome P450 1A2 Activity, Phenacetin O-Deethylation, for Assessment of Induction Potential of Drug-Metabolizing Enzymes Using HepaRG Cells.Ogasawara A,  Kato N,  Torimoto N,  Aohara F,  Ohashi R,  Yamada Y,  Taniguchi H, , , , , ,
  Drug Metab Lett., Jan 2018
  Abstract : BACKGROUND: The HepaRG cells have key drug metabolism functionalities comparable to those of primary human hepatocytes. Many studies have reported that this cell line can be used as a reliable in vitro model for human drug metabolism studies, including the assessment of cytochrome P450 (CYP) induction. OBJECTIVES: The objective of this study is to determine whether CYP mRNA level measurement is superior to the CYP enzyme activity measurement as a convenient high-throughput method for evaluating CYP induction potential using HepaRG cells. METHODS: QuantiGene Plex 2.0 Assay and LC/MS/MS. mRNA expression levels and enzyme activities of CYP1A2, CYP2B6, and CYP3A in HepaRG cells treated with prototypical inducers of each CYP isoform [omeprazole (OME) for CYP1A2, phenobarbital (PB) for CYP2B6, and rifampicin (RIF) for CYP3A] were evaluated. RESULTS: Although the activities of CYP2B6 and CYP3A was were induced by treatment with PB and RIF, we found that the activity of phenacetin O-deethylase (PHOD), which is known as a marker of the activity of CYP1A2, was also enhanced by treatment with these non-CYP1A2 inducers in HepaRG cells. Based on previously published reports, we hypothesized that the expression ratio of CYP3A to CYP1A2 is much higher in HepaRG cells than in human hepatocytes; this may result in a nonnegligible contribution of CYP3A to the PHOD reaction in HepaRG cells. Studies using CYP3A inhibitor and pregnane X receptor-knockout HepaRG cells supported this hypothesis. CONCLUSIONS: The measurement of mRNA serves as a higher reliable indicator for the evaluation of CYP induction potential when using HepaRG cells.
• Regulation of drug metabolism by the interplay of inflammatory signaling, steatosis, and xeno-sensing receptors in HepaRG cells.Tanner N,  Kubik L,  Luckert C,  Thomas M,  Hofmann U,  Zanger UM,  Bohmert L,  Lampen A,  Braeuning A
  Drug Metab Dispos, Jan 2018
  Abstract : Non-alcoholic fatty liver disease (NAFLD), characterized by triglyceride deposition in hepatocytes due to imbalanced lipid homeostasis, is of increasing concern in Western countries, with progression to non-alcoholic steatohepatitis (NASH), liver fibrosis, and cirrhosis. Previous studies suggest a complex, mutual influence of hepatic fat accumulation, NASH-related inflammatory mediators, and drug-sensing receptors regulating xenobiotic metabolism. Here, we investigated the suitability of human HepaRG hepatocarcinoma cells as a model for NAFLD and NASH. Cells were incubated for up to 14 days with an oleate/palmitate mixture (125 μM each), and/or with 10 ng/mL of the inflammatory mediator interleukin-6. Effects of these conditions on the regulation of drug metabolism were studied using xenobiotic agonists of the aryl hydrocarbon receptor (AHR), pregnane-X-receptor (PXR), constitutive androstane receptor (CAR), nuclear factor (erythroid-derived 2)-like 2 (NRF2), and peroxisome proliferator-activated receptor α (PPARα). Results underpin the suitability of HepaRG cells for NAFLD- and NASH-related research and constitute a broad-based analysis of the impact of hepatic fatty acid accumulation and inflammation on drug metabolism and its inducibility by xenobiotics. Interleukin-6 exerted pronounced negative regulatory effects on basal as well as on PXR-, CAR-, PPARα-, but not AHR-dependent induction of drug-metabolizing enzymes. This inhibition was related to diminished transactivation potential of the respective receptors, rather than to reduced transcription of nuclear receptor-encoding mRNAs. Most striking effects of interleukin-6 and/or fatty acid treatment were observed in HepaRG cells following 14 days of treatment, making these cultures appear a su
• Lipase inhibitor orlistat prevents hepatitis B virus infection by targeting an early step in the virus life cycle.Esser K,  Lucifora J,  Wettengel J,  Singethan K,  Glinzer A,  Zernecke A,  Protzer U
  Antiviral Res., Jan 2018
  Abstract : Hepatitis B Virus (HBV) is a strictly hepatotropic pathogen which is very efficiently targeted to the liver and into its host cell, the hepatocyte. The sodium taurocholate co-transporting polypeptide (NTCP) has been identified as a key virus entry receptor, but the early steps in the virus life cycle are still only barely understood. Here, we investigated the effect of lipase inhibition and lipoprotein uptake on HBV infection using differentiated HepaRG cells and primary human hepatocytes. We found that an excess of triglyceride rich lipoprotein particles in vitro diminished HBV infection and a reduced hepatic virus uptake in vivo if apolipoprotein E is lacking indicating virus transport along with lipoproteins to target hepatocytes. Moreover, we showed that HBV infection of hepatocytes was inhibited by the broadly active lipase inhibitor orlistat, approved as a therapeutic agent which blocks neutral lipid hydrolysis activity. Orlistat treatment targets HBV infection at a post-entry step and inhibited HBV infection during virus inoculation strongly in a dose-dependent manner. In contrast, orlistat had no effect on HBV gene expression or replication or when added after HBV infection. Taken together, our data indicate that HBV connects to the hepatotropic lipoprotein metabolism and that inhibition of cellular hepatic lipase(s) may allow to target early steps of HBV infection.
• Cellular and molecular effects of prolonged low-level sodium arsenite exposure on human hepatic HepaRG cells.Dreval K,  Tryndyak V,  Kindrat I,  Twaddle NC,  Orisakwe OE,  Mudalige TK,  Beland FA,  Doerge DR,  Pogribny IP
  Toxicol Sci, Jan 2018
  Abstract : Inorganic arsenic is a human carcinogen associated with several types of cancers, including liver cancer. Inorganic arsenic has been postulated to target stem cells, causing their oncogenic transformation. This is proposed to be one of the key events in arsenic-associated carcinogenesis; however, the underlying mechanisms for this process remain largely unknown. To address this question, human hepatic HepaRG cells, at progenitor and differentiated states, were continuously treated with a non-cytotoxic concentration of 1 μM sodium arsenite (NaAsO2). The HepaRG cells demonstrated active intracellular arsenite metabolism that shared important characteristic with primary human hepatocytes. Treatment of proliferating progenitor-like HepaRG cells with NaAsO2 inhibited their differentiation into mature hepatocyte-like cells, up-regulated genes involved in cell growth, proliferation, and survival, and down-regulated genes involved in cell death. In contrast, treatment of differentiated hepatocyte-like HepaRG cells with NaAsO2 resulted in enhanced cell death of mature hepatocyte-like cells, over-expression of cell death-related genes, and down-regulation of genes in the cell proliferation pathway, while biliary-like cells remained largely unaffected. Mechanistically, the cytotoxic effect of arsenic on mature hepatocyte-like HepaRG cells may be attributed to arsenic-induced dysregulation of cellular iron metabolism. The inhibitory effect of NaAsO2 on the differentiation of progenitor cells, the resistance of biliary-like cells to cell death, and the enhanced cell death of functional hepatocyte-like cells resulted in stem-cell activation. These effects favored the proliferation of liver progenitor cells that can serve as a source of initiation and driving force of arsenic-mediated liver carcinogenesis.

2017

• Mixture effects of two plant protection products in liver cell lines.Zahn E,  Wolfrum J,  Knebel C,  Heise T,  Weiß F,  Poetz O,  Marx-Stoelting P,  Rieke S
  Food Chem Toxicol, Dec 2017
  Abstract : Pesticide products contain one or more active substances as well as adjuvants, which are added for example as solvents or antioxidants. Nevertheless, only the active substances are evaluated with a comprehensive battery of mammalian toxicity tests. However, in some cases mixture effects of active substances and adjuvants may occur, leading to increased toxicity of the products. To address this issue, we investigated effects of active substances with known hepatotoxicity and two commonly used fungicides: Priori Xtra® and Adexar®. For this purpose, respective active substances individually and in combination as well as the products were applied to two human hepatoma cell lines (HepaRG and HepG2) in a broad dose range. The results of cytotoxicity analysis, nuclear receptor transactivation (AhR, CAR, PXR), mRNA and protein expression of xenobiotic metabolizing enzymes (CYP1A1, CYP2B6 and CYP3A4) allow the conclusion that active substances and plant protection products differ in terms of their in vitro toxicity. The products activate AhR, while the individual active substances as well as the combination of the active substances have no or only minor effects. The present results support the hypothesis that plant protection products may have a modified toxicity as compared to active substances alone, consequently requiring more comprehensive testing.
• Interferon sensitivity-determining region of hepatitis C virus influences virus production and interferon signaling.Sugiyama R,  Murayama A,  Nitta S,  Yamada N,  Tasaka-Fujita M,  Masaki T,  Aly HH,  Shiina M,  Ryo A,  Ishii K,  Wakita T,  Kato T
  Oncotarget., Dec 2017
  Abstract : The number of amino acid substitutions in the interferon (IFN) sensitivity-determining region (ISDR) of hepatitis C virus (HCV) NS5A is a strong predictor for the outcome of IFN-based treatment. To assess the involvement of ISDR in the HCV life cycle and to clarify the molecular mechanisms influencing IFN susceptibility, we used recombinant JFH-1 viruses with NS5A of the genotype 1b Con1 strain (JFH1/5ACon1) and with NS5A ISDR containing 7 amino acid substitutions (JFH1/5ACon1/i-7mut), and compared the virus propagation and the induction of interferon-stimulated genes (ISGs). By transfecting RNAs of these strains into HuH-7-derived cells, we found that the efficiency of infectious virus production of JFH1/5ACon1/i-7mut was attenuated compared with JFH1/5ACon1. After transfecting full-length HCV RNA into HepaRGcells, the mRNA expression of ISGs was sufficiently induced by IFN treatment in JFH1/5ACon1/i-7mut-transfected but not in JFH1/5ACon1-transfected cells. These data suggested that the NS5A-mediated inhibition of ISG induction was deteriorated by amino acid substitutions in the ISDR. In conclusion, using recombinant JFH-1 viruses, we demonstrated that HCV NS5A is associated with infectious virus production and the inhibition of IFN signaling, and amino acid substitutions in the NS5A ISDR deteriorate these functions. These observations explain the strain-specific evasion of IFN signaling by HCV.
• Adduction to arginine detoxifies aflatoxin B1 by eliminating genotoxicity and altering in vitro toxicokinetic profiles.Rushing BR, Selim MI
  Oncotarget., Dec 2017
  Abstract : Aflatoxin B1 (AFB1), a class 1 carcinogen and prominent food contaminant, is highly linked to the development of hepatocellular carcinoma (HCC) and plays a causative role in a large portion of global HCC cases. We have demonstrated that a mixture of common organic acids (citric and phosphoric acid) along with arginine can eliminate >99% of AFB1 in solution as well as on corn kernels and convert it to the AFB2a-Arg adduct, acting as a potential detoxification process for contaminated foods. Evaluation of toxicokinetic changes after AFB2a-Arg formation show that the product is highly stable in biological fluids, is not metabolized by P450 enzymes, is highly plasma protein bound, has low lipid solubility, and has poor intestinal permeability/high intestinal efflux compared to AFB1. Ames' test results show that at mutagenic concentrations of AFB1, AFB2a-Arg does not have any measurable mutagenic effect which was confirmed by DNA adduct identification by liquid chromatography-mass spectrometry. Evaluation in HepG2 and HepaRG cells showed that AFB2a-Arg did not cause any significant decreases in cell viability nor did it increase micronuclei formation when administered at toxic concentrations of AFB1. These results show that conversion of AFB1 to AFB2a-Arg is a potential strategy to detoxify contaminated foods.
• Toll-Like Receptor 7 Agonist GS-9620 Induces Prolonged Inhibition of HBV via a Type I Interferon-Dependent Mechanism.Niu C,  Li L1,  Daffis S,  Lucifora J,  Bonnin M,  Maadadi S,  Salas E,  Chu R,  Ramos H,  Livingston CM,  Beran RK,  Garg AV,  Balsitis S
  J Hepatol, Dec 2017
  Abstract : GS-9620, an oral agonist of toll-like receptor 7 (TLR7), is in clinical development for the treatment of chronic hepatitis B (CHB). GS-9620 was previously shown to induce prolonged suppression of serum viral DNA and antigens in the woodchuck and chimpanzee models of CHB. Here we investigated the molecular mechanisms that contribute to the antiviral response to GS-9620 using in vitro models of hepatitis B virus (HBV) infection. Cryopreserved primary human hepatocytes (PHH) and differentiated HepaRG (dHepaRG) cells were infected with HBV and treated with GS-9620, conditioned media from human peripheral blood mononuclear cells (PBMCs) treated with GS-9620 (GS-9620 conditioned media; GS-9620-CM), or other innate immune stimuli. The antiviral and transcriptional response to these agents was determined. GS-9620 had no antiviral activity in HBV-infected PHH, consistent with low level TLR7 mRNA expression in human hepatocytes. In contrast, GS-9620-CM induced prolonged reduction of HBV DNA, RNA, and antigen levels in PHH and dHepaRG cells via a type I interferon (IFN)-dependent mechanism. GS-9620-CM did not reduce cccDNA levels in either cell type. Transcriptional profiling demonstrated that GS-9620-CM strongly induced various HBV restriction factors - although not APOBEC3A or the Smc5/6 complex - and indicated that established HBV infection does not modulate innate immune sensing or signaling in cryopreserved PHH. GS-9620-CM also induced expression of immunoproteasome subunits and enhanced presentation of an immunodominant viral peptide in HBV-infected PHH. CONCLUSIONS: Type I IFN induced by GS-9620 durably suppressed HBV in human hepatocytes without reducing cccDNA levels. Moreover, HBV antigen presentation was enhanced, suggesting additional components of the TLR7-induced immune response played a role in the antiviral response to GS-9620 in animal models of CHB. LAY SUMMARY: GS-9620 is a drug in clinical trials for the treatment of chronic hepatitis B virus (HBV) infection. GS-9620 has previously been shown to suppress HBV in various animal models, but the underlying antiviral mechanisms were not completely understood. In this study, we determined that GS-9620 does not directly activate antiviral pathways in human liver cells, but can induce prolonged suppression of HBV via induction of an antiviral cytokine called interferon. However, interferon did not destroy the HBV genome, suggesting that other parts of the immune response (e.g. activation of immune cells that kill infected cells) also play an important role in the antiviral response to GS-9620.
• Use of HuH6 and other human-derived hepatoma lines for the detection of genotoxins: a new hope for laboratory animals?Waldherr M,  Miík M,  Ferk F,  Tomc J,  Zegura B,  Filipi M,  Mikulits W,  Mai S,  Haas O,  Huber WW,  Haslinger E1,  Knasmüller S
  Arch Toxicol, Dec 2017
  Abstract : Cell lines which are currently used in genotoxicity tests lack enzymes which activate/detoxify mutagens. Therefore, rodent-derived liver preparations are used which reflect their metabolism in humans only partly; as a consequence misleading results are often obtained. Previous findings suggest that certain liver cell lines express phase I/II enzymes and detect promutagens without activation; however, their use is hampered by different shortcomings. The aim of this study was the identification of a suitable cell line. The sensitivity of twelve hepatic cell lines was investigated in single cell gel electrophoresis assays. Furthermore, characteristics of these lines were studied which are relevant for their use in genotoxicity assays (mitotic activity, p53 status, chromosome number, and stability). Three lines (HuH6, HCC1.2, and HepG2) detected representatives of five classes of promutagens, namely, IQ and PhIP (HAAs), B(a)P (PAH), NDMA (nitrosamine), and AFB1(aflatoxin), and were sensitive towards reactive oxygen species (ROS). In contrast, the commercially available line HepaRG, postulated to be a surrogate for hepatocytes and an ideal tool for mutagenicity tests, did not detect IQ and was relatively insensitive towards ROS. All other lines failed to detect two or more compounds. HCC1.2 cells have a high and unstable chromosome number and mutated p53, these features distract from its use in routine screening. HepG2 was frequently employed in earlier studies, but pronounced inter-laboratory variations were observed. HuH6 was never used in genotoxicity experiments and is highly promising, it has a stable karyotype and we demonstrated that the results of genotoxicity experiments are reproducible.
• In vitro co-culture models to evaluate acute cytotoxicity of individual and combined mycotoxin exposures on Caco-2, THP-1 and HepaRG human cell lines.Smith MC,  Gheux A,  Coton M,  Madec S,  Hymery N,  Coton E
  Chemical Bio Interact, Dec 2017
  Abstract : Deoxynivalenol (DON) and zearalenone (ZEA) are mycotoxins primarily produced by Fusarium species and commonly co-occur in European grains. Some in vitro studies reported synergistic combined effects on cell viability reduction for these two natural food contaminants. However, most of these studies were carried out on conventional cell culture systems involving only one cell type and thus, did not accurately reflect in vivo conditions, including cell-cell communication. In this context, we developed easy bi- and tri-culture systems using the Caco-2 (intestinal epithelial cells), THP-1 (monocytes) and HepaRG (hepatocytes) human cell lines. Individual and combined cytotoxicity effects of DON and ZEA were then assessed using co-cultures during 48 h. In bi-culture systems, results showed that only the highest tested dose of ZEA (IC30) induced a significant reduction in THP-1 viability with both Caco-2 and HepaRG cells cultured in transwells above. On the contrary, only the highest tested dose of DON (IC30) significantly affected HepaRG cell viability located under the Caco-2 cell monolayer. In addition, the DON + ZEA combination seemed to induce higher cytotoxicity than each toxin alone. Mycotoxin quantification in the abluminal compartment by QTOF LC-MS suggested uptake of both mycotoxins by the different cell lines, and depending on the co-culturing cell type, thus indicating possible cell-cell interactions. Finally, in the tri-culture system, no cytotoxic effects were observed, regardless of the treatment. These findings highlighted the importance of the proposed models to better decipher toxicological impacts of mycotoxins on more complex cellular systems.
• Differential impacts of individual and combined exposures of deoxynivalenol and zearalenone on the HepaRG human hepatocyte proteome.Smith MC,  Timmins-Schiffman E,  Coton M,  Coton E,  Hymery N,  Nunn BL,  Madec S
  J Proteomics, Dec 2017
  Abstract : Numerous surveys have highlighted the natural co-occurrence of deoxynivalenol (DON) and zearalenone (ZEA) mycotoxins in food and feed. Nevertheless, data regarding cellular mechanisms involved in response to their individual and simultaneous exposures are lacking. In this study, in order to analyze how low mycotoxin doses could impact cellular physiology and homeostasis, proteomic profiles of proliferating human hepatocytes (HepaRG) exposed for 1h and 24h to low DON and ZEA cytotoxicity levels (0.2 and 20μM respectively), alone or in combination, were analyzed by LC-MS/MS. Proteome analyses of mycotoxin-treated cells identified 4000 proteins with about 1.4% and 3.7% of these proteins exhibiting a significantly modified abundance compared to controls after 1h or 24h, respectively. Analysis of the Gene Ontology biological process annotations showed that cell cycle, proliferation and/or development as well as on DNA metabolic processes were affected for most treatments. Overall, different proteins, and thus biological processes, were impacted depending on the considered mycotoxin and exposure duration. Finally, despite the important proteome changes observed following 24h exposure to both mycotoxins, only the uptake of ZEA by the cells was suggested by the mycotoxin quantification in cell supernatants. BIOLOGICAL SIGNIFICANCE: This study investigated the proteomic changes that occurred after DON and ZEA (individually and in combination) short exposures at low cytotoxicity levels in proliferating HepaRG cells using LC-MS/MS. The obtained results showed that the cellular response is time- and mycotoxin or mixture-dependent. In particular, after 1h exposure, the DON+ZEA combination led to more proteomic changes than DON or ZEA alone, whereas the opposite was observed after 24h. In addition, the significant cellular response to stress induced by ZEA after 24h exposure seemed to be reduced when combined with DON. Thus, these results supported a possible mitigation by the hepatocytes when exposed to the mycotoxin mixture for a long duration. These findings represent an essential step to further explore adaptive cell response to mycotoxin exposure using with more complex incubation kinetics and combining different "omics" tools. Moreover, as mycotoxin quantification in cell supernatants showed different behaviors for DON and ZEA, this also raises the question about how mycotoxins actually trigger the cell response.
• Modulation of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) activation by ursolic acid (UA) attenuates rifampin-isoniazid cytotoxicity.Chang HY,  Chen CJ,  Ma WC,  Cheng WK,  Lin YN,  Lee YR,  Chen JJ,  Lim YP
  Phytomedicine, Dec 2017
  Abstract : BACKGROUND: Interactions between transcriptional inducers of cytochrome P450 (CYP450) enzymes and therapeutic drugs may be prevented by antagonizing the activation of a nuclear receptor (NR), pregnane X receptor (PXR, NR1I2), thus improving therapeutic efficacy. PURPOSE: In the present study, we aim to identify that ursolic acid (UA), a widely distributed pentacyclic triterpene, may act as an effective antagonist of PXR and its sister NR receptor, constitutive androstane receptor (CAR, NR1I3). METHODS: The hepatocellular carcinoma cell line, HepG2, was used to evaluate the promoter activity of PXR and CAR target genes, CYP3A4 and CYP2B6, respectively. Catalytic activities, mRNA, and protein expression of CYP3A4 and CYP2B6 were evaluated in a differentiated HepaRG cell line. Coregulation of PXR with coregulators on CYP3A4 promoter response elements was also been characterized. RESULTS: Transient transfection assays showed that UA effectively attenuated CYP3A4 and CYP2B6 promoter activities mediated by rifampin (RIF, human PXR agonist) and CITCO (human CAR agonist). These inhibitory effects were well correlated with the expression and catalytic activities of CYP3A4 and CYP2B6. Furthermore, the interaction of co-regulators with PXR and the transcriptional complexes in the CYP3A4 promoter activity and CYP3A4 promoter xenobiotic response element (everted repeat 6, ER6), respectively, were disrupted in the presence of UA. UA showed an antagonistic effect against PXR, and reversed the cytotoxic effects of isoniazid (INH) induced by RIF. Taken together, these results show that UA inhibits the transactivation effects of PXR and CAR, and reduces the expression and function of CYP3A4 and CYP2B6. CONCLUSION: The present study suggests that UA could be a powerful agent for reducing potentially dangerous interactions between transcriptional inducers of CYP enzymes and therapeutic drugs.
• A multi-throughput multi-organ-on-a-chip system on a plate formatted pneumatic pressure-driven medium circulation platform.Satoh T,  Sugiura S,  Shin K,  Onuki-Nagasaki R,  Ishida S,  Kikuchi K,  Kakiki M,  Kanamori T
  Lab Chip, Nov 2017
  Abstract : This paper reports a multi-throughput multi-organ-on-a-chip system formed on a pneumatic pressure-driven medium circulation platform with a microplate-sized format as a novel type of microphysiological system. The pneumatic pressure-driven platform enabled parallelized multi-organ experiments (i.e. simultaneous operation of multiple multi-organ culture units) and pipette-friendly liquid handling for various conventional cell culture experiments, including cell seeding, medium change, live/dead staining, cell growth analysis, gene expression analysis of collected cells, and liquid chromatography-mass spectrometry analysis of chemical compounds in the culture medium. An eight-throughput two-organ system and a four-throughput four-organ system were constructed on a common platform, with different microfluidic plates. The two-organ system, composed of liver and cancer models, was used to demonstrate the effect of an anticancer prodrug, capecitabine (CAP), whose metabolite 5-fluorouracil (5-FU) after metabolism by HepaRG hepatic cells inhibited the proliferation of HCT-116 cancer cells. The four-organ system, composed of intestine, liver, cancer, and connective tissue models, was used to demonstrate evaluation of the effects of 5-FU and two prodrugs of 5-FU (CAP and tegafur) on multiple organ models, including cancer and connective tissue.
• DYRK1A is a regulator of S phase entry in hepatic progenitor cells.Kruitwagen HS,  Westendorp B,  Viebahn CS,  Post K,  van Wolferen ME,  Oosterhoff LA,  Egan DA,  Delabar JM,  Toussaint MJ,  Schotanus BA,  de Bruin A,  Rothuizen J,  Penning LC
  Stem Cells Dev., Nov 2017
  Abstract : Hepatic progenitor cells (HPCs) are adult liver stem cells that act as second line of defense in liver regeneration. They are normally quiescent, but in case of severe liver damage HPC proliferation is triggered by external activation mechanisms from their niche. Although several important pro-proliferative mechanisms have been described, it is not known which key intracellular regulators govern the switch between HPC quiescence and active cell cycle. We performed a high throughput kinome siRNA screen in HepaRG cells, a HPC-like cell line, and evaluated the effect on proliferation with a 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay. One hit increased the percentage of EdU-positive cells after knockdown: dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A). Although upon DYRK1A silencing the percentage of EdU and phosphorylated histone H3 positive cells was increased, total cell numbers were not increased, possibly through a subsequent delay in cell cycle progression. This phenotype was confirmed with chemical inhibition of DYRK1A using harmine and with primary HPCs cultured as liver organoids. DYRK1A inhibition impaired Dimerization Partner, RB-like, E2F and multi-vulva class B (DREAM) complex formation in HPCs and abolished its transcriptional repression on cell cycle progression. To further analyze DYRK1A function in HPC proliferation, liver organoid cultures were established from mBACtgDyrk1A mice, which harbor one extra copy of the murine Dyrk1a gene (Dyrk+++). Dyrk+++ organoids had both a reduced percentage of EdU-positive cells and reduced proliferation compared to wildtype organoids. This study provides evidence for an essential role of DYRK1A as balanced regulator of S phase entry in HPCs. An exact gene dosage is crucial, as both DYRK1A deficiency and overexpression affect HPC cell cycle progression.
• Endoplasmic reticulum stress precedes oxidative stress in antibiotic-induced cholestasis and cytotoxicity in human hepatocytes.Burban A,  Sharanek A,  Guguen-Guillouzo C,  Guillouzo A
  Free Radic Biol Med, Nov 2017
  Abstract : Endoplasmic reticulum (ER) stress has been associated with various drug-induced liver lesions but its participation in drug-induced cholestasis remains unclear. We first aimed at analyzing liver damage caused by various hepatotoxic antibiotics, including three penicillinase-resistant antibiotics (PRAs), i.e. flucloxacillin, cloxacillin and nafcillin, as well as trovafloxacin, levofloxacin and erythromycin, using human differentiated HepaRG cells and primary hepatocytes. All these antibiotics caused early cholestatic effects typified by bile canaliculi dilatation and reduced bile acid efflux within 2h and dose-dependent enhanced caspase-3 activity within 24h. PRAs induced the highest cholestatic effects at non cytotoxic concentrations. Then, molecular events involved in these lesions were analyzed. Early accumulation of misfolded proteins revealed by thioflavin-T fluorescence and associated with phosphorylation of the unfolded protein response sensors, eIF2α and/or IRE1α, was evidenced with all tested hepatotoxic antibiotics. Inhibition of ER stress markedly restored bile acid efflux and prevented bile canaliculi dilatation. Downstream of ER stress, ROS were also generated with high antibiotic concentrations. The protective HSP27-PI3K-AKT signaling pathway was activated only in PRA-treated cells and its inhibition increased ROS production and aggravated caspase-3 activity. Overall, our results demonstrate that (i) various antibiotics reported to cause cholestasis and hepatocellular injury in the clinic can also induce such effects in in vitro human hepatocytes; (ii) PRAs cause the strongest cholestatic effects in the absence of cytotoxicity; (iii) cholestatic features occur early through ER stress; (iv) cytotoxic lesions are observed later through ER stress-mediated ROS generation; and (v) activation of the HSP27-PI3K-AKT pathway protects from cytotoxic damage induced by PRAs only.
• In vitro characterization of NPS metabolites produced by human liver microsomes and the HepaRG cell line using liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis: application to furanyl fentanyl.Gaulier JM,  Richeval C,  Gicquel T,  Hugbart C,  Le Dare B,  Allorge D,  Morel I
  Curr Pharm Biotechnol, Nov 2017
  Abstract : BACKGROUND: Identification of metabolites is of importance in the challenge of new psychoactive substances (NPS) as it could improve detection window in biological matrices in clinical and forensic cases of intoxication. Considering the numerous and diverse NPS reported each year, producers increasingly appear today to be targeting non-controlled synthetic opioids, involving fentanyl derivatives such as furanyl fentanyl (Fu-F). OBJECTIVE: This work aims to investigate and compare metabolites of Fu-F using two in vitro experimental approaches. METHODS: CYP- and UGT-dependent metabolites of Fu-F were investigated by means of analyses of both human liver microsome (HLM) and hepatic (HepaRG) cell line incubates using liquid chromatography with high-resolution mass detection and, subsequently, compared and confronted to recently published data. RESULTS: Seventeen Fu-F metabolites were produced and several metabolic pathways can be postulated. HLMs and HepaRG cultures appear to be complementary: HepaRG cells produced 9 additional metabolites, but which appear to be minor in vivo metabolites. Specific* and/or abundant Fu-F metabolites are dihydrodiol-Fu-F*, norFu-F* and despropionylfentanyl. However, norFu-F seems to be inconstantly observed in in vivo cases. Furthermore, a sulfate metabolite present at significant rate in urine users was not identified here, as in another in vitro study. CONCLUSION: HLMs represent an acceptable first choice tool for a single NPS metabolism study in forensic laboratories. Dihydrodiol-Fu-F and despropionylfentanyl could be proposed as reliable metabolites to be recorded in HRMS libraries in order to improve detection of Fu-F users, although additional confrontations to in vivo data remain necessary to confirm relevant blood and urinary metabolites of Fu-F.
• The JAK1/2 inhibitor ruxolitinib reverses interleukin-6-mediated suppression of drug detoxifying proteins in cultured human hepatocytes.Febvre-James M,  Bruyere A,  Le Vee M,  Fardel O
  Drug Metab Dispos, Nov 2017
  Abstract : The inflammatory cytokine interleukin (IL)-6, which basically activates the JAK/STAT signaling pathway, is well-known to repress expression of hepatic cytochromes P-450 (CYPs) and transporters. Therapeutic proteins, like mAbs targeting IL-6 or its receptor, have been consequently demonstrated to restore full hepatic detoxification capacity, which results in inflammatory disease-related drug-drug interactions (idDDIs). In the present study, we investigated whether ruxolitinib, a small drug acting as a JAK1/2 inhibitor and currently used in the treatment of myeloproliferative neoplasms, may also counteract IL-6 repressing effects towards hepatic detoxifying systems. Ruxolitinib was found to fully inhibit IL-6-mediated repression of CYP (CYP1A2, CYP2B6 and CYP3A4) and transporter (NTCP, OATP1B1 and OCT1) mRNA levels in primary human hepatocytes and differentiated hepatoma HepaRG cells. Such effects were dose-dependent, with ruxolitinib EC50 values around 1.0-1.2 μM and thus close to ruxolitinib plasma levels which can be reached in patients. Moreover, they were associated with concomitant restoration of CYP and drug transporter activities in IL-6-exposed HepaRG cells. By contrast, ruxolitinib failed to suppress the repression of drug detoxifying protein mRNA levels caused by IL-1β. The JAK inhibitor and anti-rhumatoid arthritis compound tofacitinib was additionally found to reverse IL-6-mediated suppression of CYP and transporter mRNA expressions. Taken together, our results demonstrated that small drugs acting as JAK inhibitors, like ruxolitinib, counteract IL-6-mediated repression of drug metabolizing enzymes and drug transporters in cultured human hepatocytes. These JAK inhibitors may consequently be hypothesized to restore hepatic detoxification capacity in vivo for patients suffering from inflammatory diseases, which may in turn cause idDDIs.
• Reduced hepatitis B and D viral entry using clinically applied drugs as novel inhibitors of the bile acid transporter NTCP.Donkers JM,  Zehnder B,  van Westen GJP,  Kwakkenbos MJ,  IJzerman AP,  Oude Elferink RPJ,  Beuers U,  Urban S,  van de Graaf SFJ
  Sci Rep, Nov 2017
  Abstract : The sodium taurocholate co-transporting polypeptide (NTCP, SLC10A1) is the main hepatic transporter of conjugated bile acids, and the entry receptor for hepatitis B virus (HBV) and hepatitis delta virus (HDV). Myrcludex B, a synthetic peptide mimicking the NTCP-binding domain of HBV, effectively blocks HBV and HDV infection. In addition, Myrcludex B inhibits NTCP-mediated bile acid uptake, suggesting that also other NTCP inhibitors could potentially be a novel treatment of HBV/HDV infection. This study aims to identify clinically-applied compounds intervening with NTCP-mediated bile acid transport and HBV/HDV infection. 1280 FDA/EMA-approved drugs were screened to identify compounds that reduce uptake of taurocholic acid and lower Myrcludex B-binding in U2OS cells stably expressing human NTCP. HBV/HDV viral entry inhibition was studied in HepaRG cells. The four most potent inhibitors of human NTCP were rosiglitazone (IC50 5.1 µM), zafirlukast (IC50 6.5 µM), TRIAC (IC50 6.9 µM), and sulfasalazine (IC50 9.6 µM). Chicago sky blue 6B (IC50 7.1 µM) inhibited both NTCP and ASBT, a distinct though related bile acid transporter. Rosiglitazone, zafirlukast, TRIAC, sulfasalazine, and chicago sky blue 6B reduced HBV/HDV infection in HepaRG cells in a dose-dependent manner. Five out of 1280 clinically approved drugs were identified that inhibit NTCP-mediated bile acid uptake and HBV/HDV infection in vitro.
• The tripartite motif containing 24 acts as a novel coactivator of the constitutive active/androstane receptor.Kanno Y, Kure Y, Kobayashi S, Mizuno M, Tsuchiya Y, Yamashita N, Nemoto K, Inouye Y
  Drug Metab Dispos, Nov 2017
  Abstract : The constitutive active/androstane receptor (CAR) is a nuclear receptor that acts as a transcription factor for a variety of genes, including genes encoding xenobiotic, steroid and drug-metabolizing enzymes and transporters. Transactivation of a target gene by a transcription factor is generally mediated through the concerted and stepwise recruitment of various proteins termed co-regulators, including coactivators and corepressors. In this study, tripartite motif containing 24 (TRIM24; also known as transcriptional intermediary factor 1 alpha) was found to interact with the CAR. TRIM24 enhanced the CAR-dependent transactivation in reporter assays using the direct repeat-4 (DR4) motif, a binding site of the CAR. This enhancement was synergistically augmented in the presence of SRC1 or SRC2, both of which are coactivators of the CAR. In addition, TRIM24 was recruited to the CAR binding element of the CYP2B6 promoter together with the CAR. We also noted that knockdown of TRIM24 suppressed the CAR-induced CYP2B6 mRNA expression in HepTR/CAR and HepaRG cells and suppressed CAR-induced CYP3A4 mRNA expression in HepaRG cells but not HepTR/CAR cells. From these results, we suggest that TRIM24 is a novel coactivator of the CAR that is involved in cell- and/or promoter- selective transactivation.
• Three-Dimensional (3D) HepaRG Spheroid Model With Physiologically Relevant Xenobiotic Metabolism Competence and Hepatocyte Functionality for Liver Toxicity ScreeningRamaiahgari SC, Waidyanatha S, Dixon D, DeVito MJ, Paules RS, Ferguson SS.
  Toxicol Sci, Nov 2017
  Abstract : In the MATERIALS AND METHODS section (subsection Assessment of cytotoxicity), we stated the concentrations of all the compounds used: “All the compounds were obtained from Sigma–Aldrich, St. Louis, MO; concentrations used were shown in Supplementary Table 5.” In Supplementary Table 5, we correctly described the exposure range for acetaminophen ranging from 50 mM down to 50 µM. The RESULTS section text also correctly stated, “Acetaminophen showed a TC50 of approximately 6.1 mM (HepaRG spheroids) and approximately 5.8 mM (PHHs) after 24-h exposures, while repeated exposure of 3D HepaRG spheroids produced significantly more potent response with TC50 of approximately 2.9 mM”. However, in Figure 7, which corresponds to the text referenced above, a copy/paste error occurred for one of the curve overlays. In the final version, troglitazone “dose-response image” was copied twice in Photoshop. The correct version of the figure is shown below.
• Heparin at physiological concentration can enhance PEG-free in vitro infection with human hepatitis B virusChoijilsuren G, Jhou RS, Chou SF, Chang CJ, Yang HI, Chen YY, Chuang WL, Yu ML, Shih C.
  Sci Rep, Oct 2017
  Abstract : Hepatitis B virus (HBV) is a blood-borne pathogen responsible for chronic hepatitis, cirrhosis, and liver cancer. The mechanism of HBV entry into hepatocytes remains to be investigated. Recently, sodium taurocholate cotransporting polypeptide (NTCP) was discovered as a major HBV receptor based on an in vitro infection system using NTCP-reconstituted HepG2 cells. However, this infection system relies on the compound polyethylene glycol (4% PEG), which is not physiologically relevant to human infection. High concentration of heparin has been commonly used as an inhibitor control for in vitro infection in the field. Surprisingly, we found that heparin at physiological concentration can enhance HBV infection in a PreS1-peptide sensitive, NTCP-dependent manner in both HepaRG and HepG2-NTCP-AS cells. O-sulfation of heparin is more important for the infection enhancement than N-sulfation. This system based on the HepG2-NTCP-AS cells can support in vitro infection with HBV genotypes B and C, as well as using serum samples from HBeAg positive and negative chronic carriers. In summary, our study provides a PEG-free infection system closely resembling human natural infection. In addition, it points to a future research direction for heparin and heparin-binding host factor(s) in the blood, which are potentially involved in viral entry. To our knowledge, this is the first soluble and circulatory host factor which can enhance HBV in vitro infection.
• Elasticity-based Development of Functionally Enhanced Multicellular 3D Liver Encapsulated in Hybrid Hydrogel.Lee HJ, Son MJ, Ahn J, Oh SJ, Lee M, Kim A, Jeung YJ, Kim HG, Won M, Lim JH, Kim NS, Jung CR, Chung KS.
  Acta Biomater, Oct 2017
  Abstract : Current in vitro liver models provide three-dimensional (3-D) microenvironments in combination with tissue engineering technology and can perform more accurate in vivo mimicry than two-dimensional models. However, a human cell-based, functionally mature liver model is still desired, which would provide an alternative to animal experiments and resolve low-prediction issues on species differences. Here, we prepared hybrid hydrogels of varying elasticity and compared them with a normal liver, to develop a more mature liver model that preserves liver properties in vitro. We encapsulated HepaRG cells, either alone or with supporting cells, in a biodegradable hybrid hydrogel. The elastic modulus of the 3D liver dynamically changed during culture due to the combined effects of prolonged degradation of hydrogel and extracellular matrix formation provided by the supporting cells. As a result, when the elastic modulus of the 3D liver model converges close to that of the in vivo liver (≅ 2.3 to 5.9 kPa), both phenotypic and functional maturation of the 3D liver were realized, while hepatic gene expression, albumin secretion, cytochrome p450-3A4 activity, and drug metabolism were enhanced. Finally, the 3D liver model was expanded to applications with embryonic stem cell-derived hepatocytes and primary human hepatocytes, and it supported prolonged hepatocyte survival and functionality in long-term culture. Our model represents critical progress in developing a biomimetic liver system to simulate liver tissue remodeling, and provides a versatile platform in drug development and disease modeling, ranging from physiology to pathology. We provide a functionally improved 3D liver model that recapitulates in vivo liver stiffness. We have experimentally addressed the issues of orchestrated effects of mechanical compliance, controlled matrix formation by stromal cells in conjunction with hepatic differentiation, and functional maturation of hepatocytes in a dynamic 3D microenvironment. Our model represents critical progress in developing a biomimetic liver system to simulate liver tissue remodeling, and provides a versatile platform in drug development and disease modeling, ranging from physiology to pathology. Additionally, recent advances in the stem-cell technologies have made the development of 3D organoid possible, and thus, our study also provides further contribution to the development of physiologically relevant stem-cell-based 3D tissues that provide an elasticity-based predefined biomimetic 3D microenvironment.
• Genotoxic effects of food contact recycled paperboard extracts on two human hepatic cell linesSouton E, Severin I, LE Hegarat L, Hogeveen K, Aljawish A, Fessard V, Marie-Christine C.
  Food Addit Contam Part A Chem Anal Control Expo Risk Assess, Oct 2017
  Abstract : Food contact paperboards may be a potential source of food contamination as they can release chemicals (intentionally added or not), especially recycled paperboards. In this study, we assessed the in vitro genotoxicity of food contact paperboard samples from a manufacturer, collected at the beginning and at the end of a recycling production chain. Samples were extracted in water to mimic a wet food contact. Different genotoxic endpoints were evaluated in two human hepatic cell lines (HepG2 and HepaRG) using bioassays: γH2AX and p53 activation, primary DNA damage with the comet assay and micronucleus formation. We found that the samples from the beginning and the end of the production chain induced, with the same potency, γH2AX and p53-ser15 activation and DNA damage with the comet assay. The micronucleus assay was negative with the paperboard extract from the beginning of the chain, whereas positive data were observed for the end paperboard extract. Our resu
• Mechanisms of toxicity associated with six tyrosine kinase inhibitors in human hepatocyte cell lines.Mingard C, Paech F, Bouitbir J, Krähenbühl S.
  J Appl Toxicol, Oct 2017
  Abstract : Tyrosine kinase inhibitors have revolutionized the treatment of certain cancers. They are usually well tolerated, but can cause adverse reactions including liver injury. Currently, mechanisms of hepatotoxicity associated with tyrosine kinase inhibitors are only partially clarified. We therefore aimed at investigating the toxicity of regorafenib, sorafenib, ponatinib, crizotinib, dasatinib and pazopanib on HepG2 and partially on HepaRG cells. Regorafenib and sorafenib strongly inhibited oxidative metabolism (measured by the Seahorse-XF24 analyzer) and glycolysis, decreased the mitochondrial membrane potential and induced apoptosis and/or necrosis of HepG2 cells at concentrations similar to steady-state plasma concentrations in humans. In HepaRG cells, pretreatment with rifampicin decreased membrane toxicity (measured as adenylate kinase release) and dissipation of adenosine triphosphate stores, indicating that toxicity was associated mainly with the parent drugs. Ponatinib strongly impaired oxidative metabolism but only weakly glycolysis, and induced apoptosis of HepG2 cells at concentrations higher than steady-state plasma concentrations in humans. Crizotinib and dasatinib did not significantly affect mitochondrial functions and inhibited glycolysis only weakly, but induced apoptosis of HepG2 cells. Pazopanib was associated with a weak increase in mitochondrial reactive oxygen species accumulation and inhibition of glycolysis without being cytotoxic. In conclusion, regorafenib and sorafenib are strong mitochondrial toxicants and inhibitors of glycolysis at clinically relevant concentrations. Ponatinib affects mitochondria and glycolysis at higher concentrations than reached in plasma (but possibly in liver), whereas crizotinib, dasatinib and pazopanib showed no relevant toxicity. Mitochondrial toxicity and inhibition of glycolysis most likely explain hepatotoxicity associated with regorafenib, sorafenib and possibly pazopanib, but not for the other compounds investigated.
• miR-122 protects mice and human hepatocytes from acetaminophen toxicity by regulating CYP1A2 and CYP2E1 expression.Chowdhary V, Teng KY, Thakral S, Zhang B, Lin CH, Wani N, Bruschweiler-Li L, Zhang X, James L, Yang D, Junge N, Brüschweiler R, Lee WM, Ghoshal K.
  Am J Pathol., Oct 2017
  Abstract : Acetaminophen (APAP) toxicity is a leading cause of acute liver failure (ALF). Here, we show that miR-122 is downregulated in liver biopsies of ALF patients and in APAP treated mice. A dramatic decrease in the primary-miR-122 expression occurs in mice upon APAP overdose due to suppression of its key trans-activators, HNF4α and HNF6. More importantly, the mortality rates of both male and female liver-specific miR-122 knockout (LKO) mice were significantly higher than control mice when injected intraperitoneally with an APAP dose not lethal to the control. LKO livers exhibited higher basal expression of CYP2E1 and CYP1A2 that convert APAP to highly reactive N-acetyl-p-benzoquinone imine (NAPQI). Upregulation of Cyp1a2 primary transcript and mRNA in LKO mice correlated with the elevation of AHR and MED1, two trans-activators of Cyp1a2. Analysis of ChIP-seq data in the ENCODE database identified association of CTCF with Ahr promoter in mouse livers. Both MED1 and CTCF are validated conserved miR-122 targets. Furthermore, depletion of Ahr, Med1 or Ctcf in Mir122-/- hepatocytes reduced Cyp1a2 expression. Pulse-chase studies showed that Cyp2e1 protein level is upregulated in LKO hepatocytes. Notably, miR-122 depletion sensitized differentiated human HepaRG cells to APAP toxicity that correlated with upregulation of AHR, MED1 and CYP1A2 expression. Collectively, these results demonstrate a critical role of miR-122 in acetaminophen detoxification and implicate its therapeutic potential in ALF patients.
• Multiple microRNAs function as self-protective modules in acetaminophen-induced hepatotoxicity in humansYu D, Wu L, Gill P, Tolleson WH, Chen S, Sun J, Knox B, Jin Y, Xiao 2, Hong H, Wang Y, Ren Z, Guo L, Mei N, Guo Y, Yang X, Shi L, Chen Y, Zeng L, Dreval K, Tryndyak V, Pogribny I, Fang H, Shi T, McCullough S, Bhattacharyya S, Schnackenberg L, Mattes W, Be
  Arch Toxicol, Oct 2017
  Abstract : Acetaminophen (APAP) overdose is the leading cause of acute liver failure. Yet the mechanisms underlying adaptive tolerance toward APAP-induced liver injury are not fully understood. To better understand molecular mechanisms contributing to adaptive tolerance to APAP is an underpinning foundation for APAP-related precision medicine. In the current study, the mRNA and microRNA (miRNA) expression profiles derived from next generation sequencing data for APAP-treated (5 and 10 mM) HepaRG cells and controls were analyzed systematically. Putative miRNAs targeting key dysregulated genes involved in APAP hepatotoxicity were selected using in silico prediction algorithms, un-biased gene ontology, and network analyses. Luciferase reporter assays, RNA electrophoresis mobility shift assays, and miRNA pull-down assays were performed to investigate the role of miRNAs affecting the expression of dysregulated genes. Levels of selected miRNAs were measured in serum samples obtained from children with APAP overdose (58.6-559.4 mg/kg) and from healthy controls. As results, 2758 differentially expressed genes and 47 miRNAs were identified. Four of these miRNAs (hsa-miR-224-5p, hsa-miR-320a, hsa-miR-449a, and hsa-miR-877-5p) suppressed drug metabolizing enzyme (DME) levels involved in APAP-induced liver injury by downregulating HNF1A, HNF4A and NR1I2 expression. Exogenous transfection of these miRNAs into HepaRG cells effectively rescued them from APAP toxicity, as indicated by decreased alanine aminotransferase levels. Importantly, hsa-miR-320a and hsa-miR-877-5p levels were significantly elevated in serum samples obtained from children with APAP overdose compared to health controls. Collectively, these data indicate that hsa-miR-224-5p, hsa-miR-320a, hsa-miR-449a, and hsa-miR-877-5p suppress DME expression involved in APAP-induced hepatotoxicity and they contribute to an adaptive response in hepatocytes.
• Hepatitis B Virus Sensitivity to Interferon-? in Hepatocytes is More Associated with Cellular Interferon Response than with Viral Genotype.Shen F, Li Y, Yang Wang, Sozzi V, Revill PA, Liu J, Gao L, Yang G, Lu M, Sutter K, Dittmer U, Chen J, Yuan Z.
  Hepatology, Oct 2017
  Abstract : Interferon-α (IFN-α) is used to treat chronic HBV infection but only 20-40% of patients respond well. Clinical observations have suggested that HBV genotype is associated with the response to IFN therapy, however, its role in viral responsiveness to IFN in HBV-infected hepatocytes remain unclear. Here, we produced infectious virions of HBV genotypes A to D to infect three well-recognized cell culture-based HBV infection systems including primary human hepatocytes (PHH), differentiated HepaRG (dHepaRG) and HepG2-NTCP cells to quantitatively compare the antiviral effect of IFN-α on HBV across genotypes and cell models. The efficacy of IFN-α against HBV in hepatocyte was generally similar across genotype A2, B5, C2 and D3, however, was significantly different among the infection models as the IC50 value of IFN-α for inhibition of viral DNA replication in PHH (<20 U/ml) and dHepaRG cells were much lower than that in HepG2-NTCP cells (> 500 U/ml). Notably, even in PHH, IFN-α did not reduce HBV cccDNA at the concentrations for which viral antigens and DNA replication intermediates were strongly reduced. The three cell culture models exhibited differential cellular response to IFN-α. The genes reported to be associated with responsiveness to IFN-α in patients were robustly induced in PHH while weakly induced in HepG2-NTCP cells upon IFN-α treatment. Reduction or promotion of IFN response in PHH or HepG2-NTCP cells significantly attenuated or improved the inhibitory capacity of IFN-α on HBV replication, respectively. CONCLUSION: In the cell culture-based HBV infection models, the sensitivity of HBV to IFN-α in hepatocytes is determined more by the cell-intrinsic interferon response than by viral genotype, and improvement of the IFN response in HepG2-NTCP cells promotes the efficacy of IFN-α against HBV. This article is protected by copyright. All rights reserved.
• Toward a new and noninvasive diagnostic method of papillary thyroid cancer by using peptide vectorized contrast agents targeted to galectin-1Fanfone D, Despretz N, Stanicki D, Rubio-Magnieto J, Fossépré M, Surin M, Rorive S, Salmon I, Vander Elst L, Laurent S, Muller RN, Saussez S, Burtea C.
  Med Oncol., Oct 2017
  Abstract : The incidence of papillary thyroid cancer has increased these last decades due to a better detection. High prevalence of nodules combined with the low incidence of thyroid cancers constitutes an important diagnostic challenge. We propose to develop an alternative diagnostic method to reduce the number of useless and painful thyroidectomies using a vectorized contrast agent for magnetic resonance imaging. Galectin-1 (gal-1), a protein overexpressed in well-differentiated thyroid cancer, has been targeted with a randomized linear 12-mer peptide library using the phage display technique. Selected peptides have been conjugated to ultrasmall superparamagnetic particles of iron oxide (USPIO). Peptides and their corresponding contrast agents have been tested in vitro for their specific binding and toxicity. Two peptides (P1 and P7) were selected according to their affinity toward gal-1. Their binding has been revealed by immunohistochemistry on human thyroid cancer biopsies, and they were co-localized with gal-1 by immunofluorescence on TPC-1 cell line. Both peptides induce a decrease in TPC-1 cells' adhesion to gal-1 immobilized on culture plates. After coupling to USPIO, the peptides preserved their affinity toward gal-1. Their specific binding has been corroborated by co-localization with gal-1 expressed by TPC-1 cells and by their ability to compete with anti-gal-1 antibody. The peptides and their USPIO derivatives produce no toxicity in HepaRG cells as determined by MTT assay. The vectorized contrast agents are potential imaging probes for thyroid cancer diagnosis. Moreover, the two gal-1-targeted peptides prevent cancer cell adhesion by interacting with the carbohydrate-recognition domain of gal-1.
• Biocompatibility assessment of functionalized magnetic mesoporous silica nanoparticles in human HepaRG cells.Pisani C, Rascol E, Dorandeu C, Charnay C, Guari Y, Chopineau J, Devoisselle JM, Prat O.
  Nanotoxicology, Sep 2017
  Abstract : Magnetic mesoporous silica nanoparticles (M-MSNs) are a promising class of nanoparticles for drug delivery. However, a deep understanding of the toxicological mechanisms of action of these nanocarriers is essential, especially in the liver. The potential toxicity on HepaRG cells of pristine, pegylated (PEG), and lipid (DMPC) M-MSNs were compared. Based on MTT assay and real-time cell impedance, none of these NPs presented an extensive toxicity on hepatic cells. However, we observed by transmission electron microscopy (TEM) that the DMPC and pristine M-MSNs were greatly internalized. In comparison, PEG M-MSNs showed a slower cellular uptake. Whole gene expression profiling revealed the M-MSNs molecular modes of action in a time- and dose-dependent manner. The lowest dose tested (1.6 µg/cm2) induced no molecular effect and was defined as 'No Observed Transcriptional Effect level.' The dose 16 µg/cm2 revealed nascent but transient effects. At the highest dose (80 µg/cm2), adverse effects have clearly arisen and increased over time. The limit of biocompatibility for HepaRG cells could be set at 16 µg/cm2 for these NPs. Thanks to a comparative pathway-driven analysis, we highlighted the sequence of events that leads to the disruption of hepatobiliary system, elicited by the three types of M-MSNs, at the highest dose. The Adverse Outcome Pathway of hepatic cholestasis was implicated. Toxicogenomics applied to cell cultures is an effective tool to characterize and compare the modes of action of many substances. We propose this strategy as an asset for upstream selection of the safest nanocarriers in the framework of regulation for nanobiosafety.
• Progressive and preferential cellular accumulation of hydrophobic bile acids induced by cholestatic drugs is associated with inhibition of their amidation and sulfation.Drug Metab Dispos.
  Sharanek A, Burban A, Humbert L, Guguen-Guillouzo C, Rainteau D, Guillouzo A, Sep 2017
  Abstract : Drug-induced intrahepatic cholestasis is characterized by cellular accumulation of bile acids (BA) whose mechanisms remain poorly understood. The present study aimed to analyze early and progressive alterations of BA profiles induced by cyclosporine A, chlorpromazine, troglitazone, tolcapone, trovafloxacin and tacrolimus after 4h, 24h and 6 daily treatments of differentiated HepaRG cells. In BA-free medium the potent cholestatic drugs, cyclosporine A, chlorpromazine and troglitazone, reduced endogenous BA synthesis after 24h, while the rarely cholestatic drugs, tolcapone, trovafloxacin and tacrolimus, reduced BA synthesis only after 6 days. In the presence of physiological serum BA concentrations, cyclosporine A, chlorpromazine and troglitazone induced early and preferential cellular accumulation of unconjugated lithocholic, deoxycholic and chenodeoxycholic acids that increased 8-12- and 47-50-fold after 24h and 6 days respectively. Accumulation of these hydrophobic BAs resulted from strong inhibition of amidation and in addition, for lithocholic acid reduction of its sulfo-conjugation, and was associated with variable alterations of uptake and efflux transporters. Trovafloxacin also caused BA accumulation, especially after 6 days, while tolcapone and tacrolimus were still without effect. However, when exogenous BAs were added to the medium at cholestatic serum concentrations, a 6-day treatment with all drugs resulted in cellular BA accumulation with higher folds of chenodeoxycholic and lithocholic acids. At the tested concentration, tolcapone had the lowest effect. These results bring the first demonstration that major cholestatic drugs can cause preferential and progressive in vitro cellular accumulation of unconjugated toxic hydrophobic BAs, and bring new insights in mechanisms involved in drug-induced cellular accumulation of toxic BAs.
• A dynamic mathematical model of bile acid clearance in HepaRG cellsKaschek D, Sharanek A, Guillouzo A, Timmer J, Weaver RJ.
  Toxicol Sci, Sep 2017
  Abstract : A dynamic model based on ordinary differential equations that describes uptake, basolateral and canalicular export of taurocholic acid in human HepaRG cells is presented. The highly reproducible inter-assay experimental data were used to reliably estimate model parameters. Primary human hepatocytes were similarly evaluated to establish a mathematical model, but with notably higher inter-assay differences in taurocholic acid clearance and bile canaliculi dynamics. By use of the HepaRG cell line, the simultaneous taurocholic acid clearance associated to basolateral uptake, canalicular and sinusoidal efflux, was predicted. The mathematical model accurately reproduced the dose-dependent inhibition of taurocholic acid clearance in the presence and absence of the prototypical cholestatic drugs cyclosporine A and chlorpromazine. Rapid inhibition of taurocholic acid clearance and recovery were found to be major characteristics of cyclosporine A. Conversely, the action of chlorpromazine was described by slow onset of inhibition relative to inhibition of taurocholic acid clearance by cyclosporine A. The established mathematical model, validated by the use of these two prototypical cholestatic drugs and the integration of bile canalicular dynamics, provides an important development for the further study of human hepatobiliary function, through simultaneous temporal and vectorial membrane transport of bile acids in drug-induced cholestasis.
• Effect of methapyrilene hydrochloride on hepatic intracellular iron metabolism in vivo and in vitro.Kindrat I, Dreval K, Shpyleva S, Tryndyak V, de Conti A, Mudalige TK, Chen T, Erstenyuk AM, Beland FA, Pogribny IP.
  Toxicol Lett, Sep 2017
  Abstract : The liver, a central detoxification organ and main regulator of systemic iron homeostasis, is prone to damage by xenobiotics. In the present study, we investigated the effect of the hepatotoxicant and hepatocarcinogen methapyrilene hydrochloride on iron metabolism in rat liver in a repeat-dose in vivo toxicity study and in human HepaRG cells in vitro. Treatment of male Fischer 344 (F344) rats with methapyrilene at doses 40 and 80mg/kg body weight (bw)/day by gavage for 6 weeks resulted in changes in the expression of classic hepatotoxicity-related marker genes and iron homeostasis-related genes, especially a prominent, dose-dependent down-regulation of the transferrin (Tf) gene and an up-regulation of the ferritin, light chain (Ftl) gene. A decrease in the level of TF and an increase in the level of FTL also occurred in methapyrilene-treated differentiated HepaRG cells, indicating the existence of interspecies and in vitro-in vivo similarities in the disturbance of cellular iron homeostasis upon liver injury. In contrast, there was minimal overlap in the expression of liver toxicity-marker genes in the livers of rats and in HepaRG cells treated with methapyrilene. Importantly, the decrease of transferrin at mRNA and protein levels occurred after the treatment with a low dose of methapyrilene that exhibited minimal cytotoxicity. These results demonstrate the significance of the dysregulation of hepatic iron metabolism in the pathogenesis and mechanism of chemical-induced liver toxicity and suggest that these changes may be sensitive and useful indicators of potentially hepatotoxic chemicals.
• Hepatotoxicity of fusariotoxins, alone and in combination, towards the HepaRG human hepatocyte cell line.Smith MC, Hymery N, Troadec S, Pawtowski A, Coton E, Madec S.
  Food Chem Toxicol., Sep 2017
  Abstract : While the reality of mycotoxin co-occurrence in food commodities is now established, their effects in mixtures are not well studied. The present study investigated the individual and combined effects of deoxynivalenol (DON), nivalenol (NIV), T-2 toxin (T2), fumonisin B1 (FB1), zearalenone (ZEA) and moniliformin (MON) fusariotoxins on cell viability and cell death mechanisms in proliferating HepaRG cells, a human derived liver cell line. In addition, DON-ZEA being one of the most widespread mycotoxin mixtures in grains worldwide, its effect on the expression levels of genes encoding for sets of hepatocyte-specific functions was studied. After 48 h, T2 appeared to be the most cytotoxic tested fusariotoxins, followed by NIV, DON and ZEA. Furthermore, at low cytotoxic doses, all tested fusariotoxin mixtures (DON-MON, DON-FB1, DON-ZEA and NIV-T2) acted synergistically on cell death. Interestingly, during the first 18 h of exposure, only FB1 and ZEA alone and in combination with DON seemed to induce cell apoptosis and necrosis. At the gene level, after only 1 h, DON-ZEA combination induced expression of drug-metabolizing enzymes contrary to individual exposures. Thus, the observed synergy of fusariotoxin mixtures suggested that their simultaneous presence in food commodities can induce a toxic risk that should be better taken into consideration.
• A cell-based assay using HepaRG cells for predicting drug-induced phospholipidosis.Tomida T, Ishimura M, Iwaki M.
  J Toxicol Sci, Sep 2017
  Abstract : The utility of HepaRG cells as an in vitro cell-based assay system for predicting drug-induced phospholipidosis (PLD) was investigated. In experiment 1, 10 PLD-positive compounds and 11 PLD-negative compounds were selected. HepaRG cells were treated with each compound for 48 hr. In experiment 2, loratadine and desloratadine, a major metabolite of loratadine, were used to assess metabolic activation for PLD. HepaRG cells were treated with loratadine and desloratadine in the presence or absence of 500 μM 1-aminobenzotriazole (ABT), a broad CYP inhibitor, for 48 hr. After treatment with compounds in experiments 1 and 2, the relative fluorescence intensity (RFI) was measured using LYSO-ID Red dye to assess the PLD induction. In experiment 1, our cell-based assay system using HepaRG cells exhibited 100% sensitivity and 100% specificity for predicting drug-induced PLD. In experiment 2, loratadine increased the RFI in the PLD assay. However, the increase in the RFI was not observed in co-treatment with loratadine and ABT. In addition, desloratadine increased the RFI in the presence and absence of ABT. These results suggested that metabolic activation of loratadine may contribute to PLD in HepaRG cells. We newly demonstrated that HepaRG cells have a high ability for predicting drug-induced PLD. In addition, we newly showed that HepaRG cells may predict drug-induced PLD mediated by metabolic activation of loratadine. Thus, a cell-based assay system using HepaRG cells is a useful model for predicting drug-induced PLD.
• Virtual cell based assay simulation of intra-mitochondrial concentration in hepatocytes and cardiomyocytes.Worth AP, Louisse J, Macko P, Benito JVS, Paini A.
  Toxicol In Vitro, Sep 2017
  Abstract : In order to replace the use of animals in toxicity testing, there is a need to predict human in vivo toxic doses from concentrations that cause adverse effects in in vitro test systems. The virtual cell based assay (VCBA) has been developed to simulate intracellular concentrations as a function of time, and can be used to interpret in vitro concentration-response curves. In this study we refine and extend the VCBA model by including additional target-organ cell models and by simulating the fate and effects of chemicals at the organelle level. In particular, we describe the extension of the original VCBA to simulate chemical fate in liver (HepaRG) cells and cardiomyocytes (ICell cardiomyocytes), and we explore the effects of chemicals at the mitochondrial level. This includes a comparison of: a) in vitro results on cell viability and mitochondrial membrane potential (mmp) from two cell models (HepaRG cells and ICell cardiomyocytes); and b) VCBA simulations, including the cell and mitochondrial compartment, simulating the mmp for both cell types. This proof of concept study illustrates how the relationship between mitochondrial disruption and cell toxicity can be simulated using the VCBA.
• Editor's Highlight: Mechanistic Toxicity Tests Based on an Adverse Outcome Pathway Network for Hepatic Steatosis.Angrish MM, McQueen CA, Cohen-Hubal E, Bruno M, Ge Y, Chorley BN.
  Toxicol Sci., Sep 2017
  Abstract : Risk assessors use liver endpoints in rodent toxicology studies to assess the safety of chemical exposures. Yet, rodent endpoints may not accurately reflect human responses. For this reason and others, human-based invitro models are being developed and anchored to adverse outcome pathways to better predict adverse human health outcomes. Here, a networked adverse outcome pathway-guided selection of biology-based assays for lipid uptake, lipid efflux, fatty acid oxidation, and lipid accumulation were developed. These assays were evaluated in a metabolically competent human hepatocyte cell model (HepaRG) exposed to compounds known to cause steatosis (amiodarone, cyclosporine A, and T0901317) or activate lipid metabolism pathways (troglitazone, Wyeth-14,643, and 22(R)-hydroxycholesterol). All of the chemicals activated at least one assay, however, only T0901317 and cyclosporin A dose-dependently increased lipid accumulation. T0901317 and cyclosporin A increased fatty acid uptake, decreased lipid efflux (inferred from apolipoprotein B100 levels), and increased fatty acid synthase protein levels. Using this biologically-based evaluation of key events regulating hepatic lipid levels, we demonstrated dysregulation of compensatory pathways that normally balance hepatic lipid levels. This approach may provide biological plausibility and data needed to increase confidence in linking invitro-based measurements to chemical effects on adverse human health outcomes.
• Iron depletion induces hepatic secretion of biliary lipids and glutathione in ratsPrasnicka A, Cermanova J, Hroch M, Dolezelova E, Rozkydalova L, Smutny T, Carazo A, Chladek J, Lenicek M, Nachtigal P, Vitek L, Pavek P, Micuda S
  Biochim Biophys Acta, Sep 2017
  Abstract : Iron depletion (ID) has been shown to induce the liver expression of Cyp7a1, the rate-limiting enzyme initiating conversion of cholesterol to bile acids (BA), although the effect on bile acids metabolism and bile production is unknown. Therefore, we investigated changes in bile secretion and BA synthesis during diet-induced iron depletion (ID) in rats. ID increased bile flow along with augmented biliary excretion of bile acids, glutathione, cholesterol and phospholipids. Accordingly, we found transcriptional upregulation of the Cyp7a1, Cyp8b1, and Cyp27a1 BA synthetic enzymes, as well as induction of the Abcg5/8 cholesterol transporters in ID rat livers. In contrast, intravenous infusion of 3H-taurocholate failed to elicit any difference in biliary secretion of this compound in the ID rats. This corresponded with unchanged expression of canalicular rate-limiting transporters for BA as well as glutathione. We also observed that ID substantially changed the spectrum of BA in bile and decreased plasma concentrations of BA and cholesterol. Experiments with differentiated human hepatic HepaRG cells confirmed human CYP7A1 orthologue upregulation resulting from reduced iron concentrations. Results employing a luciferase reporter gene assay suggest that the transcriptional activation of the CYP7A1 promoter under ID conditions works independent of farnesoid X (FXR), pregnane X (PXR) and liver X (LXRα) receptors activation. It can be concluded that this study characterizes the molecular mechanisms of modified bile production as well as cholesterol as along with BA homeostasis during ID. We propose complex upregulation of BA synthesis, and biliary cholesterol secretion as the key factors affected by ID.
• Light-responsive paper strips as CO-releasing material with a colourimetric responseReddy G U, Liu J, Hoffmann P, Steinmetzer J, Görls H, Kupfer S, Askes SHC, Neugebauer U, Gräfe S, Schiller A.
  Chem Sci, Sep 2017
  Abstract : Carbon monoxide (CO) is known for its multifaceted role in human physiology, and molecules that release CO in a controlled way have been proposed as therapeutic drugs. In this work, a light-responsive CO-releasing molecule (CORM-Dabsyl) showed a strong colourimetric response upon photochemical CO-release, owing to the tight conjugation of a Mn(i) tricarbonyl centre to a dabsyl chromophoric ligand (L). Whereas the complex was very stable in the dark in nitrogen-purged aqueous media, CO-release was effectively triggered using 405 nm irradiation. CORM-Dabsyl, L and the inactive product iCORM-Dabsyl have been investigated by DFT and TD-DFT calculations. Only mild toxicity of CORM-Dabsyl was observed against LX-2 and HepaRG® human cell lines (IC50 ∼ 30 μM). Finally, to develop a CO storage and release material that is readily applicable to therapeutic situations, CORM-Dabsyl was loaded on low-cost and easily disposable paper strips, from which the light triggered CO-release was conveniently visible with the naked eye.
• Individual and combined toxicological effects of deoxynivalenol and zearalenone on human hepatocytes in in vitro chronic exposure conditions.Smith MC,  Madec S,  Pawtowski A,  Coton E,  Hymery N
  Toxicol Lett, Aug 2017
  Abstract : While numerous surveys highlighted the natural co-occurrence of mycotoxins in food, data about their toxicological combined effects is still limited. This is especially the case for chronic exposure conditions, although the latter are more representative of the mycotoxin risk associated with food consumption than acute exposure. In the present study, cell viability and gene expression levels of relevant hepatocyte-specific functions were evaluated for the HepaRG human liver cell line exposed to deoxynivalenol (DON) and/or zearalenone (ZEA) during 14, 28 and 42days at three subtoxic concentrations corresponding to i) the determined average exposure dose of French adult population, ii) the tolerable daily intake established by the Joint FAO/WHO Expert Committee and iii) the maximum level permitted by the European regulation in cereals intended for direct human consumption. For the latter, DON and DON+ZEA induced 90% cell mortality after 14 days. In addition, depending on the considered toxin or mixture, doses and exposure periods, important variations of gene expression levels were observed. Despite the fact that in vitro conditions differ from the in vivo situation, the obtained results clearly highlighted that long-term toxicological effects of chronic exposure to mycotoxin combinations should be further investigated and, if necessary, taken into consideration at the regulatory level.
• Local Anesthetics Inhibit the Growth of Human Hepatocellular Carcinoma CellsLe Gac G,  Angenard G,  Clément B,  Laviolle B,  Coulouarn C,  Beloeil H
  Anesth Analg, Aug 2017
  Abstract : Hepatocellular carcinoma (HCC) is an aggressive cancer with limited therapeutic options. Retrospective studies have shown that the administration of local anesthetics (LAs) during cancer surgery could reduce cancer recurrence. Besides, experimental studies reported that LAs could inhibit the growth of cancer cells. Thus, the purpose of this study was to investigate the effects of LAs on human HCC cells.
• AMC-Bio-Artificial Liver culturing enhances mitochondrial biogenesis in human liver cell lines: The role of oxygen, medium perfusion and 3D configuration.Adam AAA,  van Wenum M,  van der Mark VA,  Jongejan A,  Moerland PD,  Houtkooper RH,  Wanders RJA,  Oude Elferink RP,  Chamuleau RAFM,  Hoekstra R, , ,
  Mitochondrion., Aug 2017
  Abstract : Human liver cell lines, like HepaRG and C3A, acquire higher functionality when cultured in the AMC-Bio-Artificial Liver (AMC-BAL). The three main differences between BAL and monolayer culture are the oxygenation (40% vs 20%O2), dynamic vs absent medium perfusion and 3D vs 2D configuration. Here, we investigated the background of the differences between BAL-cultures and monolayers.
• New insights into HCV replication in original cells from Aedes mosquitoes.Fallecker C,  Caporossi A,  Rechoum Y,  Garzoni F,  Larrat S,  François O,  Fender P,  Morand P,  Berger I,  Petit MA,  Drouet E
  Virol J, Aug 2017
  Abstract :   The existing literature about HCV association with, and replication in mosquitoes is extremely poor. To fill this gap, we performed cellular investigations aimed at exploring (i) the capacity of HCV E1E2 glycoproteins to bind on Aedes mosquito cells and (ii) the ability of HCV serum particles (HCVsp) to replicate in these cell lines.
• The expression, induction and pharmacological activity of CYP1A2 are post-transcriptionally regulated by microRNA hsa-miR-132-5p.Chen Y,  Zeng L,  Wang Y,  Tolleson WH,  Knox B,  Chen S,  Ren Z,  Guo L,  Mei N,  Qian F,  Huang K,  Liu D,  Tong W
  Biochem Pharmacol. , Aug 2017
  Abstract : Cytochrome P450 1A2 (CYP1A2) is one of the most abundant and important drug metabolizing enzymes in human liver. However, little is known about the post-transcriptional regulation of CYP1A2, especially the mechanisms involving microRNAs (miRNAs). This study applied a systematic approach to investigate the post-transcriptional regulation of CYP1A2 by miRNAs. Candidate miRNAs targeting the 3'-untranslated region (3'-UTR) of CYP1A2 were screened in silico, resulting in the selection of sixty-two potential miRNAs for further analysis. The levels of two miRNAs, hsa-miR-132-5p and hsa-miR-221-5p, were inversely correlated with the expression of CYP1A2 mRNA transcripts in normal human liver tissue samples represented in The Cancer Genome Atlas (TCGA) dataset. The interactions between these miRNAs and cognate CYP1A2 mRNA sequences were evaluated using luciferase reporter gene studies and electrophoretic mobility shift assays, by which a direct interaction was confirmed involving hsa-miR-132-5p and a cognate binding site present in the CYP1A2 3'-UTR. Experiments by which hsa-miR-132-5p or random miRNA controls were introduced into HepG2, Huh-7 and HepaRG hepatic cell lines showed that only hsa-miR-132-5p suppressed the endogenous and lansoprazole-induced expression of CYP1A2, at biological activity, protein production, and mRNA transcript levels. Furthermore, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and lactate dehydrogenase (LDH) assays showed that hsa-miR-132-5p attenuates CYP1A2-mediated, lansoprazole-enhanced, flutamide-induced hepatic cell toxicity. Results from multilayer experiments demonstrate that hsa-miR-132-5p suppresses the expression of CYP1A2 and that this suppression is able to decrease the extent of an adverse drug-drug interaction involving lansoprazole and flutamide.
• A proteomics method using immunoaffinity fluorogenic derivatization-liquid chromatography/tandem mass spectrometry (FD-LC-MS/MS) to identify a set of interacting proteins.Nakata K,  Saitoh R,  Ishigai M,  Imai K
  Biomed Chromatogr., Aug 2017
  Abstract : Biological functions in organisms are usually controlled by a set of interacting proteins, and identifying the proteins that interact is useful for understanding the mechanism of the functions. Immunoprecipitation is a method that utilizes the affinity of an antibody to isolate and identify the proteins that have interacted in a biological sample. In this study, the FD-LC-MS/MS method, which involves fluorogenic derivatization followed by separation and quantification by HPLC and finally identification of proteins by HPLC-tandem mass spectrometry, was used to identify proteins in immunoprecipitated samples, using heat shock protein 90 (HSP90) as a model of an interacting protein in HepaRG cells. As a result, HSC70 protein, which was known to form a complex with HSP90, was isolated, together with three different types of HSP90-beta. The results demonstrated that the proposed immunoaffinity-FD-LC-MS/MS method could be useful for simultaneously detecting and identifying the proteins that interact with a certain protein.
• Short-term treatment with taurolidine is associated with liver injury.Fahrner R,  Möller A,  Press AT,  Kortgen A,  Kiehntopf M,  Rauchfuss F,  Settmacher U,  Mosig AS
  BMC Pharmacol Toxicol, Aug 2017
  Abstract : Taurolidine has been used for peritonitis, oncological and catheter-lock treatment because of its anti-inflammatory properties. It has been suggested that taurolidine has no severe side-effects, but after long-term use morphological and functional changes of the liver were reported. The aim of this study was to investigate the effect of short-term use of taurolidine on the liver.
• Aqueous extract of Phragmitis rhizoma ameliorates myelotoxicity of docetaxel in vitro and in vivo.Kim J,  Lee YJ,  Kim YA,  Cho ES,  Huh E,  Bang OS,  Kim NS
  BMC Complement Altern Med, Aug 2017
  Abstract : A variety of anticancer chemotherapeutics induce adverse side effects including myelotoxicity. Dried roots of Phragmites communis Trinius, Phragmitis rhizoma, have been clinically used in traditional folk medicine to relieve various symptoms like fever. In this study, we evaluated the protective effect of the aqueous extract of Phragmitis rhizoma (EPR) against docetaxel-induced myelotoxicity in vitro and in vivo. The in vitro myelo-protective effect of EPR was evaluated using the colony forming unit (CFU) assay with hematopoietic progenitor cells. The in vivo efficacy of EPR was evaluated in myelosuppressed C57BL/6 male mice which were induced by repeated intraperitoneal injections of 30 mg/kg docetaxel for 3 times. EPR was orally administered for 4 days to docetaxel-induced myelosuppressed C57BL/6 male mice which were induced by intraperitoneal injection of 30 mg/kg docetaxel for 3 times: Group 1 (vehicle control, n = 10), Group 2 (docetaxel plus vehicle, n = 10), Group 3 (docetaxel plus EPR 30 mg/kg, n = 10), Group 4 (docetaxel plus EPR 100 mg/kg, n = 10) and Group 5 (docetaxel plus EPR 300 mg/kg, n = 10). Whole blood counts were measured automatically, and immune organs were histologically examined. Expression of immunomodulatory cytokines was measured by quantitative real-time polymerase chain reaction or enzyme-linked immunosorbent assay. The toxicity of EPR itself was evaluated in normal human cell lines including IMR-90, foreskin fibroblast and human umbilical vein endothelial cells. The hepatotoxicity of EPR was predicted by multi-parametric assays involving cell viability, caspase 3/7 activity, GSH contents and LDH leakage using the HepaRG hepatic cell line. Co-treatment of EPR or its major component, p-hydroxycinnamic acid, increased the numbers of hematopoietic CFU counts in the docetaxel-induced in vitro myelotoxicity assay system. The in vitro protective effect of EPR against docetaxel toxicity was replicated in a myelosuppressed animal model: white blood cells, neutrophils, lymphocytes and red blood cells rebounded; bone marrow niche and structural integrity of the thymus were preserved; and the expression of immune-stimulating cytokines including IL3, IL6, SCF and GM-CSF was enhanced. Furthermore, EPR and p-hydroxycinnamic acid promoted the proliferation of primary splenocytes and thymocytes. In the toxicity assays, no remarkable signs related with toxicity were observed in all tested normal human cells and HepaRG. EPR has the potential to ameliorate docetaxel-mediated myelotoxicity in both in vitro and in vivo models. However, the identification of the responsible active components and the precise underlying myelo-protective mechanism of EPR need to be elucidated before novel drug development using EPR can precede.
• Advances in methods for characterization of hepatic urea cycle enzymatic activity in HepaRGcells using UPLC-MS/MS.Moedas MF,  Adam AAA,  Farelo MA,  IJlst L,  Chamuleau RAFM,  Hoekstra R,  Wanders RJA,  Silva MFB
  Anal Biochem, Aug 2017
  Abstract : Current methodologies for the assessment of urea cycle (UC) enzymatic activity are insufficient to accurately evaluate this pathway in biological specimens where lower UC is expected. Liver cell lines, including HepaRG, have been described to have limited nitrogen fixation through the UC, limiting their applicability as biocomponents for Bioartificial Livers (BAL). This work aims to develop novel and sensitive analytical solutions using Mass Spectrometry-based methodology to measure the activity of four UC enzymes in human liver and HepaRGcells. Activity of carbamoyl-phosphate synthetase I (CPS I), ornithine transcarbamylase (OTC), argininosuccinate lyase (ASL) and arginase (ARG I and II) was determined on homogenates from normal human liver and HepaRG cells cultured in monolayer or in the AMC-BAL. Enzyme products were determined by stable-isotope dilution UPLC-MS/MS. Activity of CPS I, OTC and ARG I/II enzymes in HepaRGmonolayer cultures was considerably lower than in human control livers albeit an increase was achieved in HepaRG-BAL cultures. Improved analytical assays developed for the study of UC enzyme activity, contributed to gain understanding of UC function in the HepaRG cell line. The decreased activity of CPS I suggests that it may be a potential rate-limiting factor underlying the low UC activity in this cell line.
• Pro-fibrotic compounds induce stellate cell activation, ECM-remodelling and Nrf2 activation in a human 3D-multicellular model of liver fibrosis.Prestigiacomo V, Weston A, Messner S, Lampart F, Suter-Dick L
  PLoS One, Jul 2017
  Abstract : BACKGROUND & AIMS: Currently most liver fibrosis research is performed in vivo, since suitable alternative in vitro systems which are able to recapitulate the cellular events leading to liver fibrosis are lacking. Here we aimed at generating a system containing cells representing the three key players of liver fibrosis (hepatocyte, Kupffer cells and stellate cells) and assess their response to pro-fibrotic compounds such as TGF-β1, methotrexate (MTX) and thioacetamide (TAA). METHODS: Human cell lines representing hepatocytes (HepaRG), Kupffer cell (THP-1 macrophages) and stellate cells (hTERT-HSC) were co-cultured using the InSphero hanging drop technology to generate scaffold-free 3D microtissues, that were treated with pro-fibrotic compounds (TGF-β1, MTX, TAA) for up to 14 days. The response of the microtissues was evaluated by determining the expression of cytokines (TNF-α, TGF-β1 and IL6), the deposition and secretion of ECM proteins and induction of gene expression of fibrosis biomarkers (e.g. αSMA). Induction of Nrf2 and Keap1, as key player of defence mechanism, was also evaluated. RESULTS: We could demonstrate that the multicellular 3D microtissue cultures could be maintained in a non-activated status, based on the low expression levels of activation markers. Macrophages were activated by stimulation with LPS and hTERT-HSC showed activation by TGF-β1. In addition, MTX and TAA elicited a fibrotic phenotype, as assessed by gene-expression and protein-deposition of ECM proteins such as collagens and fibronectin. An involvement of the antioxidant pathway upon stimulation with pro-fibrotic compounds was also observed. CONCLUSION: Here, for the first time, we demonstrate the in vitro recapitulation of key molecular and cellular events leading to liver fibrosis: hepatocellular injury, antioxidant defence response, activation of Kupffer cells and activation of HSC leading to deposition of ECM.
• Multicomponent determination of traditional Chinese medicine preparation yin-zhi-huang injection by LC-MS/MS for screening of its potential bioactive candidates using HepaRG cells.Rao Z,  Zhang F,  Zhang XY,  Zhang GQ,  Ma YR,  Zhou Y,  Qin HY,  Wu XA,  Wei YH, , , ,
  Biomed Chromatogr., Jul 2017
  Abstract : Yin-zhi-huang (YZH) injection is an injectable multiherbal prescription derived from the ancient Chinese medicine formula of Yin-chen-hao-tang, which is widely used in the clinic for the treatment of jaundice and chronic liver diseases. To date, the systematic study of the components in this multiherbal prescription still lacks suitable analytical methods that are able to simultaneously detect a broad array of components at low concentrations. In this study, a new liquid chromatography-tandem mass spectrometry method using dynamic multiple reaction monitoring mode was developed to determine multiple peaks in traditional Chinese medicine preparation YZH injection. This simple, selective and sensitive method enabled the quantification of 22 components with standard materials with a lower limit of quantification of 1.46-12.5 ng/mL in cell lysates. This method was successfully applied to celluar uptake and binding investigation of components in YZH injection. The results indicated that this strategy might be a useful approach for rapidly screening of the potential bioactive candidates from YZH injection, and the discovered candidates could be used to investigate the pharmacodynamics in further studies.
• The Association of Combined GSTM1 and CYP2C9 Genotype Status with the Occurrence of Hemorrhagic Cystitis in Pediatric Patients Receiving Myeloablative Conditioning Regimen Prior to Allogeneic Hematopoietic Stem Cell Transplantation.Uppugunduri CRS,  Storelli F,  Mlakar V,  Huezo-Diaz Curtis P,  Rezgui A,  Théorêt Y,  Marino D,  Doffey-Lazeyras F,  Chalandon Y,  Bader P,  Daali Y,  Bittencourt H,  Krajinovic M
  Front Pharmacol., Jul 2017
  Abstract :  Hemorrhagic cystitis (HC) is one of the complications of busulfan-cyclophosphamide (BU-CY) conditioning regimen during allogeneic hematopoietic stem cell transplantation (HSCT) in children. Identifying children at high risk of developing HC in a HSCT setting could facilitate the evaluation and implementation of effective prophylactic measures. In this retrospective analysis genotyping of selected candidate gene variants was performed in 72 children and plasma Sulfolane (Su, water soluble metabolite of BU) levels were measured in 39 children following treatment with BU-CY regimen. The cytotoxic effects of Su and acrolein (Ac, water soluble metabolite of CY) were tested on human urothelial cells (HUCs). The effect of Su was also tested on cytochrome P 450 (CYP) function in HepaRG hepatic cells. Cumulative incidences of HC before day 30 post HSCT were estimated using Kaplan-Meier curves and log-rank test was used to compare the difference between groups in a univariate analysis. Multivariate Cox regression was used to estimate hazard ratios with 95% confidence intervals (CIs). Multivariate analysis included co-variables that were significantly associated with HC in a univariate analysis. Cumulative incidence of HC was 15.3%. In the univariate analysis, HC incidence was significantly (p < 0.05) higher in children older than 10 years (28.6 vs. 6.8%) or in children with higher Su levels (>40 vs. HepaRG cells. Our observations suggest that pre-emptive genotyping of CYP2C9 and GSTM1 may aid in selection of more effective prophylaxis to reduce HC development in pediatric patients undergoing allogeneic HSCT. Article summary: (1) Children carrying functional alleles in GSTM1 and CYP2C9 are at high risk for developing hemorrhagic cystitis following treatment with busulfan and cyclophosphamide based conditioning regimen. (2) Identification of children at high risk for developing hemorrhagic cystitis in an allogeneic HSCT setting will enable us to evaluate and implement optimal strategies for its prevention. Trial registration: This study is a part of the trail "clinicaltrials.gov identifier: NCT01257854."  
• Induction of Human Cytochrome P450 3A4 by the Irreversible Myeloperoxidase Inactivator PF-06282999 is Mediated by the Pregnane X Receptor.Moscovitz JE, Lin Z, Johnson N, Tu M, Goosen T, Weng Y, Kalgutkar AS
  Xenobiotica, Jul 2017
  Abstract : 1. 2-(6-(5-Chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl) acetamide (PF-06282999) is a member of the thiouracil class of irreversible inactivators of human myeloperoxidase enzyme and a candidate for the treatment of cardiovascular disease. PF-06282999 is an inducer of CYP3A4 mRNA and midazolam-1'-hydroxylase activity in human hepatocytes, which is consistent with PF-06282999-dose dependent decreases in mean maximal plasma concentrations (Cmax) and area under the plasma concentration time curve (AUC) of midazolam in humans following 14-day treatment with PF-06282999. 2. In the present study, the biochemical mechanism(s) of CYP3A4 induction by PF-06282999 was studied. Incubations in reporter cells indicated that PF-06282999 selectively activated human pregnane X receptor (PXR). Treatment of human HepaRG cells with PF-06282999 led to ∼14-fold induction in CYP3A4 mRNA and 5-fold increase in midazolam-1'-hydroxylase activity, which was nullified in PXR-knock out HepaRG cells. TaqMan® gene expression analysis of human hepatocytes treated with PF-06282999 and the prototypical PXR agonist rifampin demonstrated increases in mRNA for CYP3A4 and related CYPs that are regulated by PXR. 3. Docking studies using a published human PXR crystal structure provided insights into the molecular basis for PXR activation by PF-06282999. Implementation of PXR transactivation assays in a follow-on discovery campaign should aid in the identification of back-up compounds devoid of PXR activation and CYP3A4 induction liability.
• Induction of human cytochrome P450 3A4 by the irreversible myeloperoxidase inactivator PF-06282999 is mediated by the pregnane X receptor.Moscovitz JE,  Lin Z,  Johnson N2,  Tu M,  Goosen TC,  Weng Y,  Kalgutkar AS, , , , , ,
  Xenobiotica, Jul 2017
  Abstract : 1. 2-(6-(5-Chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl) acetamide (PF-06282999) is a member of the thiouracil class of irreversible inactivators of human myeloperoxidase enzyme and a candidate for the treatment of cardiovascular disease. PF-06282999 is an inducer of CYP3A4 mRNA and midazolam-1'-hydroxylase activity in human hepatocytes, which is consistent with PF-06282999-dose dependent decreases in mean maximal plasma concentrations (Cmax) and area under the plasma concentration time curve (AUC) of midazolam in humans following 14-day treatment with PF-06282999. 2. In the present study, the biochemical mechanism(s) of CYP3A4 induction by PF-06282999 was studied. Incubations in reporter cells indicated that PF-06282999 selectively activated human pregnane X receptor (PXR). Treatment of human HepaRG cells with PF-06282999 led to ∼14-fold induction in CYP3A4 mRNA and 5-fold increase in midazolam-1'-hydroxylase activity, which was nullified in PXR-knock out HepaRG cells. TaqMan® gene expression analysis of human hepatocytes treated with PF-06282999 and the prototypical PXR agonist rifampin demonstrated increases in mRNA for CYP3A4 and related CYPs that are regulated by PXR. 3. Docking studies using a published human PXR crystal structure provided insights into the molecular basis for PXR activation by PF-06282999. Implementation of PXR transactivation assays in a follow-on discovery campaign should aid in the identification of back-up compounds devoid of PXR activation and CYP3A4 induction liability.
• Metabolism of the Marine Phycotoxin PTX-2 and Its Effects on Hepatic Xenobiotic Metabolism: Activation of Nuclear Receptors and Modulation of the Phase I Cytochrome P450.Alarcan J, Dubreil E, Huguet A, Hurtaud-Pessel D, Hessel-Pras S, Lampen A, Fessard V, Le Hegarat L.
  Toxins (Basel), Jul 2017
  Abstract : PTX-2 is a marine biotoxin frequently found in shellfish that can lead to food intoxication in humans. Information regarding PTX-2 metabolism is scarce, and little is known of its effect on xenobiotic-metabolizing enzymes (XME) or its molecular pathways. The aim of this study was consequently to examine PTX-2 Phase I metabolism using rat and human liver S9 fractions, and also to assess the capability of PTX-2: (i) to modulate the gene expression of a panel of Phase I (CYP450) and II (UGT, SULT, NAT, and GST) enzymes, as well as the Phase III or 0 (ABC and SLCO) transporters in the human hepatic HepaRG cell line using qPCR; (ii) to induce specific CYP450 in HepaRG cells measured by immunolabeling detection and the measurement of the cells' activities; and (iii) to activate nuclear receptors and induce CYP promoter activities in HEK-T and HepG2 transfected cell lines using transactivation and reporter gene assay, respectively. Our results indicate that PTX-2 hydroxylation occurred with both rat and human S9 fractions. Whereas PTX-2 mostly upregulated the gene expression of CYP1A1 and 1A2, no induction of these two CYP activities was observed. Lastly, PTX-2 did not act as an agonist of CAR or PXR. Due to its effects on some key XME, more attention should be paid to possible drug-drug interactions with phycotoxins, especially as shellfish can accumulate several phycotoxins as well as other kinds of contaminants.
• Scaling-up of a HepaRG progenitor cell based bioartificial liver: optimization for clinical application and transport.van Wenum M, Treskes P, Tang CY, Coppens EJ, Jansen K, Hendriks EJ, Camus S, van Gulik TM, Chamuleau RAFM, Hoekstra R
  IopScience, Jun 2017
  Abstract : A new generation of bioartificial livers, based on differentiated proliferative hepatocyte sources, has been developed. Several practicable and regulatory demands have to be addressed before these can be clinically evaluated. We identified three main hurdles: (1) expansion and preservation of the biocomponent, (2) development of scaled-up culture conditions and (3) transport of the device to the bedside. In this study we address these three issues for the HepaRG-progenitor cell line-loaded AMC-Bioartificial Liver. (1) HepaRG cells were expanded in large quantities and then cryopreserved or loaded directly into bioreactors. After 3 weeks of culture, key hepatic functions (ammonia/lactate elimination, apolipoprotein A1 synthesis and cytochrome P450 3A4 activity) did not differ significantly between the two groups. (2) Bioartificial livers were scaled up from 9 ml to 540 ml priming volume, with preservation of normalized hepatic functionality. Quantification of amino acid consumption revealed rapid depletion of several amino acids. (3) Whole-device cryopreservation and cooled preservation induced significant loss of hepatic functionality, whereas simulated transport from culture-facility to the bedside in a clinical-grade transport unit with controlled temperature maintenance, medium perfusion and gas supply did not affect functionality. In addition, we assessed tumorigenicity of HepaRGcells in immune-incompetent mice and found no tumor formation of HepaRG cells (n = 12), while HeLa cells induced formation of carcinomas in eight out of 12 mice in 140 days.
• Test systems in drug discovery for hazard identification and risk assessment of human drug-induced liver injury.Weaver RJ,  Betts C,  Blomme EAG,  Gerets HHJ,  Gjervig Jensen K,  Hewitt PG,  Juhila S,  Labbe G,  Liguori MJ,  Mesens N,  Ogese MO,  Persson M,  Snoeys J
  Expert Opin Drug Metab Toxicol, Jun 2017
  Abstract : The liver is an important target for drug-induced toxicities. Early detection of hepatotoxic drugs requires use of well-characterized test systems, yet current knowledge, gaps and limitations of tests employed remains an important issue for drug development. Areas Covered: The current state of the science, understanding and application of test systems in use for the detection of drug-induced cytotoxicity, mitochondrial toxicity, cholestasis and inflammation is summarized. The test systems highlighted herein cover mostly in vitro and some in vivo models and endpoint measurements used in the assessment of small molecule toxic liabilities. Opportunities for research efforts in areas necessitating the development of specific tests and improved mechanistic understanding are highlighted. Expert Opinion: Use of in vitro test systems for safety optimization will remain a core activity in drug discovery. Substantial inroads have been made with a number of assays established for human Drug-induced Liver Injury. There nevertheless remain significant gaps with a need for improved in vitro tools and novel tests to address specific mechanisms of human Drug-Induced Liver Injury. Progress in these areas will necessitate not only models fit for application, but also mechanistic understanding of how chemical insult on the liver occurs in order to identify translational and quantifiable readouts for decision-making.
• A Liver-specific Gene Expression Panel Predicts the Differentiation Status of in vitro Hepatocyte Models.Kim DS,  Ryu JW,  Son MY3,  Oh JH,  Chung KS,  Lee S, Lee JJ,  Ahn JH,  Min JS
  Hepatology., Jun 2017
  Abstract : Alternative cell sources, such as three-dimensional organoids and induced pluripotent stem cell-derived cells, might provide a potentially effective approach for both drug development applications and clinical transplantation. For example, the development of cell sources for liver cell-based therapy has been increasingly needed, and liver transplantation is performed for the treatment for patients with severe end-stage liver disease. Differentiated liver cells and three-dimensional (3D) organoids are expected to provide new cell sources for tissue models and revolutionary clinical therapies. However, conventional experimental methods confirming the expression levels of liver-specific lineage markers cannot provide complete information regarding the differentiation status or degree of similarity between liver and differentiated cell sources. Therefore, in this study, to overcome several issues associated with the assessment of differentiated liver cells and organoids, we developed a LiGEP (Liver-Specific Gene Expression Panel) algorithm that presents the degree of liver similarity as a "percentage". We demonstrated that the percentage calculated using the LiGEP algorithm was correlated with the developmental stages of in vivo liver tissues in mice, suggesting that LiGEP can correctly predict developmental stages. Moreover, 3D cultured HepaRG cells and human pluripotent stem cell (hPSC)-derived hepatocyte-like cells (HLCs) showed liver similarity scores of 59.14% and 32%, respectively, although general liver-specific markers were detected. Therefore, our study describes the first quantitative and predictive model for differentiated samples, particularly liver-specific cells or organoids, and this model can be further expanded to various tissue-specific organoids. Our LiGEP can provide useful information and insights regarding the differentiation status of in vitro liver models. This article is protected by copyright. All rights reserved.
• Inhibition of hepatitis B viral entry by nucleic acid polymers in HepaRG cells and primary human hepatocytes.Guillot C,  Martel N,  Berby F,  Bordes I,  Hantz O,  Blanchet M,  Sureau C,  Vaillant A2,  Chemin I1., , , ,
  PLoS One, Jun 2017
  Abstract : Hepatitis B virus (HBV) infection remains a major public health concern worldwide with 240 million individuals chronically infected and at risk of developing cirrhosis and hepatocellular carcinoma. Current treatments rarely cure chronic hepatitis B infection, highlighting the need for new anti-HBV drugs. Nucleic acid polymers (NAPs) are phosphorothioated oligonucleotides that have demonstrated a great potential to inhibit infection with several viruses. In chronically infected human patients, NAPs administration lead to a decline of blood HBsAg and HBV DNA and to HBsAg seroconversion, the expected signs of functional cure. NAPs have also been shown to prevent infection of duck hepatocytes with the Avihepadnavirus duck hepatitis B virus (DHBV) and to exert an antiviral activity against established DHBV infection in vitro and in vivo. In this study, we investigated the specific anti-HBV antiviral activity of NAPs in the HepaRG human hepatoma cell line and primary cultures of human hepatocytes. NAPs with different chemical features (phosphorothioation, 2'O-methyl ribose, 5-methylcytidine) were assessed for antiviral activity when provided at the time of HBV inoculation or post-inoculation. NAPs dose-dependently inhibited HBV entry in a phosphorothioation-dependent, sequence-independent and size-dependent manner. This inhibition of HBV entry by NAPs was impaired by 2'O-methyl ribose modification. NAP treatment after viral inoculation did not elicit any antiviral activity.
• Mechanisms of hepatocellular toxicity associated with new psychoactive synthetic cathinones.Luethi D,  Liechti ME,  Krähenbühl S
  Toxicology. , Jun 2017
  Abstract : Synthetic cathinones are a new class of psychostimulant substances. Rarely, they can cause liver injury but associated mechanisms are not completely elucidated. In order to increase our knowledge about mechanisms of hepatotoxicity, we investigated the effect of five frequently used cathinones on two human cell lines. Bupropion was included as structurally related drug used therapeutically. In HepG2 cells, bupropion, MDPV, mephedrone and naphyrone depleted the cellular ATP content at lower concentrations (0.2 to 1mM) than cytotoxicity occurred (0.5 to 2mM), suggesting mitochondrial toxicity. In comparison, methedrone and methylone depleted the cellular ATP pool and induced cytotoxicity at similar concentrations (≥2mM). In HepaRG cells, cytotoxicity and ATP depletion could also be demonstrated, but cytochrome P450 induction did not increase the toxicity of the compounds investigated. The mitochondrial membrane potential was decreased in HepG2 cells by bupropion, MDPV and naphyrone, confirming mitochondrial toxicity. Bupropion, but not the other compounds, uncoupled oxidative phosphorylation. Bupropion, MDPV, mephedrone and naphyrone inhibited complex I and II of the electron transport chain, naphyrone also complex III. All four mitochondrial toxicants were associated with increased mitochondrial ROS and increased lactate production, which was accompanied by a decrease in the cellular total GSH pool for naphyrone and MDPV. In conclusion, bupropion, MDPV, mephedrone and naphyrone are mitochondrial toxicants impairing the function of the electron transport chain and depleting cellular ATP stores. Since liver injury is rare in users of these drugs, affected persons must have susceptibility factors rendering them more sensitive for these drugs.
• Construction of a hepatitis B virus neutralizing chimeric monoclonal antibody recognizing escape mutants of the viral surface antigen (HBsAg).Golsaz-Shirazi F,  Amiri MM,  Farid S,  Bahadori M2,  Bohne F,  Altstetter S,  Wolff L3,  Kazemi T4,  Khoshnoodi J1,  Hojjat-Farsangi M5,  Chudy M6,  Jeddi-Tehrani M2,  Protzer U3
  Antiviral Res., Jun 2017
  Abstract : Hepatitis B virus (HBV) infection is a global burden on the health-care system and is considered as the tenth leading cause of death in the world. Over 248 million patients are currently suffering from chronic HBV infection worldwide and annual mortality rate of this infection is 686000. The "a" determinant is a hydrophilic region present in all antigenic subtypes of hepatitis B surface antigen (HBsAg), and antibodies against this region can neutralize the virus and are protective against all subtypes. We have recently generated a murine anti-HBs monoclonal antibody (4G4), which can neutralize HBV infection in HepaRG cells and recognize most of the escape mutant forms of HBsAg. Here, we describe the production and characterization of the chimeric human-murine antibody 4G4 (c-4G4). Variable region genes of heavy and light chains of the m-4G4 were cloned and fused to constant regions of human kappa and IgG1 by splice overlap extension (SOE) PCR. The chimeric antibody was expressed in Chinese Hamster Ovary (CHO)-K1 cells and purified from culture supernatant. Competition ELISA proved that both antibodies bind the same epitope within HBsAg. Antigen-binding studies using ELISA and Western blot showed that c-4G4 has retained the affinity and specificity of the parental murine antibody, and displayed a similar pattern of reactivity to 13 escape mutant forms of HBsAg. Both, the parental and c-4G4 showed a comparably high HBV neutralization capacity in cell culture even at the lowest concentration (0.6μg/ml). Due to the ability of c-4G4 to recognize most of the sub-genotypes and escape mutants of HBsAg, this antibody either alone or in combination with other anti-HBs antibodies could be considered as a potent alternative for Hepatitis B immune globulin (HBIG) as an HBV infection prophylactic or for passive immunotherapy against HBV infection
• Pooled human liver preparations, HepaRG, or HepG2 cell lines for metabolism studies of new psychoactive substances? A study using MDMA, MDBD, butylone, MDPPP, MDPV, MDPB, 5-MAPB, and 5-API as examples.Richter LHJ,  Flockerzi V,  Maurer HH,  Meyer MR
  J Pharm Biomed Anal. , Jun 2017
  Abstract : Metabolism studies play an important role in clinical and forensic toxicology. Because of potential species differences in metabolism, human samples are best suitable for elucidating metabolism. However, in the case of new psychoactive substances (NPS), human samples of controlled studies are not available. Primary human hepatocytes have been described as gold standard for in vitro metabolism studies, but there are some disadvantages such as high costs, limited availability, and variability of metabolic enzymes. Therefore, the aim of our study was to investigate and compare the metabolism of six methylenedioxy derivatives (MDMA, MDBD, butylone, MDPPP, MDPV, MDPB) and two bioisosteric analogues (5-MAPB, 5-API) using pooled human liver microsomes (pHLM) combined with cytosol (pHLC) or pooled human liver S9 fraction (pS9) all after addition of co-substrates for six phase I and II reactions. In addition, HepaRG and HepG2 cell lines were used. Results of the different in vitro tools were compared to each other, to corresponding published data, and to metabolites identified in human urine after consumption of MDMA, MDPV, or 5-MAPB. Incubations with pHLM plus pHLC showed similar results as pS9. A more cost efficient model for prediction of targets for toxicological screening procedures in human urine should be identified. As expected, the incubations with HepaRG provided better results than those with HepG2 concerning number and signal abundance of the metabolites. Due to easy handling without special equipment, incubations with pooled liver preparations should be the most suitable alternative to find targets for toxicological screening procedures for methylenedioxy derivatives and bioisosteric analogues.
• Induction of Apoptosis in HepaRG Cell Line by Aloe-Emodin through Generation of Reactive Oxygen Species and the Mitochondrial Pathway.Dong X,  Fu J,  Yin X,  Qu C,  Yang C,  He H,  Ni J
  Cell Physiol Biochem., Jun 2017
  Abstract : BACKGROUND/AIMS: Aloe-emodin (1,8-dihydroxy-3-hydroxymethyl-anthraquinone), an anthraquinone active compounds, is isolated from some traditional medicinal plants such as Rheum palmatum L. and Cassia occidentalis, which induce hepatotoxicity in rats. The aim of this study was to determine potential cytotoxic effects of aloe-emodin on HepaRG cells and to define the underlying mechanism. MTT was used to evaluate cell viability. Apoptotic cell death was analyzed via Annexin V-FITC/PI double staining. Intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were determined by flow cytometry, while the expression of apoptosis-related proteins was determined by Western blot analysis. Treatment with aloe-emodin significantly reduced cell viability and induced apoptosis in HepaRG cells in a dose- and time-dependent manner. It provoked ROS generation and depolarization of MMP in HepaRG cells when compared with controls. Aloe-emodin dose-dependently increased release of mitochondrial cytochrome c, and levels of Fas, p53, p21, Bax/Bcl-2 ratio, as well as activation of caspase-3, caspase-8, caspase-9, and subsequent cleavage of poly(ADP-ribose)polymerase (PARP). It also induced S-phase cell cycle arrest by increasing the expression of p21 and cyclin E proteins while significantly decreasing the expression of cyclin A and CDK2. These results suggest that aloe-emodin inhibits cell proliferation and induces apoptosis in HepaRG cells, most probably through a mechanism involving both Fas death pathway and the mitochondrial pathway by generation of ROS. These findings underscore the need for risk assessment of human exposure to aloe-emodin.
• Three-dimensional (3D) HepaRG Spheroid Model with Physiologically-Relevant Xenobiotic Metabolism Competence and Hepatocyte Functionality for Liver Toxicity Screening.Ramaiahgari SC,  Waidyanatha S,  Dixon D,  DeVito MJ,  Paules RS,  Ferguson SS, , , , , , ,
  Toxicol Sc, Jun 2017
  Abstract : Effective prediction of human responses to chemical and drug exposure is of critical importance in environmental toxicology research and drug development. While significant progress has been made to address this challenge using in vitro liver models, these approaches often fail due to inadequate tissue model functionality. Herein, we describe the development, optimization, and characterization of a novel three-dimensional (3D) spheroid model using differentiated HepaRG cells that achieve and maintain physiologically-relevant levels of xenobiotic metabolism (CYP1A2, CYP2B6, and CYP3A4/5). This in vitro model maintains a stable phenotype over multiple weeks in both 96- and 384-well formats, supports highly reproducible tissue-like architectures and models pharmacologically- and environmentally-important hepatic receptor pathways (i.e., AhR, CAR, PXR) analogous to primary human hepatocyte cultures. HepaRG spheroid cultures use 50-100X fewer cells than conventional two dimensional cultures, and enable the identification of metabolically-activated toxicants. Spheroid size, time in culture and culture media composition were important factors affecting basal levels of xenobiotic metabolism and liver enzyme inducibility with activators of hepatic receptors AhR, CAR and PXR. Repeated exposure studies showed higher sensitivity than traditional 2D cultures in identifying compounds that cause liver injury and metabolism-dependent toxicity. This platform combines the well-documented impact of 3D culture configuration for improved tissue functionality and longevity with the requisite throughput and repeatability needed for year-over-year toxicology screening.
• ?2-adrenergic receptor-mediated in vitro regulation of human hepatic drug transporter expression by epinephrine.Mayati A,  Moreau A,  Denizot C,  Stieger B,  Parmentier Y,  Fardel O
  Eur J Pharm Sci, Jun 2017
  Abstract : The catecholamine epinephrine is known to repress expression of hepatic drug metabolizing enzymes such as cytochromes P-450. The present study was designed to determine whether epinephrine may also target expression of main hepatic drug transporters, that play a major role in liver detoxification and are commonly coordinately regulated with drug detoxifying enzymes. Treatment of primary human hepatocytes with 10μM epinephrine for 24h repressed mRNA expression of various transporters, such as the sinusoidal influx transporters NTCP, OATP1B1, OATP2B1, OAT2, OAT7 and OCT1 and the efflux transporters MRP2, MRP3 and BSEP, whereas it induced that of MDR1, but failed to alter that of BCRP. Most of these changes in transporter mRNA levels were also found in epinephrine-exposed human highly-differentiated hepatoma HepaRG cells, which additionally exhibited reduced protein expression of OATP2B1 and MRP3, increased expression of P-glycoprotein and decreased transport activity of NTCP, OATPs and OCT1. Epinephrine effects towards transporter mRNA expression in human hepatocytes were next shown to be correlated to those of the selective β2-adrenoreceptor (ADR) agonist fenoterol, of the adenylate cyclase activator forskolin and of the cAMP analogue 8-bromo-cAMP. In addition, the non-selective β-ADR antagonist carazolol and the selective β2-ADR antagonist ICI-118,551, unlike the α-ADR antagonist phentolamine, suppressed epinephrine-mediated repressions of transporter mRNA expression. Taken together, these data indicate that epinephrine regulates in vitro expression of main hepatic drug transporters in a β2-ADR/adenylate cyclase/cAMP-dependent manner. Hepatic drug transport appears therefore as a target of the β2-adrenergic system, which may have to deserve attention for drugs interacting with β2-ADRs.
• Fluorometric evaluation of CYP3A4 expression using improved transgenic HepaRG cells carrying a dual-colour reporter for CYP3A4 and CYP3A7Takafumi Ueyama, Saori Tsuji Takemi Sugiyama, Masako Tada
  Scientific Reports, Jun 2017
  Abstract : Primary human hepatocytes are necessary to evaluate cytotoxicity, drug metabolism, and drug–druginteractions for candidate compounds in early-phase drug discovery and development. However,these analyses are often hampered by limited resources and functional or genetic variation amonglots. HepaRG human hepatocellular carcinoma cells can differentiate into mature hepatocyte-like cells(HepLCs) that possess similar metabolic activity to human hepatocytes. We previously establishedtransgenic HepaRG cells carrying a dual reporter that express red fluorescent protein (RFP) under thetranscriptional regulation of CYP3A7 in the hepatoblast-like cell state and enhanced green fluorescentprotein (EGFP) under the transcriptional regulation of CYP3A4 following HepLC differentiation. In thisstudy, we successfully isolated a subclone of transgenic CYP3A4G/7R HepaRG cells with an improvedHepLC differentiation potency. Midazolam metabolism by CYP3A4 in these HepLCs was comparableto that in wild-type HepLCs. The EGFP fluorescence intensity was greatly induced by rifampicin (RIF)treatment. There was a strong correlation between fluorometric and metabolic analyses. The foldchange in EGFP-positive cells was comparable to those in the CYP3A4 mRNA level and luminescence ofproluciferin metabolites. RIF treatment and cell proliferation increased the RFP-positive cell number.Thus, CYP3A4G/7R HepLCs provide a real-time, multiwell-based system to co-evaluate CYP3A4induction and hepatic regeneration.
• MicroRNA Hsa-miR-370-3p Suppresses the Expression and Induction of CYP2D6 by Facilitating mRNA Degradation.Zeng L, Chen Y, Wang Y, Yu LR, Knox B, Chen J, Shi T, Chen S, Ren Z, Guo L, Wu Y, Liu D, Huang K, Tong W, Yu D, Ning B
  Biochemical Pharmacology, Jun 2017
  Abstract : Cytochrome P450 2D6 (CYP2D6) participates in the metabolism of approximately 20-25% of prescribed drugs. Genetic polymorphisms influence the expression and/or activity of CYP2D6, and inter-individual differences in drug activation and elimination caused by CYP2D6 genetic variants were reported. However, little is known about the potential modulation of CYP2D6 expression by microRNAs (miRNAs). In the current study, by using in silico prediction of the stabilities of miRNA/mRNA complexes, we screened 38 miRNA candidates that may interact with the transcript of CYP2D6. An inverse correlation between the expression of miRNA hsa-miR-370-3p and the expression of CYP2D6 was observed in human liver tissue samples. Electrophoretic mobility shift assays confirmed that hsa-miR-370-3p was able to directly bind to its cognate target within the coding region of the CYP2D6 transcript. The transfection of hsa-miR-370-3p mimics into the HepG2CYP2D6 cell line, a genetically modified cell line that overexpresses exogenous CYP2D6, was able to suppress the expression of CYP2D6 significantly at both mRNA and protein levels. The transfection of hsa-miR-370-3p mimics was also able to inhibit endogenous mRNA expression and/or protein production of CYP2D6 in HepaRG cells. Furthermore, in HepaRG, HepG2, and Huh7 cells, dexamethasone-induced expression of CYP2D6 was inhibited by hsa-miR-370-3p mimics. To investigate whether the miRNA mediated suppression is caused by inhibiting protein translation or promoting mRNA degradation, an actinomycin D assay was used to measure the stability of CYP2D6 transcripts. The results indicated that hsa-miR-370-3p mimics facilitated significantly the degradation of CYP2D6 mRNA. In addition, proteomics analyses of proteins isolated from the miRNA/mRNA/protein complex suggested that a group of multifunctional proteins facilitated the interaction between hsa-miR-370-3p and CYP2D6, thereby promoting mRNA degradation.
• HBV Capsid Assembly Modulators, but not Nucleoside Analogs, Inhibit the Production of Extracellular Pregenomic RNA and Spliced RNA Variants.Liam AM, Ren S, Espiritu C, Kelly M, Lau V, Zheng L, Hartman GD, Flores OA, Klumpp K.
  Antimicrob Agents Chemother, May 2017
  Abstract : The Hepatitis B virus (HBV) core protein serves multiple essential functions in the viral life cycle and is being developed as a target for antiviral agents. Capsid assembly modulators (CAMs) are compounds that target core and misdirect capsid assembly, resulting in the suppression of HBV replication and virion production. Besides HBV DNA, circulating HBV RNA has been detected in patient serum and can be associated with treatment response. Here we studied the effect of HBV CAMs on the production of extracellular HBV RNA using infected HepaRG cells and primary human hepatocytes. Representative compounds from the sulfonamide carboxamide and heteroaryldihydropyrimidine series of CAMs were evaluated and compared to nucleos(t)ide analogs as inhibitors of the viral polymerase. Results showed that CAMs blocked extracellular HBV RNA with similar efficiencies as pgRNA encapsidation, HBV DNA replication, and Dane particles production. Nucleos(t)ide analogs inhibited viral replication and virion production, but not encapsidation or production of extracellular HBV RNA. Profiling of HBV RNA from both cultured supernatants and patient serum showed that extracellular viral RNA consisted of pgRNA and spliced pgRNA variants with internal deletion(s) but still retained the sequences at both 5' and 3' ends. Similar variants were detected in the supernatants of infected cells with and without nucleos(t)ide analog treatment. Overall, our data demonstrates that HBV CAMs represent direct antiviral agents with a differentiated profile as compared to nucleos(t)ide analogs, including the inhibition of extracellular pgRNA and spliced pgRNA.
• Activation of the aryl hydrocarbon receptor decreases rifampicin-induced CYP3A4 expression in primary human hepatocytes and HepaRGRasmussen MK, Daujat-Chavanieu M, Gerbal-Chaloin S.
  Toxicol Lett, May 2017
  Abstract : The role of the cross-talk between nuclear receptors in the regulation of Cytochrome P450 expression in the liver is well-documented. Most studies have focused on the cross-talk between the pregnane X receptor (PXR) and other receptors, such as the constitutive androstane receptor. However, cross-talk between PXRs and aryl hydrocarbon receptors (AhRs) has also been suggested, but reports regarding this cross-talk are conflicting. In the present study, we treated HepaRG and primary human hepatocytes (PHHs) with both a strong (TCDD) and weak (3-methylindole; 3MI) AhR activator to investigate their impact on PXR-regulated expression of CYP3A4. Moreover, we investigated the effect of co-activation of PXR, using rifampicin, and AhR, using TCDD and 3MI, on the regulation of CYP3A4 induction. We also investigated whether knockdown of AhR using siRNA affected the basal expression of PXR and CYP3A4 and induction of CYP3A4 by rifampicin, TCDD and 3MI. The results showed that the treatment of HepaRG cells, but not of PHHs, with AhR activators decreased mRNA expression of CYP3A4 and PXR. Moreover, in both HepaRG and PHHs, AhR activation decreased rifampicin-induced expression of CYP3A4 mRNA. Knock-down of AhR in PHHs increased both basal and rifampicin-induced expression of CYP3A4 mRNA. In conclusion, the presented results suggested that the cross-talk between PXR and AhR plays a role in the regulation of CYP3A4 gene expression.
• Isolation, purification and characterization of five active diketopiperazine derivatives from endophytic Streptomyces SUK 25 with antimicrobial and cytotoxic activities.Muhanna M. Alshaibani, Juriyati Jalil, Nik M. Sidik, Siti Junaidah Ahmad, Nurkhalida Kamal, Ruangelie Edrada-Ebel, Noraziah M. Zin
  J Microbiol Biotechnol, May 2017
  Abstract : On our search for new sources of bioactive secondary metabolites from Streptomyces sp., the ethyl acetate extracts from the endophytic Streptomyces SUK 25 afforded five active diketopiperazines compounds. The aim of this study was to characterize the bioactive compounds isolated from endophytic Streptomyces SUK 25 and evaluate its bioactivity against multiple drug resistance (MDR) bacteria such as Enterococcus raffinosus, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa and Enterobacter spp. pathogens and cytotoxic activities against the Human Hepatoma (HepaRG) cell line. The production of secondary metabolites by this strain was optimized through Thornton's media. Isolation, purification and identification of the bioactive compounds were carried out using high-performance liquid chromatography, high resolution mass liquid chromatography-mass spectrometry, fourier transform infra-red and nuclear magnetic resonance, cryopreserved HepaRG^TM cells was selected to carry out cytotoxicity. As a results endophytic Streptomyces SUK 25 produce four active diketopiperazines (DKP) compounds and an acetamide derivative, they were elucidated cyclo-(L-Val-L-Pro), cyclo-(L-Leu-L-Pro), cyclo-(L-Phe-L-Pro), cyclo-(L-Val-L-Phe) and N-(7-hydroxy-6-methyl-octyl)-acetamide. These active compounds exhibited activity against MRSA ATCC 43300 and Enterococcus raffinosus with low toxicity against human hepatoma HepaRG cells. The endophytic Streptomyces SUK 25 has the ability to produce DKP derivatives biologically active against some MDR bacteria with relatively low toxicity against HepaRG cells line.
• Mechanisms of hepatotoxicity associated with the monocyclic ?-lactam antibiotic BAL30072.Franziska Paech, Simon Messner,Jochen Spickermann,Mathias Wind,Anne-Hortense Schmitt-Hoffmann, Anne Therese WitschiBrett, A. Howell,Rachel J. Church,Jeff Woodhead, Marc Engelhardt, Stephan Krähenbühl, Email author, Martina Maurer
  Arch Toxicol, May 2017
  Abstract : BAL30072 is a new monocyclic β-lactam antibiotic under development which provides a therapeutic option for the treatment of severe infections caused by multi-drug-resistant Gram-negative bacteria. Despite the absence of liver toxicity in preclinical studies in rats and marmosets and in single dose clinical studies in humans, increased transaminase activities were observed in healthy subjects in multiple-dose clinical studies. We, therefore, initiated a comprehensive program to find out the mechanisms leading to hepatocellular injury using HepG2 cells (human hepatocellular carcinoma cell line), HepaRG cells (inducible hepatocytes derived from a human hepatic progenitor cell line), and human liver microtissue preparations. Our investigations demonstrated a concentration- and time-dependent reduction of the ATP content of BAL30072-treated HepG2 cells and liver microtissues. BAL30072 impaired oxygen consumption by HepG2 cells at clinically relevant concentrations, inhibited complexes II and III of the mitochondrial electron transport chain, increased the production of reactive oxygen species (ROS), and reduced the mitochondrial membrane potential. Furthermore, BAL 30072 impaired mitochondrial fatty acid metabolism, inhibited glycolysis, and was associated with hepatocyte apoptosis. Co-administration of N-acetyl-l-cysteine partially protected hepatocytes from BAL30072-mediated toxicity, underscoring the role of oxidative damage in the observed hepatocellular toxicity. In conclusion, BAL30072 is toxic for liver mitochondria and inhibits glycolysis at clinically relevant concentrations. Impaired hepatic mitochondrial function and inhibition of glycolysis can explain liver injury observed in human subjects receiving long-term treatment with this compound.
• A pump-free microfluidic 3D perfusion platform for the efficient differentiation of human hepatocyte-like cellsOng LJY, Chong LH, Jin L, Singh PK, Lee PS, Yu H, Ananthanarayanan A, Leo HL, Toh YC
  Biotechnol Bioeng, May 2017
  Abstract : The practical application of microfluidic liver models for in vitro drug testing is partly hampered by their reliance on human primary hepatocytes, which are limited in number and have batch-to-batch variation. Human stem cell-derived hepatocytes offer an attractive alternative cell source, although their 3D differentiation and maturation in a microfluidic platform have not yet been demonstrated. We develop a pump-free microfluidic 3D perfusion platform to achieve long-term and efficient differentiation of human liver progenitor cells into hepatocyte-like cells (HLCs). The device contains a micropillar array to immobilize cells three-dimensionally in a central cell culture compartment flanked by two side perfusion channels. Constant pump-free medium perfusion is accomplished by controlling the differential heights of horizontally-orientated inlet and outlet media reservoirs. Computational fluid dynamic simulation is used to estimate the hydrostatic pressure heads required to achieve different perfusion flow rates, which are experimentally validated by micro-particle image velocimetry, as well as viability and functional assessments in a primary rat hepatocyte model. We perform on-chip differentiation of HepaRG, a human bipotent progenitor cell, and discover that 3D microperfusion greatly enhances the hepatocyte differentiation efficiency over static 2D and 3D cultures. However, HepaRG progenitor cells are highly sensitive to the time-point at which microperfusion is applied. Isolated HepaRG cells that are primed as static 3D spheroids before being subjected to microperfusion yield a significantly higher proportion of HLCs (92%) than direct microperfusion of isolated HepaRG cells (62%). This platform potentially offers a simple and efficient means to develop highly functional microfluidic liver models incorporating human stem cell derived HLCs. This article is protected by copyright. All rights reserved.
• Evaluation of HepaRG cells for the assessment of indirect drug-induced hepatotoxicity using INH as a model substanceMann A, Pelz T, Rennert K, Mosig A, Decker M, Lupp A.
  Hum Cell, May 2017
  Abstract : HepaRG cells are widely used as an in vitro model to assess drug-induced hepatotoxicity. However, only few studies exist so far regarding their suitability to detect the effects of drugs requiring a preceding activation via the cytochrome P450 (CYP) system. A prototypic substance is the anti-tuberculosis agent INH, which is metabolized into N-acetylhydrazine, which then triggers hepatotoxicity. Therefore, the aim of the present study was to test if this effect can also be detected in HepaRG cells and if it can be counteracted by the known hepatoprotectant silibinin. For this purpose, differentiated HepaRG cells were treated with increasing concentrations of INH (0.1-100 mM) or 10 mM INH plus escalating concentrations of silibinin (1-100 µM). After 48 h of treatment, cell morphology and parameters indicating cell vitality, oxidative stress, and liver cell function were assessed. High concentrations of INH led to severe histopathological changes, reduced cell vitality and glutathione content, increased LDH and ASAT release into the medium, enhanced lipid peroxidation, and elevated cleaved caspase-3 expression. Additionally, glycogen depletion and reduced biotransformation capacity were seen at high INH concentrations, whereas at low concentrations an induction of biotransformation enzymes was noticed. Silibinin caused clear-cut protective effects, but with few parameters INH toxicity was even aggravated, most probably due to increased metabolization of INH into its toxic metabolite. In conclusion, HepaRG cells are excellently suited to evaluate the effects of substances requiring prior toxification via the CYP system, such as INH. They additionally enable the identification of complex substance interactions.
• The catechol-O-methyltransferase inhibitors tolcapone and entacapone uncouple and inhibit the mitochondrial respiratory chain in HepaRG cellsGrünig D, Felser A, Bouitbir J, Krähenbühl S.
  Toxicol In Vitro, May 2017
  Abstract : The catechol-O-methyltransferase inhibitor tolcapone causes hepatotoxicity and mitochondrial damage in animal models. We studied the interaction of tolcapone with mitochondrial respiration in comparison to entacapone in different experimental models. In HepaRG cells (human cell-line), tolcapone decreased the ATP content (estimated IC50 100±15μM) and was cytotoxic (estimated IC50 333±45μM), whereas entacapone caused no cytotoxicity and no ATP depletion up to 200μM. Cytochrome P450 induction did not increase the toxicity of the compounds. In HepaRG cells, tolcapone (not entacapone) inhibited maximal complex I- and complex II-linked oxygen consumption. In intact mouse liver mitochondria, tolcapone stimulated state 2 complex II-linked respiration and both compounds inhibited state 3 respiration of complex IV. Mitochondrial uncoupling was confirmed for both compounds by stimulation of complex I-linked respiration in the presence of oligomycin. Inhibition of complex I, II and IV for tolcapone and of complex I and IV for entacapone was directly demonstrated in disrupted mouse liver mitochondria. In HepaRG cells, tolcapone-induced inhibition of mitochondrial respiration was associated with increased lactate and ROS production and hepatocyte necrosis. In conclusion, both compounds uncouple oxidative phosphorylation and inhibit mitochondrial enzyme complexes. Tolcapone is a more potent mitochondrial toxicant than entacapone. Mitochondrial toxicity is a possible mechanism for tolcapone-associated hepatotoxicity.
• N-acetylcysteine potentiates diclofenac toxicity in Saccharomyces cerevisiae: stronger potentiation in ABC transporter mutant strainsAl-Attrache H, Chamieh H, Hamzé M, Morel I, Taha S, Abdel-Razzak Z.
  Drug Chem Toxicol, May 2017
  Abstract : Diclofenac (DCF) adverse reactions involve diverse mechanisms in different models. We recently demonstrated that DCF-induced toxicity in HepaRG decreases as they express DCF-metabolizing enzymes. DCF metabolism promotes toxicity in Saccharomyces cerevisiae expressing heterologous cytochromes-P450. N-Acetylcysteine (NAC) is used to treat diverse medical conditions due to its multiple properties (antioxidant, metal chelator, thiol-disulfide disruption). The latter property accounts for its mucolytic effects and broadens its potential molecular targets to signal transduction proteins, ABC transporters and others. Interaction of NAC with DCF effects depends on the experimental model. This study aims to investigate NAC/DCF interaction and the involvement of ABC transporters in wild type and mutant Saccharomyces cerevisiae. DCF inhibited yeast growth in a dose- and time-dependent manner and the cells started adapting to DCF 24-h post-treatment. NAC potentiated DCF-induced toxicity if added prior or parallel to DCF. Pretreatment with NAC increased its potentiation effect and compromised cells adaption to DCF. Post-treatment with NAC potentiated DCF toxicity without compromising adaptation. Moreover, mutant strains in ABC transporters Pdr5, Yor1, Bpt1 or Pdr15, were more sensitive to DCF; while mutant strains in Pdr5, Vmr1 or Pdr12 were more sensitive to NAC/DCF interaction. DCF ± NAC elicited on the mutant strain in Yap1, an oxidative stress-related protein, the same effects as on the wild type. Therefore, oxidative stress does not seem to be key actor in DCF toxicity in our model. Our hypothesis is that NAC potentiation effect is at least due to its ability to disrupt disulfide bridge in proteins required to overcome DCF toxicity in yeast.
• Polo-like-kinase 1 is a proviral host-factor for hepatitis B virus replicationDiab AM, Foca A, Fusil F, Lahlali T, Jalaguier P, Amirache F, N'Guyen L, Isorce N1, Cosset FL, Zoulim F, Andrisani OM, Durantel D1.
  Hepatology, Apr 2017
  Abstract : Chronic Hepatitis B Virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC) and current treatments for CHB and HCC are perfectible. Herein, we identified cellular Serine/Threonine Polo-like-kinase 1 (PLK1) as a positive effector of HBV replication. The aim of this study was to demonstrate the proviral role of PLK1 in HBV biosynthesis and validate PLK1 inhibition a potential antiviral strategy. To this end, we employed physiologically relevant HBV infection models of Primary Human Hepatocytes (PHH) and differentiated HepaRG cells, in conjunction with pharmacologic PLK1 inhibitors, siRNA-mediated knockdown, and overexpression of constitutively active PLK1 (PLK1CA ). In addition, humanized liver FRG mouse model was used to determine antiviral effect of PLK1 inhibitor BI-2536 on HBV infection in vivo. Lastly, in vitro PLK1 kinase assays and site-directed mutagenesis were employed to demonstrate HBV core protein (HBc) is a PLK1 substrate. We demonstrate HBV infection activated cellular PLK1 in PHH and dHepaRG cells. PLK1 inhibition by BI-2536 or siRNA-mediated knockdown suppressed, whereas overexpression of PLK1CA increased HBV DNA biosynthesis, supporting PLK1 effects on viral biosynthesis are specific, and PLK1 is a proviral cellular factor. Significantly, BI-2536 administration to HBV-infected humanized liver FRG mice strongly inhibited HBV infection, validating PLK1 as a novel antiviral target in vivo. The proviral action of PLK1 is associated with the biogenesis of the nucleocapsid, as BI-2536 leads to its decreased intracellular formation/accumulation. In this respect, our studies identified HBc as a PLK1 substrate in vitro, and mapped PLK1 phosphorylation sites on this protein. PLK1 is a proviral host factor that could be envisaged as a target for combined antiviral and anti-tumoral strategies against HBV infection and HBV mediated carcinogenesis. This article is protected by copyright. All rights reserved. © 2017 by the American Association for the Study of Liver Diseases. KEYWORDS: BI-2536; Hepatitis B virus; capsid-associated reverse transcription; core protein; polo-like-kinas
• Molecular basis of metabolism-mediated conversion of PK11195 from an antagonist to an agonist of the constitutive androstane receptorMackowiak B, Li L, Welch MA, Li D, Jones JW, Heyward S, Kane MA, Swaan PW, Wang H.
  Mol Pharmacol, Apr 2017
  Abstract : The constitutive androstane receptor (CAR) plays an important role in xenobiotic metabolism, energy homeostasis, and cell proliferation. Antagonism of CAR represents a key strategy for studying its function and may have potential clinical applications. However, specific human CAR (hCAR) antagonists are limited and conflicting data on the activity of these compounds have been reported. PK11195, a typical peripheral benzodiazepine receptor ligand, has been established as a potent hCAR deactivator in immortalized cells; whether it inhibits hCAR activity under physiologically-relevant conditions remains unclear. Here, we investigated the effects of PK11195 on hCAR in metabolically-competent human primary hepatocytes (HPH) and HepaRG cells. We show that although PK11195 antagonizes hCAR in HepG2 cells, it induces the expression of CYP2B6 and CYP3A4, targets of hCAR and the pregnane X receptor (PXR), in HPH, HepaRG, and PXR-knockout HepaRG cells. Utilizing a HPH-HepG2 co-culture model, we demonstrate that inclusion of HPH converts PK11195 from an antagonist to an agonist of hCAR, and such conversion was attenuated by potent CYP3A4 inhibitor ketoconazole. Metabolically, we show that the N-desmethyl metabolite is responsible for PK11195-mediated hCAR activation by facilitating hCAR interaction with coactivators and enhancing hCAR nuclear translocation in HPH. Structure-activity analysis revealed that N-demethylation alters the interaction of PK11195 with the binding pocket of hCAR to favor activation. Together, these results indicate that removal of a methyl group switches PK11195 from a potent antagonist of hCAR to an agonist in HPH and highlights the importance of physiologically-relevant metabolism when attempting to define the biological action of small molecules
• A Systematic Evaluation of microRNAs in Regulating Human Hepatic CYP2E1.Wang Y, Yu D, Tolleson WH, Yu LR, Green B, Zeng L, Chen Y, Chen S, Ren Z, Guo L, Tong W, Guan H, Ning B.
  Biochem Pharmacol, Apr 2017
  Abstract : Cytochrome P450 2E1 (CYP2E1) is an important drug metabolizing enzyme for processing numerous xenobiotics in the liver, including acetaminophen and ethanol. Previous studies have shown that microRNAs (miRNAs) can suppress CYP2E1 expression by binding to the 3'-untranslated region (3'-UTR) of its transcript. However, a systematic analysis of CYP2E1 regulation by miRNAs has not been described. Here, we applied in silico, in vivo, and in vitro approaches to investigate miRNAs involved in the regulation of CYP2E1. Initially, potential miRNA binding sites in the CYP2E1 mRNA transcript were identified and screened using in silico methods. Next, inverse correlations were found in human liver samples between the expression of CYP2E1 mRNA and the levels of two miRNA species, hsa-miR-214-3p and hsa-miR-942-5p. In a HepG2-derived CYP2E1 over-expression cell model, hsa-miR-214-3p exhibited strong suppression of CYP2E1 expression by targeting the coding region of its mRNA transcript, but hsa-miR-942-5p did not inhibit CYP2E1 levels. Electrophoretic mobility shift assays confirmed that hsa-miR-214-3p recruited other cellular protein factors to form stable complexes with specific sequences present in the CYP2E1 mRNA open reading frame. Transfection of HepaRG cells with hsa-miR-214-3p mimics inhibited expression of the endogenous CYP2E1 gene. Further, hsa-miR-214-3p mimics partially blocked ethanol-dependent increases in CYP2E1 mRNA and protein levels in HepG2 cells and they reduced the release of alanine aminotransferase from CYP2E1-overexpressing HepG2 cells exposed to acetaminophen. These results substantiate the suppressing effect of hsa-miR-214-3p on CYP2E1 expression.
• Initial sites of hepadnavirus integration into host genome in human hepatocytes and in the woodchuck model of hepatitis B-associated hepatocellular carcinomaChauhan R, Churchill ND, Mulrooney-Cousins PM, Michalak TI.
  Oncogenesis, Apr 2017
  Abstract : Hepatitis B virus (HBV) and the closely related woodchuck hepatitis virus (WHV) are potent carcinogens that trigger development of primary hepatocellular carcinoma (HCC). The initial sites of hepadnavirus-host genome integration, their diversity and kinetics of formation can be central to virus persistence and the initiation and progression of HCC. To recognize the nature of the very early virus-host interactions, we explored de novo infection of human hepatocyte-like HepaRG cells with authentic HBV and naive woodchucks with WHV. HepaRG were analyzed from several minutes post exposure to HBV onwards, whereas woodchuck liver biopsies at 1 or 3 h and 6 weeks post infection with WHV. Inverse PCR and clonal sequencing of the amplicons were applied to identify virus-host genomic junctions. HBV and WHV DNA and their replication intermediates became detectable in one hour after virus exposure. Concomitantly, HBV DNA integration into various host genes was detected. Notably, junctions of HBV X gene with retrotransposon sequences, such as LINE1 and LINE2, became prominent shortly after infection. In woodchucks, insertion of WHV X and preS sequences into host genome was evident at 1 and 3 h post infection (h.p.i.), confirming that hepadnavirus under natural conditions integrates into hepatocyte DNA soon after invasion. The HBV and WHV X gene enhancer II/core promotor sequence most often formed initial junctions with host DNA. Moreover, multiple virus-virus DNA fusions appeared from 1 h.p.i. onwards in both infected hepatocytes and woodchuck livers. In summary, HBV DNA integrates almost immediately after infection with a variety of host's sequences, among which tandemly repeating non-coding DNAs are common. This study revealed that HBV can engage mobile genetic elements from the beginning of infection to induce pro-oncogenic perturbations throughout the host genome. Such swift virus insertion was also evident in natural hepadnaviral infection in woodchucks.
• Impact of higher-order heme degradation products on hepatic function and hemodynamics.Seidel RA, Claudel T, Schleser FA, Ojha NK, Westerhausen M, Nietzsche S, Sponholz C, Cuperus F, Coldewey SM, Heinemann SH, Pohnert G, Trauner M, Bauer M.
  J Hepatol, Apr 2017
  Abstract : BACKGROUND& AIMS: While, traditionally, biliverdin and bilirubin are considered end products of heme catabolism, there is evidence for further degradation to diverse bioactive products. As such, Z-BOX?A and Z-BOX?B arise upon oxidation with unknown implications for hepatocellular function and integrity. We studied the impact of Z-BOX?A and B on hepatic functions and explored their alterations in health and cholestatic conditions. METHODS: Functional implications and mechanisms were investigated in rats, hepatocytic HepG2 and HepaRG cells, human immortalized hepatocytes, and isolated perfused livers. Z-BOX?A and B were determined by LC-MS/MS in acute and acute-on-chronic liver failure and hereditary unconjugated hyperbilirubinemia. RESULTS: Z-BOX?A and B are found in similar amounts in humans and rodents under physiological conditions. Serum concentrations increased ∼20-fold during cholestatic liver failure in humans and in hereditary deficiency of bilirubin glucuronidation in rats. Pharmacokinetic studies revealed shorter serum half-life of Z-BOX?A compared to its regio-isomer Z-BOX?B. While both compounds were taken up by hepatocytes, Z-BOX?A was enriched ∼100-fold and excreted in bile. Despite their reported vasoconstrictive properties in the brain vasculature, BOXes did not affect portal hemodynamics. Both Z-BOX?A and ?B showed dose-dependent cytotoxicity, affected the glutathione redox state, and differentially modulated activity of Rev-erbα and Rev-erbβ. Moreover, BOXes triggered remodeling of the hepatocellular cytoskeleton. CONCLUSIONS: Our data provide evidence that higher-order heme degradation products, namely Z-BOX?A and B, impair hepatocellular integrity and might mediate intra- and extrahepatic cytotoxic effects previously attributed to hyperbilirubinemia. LAY SUMMARY: Degradation of the blood pigment heme yields the bile pigment bilirubin and the oxidation products Z-BOX?A and Z-BOX?B. Serum concentrations of these bioactive molecules increase in jaundice and can impair liver function and integrity. Amounts of Z-BOX?A and Z-BOX?B that are observed during liver failure in humans have profound effects on hepatic function when added to cultured liver cells or infused into healthy rats. Copyright © 2017. Published by Elsevier B.V.
• Protective effects of an ethanol extract of Angelica keiskei against acetaminophen-induced hepatotoxicity in HepG2 and HepaRG cells.Choi YH, Lee HS, Chung CK, Kim EJ, Kang IJ
  Nutr Res Pract., Apr 2017
  Abstract : BACKGROUND/OBJECTIVE: Although Angelica keiskei (AK) has widely been utilized for the purpose of general health improvement among Asian, its functionality and mechanism of action. The aim of this study was to determine the protective effect of ethanol extract of AK (AK-Ex) on acute hepatotoxicity induced by acetaminophen (AAP) in HepG2 human hepatocellular liver carcinoma cells and HepaRG human hepatic progenitor cells. MATERIALS/METHODS: AK-Ex was prepared HepG2 and HepaRG cells were cultured with various concentrations and 30 mM AAP. The protective effects of AK-Ex against AAP-induced hepatotoxicity in HepG2 and HepaRG cells were evaluated using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, lactate dehydrogenase (LDH) assay, flow cytometry, and Western blotting. RESULTS: AK-Ex, when administered prior to AAP, increased cell growth and decreased leakage of LDH in a dose-dependent manner in HepG2 and HepaRG cells against AAP-induced hepatotoxicity. AK-Ex increased the level of Bcl-2 and decreased the levels of Bax, Bok and Bik decreased the permeability of the mitochondrial membrane in HepG2 cells intoxicated with AAP. AK-Ex decreased the cleavage of poly (ADP-ribose) polymerase (PARP) and the activation of caspase-9, -7, and -3. CONCLUSIONS: These results demonstrate that AK-Ex downregulates apoptosis via intrinsic and extrinsic pathways against AAP-induced hepatotoxicity. We suggest that AK could be a useful preventive agent against AAP-induced apoptosis in hepatocytes.
• Poly(malic acid) bearing Doxorubicin and N-Acetyl Galactosamine as a site-specific prodrug for targeting hepatocellular carcinoma.Venkatraj N, Nanjan MJ, Loyer P, Chandrasekar MJ, Cammas Marion S
  J Biomater Sci Polym Ed., Apr 2017
  Abstract : In the past, several systems of drug delivery carriers have been designed with a high capacity to target specific cells and/or tissues and a reduced non-specific toxicity. In this context, we synthesized and characterized novel poly(malic acid) derivatives bearing Doxorubicin (Dox), Poly(ethylene glycol) (PEG) and/or N-Acetyl Galactosamine (NAcGal) for drug delivery. These poly(malic acid) derivatives were obtained by chemical modification of the carboxylic acid lateral groups of poly(malic acid) (PMLA). The resulting nanoplatforms were evaluated for their in vitro cytotoxicity using the human HepaRG hepatoma cell line. Results reveal that the PMLA nanoplatform modified with PEG and Dox has an IC₅₀ of 936 nM corresponding to a Dox concentration of 47 nM, while the grafting of NAcGal onto the nanoplatform reduced the IC₅₀ to 527 nM corresponding to a Dox concentration of 26 nM. The presence of the targeting moiety, NAcGal, thus improves the cellular toxicity of the Dox.
• Down-regulation of cytochrome P450 2C8 by 3-methylcholanthrene in human hepatocellular carcinoma cell lines.Utgikar R, Riddick DS
  Can J Physiol Pharmacol, Apr 2017
  Abstract : The marked induction of cytochromes P450 such as CYP1A1 caused by polycyclic aromatic hydrocarbons (PAHs) like 3-methylcholanthrene (MC) is often accompanied by suppression of other hepatic P450s. The molecular mechanisms, functional consequences, and human relevance of P450 down-regulation by PAHs are poorly understood. MC suppresses mRNA levels for CYP2C8, an important human P450, in cultured human hepatocytes. To avoid hepatocyte lot-to-lot variability, we assessed <i>CYP2C8</i> regulation by MC in HepaRG cells, a terminally differentiated human hepatocellular carcinoma cell line that maintains high P450 expression. MC strongly induced CYP1A1 mRNA levels and markedly down-regulated CYP2C8 mRNA levels in HepaRG cells. Although MC also suppressed CYP2C8 mRNA levels in the HepG2 human hepatocellular carcinoma cell line, basal <i>CYP2C8</i> expression was extremely low. HepaRG cells appear to be an appropriate model system for studying the mechanisms and functional consequences of <i>CYP2C8</i> down-regulation by PAHs.
• Two-color fluorescent cytosine extension assay for the determination of global DNA methylationZhou G, Parfett C, Cummings-Lorbetskie C, Xiao GH, Desaulniers D.
  Biotechniques, Apr 2017
  Abstract : Here, we present a DNA restriction enzyme-based, fluorescent cytosine extension assay (CEA) to improve normalization and technical variation among sample-to-sample measurements. The assay includes end-labeling of parallel methylation-sensitive and methylation-insensitive DNA restriction enzyme digests along with co-purification and subsequent co-measurement of incorporated fluorescence. This non-radioactive, two-color fluorescent CEA (TCF-CEA) was shown to be a relatively rapid and accurate, with 3-fold greater precision than the one-color CEA. In addition, TCF-CEA provided an index of global DNA methylation that was sensitive to differences >5%. TCF-CEA results were highly correlated with LUminometric Methylation Assay (LUMA) results using human liver cell lines (HepG2, HepaRG, HC-04) as well as a human liver primary cell culture. Hypomethylation was observed in cells treated with the de-methylating agent 5-aza-2'-deoxycytidine. These results demonstrate that TCF-CEA provides a simple method for measuring relative degrees of global DNA methylation that could potentially be scaled up to higher-throughput formats.
• Efficient Transfection of Xenobiotic Responsive Element-biosensor plasmid using diether lipid and phosphatidylcholine liposomes in differentiated HepaRG cellsDemazeau M, Quesnot N, Ripoche N, Rauch C, Jefti? J, Morel F, Gauffre F, Benvegnu T, Loyer P
  Int J Pharm, Mar 2017
  Abstract : In this study, we evaluated cationic liposomes prepared from diether-NH2 and egg phosphatidylcholine (EPC) for in vitro gene delivery. The impact of the lipid composition, i.e. the EPC and Diether-NH2 molar ratio, on in vitro transfection efficiency and cytotoxicity was investigated using the human HEK293T and hepatoma HepaRG cells known to be permissive and poorly permissive cells for liposome-mediated gene transfer, respectively. Here, we report that EPC/Diether-NH2-based liposomes enabled a very efficient transfection with low cytotoxicity compared to commercial transfection reagents in both HEK293T and proliferating progenitor HepaRG cells. Taking advantage of these non-toxic EPC/Diether-NH2-based liposomes, we developed a method to efficiently transfect differentiated hepatocyte-like HepaRG cells and a biosensor plasmid containing a Xenobiotic Responsive Element and a minimal promoter driving the transcription of the luciferase reporter gene. We demonstrated that the luciferase activity was induced by a canonical inducer of cytochrome P450 genes, the benzo[a]pyrene, and two environmental contaminants, the fluoranthene, a polycyclic aromatic hydrocarbon, and the endosulfan, an organochlorine insecticide, known to induce toxicity and genotoxicity in differentiated HepaRG cells. In conclusion, we established a new efficient lipofection-mediated gene transfer in hepatocyte-like HepaRG cells opening new perspectives in drug evaluation relying on xenobiotic inducible biosensor plasmids.
• Mechanistic insights in cytotoxic and cholestatic potential of the endothelial receptor antagonists using HepaRG cells.Burbank MG, Sharanek A, Burban A, Mialanne H, Aerts H, Guguen-Guillouzo C, Weaver RJ, Guillouzo A
  Toxicol Sci., Mar 2017
  Abstract : Several endothelin receptor antagonists have been developed for the treatment of pulmonary arterial hypertension. Some of them have been related to clinical cases of hepatocellular injury (sitaxentan) and/or cholestasis (bosentan). We aimed to determine if ambrisentan and macitentan, in addition to bosentan and sitaxentan, could potentially cause liver damage in man by use of human HepaRG cells. Our results showed that like bosentan, macitentan induced cytotoxicity and cholestatic disorders characterized by bile canaliculi dilatation and impairment of myosin light chain kinase signaling. Macitentan also strongly inhibited taurocholic acid and carboxy-2',7'-dichlorofluorescein efflux while it had a much lower inhibitory effect on influx activity compared to bosentan and sitaxentan. Moreover, these three drugs caused decreased intracellular accumulation and parallel increased levels of total bile acids in culture media. In addition, all drugs except ambrisentan variably deregulated gene expression of bile acid transporters. By contrast, sitaxentan was hepatotoxic without causing cholestatic damage, likely via the formation of reactive metabolites and ambrisentan was not hepatotoxic. Together, our results show that some endothelin receptor antagonists can be hepatotoxic and that the recently marketed macitentan, structurally similar to bosentan, can also cause cholestatic alterations in HepaRG cells. The absence of currently known or suspected cases of cholestasis in patients suffering from pulmonary arterial hypertension treated with macitentan is rationalized by the lower therapeutic doses and Cmax, and longer receptor residence time compared to bosentan.
• An Automated Multiplexed Hepatotoxicity and CYP Induction Assay Using HepaRG Cells in 2D and 3DOtt LM, Ramachandran K, Stehno-Bittel L
  SLAS Discov., Mar 2017
  Abstract : Drug-induced liver injury (DILI) and drug-drug interactions (DDIs) are concerns when developing safe and efficacious compounds. We have developed an automated multiplex assay to detect hepatotoxicity (i.e., ATP depletion) and metabolism (i.e., cytochrome P450 1A [CYP1A] and cytochrome P450 3A4 [CYP3A4] enzyme activity) in two-dimensional (2D) and three-dimensional (3D) cell cultures. HepaRG cells were cultured in our proprietary micromold plates and produced spheroids. HepaRG cells, in 2D or 3D, expressed liver-specific proteins throughout the culture period, although 3D cultures consistently exhibited higher albumin secretion and CYP1A/CYP3A4 enzyme activity than 2D cultures. Once the spheroid hepatic quality was assessed, 2D and 3D HepaRGs were challenged to a panel of DILI- and CYP-inducing compounds for 7 days. The 3D HepaRG model had a 70% sensitivity to liver toxins at 7 days, while the 2D model had a 60% sensitivity. In both the 2D and 3D HepaRG models, 83% of compounds were predicted to be CYP inducers after 7 days of compound exposure. Combined, our results demonstrate that an automated multiplexed liver spheroid system is a promising cell-based method to evaluate DILI and DDI for early-stage drug discovery.
• Three-Dimensional Cell Cultures in Drug Discovery and DevelopmentYe Fang, Richard M. Eglen
  SLAS Discovery, Mar 2017
  Abstract : The past decades have witnessed significant efforts toward the development of three-dimensional (3D) cell cultures assystems that better mimic in vivo physiology. Today, 3D cell cultures are emerging, not only as a new tool in early drugdiscovery but also as potential therapeutics to treat disease. In this review, we assess leading 3D cell culture technologiesand their impact on drug discovery, including spheroids, organoids, scaffolds, hydrogels, organs-on-chips, and 3D bioprinting.We also discuss the implementation of these technologies in compound identification, screening, and development, rangingfrom disease modeling to assessment of efficacy and safety profiles.
• Longitudinal in vivo bioimaging of hepatocyte transcription factor activity following cholestatic liver injury in miceDelhove JM, Buckley SM, Perocheau DP, Karda R, Arbuthnot P, Henderson NC, Waddington SN, McKay T.
  Sci Rep, Feb 2017
  Abstract : Molecular mechanisms regulating liver repair following cholestatic injury remain largely unknown. We have combined a mouse model of acute cholestatic liver injury, partial bile duct ligation (pBDL), with a novel longitudinal bioimaging methodology to quantify transcription factor activity during hepatic injury and repair. We administered lentiviral transcription factor activated luciferase/eGFP reporter (TFAR) cassettes to neonatal mice enabling longitudinal TFAR profiling by continued bioimaging throughout the lives of the animals and following pBDL in adulthood. Neonatal intravascular injection of VSV-G pseudotyped lentivirus resulted in almost exclusive transduction of hepatocytes allowing analysis of hepatocyte-specific transcription factor activity. We recorded acute but transient responses with NF-κB and Smad2/3 TFAR whilst our Notch reporter was repressed over the 40 days of evaluation post-pBDL. The bipotent hepatic progenitor cell line, HepaRG, can be directed to differentiate into hepatocytes and biliary epithelia. We found that forced expression of the Notch inhibitor NUMB in HepaRG resulted in enhanced hepatocyte differentiation and proliferation whereas over-expressing the Notch agonist JAG1 resulted in biliary epithelial differentiation. In conclusion, our data demonstrates that hepatocytes rapidly upregulate NF-κB and Smad2/3 activity, whilst repressing Notch signalling. This transcriptional response to cholestatic liver injury likely promotes partial de-differentiation to allow pro-regenerative proliferation of hepatocytes.
• A transcriptomic study suggesting human iPSC-derived hepatocytes potentially offer a better in vitro model of hepatotoxicity than most hepatoma cell lines.Gao X, Liu Y
  Cell Biol Toxicol, Jan 2017
  Abstract : Hepatocytes derived from human induced pluripotent stem cells (iPSCs) hold great promise as an in vitro liver model by virtue of their unlimited long-term supply, stability and consistency in functionality, and affordability of donor diversity. However, the suitability of iPSC-derived hepatocytes (iPSC-Heps) for toxicology studies has not been fully validated. In the current study, we characterized global gene expression profiles of iPSC-Heps in comparison to those of primary human hepatocytes (PHHs) and several human hepatoma cell lines (HepaRG, HuH-7, HepG2, and HepG2/C3A). Furthermore, genes associated with hepatotoxicity, drug-metabolizing enzymes, transporters, and nuclear receptors were extracted for more detailed comparisons. Our results showed that iPSC-Heps correlate more closely to PHHs than hepatoma cell lines, suggesting that iPSC-Heps had a relatively mature hepatic phenotype that more closely resembles that of adult hepatocytes. HepaRG was the sole exception but nonetheless suffers from lack of donor diversity and poor prediction of hepatotoxicity. The effects of sex differences and DMSO treatment on gene expression of the cellular models were also investigated. Overall, the results presented in the current study suggest that iPSC-Heps represent a reproducible source of human hepatocytes and a promising in vitro model for hepatotoxicity evaluation. Further studies are needed to develop a robust protocol for hepatocyte differentiation towards a more mature adult phenotype.
• Low-dose acetaminophen induces early disruption of cell-cell tight junctions in human hepatic cells and mouse liver.Gamal W, Treskes P, Samuel K, Sullivan GJ, Siller R, Srsen V, Morgan K, Bryans A, Kozlowska A, Koulovasilopoulos A, Underwood I, Smith S, Del-Pozo J, Moss S, Thompson AI, Henderson NC, Hayes PC, Plevris JN, Bagnaninchi PO, Nelson LJ.
  Sci Rep, Jan 2017
  Abstract : Molecular mechanisms regulating liver repair following cholestatic injury remain largely unknown. We have combined a mouse model of acute cholestatic liver injury, partial bile duct ligation (pBDL), with a novel longitudinal bioimaging methodology to quantify transcription factor activity during hepatic injury and repair. We administered lentiviral transcription factor activated luciferase/eGFP reporter (TFAR) cassettes to neonatal mice enabling longitudinal TFAR profiling by continued bioimaging throughout the lives of the animals and following pBDL in adulthood. Neonatal intravascular injection of VSV-G pseudotyped lentivirus resulted in almost exclusive transduction of hepatocytes allowing analysis of hepatocyte-specific transcription factor activity. We recorded acute but transient responses with NF-κB and Smad2/3 TFAR whilst our Notch reporter was repressed over the 40 days of evaluation post-pBDL. The bipotent hepatic progenitor cell line, HepaRG, can be directed to differentiate into hepatocytes and biliary epithelia. We found that forced expression of the Notch inhibitor NUMB in HepaRG resulted in enhanced hepatocyte differentiation and proliferation whereas over-expressing the Notch agonist JAG1 resulted in biliary epithelial differentiation. In conclusion, our data demonstrates that hepatocytes rapidly upregulate NF-κB and Smad2/3 activity, whilst repressing Notch signalling. This transcriptional response to cholestatic liver injury likely promotes partial de-differentiation to allow pro-regenerative proliferation of hepatocytes.
• Transcriptional, functional and mechanistic comparisons of stem cell-derived hepatocytes, HepaRG cells and 3D human hepatocyte spheroids as predictive in vitro systems for drug-induced liver injury.Bell CC, Lauschke VM, Vorrink SU, Palmgren H, Duffin R, Andersson TB, Ingelman-Sundberg M.
  Drug Metab Dispos, Jan 2017
  Abstract : Reliable and versatile hepatic in vitro systems for the prediction of drug pharmacokinetics and toxicity are essential constituents of preclinical safety assessment pipelines for new medicines. Here, we compared three emerging cell systems, hepatocytes derived from induced pluripotent stem cells (hiPS-Hep), HepaRG cells and 3D primary human hepatocyte (PHH) spheroids at transcriptional and functional levels in a multi-center study to evaluate their potential as predictive models for drug-induced hepatotoxicity. Transcriptomic analyses revealed widespread gene expression differences between the three cell models, with 8,148 out of 17,462 analyzed genes (47%) being differentially expressed. Expression levels of genes involved in the metabolism of endogenous as well as xenobiotic compounds were significantly elevated in PHH spheroids, whereas genes involved in cell division and endocytosis were significantly upregulated in HepaRG and hiPS-Hep cells, respectively. Consequently, PHH spheroids were more sensitive to a panel of drugs with distinctly different toxicity mechanisms, an effect that was amplified by long-term exposure using repeated treatments. Importantly, toxicogenomic analyses revealed that transcriptomic changes in PHH spheroids were in compliance with cholestatic, carcinogenic or steatogenic in vivo toxicity mechanisms at clinically relevant drug concentrations. Combined, the data reveal important phenotypic differences between the three cell systems and suggest that PHH spheroids can be used for functional investigations of drug-induced liver injury in vivo in man.
• Hepatocellular Toxicity of Imidazole and Triazole Antimycotic Agents.Haegler P, Joerin L, Krähenbühl S, Bouitbir J
  Toxicol Sci., Jan 2017
  Abstract : Hepatotoxicity has been described for all antimycotic azoles currently marketed. A possible mechanism involving mitochondrial dysfunction has been postulated for ketoconazole, but not for the other azoles. The aim of the current investigations was to study the toxicity of different azoles in human cell models and to find out mechanisms of their toxicity. In HepG2 cells, posaconazole and ketoconazole were cytotoxic starting at 20 and 50 µM and decreased the cellular ATP content starting at 5 and 10 µM, respectively. In HepaRG cells, cytotoxicity started at 20 and 100 µM for posaconazole and ketoconazole, respectively, and was slightly accentuated by cytochrome P450 3A4 induction with rifampicin and 1A2 with 3-methylcholantrene. Voriconazole and fluconazole were not cytotoxic. In isolated mouse liver mitochondria, ketoconazole impaired membrane potential and complex I activity, whereas the other azoles were not toxic. In HepG2 cells exposed for 24 h, both posaconazole and ketoconazole (but not fluconazole or voriconazole) decreased the mitochondrial membrane potential, impaired the function of enzyme complexes of the electron transport chain, were associated with mitochondrial superoxide accumulation, decreased mitochondrial DNA and induced apoptosis. In HepG2 cells with mitochondrial dysfunction induced by the vitamin B12 antagonist hydroxy-cobalamin[c-lactam], cytotoxicity and/or ATP depletion was more accentuated than in untreated cells. We conclude that ketoconazole and posaconazole are mitochondrial toxicants starting at concentrations, which can be reached in vivo. Cytotoxicity and ATP depletion are more accentuated in cells with mitochondrial damage, suggesting that preexisting mitochondrial dysfunction is a susceptibility factor for hepatotoxicity associated with these drugs.
• Tailored LC-MS analysis improves the coverage of the intracellular metabolome of HepaRG cells.Cuykx M et al. Tailored LC-MS analysis improves the coverage of the intracellular metabolome of HepaRG cells.. AK390J Chromatogr A January-23;
  J Chromatogr A, Jan 2017
  Abstract : Metabolomics protocols are often combined with Liquid Chromatography-Mass Spectrometry (LC-MS) using mostly reversed phase chromatography coupled to accurate mass spectrometry, e.g. quadrupole time-of-flight (QTOF) mass spectrometers to measure as many metabolites as possible. In this study, we optimised the LC-MS separation of cell extracts after fractionation in polar and non-polar fractions. Both phases were analysed separately in a tailored approach in four different runs (two for the non-polar and two for the polar-fraction), each of them specifically adapted to improve the separation of the metabolites present in the extract. This approach improves the coverage of a broad range of the metabolome of the HepaRG cells and the separation of intra-class metabolites. The non-polar fraction was analysed using a C18-column with end-capping, mobile phase compositions were specifically adapted for each ionisation mode using different co-solvents and buffers. The polar extracts were analysed with a mixed mode Hydrophilic Interaction Liquid Chromatography (HILIC) system. Acidic metabolites from glycolysis and the Krebs cycle, together with phosphorylated compounds, were best detected with a method using ion pairing (IP) with tributylamine and separation on a phenyl-hexyl column. Accurate mass detection was performed with the QTOF in MS-mode only using an extended dynamic range to improve the quality of the dataset. Parameters with the greatest impact on the detection were the balance between mass accuracy and linear range, the fragmentor voltage, the capillary voltage, the nozzle voltage, and the nebuliser pressure. By using a tailored approach for the intracellular HepaRG metabolome, consisting of three different LC techniques, over 2200 metabolites can be measured with a high precision and acceptable linear range. The developed method is suited for qualitative untargeted LC-MS metabolomics studies.
• Impact of IL-27 on hepatocyte antiviral gene expression and function.Ramamurthy N, Boninsegna S, Adams R, Sahgal N, Lockstone H, Baban D, Marchi E, Klenerman P
  Wellcome Open Res., Jan 2017
  Abstract : Background: Interleukin (IL)-27 is a member of the IL-6/IL-12 family of cytokines. It is a potent cytokine, with potential antiviral impact, and has been shown to play a role in modulating functions of diverse cell types, including Th1, Th2, and NK and B cells, demonstrating both pro- and anti-inflammatory roles.  In hepatocytes, it is capable of inducing signal transducer and activator of transcription (STAT)1, STAT3 and interferon-stimulated genes. Methods: To address its role in viral hepatitis, the antiviral activity of IL-27 against hepatitis C virus (HCV) and hepatitis B virus (HBV) was tested in vitro using cell-culture-derived infectious HCV (HCVcc) cell culture system and the HepaRG HBV cell culture model. To further investigate the impact of IL-27 on hepatocytes, Huh7.5 cells were treated with IL-27 to analyse the differentially expressed genes by microarray analysis. Furthermore, by quantitative PCR, we analyzed the up-regulation of chemokine (CXCL)-10 in response to IL-27. Results: In both HCV and HBV infection models, we observed only a modest direct antiviral effect. Microarray analysis showed that the up-regulated genes mostly belonged to antigen presentation and DNA replication pathways, and involved strong up-regulation of CXCL-10, a gene associated with liver inflammation. Overall, gene set enrichment analysis showed a striking correlation of these genes with those up-regulated in response to related cytokines in diverse cell populations. Conclusion: Our data indicate that IL-27 can have a significant pro-inflammatory impact in vitro, although the direct antiviral effect is modest. It may have a potential impact on hepatocyte function, especially chemokine expression and antigen presentation.
• The virtual cell based assay: Current status and future perspectivesGraepel R, Lamon L, Asturiol D, Berggren E, Joossens E, Paini A, Prieto P, Whelan M, Worth A
  Toxicol In Vitro, Jan 2017
  Abstract : In order to replace the use of animals in toxicity testing, there is a need to predict in vivo toxic doses from concentrations that cause toxicological effects in relevant in vitro systems. The Virtual Cell Based Assay (VCBA) estimates time-dependent concentration of a test chemical in the cell and cell culture for a given in vitro system. The concentrations in the different compartments of the cell and test system are derived from ordinary differential equations, physicochemical parameters of the test chemical and properties of the cell line. The VCBA has been developed for a range of cell lines including BALB/c 3T3 cells, HepG2, HepaRG, lung A459 cells, and cardiomyocytes. The model can be used to design and refine in vitro experiments and extrapolate in vitro effective concentrations to in vivo doses that can be applied in risk assessment. In this paper, we first discuss potential applications of the VCBA: i) design of in vitro High Throughput Screening (HTS) experiments; ii) hazard identification (based on acute systemic toxicity); and iii) risk assessment. Further extension of the VCBA is discussed in the second part, exploring potential application to i) manufactured nanomaterials, ii) additional cell lines and endpoints, and considering iii) other opportunities.
• A novel toolbox for the in vitro assay of hepatitis D virus infectionZhao JH, Zhang YL, Zhang TY, Yuan LZ, Cheng T, Chen PJ, Yuan Q, Xia NS
  Sci Rep., Jan 2017
  Abstract : Hepatitis D virus (HDV) is a defective RNA virus that requires the presence of hepatitis B virus (HBV) for its life cycle. The in vitro HDV infection system is widely used as a surrogate model to study cellular infection with both viruses owing to its practical feasibility. However, previous methods for running this system were less efficient for high-throughput screening and large-scale studies. Here, we developed a novel method for the production of infectious HDV by adenoviral vector (AdV)-mediated transduction. We demonstrated that the AdV-based method yields 10-fold higher viral titers than the transient-transfection approach. The HDV-containing supernatant derived from AdV-infected Huh7 cells can be used as the inoculum in infectivity assays without requiring further concentration prior to use. Furthermore, we devloped a chemiluminescent immunoassay (HDV-CLEIA) to quantitatively determine intracellular HDAg with a dynamic range of 5-11,000 pg/mL. HDV-CLEIA can be used as an alternative approach to assess HDV infection. The advantages of our updated methodology were demonstrated through in vitro HDV infection of HepaRG cells and by evaluating the neutralization activity using antibodies that target various regions of the HBV/HDV envelope proteins. Together, the methods presented here com
• Netrin-1 Protects Hepatocytes Against Cell Death Through Sustained Translation During the Unfolded Protein ResponseLahlali T, Plissonnier ML, Romero-López C, Michelet M, Ducarouge B, Berzal-Herranz A, Zoulim F, Mehlen P, Parent R.
  Cell Mol Gastroenterol Hepatol, Jan 2017
  Abstract : Netrin-1, a multifunctional secreted protein, is up-regulated in cancer and inflammation. Netrin-1 blocks apoptosis induced by the prototypical dependence receptors deleted in colorectal carcinoma and uncoordinated phenotype-5. Although the unfolded protein response (UPR) triggers apoptosis on exposure to stress, it first attempts to restore endoplasmic reticulum homeostasis to foster cell survival. Importantly, UPR is implicated in chronic liver conditions including hepatic oncogenesis. Netrin-1's implication in cell survival on UPR in this context is unknown. METHODS: Isolation of translational complexes, determination of RNA secondary structures by selective 2'-hydroxyl acylation and primer extension/dimethyl sulfate, bicistronic constructs, as well as conventional cell biology and biochemistry approaches were used on in vitro-grown hepatocytic cells, wild-type, and netrin-1 transgenic mice. RESULTS: HepaRG cells constitute a bona fide model for UPR studies in vitro through adequate activation of the 3 sensors of the UPR (protein kinase RNA-like endoplasmic reticulum kinase (PERK)), inositol requiring enzyme 1α (IRE1α), and activated transcription factor 6 (ATF6). The netrin-1 messenger RNA 5'-end was shown to fold into a complex double pseudoknot and bear E-loop motifs, both of which are representative hallmarks of related internal ribosome entry site regions. Cap-independent translation of netrin 5' untranslated region-driven luciferase was observed on UPR in vitro. Unlike several structurally related oncogenic transcripts (l-myc, c-myc, c-myb), netrin-1 messenger RNA was selected for translation during UPR both in human hepatocytes and in mice livers. Depletion of netrin-1 during UPR induces apoptosis, leading to cell death through an uncoordinated phenotype-5A/C-mediated involvement of protein phosphatase 2A and death-associated protein kinase 1 in vitro and in netrin transgenic mice. CONCLUSIONS: UPR-resistant, internal ribosome entry site-driven netrin-1 translation leads to the inhibition of uncoordinated phenotype-5/death-associated protein kinase 1-mediated apoptosis in the hepatic context during UPR, a hallmark of chronic liver disease.
• CYP4F2 repression and a modified alpha-tocopherol (vitamin E) metabolism are two independent consequences of ethanol toxicity in human hepatocytes.Russo A, Bartolini D, Torquato P, Giusepponi D, Barola C, Galarini R, Birringer M, Lorkowski S, Galli F
  Toxicol In Vitro, Jan 2017
  Abstract : The expression of CYP4F2, a form of cytochrome P-450 with proposed role in α-tocopherol and long-chain fatty acid metabolism, was explored in HepG2 and HepaRG human hepatocytes during ethanol toxicity. Cytotoxicity, ROS production, and JNK and ERK1/2 kinase signaling increased in a dose and time-dependent manner during ethanol treatments; CYP4F2 gene expression decreased, while other CYP4F forms, namely 4F11 and 12, increased along with 3A4 and 2E1 isoforms. α-Tocopherol antagonized the cytotoxicity and CYP4F2 gene repression effect of ethanol in HepG2 cells. Ethanol stimulated the tocopherol-ω-hydroxylase activity and the other steps of vitamin E metabolism, which points to a minor role of CYP4F2 in this metabolism of human hepatocytes. PPAR-γ and SREBP-1c followed the same expression pattern of CYP4F2 in response to ethanol and α-tocopherol treatments. Moreover, the pharmacological inhibition of PPAR-γ synergized with ethanol in decreasing CYP4F2 protein expression, which suggests a role of this nuclear receptor in CYP4F2 transcriptional regulation. In conclusion, ethanol toxicity modifies the CYP expression pattern of human hepatic cells impairing CYP4F2 transcription and protein expression. These changes were associated with a lowered expression of the fatty acid biosynthesis regulators PPAR-γ and SREBP-1c, and with an increased enzymatic catabolism of vitamin E. CYP4F2 gene repression and a sustained vitamin E metabolism appear to be independent effects of ethanol toxicity in human hepatocytes.

2016

• Structure-activity relationship for branched oxyquinoline HIF activators: Effect of modifications to phenylacetamide "tailPoloznikov AA, Zakhariants AA, Nikulin SV, Smirnova NA, Hushpulian DM, Gaisina IN, Tonevitsky AG, Tishkov VI, Gazaryan IG.
  Biochimie, Dec 2016
  Abstract : HIF prolyl hydroxylase is a major regulator of HIF stability. Branched tail oxyquinolines have been identified as specific inhibitors of HIF prolyl hydroxylase and recently demonstrated clear benefits in various scenarios of neuronal failure. The structural optimization for branched tail oxyquinolines containing an acetamide bond has been performed in the present study using HIF1 ODD-luc reporter assay. The special attention has been paid to the length of a linker between acetamide group and phenyl ring, as well as substitutions in the phenyl ring in the other branch of the tail. The optimized version of branched tail oxyquinolines is 3-fold more potent than the original one identified before and shows a submicromolar EC50 in the reporter assay. The compounds have been studied in a "liver-on-a-chip" device to question their hepatotoxicity towards differentiated human HepaRG "hepatocytes": the absence of hepatotoxicity is observed up to 200 μM concentrations for all studied derivatives of branched tail oxyquinolines.
• Hepatitis B Virus Infection of HepaRG Cells, HepaRG-hNTCP Cells, and Primary Human Hepatocytes.Ni Y, Urban S
  Methods Mol Biol, Dec 2016
  Abstract : Investigations of virus-host interactions rely on suitable in vitro cell culture systems that efficiently support virus infection. Such systems should ideally provide conditions that resemble those of natural host cells, e.g., the cell-type specific signaling and metabolic pathways. For HBV infection, primary human hepatocytes (PHHs) are the most faithful system fulfilling these requirements but access to these cells is limited. Moreover, the reproducibility of experimental results depends on many factors including the preparation method or variability of the donors. The human liver cell line HepaRG, after differentiation, resembles PHHs with respect to many hepatocyte-specific markers including the expression of cytochrome P450 enzymes, liver-specific transcription factors, and transporter proteins like the HBV-specific receptor, sodium taurocholate co-transporting polypeptide (NTCP). HepaRG cells have also been shown to express key molecules of the innate immune system. So far, the HepaRG cell line is the only one allowing both studies on HBV/HDV infection and liver-specific drug toxicity and metabolism. The relative low susceptibility of HepaRG cells when compared with PHHs depends on various factors and can partially be overcome by constitutive expression of the receptor NTCP, allowing infection without full differentiation. Ectopic NTCP expression does not interfere with the ability of cell differentiation induced by DMSO. Here, we describe in detail how to technically perform HBV infection in vitro with these cells. The methods can be used to explore the mechanism of HBV infection and to build an antiviral screening platform suitable for evaluation of drug efficacy in cells that are metabolically close to primary human hepatocytes.
• Suppression of miR-628-3p and miR-641 is involved in rifampin-mediated CYP3A4 induction in HepaRG cells.Yan L, Liu J, Zhao Y, Nie Y, Ma X, Kan Q, Zhang L
  Pharmacogenomics, Dec 2016
  Abstract : AIM:This study aimed to explore the role of miRNAs in rifampin-mediated induction of CYP3A4 in HepaRG cells.MATERIALS & METHODS:Microarray was performed to determine the expression of miRNAs in rifampin-treated HepaRG cells, followed by bioinformatics and luciferase reporter gene assay to analyze miRNAs that directly target CYP3A4. Overexpression of miRNA mimics was used to study their effects on CYP3A4 induction.RESULTS:Forty-seven miRNAs were suppressed and 18 miRNAs were increased by rifampin (above twofold). MiR-628-3p and miR-641 repressed the 3'-UTR luciferase activity of CYP3A4. Overexpression of miR-628-3p and miR-641 showed significant decrease of CYP3A4 mRNA level as well as CYP3A4 induction by rifampin.CONCLUSION:miR-628-3p and miR-641 could directly target CYP3A4 and are negatively regulated in CYP3A4 induction by rifampin.
• Development of a Specific Substrate-Inhibitor Panel (Liver-on-a-Chip) for Evaluation of Cytochrome P450 Activity.Zakhariants AA, Burmistrova OA, Shkurnikov MY, Poloznikov AA, Sakharov DA
  Bull Exp Biol Med, Nov 2016
  Abstract : We developed a cytochrome P450 substrate-inhibitor panel for preclinical in vitro evaluation of drugs in a 3D histotypical microfluidic cell model of human liver (liver-on-a-chip technology). The concentrations of substrates and inhibitors were optimized to ensure reliable detection of the principal metabolites by HPLC-mass-spectroscopy. The selected specific substrate-inhibitor pairs, namely bupropion/2-phenyl-2-(1-piperidinyl)propane) for evaluation of CYP2B6B activity, tolbutamide/sulfaphenazole for CYP2C9, omeprazole/(+)-N-benzylnirvanol for CYP2C19, and testosterone/ketoconazole for CYP3A4, enable reliable evaluation of the drug metabolism pathway. In contrast to animal models characterized by species-specific expression profile and activity of cytochrome P450 isoforms, our in vitro model reflects the metabolism of human hepatocytes in vivo.
• Comparison of Liver Cell Models Using the Basel Phenotyping Cocktail.Berger B, Donzelli M, Maseneni S, Boess F, Roth A, Krähenbühl S, Haschke M
  Front Pharmacol, Nov 2016
  Abstract : Currently used hepatocyte cell systems for in vitro assessment of drug metabolism include hepatoma cell lines and primary human hepatocyte (PHH) cultures. We investigated the suitability of the validated in vivo Basel phenotyping cocktail (caffeine [CYP1A2], efavirenz [CYP2B6], losartan [CYP2C9], omeprazole [CYP2C19], metoprolol [CYP2D6], midazolam [CYP3A4]) in vitro and characterized four hepatocyte cell systems (HepG2 cells, HepaRG cells, and primary cryopreserved human hepatocytes in 2-dimensional [2D] culture or in 3D-spheroid co-culture) regarding basal metabolism and CYP inducibility. Under non-induced conditions, all CYP activities could be determined in 3D-PHH, CYP2B6, CYP2C19, CYP2D6, and CYP3A4 in 2D-PHH and HepaRG, and CYP2C19 and CYP3A4 in HepG2 cells. The highest non-induced CYP activities were observed in 3D-PHH and HepaRG cells. mRNA expression was at least four-fold higher for all CYPs in 3D-PHH compared to the other cell systems. After treatment with 20 μM rifampicin, mRNA increased 3- to 50-fold for all CYPs except CYP1A2 and 2D6 for HepaRG and 3D-PHH, 4-fold (CYP2B6) and 17-fold (CYP3A4) for 2D-PHH and four-fold (CYP3A4) for HepG2. In 3D-PHH at least a two-fold increase in CYP activity was observed for all inducible CYP isoforms while CYP1A2 and CYP2C9 activity did not increase in 2D-PHH and HepaRG. CYP inducibility assessed in vivo using the same phenotyping probes was also best reflected by the 3D-PHH model. Our studies show that 3D-PHH and (with some limitations) HepaRG are suitable cell systems for assessing drug metabolism and CYP induction in vitro. HepG2 cells are less suited to assess CYP induction of the 2C and 3A family. The Basel phenotyping cocktail is suitable for the assessment of CYP activity and induction also in vitro.
• Acetylated deoxycholic (DCA) and cholic (CA) acids are potent ligands of pregnane X (PXR) receptor.Carazo A, Hyrsova L, Dusek J, Chodounska H, Horvatova A, Berka K, Bazgier V, Gan-Schreier H, Chamulitrat W, Kudova E, Pavek P.
  Toxicol Lett, Nov 2016
  Abstract : The Pregnane X (PXR), Vitamin D (VDR) and Farnesoid X (FXR) nuclear receptors have been shown to be receptors of bile acids controlling their detoxification or synthesis. Chenodeoxycholic (CDCA) and lithocholic (LCA) acids are ligands of FXR and VDR, respectively, whereas 3-keto and acetylated derivates of LCA have been described as ligands for all three receptors. In this study, we hypothesized that oxidation or acetylation at position 3, 7 and 12 of bile acids DCA (deoxycholic acid), LCA, CA (cholic acid), and CDCA by detoxification enzymes or microbiome may have an effect on the interactions with bile acid nuclear receptors. We employed reporter gene assays in HepG2 cells, the TR-FRET assay with recombinant PXR and RT-PCR to study the effects of acetylated and keto bile acids on the nuclear receptors activation and their target gene expression in differentiated hepatic HepaRG cells. We demonstrate that the DCA 3,12-diacetate and CA 3,7,12-triacetate derivatives are ligands of PXR and DCA 3,12-diacetate induces PXR target genes such as CYP3A4, CYP2B6 and ABCB1/MDR1. In conclusion, we found that acetylated DCA and CA are potent ligands of PXR. Whether the acetylated bile acid derivatives are novel endogenous ligands of PXR with detoxification or physiological functions should be further studied in ongoing experiments.
• Impact of IL-27 on hepatocyte antiviral gene expression and function.Ramamurthy N, Boninsegna S, Adams R, Sahgal N, Lockstone H, Baban D, Marchi E, Klenerman P.
  Wellcome Open Res, Nov 2016
  Abstract : Background: Interleukin (IL)-27 is a member of the IL-6/IL-12 family of cytokines. It is a potent cytokine, with potential antiviral impact, and has been shown to play a role in modulating functions of diverse cell types, including Th1, Th2, and NK and B cells, demonstrating both pro- and anti-inflammatory roles.  In hepatocytes, it is capable of inducing signal transducer and activator of transcription (STAT)1, STAT3 and interferon-stimulated genes. Methods: To address its role in viral hepatitis, the antiviral activity of IL-27 against hepatitis C virus (HCV) and hepatitis B virus (HBV) was tested in vitro using cell-culture-derived infectious HCV (HCVcc) cell culture system and the HepaRG HBV cell culture model. To further investigate the impact of IL-27 on hepatocytes, Huh7.5 cells were treated with IL-27 to analyse the differentially expressed genes by microarray analysis. Furthermore, by quantitative PCR, we analyzed the up-regulation of chemokine (CXCL)-10 in response to IL-27. Results: In both HCV and HBV infection models, we observed only a modest direct antiviral effect. Microarray analysis showed that the up-regulated genes mostly belonged to antigen presentation and DNA replication pathways, and involved strong up-regulation of CXCL-10, a gene associated with liver inflammation. Overall, gene set enrichment analysis showed a striking correlation of these genes with those up-regulated in response to related cytokines in diverse cell populations. Conclusion: Our data indicate that IL-27 can have a significant pro-inflammatory impact in vitro, although the direct antiviral effect is modest. It may have a potential impact on hepatocyte function, especially chemokine expression and antigen presentation.
• Increase of Intracellular Cyclic Amp by PDE4 Inhibitors Affects HepG2 Cell Cycle Progression and SurvivalMassimi M, Cardarelli S, Galli F, Giardi MF, Ragusa F, Panera N, Cinque B, Cifone MG, Biagioni S, Giorgi M.
  J Cell Biochem, Nov 2016
  Abstract : Type 4 cyclic nucleotide phosphodiesterases (PDE4) are major members of a superfamily of enzymes (PDE) involved in modulation of intracellular signaling mediated by cAMP. Broadly expressed in most human tissues and present in large amounts in the liver, PDEs have in the last decade been key therapeutic targets for several inflammatory diseases. Recently, a significant body of work has underscored their involvement in different kinds of cancer, but with no attention paid to liver cancer. The present study investigated the effects of two PDE4 inhibitors, rolipram and DC-TA-46, on the growth of human hepatoma HepG2 cells. Treatment with these inhibitors caused a marked increase of intracellular cAMP and a dose- and time-dependent effect on cell growth. The concentrations of inhibitors that halved cell proliferation to about 50% were used for cell cycle experiments. Rolipram (10 µM) and DC-TA-46 (0.5 µM) produced a decrease of cyclin expression, in particular of cyclin A, as well as an increase in p21, p27 and p53, as evaluated by western blot analysis. Changes in the intracellular localization of cyclin D1 were also observed after treatments. In addition, both inhibitors caused apoptosis, as demonstrated by an Annexin-V cytofluorimetric assay and analysis of caspase-3/7 activity. Results demonstrated that treatment with PDE4 inhibitors affected HepG2 cell cycle and survival, suggesting that they might be useful as potential adjuvant, chemotherapeutic or chemopreventive agents in hepatocellular carcinoma. This article is protected by copyright. All rights reserved.
• Activation of nuclear receptor CAR by an environmental pollutant perfluorooctanoic acidAbe T, Takahashi M, Kano M, Amaike Y, Ishii C, Maeda K, Kudoh Y, Morishita T, Hosaka T, Sasaki T, Kodama S, Matsuzawa A, Kojima H, Yoshinari K
  Arch Toxicol, Nov 2016
  Abstract : Perfluorocarboxylic acids (PFCAs) including perfluorooctanoic acid (PFOA) are environmental pollutants showing high accumulation, thermochemical stability and hepatocarcinogenicity. Peroxisome proliferator-activated receptor α is suggested to mediate their toxicities, but the precise mechanism remains unclear. Previous reports also imply a possible role of constitutive androstane receptor (CAR), a key transcription factor for the xenobiotic-induced expression of various genes involved in drug metabolism and disposition as well as hepatocarcinogenesis. Therefore, we have investigated whether PFCAs activate CAR. In wild-type but not Car-null mice, mRNA levels of Cyp2b10, a CAR target gene, were increased by PFOA treatment. PFCA treatment induced the nuclear translocation of CAR in mouse livers. Since CAR activators are divided into two types, ligand-type activators and phenobarbital-like indirect activators, we investigated whether PFCAs are CAR ligands or not using the cell-based reporter gene assay that can detect CAR ligands but not indirect activators. As results, neither PFCAs nor phenobarbital increased reporter activities. Interestingly, in mouse hepatocytes, pretreatment with the protein phosphatase inhibitor okadaic acid prevented an increase in Cyp2b10 mRNA levels induced by phenobarbital as reported, but not that by PFOA. Finally, in human hepatocyte-like HepaRG cells, PFOA treatment increased mRNA levels of CYP2B6, a CAR target gene, as did phenobarbital. Taken together, our present results suggest that PFCAs including PFOA are indirect activators of mouse and human CAR and that the mechanism might be different from that for phenobarbital. The results imply a role of CAR in the hepatotoxicity of PFCAs.
• Induction of expression of aryl hydrocarbon receptor-dependent genes in human HepaRG cell line modified by shRNA and treated with ?-naphthoflavoneBrauze D, Zawierucha P, Kiwerska K, Bednarek K, Oleszak M, Rydzanicz M, Jarmuz-Szymczak M
  Mol Cell Biochem, Oct 2016
  Abstract : The aryl hydrocarbon receptor (AhR) mediates a variety of biological responses to ubiquitous environmental pollutants. In this study, the effects of administration of β-naphthoflavone (BNF), a potent AhR ligand, on the expression of AhR-dependent genes were examined by microarray and qPCR analysis in both, differentiated and undifferentiated HepaRG cell lines. To prove that BNF-induced changes of investigated genes were indeed AhR-dependent, we knock down the expression of AhR by stable transfection of HepaRG cells with shRNA. Regardless of genetical identity, our results clearly demonstrate different expression profiles of AhR-dependent genes between differentiated and undifferentiated HepaRG cells. Genes involved in metabolism of xenobiotics constitute only minute fraction of all genes regulated by AhR in HepaRG cells. Participation of AhR in induction of expression of genes associated with regulation of apoptosis or involved in cell proliferation as well as AhR-dependent inhibition of genes connected to cell adhesion could support suggestion of involvement of AhR not only in initiation but also in progression of carcinogenesis. Among the AhR-dependent genes known to be involved in metabolism of xenobiotics, cytochromes P4501A1 and 1B1 belong to the most inducible by BNF. On the contrary, expression of GSTA1 and GSTA2 was significantly inhibited after BNF treatment of HepaRG cells. Among the AhR-dependent genes that are not involved in metabolism of xenobiotics SERPINB2, STC2, ARL4C, and TIPARP belong to the most inducible by BNF. Our results imply involvement of Ah receptor in regulation of CYP19A1, the gene-encoding aromatase, and an enzyme responsible for a key step in the biosynthesis of estrogens.
• iTRAQ-based Membrane Proteomics Reveals Plasma Membrane Proteins Change During HepaRG Cell DifferentiationZhao M, Xu F,  Wu F, Yu D, Su N, Zhang Y, Cheng L, Xu P
  J Proteome Res, Oct 2016
  Abstract : HepaRG cell, a stabilized bipotent liver progenitor cell line, exhibits hepatocyte functions only after differentiation. However, the mechanism of transition from non-differentiated to differentiated states, accompanied by proliferation migration and differentiation, remains poorly understood, particularly those proteins residing in the plasma membrane. In this study, the membrane protein expression change of HepaRG cell during differentiation were systematically analyzed using an iTRAQ labeled quantitative membrane proteomics approach. A total of 70 membrane proteins were identified to be differentially expressed among 849 quantified membrane proteins. Function and disease clustering analysis proved that 11 of these proteins are involved in proliferation, migration and differentiation. Two key factors (MMP-14 and OCLN) were validated by qRT-PCR and western blot. Blockade of MMP-14 further demonstrated its important function during tumor cell migration. The dataset have been uploaded to ProteomeXchange with the identifier PXD004752.
• Stable overexpression of the constitutive androstane receptor reduces the requirement for culture with dimethyl sulfoxide for high drug metabolism in HepaRG cells.Van der Mark VA,  de Waart DR,  Shevchenko V,  Oude Elferink RP
  Drug Metab Dispos, Oct 2016
  Abstract : Dimethyl sulfoxide (DMSO) induces cellular differentiation and expression of drug metabolic enzymes in the human liver cell line HepaRG. However, DMSO also induces cell death and interferes with cellular activities. The aim of this study was to examine if overexpression of the constitutive androstane receptor (CAR, NR1I3), the nuclear receptor controlling various drug metabolism genes, would sufficiently promote differentiation and drug metabolism in HepaRG cells, optionally without using DMSO. By stable lentiviral overexpression of CAR, HepaRG cultures were less affected by DMSO in total protein content and obtained increased resistance to acetaminophen- and amiodarone-induced cell death. Transcript levels of CAR target genes were significantly increased in HepaRG-CAR cultures without DMSO, resulting in increased activities of cytochrome P450 (CYP) enzymes and bilirubin conjugation to levels equal or surpassing those of HepaRG cells cultured with DMSO. Unexpectedly, CAR overexpression also increased the activities of non-CAR target CYPs, as well as albumin production. In combination with DMSO treatment, CAR overexpression further increased transcript levels and activities of CAR targets. Induction of CYP1A2 and CYP2B6 remained unchanged, while CYP3A4 was reduced. Moreover, the metabolism of low clearance compounds warfarin and prednisolone was increased. In conclusion, CAR-overexpression creates a more physiologically relevant environment for studies on hepatic (drug) metabolism and differentiation in HepaRG cells without the utilization of DMSO. DMSO may still be applied to accomplish higher drug metabolism, required for sensitive assays, such as low-clearance studies and identification of (rare) metabolites, while reduced total protein content after DMSO culture is diminished by CAR overexpression. Dimethyl sulfoxide (DMSO) induces cellular differentiation and expression of drug metabolic enzymes in the human liver cell line HepaRG. However, DMSO also induces cell death and interferes with cellular activities. The aim of this study was to examine if overexpression of the constitutive androstane receptor (CAR, NR1I3), the nuclear receptor controlling various drug metabolism genes, would sufficiently promote differentiation and drug metabolism in HepaRG cells, optionally without using DMSO. By stable lentiviral overexpression of CAR, HepaRG cultures were less affected by DMSO in total protein content and obtained increased resistance to acetaminophen- and amiodarone-induced cell death. Transcript levels of CAR target genes were significantly increased in HepaRG-CAR cultures without DMSO, resulting in increased activities of cytochrome P450 (CYP) enzymes and bilirubin conjugation to levels equal or surpassing those of HepaRG cells cultured with DMSO. Unexpectedly, CAR overexpression also increased the activities of non-CAR target CYPs, as well as albumin production. In combination with DMSO treatment, CAR overexpression further increased transcript levels and activities of CAR targets. Induction of CYP1A2 and CYP2B6 remained unchanged, while CYP3A4 was reduced. Moreover, the metabolism of low clearance compounds warfarin and prednisolone was increased. In conclusion, CAR-overexpression creates a more physiologically relevant environment for studies on hepatic (drug) metabolism and differentiation in HepaRG cells without the utilization of DMSO. DMSO may still be applied to accomplish higher drug metabolism, required for sensitive assays, such as low-clearance studies and identification of (rare) metabolites, while reduced total protein content after DMSO culture is diminished by CAR overexpression. Dimethyl sulfoxide (DMSO) induces cellular differentiation and expression of drug metabolic enzymes in the human liver cell line HepaRG. However, DMSO also induces cell death and interferes with cellular activities. The aim of this study was to examine if overexpression of the constitutive androstane receptor (CAR, NR1I3), the nuclear receptor controlling various drug metabolism genes, would sufficiently promote differentiation and drug metabolism in HepaRG cells, optionally without using DMSO. By stable lentiviral overexpression of CAR, HepaRG cultures were less affected by DMSO in total protein content and obtained increased resistance to acetaminophen- and amiodarone-induced cell death. Transcript levels of CAR target genes were significantly increased in HepaRG-CAR cultures without DMSO, resulting in increased activities of cytochrome P450 (CYP) enzymes and bilirubin conjugation to levels equal or surpassing those of HepaRG cells cultured with DMSO. Unexpectedly, CAR overexpression also increased the activities of non-CAR target CYPs, as well as albumin production. In combination with DMSO treatment, CAR overexpression further increased transcript levels and activities of CAR targets. Induction of CYP1A2 and CYP2B6 remained unchanged, while CYP3A4 was reduced. Moreover, the metabolism of low clearance compounds warfarin and prednisolone was increased. In conclusion, CAR-overexpression creates a more physiologically relevant environment for studies on hepatic (drug) metabolism and differentiation in HepaRG cells without the utilization of DMSO. DMSO may still be applied to accomplish higher drug metabolism, required for sensitive assays, such as low-clearance studies and identification of (rare) metabolites, while reduced total protein content after DMSO culture is diminished by CAR overexpression.
• HDV RNA replication is associated with HBV repression and interferon-stimulated genes induction in super-infected hepatocytes.Alfaiate, Lucifora, Abeywickrama-Samarakoon, Michelet, Testoni, Cortay, Sureau, Zoulim, Dény, Durantel, , ,
  Antiviral research, Oct 2016
  Abstract : Hepatitis D virus (HDV) super-infection of Hepatitis B virus (HBV)-infected patients is the most aggressive form of viral hepatitis. HDV infection is not susceptible to direct anti-HBV drugs, and only suboptimal antiviral responses are obtained with interferon (IFN)-alpha-based therapy. To get insights on HDV replication and interplay with HBV in physiologically relevant hepatocytes, differentiated HepaRG (dHepaRG) cells, previously infected or not with HBV, were infected with HDV, and viral markers were extensively analyzed. Innate and IFN responses to HDV were monitored by measuring pro-inflammatory and interferon-stimulated gene (ISG) expression. Both mono- and super-infected dHepaRG cells supported a strong HDV intracellular replication, which was accompanied by a strong secretion of infectious HDV virions only in the super-infection setting and despite the low number of co-infected cells. Upon HDV super-infection, HBV replication markers including HBeAg, total HBV-DNA and pregenomic RNA were significantly decreased, confirming the interference of HDV on HBV. Yet, no decrease of circular covalently closed HBV DNA (cccDNA) and HBsAg levels was evidenced. At the peak of HDV-RNA accumulation and onset of interference on HBV replication, a strong type-I IFN response was observed, with interferon stimulated genes, RSAD2 (Viperin) and IFI78 (MxA) being highly induced. We established a cellular model to characterize in more detail the direct interference of HBV and HDV, and the indirect interplay between the two viruses via innate immune responses. This model will be instrumental to assess molecular and immunological mechanisms of this viral interference.
• Practical use of the Virtual Cell Based Assay: Simulation of repeat exposure experiments in liver cell linesPaini A, Mennecozzi M, Horvat T, Gerloff K, Palosaari T, Benito JV, Worth A
  Toxicol In Vitro, Oct 2016
  Abstract :   The Virtual Cell Based Assay (VCBA) was applied to simulate the long-term (repeat dose) toxic effects of chemicals, including substances in cosmetics and personal care products. The presented model is an extension of the original VCBA for simulation of single exposure and is implemented in a KNIME workflow. This work illustrates the steps taken to simulate the repeat dose effects of two reference compounds. i) Using caffeine, in vitro experimental viability data in single and repeated exposure from two human liver cell lines, HepG2 and HepaRG, were measured and used to optimize the VCBA. After which ii) amiodarone was tested and simulations were performed under repeated exposure conditions in HepaRG. The refined VCBA model can be used not only to support the design of long term in vitro experiments but also practical applications in risk assessment. Our model is a step towards the development of in silico predictive approaches to replace, refine, and reduce the in vivo repeat dose systemic toxicity studies in the assessment of human safety.Copyright © 2016. Published by Elsevier Ltd.
• Design, Synthesis, and Cytotoxicity of 5-Fluoro-2-methyl-6-(4-aryl-piperazin-1-yl) Benzoxazoles.Al-Harth,  Zoghaib WM, Pflüger M, T Schöpel M, Önder K, Reitsammer M, Hundsberger H,  Stoll R, Abdel-Jalil R
  Molecules, Sep 2016
  Abstract : To design new compounds suitable as starting points for anticancer drug development, we have synthesized a novel series of benzoxazoles with pharmaceutically advantageous piperazine and fluorine moieties attached to them. The newly synthesized benzoxazoles and their corresponding precursors were evaluated for cytotoxicity on human A-549 lung carcinoma cells and non-cancer HepaRG hepatocyes. Some of these new benzoxazoles show potential anticancer activity, while two of the intermediates show lung cancer selective properties at low concentrations where healthy cells are unaffected, indicating a selectivity window for anticancer compounds.
• Novel 3D Culture Systems for Studies of Human Liver Function and Assessments of the Hepatotoxicity of Drugs and Drug CandidatesLauschke VM,  Hendriks DF1,  Bell CC,  Andersson TB,  Ingelman-Sundberg M
  Chem Res Toxicol, Sep 2016
  Abstract : The liver is an organ with critical importance for drug treatment as the disposition and response to a given drug is often determined by its hepatic metabolism. Patient-specific factors can entail increased susceptibility to drug-induced liver injury, which constitutes a major risk for drugdevelopment programs causing attrition of promising drug candidates or costly withdrawals in postmarketing stages. Hitherto, mainly animalstudies and 2D hepatocyte systems have been used for the examination of human drug metabolism and toxicity. Yet, these models are far from satisfactory due to extensive species differences and because hepatocytes in 2D cultures rapidly dedifferentiate resulting in the loss of their hepatic phenotype and functionality. With the increasing comprehension that 3D cell culture systems more accurately reflect in vivo physiology, in the recent decade more and more research has focused on the development and optimization of various 3D culture strategies in an attempt to preserve liver properties in vitro. In this contribution, we critically review these developments, which have resulted in an arsenal of different static and perfused 3D models. These systems include sandwich-cultured hepatocytes, spheroid culture platforms, and various microfluidic liver or multiorgan biochips. Importantly, in many of these models hepatocytes maintain their phenotype for prolonged times, which allows probing the potential of newly developed chemical entities to cause chronic hepatotoxicity. Moreover, some platforms permit the investigation of drug action in specific genetic backgrounds or diseased hepatocytes, thereby significantly expanding the repertoire of tools to detect drug-induced liverinjuries. It is concluded that the development of 3D liver models has hitherto been fruitful and that systems are now at hand whose sensitivity and specificity in detecting hepatotoxicity are superior to those of classical 2D culture systems. For the future, we highlight the need to develop more integrated coculture model systems to emulate immunotoxicities that arise due to complex interactions between hepatocytes and immune cells.
• Evaluation of a novel PXR-knockout in HepaRG cellsWilliamson B, Lorbeer M, Mitchell MD, Brayman TG, Riley RJ
  Pharmacol Res Perspect, Sep 2016
  Abstract : The nuclear pregnane X receptor (PXR) regulates the expression of genes involved in the metabolism, hepatobiliary disposition, and toxicity of drugs and endogenous compounds. PXR is a promiscuous nuclear hormone receptor (NHR) with significant ligand and DNA-binding crosstalk with the constitutive androstane receptor (CAR); hence, defining the precise role of PXR in gene regulation is challenging. Here, utilising a novel PXR-knockout (KO) HepaRG cell line, real-time PCR analysis was conducted to determine PXR involvement for a range of inducers. The selective PXR agonist rifampicin, a selective CAR activator, 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO), and dual activators of CAR and PXR including phenobarbital (PB) were analyzed. HepaRG control cells (5F clone) were responsive to prototypical inducers of CYP2B6 and CYP3A4. No response was observed in the PXR-KO cells treated with rifampicin. Induction of CYP3A4 by PB, artemisinin, and phenytoin was also much reduced in PXR-KO cells, while the response to CITCO was maintained. This finding is in agreement with the abolition of functional PXR expression. The apparent EC50 values for PB were in agreement between the cell lines; however, CITCO was ~threefold (0.3 μmol/L vs. 1 μmol/L) lower in the PXR-KO cells compared with the 5F cells for CYP2B6 induction. Results presented support the application of the novel PXR-KO cells in the definitive assignment of PXR-mediated CYP2B6 and CYP3A4 induction. Utilization of such cell lines will allow advancement in composing structure activity relationships rather than relying predominantly on pharmacological manipulations and provide in-depth mechanistic evaluation.
• Opsonisation of nanoparticles prepared from poly(?-hydroxybutyrate) and poly(trimethylene carbonate)-b-poly(malic acid) amphiphilic diblock copolymers: Impact on the in vitro cell uptake by primary human macrophages and HepaRG hepatoma cells.Vene E,  Barouti G,  Jarnouen K,  Gicquel T,  Rauch C,  Ribault C,  Guillaume SM,  Cammas-Marion S,  Loyer P
  Int J Pharma, Sep 2016
  Abstract : The present work reports the investigation of the biocompatibility, opsonisation and cell uptake by human primary macrophages and HepaRG cells of nanoparticles (NPs) formulated from poly(β-malic acid)-b-poly(β-hydroxybutyrate) (PMLA-b-PHB) and poly(β-malic acid)-b-poly(trimethylene carbonate) (PMLA-b-PTMC) diblock copolymers, namely PMLA800-b-PHB7300, PMLA4500-b-PHB4400, PMLA2500-b-PTMC2800 and PMLA4300-b-PTMC1400. NPs derived from PMLA-b-PHB and PMLA-b-PTMC do not trigger lactate dehydrogenase release and do not activate the secretion of pro-inflammatory cytokines demonstrating the excellent biocompatibility of these copolymers derived nano-objects. Using a protein adsorption assay, we demonstrate that the binding of plasma proteins is very low for PMLA-b-PHB-based nano-objects, and higher for those prepared from PMLA-b-PTMC copolymers. Moreover, a more efficient uptake by macrophages and HepaRG cells is observed for NPs formulated from PMLA-b-PHB copolymers compared to that of PMLA-b-PTMC-based NPs. Interestingly, the uptake in HepaRG cells of NPs formulated from PMLA800-b-PHB7300 is much higher than that of NPs based on PMLA4500-b-PHB4400. In addition, the cell internalization of PMLA800-b-PHB7300 based-NPs, probably through endocytosis, is strongly increased by serum pre-coating in HepaRG cells but not in macrophages. Together, these data strongly suggest that the binding of a specific subset of plasmatic proteins onto the PMLA800-b-PHB7300-based NPs favors the HepaRG cell uptake while reducing that of macrophages.
• Early alterations of bile canaliculi dynamics and the ROCK/MLCK pathway are characteristics of drug-induced intrahepatic cholestasis.Burbank MG,  Burban A,  Sharanek A,  Weaver RJ,  Guguen-Guillouzo C,  Guillouzo A,
  Drug Metab Dispos, Aug 2016
  Abstract : Intrahepatic cholestasis represents 20-40% of drug-induced injuries from which a large proportion remains unpredictable. We aimed to investigate mechanisms underlying drug-induced cholestasis and improve upon its early detection using human HepaRG cells and a set of 12 cholestatic and 6 non-cholestatic drugs. Bile canaliculi (BC) dynamics, Rho/myosin-light-chain (MLC) kinase pathway implication, efflux inhibition of taurocholate, a predominant BSEP substrate, and expression of the major canalicular and basolateral bile acids transporters were analyzed. We demonstrated 12 cholestatic drugs classified on the basis of reported clinical findings caused disturbances of both BC dynamics, characterized by either dilation or constriction, and alteration of the ROCK/MLCK signalling pathway while non cholestatic compounds, by contrast, have no effect. Co-treatment with Y-27632, a ROCK inhibitor, and calmodulin, a MLCK activator, reduced BC constriction and dilatation, respectively confirming the role of these pathways in intrahepatic drug-induced cholestasis. By contrast, inhibition of taurocholate efflux and/or human BSEP overexpressed in membrane vesicles (published data) was not observed with all cholestatic drugs and moreover examples of non-cholestatic compounds reportedly found to inhibit BSEP. Transcripts levels of major bile acids transporters were determined after 24h-treatment. BSEP, NTCP and OATP-B were down-regulated with most cholestatic and some non-cholestatic drugs while deregulation of MRPs was more variable, probably mainly reflecting secondary effects. Together, our results show cholestatic drugs consistantly cause an early alteration of BC dynamics associated with modulation of the Rho/MLC kinases and these changes are more specific than efflux inhibition measurements alone as predictive non-clinical markers of drug-induced cholestasis.
• A modified multiparametric assay using HepaRG cells for predicting the degree of drug-induced liver injury risk.Tomida T,  Okamura H,  Yokoi T,  Konno Y,
  J Appl Toxicol., Aug 2016
  Abstract : The approach for predicting the degree of drug-induced liver injury (DILI) risk was investigated quantitatively in a modified multiparametric assay using HepaRG cells. Thirty-eight drugs were classified by DILI risk into five categories based on drug labels approved by the Food and Drug Administration (FDA) as follows: withdrawn (WDN), boxed warning (BW), warnings and precautions (WP), adverse reactions (AR), and no match (NM). Also, WP was classified into two categories: high and low concern. Differentiated HepaRG cells were treated with drugs for 24 h. The maximum concentration was set at 100-fold the therapeutic maximum plasma concentration (Cmax ). After treatment with drugs, the cell viability, glutathione content, caspase 3/7 activity, lactate dehydrogenase leakage and albumin secretion were measured. As modified cut-off values of each parameter, the TC50 (toxic concentration that decreased the response by 50%) and EC200 (effective concentration giving a response equal to 200% of controls) were calculated. In addition, the toxicity score (total sum score of the cytotoxic level of each parameter) was calculated. This modified multiparametric assay showed an 87% sensitivity and 87% specificity for predicting the DILI risk. The toxicity score showed a good predictive performance for WDN, BW and WP (high concern) categories [cut-off: score ≥ 1; area under a receiver operating characteristic curve (ROC-AUC): 0.88], and for WDN and BW categories (cut-off: score ≥ 3; ROC-AUC: 0.88). This study newly indicated that the degree of DILI risk might be predictable quantitatively by assessing the toxicity score in the modified multiparametric assay using HepaRG cells. 
• New screening criteria setting on evaluation of cytochrome P450 induction using HepaRG cells with multiplex branched DNA technologies in early drug discovery.Ogasawara A,  Yamada Y,  Torimoto N,  Tsuda N,  Aohara F,  Ohashi R,  Taniguchi H., , , , , ,
  Drug Metab Lett., Aug 2016
  Abstract : Cytochrome P450 (CYP) enzymes are induced by some therapeutic drugs, leading to interactions reducing drug plasma concentrations. Recently, an assessment of CYP induction using messenger RNA (mRNA) levels has shown advantages over measurement of enzymatic activity; it has a larger dynamic range of induction and enables us to measure the intrinsic induction potential of time-dependent CYP inhibitors. Therefore, we constructed a new evaluation system for CYP induction combining HepaRG cell line with multiplex branched DNA technologies to measure changes in CYP1A2, CYP2B6, and CYP3A4 mRNA expression from a cell-culture plate, and propose new criteria to evaluate induction potency of CYPs for new chemical entities in early drug discoveries. The induction potency was evaluated by calculating the concentration of test compounds that gives 10% of positive control response (R10), which is measurable even when full dose-response curves cannot be obtained. Compared with the evaluation of CYP induction in human hepatocytes, the response at R10 in HepaRG cells suggested the possibility of exhibiting induction potency for corresponding CYPs. Interestingly, the results with our in-house 109 compounds showed that several compounds induced CYP1A2 or CYP2B6 expression without upregulation of CYP3A4, suggesting that R10 is valuable in examining the induction potential of these CYP isoforms.
• Metformin Protects against Acetaminophen Hepatotoxicity by Attenuation of Mitochondrial Oxidant Stress and Dysfunction.Du K,  Ramachandran A,  Weemhoff JL,  Chavan H,  Xie Y,  Krishnamurthy P,  Jaeschke H, , , , , ,
  Toxicol Sci., Aug 2016
  Abstract : Overdose of acetaminophen (APAP) causes severe liver injury and even acute liver failure in both mice and human. A recent study by Kim et al. (2015) showed that metformin, a first-line drug to treat type 2 diabetes mellitus, protected against APAP hepatotoxicity in mice. However, its exact protective mechanism has not been well clarified. To investigate this, C57BL/6J mice were treated with 400 mg/kg APAP and 350 mg/kg metformin was given 0.5h pre- or 2h post-APAP. Our data showed that pretreatment with metformin protected against APAP hepatotoxicity, as indicated by the over 80% reduction in plasma ALT activities and significant decrease in centrilobular necrosis. Metabolic activation of APAP, as indicated by glutathione depletion and APAP-protein adducts formation, was also slightly inhibited. However, 2h post-treatment with metformin still reduced liver injury by 50%, without inhibition of adduct formation. Interestingly, neither pre- nor post-treatment of metformin inhibited c-jun N-terminal kinase (JNK) activation or its mitochondrial translocation. In contrast, APAP-induced mitochondrial oxidant stress and dysfunction were greatly attenuated in these mice. In addition, mice with 2h post-treatment with metformin also showed significant inhibition of complex I activity, which may contribute to the decreased mitochondrial oxidant stress. Furthermore, the protection was reproduced in JNK activation-absent HepaRG cells treated with 20 mM APAP followed by 0.5 or 1 mM metformin 6h later, confirming JNK-independent protection mechanisms. Thus, metformin protects against APAP hepatotoxicity by attenuating the mitochondrial oxidant stress and subsequent mitochondrial dysfunction, and may be a potential therapeutic option for APAP overdose patients.
• Accessing 3D microtissue metabolism : Lactate and oxygen monitoring in hepatocyte spheroids.Weltin A, Hammer S, Noor F, Kaminski Y, Kieninger J, Urban GA
  Biosens Bioelectron, Jul 2016
  Abstract : 3D hepatic microtissues, unlike 2D cell cultures, retain many of the in-vivo-like functionalities even after long-term cultivation. Such 3D cultures are increasingly applied to investigate liver damage due to drug exposure in toxicology. However, there is a need for thorough metabolic characterization of these microtissues for mechanistic understanding of effects on culture behaviour. We measured metabolic parameters from single human HepaRG hepatocyte spheroids online and continuously with electrochemical microsensors. A microsensor platform for lactate and oxygen was integrated in a standard 96-well plate. Electrochemical microsensors for lactate and oxygen allow fast, precise and continuous long-term measurement of metabolic parameters directly in the microwell. The demonstrated capability to precisely detect small concentration changes by single spheroids is the key to access their metabolism. Lactate levels in the culture medium starting from 50µM with production rates of 5µMh-1 were monitored and precisely quantified over three days. Parallel long-term oxygen measurements showed no oxygen depletion or hypoxic conditions in the microwell. Increased lactate production by spheroids upon suppression of the aerobic metabolism was observed. The dose-dependent decrease in lactate production caused by the addition of the hepatotoxic drug Bosentan was determined. We showed that in a toxicological application, metabolic monitoring yields quantitative, online information on cell viability, which complements and supports other methods such as microscopy. The demonstrated continuous access to 3D cell culture metabolism within a standard setup improves in vitro toxicology models in replacement strategies of animal experiments. Controlling the microenvironment of such organotypic cultures has impact in tissue engineering, cancer therapy and personalized medicine.Copyright © 2016 Elsevier B.V. All rights reserved.
• Optimization, formulation, and characterization of multiflavonoids-loaded flavanosome by bulk or sequential technique.Karthivashan G,  Masarudin MJ,  Kura AU,  Abas F,  Fakurazi S, , , , , , , ,
  Int J Nanomedicine, Jul 2016
  Abstract : This study involves adaptation of bulk or sequential technique to load multiple flavonoids in a single phytosome, which can be termed as "flavonosome". Three widely established and therapeutically valuable flavonoids, such as quercetin (Q), kaempferol (K), and apigenin (A), were quantified in the ethyl acetate fraction of Moringa oleifera leaves extract and were commercially obtained and incorporated in a single flavonosome (QKA-phosphatidylcholine) through four different methods of synthesis - bulk (M1) and serialized (M2) co-sonication and bulk (M3) and sequential (M4) co-loading. The study also established an optimal formulation method based on screening the synthesized flavonosomes with respect to their size, charge, polydispersity index, morphology, drug-carrier interaction, antioxidant potential through in vitro 1,1-diphenyl-2-picrylhydrazyl kinetics, and cytotoxicity evaluation against human hepatoma cell line (HepaRG). Furthermore, entrapment and loading efficiency of flavonoids in the optimal flavonosome have been identified. Among the four synthesis methods, sequential loading technique has been optimized as the best method for the synthesis of QKA-phosphatidylcholine flavonosome, which revealed an average diameter of 375.93±33.61 nm, with a zeta potential of -39.07±3.55 mV, and the entrapment efficiency was >98% for all the flavonoids, whereas the drug-loading capacity of Q, K, and A was 31.63%±0.17%, 34.51%±2.07%, and 31.79%±0.01%, respectively. The in vitro 1,1-diphenyl-2-picrylhydrazyl kinetics of the flavonoids indirectly depicts the release kinetic behavior of the flavonoids from the carrier. The QKA-loaded flavonosome had no indication of toxicity toward human hepatoma cell line as shown by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide result, wherein even at the higher concentration of 200 µg/mL, the flavonosomes exert >85% of cell viability. These results suggest that sequential loading technique may be a promising nanodrug delivery system for loading multiflavonoids in a single entity with sustained activity as an antioxidant, hepatoprotective, and hepatosupplement candidate.
• Effect of Circulation Parameters on Functional Status of HepaRG Spheroids Cultured in Microbioreactor.Semenova OV,  Petrov VA,  Gerasimenko TN,  Aleksandrova AV,  Burmistrova OA,  Khutornenko AA,  Osipyants AI,  Poloznikov AA,  Sakharov DA, , , ,
  Bull Exp Biol Med, Jul 2016
  Abstract : We studied the relationship between microcirculation parameters and functional status of HepaRG cells in spheroids and chose an optimal regimen within the physiologically permissible limits of mechanical impact for the cells that maintains the expression of functional genes of the liver.
• Human arylacetamide deacetylase hydrolyzes ketoconazole to trigger hepatocellular toxicity.Fukami T,  Iida A,  Konishi K,  Nakajima M, , , , , , , , ,
  Biochem Pharmacol, Jul 2016
  Abstract : Ketoconazole (KC), an antifungal agent, rarely causes severe liver injury when orally administered. It has been reported that KC is mainly hydrolyzed to N-deacetyl ketoconazole (DAK), followed by the N-hydroxylation of DAK by flavin-containing monooxygenase (FMO). Although the metabolism of KC has been considered to be associated with hepatotoxicity, the responsible enzyme(s) remain unknown. The purpose of this study was to identify the responsible enzyme(s) for KC hydrolysis in humans and to clarify their relevance to KC-induced toxicity. Kinetic analysis and inhibition studies using human liver microsomes (HLM) and recombinant enzymes revealed that human arylacetamide deacetylase (AADAC) is responsible for KC hydrolysis to form DAK, and confirmed that FMO3 is the enzyme responsible for DAK N-hydroxylation. In HLM, the clearance of KC hydrolysis occurred to the same extent as DAK N-hydroxylation, which indicates that both processes are not rate-limiting pathways. Cytotoxicity of KC and DAK was evaluated using HepaRG cells and human primary hepatocytes. Treatment of HepaRG cells with DAK for 24h showed cytotoxicity in a dose-dependent manner, whereas treatment with KC did not show due to the low expression of AADAC. Overexpression of AADAC in HepaRG cells with an adenovirus expression system elicited the cytotoxicity of KC. Cytotoxicity of KC in human primary hepatocytes was attenuated by diisopropylfluorophosphate, an AADAC inhibitor. In conclusion, the present study demonstrated that human AADAC hydrolyzes KC to trigger hepatocellular toxicity.
• Alteration of human hepatic drug transporter activity and expression by cigarette smoke condensate.Sayyed K,  Vee ML,  Abdel-Razzak Z,  Jouan E,  Stieger B,  Denizot C,  Parmentier Y,  Fardel O, , , , ,
  Toxicology, Jul 2016
  Abstract : Smoking is well-known to impair pharmacokinetics, through inducing expression of drug metabolizing enzymes. In the present study, we demonstrated that cigarette smoke condensate (CSC) also alters activity and expression of hepatic drug transporters, which are now recognized as major actors of hepatobiliary elimination of drugs. CSC thus directly inhibited activities of sinusoidal transporters such as OATP1B1, OATP1B3, OCT1 and NTCP as well as those of canalicular transporters like P-glycoprotein, MRP2, BCRP and MATE1, in hepatic transporters-overexpressing cells. CSC similarly counteracted constitutive OATP, NTCP and OCT1 activities in human highly-differentiated hepatic HepaRGcells. In parallel, CSC induced expression of BCRP at both mRNA and protein level in HepaRG cells, whereas it concomitantly repressed mRNA expression of various transporters, including OATP1B1, OATP2B1, OAT2, NTCP, OCT1 and BSEP, and enhanced that of MRP4. Such changes in transporter gene expression were found to be highly correlated to those caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin, a reference activator of the aryl hydrocarbon receptor (AhR) pathway, and were counteracted, for some of them, by siRNA-mediated AhR silencing. This suggests that CSC alters hepatic drug transporter levels via activation of the AhR cascade. Importantly, drug transporter expression regulations as well as some transporter activity inhibitions occurred for a range of CSC concentrations similar to those required for inducing drug metabolizing enzymes and may therefore be hypothesized to be relevant for smokers. Taken together, these data established human hepatic transporters as targets of cigarette smoke, which could contribute to known alteration of pharmacokinetics and some liver adverse effects caused by smoking. Smoking is well-known to impair pharmacokinetics, through inducing expression of drug metabolizing enzymes. In the present study, we demonstrated that cigarette smoke condensate (CSC) also alters activity and expression of hepatic drug transporters, which are now recognized as major actors of hepatobiliary elimination of drugs. CSC thus directly inhibited activities of sinusoidal transporters such as OATP1B1, OATP1B3, OCT1 and NTCP as well as those of canalicular transporters like P-glycoprotein, MRP2, BCRP and MATE1, in hepatic transporters-overexpressing cells. CSC similarly counteracted constitutive OATP, NTCP and OCT1 activities in human highly-differentiated hepatic HepaRGcells. In parallel, CSC induced expression of BCRP at both mRNA and protein level in HepaRG cells, whereas it concomitantly repressed mRNA expression of various transporters, including OATP1B1, OATP2B1, OAT2, NTCP, OCT1 and BSEP, and enhanced that of MRP4. Such changes in transporter gene expression were found to be highly correlated to those caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin, a reference activator of the aryl hydrocarbon receptor (AhR) pathway, and were counteracted, for some of them, by siRNA-mediated AhR silencing. This suggests that CSC alters hepatic drug transporter levels via activation of the AhR cascade. Importantly, drug transporter expression regulations as well as some transporter activity inhibitions occurred for a range of CSC concentrations similar to those required for inducing drug metabolizing enzymes and may therefore be hypothesized to be relevant for smokers. Taken together, these data established human hepatic transporters as targets of cigarette smoke, which could contribute to known alteration of pharmacokinetics and some liver adverse effects caused by smoking.
• UNUSUAL FEATURES OF SODIUM TAUROCHOLATE COTRANSPORTING POLYPEPTIDE AS A HEPATITIS B VIRUS RECEPTOR.Li J,  Zong L,  Sureau C,  Barker L,  Wands JR,  Tong S, , , , , , ,
  J Virol, Jul 2016
  Abstract : Cell culture (cc)-derived hepatitis B virus (HBV) can infect differentiated HepaRG cells, but efficient infection requires addition of polyethylene glycol (PEG) during inoculation. Identification of sodium taurocholate cotransporting polypeptide (NTCP) as an HBV receptor enabled ccHBV infection of NTCP reconstituted HepG2 cells, although very little hepatitis B surface antigen (HBsAg) is produced. We found infection by patient serum-derived HBV (sHBV), which required purification of viral particles through ultracentrifugation or PEG precipitation, was PEG independent and much more efficient in HepaRG than HepG2/NTCP cells. In contrast to hepatitis B e antigen (HBeAg), HBsAg was not a reliable marker of productive sHBV infection at early time points. Low HBsAg/HBeAg ratio by ccHBV infected HepG2/NTCP cells was attributable to dimethylsulfoxide in culture medium, NTCP overexpression, and HBV genotype D. HepG2/NTCP cells released more viral antigens than HepG2 cells following HBV genome delivery by adeno-associated virus, and stable expression of NTCP in a ccHBV producing cell line increased viral mRNAs, proteins, replicative DNA, and covalently closed circular DNA. NTCP protein expression in HepG2/NTCP cells, despite being driven by the CMV promoter, was markedly increased by dimethylsulfoxide treatment. This at least partly explains ability of DMSO to promote ccHBV infection in such cell lines. In conclusion, NTCP appeared inefficient to mediate infection by serum-derived HBV. It could promote HBV RNA transcription while inhibiting HBsAg secretion. Efficient PEG-independent sHBV infection of HepaRG cells permits comparative studies of diverse clinical HBV isolates, and will help identify additional factors on virion surface promoting attachment to hepatocytes. IMPORTANCE : Currently in vitro infection with hepatitis B virus (HBV) depends on cell culture-derived HBV inoculated in the presence of polyethylene glycol. We found patient serum-derived HBV could efficiently infect differentiated HepaRG cells independent of polyethylene glycol, which represents a more physiological infection system. Serum-derived HBV has poor infectivity in HepG2 cells reconstituted with sodium taurocholate cotransporting polypeptide (NTCP), the currently accepted HBV receptor. Moreover, HepG2/NTCP cells secreted very little hepatitis B surface antigen following infection with cell culture-derived HBV, which was attributed to NTCP overexpression, genotype D virus, and dimethylsulfoxide added to culture medium. NTCP could promote HBV RNA transcription, protein expression, and DNA replication in HepG2 cells stably transfected with HBV DNA, while dimethylsulfoxide could increase NTCP protein level despite transcriptional control by a CMV promoter. Therefore, this study revealed several unusual features of NTCP as an HBV receptor and established conditions for efficient serum virus infection in vitro.
• Metabolic fate of desomorphine elucidated using rat urine, pooled human liver preparations, and human hepatocyte cultures as well as its detectability using standard urine screening approaches.Richter LH,  Kaminski YR,  Noor F,  Meyer MR,  Maurer HH
  Anal Biocanal Chem, Jul 2016
  Abstract : Desomorphine is an opioid misused as "crocodile", a cheaper alternative to heroin. It is a crude synthesis product homemade from codeine with toxic byproducts. The aim of the present work was to investigate the metabolic fate of desomorphine in vivo using rat urine and in vitro using pooled human liver microsomes and cytosol as well as human liver cell lines (HepG2 and HepaRG) by Orbitrap-based liquid chromatography-high resolution-tandem mass spectrometry or hydrophilic interaction liquid chromatography. According to the identified metabolites, the following metabolic steps could be proposed: N-demethylation, hydroxylation at various positions, N-oxidation, glucuronidation, and sulfation. The cytochrome P450 (CYP) initial activity screening revealed CYP3A4 to be the only CYP involved in all phase I steps. UDP-glucuronyltransferase (UGT) initial activity screening showed that UGT1A1, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17 formed desomorphine glucuronide. Among the tested in vitro models, HepaRG cells were identified to be the most suitable tool for prediction of human hepatic phase I and II metabolism of drugs of abuse. Finally, desomorphine (crocodile) consumption should be detectable by all standard urine screening approaches mainly via the parent compound and/or its glucuronide assuming similar kinetics in rats and humans.
• In vitro hepatotoxicity of 'Legal X': the combination of 1-benzylpiperazine (BZP) and 1-(m-trifluoromethylphenyl)piperazine (TFMPP) triggers oxidative stress, mitochondrial impairment and apoptosis.Dias da Silva D,  Silva MJ,  Moreira P,  Martins MJ,  Valente MJ,  Carvalho F,  Bastos ML,  Carmo H, , , , ,
  Archives of toxicology, Jun 2016
  Abstract : N-Benzylpiperazine (BZP) and 1-(3-trifluoromethylphenyl)piperazine (TFMPP) are two synthetic phenylpiperazine analogues that have been frequently commercialized in combination as an alternative to ecstasy ('Legal X'). Despite reports of several clinical complications following the use of these drugs in association, few studies have been conducted so far to elucidate their combined toxicity. The present study was aimed at clarifying the cytotoxic effects of mixtures of BZP and TFMPP in vitro. Human-derived HepaRG cells and primary rat hepatocytes were exposed to the drugs, individually or combined at different mixture ratios, and cytotoxicity was assessed by the MTT assay. Mixture additivity expectations were calculated by the independent action and the concentration addition (CA) models and compared with the experimental outcomes. To delineate the mechanisms underlying the elicited effects, a range of stress endpoints was evaluated, including oxidative stress, energetic imbalance, and metabolic interactions. It was observed that primary rat hepatocytes are more sensitive than HepaRG cells to the toxicity of BZP (EC50 2.20 and 6.60 mM, respectively) and TFMPP (EC50 0.14 and 0.45 mM, respectively). For all BZP-TFMPP combinations tested, CA was the most appropriate model to predict the mixture effects. TFMPP proved to act additively with BZP to produce significant hepatotoxicity (p < 0.01). Remarkably, substantial mixture effects were observed even when each drug was present at concentrations that were harmless individually. In primary hepatocytes, a small deviation from additivity (antagonism) was observed toward the upper range of the concentration-response curve. GC/MS data suggest that a metabolic interaction may be at a play, as the mixture favors the metabolism of both substances, to a significant extent in the case of BZP (p < 0.05). Also, our results demonstrate the influence of oxidative stress and energetic imbalance on these effects (increase in RNS and ROS production, decrease in intracellular GSH/GSSG, ATP depletion and mitochondrial Δψm disruption). The present work clearly demonstrates that potentially harmful interactions among BZP and TFMPP are expected when these drugs are taken concomitantly.
• A multicenter assessment of single-cell models aligned to standard measures of cell health for prediction of acute hepatotoxicity.Sison-Young RL , Lauschke VM, Johann E, Alexandre E, Antherieu S, Aerts H, Gerets HH, Labbe G, Hoet D, Dorau M, Schofield CA, Lovatt CA, Holder JC
  Archives of toxicology, Jun 2016
  Abstract : Assessing the potential of a new drug to cause drug-induced liver injury (DILI) is a challenge for the pharmaceutical industry. We therefore determined whether cell models currently used in safety assessment (HepG2, HepaRG, Upcyte and primary human hepatocytes in conjunction with basic but commonly used endpoints) are actually able to distinguish between novel chemical entities (NCEs) with respect to their potential to cause DILI. A panel of thirteen compounds (nine DILI implicated and four non-DILI implicated in man) were selected for our study, which was conducted, for the first time, across multiple laboratories. None of the cell models could distinguish faithfully between DILI and non-DILI compounds. Only when nominal in vitro concentrations were adjusted for in vivo exposure levels were primary human hepatocytes (PHH) found to be the most accurate cell model, closely followed by HepG2. From a practical perspective, this study revealed significant inter-laboratory variation in the response of PHH, HepG2 and Upcyte cells, but not HepaRG cells. This variation was also observed to be compound dependent. Interestingly, differences between donors (hepatocytes), clones (HepG2) and the effect of cryopreservation (HepaRG and hepatocytes) were less important than differences between the cell models per se. In summary, these results demonstrate that basic cell health endpoints will not predict hepatotoxic risk in simple hepatic cells in the absence of pharmacokinetic data and that a multicenter assessment of more sophisticated signals of molecular initiating events is required to determine whether these cells can be incorporated in early safety assessment.
• Contextualizing Hepatocyte Functionality of Cryopreserved HepaRG® Cell CulturesBucher S, Jalili P, Le Guillou D, Begriche K, Rondel K, Martinais S, Zalko D, Corlu A, Robin A, Fromenty B, , , Jackson JP, Li L, Chamberlain ED, Wang H, Ferguson SS
  Drug metabolism and disposition, Jun 2016
  Abstract : Over the last decade, HepaRG® cells have emerged as a promising alternative to primary human hepatocytes (PHH) featured in over 300 research publications. Most of these reports employed freshly differentiated HepaRG® cells that require time-consuming culture (~28 days) for full differentiation. Recently, a cryopreserved, pre-differentiated format of HepaRG® cells (termed here as Cryo-HepaRG) has emerged as a new model that improves global availability and experimental flexibility; however, it is largely unknown whether this format of HepaRG® cells fully retain their hepatic characteristics. Therefore, we systematically investigated hepatocyte functionality of Cryo-HepaRG cultures in context with the range of interindividual variation observed with PHH in both sandwich culture and suspension formats. These evaluations uncovered a novel adaptation period for the Cryo-HepaRG format and demonstrated the impact of extracellular matrix on Cryo-HepaRG functionality. Pharmacologically-important drug metabolizing alleles were genotyped in HepaRG® cells and identified poor metabolizer alleles for CYP2D6, CYP2C9, and CYP3A5 which are consistent with higher frequency alleles found in individuals of Caucasian decent. We observed liver enzyme inducibility with AhR, CAR, and PXR activators comparable to that of sandwich cultured PHH. Finally, we show for the first time that Cryo-HepaRG supports proper CAR cytosolic sequestration and translocation to hepatocyte nuclei in response to phenobarbital treatment. Taken together, these data reveal important considerations when using this cell model and demonstrate that Cryo-HepaRG is suitable for metabolism and toxicology screening.
• Bisphenol a induces steatosis in HepaRG cells using a model of perinatal exposure.Bucher S, Jalili P, Le Guillou D, Begriche K, Rondel K, Martinais S, Zalko D, Corlu A, Robin A, Fromenty B
  Environmental toxicology, Jun 2016
  Abstract : Human exposure to bisphenol A (BPA) could favor obesity and related metabolic disorders such as hepatic steatosis. Investigations in rodents have shown that these deleterious effects are observed not only when BPA is administered during the adult life but also with different protocols of perinatal exposure. Whether perinatal BPA exposure could pose a risk in human is currently unknown, and thus appropriate in vitro models could be important to tackle this major issue. Accordingly, we determined whether long-term BPA treatment could induce steatosis in human HepaRGcells by using a protocol mimicking perinatal exposure. To this end, the kinetics of expression of seven proteins differentially expressed during liver development was determined during a 4-week period of cell culture required for proliferation and differentiation. By analogy with data reported in rodents and humans, our results indicated that the period of cell culture around day 15 and day 18 after seeding could be considered as the "natal" period. Consequently, HepaRG cells were treated for 3 weeks with BPA (from 0.2 to 2000 nM), with a treatment starting during the proliferating period. BPA was able to induce steatosis with a nonmonotonic dose response profile, with significant effects on neutral lipids and triglycerides observed for the 2 nM concentration. However, the expression of many enzymes involved in lipid and carbohydrate homeostasis was unchanged in exposed HepaRG cells. The expression of other potential BPA targets and enzymes involved in BPA biotransformation was also determined, giving answers as well as new questions regarding the mechanisms of action of BPA. Hence, HepaRG cells provide a valuable model that can prove useful for the toxicological assessment of endocrine disruptors on hepatic metabolisms, in particular in the developing liver. 
• The histone deacetylase inhibiting drug Entinostat induces lipid accumulation in differentiated HepaRG cellsNunn AD, Scopigno T, Pediconi N, Levrero M, Hagman H, Kiskis J, Enejder A, , , , , ,
  Scientific Reports, Jun 2016
  Abstract : Dietary overload of toxic, free metabolic intermediates leads to disrupted insulin signalling and fatty liver disease. However, it was recently reported that this pathway might not be universal: depletion of histone deacetylase (HDAC) enhances insulin sensitivity alongside hepatic lipid accumulation in mice, but the mechanistic role of microscopic lipid structure in this effect remains unclear. Here we study the effect of Entinostat, a synthetic HDAC inhibitor undergoing clinical trials, on hepatic lipid metabolism in the paradigmatic HepaRG liver cell line. Specifically, we statistically quantify lipid droplet morphology at single cell level utilizing label-free microscopy, coherent anti-Stokes Raman scattering, supported by gene expression. We observe Entinostat efficiently rerouting carbohydrates and free-fatty acids into lipid droplets, upregulating lipid coat protein gene Plin4, and relocating droplets nearer to the nucleus. Our results demonstrate the power of Entinostat to promote lipid synthesis and storage, allowing reduced systemic sugar levels and sequestration of toxic metabolites within protected protein-coated droplets, suggesting a potential therapeutic strategy for diseases such as diabetes and metabolic syndrome.
• Selecting Cells for Bioartificial Liver Devices and the Importance of a 3D Culture Environment: A Functional Comparison between the HepaRG and C3A Cell Lines.van Wenum M,  Adam AA,  Hakvoort T, Hendriks EJ,  Shevchenko V,  van Gulik TM,  Chamuleau RA,  Hoekstra R., , , , ,
  Int J Biol Sci., Jun 2016
  Abstract : Recently, the first clinical trials on Bioartificial Livers (BALs) loaded with a proliferative human hepatocyte cell source have started. There are two cell lines that are currently in an advanced state of BAL development; HepaRG and HepG2/C3A. In this study we aimed to compare both cell lines on applicability in BALs and to identify possible strategies for further improvement. We tested both cell lines in monolayer- and BAL cultures on growth characteristics, hepatic differentiation, nitrogen-, carbohydrate-, amino acid- and xenobiotic metabolism. Interestingly, both cell lines adapted the hepatocyte phenotype more closely when cultured in BALs; e.g. monolayer cultures produced lactate, while BAL cultures showed diminished lactate production (C3A) or conversion to elimination (HepaRG), and urea cycle activity increased upon BAL culturing in both cell lines. HepaRG-BALs outperformed C3A-BALs on xenobiotic metabolism, ammonia elimination and lactate elimination, while protein synthesis was comparable. In BAL cultures of both cell lines ammonia elimination correlated positively with glutamine production and glutamate consumption, suggesting ammonia elimination was mainly driven by the balance between glutaminase and glutamine synthetase activity. Both cell lines lacked significant urea cycle activity and both required multiple culture weeks before reaching optimal differentiation in BALs. In conclusion, culturing in BALs enhanced hepatic functionality of both cell lines and from these, the HepaRG cells are the most promising proliferative cell source for BAL application.
• MicroRNA hsa-miR-25-3p suppresses the expression and drug induction of CYP2B6 in human hepatocytes.Jin Y, Yu D, Tolleson WH, Knox B, Wang Y, Chen S, Ren Z, Deng H, Guo Y, Ning B
  Biochemical pharmacology, Jun 2016
  Abstract : Cytochrome P450 2B6 (CYP2B6), mainly expressed in the liver and brain, is important for processing a number of widely used drugs. Variations in CYP2B6 expression are associated with decreased drug efficacy or adverse effects in some patients. Although CYP2B6 genetic variants are associated with its differential expression, epigenetic mechanisms affecting CYP2B6 gene regulation have not been established. Sequence analysis identified 29 domains in the CYP2B6 mRNA transcript that could be subject to regulation by microRNAs. Inverse correlations were found in human hepatocytes for the levels of the microRNAs hsa-miR-504-5p and hsa-miR-25-3p compared with CYP2B6 mRNA. Reporter gene assays showed that hsa-miR-25-3p suppresses CYP2B6 expression by targeting a specific sequence in the 3'-untranslated region of the mRNA transcript. Electrophoretic mobility shift assays confirmed that hsa-miR-25-3p forms stable complexes with its cognate mRNA sequence and that it recruits cellular factors, including Ago-4. Transfection of HepaRG cells with hsa-miR-25-3p mimics inhibited expression of the endogenous CYP2B6 gene and it also decreased rifampicin-dependent induction of CYP2B6 at the mRNA and protein levels. In summary, in silico and in vitro analyses show that hsa-miR-25-3p suppresses CYP2B6 expression in human liver cells via an epigenetic mechanism.
• Differential sensitivity of metabolically competent and non-competent HepaRG cells to apoptosis induced by diclofenac combined or not with TNF-?.Al-Attrache H, Sharanek A, Burban A, Burbank M, Gicquel T, Abdel-Razzak Z, Guguen-Guillouzo C, Morel I, Guillouzo A
  toxicology letters, Jun 2016
  Abstract : The role of reactive metabolites and inflammatory stress has been largely evoked in idiosyncratic hepatotoxicity of diclofenac (DCF); however mechanisms remain poorly understood. We aimed to evaluate the influence of liver cell phenotype on the hepatotoxicity of DCFcombined or not with TNF-α using differentiated and undifferentiated HepaRG cells, and for comparison, HepG2 cells. Our results demonstrate that after a 24h-treatment metabolizing HepaRG cells were less sensitive to DCF than their undifferentiated non-metabolizing counterparts as shown by lower oxidative and endoplasmic reticulum stress responses and lower activation of caspase 9. Differentiated HepaRG cells were also less sensitive than HepG2 cells. Their lower sensitivity to DCF was related to their high content in glutathione transferases. DCF-induced apoptotic effects were potentiated by TNF-α only in death receptor-expressing differentiated HepaRG and HepG2 cells and were associated with marked activation of caspase 8. TNF-α co-treatment did not aggravate DCF-induced cholestatic features. Altogether, our results demonstrate that (i) lower sensitivity to DCF of differentiated HepaRG cells compared to their non-metabolically active counterparts was related to their high detoxifying capacity, giving support to the higher sensitivity of nonhepatic tissues than liver to this drug; (ii) TNF-α-potentiation of DCF cytotoxicity occurred only in death receptor-expressing cells.
• Human Hepatic HepaRG Cells Maintain an Organotypic Phenotype with High Intrinsic CYP450 Activity/Metabolism and Significantly Outperform Standard HepG2/C3A Cells for Pharmaceutical and Therapeutic Applications.Nelson LJ, Morgan K, Treskes P, Samuel K, Henderson CJ, LeBled C, Homer N, Helen Grant M, Hayes PC, Plevris JN
  Basic & Clinical Pharmacology & Toxicology, Jun 2016
  Abstract : Conventional in vitro human hepatic models for drug testing are based on the use of standard cell lines derived from hepatomas or primary human hepatocytes (PHHs). Limited availability, inter-donor functional variability and early phenotypic alterations of PHHs restrict their use; whilst standard cell lines such as HepG2 lack a substantial and variable set of liver-specific functions such as CYP450 activity. Alternatives include the HepG2-derivative C3A cells selected as a more differentiated and metabolically active hepatic phenotype. Human HepaRG cells are an alternative organotypic co-culture model of hepatocytes and cholangiocytes reported to maintain in vivo-like liver-specific functions, including intact Phase 1-3 drug metabolism. In this study, we compared C3A and human HepaRG cells using phenotypic profiling, CYP450 activity and drug metabolism parameters to assess their value as hepatic models for pre-clinical drug testing or therapeutics. Compared with C3As, HepaRG co-cultures, exhibit a more organotypic phenotype, including evidence of hepatic polarity with strong expression of CYP3A4, the major isoform involved in the metabolism of over 60% of marketed drugs. Significantly greater CYP450 activity and expression of CYP1A2, CYP2E1 and CYP3A4 genes inHepaRG cells (comparable with that of human liver tissue) was demonstrated. Moreover, HepaRG cells also preferentially expressed the hepatic integrin α5 β1 - an important modulator of cell behaviour including growth and survival, differentiation and polarity. Drug metabolite profiling of phenacetin (CYP1A2) and testosterone (CYP3A4) using LC-MS/MS and HPLC, respectively, revealed HepaRGs had more intact (Phase 1-2) metabolism profile. Thus, HepaRG cells significantly outperform C3A cells for potential pharmaceutical and therapeutic applications.
• Complex Approach to Xenobiotics Hepatotoxicity Testing using a Microfluidic System.Aleksandrova AV, Pul'kova NV, Sakharov DA
  Bulletin of experimental biology and medecine, Jun 2016
  Abstract : We analyzed hepatotoxicity of three drugs: acetaminophen, metformin, and isoniazid. Spheroids of differentiated HepaRG cells cultured under microfluidic conditions were used as the model. Acute toxicity of substances was assessed by analyzing cell viability, while lactate concentration in the culture medium was used as the potential marker for evaluation of chronic exposure and non-lethal side effects of xenobiotics. The results were compared with mitochondrial activity and DNA fragmentation data. The efficiency and possibility of applying the integrated approach for assessment of drug hepatotoxicity are discussed.
• Maintenance of High Cytochrome P450 Expression in HepaRG Cell Spheroids in DMSO-Free Medium.Aleksandrova AV, Burmistrova OA, Fomicheva KA, Sakharov DA
  Bulletin of experimental biology and medecine, Jun 2016
  Abstract : We studied the effects of DMSO and fibroblasts during HepaRG cell spheroid formation and conditions of their subsequent culturing on the levels of mRNA of the major cytochromes P450. A protocol of spheroid formation from differentiated HepaRG cells and their culturing in serum- and DMSO-free medium is developed.
• Characterization of hepatotoxicity mechanisms triggered by designer cathinone drugs (?-keto amphetamines).Valente MJ, Araujo AM, Bastos ML, Fernandes E, Carvahlo F, Guedes de Pinho P, Carvalho M
  Toxicological Sciences, Jun 2016
  Abstract : The use of cathinone designer drugs in recreational settings has been associated with severe toxic effects, including liver damage. The precise mechanisms by which cathinones induce hepatotoxicity and whether they act by common pathways remains to be elucidated. Herein, we assessed the toxicity of the cathinones methylone, pentedrone, 3,4-methylenedioxypyrovalerone (MDPV) and 4-methylethcathinone (4-MEC) in primary rat hepatocytes (PRH) and HepaRG cells, and compared to that of 3,4-methylenedioxymethamphetamine (MDMA). MDPV and pentedrone were significantly more toxic than MDMA, while methylone was the least cytotoxic compound. Importantly, PRH revealed to be the most sensitive experimental model and was thus used to explore the mechanisms underlying the observed toxicity. All drugs elicited the formation of reactive oxygen and nitrogen species (ROS and RNS), but more markedly for methylone, pentedrone and 4-MEC. GSH depletion was also a common effect at the highest concentration tested, whereas only MDPV and pentedrone caused significant decrease in ATP levels. The antioxidants ascorbic acid or N-acetyl-L-cysteine partially attenuated the observed cell death. All cathinones triggered significant caspase activation and apoptosis, which was partially reversed by the caspase inhibitor Ac-LETD-CHO. In conclusion, the present data shows that (i) cathinones induce in vitro hepatotoxic effects that vary in magnitude among the different analogues, (ii) oxidative stress and mitochondrial dysfunction play a role in cathinones-induced hepatic injury, and (iii) apoptosis appears to be an important pathway of cell death elicited by these novel drugs.
• Reciprocal regulation of farnesoid X receptor ? activity and hepatitis B virus replication in differentiatedHepaRG cells and primary human hepatocytes.Radreau P, Porcherot M, Ramière C, Mouzannar K , Lotteau V, André P
  FASEB journal, Jun 2016
  Abstract : Hepatitis B virus (HBV) and bile salt metabolism seem tightly connected. HBV enters hepatocytes by binding to sodium taurocholate cotransporting polypeptide (NTCP), the genome of which contains 2 active farnesoid X receptor (FXR) α response elements that participate in HBV transcriptional activity. We investigated in differentiated HepaRG cells and in primary human hepatocytes (PHHs) effects of FXR activation on HBV replication and of infection on the FXR pathway. In HepaRG, FXR agonists (6-ethyl chenodeoxycholic acid and GW4064), but no antagonist, and an FXR-unrelated bile salt inhibited viral mRNA, DNA, and protein production (IC50, 0.1-0.5 μM) and reduced covalently closed circular DNA pool size. These effects were independent of the NTCP inhibitor cyclosporine-A, which suggests inhibition occurred at a postentry step. Similar results were obtained in PHH with GW4064. Infection of these cells increased expression of FXR and modified expression of FXR-regulated genes SHP, APOA1, NTCP, CYP7A1, and CYP8B1 with a more pronounced effect in PHH than in HepaRG. FXR agonists reversed all but one of the HBV-induced FXR gene profile modifications. HBV replication and FXR regulation seem to be interdependent, and altered bile salt metabolism homeostasis might contribute to the persistence of HBV infection.-Radreau, P., Porcherot, M., Ramière, C., Mouzannar, K., Lotteau, V., André, P. Reciprocal regulation of farnesoid X receptor α activity and hepatitis B virus replication in differentiated HepaRG cells and primary human hepatocytes.
• A Rapid and Sensitive HPLC-DAD Assay to Quantify Lamotrigine, Phenytoin and Its Main Metabolite in Samples of Cultured HepaRG Cells.Ferreira A, Rodrigues M, Falcao A, Alves G
  Journal of chromatographic science, May 2016
  Abstract : A sensitive and fast high-performance liquid chromatography-diode-array detection assay was developed and validated for the simultaneous quantification of 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH), phenytoin (PHT) and lamotrigine (LTG) in samples of cultured HepaRG cells. Chromatographic separation of analytes and internal standard (IS) was achieved in ∼15 min on a C18-column, at 35°C, using acetonitrile (6%), methanol (25%) and a mixture (69%) of water-triethylamine (99.7:0.3, v/v; pH 6.0), pumped at 1 mL/min. The analytes and IS were detected at 215 or 235 nm. Calibration curves were linear with regression coefficients >0.994 over the concentration ranges of 0.1-15 µg/mL for HPPH; 0.15-30 µg/mL for PHT and 0.2-20 µg/mL for LTG. The method showed to be accurate (bias value of ±10.5 or ±17.6% in the lower limit of quantification, LLOQ) and precise (coefficient variation ≤8.1 or ≤15.4% in the LLOQ), and the absolute recovery of the analytes ranged from 62.5 to 96.9%. HepaRG cells have emerged as a very promising in vitro model to evaluate metabolic, drug interaction and/or pharmacokinetic studies, and this methodology will be suitable to support subsequent studies involving the antiepileptic drugs PHT and LTG.
• De novo HAPLN1 expression hallmarks Wnt-induced stem cell and fibrogenic networks leading to aggressive human hepatocellular carcinomas.Mebarki S, Désert R, Sulpice L, Sicard M, Desille M, Canal F, Schneider HD, Bergeat D, Turlin B, Bellaud P, Lavergne E, Le Guevel R, Corlu A
  Oncotarget, May 2016
  Abstract : About 20% hepatocellular carcinomas (HCCs) display wild-type β-catenin, enhanced Wnt signaling, hepatocyte dedifferentiation and bad outcome, suggesting a specific impact of Wnt signals on HCC stem/progenitor cells. To study Wnt-specific molecular pathways, cell fates and clinical outcome, we fine-tuned Wnt/β-catenin signaling in liver progenitor cells, using the prototypical Wnt ligand Wnt3a. Cell biology assays and transcriptomic profiling were performed in HepaRG hepatic progenitors exposed to Wnt3a after β-catenin knockdown or Wnt inhibition with FZD8_CRD. Gene expression network, molecular pathology and survival analyses were performed on HCCs and matching non-tumor livers from 70 patients by real-time PCR and tissue micro-array-based immunohistochemistry. Wnt3a reprogrammed liver progenitors to replicating fibrogenic myofibroblast-like cells displaying stem and invasive features. Invasion was inhibited by 30 nM FZD7 and FZD8 CRDs. Translation of these data to human HCCs revealed two tight gene networks associating cell surface Wnt signaling, stem/progenitor markers and mesenchymal commitment. Both networks were linked by Hyaluronan And Proteoglycan Link Protein 1 (HAPLN1), that appeared de novo in aggressive HCCs expressing cytoplasmic β-catenin and stem cell markers. HAPLN1 was independently associated with bad overall and disease-free outcome. In vitro, HAPLN1 was expressed de novo in EPCAM¯/NCAM+ mesoderm-committed progenitors, upon spontaneous epithelial-mesenchymal transition and de-differentiation of hepatocyte-like cells to liver progenitors. In these cells, HAPLN1 knockdown downregulated key markers of mesenchymal cells, such as Snail, LGR5, collagen IV and α-SMA. In conclusion, HAPLN1 reflects a signaling network leading to stemness, mesenchymal commitment and HCC progression.
• Rho-kinase/myosin light chain kinase pathway plays a key role in the impairment of bile canaliculi dynamics induced by cholestatic drugs.Sharanek A, Burban A, Burbank M, Le Guevel R, Li R, Guillouzo A, Guguen-Guillouzo C
  Scientific Reports, May 2016
  Abstract : Intrahepatic cholestasis represents a frequent manifestation of drug-induced liver injury; however, the mechanisms underlying such injuries are poorly understood. In this study of human HepaRG and primary hepatocytes, we found that bile canaliculi (BC) underwent spontaneous contractions, which are essential for bile acid (BA) efflux and require alternations in myosin light chain (MLC2) phosphorylation/dephosphorylation. Short exposure to 6 cholestatic compounds revealed that BC constriction and dilation were associated with disruptions in the ROCK/MLCK/myosin pathway. At the studied concentrations, cyclosporine A and chlorpromazine induced early ROCK activity, resulting in permanent MLC2 phosphorylation and BC constriction. However, fasudil reduced ROCK activity and caused rapid, substantial and permanent MLC2 dephosphorylation, leading to BC dilation. The remaining compounds (1-naphthyl isothiocyanate, deoxycholic acid and bosentan) caused BC dilation without modulating ROCK activity, although they were associated with a steady decrease in MLC2 phosphorylation via MLCK. These changes were associated with a common loss of BC contractions and failure of BA clearance. These results provide the first demonstration that cholestatic drugs alter BC dynamics by targeting the ROCK/MLCK pathway; in addition, they highlight new insights into the mechanisms underlying bile flow failure and can be used to identify new predictive biomarkers of drug-induced cholestasis.
• Mathematical and Experimental Model of Oxygen Diffusion for HepaRG Cell Spheroids.Aleksandrova AV, Pulkova NV, Gerasimenko TN, Anisimov NY, Tonevitskaya SA, Sakharov DA
  Bulletin of experimental biology and medecine, May 2016
  Abstract : 3D cell cultures are extensively used to study in vitro toxic effect of xenobiotics. When using multicellular spheroids, the question about their optimal size should be solved: small spheroids are difficult to manipulate, while large size of spheroids impairs the transport of nutrients and oxygen into the center. Mathematical models describing the distribution of substances in multicellular spheroids numerical procedure for solving differential equation system, which complicates their use in laboratory practice. We proposed and experimentally evaluated a new mathematical model describing oxygen distribution in HepaRG cell spheroids. Markers of functional activity were studied in spheroids of different size. The maximum size of spheroids that can be maintained in culture for 9 days without necrosis was determined.
• Transport and Toxicity of Silver Nanoparticles in HepaRG Cell Spheroids.Senyavina NV, Gerasimenko TN, Pulkova NV, Maltseva DV, , , , , , , , ,
  Bulletin of experimental biology and medecine, May 2016
  Abstract : We studied the effects of silver nanoparticles (10 nm) on HepaRG cell spheroids simulating liver tissue. The mathematical model was proposed that describes nanoparticle diffusion in a spheroid consisting of 5000 cells depending on the external nanoparticle concentration. It was demonstrated that cells in the 3D model were less sensitive to the toxic effects of nanoparticles in comparison with 2D cultures. Impaired integrity of the cell membrane did not deteriorate cell viability (according to MTT test).
• Maintenance of Hepatic Progenitor-like Characteristics of HepaRG Cells by Cultivation on VECELL Inserts"Takashi Kubo, Yukie Kuroda, Maki Hojyo, Su-Ryang Kim, Shinichiro Horiuchi, Yuko Sekino, Fabrice Morel, Anne Corlu, and Seiichi Ishida"
  AATEX, May 2016
  Abstract : Cell shape influences cell functionality. We investigated the possibility that the differentiation state ofHepaRG, a bipotential hepatic progenitor cell line, was changed by cultivation on VECELL Inserts,which consist of type I collagen-coated expanded polytetrafluoroethylene (ePTFE) mesh. HepaRGcells plated on VECELL Inserts possessed a round shape. Gene expression patterns obtained fromHepaRG cells cultured on VECELL Inserts suggested that the cells maintained their progenitorcell-like characteristics, while not losing the capacity to differentiate into hepatocytes. Retaining cellstemness is important for the expansion of progenitor cells to maintain their differentiation potency.VECELL Inserts are a novel culture apparatus that easily retains the characteristics of hepaticprogenitor cells. It may be useful for the culture and expansion of hepatic progenitor cells, and thus,provide fundamental cell source for in vitro toxicity and biotransformation assays.
• NOD1 participates in the innate immune response triggered by the hepatitis C viral polymerase.Vegna S, Gregoire D, Moreau M, Lassus P, Durantel D, Assenat E, Hibner U, Simonin Y
  journal of virology, Apr 2016
  Abstract : Hepatitis C virus (HCV) triggers innate immunity signalling in the infected cell. Replication of the viral genome is dispensable for this phenotype and we and others have recently shown that NS5B, the viral RNA dependent RNA polymerase synthesizes double stranded RNA (dsRNA) from cellular templates thus eliciting an inflammatory response, notably via activation of type I interferon and lymphotoxin β. Here we investigated intracellular signal transduction pathways involved in this process. Using HepaRG cells, a model that largely recapitulates thein vivocomplexities of the innate immunity receptors signaling, we have confirmed that NS5B triggered increased expression of the canonical pattern recognition receptors specific for dsRNA, namely RIG-I, MDA5 and TLR3. Unexpectedly, intracellular dsRNA also led to accumulation of NOD1, a receptor classically involved in recognition of bacterial peptidoglycans. NOD1 activation, confirmed by analysis of its downstream targets, was likely due to its interaction with dsRNA and was independent of RIG-I and MAVS signaling. It is likely to have a functional significance in the cellular response in the context of HCV infection since interference with NOD1 pathway severely reduced inflammatory response elicited by NS5B. IMPORTANCE: In this study we show that NOD1, a PRR that normally senses bacterial peptidoglycans, is activated by HCV viral polymerase, probably through an interaction with dsRNA, suggesting that NOD1 acts as an RNA ligand recognition receptor. In consequence, interference with NOD1-mediated signaling significantly weakens the inflammatory response to dsRNA. These results add a new level of complexity to the understanding of the crosstalk between different classes of pattern recognition receptors and may be related to certain complications of chronic hepatitis C.
• Advantageous use of HepaRG cells for the screening and mechanistic study of drug-induced steatosis.Tolosa L, Gomez-Lechon MJ, Jimenez N, Hervas D, Jover R, Donato MT
  toxicology and applied pharmacology, Apr 2016
  Abstract : Only a few in vitro assays have been proposed to evaluate the steatotic potential of new drugs. The present study examines the utility of HepaRG cells as a cell-based assay system for screening drug-induced liver steatosis. A high-content screening assay was run to evaluate multiple toxicity-related cell parameters in HepaRG cells exposed to 28 compounds, including drugs reported to cause steatosis through different mechanisms and non-steatotic compounds. Lipid content was the most sensitive parameter for all the steatotic drugs, whereas no effects on lipid levels were produced by non-steatotic compounds. Apart from fat accumulation, increased ROS production and altered mitochondrial membrane potential were also found in the cells exposed to steatotic drugs, which indicates that all these cellular events contributed to drug-induced hepatotoxicity. These findings are of clinical relevance as most effects were observed at drug concentrations under 100-fold of the therapeutic peak plasmatic concentration. HepaRG cells showed increased lipid overaccumulation vs. HepG2 cells, which suggests greater sensitivity to drug-induced steatosis. An altered expression profile of transcription factors and the genes that code key proteins in lipid metabolism was also found in the cells exposed to drugs capable of inducing liver steatosis. Our results generally indicate the value of HepaRG cells for assessing the risk of liver damage associated with steatogenic compounds and for investigating the molecular mechanisms involved in drug-induced steatosis.
• Novel roles for AhR and ARNT in the regulation of alcohol dehydrogenases in human hepatic cells.Attignon EA, Leblanc AF, Le Grand B, Duval C, Aggerbeck M, Rouach H, Blanc EB, , , , , ,
  Archives of toxicology, Apr 2016
  Abstract : The mechanisms by which pollutants participate in the development of diverse pathologies are not completely understood. The pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) activates the AhR (aryl hydrocarbon receptor) signaling pathway. We previously showed that TCDD (25 nM, 30 h) decreased the expression of several alcohol metabolism enzymes (cytochrome P450 2E1, alcohol dehydrogenases ADH1, 4 and 6) in differentiated human hepatic cells (HepaRG). Here, we show that, as rapidly as 8 h after treatment (25 nM TCDD) ADH expression decreased 40 % (p < 0.05). ADH1 and 4 protein levels decreased 40 and 27 %, respectively (p < 0.05), after 72 h (25 nM TCDD). The protein half-lives were not modified by TCDD which suggests transcriptional regulation of expression. The AhR antagonist CH-223191 or AhR siRNA reduced the inhibitory effect of 25 nM TCDD on ADH1A, 4 and 6 expression 50-100 % (p < 0.05). The genomic pathway (via the AhR/ARNT complex) and not the non-genomic pathway involving c-SRC mediated these effects. Other AhR ligands (3-methylcholanthrene and PCB 126) decreased ADH1B, 4 and 6 mRNAs by more than 78 and 55 %, respectively (p < 0.01). TCDD also regulated the expression of ADH4 in the HepG2 human hepatic cell line, in primary human hepatocytes and in C57BL/6J mouse liver. In conclusion, activation of the AhR/ARNT signaling pathway by AhR ligands represents a novel mechanism for regulating the expression of ADHs. These effects may be implicated in the toxicity of AhR ligands as well as in the alteration of ethanol or retinol metabolism and may be associated further with higher risk of liver diseases or/and alcohol abuse disorders.
• The HepaRG cell line, a superior in vitro model to L-02, HepG2 and hiHeps cell lines for assessing drug-induced liver injury.Wu Y, Geng XC, Wang JF, Miao YF,  Lu YL,  Li B
  Cell Biol Toxicol., Mar 2016
  Abstract : Drug-induced liver injury (DILI) is a leading cause of discontinuation of new drug approval or withdrawal of marketed medicine based on safety due to organ vulnerability. The aim of this research is to investigate the potential abilities of four different in vitro cell models (L-02, HepG2, HepaRG, and hiHeps cell lines) in assessing marketed drugs labeled with apparently different types of liver injury. A total of 17 drugs with versatile pharmacological profiles were chosen, of which, 14 drugs are recognized as DILI agents and 3 drugs are DILI irrelevant. Preliminary cellular screening assays indicated that the HepaRG cell line had an advantage over other cell lines in predicting drugs associated with DILI in vitro as it had the highest Youden's index (71.4 %). A multi-parametric screening assay showed that oxidative stress, mitochondrial damage, and disorders of neutral lipid metabolism were changed notably in the HepaRG cell line after DILI-related drugs exposure, accounting for its high sensitivity in comparison with other three cell lines. In addition, aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and malate dehydrogenase (MDH) all correlated with the cytotoxic effects of diclofenac sodium (p < 0.05), buspirone hydrochloride (p < 0.01), and danazol (p < 0.01) in the HepaRG cell line. We conclude that the HepaRG cell line is a superior in vitro cell model to other three cell lines for evaluating drugs with DILI potential.
• Grouping Thirty Four Chemicals Based on Mode of Action Using Connectivity Mapping.De Abrew KN,  Kainkaryam RM, Shan YK, Overmann GJ,  Settivari RS, Wang X, Xu j, Adams RL, Tiesman JP,  Carney EW, Naciff JM, Daston GP
  Toxicol Science, Mar 2016
  Abstract : Connectivity mapping (CMap) is a method used in the pharmaceutical industry to find connections between small molecules, disease states and genes. The concept can be applied to a predictive toxicology paradigm to find connections between chemicals, adverse events and genes. In order to assess the applicability of the technique for predictive toxicology purposes, we performed gene array experiments on 34 different chemicals: Bisphenol A, Genistein, Ethinyl-estradiol, Tamoxifen, Clofibrate, Dehydorepiandrosterone, Troglitazone, diethylhexyl phthalate, Flutamide, Trenbolone, Phenobarbital, Retinoic acid, Thyroxine, 1α,25-Dihydroxyvitamin D3, Clobetasol, Farnesol, Chenodeoxycholic acid, Progesterone, RU486, Ketoconazole, Valproic acid, Desferrioxamine, Amoxicillin, 6-aminonicotinamide, Metformin, Phenformin, Methotrexate, Vinblastine, ANIT (1-Naphthyl isothiocyanate), Griseofulvin, Nicotine, Imidacloprid, Vorinostat, 2,3,7,8-Tetrachloro-dibenzo-p-dioxin (TCDD) at the 6, 24 and 48 h time points for 3 different concentrations in the 4 cell lines: MCF-7, Ishikawa, HepaRG and HepG2. The 34 chemicals were grouped in to predefined MOA based chemical classes based on current literature. CMap was used to find linkages between each chemical and between chemical classes. Cell line specific linkages were compared to each other and to test if the method was platform and user independent a similar analysis was performed against publicly available data. The study showed that the method can group chemicals based on MOAs and the inter chemical class comparison alluded to connections between MOAs that were not predefined. Comparison to the publicly available data showed that the method is user and platform independent. The results provide an example of an alternate data analysis process for high content data, beneficial for predictive toxicology, especially when grouping chemicals for read across purposes.
• Antiviral activity of various interferons and pro-inflammatory cytokines in non-transformed cultured hepatocytes infected with hepatitis B virus.Isorce N, Testoni B, Locatelli M, Fresquet J, Rivoire M, Luangsay S, Zoulim F, Durantel D
  Antiviral research, Mar 2016
  Abstract : In HBV-infected patients, therapies with nucleoside analogues or IFNα remain ineffective in eradicating the infection. Our aim was to re-analyze the anti-HBV activity of a large panel of IFNs and cytokines in vitro using non-transformed cultured hepatocytes infected with HBV, to identify new immune-therapeutic options. HepaRG cells and primary human hepatocytes were infected with HBV and, when infection was established, treated with various concentrations of different IFNs or inflammatory cytokines. Viral parameters were evaluated by quantifying HBV nucleic acids by qPCR and Southern Blot, and secreted HBV antigens were evaluated using ELISA. The cytokines tested were IFNβ, IFNγ, IFNλ, TNFα, IL-6, IL-1β, IL-18 as well as nucleos(t)ide analogues tenofovir and ribavirin. Cytokines and drugs, with the exception of IL-18 and ribavirin, exhibited a suppressive effect on HBV replication at least as strong as, but often stronger than, IFNα. The cytokine presenting the highest effect on HBV DNA was IL-1β, which exerted its inhibition within picomolar range. Importantly, we noticed differential effects on other parameters (HBV RNA, HBeAg, HBsAg) between both IFNs and inflammatory cytokines, thus suggesting different mechanisms of action. The combination of IL-1β and already used therapies, i.e. IFNα or tenofovir, demonstrated a stronger or similar anti-HBV activity. IL-1β was found to have a very potent antiviral effect against HBV in vitro. HBV was previously shown to promptly inhibit IL-1β production in Kupffer cells. Strategies aiming at unlocking this inhibition and restoring local production of IL-1β may help to further inhibit HBV replication in vivo.
• Engineering EMT using 3D micro-scaffold to promote hepatic functions for drug hepatotoxicity evaluation.Wang J, Chen F, Liu L, Qi C, Wang B, Yan X, Huang C, Hou W, Zhang MQ, Chen Y, Du Y
  Biomaterials, Mar 2016
  Abstract : Accompanied by decreased hepatic functions, epithelial-mesenchymal transition (EMT) was observed in two dimensional (2D) cultured hepatocytes with elongated morphology, loss of polarity and weakened cell-cell interaction, while upgrading to 3D culture has been considered as significant improvement of its 2D counterpart for hepatocyte maintenance. Here we hypothesize that 3D culture enhances hepatic functions through regulating the EMT status. Biomaterial-engineered EMT was achieved by culturing HepaRG as 3D spheroids (SP-3D) or 3D stretched cells (ST-3D) in non-adherent and adherent micro-scaffold respectively. In SP-3D, constrained EMT of HepaRG, a hepatic stem cell line, as represented by increased epithelial markers and decreased mesenchymal markers, was echoed by improved hepatic functions. To investigate the relationship between EMT status and hepatic functions, time-series RNA-Seq and gene network analysis were used for comparing different cell culture models, which identified histone deacetylases (HDACs) as key mediating factors. Protein analysis confirmed that high HDAC activity was correlated with high expression of Cadherin-1 (CDH1) and hepatic function genes, which were decreased upon HDAC inhibitor treatment in SP-3D, suggesting HDACs may play positive role in regulating EMT and hepatic functions. To illustrate the application of 3D micro-scaffold culture in drug safety evaluation, hepatotoxicity and metabolism assays of two hepatotoxins (i.e. N-acetyl-p-aminophenol and Doxorubicin) were performed and SP-3D showed more biomimetic toxicity response, indicating regulation of EMT as a vital consideration in designing 3D hepatocyte culture configuration.
• Modulation of CYP3A4 activity alters the cytotoxicity of lipophilic phycotoxins in human hepatic HepaRG cells.Ferron PJ , Hogeveen K, De Sousa G, Rahmani R, Dubreil E, Fessard V, Le Hegarat L
  Toxicology in vitro, Mar 2016
  Abstract : The aim of this study was to investigate (i) the cytotoxic effects of lipophilic phycotoxins, including okadaic acid (OA) and dinophysistoxin-1 and -2 (DTX-1 and DTX-2), pectenotoxin-2 (PTX-2), yessotoxin (YTX), spirolide (SPX), and azaspiracids-1, -2 and -3 (AZA-1, AZA-2 and AZA-3), in humanHepaRG cells using a multiparametric high content analysis approach, (ii) the ability of nine lipophilic phycotoxins to act as PXR agonists in a HepG2-PXR cell line, (iii) their potential to induce CYP450 activity, and (iv) the role of CYP3A4 in cytotoxicity induced by lipophilic phycotoxins. Our results indicate that while OA, DTX-1 and DTX-2 activated PXR-dependent transcriptional activity in HepG2 cells, no increase of CYP450 (1A2, 3A4, 2C9, 2C19) activities were observed in HepaRG cell following a 72 hour treatment with these toxins. Multiparametric analysis showed that OA, DTX-1, DTX-2, and PTX-2 were highly cytotoxic in HepaRG cells; inducing cell loss, activation of caspase-3 and γ-H2AX formation. However, no toxicity was observed for YTX, SPX, and AZAs. Moreover, we found that inhibition of CYP3A4 activity by ketoconazole enhances the toxic effects of OA, DTX-1, DTX-2, and PTX-2 in HepaRG cells. Taken together, these results suggest that CYP3A4-mediated metabolism of some lipophilic phycotoxins decreases their in vitro toxicity.
• Establishment of a Drug-Induced, Bile Acid-Dependent Hepatotoxicity Model Using HepaRG Cells.Susukida T, Sekine S, Nozaki M, Tokizono M, Oizumi K, Horie T, Ito K
  Journal of pharmaceutical sciences, Mar 2016
  Abstract : Bile acid (BA) retention within hepatocytes is an underlying mechanism of cholestatic drug-induced liver injury (DILI). We previously developed an assay using sandwich-cultured human hepatocytes (SCHHs) to evaluate drug-induced hepatocyte toxicity accompanying intracellular BA accumulation. However, due to shortcomings commonly associated with the use of primary human hepatocytes (e.g., limited availability, lot-to-lot variability, and high cost), we examined if the human hepatic stem cell line, HepaRG, might also be applicable to our assay system. Consequently, mRNA expression levels of human BA efflux and uptake transporters were lower in HepaRG cells than in SCHHs but higher than in HepG2 human hepatoma cells. Nevertheless, HepaRG cells and SCHHs showed similar toxicity responses to 22 selected drugs, including cyclosporine A (CsA). CsA (10 μM) was cytotoxic toward HepaRG cells in the presence of BAs and also reduced the biliary efflux rate of [3H]taurocholic acid from 38.5% to 19.2%. Therefore, HepaRG cells are useful for the evaluation of BA-dependent drug toxicity caused by biliary BA efflux inhibition. Regardless, the prediction accuracy for cholestatic DILI risk was poor for HepaRG cells versus SCHHs, suggesting that our DILI model system requires further improvements to increase the utility of HepaRG cells as a preclinical screening tool.
• HBV culture and infectious systems.Hayes CN Chayama K
  Hepatology international, Mar 2016
  Abstract : While an effective vaccine against hepatitis B virus (HBV) has long been available, chronic HBV infection remains a severe global public health concern. Current treatment options have limited effectiveness, and long-term therapy is required to suppress HBV replication; however, complete elimination of the virus is rare. The lack of suitable animal models and infection systems has hindered efforts to unravel the HBV life cycle, particularly the early events in HBV entry, which appear to be highly species- and tissue-specific. Human primary hepatocytes remain the gold standard for HBV replication studies but are limited by availability and variability. While the HepaRG cell line is permissive for HBV replication, other hepatoma cell lines such as HepG2 do not support HBV replication. The recent discovery of sodium taurocholate transporting peptide (NTCP) as a primary receptor for HBV binding has led to the development of replication-competent cell lines such as HepG2-NTCP. Human hepatocytes grown in chimeric mice have provided another approach that allows primary human hepatocytes to be used while overcoming many of their limitations. Although the difficulty in developing HBV infection systems has hindered development of effective treatments, the variability and limited replication efficiency among cell lines point to additional liver-specific factors involved in HBV infection. It is hoped that HBV infection studies will lead to novel drug targets and therapeutic options for the treatment of chronic HBV infection.
• The pregnane X receptor down-regulates organic cation transporter 1 (SLC22A1) in human hepatocytes by squelching SRC-1 coactivator.Hyrsova L, Smutny T, Carazo A, Moravcik S, Mandikova J, Trejtnar F, Gerbal-Chaloin S, Pavek P
  British journal of pharmacology, Feb 2016
  Abstract : BACKGROUND AND PURPOSE:The organic cation transporter 1 (OCT1, SLC22A1) transports organic cationic drugs into hepatocytes. The high hepatic expression of OCT1 is controlled by the HNF4α and USF transcription factors. The pregnane X receptor (PXR), a ligand-dependent nuclear receptor, mediates the induction of principal xenobiotic metabolizing enzymes and drug transporters in the liver. No xenobiotic handling gene, however, has as of yet been systematically described to be down-regulated through PXR activation. EXPERIMENTAL APPROACH: The study examines the regulation of OCT1 transporter expression by activation of PXR in hepatocyte cells. KEY RESULTS:The significant down-regulation of OCT1 mRNA in human hepatocytes along with reduced [3H]MPP+ accumulation in differentiated HepaRG cells after treatment with rifampicin was found. PXR ligands rifampicin and hyperforin as well as the constitutively active PXR mutant T248D significantly suppressed the activity of the 1.8 kb OCT1 promoter construct in gene reporter assays. The silencing of both PXR and HNF4α in HepaRG cells abrogated the PXR ligand-mediated down-regulation of OCT1 expression. The mutation of HNF4α and USF1 (E-box) responsive elements reversed the PXR-mediated inhibition in gene reporter assays. Chromatin immunoprecipitation assays indicate that PXR activation sequestrates the SRC-1 coactivator from the HNF4α response element and E-box of the OCT1 promoter. Consistently, the exogenous overexpression of the SCR-1, but not the PGC1α, coactivator relieved the PXR-mediated repressive effect on OCT1 transactivation.
• Disruption of BSEP Function in HepaRG cells Alters Bile Acid Disposition and is a susceptive factor to Drug-Induced Cholestatic Injury.Qiu X, Zhang Y, Liu T, Shen H, Xiao Y, Bourner M, Pratt J, Thompson D, Marathe P, Humphreys WG, Lai Y
  Molecular pharmaceutics, Feb 2016
  Abstract : In the present study we characterized in vitro biosynthesis and disposition of bile acids (BAs), and hepatic transporter expression followed by ABCB11 (BSEP) gene knockout in HepaRG cells (HepaRG-KO cells). BSEP KO in HepaRG cells led to time- dependent BA accumulation, resulting in reduced biosynthesis of BAs and altered BA disposition. In HepaRG-KO cells, the expression of NTCP, OATP1B1, OATP2B1, BCRP, P-gp and MRP2 were reduced, while MRP3 and OCT1 were up-regulated. As a result, BSEP KO altered the disposition of BAs and subsequently underwent adaptive regulations of BA synthesis and homeostasis to enable healthy growth of the cells. While BSEP inhibitors caused no or slight increase of BAs in HepaRG wild type cells (HepaRG-WT cells), excessive intracellular accumulation of BAs was observed in HepaRG-KO cells exposed to bosentan and troglitazone, but not dipyridamole. LDH release in the medium was remarkably increased in HepaRG-KO cultures exposed to troglitazone (50 μM), suggesting drug-induced cellular injury. The results revealed that functional impairment of BSEP predisposes the cells to altered BA disposition and is a susceptive factor to drug-induced cholestatic injury. In total, BSEP inhibition might trigger the processes, but not a sole determinant of cholestatic cellular injury. As Intracellular BA accumulation is determined by BSEP function and the subsequent adaptive gene regulation, assessment of intracellular BA accumulation inHepaRG-KO cells could be a useful approach to evaluate drug-induced liver injury (DILI) potentials of drugs that could disrupt other BA homeostasis pathways beyond BSEP inhibition.
• High content analysis assay for prediction of human hepatotoxicity in HepaRG and HepG2 cells.Saito J, Okamura A, Takeuchi K, Hanioka K, Okada A, Ohata T
  Toxicology in vitro, Feb 2016
  Abstract : Drug-induced liver injury (DILI) results in the termination of drug development or withdrawal of a drug from the market. The establishment of a predictive, high-throughput preclinical test system to evaluate potential clinical DILI is therefore required. Here, we established a high content analysis (HCA) assay in human hepatocyte cell lines such as the HepaRG with normal expression levels of CYP enzymes and HepG2 with extremely low expression levels of CYP enzymes. Clinical DILI or non-DILI compounds were evaluated for reactive oxygen species (ROS) production, glutathione (GSH) consumption, and mitochondrial membrane potential (MMP) attenuation. A proportion of DILI compounds induced ROS generation, GSH depletion, and MMP dysfunction, which was consistent with reported mechanisms of DILI of these compounds. In particular, DILI compounds that deplete GSH via reactive metabolites exhibited a more marked decrease in intracellular GSH or increase in ROS production in HepaRG cells than in HepG2 cells. Comparison of the two cell lines with different levels of CYP expression might help clarify the contribution of metabolism to hepatocyte toxicity. These results suggest that the HCA assay in HepaRG and HepG2 cells might help improve the accuracy of evaluating clinical DILI potential during drug screening.
• Comparative analysis of 3D culture methods on human HepG2 cells.Luckert C, Schulz C, Lehmann N, Thomas M, Hofmann U, Hammad S, Hengstler JG, Braeuning A, Lampen A, Hessel S
  Archives of toxicology, Feb 2016
  Abstract : Human primary hepatocytes represent a gold standard in in vitro liver research. Due to their low availability and high costs alternative liver cell models with comparable morphological and biochemical characteristics have come into focus. The human hepatocarcinoma cell line HepG2 is often used as a liver model for toxicity studies. However, under two-dimensional (2D) cultivation conditions the expression of xenobiotic-metabolizing enzymes and typical liver markers such as albumin is very low. Cultivation for 21 days in a three-dimensional (3D) Matrigel culture system has been reported to strongly increase the metabolic competence of HepG2 cells. In our present study we further compared HepG2 cell cultivation in three different 3D systems: collagen, Matrigel and Alvetex culture. Cell morphology, albumin secretion, cytochrome P450 monooxygenase enzyme activities, as well as gene expression of xenobiotic-metabolizing and liver-specific enzymes were analyzed after 3, 7, 14, and 21 days of cultivation. Our results show that the previously reported increase of metabolic competence of HepG2 cells is not primarily the result of 3D culture but a consequence of the duration of cultivation. HepG2 cells grown for 21 days in 2D monolayer exhibit comparable biochemical characteristics, CYP activities and gene expression patterns as all 3D culture systems used in our study. However, CYP activities did not reach the level of HepaRG cells. In conclusion, the increase of metabolic competence of the hepatocarcinoma cell line HepG2 is not due to 3D cultivation but rather a result of prolonged cultivation time.
• Hepatic differentiation of human pluripotent stem cells on human liver progenitor HepaRG-derived acellular matrix.Kanninen LK, Porola P, Niklander J, Malinen MM, Corlu A, Guguen-Guillouzo C, Urtti A , Yliperttula ML, Lou YR
  Experimental cell research , Feb 2016
  Abstract : Human hepatocytes are extensively needed in drug discovery and development. Stem cell-derived hepatocytes are expected to be an improved and continuous model of human liver to study drug candidates. Generation of endoderm-derived hepatocytes from human pluripotent stem cells (hPSCs), including human embryonic stem cells and induced pluripotent stem cells, is a complex, challenging process requiring specific signals from soluble factors and insoluble matrices at each developmental stage. In this study, we used human liver progenitor HepaRG-derived acellular matrix (ACM) as a hepatic progenitor-specific matrix to induce hepatic commitment of hPSC-derived definitive endoderm (DE) cells. The DE cells showed much better attachment to the HepaRG ACM than other matrices tested and then differentiated towards hepatic cells, which expressed hepatocyte-specific makers. We demonstrate that Matrigel overlay induced hepatocyte phenotype and inhibited biliary epithelial differentiation in two hPSC lines studied. In conclusion, our study demonstrates that the HepaRG ACM, a hepatic progenitor-specific matrix, plays an important role in the hepatic differentiation of hPSCs.
• Determination of Human Hepatocyte Intrinsic Clearance for Slowly Metabolized Compounds: Comparison of a Primary Hepatocyte/Stromal Cell Co-Culture with Plated Primary Hepatocytes and HepaRG.Bonn B, Svanberg P, Janefeldt A, Hultman I, Grime KH
  Drug metabolism and disposition, Feb 2016
  Abstract : A key requirement in Drug Discovery is to accurately define intrinsic clearance (CLint) values of less than 1 μL/min/106hepatocytes. This requires assays that allow for longer incubation time as a complement to suspended hepatocytes. This study assessed the effectiveness of plated HepaRG cells, plated primary human hepatocytes (PHH) and the HμREL® human hepatocyte/stromal cell co-culture for determination of low CLint values. The investigation demonstrated that the systems were capable of providing statistically significant CLint estimations down to 0.2 μL/min/106 cells. The HμREL® assay provided a higher level of reproducibility, with repeat significant CLint values being defined in a minimum of triplicate consecutive assays for 6/7 of the low CLint compounds, compared to 4/7 for PHH and 2/7 for HepaRG. The assays were also compared with a suspension assay using drugs with higher CLint values and diverse enzymology. The CLint values from the PHH and HμREL® assays were similar to those defined by a hepatocyte suspension assay, indicating that they can be used inter-changeably alongside a standard assay. Finally, data from these two assays could also predict in vivo hepatic metabolic CLint to within 3-fold for greater than 70% of the compounds tested, with average fold errors (AFE) of 1.6 and 2.3 respectively; whereas the HepaRG data was predictive to within 3-fold for only 50% of compounds (AFE 2.9). In summary all systems have utility for low CLint determination, but the HμREL® co-culture appears slightly superior regarding overall assay performance.
• Activation of the constitutive androstane receptor increases the therapeutic index of CHOP in lymphoma treatmentHedrich WD, Xiao J, Heyward S, Zhang Y, Zhang J, Baer MR, Hassan HE, Wang H
  Molecular Cancer Therapeutics, Jan 2016
  Abstract : The constitutive androstane receptor (CAR, NR1i3) is a key regulator of CYP2B6, the enzyme predominantly responsible for the biotransformation of cyclophosphamide (CPA) to its pharmacologically active metabolite, 4-hydroxycyclophosphamide (4-OH-CPA). Previous studies from our laboratory illustrated that CAR activation increases the formation of 4-OH-CPA; however, CPA is rarely utilized clinically outside of combination therapies. Here, we hypothesize that including a selective human CAR activator with the CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) regimen can improve the efficacy without exacerbating off-target toxicity of this regimen in non-Hodgkin lymphoma treatment. In this study, we have developed a novel multi-organ co-culture system containing human primary hepatocytes for hepatic metabolism, lymphoma cells as a model target for CHOP, and cardiomyocytes as a major site of off-target toxicity associated with this regimen. We found that a selective human CAR activator, CITCO (6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime), altered expression of key drug-metabolizing enzymes and transporters in human hepatocytes, which positively impacts the metabolic profile of CHOP. Co-administration of CITCO and CHOP in the co-culture model led to significantly enhanced cytotoxicity in lymphoma cells but not in cardiomyocytes. Moreover, the beneficial effects of CITCO were abrogated when CAR knockout HepaRG cells were used in the co-culture model. Importantly, synergistic anticancer effects were observed between CITCO and CHOP, in that inclusion of CITCO alongside the CHOP regimen offers comparable antineoplastic activity toward lymphoma cells at significantly reduced drug concentrations and the decreased CHOP load attenuates cardiotoxicity. Overall, these findings provide a potentially promising novel strategy for facilitating CHOP-based chemotherapy.
• Splicing regulator SLU7 preserves survival of hepatocellular carcinoma cells and other solid tumors via oncogenic miR-17-92 cluster expressionUrastun R, Elizalde M, Azkona M, Latasa MU, Garcia-Irigoyen O, Uriate I, Fernandez-Barrena MG, Vicent S, Alonso MM, Muntané J, Prieto J, Avila MA, Berasain C
  Oncogene, Jan 2016
  Abstract : Resisting death is a central hallmark of cancer cells. Tumors rely on a number of genetic mechanisms to avoid apoptosis, and alterations in mRNA alternative splicing are increasingly recognized to have a role in tumorigenesis. In this study, we identify the splicing regulator SLU7 as an essential factor for the preservation of hepatocellular carcinoma (HCC) cells viability. Compared with hepatocytes, SLU7 expression is reduced in HCC cells; however, further SLU7 depletion triggered autophagy-related cellular apoptosis in association with the overproduction of reactive oxygen species. Remarkably, these responses were not observed in primary human hepatocytes or in the well-differentiated HepaRG cell line. Mechanistically, we demonstrate that SLU7 binds the C13orf25 primary transcript in which the polycistronic oncomir miR-17-92 cluster is encompassed, and is necessary for its processing and expression. SLU7 knockdown altered the splicing of the C13orf25 primary transcript, and markedly reduced the expression of its miR-17, miR-20 and miR-92a constituents. This led to the upregulation of CDKN1A (P21) and BCL2L11 (BIM) expression, two bona fide targets of the miR-17-92 cluster and recognized mediators of its pro-survival and tumorigenic activity. Interestingly, altered splicing of miR-17-92 and downregulation of miR-17 and miR-20 were not observed upon SLU7 knockdown in non-transformed hepatocytes, but was found in other (HeLa, H358) but not in all (Caco2) non-hepatic tumor cells. The functional relevance of miR-17-92 dysregulation upon SLU7 knockdown was established when oxidative stress, autophagy and apoptosis were reversed by co-transfection of HCC cells with a miR-17 mimic. Together, these findings indicate that SLU7 is co-opted by HCC cells and other tumor cell types to maintain survival, and identify this splicing regulator as a new determinant for the expression of the oncogenic miR-17-92 cluster. This novel mechanism may be exploited for the development of antitumoral strategies in cancers displaying such SLU7-miR-17-92 crosstalk.Oncogene advance online publication, 25 January 2016; doi:10.1038/onc.2015.517.
• Sp1-mediated ectopic expression of T-cell lymphoma invasion and metastasis 2 in hepatocellular carcinoma.Yen WH, Ke WS, Hung JJ, Chen TM, Chen JS, Sun HS
  Cancer Med, Jan 2016
  Abstract : T-cell lymphoma invasion and metastasis 2 (TIAM2) is a neuron-specific protein that has been found ectopically expressed in hepatocellular carcinoma (HCC). Results from clinical specimens and cellular and animal models have shown that the short form of TIAM2 (TIAM2S) functions as an oncogene in the tumorigenesis of liver cancer. However, the regulation of TIAM2S ectopic expression in HCC cells remains largely unknown. This study aimed to identify the mechanism underlying the ectopic expression of TIAM2S in liver cancer cells. In this report, we provide evidence illustrating that Sp1 binds directly to the GC box located in the TIAM2S core promoter. We further demonstrated that overexpression of Sp1 inHepaRG cells promotes endogenous TIAM2S mRNA and protein expressions, and knockdown of Sp1 in 2 HCC cell lines, HepG2 and PLC/PRF/5, led to a substantial reduction in TIAM2S mRNA and protein in these cells. Of 60 paired HCC samples, 70% showed a significant increase (from 1.1- to 3.6-fold) in Sp1 protein expression in the tumor cells. The elevated Sp1 expression was highly correlated with both TIAM2S mRNA and protein expressions in these samples. Together, these results illustrate that Sp1 positively controls TIAM2S transcription and that Sp1-mediated transcriptional activation is essential for TIAM2S ectopic expression in liver cancer cells.
• Netrin-1 Protects Hepatocytes Against Cell Death Through Sustained Translation During the Unfolded Protein ResponseLahlali T, Plissonnier ML, Romero-López C, Michelet M, Ducarouge B, Berzal-Herranz A, Zoulim F, Mehlen P, Parent R.
  Cell Mol Gastroenterol Hepatol, Jan 2016
  Abstract : Netrin-1, a multifunctional secreted protein, is up-regulated in cancer and inflammation. Netrin-1 blocks apoptosis induced by the prototypical dependence receptors deleted in colorectal carcinoma and uncoordinated phenotype-5. Although the unfolded protein response (UPR) triggers apoptosis on exposure to stress, it first attempts to restore endoplasmic reticulum homeostasis to foster cell survival. Importantly, UPR is implicated in chronic liver conditions including hepatic oncogenesis. Netrin-1's implication in cell survival on UPR in this context is unknown. METHODS: Isolation of translational complexes, determination of RNA secondary structures by selective 2'-hydroxyl acylation and primer extension/dimethyl sulfate, bicistronic constructs, as well as conventional cell biology and biochemistry approaches were used on in vitro-grown hepatocytic cells, wild-type, and netrin-1 transgenic mice. RESULTS: HepaRG cells constitute a bona fide model for UPR studies in vitro through adequate activation of the 3 sensors of the UPR (protein kinase RNA-like endoplasmic reticulum kinase (PERK)), inositol requiring enzyme 1α (IRE1α), and activated transcription factor 6 (ATF6). The netrin-1 messenger RNA 5'-end was shown to fold into a complex double pseudoknot and bear E-loop motifs, both of which are representative hallmarks of related internal ribosome entry site regions. Cap-independent translation of netrin 5' untranslated region-driven luciferase was observed on UPR in vitro. Unlike several structurally related oncogenic transcripts (l-myc, c-myc, c-myb), netrin-1 messenger RNA was selected for translation during UPR both in human hepatocytes and in mice livers. Depletion of netrin-1 during UPR induces apoptosis, leading to cell death through an uncoordinated phenotype-5A/C-mediated involvement of protein phosphatase 2A and death-associated protein kinase 1 in vitro and in netrin transgenic mice. CONCLUSIONS: UPR-resistant, internal ribosome entry site-driven netrin-1 translation leads to the inhibition of uncoordinated phenotype-5/death-associated protein kinase 1-mediated apoptosis in the hepatic context during UPR, a hallmark of chronic liver disease.
• Reduced cardiolipin content decreases respiratory chain capacities and increases ATP synthesis yield in the human HepaRG cells.Peyta L, Jarnouen K, Pinault M, Guimaraes C, de Barros JP, Chevalier S, Dumas JF, Maillot F1, Hatch GM1, Loyer P, Servais S
  Biochim Biophys Acta, Jan 2016
  Abstract : Cardiolipin (CL) is a unique mitochondrial phospholipid potentially affecting many aspects of mitochondrial function/processes, i.e. energy production through oxidative phosphorylation. Most data focusing on implication of CL content and mitochondrial bioenergetics were performed in yeast or in cellular models of Barth syndrome. Previous work reported that increase in CL content leads to decrease in liver mitochondrial ATP synthesis yield. Therefore the aim of this study was to determine the effects of moderate decrease in CL content on mitochondrial bioenergetics in human hepatocytes. For this purpose, we generated a cardiolipin synthase knockdown (shCLS) in HepaRG hepatoma cells showing bioenergetics features similar to primary human hepatocytes. shCLS cells exhibited a 55% reduction in CLS gene and a 40% decrease in protein expression resulting in a 45% lower content in CL compared to control (shCTL) cells. Oxygen consumption was significantly reduced in shCLS cells compared to shCTL regardless of substrate used and energy state analyzed. Mitochondrial low molecular weight supercomplex content was higher in shCLS cells (+60%) compared to shCTL. Significant fragmentation of the mitochondrial network was observed in shCLS cells compared to shCTL cells. Surprisingly, mitochondrial ATP synthesis was unchanged in shCLS compared to shCTL cells but exhibited a higher ATP:O ratio (+46%) in shCLS cells. Our results suggest that lowered respiratory chain activity induced by moderate reduction in CL content may be due to both destabilization of supercomplexes and mitochondrial network fragmentation. In addition, CL content may regulate mitochondrial ATP synthesis yield.
• Functional polymer-dependent 3D culture accelerates the differentiation of HepaRG cells into mature hepatocytes.Higuchi Y, Kawai K, Kanaki T, Yamazaki H, Chesné C, Guguen-Guillouzo C, Suemizu H
  Hepathology research, Jan 2016
  Abstract : The hepatoma-derived cell line HepaRG is regarded as an in vitro model of drug metabolism because fully differentiated HepaRG cells demonstrate functional metabolic responses comparable to those of primary human hepatocytes. Recently, it was demonstrated that the three-dimensional (3D) culture of HepaRG cells enhanced their metabolic functions and toxicological responses. We approached the mechanisms underlying these enhancement effects.  
• Effects of Hypericum perforatum extract and its main bioactive compounds on the cytotoxicity and expression of CYP1A2 and CYP2D6 in hepatic cells.Silva SM, Martinho A, Moreno I, Granadeiro LB, Alves G, Duarte AP, Domingues F, Gallardo E
  Life Sciences, Jan 2016
  Abstract : AIMS: Hypericum perforatum (H. perforatum) is one of the most used medicinal plants. However, it has been associated with relevant interactions with several drugs. This situation is probably mediated by cytochrome P450 enzymes (CYP450), namely the 1A2 (CYP1A2) and 2D6 (CYP2D6) isoforms This study aims to assess the cytotoxic and CYP1A2 and CYP2D6 inductive and/or inhibitory effects of a H. perforatum extract and its main bioactive components in hepatic cell lines. MAIN METHODS: A MTT proliferation assay was performed in WRL-68, HepG2 and HepaRG cells after exposition to different concentrations of H. perforatum extract, hypericin and hyperforin for 24 and 72h. Then, a real-time PCR analysis was accomplished after incubating the cells with these products evaluating the relative CYP1A2 and CYP2D6 expression. KEY FINDINGS: These products have relevant cytotoxicity at a 10μM concentration and it was also demonstrated for the first time that H. perforatum can lead to a significant CYP1A2 and CYP2D6 induction in all cell lines. Moreover, hypericin seems to induce CYP1A2 in HepG2 cells and to inhibit its expression in HepaRG cells while hyperforin induced CYP1A2 in HepG2 and in WRL-68 cells. Additionally, hypericin and hyperforin induce CYP2D6 in HepG2 cells but inhibits its expression in HepaRG and in WRL-68 cells. SIGNIFICANCE: This study not only evidenced that H. perforatum extract and two of its bioactive components can have toxic effects in hepatic cell lines but also emphasized the potential risk of the consumption of H. perforatum with CYP1A2- and CYP2D6-metabolized drugs.  
• Interferon-? and Tumor Necrosis Factor-? Produced by T Cells Reduce the HBV Persistence Form, cccDNA, Without Cytolysis.Xia Y, Stadler D, Lucifora J, Reisinger F, Webb D, Hösel M, Michler T, Wisskirchen K, Cheng X, Zhang K, Chou WM, Wettengel JM, Malo A
  Gastroenterology, Jan 2016
  Abstract : BACKGROUND & AIMS: Viral clearance involves immune cell cytolysis of infected cells. However, studies of hepatitis B virus (HBV) infection in chimpanzees have indicated that cytokines released by T cells also can promote viral clearance via noncytolytic processes. We investigated the noncytolytic mechanisms by which T cells eliminate HBV from infected hepatocytes. METHODS: We performed a cytokine enzyme-linked immunosorbent assay of serum samples from patients with acute and chronic hepatitis B. Liver biopsy specimens were analyzed by in situ hybridization. HepG2-H1.3 cells, HBV-infected HepaRG cells, and primary human hepatocytes were incubated with interferon-γ (IFNγ) or tumor necrosis factor-α (TNF-α), or co-cultured with T cells. We measured markers of HBV replication, including the covalently closed circular DNA (cccDNA). RESULTS: Levels of IFNγ and TNF-α were increased in serum samples from patients with acute vs chronic hepatitis B and controls. In human hepatocytes with stably replicating HBV, as well as in HBV-infected primary human hepatocytes or HepaRG cells, IFNγ and TNF-α each induced deamination of cccDNA and interfered with its stability; their effects were additive. HBV-specific T cells, through secretion of IFNγ and TNF-α, inhibited HBV replication and reduced cccDNA in infected cells without the direct contact required for cytolysis. Blocking IFNγ and TNF-α after T-cell stimulation prevented the loss of cccDNA. Deprivation of cccDNA required activation of nuclear APOBEC3 deaminases by the cytokines. In liver biopsy specimens from patients with acute hepatitis B, but not chronic hepatitis B or controls, hepatocytes expressed APOBEC3A and APOBEC3B. CONCLUSIONS: IFNγ and TNF-α, produced by T cells, reduce levels of HBV cccDNA in hepatocytes by inducing deamination and subsequent cccDNA decay.

2015

• A human monoclonal antibody against small envelope protein of hepatitis B virus with potent neutralization effect.Wang W, Sun L, Li T, Ma Y, Li J, Liu Y, Li M, Wang L, Li C, Xie Y, Wen Y, Liang M, Chen L
  MAbs, Dec 2015
  Abstract : Hepatitis B virus (HBV) produces large (L), middle (M), and small (S) envelope proteins, alternatively referred to as hepatitis B surface antigen (HBsAg). Currently, yeast-derived S protein serves as the preventive vaccine, while hepatitis B immune globulin (HBIG) concentrated from pooled plasma of vaccine recipients is employed for post-exposure prophylaxis. However, only a small proportion of the antibodies in HBIG are HBV specific. In the present study, a human monoclonal anti-S antibody (G12) was developed, produced under GLP conditions, and subjected to a panel of functional assays. In vitro results demonstrated high affinity of G12 for the S protein (KD=7.56 nM). It reacted with envelope proteins of all 7 HBV genotypes tested (A-F, H) by immunofluorescent staining, and more than 97% of HBsAg-positive patient serum samples by enzyme-linked immunosorbent assay. G12 recognized a conformational epitope, although the exact sequence remains unknown. Strikingly, G12 was at least 1,000-fold more potent than HBIG in neutralizing HBV infectivity in both HepaRG cell line and HepG2 cells reconstituted with the HBV receptor. In a transgenic mouse model of HBV persistence, a single peritoneal injection of G12 markedly diminished serum HBsAg titers in all seven mice, which was sustained for the observation period of 144 days in mice with low pre-treatment levels. While the therapeutic potential of G12 warrants further investigation using a large number of animals, G12 is a potent neutralizing human monoclonal antibody and a promising candidate to replace or supplement HBIG in the prevention of HBV infection.
• Evaluation of cytochrome P450 inductions by anti-epileptic drug oxcarbazepine, 10-hydroxyoxcarbazepine, and carbamazepine using human hepatocytes and HepaRG cellsSugiyama I, Murayama N, Kuroki A, Kota J, Iwano S, Yamazaki H, Hirota T
  Xenobiotica, Dec 2015
  Abstract : Anti-epileptic drug oxcarbazepine is structurally related to carbamazepine, but has reportedly different metabolic pathway. Auto-induction potentials of oxcarbazepine, its pharmacologically active metabolite 10-hydroxyoxcarbazepine and carbamazepine were evaluated by cytochrome P450 (CYP) 1A2, CYP2B6 and CYP3A4 mRNA levels and primary metabolic rates using human hepatocytes and HepaRG cells. For the CYP1A2 the induction potential determined as the fold change in mRNA levels was 7.2 (range: 2.3-11.5) and 10.0 (6.2-13.7) for oxcarbazepine and carbamazepine, respectively, while 10-hydroxyoxcarbazepine did not induce. The fold change in mRNA levels for CYP2B6 was 11.5 (3.2-19.3), 7.0 (2.5-10.8) and 14.8 (3.1-29.1) for oxcarbazepine, 10-hydroxyoxcarbazepine and carbamazepine, respectively. The fold change for CYP3A4 induction level by oxcarbazepine, 10-hydroxyoxcarbazepine and carbamazepine was 3.5 (1.2-7.4), 2.7 (0.8-5.7) and 8.3 (3.5-14.5), respectively. The data suggest lower induction potential of oxcarbazepine and 10-hydroxyoxcarbazepine relative to carbamazepine. The results in HepaRG cells showed similar trend as the human hepatocytes. After incubation for 72 h in hepatocytes and HepaRG cells, auto-induction was evident for only carbamazepine metabolism. The 10-keto group instead of double bond at C10 position is evidently a determinant factor for limited auto-induction of P450 enzymes by oxcarbazepine.
• A cellular model to study drug-induced liver injury in nonalcoholic fatty liver disease: application to acetaminophen.Michaut A, Le Guillou D, Moreau C, Bucher S, McGill MR, Martinais S, Gicquel T, Morel I, Robin MA, Jaeschke H, Fromenty B
  Toxicology and Applied Pharmacology, Dec 2015
  Abstract : Obesity and nonalcoholic fatty liver disease (NAFLD) can increase susceptibility to hepatotoxicity induced by some xenobiotics including drugs, but the involved mechanisms are poorly understood. For acetaminophen (APAP), a role of hepatic cytochrome P450 2E1 (CYP2E1) is suspected since the activity of this enzyme is consistently enhanced during NAFLD. The first aim of our study was to set up a cellular model of NAFLD characterized not only by triglyceride accumulation but also by higher CYP2E1 activity. To this end, human HepaRG cells were incubated for one week with stearic acid or oleic acid, in the presence of different concentrations of insulin. Although cellular triglycerides and the expression of lipid-responsive genes were similar with both fatty acids, CYP2E1 activity was significantly increased only by stearic acid. CYP2E1 activity was reduced by insulin and this effect was reproduced in cultured primary human hepatocytes. Next, APAP cytotoxicity was assessed in HepaRG cells with or without lipid accretion and CYP2E1 induction. Experiments with a large range of APAP concentrations showed that the loss of ATP and glutathione was almost always greater in the presence of stearic acid. In cells pretreated with the CYP2E1 inhibitor chlormethiazole, recovery of ATP was significantly higher in the presence of stearate with low (2.5 mM) or high (20 mM) concentrations of APAP. Levels of APAP-glucuronide were significantly enhanced by insulin. Hence, HepaRG cells can be used as a valuable model of NAFLD to unveil important metabolic and hormonal factors which can increase susceptibility to drug-induced hepatotoxicity.
• Gemcitabine and Oxaliplatin, but Not Sorafenib or Paclitaxel, Have a Synergistic Effect with Yttrium-90 in Reducing Hepatocellular Carcinoma and Cholangiocarcinoma Cell Line Viability.Edeline J, Coulouarn C, Crouzet L, Pracht M, Lepareur N, Clément B, Garain E,
  Journal of Vascular and Interventional radiology, Dec 2015
  Abstract : Synergy between yttrium-90 ((90)Y) and antineoplastic drugs was investigated. Viability of HepaRG (hepatocellular carcinoma) and HuCCT1 (cholangiocarcinoma) cells was studied through a tetrazolium dye reduction assay. A combination index (CI) was calculated, with CI < 1 denoting synergy and CI > 1 denoting antagonism. In HepaRG cells, gemcitabine showed synergy with (90)Y (CI = 0.70 [95% confidence interval = 0.65-0.75]), whereas oxaliplatin (CI = 1.15 [1.08-1.21]), paclitaxel (CI = 1.26 [1.15-1.37]), and sorafenib (CI = 1.77 [1.65-1.89]) showed antagonism. In HuCCT1 cells, gemcitabine (CI = 0.54 [0.50-0.58]) and oxaliplatin (CI = 0.86 [0.82-0.90]) showed synergy with (90)Y, whereas paclitaxel (CI = 1.18 [1.09-1.27]) and sorafenib (CI = 1.21 [1.12-1.30]) showed antagonism. These results suggest that gemcitabine and oxaliplatin should be tested in combination with (90)Y radioembolization for treatment of liver cancer.
• Biokinetics in repeated-dosing in vitro drug toxicity studiesKramer NI, Di Consiglio E, Blaauboer BJ, Testai E
  Toxicology in vtro, Dec 2015
  Abstract : The aim of the EU FP7 Predict-IV project was to improve the predictivity of in vitro assays for unwanted effects of drugs after repeated dosing. The project assessed the added benefit of integrating long-lived in vitro organotypic cell systems with 'omics' technologies and in silico modelling, including systems biology and pharmacokinetic assessments. RPTEC/TERT1 kidney cells, primary rat and human hepatocytes, HepaRG liver cells and 2D and 3D primary brain cultures were dosed daily or every other day for 14days to a selection of drugs varying in their mechanism of pharmacological action. Since concentration-effect relationships not only depend on the activity of the drug or the sensitivity of the target, but also on the distribution of compounds in the in vitro system, the concentration of a selection of drugs in cells, microtitre plate plastic and medium was measured over time. Results, reviewed in this paper, indicate that lipophilic drugs bind significantly to plastic labware. A few drugs, including less lipophilic drugs, bind to cell-attachment matrices. Chemicals that reach high concentrations in cells, including cyclosporin A and amiodarone, significantly accumulate over time after repeated dosing, partly explaining their increased toxicity after repeated dosing, compared to a single dose.
• Protein Kinase C-Independent Inhibition of Organic Cation Transporter 1 Activity by the Bisindolylmaleimide Ro 31-8220Mayati A, Bruyere A, Moreau A, Jouan E, Denizot C, Parmentier Y, Fardel O
  Plos one, Dec 2015
  Abstract : Ro 31–8220 is a potent protein kinase C (PKC) inhibitor belonging to the chemical class of bisindolylmaleimides (BIMs). Various PKC-independent effects of Ro 31–8220 have however been demonstrated, including inhibition of the ATP-binding cassette drug transporter breast cancer resistance protein. In the present study, we reported that the BIM also blocks activity of the solute carrier organic cation transporter (OCT) 1, involved in uptake of marketed drugs in the liver, in a PKC-independent manner. Ro 31–8220, in contrast to other pan-PKC inhibitors such as staurosporine and chelerythrine, was thus shown tocis-inhibit uptake of the reference OCT1 substrate tetraethylammonium in OCT1-transfected HEK293 cells in a concentration-dependent manner (IC50 = 0.18 μM) and without altering membrane expression of OCT1. This blockage of OCT1 was also observed in human hepatic HepaRG cells that constitutionally express OCT1. It likely occurred through a mixed mechanism of inhibition. Ro 31–8220 additionally trans-inhibited TEA uptake in OCT1-transfected HEK293 cells, which likely discards a transport of Ro 31–8220 by OCT1. Besides Ro 31–8220, 7 additional BIMs, including the PKC inhibitor LY 333531, inhibited OCT1 activity, whereas 4 other BIMs were without effect. In silico analysis of structure-activity relationships next revealed that various molecular descriptors, especially 3D-WHIM descriptors related to total size, correspond to key physico-chemical parameters for inhibition of OCT1 activity by BIMs. In addition to activity of OCT1, Ro 31–8220 inhibited those of other organic cation transporters such as multidrug and toxin extrusion protein (MATE) 1 and MATE2-K, whereas, by contrast, it stimulated that of OCT2. Taken together, these data extend the nature of cellular off-targets of the BIM Ro 31–8220 to OCT1 and other organic cation transporters, which has likely to be kept in mind when using Ro 31–8220 and other BIMs as PKC inhibitors in experimental or clinical studies.
• Development of an oxygenation culture method for activating the liver-specific functions of HepG2 cells utilizing a collagen vitrigel membrane chamber.Oshikata-Miyazaki A, Takezawa T
  Cytotechnology, Dec 2015
  Abstract : We recently developed a collagen vitrigel membrane (CVM) chamber possessing a scaffold composed of high-density collagen fibrils. In this study, we first confirmed that the advantage of CVM chamber in comparison to the traditional culture chamber with porous polyethylene terephthalate membrane is to preserve a culture medium poured in its inside even though the under side is not a liquid phase but solid and gas phases. Subsequently, we designed three different culture systems to grow HepG2 cells in a culture medium (liquid phase) on the CVM which the under side is a culture medium, a plastic surface (solid phase) or 5 % CO2 in air (gas phase) and aimed to develop a brief culture method useful for activating the liver-specific functions and analyzing the pharmacokinetics of fluorescein diacetate. HepG2 cells cultured for 2 days on the liquid-solid interface and subsequently for 1 day on the liquid-gas interface represented excellent cell viability and morphology in comparison to the others, and remarkably improved albumin secretion and urea synthesis to almost the same level of freshly isolated human hepatocytes and CYP3A4 activity to about half the level of differentiated HepaRG cells. Also, the cells rapidly absorbed fluorescein diacetate, distributed it in cytosol, metabolized it into fluorescein, and speedily excreted fluorescein into both bile canaliculus-like networks and extracellular solution. These data suggest that hepatic structure and functions of monolayered HepG2 cells can be induced within a day after the oxygenation from beneath the CVM.
• In vitro metabolism of the cyanotoxin cylindrospermopsin in HepaRG cells and liver tissue fractionsKittler K, Hurtaud Pessel D, Maul R, Kolrep F, Fessard V
  toxicon, Dec 2015
  Abstract : No evidence for phase I metabolites of the cyanotoxin cylindrospermopsin (CYN) was given using HepaRG cells and different liver tissue fractions when studying metabolic conversion. Although the application of ketoconazole, a CYP3A4 inhibitor, led to a decreased cytotoxicity of CYN, no metabolites were detected applying high resolution mass spectrometry. Quantification of non-modified CYN led to recovery rates of almost 100%. Consequently, reduction of CYN toxicity in the presence of metabolism inhibiting agents must be attributed to alternative pathways.  
• Metabolomics analysis of the toxicity pathways of triphenyl phosphate in HepaRG cells and comparison to oxidative stress mechanisms caused by acetaminophenVan den Eede N, Cuykx M, Rodrigues RM, Laukens K, Neels H, Covaci A5, Vanhaecke T,
  taxicology in vitro, Dec 2015
  Abstract : Since the publication of REACH guidelines, the need for in vitro tools for toxicity testing has increased. We present here the development of a hepatotoxicity testing tool using human HepaRG cell cultures and metabolomics. HepaRG cells were exposed to either 4mM acetaminophen (APAP) as reference toxicant for oxidative stress or 50μM triphenyl phosphate (TPHP) as toxicant with unknown toxicity pathways (TPs). After 72h exposure, cells were subjected to quenching and liquid-liquid extraction which resulted in a polar and an apolar fraction. Analysis of fractions was performed by ultrahigh performance liquid chromatography-high resolution tandem mass spectrometry (UHPLC-QTOF-MS). Significantly up or down regulated metabolites were selected by univariate statistics prior to identification. In order to obtain robust and specific TP biomarkers, the experiment was also repeated using a different culture medium composition to assess which metabolites show consistent changes. Potential biomarkers belonging to different TPs were found for APAP and TPHP. For APAP, the biomarkers were related to a decrease in unsaturated phospholipids, and for TPHP to an accumulation of phosphoglycerolipids and increase of palmitoyl lysophosphatidylcholine. This first proof-of-concept opens new perspectives for the analysis of other (reference) toxicants with different TPs and it can be used to expand the in vitro tool for hepatotoxicity screening of various compounds.
• MicroRNA responses to the genotoxic carcinogens aflatoxin B1 and benzo[a]pyrene in human HepaRG cellsMarrone AK, Tryndyak V, Beland FA, Pogribny Ip
  Toxicological Sciences, Nov 2015
  Abstract : Recent advances in toxicogenomics present an opportunity to develop new in vitro testing methodologies to identify human carcinogens. We have investigated microRNA expression responses to the treatment of human liver HepaRG cells with the human genotoxic carcinogens aflatoxin B1 (AFB1) and benzo[a]pyrene (B[a]P), and the structurally similar compounds aflatoxin B2 (AFB2) and benzo[e]pyrene (B[e]P) that exhibit minimal carcinogenic potential. We demonstrate that treatment of HepaRG cells with AFB1 or B[a]P resulted in specific changes in the expression of miRNAs as compared to their non-carcinogenic analogues, particularly in a marked over-expression of miR-410. An additional novel finding is the dose- and time-dependent inhibition of miR-122 in AFB1-treated HepaRG cells. Mechanistically, the AFB1-induced down-regulation of miR-122 was attributed to inhibition of the HNF4A/miR-122 regulatory pathway. These results demonstrate that HepaRG cells can be used to investigate miRNA responses to xenobiotic exposure, and illustrate the existence of early non-genotoxic events, in addition to a well-established genotoxic mode of action changes, in the mechanism of AFB1 and B[a]P carcinogenicity. Published by Oxford University Press on behalf of the Society of Toxicology 2015. This work is written by US Government employees and is in the public domain in the US.
• Hepatitis B virus efficiently infects non-adherent hepatoma cells via human sodium taurocholate cotransporting polypeptideOkuyama Dobashi K, Kasai H, Tanaka T, Yasmahita A, Yasumoto J, Chen W, Okamoto T, Maekawa S, Watashi K, Wakita T, Ryo A, Suzuki T, Matsuura Y
  Scientefic reports, Nov 2015
  Abstract : Sodium taurocholate cotransporting polypeptide (NTCP) has been reported as a functional receptor for hepatitis B virus (HBV) infection. However, HBV could not efficiently infect HepG2 cells expressing NTCP (NTCP-HepG2 cells) under adherent monolayer-cell conditions. In this study, NTCP was mainly detected in the basolateral membrane region, but not the apical site, of monolayer NTCP-HepG2 cells. We hypothesized that non-adherent cell conditions of infection would enhance HBV infectivity. Non-adherent NTCP-HepG2 cells were prepared by treatment with trypsin and EDTA, which did not degrade NTCP in the membrane fraction. HBV successfully infected NTCP-HepG2 cells at a viral dose 10 times lower in non-adherent phase than in adherent phase. Efficient infection of non-adherent NTCP-HepG2 cells with blood-borne or cell-culture-derived HBV was observed and was remarkably impaired in the presence of the myristoylated preS1 peptide. HBV could also efficiently infect HepaRG cells under non-adherent cell conditions. We screened several compounds using our culture system and identified proscillaridin A as a potent anti-HBV agent with an IC50 value of 7.2 nM. In conclusion, non-adherent host cell conditions of infection augmented HBV infectivity in an NTCP-dependent manner, thus providing a novel strategy to identify anti-HBV drugs and investigate the mechanism of HBV infection.
• Novel human hepatic organoid model enables testing of drug-induced liver fibrosis in vitroLeite SB, Roosens T, El Taghdouini A, Mannaerts I, Smout AJ, Najimi M, Sokal E, Noor F, Chesne C, van Grunsven LA
  Biomaterials., Nov 2015
  Abstract :   Current models for in vitro fibrosis consist of simple mono-layer cultures of rodent hepatic stellate cells (HSC), ignoring the role of hepatocyte injury. We aimed to develop a method allowing the detection of hepatocyte-mediated and drug-induced liver fibrosis. We used HepaRG (Hep) and primary human HSCs cultured as 3D spheroids in 96-well plates. These resulting scaffold-free organoids were characterized for CYP induction, albumin secretion, and hepatocyte and HSC-specific gene expression by qPCR. The metabolic competence of the organoid over 21 days allows activation of HSCs in the organoid in a drug- and hepatocyte-dependent manner. After a single dose or repeated exposure for 14 days to the pro-fibrotic compounds Allyl alcohol and Methotrexate, hepatic organoids display fibrotic features such as HSC activation, collagen secretion and deposition. Acetaminophen was identified by these organoids as an inducer of hepatotoxic-mediated HSC activation which was confirmed in vivo in mice. This novel hepatic organoid culture model is the first that can detect hepatocyte-dependent and compound-induced HSC activation, thereby representing an important step forward towards in vitro compound testing for drug-induced liver fibrosis.
• Toxicity of Carboxylic Acid-Containing Drugs: The Role of Acyl Migration and CoA Conjugation InvestigatedLassila T, Hokkanen J, Matilla S , Turpeinen M, Tolonen A
  Chemical Research in Technology , Nov 2015
  Abstract : Many carboxylic acid-containing drugs are associated with idiosyncratic drug toxicity (IDT), which may be caused by reactive acyl glucuronide metabolites. The rate of acyl migration has been earlier suggested as a predictor of acyl glucuronide reactivity. Additionally, acyl Coenzyme A (CoA) conjugates are known to be reactive. Here, 13 drugs with a carboxylic acid moiety were incubated with human liver microsomes to produce acyl glucuronide conjugates for the determination of acyl glucuronide half-lives by acyl migration and with HepaRG cells to monitor the formation of acyl CoA conjugates, their further conjugate metabolites, and trans-acylation products with glutathione. Additionally, in vitro cytotoxicity and mitochondrial toxicity experiments were performed with HepaRG cells to compare the predictability of toxicity. Clearly, longer acyl glucuronide half-lives were observed for safe drugs compared to drugs that can cause IDT. Correlation between half-lives and toxicity classification increased when "relative half-lives," taking into account the formation of isomeric AG-forms due to acyl migration and eliminating the effect of hydrolysis, were used instead of plain disappearance of the initial 1-O-β-AG-form. Correlation was improved further when a daily dose of the drug was taken into account. CoA and related conjugates were detected primarily for the drugs that have the capability to cause IDT, although some exceptions to this were observed. Cytotoxicity and mitochondrial toxicity did not correlate to drug safety. On the basis of the results, the short relative half-life of the acyl glucuronide (high acyl migration rate), high daily dose and detection of acyl CoA conjugates, or further metabolites derived from acyl CoA together seem to indicate that carboxylic acid-containing drugs have a higher probability to cause drug-induced liver injury (DILI).
• Formation of GSH-trapped reactive metabolites in human liver microsomes, S9 fraction, HepaRG-cells, and human hepatocytes.Lassila T, Rousu T, Mattila S, Chesné C, Pelkonen O, Turpeinen M, Tolonen A
  Journal of Pharmaceutical and Biomedical Analysis, Nov 2015
  Abstract : The objective was to compare several in vitro human liver-derived subcellular and cellular incuba-tion systems for the formation of GSH-trapped reactive metabolites. Incubations of pooled human livermicrosomes, human liver S9 fractions, HepaRG-cells, and human hepatocytes were performed with glu-tathione as a trapping agent. Experiments with liver S9 were performed under two conditions, using onlyNADPH and using a full set of cofactors enabling also conjugative metabolism. Ten structurally differ-ent compounds were used as a test set, chosen as either “positive” (ciprofloxacin, clozapine, diclofenac,ethinyl estradiol, pulegone, and ticlopidine) or “negative” (caffeine, citalopram, losartan, montelukast)compounds, based on their known adverse reactions on liver or bone marrow. GSH conjugates wereobserved for seven of the ten compounds; while no conjugates were observed for caffeine, citalopram,or ciprofloxacin. Hepatocyte and HepaRG assays produced a clearly lower number and lower relativeabundance of GSH conjugates compared to assays with microsomes and S9 fractions. The major GSHconjugates were different for many compounds in cellular subfractions and cell-based systems. Hepato-cytes generally produced a higher number of GSH conjugates than HepaRG cells, although the differenceswere minor. The results show that the hepatic enzyme system used for screening of GSH-trapped reactivemetabolites do have a high impact on the results, and results between different systems are comparableonly qualitatively.
• A novel cell-based assay for the evaluation of immune- and inflammatory-related gene expression as biomarkers for the risk assessment of drug-induced liver injury.Oda S, Matsuo K, Nakajima A, Yokoi T
  Toxicology Letters, Nov 2015
  Abstract : Drug-induced liver injury (DILI) is a major problem in drug development. Although some in vitro methods assessing DILI risk that utilize hepatic cell death or cellular stress as markers have been developed, the predictive ability of these tests is low. In this study, we sought to develop a novel cell-based assay for the risk assessment of DILI that considers drug metabolism as well as immune- and inflammatory-related gene expression. To accomplish this goal, human hepatoma HepaRG or HepG2 cells were treated with 96 drugs with different clinical DILI risks. The conditioned media were subsequently used to treat human promyelocytic leukemia HL-60 cells, and the mRNA expression levels of immune- and inflammatory-related genes in the cells were measured. An area under the receiver operating characteristic curve (ROC-AUC) was calculated to evaluate the predictive performance of the mRNA levels as markers to discriminate DILI risk. The expression of interleukin-8 (IL-8) in HL-60 cells treated with conditioned media from HepaRG cells (HL-60/HepaRG) exhibited the highest ROC-AUC value of 0.758, followed by the expression of IL-1β in HL-60/HepaRG (ROC-AUC: 0.726). Notably, the ROC-AUC values of these genes were higher in HL-60/HepaRG than in HL-60/HepG2, which suggests that HL-60/HepaRG has a higher potential for detecting the metabolic activation of drugs. An integrated score calculated from the levels of S100 calcium-binding protein A9 (S100A9), IL-1β, and IL-8 more precisely determined the DILI risks than individual gene expression did. The developed cell-based assay that utilizes immune-related gene expression would aid in the assessment of potential DILI risks
• Peroxisome proliferator-activated receptor alpha, PPAR?, directly regulates transcription of cytochrome P450 CYP2C8Thomas M, Winter S, Klumpp B, Turpeinen M, Klein K, Schwab M, Zanger UM
  Frontiers in pharmacology , Nov 2015
  Abstract : The cytochrome P450, CYP2C8, metabolizes more than 60 clinically used drugs as well as endogenous substances including retinoic acid and arachidonic acid. However, predictive factors for interindividual variability in the efficacy and toxicity of CYP2C8 drug substrates are essentially lacking. Recently we demonstrated that peroxisome proliferator-activated receptor alpha (PPARα), a nuclear receptor primarily involved in control of lipid and energy homeostasis directly regulates the transcription of CYP3A4. Here we investigated the potential regulation of CYP2C8 by PPARα. Two linked intronic SNPs in PPARα (rs4253728, rs4823613) previously associated with hepatic CYP3A4 status showed significant association with CYP2C8 protein level in human liver samples (N = 150). Furthermore, siRNA-mediated knock-down of PPARα in HepaRG human hepatocyte cells resulted in up to ∼60 and ∼50% downregulation of CYP2C8 mRNA and activity, while treatment with the PPARα agonist WY14,643 lead to an induction by >150 and >100%, respectively. Using chromatin immunoprecipitation scanning assay we identified a specific upstream gene region that is occupied in vivo by PPARα. Electromobility shift assay demonstrated direct binding of PPARα to a DR-1 motif located at positions –2762/–2775 bp upstream of the CYP2C8 transcription start site. We further validated the functional activity of this element using luciferase reporter gene assays in HuH7 cells. Moreover, based on our previous studies we demonstrated that WNT/β-catenin acts as a functional inhibitor of PPARα-mediated inducibility of CYP2C8 expression. In conclusion, our data suggest direct involvement of PPARα in both constitutive and inducible regulation of CYP2C8 expression in human liver, which is further modulated by WNT/β-catenin pathway. PPARA gene polymorphism could have a modest influence on CYP2C8 phenotype.
• Comparative Analysis and Functional Characterization of HC-AFW1 Hepatocarcinoma Cells: Cytochrome P450 Expression and Induction by Nuclear Receptor Agonists.Braeuning A, Thomas M, Hofmann U, Vetter S, Zeller E, Petzuch B, Johänning J, Schroth W, Weiss TS, Zanger UM, Schwarz M
  Drug metabolism and disposition, Nov 2015
  Abstract : Enzymatic conversion of most xenobiotic compounds is accomplished by hepatocytes in the liver, which are also an important target for the manifestation of the toxic effects of foreign compounds. Most cell lines derived from hepatocytes lack important toxifying or detoxifying enzymes or are defective in signaling pathways that regulate expression and activity of these enzymes. On the other hand, the use of primary human hepatocytes is complicated by scarce availability of cells and high interdonor variability. Thus, analyses of drug metabolism and hepatotoxicity in vitro are a difficult task. The cell line HC-AFW1 was isolated from a pediatric hepatocellular carcinoma and so far has been used for tumorigenicity and chemotherapy resistance studies. Here, a comprehensive characterization of xenobiotic metabolism in HC-AFW1 cells is presented along with studies on the functionality of the most important transcriptional regulators of drug-metabolizing enzymes. Results from HC-AFW1 cells were compared with commercially available HepaRG cells and cultured primary human hepatocytes. Data show that the nuclear receptors and xenosensors AHR (aryl hydrocarbon receptor), CAR (constitutive androstane receptor), PXR (pregnane-X-receptor), NRF2 [nuclear factor (erythroid-derived 2)-like 2], and PPARα (peroxisome proliferator-activated receptor α) are functional in HC-AFW1 cells, comparable to HepaRG and primary cells. HC-AFW1 cells possess considerable activities of different cytochrome P450 enzymes, which, however, are lower than corresponding enzyme activities in HepaRG cells or primary hepatocytes. In summary, HC-AFW1 are a new promising tool for studying the mechanisms of the regulation of drug metabolism in human liver cells in vitro.
• Genome-wide analysis of human constitutive androstane receptor (CAR) transcriptome in wild-type and CAR-knockout HepaRG cells.Li D, Mackowiak B, Brayman TG, Mitchell M, Zhang L, Huang SM, Wang H
  Biochemical pharmacology, Nov 2015
  Abstract : The constitutive androstane receptor (CAR) modulates the transcription of numerous genes involving drug metabolism, energy homeostasis, and cell proliferation. Most functions of CAR however were defined from animal studies. Given the known species difference of CAR and the significant cross-talk between CAR and the pregnane X receptor (PXR), it is extremely difficult to decipher the exact role of human CAR (hCAR) in gene regulation, relying predominantly on pharmacological manipulations. Here, utilizing a newly generated hCAR-knockout (KO) HepaRG cell line, we carried out RNA-seq analysis of the global transcriptomes in wild-type (WT) and hCAR-KO HepaRG cells treated with CITCO, a selective hCAR agonist, phenobarbital (PB), a dual activator of hCAR and hPXR, or vehicle control. Real-time PCR assays in separate experiments were used to validate RNA-seq findings. Our results indicate that genes encoding drug-metabolizing enzymes are among the main clusters altered by both CITCO and PB. Specifically, CITCO significantly changed the expression of 135 genes in an hCAR-dependent manner, while PB altered the expression of 227 genes in WT cells of which 94 were simultaneously modulated in both cell lines reflecting dual effects of PB on hCAR/PXR. Notably, we found that many genes promoting cell proliferation and tumorigenesis were up-regulated in hCAR-KO cells, suggesting that hCAR may play an important role in cell growth that differs from mouse CAR. Together, our results reveal both novel and known targets of hCAR and support the role of hCAR in maintaining the homeostasis of metabolism and cell proliferation in the liver.
• MicroRNA hsa-miR-29a-3p modulates CYP2C19 in human liver cells.Yu D, Green B, Tolleson WH, Jin Y, Mei N, Guo Y, Deng H, Pogribny I, Ning B, , , ,
  Biochemicol pharmacology, Nov 2015
  Abstract : Cytochrome P450 2C19 (CYP2C19) is involved in the metabolism of many drugs. Extensive studies have demonstrated that genetic variants and endogenous and environmental factors play important roles in the expression of CYP2C19. However, the role of microRNAs (miRNAs) in controlling CYP2C19 expression has not been investigated completely. In the present study, we performed in silico analysis to rank putative miRNA/CYP2C19 hybrids with regards to the predicted stabilities of their duplexes and then we applied a series of biochemical and molecular assays to elucidate the underlying functional mechanisms for the regulation of CYP2C19 by miRNAs. In silico analysis indicated that hsa-miR-23a-3p and hsa-miR-29a-3p target the coding region of CYP2C19 with hybrid stabilities of -27.5kcal/mol and -23.3kcal/mol, respectively. RNA electrophoresis mobility shift assays showed that both hsa-miR-23a-3p and hsa-miR-29a-3p miRNAs were able to bind directly to their cognate targets in the CYP2C19 transcript. Further, a significant inverse correlation was found between chemically-induced up-regulation of hsa-miR-29a-3p and CYP2C19 expression inHepaRG cells. In addition, inverse correlations were also observed in human liver tissue samples between the level of CYP2C19 mRNA expression and both hsa-miR-23a-3p and hsa-miR-29a-3p levels. All these results demonstrated the suppressing role of hsa-miR-29a-3p on CYP2C19 expression.
• Regulation of hepatic cardiolipin metabolism by TNF: Implication in cancer cachexiaPeyta L, Jarnouen K, Pinault M, Coulouarn C, Guimaraes C, Goupille C, de Barros JP, Chevalier S, Dumas JF, Maillot F1, Hatch GM1, Loyer P, Servais S
  biochmica biophysica acta, Nov 2015
  Abstract : Cardiolipin (CL) content accumulation leads to an increase in energy wasting in liver mitochondria in a rat model of cancer cachexia in which tumor necrosis factor alpha (TNFα) is highly expressed. In this study we investigated the mechanisms involved in liver mitochondria CL accumulation in cancer cachexia and examined if TNFα was involved in this process leading to mitochondrial bioenergetics alterations. We studied gene, protein expression and activity of the main enzymes involved in CL metabolism in liver mitochondria from a rat model of cancer cachexia and in HepaRGhepatocyte-like cells exposed to 20 ng/ml of TNFα for 12 h. Phosphatidylglycerolphosphate synthase (PGPS) gene expression was increased 2.3-fold (p
• Evaluation of CYP3A4 inhibition and hepatotoxicity using DMSO-treated human hepatoma HuH-7 cellsLiu Y, Flynn TJ, Xia M, Wiesenfeld PL, Ferguson MS
  Cell biology and toxicology, Oct 2015
  Abstract : A human hepatoma cell line (HuH-7) was evaluated as a metabolically competent cell model to investigate cytochrome P450 3A4 (CYP3A4) inhibition, induction, and hepatotoxicity. First, CYP3A4 gene expression and activity were determined in HuH-7 cells under three culture conditions: 1-week culture, 3-week culture, or 1 % dimethyl sulfoxide (DMSO) treatment. HuH-7 cells treated with DMSO for 2 weeks after confluence expressed the highest CYP3A4 gene expression and activity compared to the other two culture conditions. Furthermore, CYP3A4 activity in DMSO-treated HuH-7 cells was compared to that in a human hepatoma cell line (HepG2/C3A) and human bipotent progenitor cell line (HepaRG), which yielded the following ranking: HepaRG > DMSO-treated HuH-7 >> HepG2/C3A cells. The effects of three known CYP3A4 inhibitors were evaluated using DMSO-treated HuH-7 cells. CYP3A4 enzyme inhibition in HuH-7 cells was further compared to human recombinant CYP3A4, indicating similar potency for reversible inhibitors (IC 50 within 2.5-fold), but different potency for the irreversible inhibitor. Next, induction of CYP3A4 activity was compared between DMSO-treated HuH-7 and HepaRG cells using two known inducers. DMSO-treated HuH-7 cells yielded minimal CYP3A4 induction compared to that in the HepaRG cells after 48-h treatments. Finally, the cytotoxicity of five known hepatotoxicants was evaluated in DMSO-treated HuH-7, HepG2/C3A, and HepaRG cells, and significant differences in cytotoxic sensitivity were observed. Overall, DMSO-treated HuH-7 cells are a valuable model for medium- or high-throughput screening of chemicals for CYP3A4 inhibition and hepatotoxicity.
• MicroRNA changes, activation of progenitor cells and severity of liver injury in mice induced by choline and folate deficiency.Tryndyak VP, Marrone AK, Latendresse JR, Muskhelishvili L, Beland FA, Pogribny IP, , , , , , ,
  The journal of nutritional biochemistry , Oct 2015
  Abstract : Dietary deficiency in methyl-group donors and cofactors induces liver injury that resembles many pathophysiological and histopathological features of human nonalcoholic fatty liver disease (NAFLD), including an altered expression of microRNAs (miRNAs). We evaluated the consequences of a choline- and folate-deficient (CFD) diet on the expression of miRNAs in the livers of male A/J and WSB/EiJ mice. The results demonstrate that NAFLD-like liver injury induced by the CFD diet in A/J and WSB/EiJ mice was associated with marked alterations in hepatic miRNAome profiles, with the magnitude of miRNA expression changes being greater in WSB/EiJ mice, the strain characterized by the greatest severity of liver injury. Specifically, WSB/EiJ mice exhibited more prominent changes in the expression of common miRNAs as compared to A/J mice and distinct miRNA alterations, including the overexpression of miR-134, miR-409-3p, miR-410 and miR-495 miRNAs that were accompanied by an activation of hepatic progenitor cells and fibrogenesis. This in vivo finding was further confirmed by in vitro experiments showing an overexpression of these miRNAs in undifferentiated progenitor hepatic HepaRG cells compared to in fully differentiated HepaRG cells. Additionally, a marked elevation of miR-134, miR-409-3p, miR-410 and miR-495 was found in plasma of WSB/EiJ mice fed the CFD diet, while none of the miRNAs was changed in plasma of A/J mice. These findings suggest that miRNAs may be crucial regulators responsible for the progression of NAFLD and may be useful as noninvasive diagnostic indicators of the severity and progression of NAFLD.
• Toxicogenomics-based prediction of acetaminophen-induced liver injury using human hepatic cell systemsRodriques RM, Heymans A , De Boe V, Sachinidis A, Chaudhari U , Govaere O, Roskams T, Vanhaecke T, Rogier V, De Kock J
  Texicology Letters, Oct 2015
  Abstract : Primary human hepatocytes (hHEP), human HepaRG and HepG2 cell lines are the most used human liver-based in vitro models for hepatotoxicity testing, including screening of drug-induced liver injury (DILI)-inducing compounds. hHEP are the reference hepatic in vitro system, but their availability is limited and the cells available for toxicology studies are often of poor quality. Hepatic cell lines on the other hand are highly proliferative and represent an inexhaustible hepatic cell source. However, these hepatoma-derived cells do not represent the population diversity and display reduced hepatic metabolism. Alternatively, stem cell-derived hepatic cells, which can be produced in high numbers and can differentiate into multiple cell lineages, are also being evaluated as a cell source for in vitro hepatotoxicity studies. Human skin-derived precursors (hSKP) are post-natal stem cells that, after conversion towards hepatic cells (hSKP-HPC), respond to hepatotoxic compounds in a comparable way as hHEP. In the current study, four different human hepatic cell systems (hSKP-HPC, hHEP, HepaRG and HepG2) are evaluated for their capacity to predict hepatic toxicity. Their hepatotoxic response to acetaminophen (APAP) exposure is compared to data obtained from patients suffering from APAP-induced acute liver failure (ALF). The results indicate that hHEP, HepaRG and hSKP-HPC identify comparable APAP-induced hepatotoxic functions and that HepG2 cells show the slightest hepatotoxic response. Pathway analyses further points out that HepaRG cells show the highest predicted activation of the functional genes related to 'damage of liver', followed by hSKP-HPC and hHEP cells that generated similar results. HepG2 did not show any activation of this function.
• Evaluation of Normalization Methods To Predict CYP3A4 Induction in Six Fully Characterized Cryopreserved Human Hepatocyte Preparations and HepaRG CellsVermet H, Raoust N, Ngo R, Esserméant L, Klieber S, Fabre G, Boulenc X,
  Drug Metab Dispos, Oct 2015
  Abstract : Prediction of drug-drug interactions due to cytochrome P450 isoform 3A4 (CYP3A4) overexpression is important because this CYP isoform is involved in the metabolism of about 30% of clinically used drugs from almost all therapeutic categories. Therefore, it is mandatory to attempt to predict the potential of a new compound to induce CYP3A4. Among several in vitro-in vivo extrapolation methods recently proposed in the literature, an approach using a scaling factor, called a d factor, for a given hepatocyte batch to provide extrapolation between in vitro induction data and clinical outcome has been adopted by leading health authorities. We challenged the relevance of the calibration factor determined using a set of 15 well-known clinical CYP3A4 inducers or the potent CYP3A4 inducer rifampicin only. These investigations were conducted using six batches of human hepatocytes and an established HepaRG cell line. Our findings show that use of a calibration factor is preferable for clinical predictions, as shown previously by other investigators. Moreover, the present results also suggest that the accuracy of prediction through calculation of this factor is sufficient when rifampicin is considered alone, and the use of a larger set of fully characterized CYP3A4 clinical inducers is not required. For the established HepaRG cell line, the findings obtained in three experiments using a single batch of cells show a good prediction accuracy with or without the d factor. Additional investigations with different batches of HepaRG cell lines are needed to confirm these results.
• Protein kinase C-dependent regulation of human hepatic drug transporter expressionMayati A, Le Vee M, Moreau A, Jouan E, Bucher S, Stieger B, Denizot C, Parmentier Y, Fardel O
  Biochem Pharmacol, Oct 2015
  Abstract : Hepatic drug transporters are now recognized as major actors of hepatobiliary elimination of drugs. Characterization of their regulatory pathways is therefore an important issue. In this context, the present study was designed to analyze the potential regulation of human hepatic transporter expression by protein kinase C (PKC) activation. Treatment by the reference PKC activator phorbol 12-myristate 13-acetate (PMA) for 48h was shown to decrease mRNA expression of various sinusoidal transporters, including OATP1B1, OATP2B1, NTCP, OCT1 and MRP3, but to increase that of OATP1B3, whereas mRNA expression of canalicular transporters was transiently enhanced (MDR1), decreased (BSEP and MRP2) or unchanged (BCRP) in human hepatoma HepaRG cells. The profile of hepatic transporter mRNA expression changes in PMA-treated HepaRG cells was correlated to that found in PMA-exposed primary human hepatocytes and was similarly observed in response to the PKC-activating marketed drug ingenol mebutate. It was associated with concomitant repression of OATP1B1 and OATP2B1 protein expression and reduction of OATP, OCT1, NTCP and MRP2 activity. The use of chemical PKC inhibitors further suggested a contribution of novel PKCs isoforms to PMA-mediated regulations of transporter mRNA expression. PMA was finally shown to cause epithelial-mesenchymal transition (EMT) in HepaRG cells and exposure to various additional EMT inducers, i.e., hepatocyte growth factor, tumor growth factor-β1 or the HNF4α inhibitor BI6015, led to transporter expression alterations highly correlated to those triggered by PMA. Taken together, these data highlight PKC-dependent regulation of human hepatic drug transporter expression, which may be closely linked to EMT triggered by PKC activation.
• Human hepatoma cell lines on gas foaming templated alginate scaffolds for in vitro drug-drug interaction and metabolism studiesStampella A, Rizzitelli G, Donati F, Mazzarino M, Torre X, Bortrè F, Giardi MF, Dentini M, Barbetta A, Massimi M
  Toxicol In Vitro, Oct 2015
  Abstract : Liver in vitro systems that allow reliable prediction of major human in vivo metabolic pathways have a significant impact in drug screening and drug metabolism research. In the present study, a novel porous scaffold composed of alginate was prepared by employing a gas-in-liquid foaming approach. Galactose residues were introduced on scaffold surfaces to promote cell adhesion and to enhance liver specific functions of the entrapped HepG2/C3A cells. Hepatoma cells in the gal-alginate scaffold showed higher levels of liver specific products (albumin and urea) and were more responsive to specific inducers (e.g. dexamethasone) and inhibitors (e.g. ketoconazole) of the CYP3A4 system than in conventional monolayer culture. HepG2/C3A cells were also more efficient in terms of rapid elimination of testosterone, used as a model substance, at rates comparable to those of in vivo excretion. In addition, an improvement in metabolism of testosterone, in terms of phase II metabolite formation, was also observed when the more differentiated HepaRG cells were used. Together the data suggest that hepatocyte/gas templated alginate-systems provide an innovative high throughput platform for in vitro drug metabolism and drug-drug interaction studies, with broad fields of application, and might provide a valid tool for minimizing animal use in preclinical testing of human relevance.
• Lack of Direct Cytotoxicity of Extracellular ATP against Hepatocytes: Role in the Mechanism of Acetaminophen Hepatotoxicity.Xie Y, Woolbright BL, Kos M, McGill MR, Dorko K, Kumer SC, Schmitt TM, Jaeschke H
  Journal of Clinical and Translational Research, Sep 2015
  Abstract : Background Acetaminophen (APAP) hepatotoxicity is a major cause of acute liver failure in many countries. Mechanistic studies in mice and humans have implicated formation of a reactive metabolite, mitochondrial dysfunction and oxidant stress as critical events in the pathophysiology of APAP-induced liver cell death. It was recently suggested that ATP released from necrotic cells can directly cause cell death in mouse hepatocytes and in a hepatoma cell line (HepG2). Aim To assess if ATP can directly cause cell toxicity in hepatocytes and evaluate their relevance in the human system. Methods Primary mouse hepatocytes, human HepG2 cells, the metabolically competent human HepaRG cell line and freshly isolated primary human hepatocytes were exposed to 10-100 μM ATP or ATγP in the presence or absence of 5-10 mM APAP for 9-24 h. Results ATP or ATγP was unable to directly cause cell toxicity in all 4 types of hepatocytes. In addition, ATP did not enhance APAP-induced cell death observed in primary mouse or human hepatocytes, or in HepaRG cells as measured by LDH release and by propidium iodide staining in primary mouse hepatocytes. Furthermore, addition of ATP did not cause mitochondrial dysfunction or enhance APAP-induced mitochondrial dysfunction in primary murine hepatocytes, although ATP did cause cell death in murine RAW macrophages. Conclusions It is unlikely that ATP released from necrotic cells can significantly affect cell death in human or mouse liver during APAP hepatotoxicity. Relevance for Patients Understanding the mechanisms of APAP-induced cell injury is critical for identifying novel therapeutic targets to prevent liver injury and acute liver failure in APAP overdose patients.
• In silico modeling for the prediction of dose and pathway related adverse effects in humans from in vitro repeated-dose studies.Klein S, Maggioni S, Bucher J, Mueller D, Niklas J, Shevchenko V, Mauch K, Heinzle E, Noor F
  Toxicological Sciences, Sep 2015
  Abstract : Long-term repeated-dose toxicity is mainly assessed in animals despite poor concordance of animal data with human toxicity. Advanced human in vitro systems e.g. with metabolically competent HepaRG cells, are used for toxicity screening. Extrapolation of in vitro toxicity to in vivo effects (IVIVE) is possible by reverse dosimetry using pharmacokinetic (PK) modeling. We assessed long-term repeated-dose toxicity of bosentan and valproic acid (VPA) in HepaRG cells under serum-free conditions. Upon 28 day exposure, the EC50 values for bosentan and VPA decreased by 21 and 33 fold respectively. Using EC10 as lowest threshold of toxicity in vitro, we estimated the oral equivalent doses for both test compounds using a simplified PK model for IVIVE. The model predicts that bosentan is safe at the considered dose under the assumed conditions upon 4 weeks exposure. For VPA, hepatotoxicity is predicted for 4 and 47% of the virtual population at the maximum recommended daily dose after 3 and 4 weeks of exposure respectively. We also investigated the changes in the central carbon metabolism of HepaRG cells exposed to orally bioavailable concentrations of both drugs. These concentrations are below the 28 day EC10 and induce significant changes especially in glucose metabolism and urea production. These metabolic changes may have a pronounced impact in susceptible patients such as those with compromised liver function and urea cycle deficiency leading to idiosyncratic toxicity. The combination of modeling based on in vitro repeated-dose data and metabolic changes allows the prediction of human relevant in vivo toxicity with mechanistic insights.
• A microfluidically perfused three dimensional human liver modelRennert K, Steinborn S, Groger M, Ungerbock B, Jank AM, Ehgartner J, Nietzsche S, Dinger J, Kiehntopf M, Funke H, Peters F, Lupp A, Gartner C
  Biomaterials, Sep 2015
  Abstract : Within the liver, non-parenchymal cells (NPCs) are critically involved in the regulation of hepatocyte polarization and maintenance of metabolic function. We here report the establishment of a liver organoid that integrates NPCs in a vascular layer composed of endothelial cells and tissue macrophages and a hepatic layer comprising stellate cells co-cultured with hepatocytes. The three-dimensional liver organoid is embedded in a microfluidically perfused biochip that enables sufficient nutrition supply and resembles morphological aspects of the human liver sinusoid. It utilizes a suspended membrane as a cell substrate mimicking the space of Disse. Luminescence-based sensor spots were integrated into the chip to allow online measurement of cellular oxygen consumption. Application of microfluidic flow induces defined expression of ZO-1, transferrin, ASGPR-1 along with an increased expression of MRP-2 transporter protein within the liver organoids. Moreover, perfusion was accompanied by an increased hepatobiliary secretion of 5(6)-carboxy-2',7'-dichlorofluorescein and an enhanced formation of hepatocyte microvilli. From this we conclude that the perfused liver organoid shares relevant morphological and functional characteristics with the human liver and represents a new in vitro research tool to study human hepatocellular physiology at the cellular level under conditions close to the physiological situation.
• Hepatotoxicity of piperazine designer drugs: Comparison of different in vitro modelsDias-da-Silva D, Arbo MD, Valente MJ, Bastos ML, Carmo H
  Toxicol in vitro, Aug 2015
  Abstract : Piperazine derived drugs emerged on the drug market in the last decade. The aim of this study was to investigate in vitro the potential hepatotoxicityof the designer drugs N-benzylpiperazine (BZP), 1-(3-trifluoromethylphenyl)piperazine (TFMPP), 1-(4-methoxyphenyl)piperazine (MeOPP) and 1-(3,4-methylenedioxybenzyl)piperazine (MDBP) in two human hepatic cell lines (HepaRG and HepG2) and in primary rat hepatocytes. Cell death was evaluated by the MTT assay, after 24 h-incubations. Among the tested drugs, TFMPP was the most cytotoxic. HepaRG cells and primary hepatocytes revealed to be the most and the least resistant cellular models, respectively. To ascertain whether the CYP450 metabolism could explain their higher susceptibility, primary hepatocytes were co-incubated with the piperazines and the CYP450 inhibitors metyrapone and quinidine, showing that CYP450-mediated metabolism contributes to the detoxification of these drugs. Additionally, the intracellular contents of reactive species, ATP, reduced (GSH) and oxidized (GSSG) glutathione, changes in mitochondrial membrane potential (Δψm) and caspase-3 activation were further evaluated in primary cells. Overall, an increase in reactive species formation, followed by intracellular GSH and ATP depletion, loss of Δψm and caspase-3 activation was observed for all piperazines, in a concentration-dependent manner. In conclusion, piperazine designer drugs produce hepatic detrimental effects that can vary in magnitude among the different analogues.
• Evaluation of genotoxicity using automated detection of ?H2AX in metabolically competent HepaRG cellsQuesnot N, Rondel K, Audebert M, Martinais S, Glaise D, Morel F, Loyer P, Robin MA,
  Mutagensis, Aug 2015
  Abstract : The in situ detection of γH2AX was recently reported to be a promising biomarker of genotoxicity. In addition, the human HepaRG hepatoma cells appear to be relevant for investigating hepatic genotoxicity since they express most of drug metabolizing enzymes and a wild type p53. The aim of this study was to determine whether the automated in situ detection of γH2AX positive HepaRG cells could be relevant for evaluation of genotoxicity after single or long-term repeated in vitro exposure compared to micronucleus assay. Metabolically competent HepaRG cells were treated daily with environmental contaminants and genotoxicity was evaluated after 1, 7 and 14 days. Using these cells, we confirmed the genotoxicity of aflatoxin B1 and benzo(a)pyrene and demonstrated that dimethylbenzanthracene, fipronil and endosulfan previously found genotoxic with comet or micronucleus assays also induced γH2AX phosphorylation. Furthermore, we showed that fluoranthene and bisphenol A induced γH2AX while no effect had been previously reported in HepG2 cells. In addition, induction of γH2AX was observed with some compounds only after 7 days, highlighting the importance of studying long-term effects of low doses of contaminants. Together, our data demonstrate that automated γH2AX detection in metabolically competent HepaRG cells is a suitable high-through put genotoxicity screening assay.
• Bile Acid-Induced Toxicity in HepaRG Cells Recapitulates the Response in Primary Human HepatocytesWoolbright B, MacGil MR, Yan H, Jaeschke H, , , , , , , , ,
  Basic Clin Pharmacol Toxicol., Jul 2015
  Abstract : Cholestatic liver injury is a pathological component of numerous disease states. Much of the current literature on cholestatic liver injury is derived from in vitro studies using rodent hepatocytes or cell lines transfected with bile acid (BA) uptake transporters. While these studies demonstrate BA-driven apoptosis, it is debatable whether these models reflect the human pathophysiology, as primary human hepatocytes undergo primarily necrosis. HepaRGcells are a bipotential, human hepatoma line that express apical and basolateral BA transporters. Thus, we sought to determine whether HepaRG cells could replicate the response of primary human hepatocytes to BA exposure in vitro. HepG2 cells, primary murine hepatocytes (PMH) or HepaRG cells, were exposed to taurocholic acid (TCA), or glycochenodeoxycholate (GCDC) and lactate dehydrogenase release were measured to determine cell death. Cell death occurred dose-responsively in HepaRG cells when exposed to GCDC; however, HepG2 cells died acutely only at very high concentrations of GCDC. In HepaRG cells, pre-treatment with the caspase inhibitor z-VD-FMK had no effect on cell death, indicating a lack of apoptotic cell death, and while c-jun N-terminal kinase (JNK) protein was activated by GCDC treatment in HepaRG cells, the inhibition of JNK did not protect. Although previous data indicate that TCA stimulates pro-inflammatory gene induction in PMH, there was no change in gene expression after TCA stimulation in HepaRG cells, which mimicked previous data found in primary human hepatocytes. These data provide evidence for HepaRG cells as a new model for the study of the effect of BA on human hepatocytes.
• Kinetics and dynamics of cyclosporine A in three hepatic cell culture systemsBellwon P, Truisi GL, Bois FY, Wilmes A, Schmitt TM, Savary CC, Parmentier C, Hewitt PG, Schmal O, Josse R, Richert L, Guillouzo A, Mueller SO
  Toxical in vitro, Jul 2015
  Abstract : In vitro experiments have a high potential to improve current chemical safety assessment and reduce the number of animals used. However, most studies conduct hazard assessment alone, largely ignoring exposure and kinetic parameters. Therefore, in this study the kinetics of cyclosporine A (CsA) and the dynamics of CsA-induced cyclophilin B (Cyp-B) secretion were investigated in three widely used hepatic in vitro models: primary rat hepatocytes (PRH), primary human hepatocytes (PHH) and HepaRG cells. Cells were exposed daily to CsA for up to 14days. CsA in cells and culture media was quantified by LC-MS/MS and used for pharmacokinetic modeling. Cyp-B was quantified by western blot analysis in cells and media. All cell systems took up CsA rapidly from the medium after initial exposure and all showed a time- and concentration-dependent Cyp-B cellular depletion and extracellular secretion. Only in PRH an accumulation of CsA over 14days repeated exposure was observed. Donor-specific effects in CsA clearance were observed in the PHH model and both PHH and HepaRG cells significantly metabolized CsA, with no bioaccumulation being observed after repeated exposure. The developed kinetic models are described in detail and show that all models under-predict the in vivo hepatic clearance of CsA, but to different extents with 27-, 24- and 2-fold for PRH, PHH and HepaRG cells, respectively. This study highlights the need for more attention to kinetics in in vitro studies.
• Expression and Functionality of Toll- and RIG-like receptors in HepaRG Cells.Luangsay S, Ait-Goughoulte M, Michelet M, Floriot O, Bonnin M, Gruffaz M, Rivoire M, Fletcher S, Javanbakht H, Lucifora J, Zoulim F, Durantel D
  J Hepatol., Jul 2015
  Abstract : BACKGROUND & AIMS: HepaRG cells are considered as the best surrogate model to primary human hepatocyte (PHH) culture to investigate host-pathogen interactions. Yet their innate immune functions remain unknown. In this study, we studied the expression and functionality of Toll-like (TLR) and retinoic-acid-inducible gene-1 (RIG-I)-like (RLR) receptors in these cells.METHODS: Gene and protein expression levels of TLR-1 to 9 and RLR in HepaRG were mainly compared to PHH, by RT-qPCR, FACS, and Western Blotting. Their functionality was assessed, by measuring the induction of toll/rig-like themselves and several target innate gene expressions, as well as the secretion of IL-6, IP-10, and type-I interferon (IFN), upon agonist stimulation. Their functionality was also evidenced, by measuring the antiviral activity of some TLR/RLR agonists against hepatitis B virus (HBV) infection.RESULTS: The basal gene and protein expression profile of TLR/RLR in HepaRG cells was similar to PHH. Most of receptors, except for TLR-7 and 9, were expressed as proteins and functionally active as evidenced by the induction of some innate genes, as well as by secretion of IL-6 and IP-10, upon agonist stimulation. The highest levels of IL-6 and IP-10 secretion were obtained by TLR-2 and TLR-3 agonisation respectively. The highest preventive anti-HBV activity was obtained following TLR-2, TLR-4 or RIG-I/MDA-5 stimulations, which correlated with their high capacity to produce both cytokines.CONCLUSIONS: Our results indicate that HepaRG cells express a similar pattern of functional TLR/RLR as compared to PHH, thus qualifying HepaRG cells as a surrogate model to study pathogen interactions with hepatocyte innate system.
• HBx relieves chromatin-mediated transcriptional repression of hepatitis B viral cccDNA involving SETDB1 histone methyltransferase.Rivière L, Gerossier L, Ducroux A, Dion S, Deng Q, Michel ML, Buendia MA, Hantz O, Neuveut C
  J Hepatol., Jul 2015
  Abstract : BACKGROUNDS AND AIMS: Maintenance of the covalently closed circular HBV DNA (cccDNA) that serves as a template for HBV transcription is responsible for the failure of antiviral therapies. While studies in chronic hepatitis patients have shown that high viremia correlates with hyperacetylation of cccDNA-associated histones, the molecular mechanisms controlling cccDNA stability and transcriptional regulation are still poorly understood. This study aimed to decipher the role of chromatin and chromatin modifier proteins on HBV transcription.METHODS: We analyzed the chromatin structure of actively transcribed or silenced cccDNA by infecting primary human hepatocytes and differenciated hepaRG cells with wild type virus or virus deficient (HBV X-) for the expression of Hepatitis B Virus X protein (HBx), that is required for HBV expression.RESULTS: In the absence of HBx, HBV cccDNA was transcriptionally silenced with the concomitant decrease of histone 3 (H3) acetylation and H3K4me3, increase of H3 di- and tri-methylation (H3K9me) and the recruitment of heterochromatin protein 1 factors (HP1) that correlate with condensed chromatin. SETDB1 was found to be the main histone methyltransferase responsible for the deposition of H3K9me3 and HBV repression. Finally, full transcriptional reactivation of HBVX- upon HBx re-expression correlated with an increase of histone acetylation and H3K4me3, and a concomitant decrease of HP1 binding and of H3K9me3 on the cccDNA.CONCLUSION: Upon HBV infection, cellular mechanisms involving SETDB1-mediated H3K9me3 and HP1 induce silencing of HBV cccDNA transcription through modulation of chromatin structure. HBx is able to relieve this repression and allow the establishment of active chromatin
• Applications of human hepatitis B virus preS domain in bio- and nanotechnology.Toita R, Kawano T, Kang JH, Murata M
  World J Gastroenterol., Jul 2015
  Abstract : Human hepatitis B virus (HBV) is a member of the family Hepadnaviridae, and causes acute and chronic infections of the liver. The hepatitis B surface antigen (HBsAg) contains the large (L), middle (M), and small (S) surface proteins. The L protein consists of the S protein, preS1, and preS2. In HBsAg, the preS domain (preS1 + preS2) plays a key role in the infection of hepatocytic cells by HBV and has several immunogenic epitopes. Based on these characteristics of preS, several preS-based diagnostic and therapeutic materials and systems have been developed. PreS1-specific monoclonal antibodies (e.g., MA18/7 and KR127) can be used to inhibit HBV infection. A myristoylated preS1 peptide (amino acids 2-48) also inhibits the attachment of HBV to HepaRG cells, primary human hepatocytes, and primary tupaia hepatocytes. Antibodies and antigens related to the components of HBsAg, preS (preS1 + preS2), or preS1 can be available as diagnostic markers of acute and chronic HBV infections. Hepatocyte-targeting delivery systems for therapeutic molecules (drugs, genes, or proteins) are very important for increasing the clinical efficacy of these molecules and in reducing their adverse effects on other organs. The selective delivery of diagnostic molecules to target hepatocytic cells can also improve the efficiency of diagnosis. In addition to the full-length HBV vector, preS (preS1 + preS2), preS1, and preS1-derived fragments can be useful in hepatocyte-specific targeting. In this review, we discuss the literature concerning the applications of the HBV preS domain in bio- and nanotechnology.
• Hepatic TLR4 Signaling in Obese NAFLDSharifnia T, Antoun J, Verriere TG, Suarez G, Wattacheril J, Wilson KT, Peek RM Jr, Abumrad NN, Flynn CR
  Am J Physiol Gastrointest Liver Physiol., Jun 2015
  Abstract : Nonalcoholic fatty liver disease (NAFLD) occurs frequently in the setting of metabolic syndrome, but the factors leading to nonalcoholic steatohepatitis (NASH) are not fully understood. This study investigated toll-like receptor 4 (TLR4) signaling in human liver with the goal of delineating whether activation of this pathway segregates those with nonalcoholic fatty liver (NAFL) from those with NASH. Experiments were performed using liver biopsy tissue obtained from Class III obese subjects undergoing bariatric surgery, and extended to an immortalized human hepatocyte HepaRG cell line and primary human hepatocytes. The bacterial endotoxin lipopolysaccharide (LPS) and total free fatty acid levels were significantly increased in plasma of NASH patients. TLR4 mRNA levels were significantly increased in subjects with NASH compared to NAFL as was IRF3 in the myeloid differentiation factor 88- (Myd88-) independent signaling pathway. In HepaRG cells, NF-?B nuclear translocation and functional activity increased following treatment with the fatty acid, palmitate, and following exposure to LPS, when compared to hepatocytes stimulated with a lipogenic treatment that induced de novo lipogenesis. Palmitate- and LPS-induction of NF-?B activity was partially attenuated by chemical- or siRNA-mediated inhibition of TLR4. Expression of TLR4 and its downstream mediators was up-regulated with palmitate and LPS. Similar results were observed using primary human hepatocytes from a lean donor. Interestingly, NF-?B activity assays showed obese donor hepatocytes were resistant to chemical TLR4 inhibition.CONCLUSION:TLR4 expression is up-regulated in a large cohort of NASH patients, when compared to those with NAFL, and this occurs within the setting of increased LPS and fatty acids.
• A four-organ-chip for interconnected long-term co-culture of human intestine, liver, skin and kidney equivalentsMaschmeyer I, Lorenz A, Schimek K, Hasenberg T, Ramme A, Hübner J, Lindner M, Drewell C, Bauer S, Thomas A, Sisoli Sambo N, Sonntag F, Lauster R, Marx U
  Royal Society of Chemical, May 2015
  Abstract : Systemic absorption and metabolism of drugs in the small intestine, metabolism by the liver as well as excretion by the kidney are key determinants of efficacy and safety for therapeutic candidates. However, these systemic responses of applied substances lack in most in vitro assays. In this study, a microphysiological system maintaining the functionality of four organs over 28 days in co-culture has been established at a minute but standardized microsystem scale. Preformed human intestine and skin models have been integrated into the four-organ-chip on standard cell culture inserts at a size 100,000-fold smaller than their human counterpart organs. A 3D-based spheroid, equivalent to ten liver lobules, mimics liver function. Finally, a barrier segregating the media flow through the organs from fluids excreted by the kidney has been generated by a polymeric membrane covered by a monolayer of human proximal tubule epithelial cells. A peristaltic onchip micropump ensures pulsatile media flow interconnecting the four tissue culture compartments through microfluidic channels. A second microfluidic circuit ensures drainage of the fluid excreted through the kidney epithelial cell layer. This four-organ-chip system assures near to physiological fluid-to-tissue ratios. In-depth metabolic and gene analysis revealed the establishment of reproducible homeostasis among the co-cultures within two to four days, sustainable over at least 28 days independent of the individual human cell line or tissue donor background used for each organ equivalent. Lastly, 3D imaging two-photon microscopy visualised details of spatiotemporal segregation of the two microfluidic flows by proximal tubule epithelia. To our knowledge, this study is the first approach to establish a system for in vitro microfluidic ADME profiling and repeated dose systemic toxicity testing of drug candidates over 28 days.
• The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture.Materne EM, Maschmeyer I, Lorenz AK, Horland R, Schimek KM, Busek M, Sonntag F, Lauster R, Marx U
  J Vis Exp., May 2015
  Abstract : The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments, as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier, more quantifiable extracellular relay of signals to the cells. For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.
• Viral Entry of Hepatitis B and D Viruses and Bile Salts Transportation Share Common Molecular Determinants on Sodium Taurocholate Cotransporting PolypeptideYan X, Wang J, Zhu L, Lowrey JJ, Zhang Y, Hou W, Dong J, Du Y
  Lab Chip., May 2015
  Abstract : Hydrogel as three-dimensional (3D) substrate has been employed in miniaturized high throughput protein detection platforms to increase the number of effective antibodies and signal augmentation. However, the high water content of the hydrogel can dilute samples and create barrier to mass transfer, limiting hydrogel height to several microns in most platforms. Moreover, these platforms cannot achieve widespread use in common laboratories as they usually rely heavily on expensive robotic liquid handlers and custom-built components. Here we developed a ready-to-use, easy to store and handle, versatile and multiplex-able 3D scaffold-based immunoassay chip (3D immunoChip) possible for high throughput protein quantification using bench-top equipment in common laboratories. Sample dilution, mass transfer, signal scattering and storage problems can be avoided by using dry scaffolds that regain transparency upon rehydration. When combined with hydrophilic-hydrophobic patterned reagent loading slides, manual high throughput handling of samples can be achieved. As these micro-scaffolds are patterned without barriers in between, simultaneous and effortless washing of all the reaction zones is possible in a Petri dish. Such features aid the 3D immunoChip in saving up to 100 times reagent and about 6 times labour. The 3D immunoChip is able to detect albumin (ALB), as a model analyte, from 5 ng mL-1 to 1000 ng mL-1, making it comparable to the commercialized ELISA kit based on a 96-well plate (0.22-400 ng mL-1). This thus enables the 3D immunoChip to directly detect ALB secreted by HepaRG cells cultured in a 3D cell culture array chip for high throughput drug hepatotoxicity evaluation, which could potentially accelerate drug screening.
• Drug biokinetic and toxicity assessments in rat and human primary hepatocytes and HepaRG cells within the EU-funded Predict-IV project.Mueller SO, Guillouzo A, Hewitt PG, Richert L
  Toxicol In Vitro, May 2015
  Abstract : The overall aim of Predict-IV (EU-funded collaborative project #202222) was to develop improved testing strategies for drug safety in the late discovery phase. One major focus was the prediction of hepatotoxicity as liver remains one of the major organ leading to failure in drug development, drug withdrawal and has a poor predictivity from animal experiments. In this overview we describe the use and applicability of the three cell models employed, i.e., primary rat hepatocytes, primary human hepatocytes and the human HepaRG cell line, using four model compounds, chlorpromazine, ibuprofen, cyclosporine A and amiodarone. This overview described the data generated on mode of action of liver toxicity after long-term repeat-dosing. Moreover we have quantified parent compound and its distribution in various in vitro compartments, which allowed us to develop biokinetic models where we could derive real exposure concentrations in vitro. In conclusion, the complex data set enables quantitative measurements that proved the concept that we can define human relevant free and toxic exposure levels in vitro. Further compounds have to be analyzed in a broader concentration range to fully exploit these promising results for improved prediction of hepatotoxicity and hazard assessment for humans.
• Three-dimensional (3D) spheroid cultures improve the metabolic gene expression profiles of HepaRG cells.Takahashi Y, Hori Y, Yamamoto T, Urashima T, Ohara Y, Tanaka H
  Biosci Rep., May 2015
  Abstract : Three-dimensional (3D) cultures are considered to be an effective method for toxicological studies; however, little evidence has been reported whether 3D cultures have an impact on hepatocellular physiology regarding lipid or glucose metabolism. Here, we conducted physiological characterization of hepatoma cell lines HepG2 and HepaRG cells cultured in 3D conditions using a hanging drop method to verify the effect of culture environment on cellular responses. ApoB as well as albumin secretion was augmented by 3D cultures. Expression of genes related to not only drug, but also glucose and lipid metabolism were significantly enhanced in 3D cultured HepaRG spheroids. Furthermore, mRNA levels of CYP enzymes following exposure to corresponding inducers increased under the 3D condition. These data suggest that this simple 3D culture system without any special biomaterials can improve liver-specific characteristics including lipid metabolism. Considering that the system enableshigh-throughput assay, it may become a powerful tool for compound screening concerning hepatocellular responses in order to identify potential drugs.
• Multiparametric assay using HepaRG cells for predicting drug-induced liver injury.Tomida T, Okamura H, Satsukawa M, Yokoi T, Konno Y
  Toxicol Lett., May 2015
  Abstract : The utility of HepaRG cells as an in vitro cell-based assay system for assessing drug-induced liver injury (DILI) risk was investigated. Seventeen DILI-positive and 15 DILI-negative drugs were selected for the assay. HepaRG cells were treated with each drug for 24h at concentrations that were 1.6-, 6.3-, 25-, and 100-fold the therapeutic maximum plasma concentration (Cmax). After treatment, the cell viability, glutathione content, caspase 3/7 activity, lipid accumulation, leakage of lactate dehydrogenase, and albumin secretion were measured. The sensitivity and specificity were calculated to assess the ability of the assay to predict DILI. Our multiparametric assay using HepaRG cells exhibited a 67% sensitivity and 73% specificity at a 100-fold concentration of Cmax and a 41% sensitivity and 87% specificity at a 25-fold concentration of Cmax. When a 25-fold Cmax cut-off was applied, approximately 70% of drugs exhibiting positive responses were classified into the high DILI risk category. HepaRG cells distinguished relatively safe drugs from their high-risk analogs. Our study indicates that HepaRG cells may be of use to (1) prioritize drug analogs, (2) analyze the mechanism of DILI, and (3) assess the risk for DILI in the early drug discovery stage.
• Allopurinol and 5-aminosalicylic acid influence thiopurine-induced hepatotoxicity in vitro.Broekman MM, Roelofs HM, Wong DR, Kerstholt M, Leijten A, Hoentjen F, Peters WH, Wanten GJ, de Jong DJ
  Cell Biol Toxicol., May 2015
  Abstract : INTRODUCTION: The use of thiopurines is frequently accompanied by hepatotoxicity. Studies on hepatocyte cultures showed a time- and dose-dependent increase of thiopurine toxicity. 5-Aminosalicylic acid (5-ASA) and allopurinol can influence thiopurine metabolism; however, it is unknown whether this affects in vitro cytotoxicity.METHODS: Human hepatoma cells (Huh7, HepG2 and HepaRG) were incubated with increasing concentrations of thiopurines, 5-ASA or allopurinol. Water-soluble tetrazolium salt-1 (WST-1) cytotoxicity assays were used to calculate cell survival curves and half maximal inhibitory concentrations (IC50). Combination experiments with thiopurines with a fixed dose of 200 μM 5-ASA or 100 μM allopurinol were conducted in HepaRG cells. Caspase-3/7 activation was evaluated, and single cell electrophoresis analysis was performed.RESULTS: A time- and dose-related cytotoxic effect was seen with azathioprine (AZA) in all hepatoma cells, whereas Huh7 and HepG2 cells did not show toxicity to 6-mercaptopurine (6-MP). HepaRG cells expressed the highest levels of drug metabolising enzymes, and therefore, combination experiments were conducted in HepaRG cells. Addition of a non-toxic dose of allopurinol resulted in a twofold to threefold increased cytotoxicity of all thiopurines, which seemed to be mediated by apoptosis/DNA damage.CONCLUSION: The addition of allopurinol to thiopurines leads to a two-threefold increased cytotoxicity in HepaRG cells.
• Time course of acetaminophen-protein adducts and acetaminophen metabolites in circulation of overdose patients and in HepaRG cells.Xie Y, McGill MR, Cook SF, Sharpe MR, Winefield RD, Wilkins DG, Rollins DE, Jaeschke H
  Xenobiotica, Apr 2015
  Abstract : It has been suggested that acetaminophen (APAP)-protein adducts can be measured in circulation to diagnose APAP-induced liver injury. However, the full-time course of plasma adducts has not been studied specifically in early-presenting overdose patients. In fact, surprisingly little work has been done on the metabolism of APAP after overdose in general. 2. We measured APAP, five APAP metabolites and APAP-protein adducts in plasma samples from early- and late-presenting overdose patients, and APAP-protein adducts in culture medium from HepaRG cells. 3. In contrast to earlier rodents studies, we found that APAP-protein adducts were lower at early time points and peaked around the time of peak liver injury, suggesting that these adduct levels may take longer to become elevated or remain elevated than previously thought. 4. APAP and its major metabolites were elevated in plasma at early time points and rapidly decreased. 5. Although clinical measurement of APAP-protein adducts holds promise as a diagnostic tool, we suggest caution in its interpretation in very early-presenting patients. Our data also support the idea that sulfation is saturated even at low doses but glucuronidation has a much higher capacity, highlighting the importance of glucuronidation in APAP metabolism.
• Hepatotoxicity of piperazine designer drugs: comparison of different in vitro models.Dias-da-Silva D, Arbo MD, Valente MJ, Bastos ML, Carmo H
  Toxicol. In Vitro, Apr 2015
  Abstract : Piperazine derived drugs emerged on the drug market in the last decade. The aim of this study was to investigate in vitro the potential hepatotoxicity of the designer drugs N-benzylpiperazine (BZP), 1-(3-trifluoromethylphenyl)piperazine (TFMPP), 1-(4-methoxyphenyl)piperazine (MeOPP) and 1-(3,4-methylenedioxybenzyl)piperazine (MDBP) in two human hepatic cell lines (HepaRG and HepG2) and in primary rat hepatocytes. Cell death was evaluated by the MTT assay, after 24h-incubations. Among the tested drugs, TFMPP was the most cytotoxic. HepaRG cells and primary hepatocytes revealed to be the most and the least resistant cellular models, respectively. To ascertain whether the CYP450 metabolism could explain their higher susceptibility, primary hepatocytes were co-incubated with the piperazines and the CYP450 inhibitors metyrapone and quinidine, showing that CYP450-mediated metabolism contributes to the detoxification of these drugs. Additionally, the intracellular contents of reactive species, ATP, reduced (GSH) and oxidized (GSSG) glutathione, changes in mitochondrial membrane potential (Δψm) and caspase-3 activation were further evaluated in primary cells. Overall, an increase in reactive species formation, followed by intracellular GSH and ATP depletion, loss of Δψm and caspase-3 activation was observed for all piperazines, in a concentration-dependent manner. In conclusion, piperazine designer drugs produce hepatic detrimental effects that can vary in magnitude among the different analogues.
• The aryl hydrocarbon receptor-dependent disruption of contact inhibition in rat liver WB-F344 epithelial cells is linked with induction of survivin, but not with inhibition of apoptosis.Svobodová J, Kabátková M, ?merdová L, Brenerová P, Dvo?ák Z, Machala M, Vondrá?ek J
  Toxicology, Apr 2015
  Abstract : Inhibition of apoptosis by the ligands of the aryl hydrocarbon receptor (AhR) has been proposed to play a role in their tumor promoting effects on liver parenchymal cells. However, little is presently known about the impact of toxic AhR ligands, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on apoptosis in other liver cell types, such as in liver epithelial/progenitor cells. In the present study, we focused on the effects of TCDD on apoptosis regulation in a model of liver progenitor cells, rat WB-F344 cell line, during the TCDD-elicited release from contact inhibition. The stimulation of cell proliferation in this cell line was associated with deregulated expression of a number of genes known to be under transcriptional control of the Hippo signaling pathway, a principal regulatory pathway involved in contact inhibition of cell proliferation. Interestingly, we found that mRNA and protein levels of survivin, a known Hippo target, which plays a role both in cell division and inhibition of apoptosis, were significantly up-regulated in rat liver epithelial cell model, as well as in undifferentiated human liver HepaRG cells. Using the short interfering RNA-mediated knockdown, we confirmed that survivin plays a central role in cell division of WB-F344 cells. When evaluating the effects of TCDD on apoptosis induction by camptothecin, a genotoxic topoisomerase I inhibitor, we observed that the pre-treatment of WB-F344 cells with TCDD increased number of cells with apoptotic nuclear morphology, and it potentiated cleavage of both caspase-3 and poly(ADP-ribose) polymerase I. This indicated that despite the observed up-regulation of survivin, apoptosis induced by the genotoxin was potentiated in the model of rat liver progenitor cells. The present results indicate that, unlike in hepatocytes, AhR agonists may not prevent induction of apoptosis elicited by DNA-damaging agents in a model of rat liver progenitor cells.
• BK/TD models for analyzing in vitro impedance data on cytotoxicity.Teng S, Barcellini-Couget S, Beaudouin R, Brochot C, Desousa G, Rahmani R, Pery A
  Toxicol Lett., Mar 2015
  Abstract : The ban of animal testing has enhanced the development of new in vitro technologies for cosmetics safety assessment. Impedance metrics is one such technology which enables monitoring of cell viability in real time. However, analyzing real time data requires moving from static to dynamic toxicity assessment. In the present study, we built mechanistic biokinetic/toxicodynamic (BK/TD) models to analyze the time course of cell viability in cytotoxicity assay using impedance. These models account for the fate of the tested compounds during the assay. BK/TD models were applied to analyze HepaRG cell viability, after single (48h) and repeated (4 weeks) exposures to three hepatotoxic compounds (coumarin, isoeugenol and benzophenone-2). The BK/TD models properly fit the data used for their calibration that was obtained for single or repeated exposure. Only for one out of the three compounds, the models calibrated with a single exposure were able to predict repeated exposure data. We therefore recommend the use of long-term exposure in vitro data in order to adequately account for chronic hepatotoxic effects. The models we propose here are capable of being coupled with human biokinetic models in order to relate dose exposure and human hepatotoxicity.
• Forced expression of PDX-1 gene makes hepatoma cells to acquire glucose-responsive insulin secretion while maintaining hepatic characteristic.Hashimoto H, Higuchi Y, Kawai K
  Cell Mol Biol, Mar 2015
  Abstract : Evidence shows that forced expression of the PDX1 gene converts hepatoma cells, mouse liver epithelial cells (MLECs) and HepaRG cells, into insulin—;producing cells, β —cells, or islets of Langerhans. However, no reports have investigated the characteristics of mouse or human hepatocytes introduced with the PDX1 gene over prolonged observation periods. In this study, we immunohistologically and molecularly investigated the alternative processes induced by PDX1 gene introduction in mouse and human hepatocytes over prolonged observation periods using immunocytochemistry, immunofluorescence, polymerase chain reaction (PCR), Western blotting, and flow cytometry (FCM) analysis. Immunocytochemical and immunofluorescent observations showed that MLECs and HepaRG cells on 2 and 21 days after introduction of the PDX1 gene comprised cells double—positive for insulin and albumin. Additionally, they showed MAFA expression and glucose—responsive insulin secretion with glucokinase expression. However mouse embryonic fibroblasts introduced with PDX1—GFP could not acquire glucose—responsive insulin secretion and glucokinase expression. Subsequently, we hypothesized that the number of albumin—positive MLECs and HepaRG cells would decrease after introduction of PDX1 due to the conversion of MLECs and HepaRG cells into insulin—producing cells. However, FCM analysis indicated that the number of albumin—positive MLECs and HepaRG cells was not altered by the introduction of PDX1. We thought that MLECs and HepaRG cells introduced with the PDX1 gene could acquire a functional insulin secretory capacity without conversion to β—cells, or islets of Langerhans, and the acquisition could need glucokinase expression.
• Inflammation-Associated microRNA-130b Downregulates Cytochrome P450 Activities and Directly Targets CYP2C9.Rieger JK, Reutter S, Hofmann U, Schwab M, Zanger UM
  Drug Metabolism Dispos., Mar 2015
  Abstract : Expression of genes involved in absorption, distribution, metabolism and excretion (ADME) of drugs is collectively impaired during pathophysiological conditions such as cholestasis and inflammation. The mechanism of coordinated ADME gene downregulation remains unclear. In our previous study strongly elevated levels of microRNAs (miRNA) miR-21, miR-34a, and miR-130b in cholestatic liver and of miR-21 and miR-130b during inflammation were observed. Using HepaRG cells, which retain many functional characteristics of human hepatocytes, we investigated the potential of these miRNAs to downregulate ADME genes. Cells were transfected with the corresponding miRNA mimics, chemically modified double-stranded RNAs that mimic endogenous miRNAs, followed by mRNA profiling by quantitative RT-PCR. Enzyme activities of six cytochromes P450 (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP3A4) were determined with a liquid chromatography-tandem mass spectrometric cocktail assay. While miR-21 and miR-34a showed little effects, transfection of miR-130b lead to significantly lower expression of nuclear receptors CAR and FXRa, the CYPs 1A1, 1A2, 2A6, 2C8, 2C9, and 2C19, as well as GSTA2. Furthermore, miR-130b negatively affected activity levels of all measured P450s by more than 40%. Reporter gene assays employing the CYP2C9 3'UTR confirmed direct regulation by miR-130b. These data support miR-130b as a potential negative regulator of drug metabolism by directly and/or indirectly affecting the expression of several ADME genes. This may be of relevance in pathophysiological conditions such as cholestasis and inflammation, which are associated with increased miR-130b expression.The American Society for Pharmacology and Experimental Therapeutics.
• Prediction of Gestational Age-Dependent Induction of In Vivo Hepatic CYP3A Activity Based on HepaRG Cells and Human Hepatocytes.Zhang Z, Farooq M, Prasad B, Grepper S, Unadkat JD
  Drug Metabolism Dispos., Mar 2015
  Abstract : In pregnant women, CYP3A activity increases by 100% during the third trimester (T3). Due to logistical and ethical constraints, little is known about the magnitude of CYP3A induction during the first (T1) and second (T2) trimesters. Our laboratory has shown that sandwich-cultured human hepatocytes (SCHH) and HepaRG cells have the potential to predict the magnitude of in vivo induction of CYP3A activity likely to be observed in T1 and T2. Therefore, we incubated SCHH and HepaRG cells with plasma concentrations of various pregnancy-related hormones (PRHs, individually or in combination) observed during T1, T2, or T3 in pregnant women. Then, CYP3A activity was measured by 1'-OH-midazolam formation. In all three trimesters, only cortisol (C) consistently and significantly induced CYP3A activity, while other individual hormones (progesterone, estradiol or growth hormones) failed to induce CYP3A activity. At physiologically relevant 1X plasma concentrations, the magnitude of CYP3A induction by C or the combination of all PRHs did not change significantly with gestational age. The pattern of induction of CYP3A activity in SCHH by the hormones was similar to that in HepaRG cells. Based on these data, we conclude that C remains the major inducer of CYP3A activity earlier in gestation. Moreover, we predict that the magnitude of CYP3A induction during T1 and T2 will be similar to that observed during T3 (~100% increase vs. postpartum). This prediction is consistent with the observation of similar increase in T2 and T3 oral clearance of indinavir (a CYP3A cleared drug) vs. postpartum.The American Society for Pharmacology and Experimental Therapeutics.
• Progenitor cell expansion and impaired hepatocyte regeneration in explanted livers from alcoholic hepatitisDubuquoy L, Louvet A, Lassailly G, Truant S, Boleslawski E, Artru F, Maggiotto F, Gantier E, Buob D, Leteurtre E , Cannesson A, Dharancy S, Moreno C, Pruvot FR, Bataller R, Mathurin P
  Hepatology, Mar 2015
  Abstract : Objective: In alcoholic hepatitis (AH), development of targeted therapies is crucial and requires improved knowledge of cellular and molecular drivers in liver dysfunction. The unique opportunity of using explanted livers from patients with AH having undergone salvage liver transplantation allowed to perform more in-depth molecular translational studies.Design: We studied liver explants from patients with AH submitted to salvage transplantation (n=16), from patients with alcoholic cirrhosis without AH (n=12) and fragments of normal livers (n=16). Hepatic cytokine content was quantified. Hepatocyte function and proliferation and the presence of hepatic progenitor cells (HPCs) were evaluated by immunohistochemistry, western blot or quantitative PCR. Mitochondrial morphology was evaluated by electron microscopy.Results: Livers from patients with AH showed decreased cytokine levels involved in liver regeneration (tumour necrosis factor α and interleukin-6), as well as a virtual absence of markers of hepatocyte proliferation compared with alcoholic cirrhosis and normal livers. Electron microscopy revealed obvious mitochondrial abnormalities in AH hepatocytes. Importantly, livers from patients with AH showed substantial accumulation of HPCs that, unexpectedly, differentiate only into biliary cells. AH livers predominantly express laminin (extracellular matrix protein favouring cholangiocyte differentiation); consequently, HPC expansion is inefficient at yielding mature hepatocytes.Conclusions: AH not responding to medical therapy is associated with lack of expression of cytokines involved in liver regeneration and profound mitochondrial damage along with lack of proliferative hepatocytes. Expansion of HPCs is inefficient to yield mature hepatocytes. Manoeuvres aimed at promoting differentiation of HPCs into mature hepatocytes should be tested in AH.
• Interleukin 6 inhibits HBV entry through NTCP down regulation.Bouezzedine F, Fardel O, Gripon P
  Virology, Mar 2015
  Abstract : Hepatitis B virus (HBV) infection is a major public health problem. Recently, the human liver bile acid transporter Na+/taurocholate cotransporting polypeptide (NTCP) has been identified as an HBV specific receptor. NTCP expression is known to be strongly regulated by IL-6. This study was aimed at characterizing the effect of IL-6 on HBV entry. HBV entry was inhibited by up to 90% when cells were pretreated with IL-6 as shown by a strong inhibition of long term HBsAg secretion. This effect was confirmed by showing a severe reduction of intracellular HBV cccDNA. In parallel, we observed a 98% decrease in NTCP mRNA steady state level and an 80% reduction in NTCP-mediated taurocholate uptake. IL-6-mediated inhibition of NTCP-mediated taurocholate uptake and viral entry exhibited similar dose-dependence and kinetics while restoration of NTCP expression suppressed the inhibitory effect of IL-6. NTCP-mediated HBV entry is therefore markedly inhibited by IL-6.
• Characterizing the mechanism of thiazolidinedione-induced hepatotoxicity: an in vitro model in mitochondria.Hu D, Wu CQ, Li ZJ, Liu Y, Fan X, Wang QJ, Ding RG
  Toxicol Appl Pharmacol., Mar 2015
  Abstract : OBJECTIVE: To characterize the mechanism of action of thiazolidinedione- (TZD) induced liver mitochondrial toxicity caused by troglitazone, rosiglitazone, and pioglitazone in HepaRG cells.Methods: Human hepatoma cells (HepaRG) were treated with troglitazone, rosiglitazone, or pioglitazone (12.5, 25, and 50μM) for 48hours. The Seahorse Biosciences XF24 Flux Analyzer was used to measure mitochondrial oxygen consumption. The effect of TZDs on reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were detected by flow cytometry. The mitochondrial ultrastructure of HepaRG cells was observed under a transmission electrical microscope (TEM), mtDNA content was evaluated by real-time PCR, and ATP content and mitochondrial respiratory chain(MRC)complex I, II, III, IV activity were measured via chemiluminescence. Results were considered statistically significant at p<0.05. RESULTS: Among the three drugs, troglitazone exhibited the highest potency, followed by rosiglitazone, and then pioglitazone. The TZDs caused varying degrees of mitochondrial respiratory function disorders including decreases in oxygen consumption, MRC activity, and ATP level, and an elevation in ROS level. TZD treatment resulted in mtDNA content decline, reduction in MMP, and alterations of mitochondrial structure. CONCLUSION: All investigated TZDs show a certain degree of mitochondrial toxicity, with troglitazone exhibiting the highest potency. The underlying mechanism of TZD-induced hepatotoxicity may be associated with alterations in mitochondrial respiratory function disorders, oxidative stress, and changes in membrane permeability. These parameters may be used early in drug development to further optimize risk: benefit profiles.
• Integration of metabolic activation with a predictive toxicogenomics signature to classify genotoxic versus nongenotoxic chemicals in human TK6 cells.Buick JK, Moffat I, Williams A, Swartz CD, Recio L, Hyduke DR, Li HH, Fornace AJ Jr, Aubrecht J, Yauk CL
  Environ Mol Mutagen., Mar 2015
  Abstract : The use of integrated approaches in genetic toxicology, including the incorporation of gene expression data to determine the molecular pathways involved in the response, is becoming more common. In a companion article, a genomic biomarker was developed in human TK6 cells to classify chemicals as genotoxic or nongenotoxic. Because TK6 cells are not metabolically competent, we set out to broaden the utility of the biomarker for use with chemicals requiring metabolic activation. Specifically, chemical exposures were conducted in the presence of rat liver S9. The ability of the biomarker to classify genotoxic (benzo[a]pyrene, BaP; aflatoxin B1, AFB1) and nongenotoxic (dexamethasone, DEX; phenobarbital, PB) agents correctly was evaluated. Cells were exposed to increasing chemical concentrations for 4 hr and collected 0 hr, 4 hr, and 20 hr postexposure. Relative survival, apoptosis, and micronucleus frequency were measured at 24 hr. Transcriptome profiles were measured with Agilent microarrays. Statistical modeling and bioinformatics tools were applied to classify each chemical using the genomic biomarker. BaP and AFB1 were correctly classified as genotoxic at the mid- and high concentrations at all three time points, whereas DEX was correctly classified as nongenotoxic at all concentrations and time points. The high concentration of PB was misclassified at 24 hr, suggesting that cytotoxicity at later time points may cause misclassification. The data suggest that the use of S9 does not impair the ability of the biomarker to classify genotoxicity in TK6 cells. Finally, we demonstrate that the biomarker is also able to accurately classify genotoxicity using a publicly available dataset derived from human HepaRG cells. 
• Aberrant DNA methylation of imprinted loci in hepatocellular carcinoma and after in vitro exposure to common risk factors.Lambert MP, Ancey PB, Esposti DD, Cros MP, Sklias A, Scoazec JY, Durantel D, Hernandez-Vargas H, Herceg Z
  Clin Epigenetics, Feb 2015
  Abstract : BACKGROUND:Hepatocellular carcinoma (HCC) is among the most frequent human malignancies and a major cause of cancer-related death worldwide. It is characterized by late detection and fast progression, and it is believed that epigenetic disruption may be one of the molecular mechanisms leading to hepatocarcinogenesis. Previous studies from our group revealed that HCC tumors exhibit specific DNA methylation signatures associated with major risk factors and tumor progression. Imprinted genes are mono-allelically expressed in a parent-of-origin-dependent manner and have been suggested to be more susceptible to deregulation in cancer. To test this notion, we performed a targeted analysis of DNA methylation in known imprinted genes, using HCC samples and in vitro models of carcinogenic exposure.RESULTS:Analysis of HCC DNA methylation in two independent datasets showed that differentially methylated loci are significantly enriched in imprinted genes. Most of the promoters of imprinted genes were found hypomethylated in HCC tumors compared to surrounding tissues, contrasting with the frequent promoter hypermethylation observed in tumors. We next investigated the status of methylation of the imprinting control region (ICR) of different imprinted clusters and found that the 15q11-13 ICR was significantly hypomethylated in tumors relative to their surrounding tissues. In addition, expression of imprinted genes within this cluster was frequently deregulated in a gene-specific manner, suggesting distinct mechanisms of regulation in this region. Finally, primary human hepatocytes and hepatocyte-like HepaRG cells displayed higher methylation variability in certain imprinted loci after natural hepatitis B virus (HBV) infection and after lipid accumulation, respectively.CONCLUSION:The methylation status of a large panel of imprinted genes was found deregulated in HCC, suggesting a major role of this mechanism during hepatocarcinogenesis. In vitro models support the hypothesis of imprinted gene methylation as a potential marker of environmental exposures.
• Comparative localization and functional activity of the main hepatobiliary transporters in HepaRG cells and primary human hepatocytes.Bachour-El Azzi P, Sharanek A, Burban A, Li R, Le Guével R, Abdel-Razzak Z, Stieger B, Guguen-Guillouzo C, Guillouzo A
  Toxicol Sci., Feb 2015
  Abstract : The role of hepatobiliary transporters in drug-induced liver injury remains poorly understood. Various in vivo and in vitro biological approaches are currently used for studying hepatic transporters; however, appropriate localization and functional activity of these transporters are essential for normal biliary flow and drug transport. Human hepatocytes (HH) are considered as the most suitable in vitro cell model but erratic availability and inter-donor functional variations limit their use. In the present work, we aimed to compare localization of influx and efflux transporters and their functional activity in differentiated human HepaRG hepatocytes with fresh HH in conventional (CCHH) and sandwich (SCHH) cultures. All tested influx and efflux transporters were correctly localized to canalicular (BSEP, MRP2, MDR1, MDR3) or basolateral (NTCP, MRP3) membrane domains and were functional in all models. Contrary to other transporters, NTCP and BSEP were less abundant and active in HepaRG cells, cellular uptake of taurocholate was 2.2- and 1.4-fold and bile excretion index 2.8-and 2.6- fold lower, than in SCHH and CCHH respectively. However, when taurocholate canalicular efflux was evaluated in standard and divalent cation-free conditions in buffers or cell lysates, the difference between the three models did not exceed 9.3%. Interestingly, cell imaging showed higher bile canaliculi contraction/relaxation activity in HepaRG hepatocytes and larger bile canaliculi networks in SCHH. Altogether, our results bring new insights in mechanisms involved in bile acids accumulation and excretion in HH and suggest that HepaRG cells represent a suitable model for studying hepatobiliary transporters and drug-induced cholestasis.
• Cell-specific biotransformation of benzophenone-2 and bisphenol-S in zebrafish and human in vitro models used for toxicity and estrogenicity screening.Le Fol V, Ait-Aissa S, Cabaton N, Dolo L, Grimaldi M, Balaguer P, Perdu E, Debrauwer L, Brion F, Zalko D
  Environ Sci Technol., Feb 2015
  Abstract : Several human and fish bioassays have been designed to characterize the toxicity and the estrogenic activity of chemicals. However, their biotransformation capability (bio-activation/detoxification processes) is rarely reported, although this can influence the estrogenic potency of test compounds. The fate of two estrogenic chemicals, the UV filter benzophenone-2 (BP2) and the bisphenol A substitute bisphenol S (BPS) was deciphered in eight human and zebrafish in vitro cell models, encompassing hepatic and mammary cellular contexts. BP2 and BPS were metabolized into a variety of gluco- and sulfo-conjugated metabolites. Similar patterns of BP2 and BPS biotransformation were observed among zebrafish models (primary hepatocytes, ZFL and ZELH-zfER cell lines). Interestingly, metabolic patterns in zebrafish models and in the human hepatic cell line HepaRG shared many similarities, while biotransformation rates in cell lines widely used for estrogenicity testing (MELN and T47D-KBLuc) were quantitatively low and qualitatively different. This study provides new data on the comparative metabolism of BP2 and BPS in human and fish cellular models that will help characterize their metabolic capabilities, and underlines the relevance of using in vitro zebrafish-based bioassays when screening for endocrine disrupting chemicals.
• Sgo1 is a potential therapeutic target for hepatocellular carcinoma.Wang LH, Yen CJ, Li TN, Elowe S, Wang WC, Wang LH
  Oncotarget., Jan 2015
  Abstract : Shugoshin-like protein 1 (Sgo1) is an essential protein in mitosis; it protects sister chromatid cohesion and thereby ensures the fidelity of chromosome separation. We found that the expression of Sgo1 mRNA was relatively low in normal tissues, but was upregulated in 82% of hepatocellular carcinoma (HCC), and correlated with elevated alpha-fetoprotein and early disease onset of HCC. The depletion of Sgo1 reduced cell viability of hepatoma cell lines including HuH7, HepG2, Hep3B, and HepaRG. Using time-lapse microscopy, we showed that hepatoma cells were delayed and ultimately die in mitosis in the absence of Sgo1. In contrast, cell viability and mitotic progression of immortalized cells were not significantly affected. Notably, mitotic cell death induced upon Sgo1 depletion was suppressed upon inhibitions of cyclin-dependent kinase-1 and Aurora kinase-B, or the depletion of mitotic arrest deficient-2. Thus, mitotic cell death induced upon Sgo1 depletion in hepatoma cells is mediated by persistent activation of the spindle assembly checkpoint. Together, these results highlight the essential role of Sgo1 in the maintenance of a proper mitotic progression in hepatoma cells and suggest that Sgo1 is a promising oncotarget for HCC.
• Detection of main metabolites of XLR-11 and its thermal degradation product in human hepatoma HepaRG cells and human urine.Kanamori T, Kanda K, Yamamuro T, Kuwayama K, Tsujikawa K, Iwata YT, Inoue T
  Drug Test Anal., Jan 2015
  Abstract : The metabolism of (1-(5-fluoropentyl)-1H-indol-3-yl)(2,2,3,3-tetramethylcyclopropyl)methanone (XLR-11), a novel synthetic cannabinoid, was studied using a HepaRG cell culture. The HepaRG cells were incubated with the drug for 48 hours and the metabolites were extracted from the culture medium by liquid-liquid extraction. The extract was analyzed by liquid chromatography/mass spectrometry to detect the metabolites. N-(5-Hydroxypentyl) metabolite and N-pentanoic acid metabolite were identified in the culture medium of XLR-11, and several other metabolites, presumably formed by oxidation of the first two metabolites and XLR-11, were detected. The extract of an XLR-11 user's urine was also analyzed; however, the metabolites detected in the urine were different from XLR-11 metabolites in the medium. A metabolic experiment with the thermal degradation product of XLR-11, XLR-11 degradant, using HepaRG cells revealed that the urinary metabolites were almost identical to the XLR-11 degradant metabolites. These findings suggest that most of the XLR-11 was degraded by heating when the user smoked the herbal product containing XLR-11. Copyright © 2015 John Wiley & Sons, Ltd.
• Understanding the biokinetics of ibuprofen after single and repeated treatments in rat and human in vitro liver cell systems.Truisi GL, Consiglio ED, Parmentier C, Savary CC, Pomponio G, Bois F, Lauer B, Jossé R, Hewitt PG, Mueller SO, Richert L, Guillouzo A, Testai E
  Toxicology Lett., Jan 2015
  Abstract : Common in vitro toxicity testing often neglects the fate and intracellular concentration of tested compounds, potentially limiting the predictability of in vitro results for in vivo extrapolation. We used in vitro long-term cultures of primary rat (PRH) and human hepatocytes (PHH) and HepaRG cells to characterise and model the biokinetic profile of ibuprofen (IBU) after single and daily repeated exposure (14 days) to two concentrations. A cross-model comparison was carried out at 100μM, roughly corresponding to the human therapeutic plasma concentration. Our results showed that IBU uptake was rapid and a dynamic equilibrium was reached within 1 or 2 days. All three cell systems efficiently metabolised IBU. In terms of species-differences, our data mirrored known in vivo results. Although no bioaccumulation was observed, IBU intracellular concentration was higher in PRH due to a 10-fold lower metabolic clearance compared to the human-derived cells. In HepaRG cells, IBU metabolism increased over time, but was not related to the treatment. In PHH, a low CYP2C9 activity, the major IBU-metabolising CYP, led to an increased cytotoxicity. A high inter-individual variability was seen in PHH, whereas HepaRG cells and PRH were more reproducible models. Although the concentrations of IBU in PRH over time differed from the concentrations found in human cells under similar exposure conditions.
• Long-Term Culture of Genome-Stable Bipotent Stem Cells from Adult Human LiverHuch M, Gehart H, Boxtel R, Hamer K, Blokzijl K, Verstegen M, Ellis E, Wenum M, Fuchs S, de Ligt J, van de Wetering M, Sasaki N, Boers S, Kemperman H, de Jonge J, N.M. Ijzermans J, Nieuwenhuis E, Hoekstra R, Strom S, Vries R, van der Laan L, Cuppen E...
  Cells, Jan 2015
  Abstract : HepaRG mentioned in Fig. 6G Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human liver. The expanded cells are highly stable at the chromosome and structural level, while single base changes occur at very low rates. The cells can readily be converted into functional hepatocytes in vitro and upon transplantation in vivo. Organoids from a1-antitrypsin deficiency and Alagille syndrome patients mirror the in vivo pathology. Clonal long-term expansion of primary adult liver stem cells opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine, and gene therapy.
• Transcriptomic analysis of untreated and drug-treated differentiated HepaRG cells over a 2-week period.Savary CC, Jiang X, Aubry M, Jossé R, Kopp-Schneider A, Hewitt P, Guillouzo A
  Toxicology In Vitro, Jan 2015
  Abstract : Previous works have shown that differentiated human HepaRG cells can exhibit drug metabolism activities close to those of primary human hepatocytes for several weeks at confluence. The present study was designed to evaluate their long-term functional stability and their response to repeated daily drug treatments over a 14-day period, using a transcriptomic approach. Our data show that less than 1% of the expressed genes were markedly deregulated over this two weeks period and mainly included down-regulation of genes related to the cell cycle and from 3days, overexpression of genes involved in xenobiotic and lipid metabolism. After daily treatment with the three PPAR agonists, fenofibrate, troglitazone and rosiglitazone qualitative and/or quantitative changes in gene profiling were observed depending on the compound and duration of treatment. The highest increase in the number of deregulated genes as a function of drug treatment was seen with rosiglitazone. The most up-regulated genes common across the three compounds were mainly related to lipid and xenobiotic metabolisms. All the data support the conclusion that human HepaRG cells have an exceptional functional stability at confluence and that they are suitable for investigations on chronic effects of drugs and other chemicals.

2014

• In vitro kinetics of amiodarone and its major metabolite in two human liver cell models after acute and repeated treatments.Pomponio G, Savary C, Parmentier C, Bois F, Guillouzo A, Romanelli L, Richert L, Di Consiglio E, Testai E
  Toxicol In Vitro., Dec 2014
  Abstract : The limited value of in vitro toxicity data for the in vivo extrapolation has been often attributed to the lack of kinetic data. Here the in vitro kinetics of amiodarone (AMI) and its mono-N-desethyl (MDEA) metabolite was determined and modelled in primary human hepatocytes (PHH) and HepaRG cells, after single and repeated administration of clinically relevant concentrations. AMI bioavailability was influenced by adsorption to the plastic and the presence of protein in the medium (e.g. 10% serum protein reduced the uptake by half in HepaRG cells). The cell uptake was quick (within 3h), AMI metabolism was efficient and a dynamic equilibrium was reached in about a week after multiple dosing. In HepaRG cells the metabolic clearance was higher than in PHH and increased over time, as well as CYP3A4. The interindividual variability in MDEA production in PHHs was not proportional to the differences in CYP3A4 activities, suggesting the involvement of other CYPs and/or AMI-related CYP inhibition. After repeated treatment AMI showed a slight potential for bioaccumulation, whereas much higher intracellular MDEA levels accumulated over time, especially in the HepaRG cells, associated with occurrence of phospholipidosis. The knowledge of in vitro biokinetics is important to transform an actual in vitro concentration-effect into an in vivo dose-effect relationship by using appropriate modelling, thus improving the in vitro-to-in vivo extrapolation.
• Human HepaRG cells can be cultured in hanging-drop plates for cytochrome P450 induction and function assays Murayama N, Usui T, Slawny N, Chesné C, Yamazaki H
  Drug Metabolism Letters, Dec 2014
  Abstract : Recent guidance/guidelines for industry recommend that cytochrome P450 induction be assessed using human hepatocyte enzyme activity and/or mRNA levels to evaluate potential drug–drug interactions.  To evaluate time-dependent cytochrome P450 induction precisely, induction of CYP1A2, CYP2B6, and CYP3A4 mRNA was confirmed (> 2-fold) by treatment with omeprazole, phenobarbital, and rifampicin, respectively, for 24 or 48 h on day 3 from the start of culture.  After 24 h, the fold induction of CYP1A2 with 3.6 and 1.8 × 10⁴ HepaRG cells per well was lower than that for 7.2 × 10⁴ cells. CYP1A2 induction levels at 24 h were higher than those after 48 h.  In contrast, higher CYP2B6 inductions were confirmed after 48 h exposure than after 24 h, independent of the number of cells per well.  To help reduce the use of human cryopreserved hepatocytes, typical P450-dependent enzyme activities were investigated in human HepaRG cells cultured in commercial hanging-drop plates.  Newly designed 96-well hanging-drop plates were capable of maintaining human CYP3A-dependent midazolam hydroxylation activities for up to 4 days using only 10% of the recommended initial 7.2 × 10⁴ cells per well.  Favorable HepaRG function using hanging-drop plates was confirmed by detecting 1′-hydroxymidazolam O-glucuronide on day 3, suggesting an improvement over traditional control plates in which this metabolite can be detected for 24-well plates.  These results suggest that the catalytic function and/or induction of CYP1A2, CYP2B6, and CYP3A4 can be readily assessed with reduced numbers of starting HepaRG cells cultured in three-dimensional cultures in drops prepared with hanging-drop plates.
• Design, synthesis and biological evaluation of a class of bioisosteric oximes of the novel dual peroxisome proliferator-activated receptor a/y ligand LT175Piemontese L, Fracchiolla G, Carrieri A, Parente M, Laghezza A, Carbonara G, Sblano S , Tauro M, Gilardi F, Tortorella P, Lavecchia A, Crestani M, Desvergne B, Loiodice F
  Journal of Medicinal Chemistry, Dec 2014
  Abstract : The effects resulting from the introduction of an oxime group in place of the distal aromatic ring of the diphenyl moiety of LT175, previously reported as a PPARα/γ dual agonist, have been investigated. This modification allowed the identification of new bioisosteric ligands with fairly good activity on PPARα and fine-tuned moderate activity on PPARγ. For the most interesting compound (S)-3, docking studies in PPARα and PPARγ provided a molecular explanation for its different behavior as full and partial agonist of the two receptor isotypes, respectively. A further investigation of this compound was carried out performing gene expression studies on HepaRG cells. The results obtained allowed to hypothesize a possible mechanism through which this ligand could be useful in the treatment of metabolic disorders. The higher induction of the expression of some genes, compared to selective agonists, seems to confirm the importance of a dual PPARα/γ activity which probably involves a synergistic effect on both receptor subtypes.
• Expression and characterization of myristoylated preS1-conjugated nanocages for targeted cell delivery.Murata M, Piao JS, Narahara S, Kawano T, Hamano N, Kang JH, Asai D, Ugawa R, Hashizume M
  Protein Expr Purif., Dec 2014
  Abstract : Lipid modification of proteins plays key roles in cellular signaling pathways. We describe the development of myristoylated preS1-nanocages (myr-preS1-nanocages) that specifically target human hepatocyte-like HepaRG cells in which a specific receptor-binding peptide (preS1) is joined to the surface of naturally occurring ferritin cages. Using a genetic engineering approach, the preS1 peptide was joined to the N-terminal regions of the ferritin cage via flexible linker moieties. Myristoylation of the preS1 peptide was achieved by co-expression with yeast N-myristoyltransferase-1 in the presence of myristic acid in Escherichia coli cells. The myristoylated preS1-nanocages exhibited significantly greater specificity for human hepatocyte-like HepaRG cells than the unmyristoylated preS1-nanocages. These results suggest that the lipid-modified nanocages have great potential for effective targeted delivery to specific cells.
• HepaRG culture in tethered spheroids as an in vitro three-dimensional model for drug safety screeningWang Z, Luo X, Anene-Nzelu C, Yu Y, Hong X, Singh N, Xia L, Liu S, Yu H
  Journal of applied toxicology, Dec 2014
  Abstract : Conventional two-dimensional (2D) monolayer cultures of HepaRG cells allow in vitro maintenance of many liverspecific functions. However, cellular dedifferentiation and functional deterioration over an extended culture period in the conventional 2D HepaRG culture have hampered its applications in drug testing. To address this issue, we developed tethered spheroids of HepaRG cells on Arg–Gly–Asp (RGD) and galactose-conjugated substratum with an optimized hybrid ratio as an in vitro three-dimensional (3D) human hepatocyte model. The liver-specific gene expression level and drug metabolizing enzyme activities in HepaRG-tethered spheorids were markedly higher than those in 2D cultures throughout the culture period of 7 days. The inducibility of three major cytochrome P450 (CYP) enzymes, namely CYP1A2, CYP2B6 and CYP3A4, was improved in both mRNA and activity level in tethered spheroids. Drug-induced cytotoxic responses to model hepatotoxins (acetaminophen, chlorpromazine and ketoconazole) in tethered spheroids were comparable to 2D cultures as well as other studies in the literature. Our results suggested that the HepaRG-tethered spheroid would be an alternative in vitro model suitable for drug safety screening. Copyright © 2014 John Wiley & Sons, Ltd.
• Hepatitis B virus polymerase disrupts K63-linked ubiquitination of STING to block innate cytosolic DNA-sensing pathways.Liu Y, Li J, Chen J, Wang W, Du X, Song W, Zhang W, Lin L, Yuan Z
  J Virol., Dec 2014
  Abstract : The cellular innate immune system recognizing pathogen infection is essential for host defense against viruses. In parallel, viruses have developed a variety of strategies to evade the innate immunity. The hepatitis B virus (HBV), a DNA virus that causes chronic hepatitis, has been shown to inhibit the RNA helicase RIG-I-mediated interferon (IFN) induction. However, it is still unknown whether HBV could affect the host DNA-sensing pathways. Here we report that in transient HBV-transfected Huh7 cells, HBV stably producing cell line HepAD38, and HBV-infected HepaRG cells and primary human hepatocytes, HBV markedly interfered with IFN-β induction and antiviral immunity mediated by STING, which has been identified as a central factor in foreign DNA recognition and antiviral innate immunity. Screening analysis demonstrated that the viral polymerase (Pol), but not other HBV-encoded proteins, was able to inhibit STING-stimulated interferon regulatory factor 3 (IRF3) activation and IFN-β induction. Moreover, the reverse transcriptase (RT) and the ribonuclease H (RH) domains of Pol were identified to be responsible for the inhibitory effects. Furthermore, Pol was shown to physically associate with STING and dramatically decrease the K63-linked polyubiquitination of STING via its RT domain without altering the expression level of STING. Taken together, these observations suggest that besides its inherent catalytic function, Pol has a role in suppression of IFN-β production by direct interaction with STING and subsequent disruption of its K63-linked ubiquitination, providing a new mechanism for HBV to counteract the innate DNA-sensing pathways
• Activating and inhibitory functions of WNT/B-catenin in the induction of cytochromes P450 by nuclear receptors in HepaRG cellsThomas M, Bayha C, Vetter S, Hofmann U, Schwarz M, Zanger UM, Braeuning A
  MolPharmacol, Dec 2014
  Abstract : The WNT/β-catenin signaling pathway has been identified as an important endogenous regulator of hepatic cytochrome P450 (CYP) expression in mouse liver. In particular, it is involved in the regulation of CYP expression in response to exposure to xenobiotic agonists of the nuclear receptors CAR, AhR, and Nrf2. In order to systematically elucidate the impact of the WNT/β-catenin pathway on the regulation and inducibility of major human cytochrome P450 enzymes, HepaRG cells were treated with either the WNT/β-catenin signaling pathway agonist, WNT3a, or with siRNA directed against β-catenin, alone or in combination with a panel of activating ligands for AhR (2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD), CAR (6-(4-Chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime, CITCO), PXR (rifampicin) and PPARα (4-Chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid, WY14,643). Assessment of CYP gene expression and enzymatic activity following downregulation or activation of the WNT/β-catenin pathway revealed a requirement of β-catenin in the AhR-, CAR- and PXR-mediated induction of CYP1A, CYP2B6 and CYP3A4 (for CAR and PXR) and additionally of CYP2C8 (for PXR) gene expression. In contrast, activation of the WNT/β-catenin pathway prevented PPARα-mediated induction of CYP1A, CYP2C8 and CYP3A4 genes, suggesting a dominant-negative role of β-catenin in PPARα-mediated regulation of these genes. Our data indicate a significant impact of the WNT/β-catenin pathway on the regulation of cytochromes P450 enzymes in human hepatocytes and reveal a novel crosstalk between β-catenin and PPARα signaling pathways in the regulation of CYP expression.
• Organocatalysis Paradigm Revisited: Are Metal-free Catalysts really Harmless?Nachtergael A, Coulembier O, Dubois P, Helvenstein M, Duez P, Blankert B, Mespouille L
  Biomacromolecules, Dec 2014
  Abstract : Catalysts are commonly used in polymer synthesis. Traditionally, catalysts used to be metallic compounds but some studies have pointed out their toxicity for human health and environment and the removal of metal impurities from synthetic polymer is quite expensive. To address these issues, organocatalysts have been intensively synthetized and are now widely used in ring-opening polymerization (ROP) reactions. However, for most of them, there is not any evidence of their safety. The present study attempts to assess if well-established organo-based ROP catalysts used for the preparation of FDA-approved polyesters may present or not a certain level of cytotoxicity. In vitro toxicity is evaluated using a methyl-thiazol-tetrazolium (MTT) cytotoxicity assay on two cell models (FHs74Int and HepaRG). Among the investigated organocatalysts, only functionalized thiourea shows an important cytotoxicity on both cell models. 4-dimethylaminopyridine (DMAP), 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) and meta-(trimethylammonio)phenolate betaine show cytotoxicity against HepaRG cell line only at a high concentration.
• A Systematic Comparison of the Impact of Inflammatory Signaling on ADME Gene Expression and Activity in Primary Human Hepatocytes and HepaRG Cells.Klein M, Thomas M, Hofmann U, Seehofer D, Damm G, Zanger UM
  Drug Metab Dispos., Dec 2014
  Abstract : Inflammatory processes are associated with compromised metabolism and elimination of drugs in the liver, largely mediated by proinflammatory cytokines, such as interleukin-6 (IL-6). The HepaRG cell line is an established surrogate for primary human hepatocytes (PHH) in drug metabolism and toxicity studies. However, the impact of inflammatory signaling on HepaRG cells has not been well characterized. In this study, the response of primary human hepatocytes and HepaRG cells to IL-6 was comparatively analyzed. For this purpose, broad spectrum gene expression profiling, including acute phase response genes and a large panel of drug metabolizing enzyme and transporter (DMET) genes as well as their modifiers and regulators, was conducted in combination with cytochrome P450 activity measurements. Exposure of PHH and HepaRG cells to IL-6 resulted in highly similar coordinated reduction of DMET mRNA, including major ABCs, CYPs, GSTs, UGTs, and SLCs. Enzyme activities of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP3A4 were reduced upon 48-72 hours exposure to IL-6 in PHH and HepaRG. However, while these effects were not significant in PHH due to large interindividual donor variability, the impact on HepaRG was more pronounced and highly significant, thus emphasizing the advantage of HepaRG as a more reproducible model system. Exposure of HepaRG cells to interleukin-1β and tumor necrosis factor α resulted in similar effects on gene expression and enzyme activities. The present study emphasizes the role of proinflammatory cytokines in the regulation of drug metabolism and supports the use of HepaRG in lieu of PHH to minimize subject variability.The American Society for Pharmacology and Experimental Therapeutics.
• Biokinetics of chlorpromazine in primary rat and human hepatocytes and human HepaRG cells after repeated exposure.Broeders JJ, Parmentier C, Truisi GL, Jossé R, Alexandre E, Savary CC, Hewitt PG, Mueller SO, Guillouzo A, Richert L, van Eijkeren JC, Hermens JL, Blaauboer BJ
  Toxicol. In Vitro, Dec 2014
  Abstract : Since drug induced liver injury is difficult to predict in animal models, more representative tests are needed to better evaluate these effects in humans. Existing in vitro systems hold great potential to detect hepatotoxicity of pharmaceuticals. In this study, the in vitro biokinetics of the model hepatotoxicant chlorpromazine (CPZ) were evaluated in three different liver cell systems after repeated exposure in order to incorporate repeated-dose testing into an in vitro assay. Primary rat and human hepatocytes, cultured in sandwich configuration and the human HepaRG cell line were treated daily with CPZ for 14days. Samples were taken from medium, cells and well plastic at specific time points after the first and last exposure. The samples were analysed by HPLC-UV to determine the amount of CPZ in these samples. Based on cytotoxicity assays, the three models were tested at 1-2μM CPZ, while the primary rat hepatocytes and the HepaRG cell line were in addition exposed to a higher concentration of 15-20μM. Overall, the mass balance of CPZ decreased in the course of 24h, indicating the metabolism of the compound within the cells. The largest decrease in parent compound was seen in the primary cultures; in the HepaRG cell cultures the mass balance only decreased to 50%. CPZ accumulated in the cells during the 14-day repeated exposure. Possible explanations for the accumulation of CPZ are a decrease in metabolism over time, inhibition of efflux transporters or binding to phospholipids. The biokinetics of CPZ differed between the three liver cell models and were influenced by specific cell properties as well as culture conditions. These results support the conclusion that in vitro biokinetics data are necessary to better interpret chemical-induced cytotoxicity data.
• Metoronidazole reduces the expression of cytochrome P450 enzymes in HepaRG cells and cryopreserved human hepatocytes.Kudo T, Endo Y, Taguchi R, Yatsu M, Ito K
  Xenobiotica, Dec 2014
  Abstract : 1. Blood levels of S-warfarin have been reported to be increased by concomitant administration of metronidazole (MTZ), an antiprotozoal imidazole derivative. 2. To elucidate the mechanism of this interaction and to identify other possible drug-drug interactions, we conducted an in vitro study with the human hepatoma HepaRG cells and cryopreserved human hepatocytes on the ability of MTZ to reduce the expression of cytochrome P450 (CYP) as well as nuclear receptors that regulate the expression of these enzymes. 3. HepaRG cells and cryopreserved human hepatocytes were treated with MTZ (20 to 500 µM) and were then analyzed by real-time RT-PCR to determine mRNA levels of drug-metabolizing enzymes and nuclear receptors. 4. In both cells, the expressions of CYP2C8, CYP2C9, CYP3A4 and constitutive androstane receptor (CAR) were decreased by MTZ treatment. Particularly, in HepaRG cells, their mRNA levels were decreased by MTZ treatment in a concentration-dependent manner. 5. Our findings suggest that the interaction between MTZ and S-warfarin may be due to the MTZ-induced down-regulation of CYP2C9, the primary enzyme responsible for S-warfarin hydroxylation, and CAR, which regulates CYP2C9 expression. We also found that MTZ use may alter the disposition of drugs metabolized by the CYP isozymes investigated.
• In vitro-to-in vivo correlation of the skin penetration, liver clearance and hepatotoxicity of caffeine.Gajewska M, Paina A, Sala Benito JV, Burton J, Worth A, Urani C, Briesen H, Schramm KW
  Food Chem Toxicol., Dec 2014
  Abstract : This work illustrates the use of Physiologically-Based Toxicokinetic (PBTK) modelling for the healthy Caucasian population in in vitro-to-in vivo correlation of kinetic measures of caffeine skin penetration and liver clearance (based on literature experiments), as well as dose metrics of caffeine-induced measured HepaRG toxicity. We applied a simple correlation factor to quantify the in vitro and in vivo differences in the amount of caffeine permeated through the skin and concentration-time profiles of caffeine in the liver. We developed a multi-scale computational approach by linking the PBTK model with a Virtual Cell-Based Assay to relate an external oral and dermal dose with the measured in vitro HepaRG cell viability. The results revealed higher in vivo skin permeation profiles than those determined in vitro using identical exposure conditions. Liver clearance of caffeine derived from in vitro metabolism rates was found to be much slower than the optimised in vivo clearance with respect to caffeine plasma concentrations. Finally, HepaRG cell viability was shown to remain almost unchanged for external caffeine doses of 5-400 mg for both oral and dermal absorption routes. We modelled single exposure to caffeine only.
• Intracellular accumulation of subviral HBsAg particles and diminished Nrf2 activation in HBV genotype G expressing cells lead to an increased ROI level.Peiffer KH, Akhras S, Himmelsbach K, Hassemer M, Finkernagel M, Carra G, Nuebling M, Chudy M, Niekamp H, Glebe D, Sarrazin C, Zeuzem S, Hildt E
  Journal of Hepatology, Dec 2014
  Abstract : Background and aims: Hepatitis B virus genotype G (HBV/G) is characterized by a lack of HBeAg secretion and very low HBsAg secretion. This study aims to analyze HBV/G in comparison to HBV genotype A2 regarding morphogenesis and release of HBV-derived particles and to characterize factors contributing to HBV/G-associated pathogenesis.Methods: HBV/G and HBV/A expressing hepatoma cells and infected HepaRG cells were analyzed by confocal laser scanning microscopy, western blot analysis, real-time PCR, density gradient centrifugation and electron microscopy. Moreover modulation of the transcription factors Nrf2 and AP-1 was analyzed.Results: While the release of viral particles is not affected in HBV/G replicating cells, the secretion of subviral particles is impaired although subviral particles are produced in high amounts. These subviral particles that display an increased density and a predominantly filamentous morphology accumulate at the endoplasmic reticulum. The PreS1PreS2 domain of genotype G that forms aggregates causes the block in HBsAg-secretion at the ER and leads to decreased transcriptional activator function of LHBs. Intracellular accumulation of HBsAg and impaired induction of the cytoprotective transcription factor Nrf2 lead to an elevated level of ROI that results in activation of JNK and thereby to Ser-phosphorylation of IRS-1 that is known to impair insulin signaling, a key factor for liver regeneration.Conclusions: Although competent for release of viral particles, secretion of subviral particles is impaired in HBV/G expressing cells leading to ER-stress. Since in parallel HBV-inducedNrf2 activation is diminished, the capacity to inactivate ROIs is decreased. This might confer to genotype-specific pathogenesis.
• The role of the c-Jun N-terminal kinases 1/2 and receptor-interacting protein kinase 3 in furosemide-induced liver injury.McGill MR, Du K, Xie Y, Bajt ML, Ding WX, Jaeschke H
  Xenobiotica, Nov 2014
  Abstract : 1. The mechanisms of furosemide (FS) hepatotoxicity were explored in mice. Specifically, C57Bl/6 J mice were treated with 500 mg FS/kg bodyweight, and c-Jun N-terminal kinase (JNK) activation and receptor-interacting protein kinase 3 (RIP3) expression were measured by western blotting. Co-treatment with FS and the JNK inhibitor SP600125 was also performed, and FS-induced liver injury was compared in wild-type and RIP3 knockout (KO) mice. 2. JNK phosphorylation and RIP3 expression were increased in livers from the FS-treated mice as early as 6 h after treatment and persisted until at least 24 h. JNK phosphorylation was also observed in primary mouse hepatocytes and human HepaRG cells treated with FS. 3. Phosphorylated JNK translocated into mitochondria in livers, but no evidence of mitochondrial damage was observed. 4. SP600125-treated mice, SP600125 co-treated primary mouse hepatocytes and RIP3 KO mice were not protected against FS hepatotoxicity. These data show that, although JNK activation and RIP3 expression are induced by FS, neither contributes to the liver injury.
• Biotransformation in vitro: An essential consideration in the quantitative in vitro-to-in vivo extrapolation (QIVIVE) of toxicity dataWilk-Zasadna I,Bernasconi C, Pelkonen O, Coecke S
  Toxicology, Nov 2014
  Abstract : Early consideration of the multiplicity of factors that govern the biological fate of foreign compounds in living systems is a necessary prerequisite for the quantitative in vitro–in vivo extrapolation (QIVIVE) of toxicity data. Substantial technological advances in in vitro methodologies have facilitated the study of in vitro metabolism and the further use of such data for in vivo prediction. However, extrapolation to in vivo with a comfortable degree of confidence, requires continuous progress in the field to address challenges such as e.g.,in vitro evaluation of chemical–chemical interactions, accounting for individual variability but also analytical challenges for ensuring sensitive measurement technologies. This paper discusses the current status of in vitro metabolism studies for QIVIVE extrapolation, serving today’s hazard and risk assessment needs. A short overview of the methodologies for in vitro metabolism studies is given. Furthermore, recommendations for priority research and other activities are provided to ensure further widespread uptake of in vitro metabolism methods in 21st century toxicology. The need for more streamlined and explicitly described integrated approaches to reflect the physiology and the related dynamic and kinetic processes of the human body is highlighted i.e., using in vitro data in combination with in silico approaches.
• Live spheroid formation recorded with light sheet-based fluorescence microscopy.Pampaloni F, Richa R, Ansari N, Stelzer EH.
  Methods Mol Biol., Nov 2014
  Abstract : We provide a detailed protocol for a three-dimensional long-term live imaging of cellular spheroids with light sheet-based fluorescence microscopy. The protocol allows the recording of all phases of spheroid formation in three dimensions, including cell proliferation, aggregation, and compaction. We employ the human hepatic cell line HepaRG transfected with the fusion protein H2B-GFP, i.e., a fluorescing histone. The protocol allows monitoring the effect of drugs or toxicants.
• A trial study on proteomics fingerprint analysis of HepaRG cells by FD-LC-MS/MSKazuhiro Imai, Katsunori Nakata, Tomoko Ichibangase, Ryoichi Saitoh , Masaki Ishigai
  Analyst, Nov 2014
  Abstract : A proteomics profile analysis was performed on a human hepatocyte carcinoma cell line (HepaRG) using the FD-LC-MS/MS method. One hundred fifty eight proteins were identified for the first time and 10 were specific to human hepatocytes. These proteins are a “proteomics fingerprint” that can be used to characterize HepaRG cells.
• Bioartificial livers in vitro and in vivo: tailoring biocomponents to the expanding variety of applicationsWenum M, Chamuleau RA, Gulik T, Siliakus A, Seppen J, Hoekstra H
  Expert Opinion, Nov 2014
  Abstract : Introduction: Bioartificial livers (BALs) were originally developed to treat patients suffering from severe liver failure and relied on primary hepatocytes or on hepatoblastoma-derived cell lines. Currently, new in vitro BAL applications are emerging, including drug toxicity testing, disease modeling and basic clinical research, and in recent years, advances in the field of stem cell biology have resulted in potential alternative cell sources.Areas covered: This review identifies the demands of clinical and in vitro BAL applications to their biocomponent and summarizes the functionality and developmental state of BAL technology and cell types currently available. Relevant studies identified by searching the MEDLINE database until April 2014 were reviewed, supplemented with some of our own unpublished data.Expert opinion: BALs have the potential to meet demands currently left unmet in both clinical and in vitro applications. All the reviewed biocomponents show limitations towards one or more BAL applications. However, the generation of stem cell-derived hepatocyte-like cells is progressing rapidly, so the criteria for patient-specific drug toxicity screening and disease modeling are probably met in the near future. HepaRG cells are the most promising biocomponent for clinical BAL, based on their proliferative and differentiation capacity.
• HepaRG cells as human relevant in vitro model to study the effects of inflammatory stimuli on Cytochrome P450 isoenzymesRubin K, Janefeldt A, Andersson L, Berke Z, Grime K, Andersson TB
  Drug Metab Dispos. , Nov 2014
  Abstract : The suppression of hepatic Cytochrome P450 (P450) expression during inflammatory and infectious diseases and the relief of this suppression by successful disease treatment have been previously demonstrated to impact drug disposition. To address this clinically relevant phenomenon preclinically, the effect of pro-inflammatory cytokines on P450 isoenzymes in human hepatocytes has been examined by several researchers. In the present study we used the human hepatoma cell line HepaRG and cryopreserved primary human hepatocytes to investigate the effects of various inflammatory stimuli on P450 levels with the aim of further characterizing HepaRG cells as a useful surrogate for primary hepatocytes. In this study, HepaRG cells were exposed to bacterial lipopolysaccharide (LPS), interleukin-6 (IL-6) and interleukin-18 (IL-18) for 48 or 72 hours. The effects on CYP1A2, CYP2B6 and CYP3A4 mRNA and catalytic activity (phenacetin-O-deethylase, bupropion-hydroxylase and midazolam-1´-hydroxylase) were measured. Cryopreserved pooled plateable hepatocytes were also exposed to IL-6 or IL-18 for 48 hours and the effects on CYP1A2, CYP2B6 and CYP3A4 mRNA levels were measured. The exposure of HepaRG cells to IL-6 and LPS resulted in suppression of and CYP1A2, CYP2B6 and CYP3A4 mRNA levels as well catalytic activities. However, no suppression of P450 activities or mRNA levels was observed after exposure to IL-18. Similar results on CYP1A2, CYP2B6 and CYP3A4 mRNA levels were observed with primary hepatocytes. The present study indicates that different pro-inflammatory mediators influence the expression of P450 differentially and that HepaRG cells may be used as an alternative to human hepatocytes for studies on cytokine-mediated suppression of drug-metabolizing enzymes.
• Regulation of Hepatic Drug Transporter Activity and Expression by Organochlorine PesticidesBucher S, Le Vee M, Jouan E, Fardel O
  J BIOCHEM MOLECULAR TOXICOLOGY, Nov 2014
  Abstract : Organochlorine (OC) pesticides constitute a major class of persistent and toxic organic pollutants, known to modulate drug-detoxifying enzymes. In the present study, OCs were demonstratedto also alter the activity and expression of human hepatic drug transporters. Activity of the sinusoidal influx transporter OCT1 (organic cation transporter 1) was thus inhibited by endosulfan, chlordane, heptachlor, lindane, and dieldrine, but not by dichlorodiphenyltrichloroethane isomers, whereas those of the canalicular efflux pumps MRP2 (multidrug resistance-associated protein 2) and BCRP (breast cancer resistance protein) were blocked by endosulfan, chlordane, heptachlor, and chlordecone; this latter OC additionally inhibited the multidrug resistance gene 1 (MDR1)/P-glycoprotein (P-gp) activity. OCs, except endosulfan, were next found to induce MDR1/P-gp and MRP2 mRNA expressions in hepatoma HepaRG cells; some of them also upregulated BCRP. By contrast, expression of sinusoidal transporters was not impaired (organic anion-transporting polypeptide (OATP) 1B1 and OATP2B1) or was downregulated (sodium taurocholate co-transporting polypeptide (NTCP) and OCT1). Such regulations of drug transporter activity and expression, depending on the respective nature of OCs and transporters, may contribute to the toxicity of OC pesticides
• Alisporivir Inhibition of Hepatocyte Cyclophilins Reduces HBV Replication and Hepatitis B Surface Antigen Production Phillips S, Chokshi S, Chatterji U, Riva A, Bobardt M, Williams R, Gallay P, V. Naoumov N
  Gastroenterology, Oct 2014
  Abstract : BACKGROUND & AIMS: Cyclophilins are host factors required for hepatitis C virus (HCV) replication. Cyclophilin inhibitors such as alisporivir have shown strong anti-HCV activity in vitro and in clinical studies. However, little is known about whether hepatocyte cyclophilins are involved in the hepatitis B virus (HBV) life cycle. We investigated the effects of 2 cyclophilin inhibitors (alisporivir and NIM811) on HBV replication and hepatitis B surface antigen (HBsAg) production in cell lines. METHODS: Liver-derived cell lines producing full-length HBV and HBsAg particles, due to stable (HepG2215) or transient (HuH-7) transfection, or infected with HBV (HepaRG cells), were incubated with alisporivir or NIM811 alone, or alisporivir in combination with a direct antiviral (telbivudine). The roles of individual cyclophilins in drug response was evaluated by small interfering RNA knockdown of cyclophilin (CYP)A, CYPC, or CYPD in HepG2215 cells, or CYPA knockdown in HuH-7 cells. The kinetics of antiviral activity were assessed based on levels of HBV DNA and HBsAg and Southern blot analysis. RESULTS: In HepG2215, HuH-7, and HepaRG cells, alisporivir reduced intracellular and secreted HBV DNA, in a dose-dependent manner. Knockdown of CYPA, CYPC, or CYPD (reduced by 80%) significantly reduced levels of HBV DNA and secreted HBsAg. Knockdown of CYPA significantly reduced secretion of HBsAg, leading to accumulation of intracellular HBsAg; the addition of alisporivir greatly reduced levels of HBsAg in these cells. The combination of alisporivir and telbivudine had greater antiviral effects than those of telbivudine or alisporivir alone. CONCLUSIONS: Alisporivir inhibition of cyclophilins in hepatocyte cell lines reduces replication of HBV DNA and HBsAg production and secretion. These effects are potentiated in combination with direct antiviral agents that target HBV DNA polymerase.
• Expression of Stress-Dependent Genes in Hepatocytes Spheroids after Cisplatin Treatment.Rusanov AL, Pul'kova NV, Klonova MG, Fomicheva KA, Kozhin PM, Sevast'yanova MA, Shkurnikov MY
  Bull Exp Biol Med., Sep 2014
  Abstract : Effects of cisplatin on the hepatic HepaRG cells cultured in spheroids were estimated using biochemical, cytofluorometric, and molecular methods. Hepatocyte viability and expression of mRNA of stress-dependent genes involved in the cell response to toxic agent cisplatin underwent time- and dose-dependent changes. Activation of oxidative stress was observed at the early stages of incubation (3 h) followed by induction of apoptosis after prolonging the incubation to 24 h. The prospects of using HepaRG cells cultured in spheroids for estimation of drug toxicity by variations in the expression of stress-dependent genes were demonstrated. An increase in expression of genes of GSR and HSPA1A proteins at the early stages of incubation with cisplatin can serve as a marker of the cytotoxic effects of cisplatin and other agents with similar mechanisms of action.
• Comparative evaluation of N-acetylcysteine and N-acetylcysteineamide in acetaminophen-induced hepatotoxicity in human hepatoma HepaRG cells.Tobwala S, Khayyat A, Fan W, Ercal N
  Exp Biol Med, Sep 2014
  Abstract : Acetaminophen (N-acetyl-p-aminophenol, APAP) is one of the most widely used over-the-counter antipyretic analgesic medications. Despite being safe at therapeutic doses, an accidental or intentional overdose can result in severe hepatotoxicity; a leading cause of drug-induced liver failure in the U.S. Depletion of glutathione (GSH) is implicated as an initiating event in APAP-induced toxicity. N-acetylcysteine (NAC), a GSH precursor, is the only currently approved antidote for an APAP overdose. Unfortunately, fairly high doses and longer treatment times are required due to its poor bioavailability. In addition, oral and intravenous administration of NAC in a hospital setting are laborious and costly. Therefore, we studied the protective effects of N-acetylcysteineamide (NACA), a novel antioxidant, with higher bioavailability and compared it with NAC in APAP-induced hepatotoxicity in a human-relevant in vitro system, HepaRG. Our results indicated that exposure of HepaRG cells to APAP resulted in GSH depletion, reactive oxygen species (ROS) formation, increased lipid peroxidation, mitochondrial dysfunction (assessed by JC-1 fluorescence), and lactate dehydrogenase release. Both NAC and NACA protected against APAP-induced hepatotoxicity by restoring GSH levels, scavenging ROS, inhibiting lipid peroxidation, and preserving mitochondrial membrane potential. However, NACA was better than NAC at combating oxidative stress and protecting against APAP-induced damage. The higher efficiency of NACA in protecting cells against APAP-induced toxicity suggests that NACA can be developed into a promising therapeutic option for treatment of an APAP overdose.
• Reversible immortalization of human hepatocytes mediated by retroviral transfer and site-specific recombinationMeng FY, Liu L, Yang FH, Li CY, Liu J, Zhou P
  Journal of Gastroenterology, Sep 2014
  Abstract : AIM: To establish a method for the reversible immortalization of human hepatocytes, which may offer a good and safe source of hepatocytes for practical applications.METHODS: We successfully isolated primary human hepatocytes from surgically resected liver tissue taken from a patient with liver hemangiomas. The freshly isolated cells were then immortalized with retroviral vector SSR#69 expressing simian virus 40 large T antigen (SV40T) and hygromycin-resistance genes flanked by paired loxP recombination targets.RESULTS: The freshly isolated hepatocytes with high viability (85%) were successfully immortalized using retroviral gene transfer of SV40T. SV40T in the immortalized cells was then excised by Cre/loxP site-specific recombination. This cell population exhibited the characteristics of differentiated hepatocytes.CONCLUSION: We successfully established reversibly immortalized human hepatocytes, which will provide an unlimited supply of cells for practical applications.
• Diversified expression of aryl hydrocarbon receptor dependent genes in human laryngeal squamous cell carcinoma cell lines treated with ?-naphthoflavone.Brauze D, Fijalkiewicz K, Szaumkessel M, Kiwerska K, Bednarek K, Rydzanicz M, Richter J, Grenman R, Jarmuz-Szymczak M
  Toxicol Lett., Sep 2014
  Abstract : The aryl hydrocarbon receptor (AhR) mediates a variety of biological responses to ubiquitous environmental pollutants. In this study the effect of administration of β-naphthoflavone (BNF), potent AhR ligand, on the expression of AhR, AhRR, CYP1A1, CYP1A2, CYP1B1, NQO1, GSTA1, ALDH3A1 and UGT1A genes encoding the enzymes controlled by AhR were examined in thirteen laryngeal tumor cell lines and in HepaRG cell line. The analyzed cell lines were derived from patients with squamous laryngeal cancer, with history of cigarette smoking and without signs of human papillomavirus types 16 and 18 infection in investigated cells. Quantitative real-time RT-PCR analysis revealed huge interindividual differences in expression of genes from AhR regulatory network. Our results strongly suggest predominant effect of DNA methylation on induction of CYP1A1 expression by AhR ligands as well. Our results indicate that differentiated HepaRG cell line appeared to be very good substitute for human liver in studies on xenobiotic metabolism by AhR regulated enzymes.
• Inhibition of Cytochrome P450 2C9 Expression and Activity In Vitro by Allyl Isothiocyanate.Lim YP, Chen WC, Cheng CH, Ma WC, Lin YH, Chen CY, Hung DZ, Chen JJ, Yokoi T, Nakajima M, Chen CJ
  Planta Med, Sep 2014
  Abstract : The growing interest in the use of natural herbal products and dietary supplements to treat and prevent diseases raises the question of medicinal drug safety. Allyl isothiocyanate, a hydrolysis product of a glucosinolate, sinigrin, has multiple beneficial properties, and based on this fact, allyl isothiocyanate-containing dietary supplements have been developed. To date, no studies of the effects of this compound on the cytochrome P450 2C9 have been reported. In this study, we found that allyl isothiocyanate reduced catalytic activity, messenger ribonucleic acid, and protein expression of cytochrome P450 2C9 in HepaRG cells. An investigation of the transcriptional activity of the pregnane X receptor and the constitutive androstane receptor revealed that allyl isothiocyanate disrupted the transcriptional coregulation effects of the pregnane X receptor/constitutive androstane receptor with several important coregulators and interfered with the assembly of transcriptional complexes of the cytochrome P450 2C9 pregnane X receptor/constitutive androstane receptor-response element. The decrease of cytochrome P450 2C9 expression and activity mediated by allyl isothiocyanate suggested that this agent could alter the metabolism of drugs metabolized by cytochrome P450 2C9. This may cause food/dietary supplement-drug interactions or alter the therapeutic effects, and even the toxicity of drugs coadministered with allyl isothiocyanate. Since the consumption of allyl isothiocyanate-containing food/dietary supplements continues to increase, it is important to predict and ultimately avoid interactions with concomitant drugs. It is required that these possible pharmacokinetic interactions be characterized and the recommendations available to patients and healthcare professionals be improved.
• HepaRG cell line as an in vitro model for screening drug-drug interactions mediated by metabolic induction: amiodarone used as a model substance.Ferreira A, Rodrigues M, Silvestre S, Falcão A, Alves G
  Toxicol In Vitro., Aug 2014
  Abstract : The prediction of drug-drug interactions mediated by the induction or inhibition of cytochrome P450 enzymes is of great relevance in the development of new drugs. Due to the fact that HepaRG, a new human cell line derived from a hepatocellular carcinoma, is being considered a promising model to evaluate the in vitro metabolism of drugs, it was herein used for investigating metabolic-based drug-drug interactions mediated by metabolic induction. In this study, rifampicin and phenytoin were used as probe inducers and amiodarone (AM) as a model drug. HepaRG cells were firstly seeded in the supplemented Williams' E, and then differentiated in the same culture medium, supplemented with 2% dimethyl sulfoxide for 2 weeks. For metabolic induction studies AM was incubated during 12 h in HepaRG cells which were pre-incubated with phenytoin or rifampicin for 72 h. The concentrations of AM and its main metabolite, mono-N-desethylamiodarone, were quantified by HPLC-DAD. This study evidenced that rifampicin and phenytoin are powerful inducers of the metabolism of AM, including at therapeutic drug concentrations. These experimental findings demonstrated, for the first time, the applicability of HepaRG cells as a useful in vitro model for the prediction of metabolic-based drug-drug interactions, namely those mediated by metabolic induction. Thus, this model could potentially be a worthy alternative to the primary human hepatocytes.Copyright © 2014 Elsevier Ltd. All rights reserved.
• High Content Imaging and Analysis Enables Quantitative In Situ Assessment of CYP3A4 using Cryopreserved Differentiated HepaRG Cells.Ranade AR, Melinda S. Wilson, Amy M. McClanahan, Andrew J. Ball
  -, Aug 2014
  Abstract : High-throughput imaging-based hepatotoxicity studies capable of analyzing individual cells in situ hold enormous promise for drug safety testing, but are frequently limited by a lack of sufficient metabolically-competent human cells. This study examined cryopreserved HepaRG™ cells, a human liver cell line which differentiates into both hepatocytes and biliary epithelial cells, to determine if these cells may represent a suitable metabolicallycompetent cellular model for novel High Content Analysis (HCA) applications. Characterization studies showed that these cells retain many features characteristic of primary human hepatocytes and display significant CYP3A4 and CYP1A2 induction, unlike the HepG2 cell line commonly utilized for HCA studies. Furthermore, this study demonstrates that CYP3A4 induction can be quantified via a simple image analysis-based method, using HepaRG™ cells as a model system. Additionally, data demonstrate that the hepatocyte and biliary epithelial subpopulations characteristic of HepaRG™ cultures can be separated during analysis simply on the basis of nuclear size measurements. Proof of concept studies with fluorescent cell function reagents indicated that further multiparametric image-based assessment is achievable with HepaRG™. In summary, image-based screening of metabolically competent human hepatocyte models cells such as HepaRG™ offers novel approaches for hepatotoxicity assessment and improved drug screening tools.
• HepaRG microencapsulated spheroids in DMSO-free culture: novel culturing approaches for enhanced xenobiotic and biosynthetic metabolism.Rebelo SP, Costa R, Estrada M, Shevchenko V, Brito C, Alves PM
  Arch Toxicol., Aug 2014
  Abstract : The need for models that recapitulate liver physiology is perceived for drug development, study of liver disease and bioartificial liver support. The bipotent cell line HepaRG constitutes an efficient surrogate of liver function, yet its differentiated status relies on high concentrations of DMSO, which may compromise the study of drug metabolism and limit the applicability of this hepatic model. Herein, we present a three-dimensional (3D) strategy for the differentiation of HepaRG based on alginate microencapsulation of cell spheroids and culture in dimethyl sulfoxide (DMSO)-free conditions. A ratio of 2.9:1 hepatocyte-like to biliary-like cells was obtained in the 3D culture, with an improvement of 35.9 % in the hepatocyte differentiation when compared with two-dimensional (2D) cultures. The expression of the hepatic identity genes HNF4α and PXR in 3D cultures was comparable to 2D differentiated cultures, while the expression of homeostatic-associated genes albumin and carbamoyl phosphate synthase 1 was higher in 3D. Moreover, the spheroids presented a polarized organization, exhibiting an interconnected bile canalicular network and excretory functionality, assessed by specific activity of MRP2. Importantly, despite variability in basal gene expression levels, the activity of the phase I enzymes cytochrome P450 family 3, subfamily A, polypeptide 4 and cytochrome P450 family 1, subfamily A, polypeptide 2 upon induction was comparable to differentiated 2D cultures and albumin production and ammonia detoxification were enhanced in 3D. The presented model is suitable for toxicological applications, as it allows high throughput analysis of multiple compounds in a DMSO-free setting. Due to the high xenobiotic metabolism and maintenance of biosynthetic functions, the applicability of this model might be broadened to understand liver physiology and for bioartificial liver applications.
• Inflammatory cytokines promote the retrodifferentiation of tumor-derived hepatocyte-like cells to progenitor cells.Dubois-Pot-Schneider H, Fekir K, Coulouarn C, Glaise D, Aninat C, Jarnouen K, Le Guével R, Kubo T, Ishida S, Morel F, Corlu A
  Hepatology, Aug 2014
  Abstract : Human hepatocellular carcinoma (HCC) heterogeneity promotes recurrence and resistance to therapies. Recent studies have reported that HCC may be derived not only from adult hepatocytes and hepatoblasts but also hepatic stem/progenitors. In this context, HepaRG cells may represent a suitable cellular model to study stem/progenitor cancer cells and the retrodifferentiation of tumor-derived hepatocyte-like cells. Indeed, they differentiate into hepatocyte- and biliary-like cells. Moreover, tumor-derived HepaRG hepatocyte-like cells (HepaRG-tdHep) differentiate into both hepatocyte- and biliary-like cells through a hepatic progenitor. In this study we report the mechanisms and molecular effectors involved in the retrodifferentiation of HepaRG-tdHep into bipotent progenitors. Gene expression profiling was used to identify genomic changes during the retrodifferentiation of HepaRG-tdHep into progenitors. We demonstrated that gene expression signatures related to a poor-prognosis HCC subclass, proliferative progenitors or embryonic stem cells were significantly enriched in HepaRG progenitors derived from HepaRG-tdHep. HepaRG-tdHep retrodifferentiation is mediated by crosstalk between transforming growth factor beta 1 (TGFβ1) and inflammatory cytokine pathways (e.g., tumor necrosis factor alpha (TNFα) and interleukin 6 (IL6)). Signatures related to TNFα, IL6 and TGFβ activation pathways are induced within the first hour of retrodifferentiation. Moreover, specific activation or inhibition of these signaling pathways allowed us to determine that TNFα and IL6 contribute to the loss of hepatic-specific marker expression and that TGFβ1 induces an epithelial-to-mesenchymal transition of HepaRG-tdHep. Interestingly, the retrodifferentiation process is blocked by the histone deacetylase inhibitor trichostatin A, opening new therapeutic opportunities. Conclusion: Cancer progenitor cells (or metastasis progenitors) may derive from tumor-derived hepatocyte-like cells in an inflammatory environment that is frequently associated with HCC. (Hepatology 2014;).Copyright © 2014 American Association for the Study of Liver Diseases.
• Dual-colour fluorescence imaging to monitor CYP3A4 and CYP3A7 expression in human hepatic carcinoma HepG2 and HepaRG cells.Tsuji S, Kawamura F, Kubiura M, Hayashi A, Ohbayashi T, Kazuki Y, Chesné C, Oshimura M, Tada M
  PloS One, Aug 2014
  Abstract : Human adult hepatocytes expressing CYP3A4, a major cytochrome P450 enzyme, are required for cell-based assays to evaluate the potential risk of drug-drug interactions caused by transcriptional induction of P450 enzymes in early-phase drug discovery and development. However, CYP3A7 is preferentially expressed in premature hepatoblasts and major hepatic carcinoma cell lines. The human hepatocellular carcinoma cell line HepaRG possesses a high self-renewal capacity and can differentiate into hepatic cells similar to human adult hepatocytes in vitro. Transgenic HepaRG cells, in which the expression of fluorescent reporters is regulated by 35 kb regulatory elements of CYP3A4, have a distinct advantage over human hepatocytes isolated by collagenase perfusion, which are unstable in culture. Thus, we created transgenic HepaRG and HepG2 cells by replacing the protein-coding regions of human CYP3A4 and CYP3A7 with enhanced green fluorescent protein (EGFP) and DsRed reporters, respectively, in a bacterial artificial chromosome vector that included whole regulatory elements. The intensity of DsRed fluorescence was initially high during the proliferation of transgenic HepaRG cells. However, most EGFP-positive cells were derived from those in which DsRed fluorescence was extinguished. Comparative analyses in these transgenic clones showed that changes in the total fluorescence intensity of EGFP reflected fold changes in the mRNA level of endogenous CYP3A4. Moreover, CYP3A4 induction was monitored by the increase in EGFP fluorescence. Thus, this assay provides a real-time evaluation system for quality assurance of hepatic differentiation into CYP3A4-expressing cells, unfavourable CYP3A4 induction, and fluorescence-activated cell sorting-mediated enrichment of CYP3A4-expressing hepatocytes based on the total fluorescence intensities of fluorescent reporters, without the need for many time-consuming steps.
• Nicotine, cotinine, and &beta;-nicotyrine inhibit NNK-induced DNA-strand break in the hepatic cell line HepaRG.Ordonez P, Sierra AB, Camacho OM, Baxter A, Banerjee A, Waters D, Minet E
  Toxicol In Vitro, Jul 2014
  Abstract : Recent in vitro work using purified enzymes demonstrated that nicotine and/or a nicotine metabolite could inhibit CYPs (CYP2A6, 2A13, 2E1) involved in the metabolism of the genotoxic tobacco nitrosamine NNK. This observation raises the possibility of nicotine interaction with the mechanism of NNK bioactivation. Therefore, we hypothesized that nicotine or a nicotine metabolite such as cotinine might contribute to the inhibition of NNK-induced DNA strand breaks by interfering with CYP enzymes. The effect of nicotine and cotinine on DNA strand breaks was evaluated using the COMET assay in CYP competent HepaRG cells incubated with bioactive CYP-dependent NNK and CYP-independent NNKOAc (4-(acetoxymethylnitrosoamino)-1-(3-pyridyl)-1-butanone). We report a dose-dependent reduction in DNA damage in hepatic-derived cell lines in the presence of nicotine and cotinine. Those results are discussed in the context of the in vitro model selected.Copyright © 2014. Published by Elsevier Ltd.
• Antioxidant potential of Sutherlandia frutescens and its protective effects against oxidative stress in various cell cultures.Tobwala S, Fan W, Hines CJ, Folk WR, Ercal N
  BMC Complement Altern Med., Jul 2014
  Abstract : BACKGROUND:Sutherlandia frutescens (L.) R.Br. (SF) is a South African plant that is widely used to treat stress, infections, cancer, and chronic diseases, many of which involve oxidative stress. The aim of the study was to quantitatively assess the antioxidant potential of SF extracts in cell-free system as well as in cell lines.METHODS:Dried SF vegetative parts were extracted using six different solvents, and the extracts were assessed for total phenolic and flavonoid contents, total reducing power, iron chelating capacity, and free radical scavenging power, including, scavenging of hydroxyl radicals, superoxide anions, nitric oxide, and hydrogen peroxide. We further investigated the freeze-dried hot water extract of SF (SFE) to assess its effect against oxidative stress induced by tert-butyl hydroperoxide (t-BHP), an organic peroxide. Three different cell lines: Chinese hamster ovary (CHO), human hepatoma (HepaRG), and human pulmonary alveolar carcinoma (A549) cells, were employed to determine cell viability, intracellular reactive oxygen species (ROS) levels, and reduced to oxidized glutathione levels (GSH/GSSG).RESULTS:The results indicated that: (1) SF extracts have significant antioxidant potential that is dependent upon the nature of the extraction solvent and (2) SFE protects against tBHP-induced oxidative stress in cells by scavenging ROS and preserving intracellular GSH/GSSG.CONCLUSION:Oxidative stress is implicated in a number of disorders, and due to the public's concerns about synthetic antioxidants, various natural antioxidants are being explored for their therapeutic potential. Our findings support claims for S. frutescens being a promising adjunctive therapeutic for oxidative stress-related health problems.
• Liver X receptor &alpha; (LXR&alpha; /NR1H3) regulates differentiation of hepatocyte-like cells via reciprocal regulation of HNF4 &alpha;Chen KT, Pernelle K, Tsai YH, Wu YH, Hsieh JY, Liao KH, Guguen-Guillouzo C, Wang HW
  J Hepatol., Jul 2014
  Abstract : BACKGROUND & AIMS:Hepatocyte-like cells differentiated from different stem cell source are considered to have a range of possible therapeutic applications, including drug discovery, metabolism disease modeling and cell transplantation. However, little is known about how stem cells differentiate into mature and functional hepatocytes.METHODS:Using transcriptomic screening, a transcriptional factor, liver x receptor α (NR1H3) was identified as increased during HepaRG cell hepatogenesis; this protein is also up-regulated during embryonic stem cells and induced pluripotent stem cells differentiation.RESULTS:Overexpressing NR1H3 in human HepaRG cells promoted hepatic maturation, and these hepatocyte-like cells exhibit various functions associated with mature hepatocytes, including cytochrome P450 (CYP) enzyme activity, the secretion of urea and albumin, up regulation of hepatic-specific transcripts and an increase in glycogen storage. Importantly, the NR1H3-derived hepatocyte-like cells were able to rescued lethal fulminant hepatic failure using a non-obese diabetic severe combined immunodeficient mouse model.CONCLUSIONS:In this study, we found out that NR1H3 accelerates hepatic differentiation through a HNF4α-dependent reciprocal network. This contribute in hepatogenesis and is therapeutically beneficial to liver disease.Copyright © 2014. Published by Elsevier B.V.
• Rapid Prototyping of Chitosan-Coated Alginate Scaffolds Through the Use of a 3D Fiber Deposition Technique.Cristina Colosia, Marco Costantinia, Roberta Latinia, Serena Ciccarellia, Alessandra Stampellac, Andrea Barbettaa, Mara Massimib, Laura Conti Devirgiliisc, Mariella Dentini
  J Mat Chem, Jul 2014
  Abstract : Three dimensional, periodic scaffolds of chitosan-coated alginate are fabricated in a layer-by-layer fashion by rapid prototyping. A novel dispensing system based on two coaxial needles deliver simultaneously an alginate and calcium chloride solutions permitting the direct deposition of alginate fibers according to any designed pattern. Coating of the alginate fiber with chitosan and subsequent cross-linking with EDC and genipin assured the endurance of the scaffold in culture environment for a prolonged period of time. The cross-linking protocol adopted, imparted to the scaffold a hierarchical chemical structure as evidenced by Confocal Laser Microscopy and FTIR spectroscopy.  The core of the fibers making up the scaffold is represented by alginate chains cross-linked by ester bonds only, the periphery of the fiber are constituted by an inter-polyelectrolyte complex of alginate and chitosan cross-linked in all pair combinations. Fibers belonging to adjacent layers are glued together by the chitosan coating. Mechanical behavior of the scaffolds characterized by different layouts of deposition was determined revealing anisotropic  properties. The biocompatibility and capability of the scaffolds to sustain hepatocytes (HepaRG) cultures was demonstrated. Typical hepatic function such as albumin and urea secretion and induction of CYP3A4 enzyme activity following drug administration were excellent, thus proving the potentials of these constructs in monitoring liver specific function.
• Antidiabetic effects of the Cimicifuga racemosa extract Ze 450 in vitro and in vivo in ob/ob mice.Moser C, Vickers SP, Brammer R, Cheetham SC, Drewe J
  Phytomedicine, Jul 2014
  Abstract : INTRODUCTION:It was the aim of the present experiments to examine potential antidiabetic effects of the Cimicifuga racemosa extract Ze 450.METHODS:Ze 450 and some of its components (23-epi-26-deoxyactein, protopine and cimiracemoside C) were investigated in vitro for their effects on AMP-activated protein kinase (AMPK) compared to metformin in HepaRG cells. Ze 450 (given orally (PO) and intraperitonally (IP)), metformin (PO) and controls were given over 7 days to 68 male ob/ob mice. Glucose and insulin concentrations were measured at baseline and during an oral glucose tolerance test (OGTT).RESULTS:Ze 450 and its components activated AMPK to the same extent as metformin. In mice, Ze 450 (PO/IP) decreased significantly average daily and cumulative weight gain, average daily food and water intake, while metformin had no effect. In contrast to metformin, PO Ze 450 virtually did not change maximum glucose levels during OGTT, however, prolonged elimination. Ze 450 administered PO and IP decreased significantly post-stimulated insulin, whereas metformin did not. HOMA-IR index of insulin resistance improved significantly after IP and PO Ze 450 and slightly after metformin. In summary, the results demonstrate that Ze 450 reduced significantly body weight, plasma glucose, improved glucose metabolism and insulin sensitivity in diabetic ob/ob mice. In vitro experiments suggest that part of the effects may be related to AMPK activation.CONCLUSIONS:Ze 450 may have utility in the treatment of type 2 diabetes. However, longer term studies in additional animal models or patients with disturbed glucose tolerance or diabetes may be of use to investigate this further.Copyright © 2014 Elsevier GmbH. All rights reserved.
• Impact of Inflammation on Chlorpromazine-induced Cytotoxicity and Cholestatic Features in HepaRG Cells.Bachour-El Azzi P, Sharanek A, Abdel-Razzak Z, Antherieu S, Al-Attrache H, Savary C, Lepage S, Morel I, Labbe G, Guguen-Guillouzo C, Guillouzo A
  Drug Metab Dispos., Jul 2014
  Abstract : Several factors are thought to be implicated in the occurrence of idiosyncratic adverse drug reactions. The present work aimed to question as to whether inflammation is a determinant factor in hepatic lesions induced by chlorpromazine (CPZ) using the human HepaRG cell line. An inflammation state was induced by a 24 hour-exposure to pro-inflammatory cytokines IL-6 and IL-1β; then the cells were simultaneously treated with CPZ and/or cytokine for 24 hours or daily for 5 days. The inflammatory response was assessed by induction of C-reactive protein and IL-8 transcripts and proteins as well as inhibition of CPZ metabolism and down-regulation of CYP3A4 and CYP1A2 transcripts, two major CYPs involved in its metabolism. Most effects of co-treatments with cytokines and CPZ were amplified or only observed after 5 daily treatments; they mainly included increased cytotoxicity and overexpression of oxidative stress-related genes, decreased Na+-taurocholate cotransporting polypeptide (NTCP) mRNA levels and activity, a key transporter involved in bile acids uptake, and deregulation of several other transporters. However, CPZ-induced inhibition of taurocholic acid efflux and pericanalicular F-actin distribution were not affected. In addition, a time-dependent induction of phospholipidosis, was noticed in CPZ-treated cells, without obvious influence of the inflammatory stress. In summary, our results show that an inflammatory state induced by pro-inflammatory cytokines, increased cytotoxicity and enhanced some cholestatic features induced by the idiosyncratic drug CPZ in HepaRG cells. These changes, together with inhibition of CYP activities, could have important consequences if extrapolated to the in vivo situation.The American Society for Pharmacology and Experimental Therapeutics.
• Strategies to Inhibit Entry of HBV and HDV Into Hepatocytes.Urban S, Bartenschlager R, Kubitz R, Zoulim F
  Gastroenterology, Jul 2014
  Abstract : Although there has been much research into the pathogenesis and treatment of hepatitis B virus (HBV) and hepatitis D virus (HDV) infections, we still do not completely understand how these pathogens enter hepatocytes. This is because in vitro infection studies have only been performed in primary human hepatocytes. Development of a polarizable, HBV-susceptible human hepatoma cell line and studies of primary hepatocytes from Tupaia belangeri have provided important insights into the viral and cellular factors involved in virus binding and infection. The large envelope (L) protein on the surface of HBV and HDV particles has many different functions and is required for virus entry. The L protein mediates attachment of virions to heparan sulfate proteoglycans on the surface of hepatocytes. The myristoylated N-terminal preS1 domain of the L protein subsequently binds to the sodium taurocholate cotransporting polypeptide (NTCP, encoded by SLC10A1), the recently identified bona fide receptor for HBV and HDV. The receptor functions of NTCP and virus entry are blocked, in vitro and in vivo, by Myrcludex B, a synthetic N-acylated preS1 lipopeptide. Currently, the only agents available to treat chronic HBV infection target the viral polymerase, and no selective therapies are available for HDV infection. It is therefore important to study the therapeutic potential of virus entry inhibitors, especially when combined with strategies to induce immune-mediated killing of infected hepatocytes.Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.
• Detection of Dichlorvos Adducts in a Hepatocyte Cell Line.Bui-Nguyen TM, Dennis WE, Jackson DA, Stallings JD, Lewis JA
  J Proteome Res., Jun 2014
  Abstract : The toxicity of dichlorvos (DDVP), an organophosphate (OP) pesticide, classically results from modification of the serine in the active sites of cholinesterases. However, DDVP also forms adducts on unrelated targets such as transferrin and albumin, suggesting that DDVP could cause perturbations in cellular processes by modifying non-cholinesterase targets. Here, we identify novel DDVP-modified targets in lysed human hepatocyte-like cells (HepaRG) using a direct liquid chromatography-mass spectrometry (LC-MS) assay of cell lysates incubated with DDVP or using a competitive pull-down experiments with a biotin-linked organophosphorus compound (10-fluoroethoxyphosphinyl-N-biotinamidopentyldecanamide; FP-biotin), which competes with DDVP for similar binding sites. We show that DDVP forms adducts to several proteins important for the cellular metabolic pathways and differentiation, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin. We validated the results using purified proteins and enzymatic assays. The study not only identified novel DDVP-modified targets but also suggested that the modification directly inhibits the enzymes. The current approach provides information for future hypothesis-based studies to understand the underlying mechanism of toxicity of DDVP in non-neuronal tissues.
• Different Dose-Dependent Mechanisms are Involved in Early Cyclosporine A-Induced Cholestatic Effects in HepaRG Cells.Sharanek A, Bachour-El Azzi P, Al-Attrache H, Savary CC, Humbert L, Rainteau D, Guguen-Guillouzo C, Guillouzo A
  Toxicol Sci., Jun 2014
  Abstract : Mechanisms involved in drug-induced cholestasis in humans remain poorly understood. Although cyclosporine A (CsA) and tacrolimus (FK506) share similar immunosuppressive properties, only CsA is known to cause dose-dependent cholestasis. Here, we have investigated the mechanisms implicated in early cholestatic effects of CsA using the differentiated human HepaRG cell line. Inhibition of efflux and uptake of taurocholate was evidenced as early as 15 minutes and 1 hour respectively after addition of 10 μM CsA; it peaked at around 2 hours and was reversible. These early effects were associated with generation of oxidative stress and deregulation of cPKC pathway. At higher CsA concentrations (≥50 μM) alterations of efflux and uptake activities were enhanced and became irreversible, pericanalicular F-actin microfilaments were disorganized and bile canaliculi were constricted. These changes were associated with induction of endoplasmic reticulum stress that preceded generation of oxidative stress. Concentration-dependent changes were observed on total bile acid disposition, which were characterized by an increase and a decrease in culture medium and cells respectively, after a 24-hour treatment with CsA. Accordingly, genes encoding hepatobiliary transporters and bile acid synthesis enzymes were differently deregulated depending on CsA concentration. By contrast, FK506 induced limited effects only at 25-50 μM and did not alter bile canaliculi. Our data demonstrate involvement of different concentration-dependent mechanisms in CsA-induced cholestasis and point out a critical role of endoplasmic reticulum stress in the occurrence of the major cholestatic features.
• Reflection Coefficient S11 Related Measurement System for Label-Free Cell Seeding Analysis and Drug Testing in a Three-Dimensional (3D) Cell Culture Model.Lornejad-Schäfer MR, Hilber W, Schäfer C
  J Biosciences, Jun 2014
  Abstract : Context: With regard to two-dimensional (2D) cell cultures in flat dishes, most of the three-dimensional (3D) cell cultures have advantages and gain importance in life sciences. But, their higher complexity requires adaptation of existing methods or different measurement systems in order to achieve accurate results. Bio-Impedance spectroscopy (Bio-IS) is a label- free and non-destructive method that is considered for analysis of 3D cell models.  Purpose: We design a new Bio-IS measurement system for a 3D liver cell model to assess cell seeding and drug effects. Experiment Methods: We measured reflection coefficient S11 with differently (disc- and needle-like) shaped electrodes, which are in defined contact with the 3D liver cell model, metabolic activity (MTT test), and cytotoxicity (LDH assay). Results: We demonstrate that reflection coefficient S11 may be utilized to assess the 3D liver cell system in the frequency range of β-dispersion at 73 MHz only by means of the needle-like electrodes. This enables a reliable label-free and non-destructive determination of cell seeding and drug effects in the 3D liver cell culture model. The physical results have been verified by destructive biochemical methods (LDH and MTT assay).Conclusion: Our Bio-IS system for 3D liver cell models using needle-like electrodes enables label-ree and nondestructive determination of cell seeding and drug effects.
• Critical differences in toxicity mechanisms in induced pluripotent stem cell-derived hepatocytes, hepatic cell lines and primary hepatocytes.Sjogren AK, Liljevald M, Glinghammar B, Sagemark J, Li XQ, Jonebring A, Cotgreave I, Brolén G, Andersson TB
  Arch Toxicol, Jun 2014
  Abstract : Human-induced pluripotent stem cell-derived hepatocytes (hiPSC-Hep) hold great potential as an unlimited cell source for toxicity testing in drug discovery research. However, little is known about mechanisms of compound toxicity in hiPSC-Hep. In this study, modified mRNA was used to reprogram foreskin fibroblasts into hiPSC that were differentiated into hiPSC-Hep. The hiPSC-Hep expressed characteristic hepatic proteins and exhibited cytochrome P450 (CYP) enzyme activities. Next, the hiPSC-Hep, primary cryopreserved human hepatocytes (cryo-hHep) and the hepatic cell lines HepaRG and Huh7 were treated with staurosporine and acetaminophen, and the toxic responses were compared. In addition, the expression of genes regulating and executing apoptosis was analyzed in the different cell types. Staurosporine, an inducer of apoptosis, decreased ATP levels and activated caspases 3 and 7 in all cell types, but to less extent in Huh7. Furthermore, a hierarchical clustering and a principal component analysis (PCA) of the expression of apoptosis-associated genes separated cryo-hHep from the other cell types, while an enrichment analysis of apoptotic pathways identified hiPSC-Hep as more similar to cryo-hHep than the hepatic cell lines. Finally, acetaminophen induced apoptosis in hiPSC-Hep, HepaRG and Huh7, while the compound initiated a direct necrotic response in cryo-hHep. Our results indicate that for studying compounds initiating apoptosis directly hiPSC-Hep may be a good alternative to cryo-hHep. Furthermore, for compounds with more complex mechanisms of toxicity involving metabolic activation, such as acetaminophen, our data suggest that the cause of cell death depends on a balance between factors controlling death signals and the drug-metabolizing capacity.
• Effect and mechanisms of curdlan sulfate on inhibiting HBV infection and acting as an HB vaccine adjuvant.Li P, Tan H, Xu D, Yin F, Cheng Y, Zhang X, Liu Y, Wang F
  Carbohydr Polym, Jun 2014
  Abstract : In this study, the effect and mechanisms of curdlan sulfate (CS3) on hepatitis B virus (HBV) infection and promoting immune response of the mice immunized with recombinant hepatitis B surface protein (HBsAg) were investigated. The results showed that CS3 could inhibit HBV infection of HepG2 and HepaRG cells, especially the process of HBV particle binding to the cell surfaces. The surface plasmon response (SPR) technology indicated that CS3 could bind with recombinant HBsAg and the binding ability depended on the content of sulfate groups on the polysaccharide chains. Co-administration of CS3 to BALB/c mice immunized with HBsAg significantly enhanced the influx of macrophages and dendritic cells in spleen, increased antigen-specific CD4(+) and CD8(+) cell numbers, and promoted splenocyte proliferation. The titer of HBsAg-specific antibodies was also augmented by use of CS3 as a vaccine adjuvant. The higher expression of interferon (IFN)-γ, lower expression of interleukin (IL)-4, and higher IgG2a/IgG1 ratio within the anti-HBsAg antibodies in mice immunized with HBsAg plus CS3 than those in mice receiving HBsAg alone indicated that CS3 induced a shift toward a Th1-biased immune response. These results presented that CS3 could be developed as an immunotherapy agent or vaccine adjuvant for HBV infection treatment or prevention.
• Testing chemical carcinogenicity by using a transcriptomics HepaRG-Based model?Doktorova T, Yildirimman R, Ceelen L, Vilardell M, Vanhaecke T, Vinken M, Ates G, Heymans A, Gmuender H, Bort R, Corvi R, Phrakonkham P, Li R
  EXCLI Journal, Jun 2014
  Abstract : The EU FP6 project carcinoGENOMICS explored the combination of toxicogenomics and in vitro cell culture models for identifying organotypical genotoxic- and non-genotoxic carcinogen-specific gene signatures. Here the performance of its gene classifier, derived from exposure of metabolically competent human HepaRG cells to prototypical non-carcinogens (10 compounds) and hepatocarcinogens (20 compounds), is reported. Analysis of the data at the gene and the pathway level by using independent biostatistical approaches showed a distinct separation of genotoxic from non-genotoxic hepatocarcinogens and non-carcinogens (up to 88 % correct prediction). The most characteristic pathway responding to genotoxic exposure was DNA damage. Interlaboratory reproducibility was assessed by blindly testing of three compounds, from the set of 30 compounds, by three independent laboratories. Subsequent classification of these compounds resulted in correct prediction of the genotoxicants. As expected, results on the non genotoxic carcinogens and the non-carcinogens were less predictive. In conclusion, the combination of transcriptomics with the HepaRG in vitro cell model provides a potential weight of evidence approach for the evaluation of the genotoxic potential of chemical substances.
• Mechanisms of acetaminophen-induced cell death in primary human hepatocytes.Xie Y, McGill MR, Dorko K, Kumer SC, Schmitt TM, Forster J, Jaeschke H
  Toxicol Appl Pharmacol., Jun 2014
  Abstract : Acetaminophen (APAP) overdose is the most prevalent cause of drug-induced liver injury in western countries. Numerous studies have been conducted to investigate the mechanisms of injury after APAP overdose in various animal models; however, the importance of these mechanisms for humans remains unclear. Here we investigated APAP hepatotoxicity using freshly isolated primary human hepatocytes (PHH) from either donor livers or liver resections. PHH were exposed to 5mM, 10mM or 20mM APAP over a period of 48h and multiple parameters were assessed. APAP dose-dependently induced significant hepatocyte necrosis starting from 24h, which correlated with the clinical onset of human liver injury after APAP overdose. Interestingly, cellular glutathione was depleted rapidly during the first 3h. APAP also resulted in early formation of APAP-protein adducts (measured in whole cell lysate and in mitochondria) and mitochondrial dysfunction, indicated by the loss of mitochondrial membrane potential after 12h. Furthermore, APAP time-dependently triggered c-Jun N-terminal kinase (JNK) activation in the cytosol and translocation of phospho-JNK to the mitochondria. Both co-treatment and post-treatment (3h) with the JNK inhibitor SP600125 reduced JNK activation and significantly attenuated cell death at 24h and 48h after APAP. The clinical antidote N-acetylcysteine offered almost complete protection even if administered 6h after APAP and a partial protection when given at 15h. Conclusion: These data highlight important mechanistic events in APAP toxicity in PHH and indicate a critical role of JNK in the progression of injury after APAP in humans. The JNK pathway may represent a therapeutic target in the clinic.
• Interactions of Endosulfan and Methoxychlor Involving CYP3A4 and CYP2B6 in Human HepaRG Cells.Savary CC, Josse R, Guillet F, Bruyere A, Robin MA, Guillouzo A
  Drug Metab Dispo, May 2014
  Abstract : Humans are usually simultaneously exposed to several pesticides; consequently, combined actions between pesticides themselves or between pesticides and other chemicals need to be addressed in the risk assessment. Many pesticides are efficient activators of PXR and/or CAR, two major nuclear receptors that are also activated by other substrates. In the present work, we searched for interactions between endosulfan and methoxychlor, two organochlorine pesticides whose major routes of metabolism involve CAR- and PXR-regulated CYP3A4 and CYP2B6, and whose mechanisms of action remain poorly understood in humans. For this purpose, HepaRG cells were treated with both pesticides separately or in mixture for 24h or 2 weeks at concentrations relevant to human exposure levels. In combination they exerted synergistic cytotoxic effects. Whatever the duration of treatment both compounds increased CYP3A4 and CYP2B6 mRNA levels while they differently affected their corresponding activities. Endosulfan exerted a direct reversible inhibition of CYP3A4 activity that was confirmed in human liver microsomes. By contrast, methoxychlor induced this activity. The effects of the mixture on CYP3A4 activity were equal to the sum of those of each individual compound, suggesting an additive effect of each pesticide. Despite CYP2B6 activity was unchanged and increased with endosulfan and methoxychlor respectively, no change was observed with their mixture, supporting an antagonistic effect. Altogether, our data suggest that CAR and PXR activators endosulfan and methoxychlor can interact together and with other exogenous substrates in human hepatocytes. Their effects on CYP3A4 and CYP2B6 activities could have important consequences if extrapolated to the in vivo situation.The American Society for Pharmacology and Experimental Therapeutics.
• Splicing regulator SLU7 is essential for maintaining liver homeostasisElizalde M,Urtasun R,Akzona M,Latasa MU,Goni S,Garcia-Irigoyen O,Uriarte I,Segura V,Collantes M,Di Scala M,Lujambio A,Prieto J,Avila MA, Berasain C
  J Clinical Investigation, May 2014
  Abstract : A precise equilibrium between cellular differentiation and proliferation is fundamental for tissue homeostasis. Maintaining this balance is particularly important for the liver, a highly differentiated organ with systemic metabolic functions that is endowed with unparalleled regenerative potential. Carcinogenesis in the liver develops as the result of hepatocellular de-differentiation and uncontrolled proliferation. Here, we identified SLU7, which encodes a pre-mRNA splicing regulator that is inhibited in hepatocarcinoma, as a pivotal gene for hepatocellular homeostasis. SLU7 knockdown in human liver cells and mouse liver resulted in profound changes in pre-mRNA splicing and gene expression, leading to impaired glucose and lipid metabolism, refractoriness to key metabolic hormones, and reversion to a fetal-like gene expression pattern. Additionally, loss of SLU7 also increased hepatocellular proliferation and induced a switch to a tumor-like glycolytic phenotype. Slu7 governed the splicing and/or expression of multiple genes essential for hepatocellular differentiation, including serine/arginine-rich splicing factor 3 (Srsf3) and hepatocyte nuclear factor 4α (Hnf4α), and was critical for cAMP-regulated gene transcription. Together, out data indicate that SLU7 is central regulator of hepatocyte identity and quiescence.
• Generation of functional cholangiocyte-like cells from human pluripotent stem cells and HepaRG cells.Dianat N, Dubois-Pot-Schneider H, Steichen C, Desterke C, Leclerc P, Raveux A, Combettes L, Weber A, Corlu A, Dubart-Kupperschmitt A
  Hepatology, Apr 2014
  Abstract : Cholangiocytes are biliary epithelial cells, which, like hepatocytes, originate from hepatoblasts during embryonic development. In this study, we investigated the potential of human embryonic stem cells (hESCs) to differentiate into cholangiocytes and we report a new approach, which drives differentiation of hESCs toward the cholangiocytic lineage using feeder-free and defined culture conditions. After differentiation into hepatic progenitors, hESCs were differentiated further into cholangiocytes using growth hormone, epidermal growth factor, Interleukin-6 and then sodium taurocholate. These conditions also allowed us to generate cholangiocytes from HepaRG-derived hepatoblasts. hESC- and HepaRG-derived cholangiocyte-like cells expressed markers of cholangiocytes including Cytokeratin 7 and Osteopontin, and transcription factors SOX9 and Hepatocyte nuclear factor 6. The cells also displayed specific proteins important for cholangiocyte functions including cystic fibrosis transmembrane conductance regulator, secretin receptor and nuclear receptors. They formed primary cilia and also responded to hormonal stimulation by increase of intracellular Ca2+ . We demonstrated by integrative genomics that the expression of genes, which signed hESC- or HepaRG-cholangiocytes separates hepatocytic lineage from cholangiocyte lineage. When grown in a 3D matrix, cholangiocytes developed epithelial/apicobasal polarity and formed functional cysts and biliary ducts. In addition, we showed that cholangiocyte-like cells could also be generated from human induced pluripotent stem cells, demonstrating the efficacy of our approach with stem/progenitor cells of diverse origins. Conclusion: We have developed a robust and efficient method for differentiating pluripotent stem cells into cholangiocyte-like cells, which display structural and functional similarities with bile duct cells in normal liver. These cells will be useful for the in vitro study of the molecular mechanisms of bile duct development and have important potential for therapeutic strategies, including bioengineered liver approaches.
• Differentiation of liver progenitor cell line to functional organotypic cultures in 3D nanofibrillar cellulose and hyaluronan-gelatin hydrogels.Malinen MM, Kanninen LK, Corlu A, Isoniemi HM, Lou YR, Yliperttula ML, Urtti AO
  Biomaterials, Apr 2014
  Abstract : Physiologically relevant hepatic cell culture models must be based on three-dimensional (3D) culture of human cells. However, liver cells are generally cultured in two-dimensional (2D) format that deviates from the normal in vivo morphology. We generated 3D culture environment for HepaRG liver progenitor cells using wood-derived nanofibrillar cellulose (NFC) and hyaluronan-gelatin (HG) hydrogels. Culture of undifferentiated HepaRG cells in NFC and HG hydrogels induced formation of 3D multicellular spheroids with apicobasal polarity and functional bile canaliculi-like structures, structural hallmarks of the liver tissue. Furthermore, hepatobiliary drug transporters, MRP2 and MDR1, were localized on the canalicular membranes of the spheroids and vectorial transport of fluorescent probes towards the biliary compartment was demonstrated. Cell culture in 3D hydrogel supported the mRNA expression of hepatocyte markers (albumin and CYP3A4), and metabolic activity of CYP3A4 in the HepaRG cell cultures. On the contrary, the 3D hydrogel cultures with pre-differentiated HepaRG cells showed decreasing expression of albumin and CYP3A4 transcripts as well as CYP3A4 activity. It is concluded that NFC and HG hydrogels expedite the hepatic differentiation of HepaRG liver progenitor cells better than the standard 2D culture environment. This was shown as improved cell morphology, expression and localization of hepatic markers, metabolic activity and vectorial transport. The NFC and HG hydrogels are promising materials for hepatic cell culture and tissue engineering.
• Hepatitis B and D Viruses Exploit Sodium Taurocholate Co-transporting Polypeptide for Species-specific Entry into Hepatocytes.Ni Y, Lempp FA, Mehrle S, Nkongolo S, Kaufman C, Fälth M, Stindt J, Königer C, Nassal M, Kubitz R, Sültmann H, Urban S
  Gastroenterology, Apr 2014
  Abstract : BACKGROUND & AIMS: Hepatitis B and D viruses (HBV and HDV) are human pathogens with restricted host ranges and high selectivity for hepatocytes; the HBV L-envelope protein interacts specifically with a receptor on these cells. We aimed to identify this receptor and analyze whether it is the recently described sodium-taurocholate co-transporter polypeptide (NTCP), encoded by the SLC10A1 gene. METHODS: To identify receptor candidates we compared gene expression patterns between differentiated HepaRG cells, which express the receptor, and naïve cells, which do not. Receptor candidates were evaluated by small hairpin (sh)RNA silencing in HepaRG cells; the ability of receptor expression to confer binding and infection were tested in transduced hepatoma cell lines. We used interspecies domain swapping to identify motifs for receptor-mediated host discrimination of HBV and HDV binding and infection. RESULTS: Bioinformatic analyses of comparative expression arrays confirmed that NTCP, which was previously identified through a biochemical approach is a bona fide receptor for HBV and HDV. NTCPs from rat, mouse, and human bound MyrB, a peptide ligand derived from the HBV L-protein. MyrB blocked NTCP transport of bile salts; shRNA- mediated knockdown of NTCP in HepaRG cells prevented their infection by HBV or HDV. Expression of human but not mouse NTCP in HepG2 and HuH7 cells conferred a limited cell-type related and virus-dependent susceptibility to infection; these limitations were overcome when cells were cultured with DMSO. We identified 2 short sequence motifs in human NTCP that were required for species-specific binding and infection by HBV and HDV. CONCLUSIONS: Human NTCP is a specific receptor for HBV and HDV. NTCP-expressing cell lines can be efficiently infected with these viruses, and might be used in basic research and high-throughput screening studies. Mapping of motifs in NTCPs have increased our understanding of the species specificities of HBV and HDV, and could lead to small animal models for studies of viral infection and replication.
• Comparative study of the effects of anti-tuberculosis drugs and antiretroviral drugs on CYP3A4 and P-glycoprotein.Horita Y, Doi N
  Antimicrob Agents Chemother, Mar 2014
  Abstract : Predicting drug-drug interactions (DDIs) related to cytochrome P450 (CYP) such as CYP3A4 and one of the major drug transporters, P-glycoprotein (P-gp), is crucial for the development of future chemotherapeutic regimens to treat tuberculosis (TB) and TB/AIDS co-infection cases. We evaluated the effects of 30 anti-TB drugs, novel candidates, macrolides, and representative antiretroviral drugs on human CYP3A4 activity using a commercially available screening kit for CYP3A4 inhibitors and a human hepatocyte, HepaRG. Moreover, in order to estimate the interactions of these drugs with human P-gp, screening for substrates was performed. For some substrates, P-gp inhibition tests were carried out using P-gp-expressing MDCK cells. As a result, almost all the compounds showed the expected effects on human CYP3A4 both in the in vitro screening and HepaRG cells. Importantly, the unproved mechanisms of DDIs caused by WHO group-five drugs, thioamides, and p-aminosalicylic acid were elucidated. Intriguingly, CFZ exhibited weak inductive effects on CYP3A4 at more than 0.25 μM in HepaRG, while the inhibitory effect was observed at 1.69 μM in the in vitro screening, suggesting that CFZ autoinduces CYP3A4 in the human liver. Our method based on one of the pharmacokinetics parameters in humans provides more practical information associated with not only DDIs but also drug metabolism.
• Evaluation of Impedance-Based Label-Free Technology as a Tool for Pharmacology and Toxicology InvestigationsAtienzar FA, Gerets H, Tilmant K, Toussaint G, Dhalluin S
  Biosensors, Mar 2014
  Abstract : The use of label-free technologies based on electrical impedance is becoming more and more popular in drug discovery. Indeed, such a methodology allows the continuous monitoring of diverse cellular processes, including proliferation, migration, cytotoxicity and receptor-mediated signaling. The objective of the present study was to further assess the usefulness of the real-time cell analyzer (RTCA) and, in particular, the xCELLigence platform, in the context of early drug development for pharmacology and toxicology investigations. In the present manuscript, four cellular models were exposed to 50 compounds to compare the cell index generated by RTCA and cell viability measured with a traditional viability assay. The data revealed an acceptable correlation (ca. 80%) for both cell lines (i.e., HepG2 and HepaRG), but a lack of correlation (ca. 55%) for the primary human and rat hepatocytes. In addition, specific RTCA profiles (signatures) were generated when HepG2 and HepaRG cells were exposed to calcium modulators, antimitotics, DNA damaging and nuclear receptor agents, with a percentage of prediction close to 80% for both cellular models. In a subsequent experiment, HepG2 cells were exposed to 81 proprietary UCB compounds known to be genotoxic or not. Based on the DNA damaging signatures, the RTCA technology allowed the detection of ca. 50% of the genotoxic compounds (n = 29) and nearly 100% of the non-genotoxic compounds (n = 52). Overall, despite some limitations, the xCELLigence platform is a powerful and reliable tool that can be used in drug discovery for toxicity and pharmacology studies.
• Specific and Nonhepatotoxic Degradation of Nuclear Hepatitis B virus cccDNAJulie Lucifora, Yuchen Xia, Florian Reisinger, Ke Zhang, Daniela Stadler, Xiaoming Cheng, Martin F. Sprinzl, Herwig Koppensteiner, Zuzanna Makowska, Tassilo Volz, Caroline Remouchamps, Wen-Min Chou, Wolfgang E. Thasler, Norbert Hüser, David Durantel, T. J
  Science AAAS, Mar 2014
  Abstract : Current antiviral agents can control but not eliminate hepatitis B virus (HBV), because HBV establishes a stable nuclear covalently closed circular DNA (cccDNA). Interferon-α treatment can clear HBV but is limited by systemic side effects. We describe how interferon-α can induce specific degradation of the nuclear viral DNA without hepatotoxicity and propose lymphotoxin-β receptor activation as a therapeutic alternative. Interferon-α and lymphotoxin-β receptor activation up-regulated APOBEC3A and APOBEC3B cytidine deaminases, respectively, in HBV-infected cells, primary hepatocytes, and human liver needle biopsies. HBV core protein mediated the interaction with nuclear cccDNA, resulting in cytidine deamination, apurinic/apyrimidinic site formation, and finally cccDNA degradation that prevented HBV reactivation. Genomic DNA was not affected. Thus, inducing nuclear deaminases—for example, by lymphotoxin-β receptor activation—allows the development of new therapeutics that, in combination with existing antivirals, may cure hepatitis B.
• Menthol reduces the anticoagulant effect of warfarin by inducing cytochrome P450 2C expression.Hoshino M, Ikarashi N, Tsukui M, Kurokawa A, Naito R, Suzuki M, Yokobori K, Ochiai T, Ishii M, Kusunoki Y
  Eur J Pharm Sci, Mar 2014
  Abstract : Recently, it was reported that the anticoagulant effect of warfarin was reduced when patients receiving warfarin also took menthol. The purpose of this study is to reveal the mechanism of this reduced anticoagulant effect of warfarin from the pharmacokinetic point of view. Warfarin was orally administered to mice 24h after the administration of menthol for 2days, and the plasma warfarin concentration was measured. In the menthol administration group, the area under the blood concentration time curve of warfarin was decreased by approximately 25%, while total clearance was increased to 1.3-fold compared to the control group. The hepatic cytochrome P450 (CYP) 2C protein expression level in the menthol administration group was significantly increased compared to that in the control group. An increase in the nuclear translocation of constitutive androstane receptor (CAR) was also observed. The addition of menthol to human hepatic cells, HepaRG cells, caused an increase in the mRNA expression level of CYP2C9. The results of this study revealed that menthol causes an increase in CYP2C expression levels in the liver, which leads to an enhancement of warfarin metabolism, resulting in a decreased anticoagulant effect of warfarin. It was also suggested that menthol acted directly on the liver and increased the expression level of CYP2C by enhancing the nuclear translocation of CAR.
• Evaluation of 23 lots of commercially available cryopreserved hepatocytes for induction assays of human cytochromes P450.Yajima K, Uno Y, Murayama N, Uehara S, Shimizu M, Nakamura C, Iwasaki K, Utoh M, Yamazaki H
  Drug metab dispo, Feb 2014
  Abstract : Due to the importance of in vitro P450 induction assay to assess the possible drug-drug interaction events, the recent US FDA draft guidance and EMA guideline recommend to assess P450 induction using fresh or cryopreserved hepatocytes at mRNA level and/or enzyme activity level. Although cryopreserved hepatocytes are commercially available for P450 induction assays, feasibility and practicability of these hepatocytes have not been fully investigated. In this study, a total 23 lots of human cryopreserved hepatocytes were treated with 3 typical inducers (omeprazole, phenobarbital, and rifampicin), and induction of CYP1A2, CYP2B6, and CYP3A4 enzyme activity was measured. In 8 of these 23 hepatocyte lots, induction of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP3A4 mRNA were also analyzed. The results revealed that CYP1A2, CYP2B6, and CYP3A4 were induced (> 2.0-fold) by omeprazole, phenobarbital, and rifampicin, respectively, in all the hepatocyte lots tested at enzyme activity level (23 lots) and mRNA level (8 lots). In contrast, of the 8 hepatocyte lots treated with rifampicin, CYP2C8 and CYP2C9 mRNA were not induced in 5 and 2 hepatocyte lots, respectively, and CYP2C19 mRNA was not induced in any of the 8 hepatocyte lots tested. These results suggest that induction of CYP1A2, CYP2B6, and CYP3A4 can be readily assessed, but evaluation for CYP2C mRNA induction might not be feasible, using commercially available human cryopreserved hepatocytes.
• NTCP and Beyond: Opening the Door to Unveil Hepatitis B Virus Entry.Watashi K, Urban S, Li W, Wakita T
  Int J Mol Sci., Feb 2014
  Abstract : Chronic hepatitis B virus (HBV) infection, affecting approximately 240 million people worldwide, is a major public health problem that elevates the risk of developing liver cirrhosis and hepatocellular carcinoma. Given that current anti-HBV drugs are limited to interferon-based regimens and nucleos(t)ide analogs, the development of new anti-HBV agents is urgently needed. The viral entry process is generally an attractive target implicated in antiviral strategies. Using primary cells from humans and Tupaia belangeri, as well as HepaRG cells, important determinants of viral entry have been achieved. Recently, sodium taurocholate cotransporting polypeptide (NTCP) was identified as an HBV entry receptor and enabled the establishment of a susceptible cell line that can efficiently support HBV infection. This finding will allow a deeper understanding of the requirements for efficient HBV infection, including the elucidation of the molecular entry mechanism. In addition, pharmacological studies suggest that NTCP is able to serve as a therapeutic target. This article summarizes our current knowledge on the mechanisms of HBV entry and the role of NTCP in this process.
• Persistence and activation of malaria hypnozoites in long-term primary hepatocyte cultures.Laurent Dembélé, Jean-François Franetich, Audrey Lorthiois, Audrey Gego, Anne-Marie Zeeman, Clemens H M Kocken, Roger Le Grand, Nathalie Dereuddre-Bosquet, Geert-Jan van Gemert, Robert Sauerwein, Dominique Mazier
  Nat med, Feb 2014
  Abstract : Malaria relapses, resulting from the activation of quiescent hepatic hypnozoites of Plasmodium vivax and Plasmodium ovale, hinder global efforts to control and eliminate malaria. As primaquine, the only drug capable of eliminating hypnozoites, is unsuitable for mass administration, an alternative drug is needed urgently. Currently, analyses of hypnozoites, including screening of compounds that would eliminate them, can only be made using common macaque models, principally Macaca rhesus and Macaca fascicularis, experimentally infected with the relapsing Plasmodium cynomolgi. Here, we present a protocol for long-term in vitro cultivation of P. cynomolgi-infected M. fascicularis primary hepatocytes during which hypnozoites persist and activate to resume normal development. In a proof-of-concept experiment, we obtained evidence that exposure to an inhibitor of histone modification enzymes implicated in epigenetic control of gene expression induces an accelerated rate of hypnozoite activation. The protocol presented may further enable investigations of hypnozoite biology and the search for compounds that kill hypnozoites or disrupt their quiescence.
• Adverse outcome pathway-based screening strategies for an animal-free safety assessment of chemicals.Landesmann B, Mennecozzi M, Berggren E, Whelan M
  Altern Lab Anim, Feb 2014
  Abstract : Currently, the assessment of risk to human health from exposure to manufactured chemicals is mainly based on experiments performed on living animals (in vivo). Substantial efforts are being undertaken to develop alternative solutions to in vivo toxicity testing. This new paradigm, based on the Mode-of-Action (MoA) framework, postulates that any adverse human health effect caused by exposure to an exogenous substance can be described by a series of causally-linked biochemical or biological key events with measurable parameters. The elaboration of mechanistic knowledge through literature research is necessary for a MoA-driven design of integrated testing strategies using in vitro methods for in vivo predictions. The objective of our ongoing research is to demonstrate the feasibility of an integrated approach to predict human toxicity following the Adverse Outcome Pathway (AOP) framework. In our previous work on MoA with the HepaRG cell model, we developed a strategy to identify chemicals that were hepatotoxic. This pioneered an innovative way of using data from in vitro experiments to group chemicals based on their MoA, which is likely to be an important step in a toxicity testing strategy.
• PPAR agonists reduce steatosis in oleic acid-overloaded HepaRG cells.Rogue A, Anthérieu S, Vluggens A, Umbdenstock T, Claude N, Moureyre-Spire C, Weaver R, Guillouzo A
  Nature Med, Feb 2014
  Abstract : Although non-alcoholic fatty liver disease (NAFLD) is currently the most common form of chronic liver disease there is no pharmacological agent approved for its treatment. Since peroxisome proliferator-activated receptors (PPARs) are closely associated with hepatic lipid metabolism, they seem to play important roles in NAFLD. However, the effects of PPAR agonists on steatosis that is a common pathology associated with NAFLD, remain largely controversial. In this study, the effects of various PPAR agonists, i.e. fenofibrate, bezafibrate, troglitazone, rosiglitazone, muraglitazar and tesaglitazar on oleic acid-induced steatotic HepaRG cells were investigated after a single 24-hour or 2-week repeat treatment. Lipid vesicles stained by Oil-Red O and triglycerides accumulation caused by oleic acid overload, were decreased, by up to 50%, while fatty acid oxidation was induced after 2-week cotreatment with PPAR agonists. The greatest effects on reduction of steatosis were obtained with the dual PPAR?/γ agonist muraglitazar. Such improvement of steatosis was associated with up-regulation of genes related to fatty acid oxidation activity and down-regulation of many genes involved in lipogenesis. Moreover, modulation of expression of some nuclear receptor genes, such as FXR, LXRα and CAR, which are potent actors in the control of lipogenesis, was observed and might explain repression of de novo lipogenesis. Conclusion: Altogether, our in vitro data on steatotic HepaRG cells treated with PPAR agonists correlated well with clinical investigations, bringing a proof of concept that drug-induced reversal of steatosis in human can be evaluated in in vitro before conducting long-term and costly in vivo studies in animals and patients.
• CYP3A4 activity reduces the cytotoxic effects of okadaic acid in HepaRG cells.Kittler K,Fessard V,Maul R,Hurtaud-Pessel D
  Arch Toxicol., Feb 2014
  Abstract : The biotoxin okadaic acid (OA), produced by dinoflagellates in marine environment, can accumulate in sponges and shellfish. Consumption of contaminated shellfish induces acute toxic effects such as diarrhea, nausea, vomiting, and abdominal pain. CYP3A4, one of the most important human xenobiotic metabolizing enzymes, is supposed to be involved in the metabolism of OA. Aim of our study was to evaluate the role of CYP3A4 in OA in vitro metabolism as well as in cell cytotoxicity in parallel. Therefore, a metabolic competent HepaRG cell line was exposed to OA with and without addition of the CYP3A4 inhibitor ketoconazole. Without the inhibitor, two mono-hydroxylated metabolites could be identified, whereas in its presence, no metabolites could be detected. Confirmation of the formed metabolites was accomplished by measuring the exact masses and investigating the fragmentation pattern. Data obtained from cytotoxicity assays showed that OA cytotoxicity is reduced when CYP3A4 is active. Thus, hydroxylation appears to be a crucial step for metabolic OA detoxification.
• Thalidomide Increases Human Hepatic Cytochrome P450 3A Enzymes by Direct Activation of Pregnane X Receptor.Murayama N, Beuningen R, Suemizu H, Guguen-Guillouzo C, Shibata N, Yajima K, Utoh M, Chesné C, Yamazaki H, Shimizu M
  Chem Res Toxicol, Jan 2014
  Abstract : Heterotropic cooperativity of human cytochrome P450 (P450) 3A4/3A5 by the teratogen thalidomide was recently demonstrated using the model substrate midazolam in various in vitro and in vivo models (Yamazaki, H. et al. (2013) Chem. Res. Toxicol. 26, 486-489). Chimeric mice with humanized liver also displayed enhanced midazolam clearance upon pre-treatment with orally administered thalidomide, presumably because of human P450 3A induction. In the current study, we further investigated the regulation of human hepatic drug metabolizing enzymes. Thalidomide enhanced levels of P450 3A4 and 2B6 mRNA, protein expression, and/or oxidation activity in human hepatocytes, indirectly suggesting activation of upstream transcription factors involved in detoxication, e.g. the nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR). A key event after ligand binding is an alteration of nuclear receptor conformation and recruitment of co-regulator proteins that alter chromatin accessibility of target genes. To investigate direct engagement and functional alteration of PXR and CAR by thalidomide, we utilized a peptide microarray with 154 co-regulator-derived nuclear receptor-interaction motifs and co-regulator- and nuclear receptor boxes, which serves as a sensor for nuclear receptor conformation and activity status as a function of ligand. Thalidomide and its human proximate metabolite 5-hydroxythalidomide displayed significantmodulation of co-regulator interaction with PXR and CAR ligand-binding domains, similar to established agonists for these receptors. These results collectively suggest that thalidomide acts as a ligand for PXR and CAR and causes enzyme induction leading to increased P450 enzyme activity. The possibilities of drug interactions during thalidomide therapy in humans require further evaluation.
• Absence of in vitro genotoxicity potential of the mycotoxin deoxynivalenol in bacteria and in human TK6 and HepaRG cell lines.Takakura N, Nesslany F, Fessard V, Hegarat LL
  Food Chem Toxicol., Jan 2014
  Abstract : Deoxynivalenol (DON) is the major mycotoxin detected in cereal foods and a risk for human health following DON ingestion could not be excluded due to high level exposure. In this light, the hazard of DON must be carefully evaluated. Therefore, the aim of this study is to perform in vitro genotoxicity tests with DON using the Salmonella typhimurium reverse mutation assay (Ames'test), the comet assay and the micronucleus test in accordance with the OECD test guideline 487 in two human cell lines: the lymphoblastoid TK6 and the hepatoma HepaRG cells. DON gave negative results in the Ames'test performed, both with and without rat liver S9 on three strains TA98, TA100 and TA102. DON elicited cytotoxicity in TK6 and HepaRG cells but did not induce primary DNA damage. DON failed also to induce MN formation in TK6 cells with or without human and rat liver S9. After 24h of treatment, DON induced micronucleus formation in TK6 cells but only at concentrations producing more than 55±5% cytotoxicity. In HepaRG cells, DON highly increased the caspase-3/7 activity but no micronucleus induction was observed. Taken together, our results suggest that DON could be considered as a non in vitro genotoxin.
• Viral Entry of Hepatitis B and D Viruses and Bile Salts Transportation Share Common Molecular Determinants on Sodium Taurocholate Cotransporting Polypeptide Yan H, Peng B, Liu Y, Xu G, He W, Ren B, Jing Z, Sui J, Li W
  J Virol, Jan 2014
  Abstract : The liver bile acids transporter sodium taurocholate cotransporting polypeptide (NTCP) is responsible for the majority of sodium-dependent bile salts uptake by hepatocytes. NTCP also functions as a cellular receptor for viral entry of hepatitis B virus (HBV) and hepatitis D virus (HDV) through a specific interaction between NTCP and the pre-S1 domain of HBV large envelope protein. However, it remains unknown if these two functions of NTCP are independent or if they interfere with each other. Here we show that binding of the pre-S1 domain to human NTCP blocks taurocholate uptake by the receptor; conversely, some bile acid substrates of NTCP inhibit HBV and HDV entry. Mutations of NTCP residues critical for bile salts binding severely impair viral infection by HDV and HBV; to a lesser extent, the residues important for sodium binding also inhibit viral infection. The mutation S267F, corresponding to a single nucleotide polymorphism (SNP) found in about 9% of the East Asian population,renders NTCP without either taurocholate transporting activity or the ability to support HBV or HDV infection in cell culture. These results demonstrate that molecular determinants critical for HBV and HDV entry overlap with that for bile salts uptake by NTCP, indicating that viral infection may interfere with the normal function of NTCP, and bile acids and their derivatives hold the potential for further development into antiviral drugs.
• Performance of comet and micronucleus assays in metabolic competent HepaRG cells to predict in vivo geno toxicity.Le Hegarat L, Mourot A, Huet S, Vasseur L, Camus S, Chesne C, Fessard V.
  Toxcixol SCI, Jan 2014
  Abstract : Genetic toxicity information is critical for the safety assessment of all xenobiotics. In the absence of carcinogenicity data, genetic toxicity studies may be used to draw conclusions about the carcinogenicity potential of chemicals. However, current in vitro assays have many limitations as they produce a high rate of irrelevant positive data and possible false negative data due to the weakness of the in vitro models used. Based on the knowledge that the majority of human genotoxic carcinogens require metabolic activation to become genotoxic, it is necessary to develop in vitro cell models that mimic human liver metabolism to replace the use of liver S9 fraction, which, though helpful for predicting the potential carcinogenicity of chemicals in rodents, is questionable in humans. We therefore investigate whether the recently described human hepatoma HepaRG cells, which express the major characteristics of liver functions similarly to primary human hepatocytes, could be a suitable model for human genotoxicity assessment. We determine the performance of comet and micronucleus assays in HepaRG cells to predict in vivo genotoxins based on the list of compounds published by ECVAM. Twenty compounds were tested in HepaRG cells with comet and micronucleus assays over a 24-hour period. The specificity, the sensitivity and the accuracy of the two tests were determined. We found that the comet assay had higher specificity (100%) than the MN test (80%), whereas the latter was far more sensitive (73%) than the former (44%), resulting nonetheless in an accuracy of 72% for the comet assay and 75% for the MN test. Taken together, our data suggest that the HepaRG cell line can be of use in genetic toxicology and that efforts to develop competent human liver cell models should be increased.
• Human Placental Lactogen Induces CYP2E1 Expression Via PI 3-kinase Pathway in Female Human Hepatocytes.Lee JK, Chung HJ, Fischer L, Fischer J, Gonzalez FJ, Jeong H.
  Drug Metab Dispos., Jan 2014
  Abstract : Predictive in vitro models alternative to in vivo animal will have a significant impact in toxicology. Conventional 2D models do not reflect the complexity of a 3D organ resulting in discrepancies between experimental in vitro and in vivo data. Using 3D HepaRG organotypic cultures we tested four drugs (aflatoxin B1, amiodarone, valproic acid and chlorpromazine) for toxic effects and compared the results with 2D HepaRG and HepG2 cultures. We show that 3D HepaRG cultures are more sensitive than the other tested cultures to aflatoxin B1 which is only toxic upon metabolic activation in the liver. We observed that CYP3A4 activity is higher in the 3D HepaRG cultures compared to the 2D HepaRG cultures. Furthermore, we investigated repeated dose toxicity of chlorpromazine and assessed its effects on glucose and lactate metabolism. Sub-toxic concentrations of chlorpromazine induced significant metabolic changes in both 2D and 3D HepaRG cultures upon acute and repeated dose (3 doses) exposure. In summary, our data support the hypothesis that 3D cell culture models better mimic the in vivo tissue and improve cellular functionality. The 3D HepaRG organotypic cultures represent a high throughput system for drug toxicity screening. This system is therefore a promising tool in preclinical testing of human relevance which can allow reducing and/or replacing animal testing for drug adverse effects.
• Regulation of Hepatic Drug Transporter Activity and Expression by Organochlorine Pesticides.Bucher S, Le Vee M, Jouan E, Fardel O
  J Biochem Mol Toxicol, Jan 2014
  Abstract : Organochlorine (OC) pesticides constitute a major class of persistent and toxic organic pollutants, known to modulate drug-detoxifying enzymes. In the present study, OCs were demonstrated to also alter the activity and expression of human hepatic drug transporters. Activity of the sinusoidal influx transporter OCT1 (organic cation transporter 1) was thus inhibited by endosulfan, chlordane, heptachlor, lindane, and dieldrine, but not by dichlorodiphenyltrichloroethane isomers, whereas those of the canalicular efflux pumps MRP2 (multidrug resistance-associated protein 2) and BCRP (breast cancer resistance protein) were blocked by endosulfan, chlordane, heptachlor, and chlordecone; this latter OC additionally inhibited the multidrug resistance gene 1 (MDR1)/P-glycoprotein (P-gp) activity. OCs, except endosulfan, were next found to induce MDR1/P-gp and MRP2 mRNA expressions in hepatoma HepaRG cells; some of them also upregulated BCRP. By contrast, expression of sinusoidal transporters was not impaired (organic anion-transporting polypeptide (OATP) 1B1 and OATP2B1) or was downregulated (sodium taurocholate co-transporting polypeptide (NTCP) and OCT1). Such regulations of drug transporter activity and expression, depending on the respective nature of OCs and transporters, may contribute to the toxicity of OC pesticides.

2013

• Cyclosporin A and its analogs inhibit hepatitis B virus entry into cultured hepatocytes through targeting a membrane transporter NTCP.Watashi K, Sluder A, Daito T, Matsunaga S, Ryo A, Nagamori S, Iwamoto M, Nakajima S, Tsukuda S, Borroto-Esoda K, Sugiyama M, Tanaka Y, Kanai Y
  Hepatology, Dec 2013
  Abstract : Chronic hepatitis B virus (HBV) infection is a major public health problem worldwide. Although nucleos(t)ide analogs inhibiting viral reverse transcriptase are clinically available as anti-HBV agents, emergence of drug resistant virus highlights the need for new anti-HBV agents interfering with other targets. Here we report that cyclosporin A (CsA) can inhibit HBV entry into cultured hepatocytes. The anti-HBV effect of CsA was independent of binding to cyclophilin and calcineurin. Rather, blockade of HBV infection correlated with the ability to inhibit the transporter activity of sodium taurocholate cotransporting polypeptide (NTCP). We also found that HBV infection susceptible cells, differentiated HepaRG cells and primary human hepatocytes expressed NTCP, while non-susceptible cell lines did not. A series of compounds targeting NTCP could inhibit HBV infection. CsA inhibited the binding between NTCP and large envelope protein in vitro. Evaluation of CsA analogs identified a compound with higher anti-HBV potency, having an IC₅₀ < 0.2 μM. This study provides a proof of concept for the novel strategy to identify anti-HBV agents by targeting the candidate HBV receptor, NTCP, utilizing CsA as a structural platform.
• Cellular impact of combinations of endosulfan, atrazine, and chlorpyrifos on human primary hepatocytes and HepaRG cells after short and chronic exposures.Nawaz A, Razpotnik A, Rouimi P, de Sousa G, Cravedi JP, Rahmani R
  Cell Biol Toxicol, Dec 2013
  Abstract : Chronic exposure to low doses of pesticides present in the environment is increasingly suspected to cause major health issues to humans. Toxicological evaluations become more complex when the exposure concerns chemical combinations. Atrazine, chlorpyrifos, and endosulfan are pesticides used worldwide in agriculture and are therefore currently found at residual levels in food and the environment, even in countries in which they are now banned. Our study aimed to use Real-Time Cell Impedance Analyzer to investigate changes in phenotypical status of primary human hepatocytes and differentiated HepaRG cells induced by short and chronic exposures to these three chemicals. In contrast to the traditionally used endpoint cytotoxicity test, this technology allows kinetic measurements in real-time throughout the entire experiment. Our data show significantly higher cytotoxic effects of mixtures as compared to individual pesticides and a greater susceptibility of human hepatocytes as compared to HepaRG to short-term exposure (24 h). Repeated exposure over 2 weeks to endosulfan and endosulfan-containing mixture induced HepaRG cell death in a time- and dose-dependent manner. Of the typical genes involved in metabolism and cell-response to xenobiotics, we found an exposure time- and condition-dependent deregulation of the expression of CYP3A4 and UGT1A in HepaRG cells exposed to low doses of pesticides and mixtures. Our data demonstrate the usefulness of real-time cell monitoring in long-term toxicological evaluations of co-exposure to xenobiotics. In addition, they support but at the same time highlight certain limitations in the use of HepaRG cells as the gold standard liver cell model in toxicity studies.
• Evaluation and identification of hepatitis B virus entry inhibitors using HepG2 cells overexpressing a membrane transporter NTCP.Iwamoto M, Watashi K, Aly HH, Fukasawa M, Fujimoto A, Suzuki R, Aizaki H, Ito T, Koiwai O, Kusuhara H, Wakita T
  Biochem Biophys Res Commun, Dec 2013
  Abstract : Hepatitis B virus (HBV) entry has been analyzed using infection-susceptible cells, including primary human hepatocytes, primary tupaia hepatocytes, and HepaRG cells. Recently, the sodium taurocholate cotransporting polypeptide (NTCP) membrane transporter was reported as an HBV entry receptor. In this study, we established a strain of HepG2 cells engineered to overexpress the human NTCP gene (HepG2-hNTCP-C4 cells). HepG2-hNTCP-C4 cells were shown to be susceptible to infection by blood-borne and cell culture-derived HBV. HBV infection was facilitated by pretreating cells with 3% dimethyl sulfoxide permitting nearly 50% of the cells to be infected with HBV. Knockdown analysis suggested that HBV infection of HepG2-hNTCP-C4 cells was mediated by NTCP. HBV infection was blocked by an anti-HBV surface protein neutralizing antibody, by compounds known to inhibit NTCP transporter activity, and by cyclosporin A and its derivatives. The infection assay suggested that cyclosporin B was a more potent inhibitor of HBV entry than was cyclosporin A. Further chemical screening identified oxysterols, oxidized derivatives of cholesterol, as inhibitors of HBV infection. Thus, the HepG2-hNTCP-C4 cell line established in this study is a useful tool for the identification of inhibitors of HBV infection as well as for the analysis of the molecular mechanisms of HBV infection.
• Impact of isomalathion on malathion cytotoxicity and genotoxicity in human HepaRG cells.Josse R, Sharanek A, Savary C, Guillouzo A
  Chem Biol Interact, Dec 2013
  Abstract : Isomalathion is a major impurity of technical grade malathion, one of the most abundantly applied insecticides; however little is known about its hepatotoxicity. In the present study, cytotoxicity and genotoxicity of malathion and isomalathion either individually or in combination, were assessed using the metabolically competent human liver HepaRG cell line. Isomalathion reduced cell viability starting at a 100 lM concentration after a 24 h exposure. It also significantly induced caspase-3 activity in a dosedependent manner starting at 5 lM. On the contrary, even at concentrations as high as 500 lMmalathion affected neither cell viability nor caspase-3 activity. Moreover, co-exposure of both compounds resulted in decreased toxicity of isomalathion. By contrast, malathion and isomalathion either separately or in combination, slightly induced micronuclei formation at low concentrations and had additive genotoxic effects when combined at 25 lM. Individually or combined isomalathion directly inhibited activity of carboxyesterases which are involved in detoxication of malathion. In addition, transcripts of CYP2B6 and CYP3A4, two CYPs responsible for malathion phase I metabolism, were strongly induced by the mixture while isomalathion alone only moderately decreased CYP1A2 and increased CYP2B6 transcripts. However, these CYPs were not altered at the protein or activity levels. Taken altogether, our results showed that isomalathion was much more cytotoxic than malathion while both compounds had comparable genotoxic effects in HepaRG hepatocytes at low concentrations and brought further support to the importance of considering impurities and interactions during evaluation of health risks of pesticides.
• Monoclonal antibodies to various epitopes of HBs antigen inhibit HBV infection.Golsaz Shirazi F, Mohammadi H, Amiri MM, Singethan K, Xia Y, Bayat AA, Bahadori M, Rabbani H, Jeddi-Tehrani M, Protzer U, Shokri F
  J Gastroenterol Hepatol, Dec 2013
  Abstract : BACKGROUND AND AIM: Antibodies against the "a" determinant of hepatitis B surface antigen (HBsAg) are able to neutralize circulating HBV particles and to prevent HBV infection. It has been proposed, that a single amino acid exchange may allow the virus to escape the immune response. We used a set of monoclonal antibodies (MAbs) to investigate whether a single mutation may account for virus escape from humoral immunity. METHODS: Nine murine HBsAg specific MAbs were raised. Reactivity of all antibodies with 14 recombinant mutants of HBsAg was assessed by ELISA. HBV infection of HepaRG cells was used to evaluate viral neutralization capacity of MAbs in vitro. RESULTS: All MAbs were able to inhibit the establishment of HBV infection in a dose dependent fashion, but recognition of HBsAg variants varied. The MAbs were classified into 3 subgroups based on their pattern of reactivity to the HBsAg variants. Accordingly, three MAbs showed weak reactivity (
• A bispecific antibody against two different epitopes on hepatitis B surface antigen has potent hepatitis B virus neutralizing activity.Tan W,Meng Y,Li H,Chen Y,Han S,Zeng J,Huang A,Li B,Zhang Y,Guo Y
  MAbs., Nov 2013
  Abstract : Treatment of chronic hepatitis B virus (HBV) infection with interferon and viral reverse transcriptase inhibitor regimens results in poor viral clearance, loss of response, and emergence of drug-resistant mutant virus strains. These problems continue to drive the development of new therapeutic approaches to combat HBV. Here, we engineered a bispecific antibody using two monoclonal antibodies cloned from hepatitis B surface antigen (HBsAg)-specific memory B cells from recombinant HBsAg-vaccinated healthy volunteers. Next, we evaluated its efficacy in neutralizing HBV in HepaRG cells. This bispecific antibody, denoted as C4D2-BsAb, had superior HBV-neutralizing activity compared with the combination of both parental monoclonal antibodies, possibly through steric hindrance or induction of HBsAg conformational changes. Moreover, C4D2-BsAb has superior endocytotic characteristics into hepatocytes, which inhibits the secretion of HBsAg. These results suggest that the anti-HBsAg bispecific antibody may be an effective treatment method against HBV infection.
• Cyclosporin A inhibits Hepatitis B and Hepatitis D Virus entry by Cyclophilin-independent interference with the NTCP receptor.Nkongolo S, Lempp FA, Ni Y, Kaufman C, Lindner T, Esser-Nobis K, Lohmann V, Mier W, Mehrle S, Urban S
  J Hepatol, Nov 2013
  Abstract : BACKGROUND & AIMS: Chronic hepatitis B and hepatitis D are global health problems caused by the human hepatitis B and hepatitis D virus. The myristoylated preS1 domain of the large envelope protein mediates specific binding to hepatocytes by sodium taurocholate co-transporting polypeptide (NTCP). NTCP is a bile salt transporter known to be inhibited by cyclosporin A. This study aimed to characterize the effect of cyclosporin A on HBV/HDV infection.METHODS: HepaRG cells, primary human hepatocytes, and susceptible NTCP-expressing hepatoma cell lines were applied for infection experiments. The mode of action of cyclosporin A was studied by comparing the effect of different inhibitors, cyclophilin A/B/C-silenced cell lines as well as NTCP variants and mutants. Bile salt transporter and HBV receptor functions were investigated by taurocholate uptake and quantification of HBVpreS binding.RESULTS: Cyclosporin A inhibited hepatitis B and D virus infections during and - less pronounced - prior to virus inoculation. Binding of HBVpreS to NTCP was blocked by cyclosporin A concentrations at 8μM. An NTCP variant deficient in HBVpreS binding but competent for bile salt transport showed resistance to cyclosporin A. Silencing of cyclophilins A/B/C did not abrogate transporter and receptor inhibition. In contrast, tacrolimus, a cyclophilin-independent calcineurin inhibitor, was inactive.CONCLUSIONS: HBV and HDV entry via sodium taurocholate co-transporting polypeptide is inhibited by cyclosporin A. The interaction between the drug and the viral receptor is direct and overlaps with a functional binding site of the preS1 domain, which mediates viral entry.
• Studies on the Role of Metabolic Activation in Tyrosine Kinase Inhibitor (TKI)-dependent Hepatotoxicity: Induction of CYP3A4 Enhances the Cytotoxicity of Lapatinib in HepaRG Cells.Hardy KD, Wahlin MD, Papageorgiou I, Unadkat J, Rettie AE, Nelson SD
  Drug metab Dispos, Nov 2013
  Abstract : Idiosyncratic hepatotoxicity has been associated with the oral tyrosine kinase inhibitor lapatinib, which is used in metastatic breast cancer therapy. Lapatinib is extensively metabolized by cytochrome P450 3A4/5 to yield an O-debenzylated metabolite, which can undergo further oxidation to a reactive quinone imine. A recent clinical study reported that concomitant use of lapatinib with dexamethasone increased the incidence of hepatotoxicity in metastatic breast cancer patients treated with lapatinib, and so we hypothesized that induction of CYP3A enhances the bioactivation of lapatinib to reactive intermediates that contribute to hepatotoxicity. Therefore, we examined the effect of CYP3A4 induction on the cytotoxicity and metabolism of lapatinib in the HepaRG human hepatic cell line. Differentiated HepaRG cells were pretreated with dexamethasone (100 μM) or the prototypical CYP3A4 inducer rifampicin (4 μM) for 72 hours, followed by incubation with lapatinib (0-100 μM) for 24 hours. Cell viability was monitored using WST-1 assays, and metabolites were quantified by liquid chromatography coupled to tandem mass spectrometry. Induction of CYP3A4 by dexamethasone or rifampicin enhanced lapatinib-induced cytotoxicity, compared with treatment with lapatinib alone. A direct comparison of the cytotoxicity of lapatinib versus O-debenzylated lapatinib demonstrated that the O-debenzylated metabolite was significantly more cytotoxic than lapatinib itself. Furthermore, pretreatment with 25 μM l-buthionine sulfoximine to deplete intracellular glutathione markedly enhanced lapatinib cytotoxicity. Cytotoxicity was correlated with increased formation of O-debenzylated lapatinib and cysteine adducts of the putative quinone imine intermediate. Collectively, these data suggest that CYP3A4 induction potentiates lapatinib-induced hepatotoxicity via increased reactive metabolite formation.
• 3D organotypic HepaRG cultures as in vitro model for acute and repeated dose toxicity studies.Mueller D., Krämer L., Hoffmann E., Klein S., Noor F.
  Toxicolgy in Vitro, Oct 2013
  Abstract : Predictive in vitro models alternative to in vivo animal will have a significant impact in toxicology. Conventional 2D models do not reflect the complexity of a 3D organ resulting in discrepancies between experimental in vitro and in vivo data. Using 3D HepaRG organotypic cultures we tested four drugs (aflatoxin B1, amiodarone, valproic acid and chlorpromazine) for toxic effects and compared the results with 2D HepaRG and HepG2 cultures. We show that 3D HepaRG cultures are more sensitive than the other tested cultures to aflatoxin B1 which is only toxic upon metabolic activation in the liver. We observed that CYP3A4 activity is higher in the 3D HepaRG cultures compared to the 2D HepaRG cultures. Furthermore, we investigated repeated dose toxicity of chlorpromazine and assessed its effects on glucose and lactate metabolism. Sub-toxic concentrations of chlorpromazine induced significant metabolic changes in both 2D and 3D HepaRG cultures upon acute and repeated dose (3 doses) exposure. In summary, our data support the hypothesis that 3D cell culture models better mimic the in vivo tissue and improve cellular functionality. The 3D HepaRG organotypic cultures represent a high throughput system for drug toxicity screening. This system is therefore a promising tool in preclinical testing of human relevance which can allow reducing and/or replacing animal testing for drug adverse effects.
• A dynamic multi-organ-chip for long-term cultivation and substance testing proven by 3D human liver and skin tissue co-culture.Wagner I, Materne EM, Brincker S, Süssbier U, Frädrich C, Busek M, Sonntag F, Sakharov DA,Trushkin EV, Tonevitsky AG
  Lab Chip, Sep 2013
  Abstract : Current in vitro and animal tests for drug development are failing to emulate the systemic organ complexity of the human body and, therefore, to accurately predict drug toxicity. In this study, we present a multi-organ-chip capable of maintaining 3D tissues derived from cell lines, primary cells and biopsies of various human organs. We designed a multi-organ-chip with co-cultures of human artificial liver microtissues and skin biopsies, each a (1)/100,000 of the biomass of their original human organ counterparts, and have successfully proven its long-term performance. The system supports two different culture modes: i) tissue exposed to the fluid flow, or ii) tissue shielded from the underlying fluid flow by standard Transwell® cultures. Crosstalk between the two tissues was observed in 14-day co-cultures exposed to fluid flow. Applying the same culture mode, liver microtissues showed sensitivity at different molecular levels to the toxic substance troglitazone during a 6-day exposure. Finally, an astonishingly stable long-term performance of the Transwell®-based co-cultures could be observed over a 28-day period. This mode facilitates exposure of skin at the air-liquid interface. Thus, we provide here a potential new tool for systemic substance testing.
• Long-term maintenance of HepaRG cells in serum-free conditions and application in a repeated dose study.Klein S, Mueller D, Schevchenko V, Noor F.
  J Appl Toxicol., Sep 2013
  Abstract : The bioactivation of pro-toxicants is the biological process through which some chemicals are metabolized into reactive metabolites. Therefore, in vitro toxicological evaluation should ideally be conducted in cell systems retaining adequate metabolic competency and relevant to the route of exposure. The respiratory tract is the primary route of exposure to inhaled pro-toxicants and lung-derived BEAS-2B cell line has been considered as a potentially suitable model for in vitro toxicology testing. However, its metabolic activity has not been characterized. We performed a gene expression analysis for 41 metabolism-related genes and compared the profile with liver- and lung-derived cell lines (HepaRG, HepG2 and A549). To confirm that mRNA expression was associated with the corresponding enzyme activity, we used a series of metabolic substrates of CYPs (CYP1A1/1B1, CYP1A2, CYP2A6/2A13 and CYP2E1) known to bioactivate inhaled pro-toxicants. CYP activities were compared between BEAS-2B, HepaRG, HepG2, and A549 cells and published literature on primary bronchial epithelium cells (HBEC). We found that in contrast to HBEC, BEAS-2B and A549 have limited CYP activity which was in agreement with their CYP gene expression profile. Control cell lines such as HepG2 and HepaRG were metabolically active for the tested CYPs. We recommend that similar strategies can be used to select suitable cell systems in the context of pro-toxicant assessment.
• Profiling the Impact of Medium Formulation on Morphology and Functionality of Primary Hepatocytes in vitro.Nelson LJ, Treskes P, Howie AF, Walker SW., Hayes PC, Prelvis JN
  Scientific Reports, Sep 2013
  Abstract : The characterization of fully-defined in vitro hepatic culture systems requires testing of functional and morphological variables to obtain the optimal trophic support, particularly for cell therapeutics including bioartificial liver systems (BALs). Using serum-free fully-defined culture medium formulations, we measured synthetic, detoxification and metabolic variables of primary porcine hepatocytes (PPHs)--integrated these datasets using a defined scoring system and correlated this hepatocyte biological activity index (HBAI) with morphological parameters. Hepatic-specific functions exceeded those of both primary human hepatocytes (PHHs) and HepaRG cells, whilst retaining biotransformation potential and in vivo-like ultrastructural morphology, suggesting PPHs as a potential surrogate for PHHs in various biotech applications. The HBAI permits assessment of global functional capacity allowing the rational choice of optimal trophic support for a defined operational task (including BALs, hepatocellular transplantation, and cytochrome P450 (CYP450) drug metabolism studies), mitigates risk associated with sub-optimal culture systems, and reduces time and cost of research and therapeutic applications.
• The human hepatic cell line HepaRG as a possible cell source for the generation of humanized liver TK-NOG mice.Higuchi Y, Kawai K, Yamazaki H, Nakamura M, Bree F, Guguen-Guillouzo C, Suemizu H
  Xenobiotica, Sep 2013
  Abstract : Humanized-liver mice, in which the liver has been repopulated with human hepatocytes, have been used to study aspects of human liver physiology such as drug metabolism, toxicology and hepatitis infection. However, the procurement of human hepatocytes is a major problem in producing humanized-liver mice because of the finite nature of the patient-derived resource. 2. In order to overcome this limitation, the human hepatic cell line HepaRG® were evaluated as promising donor cells for liver reconstitution in the TK-NOG mouse model. 3. We demonstrate that, in vivo, transplanted confluent culture or differentiated HepaRG® cells proliferated and differentiated toward both hepatocyte-like and biliary-like cells within the recipient liver. In contrast, proliferative HepaRG® cells could engraft TK-NOG mouse liver but could differentiate only toward biliary-like cells. The differentiation to hepatocyte-like cells was characterized by the detection of human albumin in the recipient mouse serum and was confirmed by immunohistochemical staining for human leukocyte antigen, human albumin, cytochrome P450 3A4, and multidrug resistance-associated protein 2. Biliary-like cells were characterized by positive staining for cytokeratin-19. 4. These results indicated that the differentiated HepaRG® cells are a possible cell source for generating humanized-liver mice, which are a useful model for in vivo studies of liver physiology.
• Polarized expression of drug transporters in differentiated human hepatoma HepaRG cells.Le Vee M, Noel G, Jouan E, Stieger B, Fardel O
  Toxicology in vitro, Sep 2013
  Abstract : The HepaRG cell line is a well-differentiated human hepatoma cell line proposed as a surrogate for human hepatocytes, especially for hepatic detoxification studies. Polarized status of drug transporters, i.e., their coordinated location at sinusoidal or canalicular membranes, which represents a key hallmark of hepato-biliary drug transport, remains however incompletely documented in HepaRG cells. The present study was therefore designed to analyze transporter location in HepaRG cells, which exhibit mRNA expressions of most of hepatic transporters. HepaRG cells were demonstrated, through immunofluorescence staining, to express several drug transporters at their sinusoidal pole, especially the influx transporters organic anion transporting polypeptide (OATP) 1B1, OATP2B1 and organic cation transporter (OCT) 1 and the efflux transporter multidrug resistance-associated protein (MRP) 3. In addition, the efflux transporters P-glycoprotein and MRP2 were detected at the canalicular pole of HepaRG cells. Moreover, saturable uptake of reference substrates for the sinusoidal transporters sodium-taurocholate cotransporting polypeptide, OATPs and OCT1 and canalicular secretion of reference substrates for the efflux transporters bile salt export pump and MRP2 were observed. This polarized and functional expression of various sinusoidal and canalicular transporters in HepaRG cells highlights the interest of using these hepatoma cells in xenobiotic transport studies.
• Iron-mediated effect of alcohol on hepatocyte differentiation in HepaRG cells.Do TH, Gaboriau F, Cannie I, Batusanski F, Ropert M, Moirand R, Brissot P, Loreal O, Lescoat G
  Chem biol interact, Sep 2013
  Abstract : The development of alcoholic liver diseases depends on the ability of hepatocyte to proliferate and differentiate in the case of alcohol-induced injury. Our previous work showed an inhibitory effect of alcohol on hepatocyte proliferation. However, the effect of alcohol on hepatocyte differentiation has not yet been precisely characterized. In the present study, we evaluated the effect of alcohol on hepatocyte differentiation in relationship with changes of iron metabolism in HepaRG cells. This unique bipotent human cell line can differentiate into hepatocytes and biliary epithelial cells, paralleling liver development. Results showed that alcohol reduced cell viability, total protein level and enhanced hepatic enzymes leakage in differentiated HepaRG cells. Moreover, it caused cell enlargement, decreased number of hepatocyte and expression of C/EBPα as well as bile canaliculi F-actin. Alcohol increased expression of hepatic cell-specific markers and alcohol-metabolizing enzymes (ADH2, CYP2E1). This was associated with a lipid peroxidation and an iron excess expressed by an increase in total iron content, ferritin level, iron uptake as well as an overexpression of genes involved in iron transport and storage. Alcohol-induced hepatoxicity was amplified by exogenous iron via exceeding iron overload. Taken together, our data demonstrate that in differentiated hepatocytes, alcohol reduces proliferation while increasing expression of hepatic cell-specific markers. Moreover, iron overload could be one of the underlying mechanisms of effect of alcohol on the whole differentiation process of hepatocytes.
• Interleukin-1 and tumor necrosis factor-alpha trigger restriction of hepatitis B virus infection via a cytidine deaminase AID.Watashi K, Liang G, Iwamoto M, Marusawa H, Uchida N, Daito T, Kitamura K, Maramatsu M, Ohashi H, Kiyohara T, Suzuki R, Li J, Tong S
  J Biol Chem, Sep 2013
  Abstract : Virus infection is restricted by intracellular immune responses in host cells, and this is typically modulated by stimulation of cytokines. The cytokines and host factors that determine the host cell restriction against hepatitis B virus (HBV) infection are not well understood. We screened 36 cytokines and chemokines to determine which were able to reduce the susceptibility of HepaRG cells to HBV infection. Here, we found that pretreatment with IL-1β and TNFα remarkably reduced the host cell susceptibility to HBV infection. This effect was mediated by activation of the NF-κB signaling pathway. A cytidine deaminase, activation-induced cytidine deaminase (AID), was up-regulated by both IL-1β and TNFα in a variety of hepatocyte cell lines and primary human hepatocytes. Another deaminase APOBEC3G was not induced by these proinflammatory cytokines. Knockdown of AID expression impaired the anti-HBV effect of IL-1β, and overexpression of AID antagonized HBV infection, suggesting that AID was one of the responsible factors for the anti-HBV activity of IL-1/TNFα. Although AID induced hypermutation of HBV DNA, this activity was dispensable for the anti-HBV activity. The antiviral effect of IL-1/TNFα was also observed on different HBV genotypes but not on hepatitis C virus. These results demonstrate that proinflammatory cytokines IL-1/TNFα trigger a novel antiviral mechanism involving AID to regulate host cell permissiveness to HBV infection.
• Evaluation of Normalization Strategies Used in Real-Time Quantitative PCR Experiments in HepaRG Cell Line Studies.Ceelen L, De Craene J, De Spiegelaere W
  Clin Chem, Sep 2013
  Abstract : background:The HepaRG cell line is widely used as an alternative for primary human hepatocytes for numerous applications, including drug screening, and is progressively gaining importance as a human-relevant cell source. Consequently, increasing numbers of experiments are being performed with this cell line, including real-time quantitative PCR (RT-qPCR) experiments for gene expression studies.Content:When RT-qPCR experiments are performed, results are reliable only when attention is paid to several critical aspects, including a proper normalization strategy. Therefore, in 2011 we determined the most optimal reference genes for gene expression studies in the HepaRG cell system, according to the MIQUE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines. This study additionally provided clear evidence that the use of a single reference gene [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein S18 (RPS18), or actin, beta (ACTB)] was insufficient for normalization in HepaRG cells. Our screening of relevant studies published after our study suggested that the findings of our study were completely ignored.Summary:In none of the 24 reviewed studies was a proper normalization method used. Only 1 reference gene was included for normalization in 21 out of the 24 reported studies we screened, with RPS18 and GAPDH used most frequently, followed by hypoxanthine phosphoribosyltransferase 1 (HPRT1), recombinant human glucuronidase, beta (GUSB) (hGus), β-2 microglobin (B2M), and acidic ribosomal phosphoprotein P0 (36B4). For 2 studies the use of multiple reference genes (2 and 3) was reported, but these had not been prevalidated for expression stability in HepaRG cells. In 1 study, there was no evidence that any reference gene had been used. Current RT-qPCR gene expression studies in HepaRG cells are being performed without adequate consideration or evaluation of reference genes. Such studies can yield erroneous and biologically irrelevant results.
• Metabolic characterization of cell systems used in in vitro toxicology testing: Lung cell system BEAS-2B as a working example.Garcia-Canton C, Minet E, Anadon A, Meredith C
  Toxicol In vitro, Sep 2013
  Abstract : The bioactivation of pro-toxicants is the biological process through which some chemicals are metabolized into reactive metabolites. Therefore, in vitro toxicological evaluation should ideally be conducted in cell systems retaining adequate metabolic competency and relevant to the route of exposure. The respiratory tract is the primary route of exposure to inhaled pro-toxicants and lung-derived BEAS-2B cell line has been considered as a potentially suitable model for in vitro toxicology testing. However, its metabolic activity has not been characterized. We performed a gene expression analysis for 41 metabolism-related genes and compared the profile with liver- and lung-derived cell lines (HepaRG, HepG2 and A549). To confirm that mRNA expression was associated with the corresponding enzyme activity, we used a series of metabolic substrates of CYPs (CYP1A1/1B1, CYP1A2, CYP2A6/2A13 and CYP2E1) known to bioactivate inhaled pro-toxicants. CYP activities were compared between BEAS-2B, HepaRG, HepG2, and A549 cells and published literature on primary bronchial epithelium cells (HBEC). We found that in contrast to HBEC, BEAS-2B and A549 have limited CYP activity which was in agreement with their CYP gene expression profile. Control cell lines such as HepG2 and HepaRG were metabolically active for the tested CYPs. We recommend that similar strategies can be used to select suitable cell systems in the context of pro-toxicant assessment.
• Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME.Godoy P, Hewitt NJ, Albrecht U, Andersen ME, Ansari N, Bhattacharya S, Bode JG, Bolleyn J, Borner C, Böttger J, Braeuning A, Budinsky RA, Burkhardt B, Cameron NR, Camussi G, Cho CS, Choi YJ, Craig Rowlands J, Dahmen U, Damm G, Dirsch O, Donato MT, Dong J
  Arch Toxicol., Aug 2013
  Abstract : This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell-derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.
• In vitro drug metabolism testing using blood-monocyte derivatives.Gomez-Icazbalceta G, Gonzalez Sanchez I, Moreno J, Cerbon MA, Cervantes A
  Expert Opin Drug Metab Toxicol, Aug 2013
  Abstract : "INTRODUCTION: Monocytes and their cell derivatives can participate in drug metabolism. These cells express different Phase-I or -II drug metabolizing enzymes and can be differentiated into neo-hepatocytes (NeoHep) and represent a promising alternative strategy to test drug metabolism. This is particularly useful as primary human hepatocytes (PHH), are difficult to obtain and maintain in culture. AREAS COVERED: The authors analyze the use of blood monocytes and their derivatives for the study of drug metabolism. They also compare them to the in vitro ability of cells from different sources including: PHH, immortalized hepatocytes, tumor cell lines and NeoHep. EXPERT OPINION: The use of monocytes, macrophages, dendritic or Kupffer cells, to test drug metabolism, has serious limitations because these cells express lower levels of cytochrome P450 enzymes than PHH. The best available option, to replace PHH, have been tumor cell lines such as HepaRG, as well as immortalized hepatocytes from adult or fetal sources. Monocyte-derived NeoHep cells are novel and easily accessible cells, which express many drug metabolizing enzymes at levels comparable to PHH. These cells allow drug evaluation under a diverse genetic background. While these cells are in the early stages of evaluation and do need to be examined more thoroughly, they constitute a promising new tool for in vitro drug testing. "
• Cyclosporine A treated in vitro models induce cholestasis response through comparison of phenotype-directed gene expression analysis of in vivo Cyclosporine A-induced cholestasis.Kienhuis AS, Vitins AP, Pennings JL, Pronk TE, Speksnijder EN, Roodbergen N, Van Delft JH, Luijten M, Van der ven LT
  Toxicol Lett, Aug 2013
  Abstract : In vitro models for hepatotoxicity testing are a necessity for advancement of toxicological research. Assessing the in vitro response requires in vivo validated gene sets reflective of the hepatotoxic phenotype. Cholestasis, the impairment of bile flow, is induced in C57BL/6J mice treated with cyclosporine A (CsA) to identify phenotype reflective gene sets. CsA treatment through oral gavage for 25 days induced cholestasis, as confirmed by histopathology and serum chemistry. Over 1, 4, and 11 days of CsA exposure gradual increases in serum markers were correlated to gene expression. This phenotype-directed analysis identified gene sets specific to the onset and progression of cholestasis, such as PPAR related processes and drug metabolism, by circumventing other effects of CsA, such as immunosuppression, found in dose*time group analysis. In vivo gene sets are enriched in publicly available data sets of CsA-treated HepaRG and primary mouse hepatocytes. However, genes identified within these gene sets did not overlap between in vivo and in vitro. In vitro regulated genes represent the initial response to cholestasis, whereas in vivo genes represent the later adaptive response. We conclude that the applicability of in vitro models for hepatotoxicity testing fully depends on a solid in vivo phenotype anchored analysis.
• Comparative Proteomics Reveals Novel Components at the Plasma Membrane of Differentiated HepaRG Cells and Different Distribution in Hepatocyte- and Biliary-Like Cells.Petrareanu C, Macovei A, Sokolawska, Woods AG, Lazar C, Radu GL, Darie CC, Branza-Nichita N
  PloS One, Aug 2013
  Abstract : Hepatitis B virus (HBV) is a human pathogen causing severe liver disease and eventually death. Despite important progress in deciphering HBV internalization, the early virus-cell interactions leading to infection are not known. HepaRG is a human bipotent liver cell line bearing the unique ability to differentiate towards a mixture of hepatocyte- and biliary-like cells. In addition to expressing metabolic functions normally found in liver, differentiated HepaRG cells support HBV infection in vitro, thus resembling cultured primary hepatocytes more than other hepatoma cells. Therefore, extensive characterization of the plasma membrane proteome from HepaRG cells would allow the identification of new cellular factors potentially involved in infection. Here we analyzed the plasma membranes of non-differentiated and differentiated HepaRG cells using nanoliquid chromatography-tandem mass spectrometry to identify the differences between the proteomes and the changes that lead to differentiation of these cells. We followed up on differentially-regulated proteins in hepatocytes- and biliary-like cells, focusing on Cathepsins D and K, Cyclophilin A, Annexin 1/A1, PDI and PDI A4/ERp72. Major differences between the two proteomes were found, including differentially regulated proteins, protein-protein interactions and intracellular localizations following differentiation. The results advance our current understanding of HepaRG differentiation and the unique properties of these cells.
• Rapid fabricating technique for multi-layered human hepatic cell sheets by forceful contraction of the fibroblast monolayer.Sakai Y, Koike M, Hasegawa H, Yamnouchi K, Soyama A, Takatsuki M, Kuroki T, Oshaki K, Okano T, Egushi S
  PloS One, Jul 2013
  Abstract : Cell sheet engineering is attracting attention from investigators in various fields, from basic research scientists to clinicians focused on regenerative medicine. However, hepatocytes have a limited proliferation potential in vitro, and it generally takes a several days to form a sheet morphology and multi-layered sheets. We herein report our rapid and efficient technique for generating multi-layered human hepatic cell (HepaRG® cell) sheets using pre-cultured fibroblast monolayers derived from human skin (TIG-118 cells) as a feeder layer on a temperature-responsive culture dish. Multi-layered TIG-118/HepaRG cell sheets with a thick morphology were harvested on day 4 of culturing HepaRG cells by forceful contraction of the TIG-118 cells, and the resulting sheet could be easily handled. In addition, the human albumin and alpha 1-antitrypsin synthesis activities of TIG-118/HepaRG cells were approximately 1.2 and 1.3 times higher than those of HepaRG cells, respectively. Therefore, this technique is considered to be a promising modality for rapidly fabricating multi-layered human hepatocyte sheets from cells with limited proliferation potential, and the engineered cell sheet could be used for cell transplantation with highly specific functions.
• Baseline and genotoxic compound induced gene expression profiles in HepG2 and HepaRG compared to primary human hepatocytes.Jetten MJ, Kleinjans JC, Claessen SM, Chesné C, Van Deft JH
  Toxicol in vitro, Jul 2013
  Abstract : Efforts are put into developing toxicogenomics-based toxicity testing methods using in vitro human cell models for improving human risk assessment/replacing animal models. Human in vitro liver models include HepG2, HepaRG and primary human hepatocytes (PHH). Studies on comparability/applicability of these cell types mainly focus on assessing baseline biotransformation capacities/cytochrome P450-inducibility, but compound-induced gene expression profiles are at least as important. Therefore, we compared baseline and aflatoxin B1- and benzo(α)pyrene-induced gene expression profiles in HepG2, HepaRG and PHH (11-13 donors). At baseline, all liver models differ from each other with respect to whole genome gene expression levels. PHH show profound inter-individual differences, and are most similar to HepaRG. After compound exposure, induced gene expression profiles are more similar between cell models, especially for benzo(α)pyrene. Pathways involved in compound metabolism are induced in all 3 models, while others are more pronounced in a specific cell model. Examples are transcriptomic modifications of carbohydrate-related genes (HepaRG) and of receptor-related genes (PHH) after benzo(α)pyrene exposure, and of cell cycle-related genes (HepG2) after aflatoxin B1 exposure. PHH gene expression responses are the most heterogeneous. In conclusion, at base line level PHH are more similar to HepaRG than to HepG2, but for toxicogenomics applications both cell lines perform equally well in comparison to PHH.
• Degradable and biocompatible nanoparticles decorated with cyclic RGD peptide for efficient drug delivery to hepatoma cells in vitro.Loyer P, Bedhouche W, Huang ZW, Cammas-Marion S
  Int J Pharm, Jun 2013
  Abstract : Amphiphilic derivatives of poly(benzyl malate) were synthesized and characterized with the aim of being used as degradable and biocompatible building blocks for the design of functional nanoparticles (NPs). An anti-cancer model drug, doxorubicin, has been successfully encapsulated into the prepared NPs and its release profile has been evaluated in water and in culture medium. NPs bearing biotin molecules were prepared either for site-specific drug delivery via the targeting of biotin receptors overexpressed on the surface of several cancer cells, or for grafting biotinylated cyclic RGD peptide onto their surface using the strong and highly specific interactions between biotin and the streptavidin protein. We have shown that this binding did not affect dramatically the physico-chemical properties of the corresponding NPs. Cyclic RGD grafted fluorescent NPs were more efficiently uptaken by the HepaRG hepatoma cells than biotinylated fluorescent NPs. Furthermore, the targeting of HepaRG hepatoma cells with NPs bearing cyclic RGD was very efficient and much weaker for HeLa and HT29 cell lines confirming that cyclic RGD is a suitable targeting agent for liver cells. Our results also provide a new mean for rapid screening of short hepatotropic peptides in order to design NPs showing specific liver targeting properties.
• Regulation of hepatitis B virus infection by Rab5, Rab7, and the endolysosomal compartment.Macovei A, Petrareanu C, Lazar C, Florian P, Branza-Nichita N
  J virol, Jun 2013
  Abstract : Despite important progress toward deciphering human hepatitis B virus (HBV) entry into host cells, many aspects of the early steps of the life cycle remained completely obscure. Following endocytosis, HBV must travel through the complex network of the endocytic pathway to reach the cell nucleus and initiate replication. In addition to guiding the viral particles to the replication site, the endosomal vesicles may play a crucial role in infection, providing the appropriate environment for virus uncoating and nucleocapsid release. In this work, we investigated the trafficking of HBV particles internalized in permissive cells. Expression of key Rab proteins, involved in specific pathways leading to different intracellular locations, was modulated in HepaRG cells, using a stable and inducible short hairpin RNA (shRNA) expression system. The trafficking properties of the newly developed cells were demonstrated by confocal microscopy and flow cytometry using specific markers. The results showed that HBV infection strongly depends on Rab5 and Rab7 expression, indicating that HBV transport from early to mature endosomes is required for a step in the viral life cycle. This may involve reduction of disulfide bond-linked envelope proteins, as alteration of the redox potential of the endocytic pathway resulted in inhibition of infection. Subcellular fractionation of HBV-infected cells showed that viral particles are further transported to lysosomes. Intriguingly, infection was not dependent on the lysosomal activity, suggesting that trafficking to this compartment is a "dead-end" route. Together, these data add to our understanding of the HBV-host cell interactions controlling the early stages of infection.
• Increased hepatic functionality of the human hepatoma cell line HepaRG cultured in the AMC bioreactor.Nibourg G, Hoekstra R, van der Hoeven T., Ackermans M, Hakvoort TB, van Gulik T, Chamuleau R
  Int J Biochem Cell Biol., Jun 2013
  Abstract : The clinical application of a bioartificial liver (BAL) depends on the availability of a human cell source with high hepatic functionality, such as the human hepatoma cell line HepaRG. This cell line has demonstrated high hepatic functionality, but the effect of BAL culture on its functionality in time is not known. Therefore, we studied the characteristics of the HepaRG-AMC-BAL over time, and compared the functionality of the HepaRG-AMC-BAL with monolayer cultures of HepaRG cells, normalized for protein (bioactive mass) and DNA (cell number). Histological analysis of 14-day-old BALs demonstrated functional heterogeneity similar to that of monolayer cultures. Hepatic functionality of the HepaRG-AMC-BALs increased during 2-3 weeks of culture. The majority of the measured protein-normalized hepatic functions were already higher in day 14 BAL cultures compared to monolayer cultures, including ammonia elimination (3.2-fold), urea production (1.5-fold), conversion of (15)N-ammonia into (15)N-urea (1.4-fold), and cytochrome P450 3A4 activity (7.9-fold). Lactate production in monolayer cultures switched into lactate consumption in the BAL cultures, a hallmark of primary hepatocytes. When normalized for DNA, only cytochrome P450 3A4 activity was 2.5-fold higher in the BAL cultures compared to monolayer cultures and lactate production switched to consumption, whereas urea production and (15)N-urea production were 1.5- to 2-fold lower. The different outcomes for protein and DNA normalized functions probably relate to a smaller cell volume of HepaRG cells when cultured in the AMC-BAL. Cell damage was 4-fold lower in day 14 BAL cultures compared to monolayer cultures. Transcript levels of cytochrome P450 1A2, 2B6, 3A4 and 3A7 genes and of regulatory genes hepatic nuclear factor 4α and pregnane X receptor increased in time in BAL cultures and reached higher levels than in monolayer cultures. Lastly, metabolism of amino acids, particularly the alanine consumption and ornithine production of HepaRG-AMC-BALs more resembled that of primary hepatocytes than monolayer HepaRG cultures. We conclude therefore that BAL culture of HepaRG cells increases its hepatic functionality, particularly when normalized for biomass, both over time, and compared to monolayer, and this is associated with a reduction in cell damage, upregulation of both regulatory and structural hepatic genes, and changes in amino-acid metabolism. These results underline the potential of HepaRG cells for BAL application.
• Highly efficient Sirna and Gene transfer into Hepatocyte-like HepaRG Cells and primary human Hepatocytes : New Means for Drug Metabolism and Toxicity Studies.Laurent V, Glaise D, Nubel T, Gilot D, Corlu A, Loyer P
  Methods Mol Biol, May 2013
  Abstract : The metabolically competent hepatocyte-like human HepaRG cells represent a suitable alternative in vitro cell model to human primary hepatocytes. Here, we describe the culture procedure required to expand progenitor HepaRG cells and to differentiate them into hepatocyte-like cells. Transient transfection of gene and siRNA into cultured cells, using nonviral strategies, is an invaluable technique to decipher gene functions. In this chapter, we detail transfection protocols for efficient transfer of plasmid DNA or siRNAs into proliferating progenitor or quiescent differentiated HepaRG cells as well as into primary hepatocytes.
• Characterization of the anti-HBV activity of HLP1-23, a human lactoferrin-derived peptide.Florian P, Macovei A, Lazar C, Milac AL, Sokolowska I, Darie CC, Evans RW, Roseanu A, Branza-Nichita N
  J med Virol, May 2013
  Abstract : Lactoferrin (Lf) was shown to exhibit its antiviral activity at an early phase of viral infection and a mechanism whereby the protein interacts with host cell surface molecules has been suggested. In this study, human Lf (HLf) and seven HLf-derived synthetic peptides (HLP) corresponding to the N-terminal domain of the native protein (1-47 amino acids sequence) were assayed for their capacity to prevent hepatitis B virus (HBV) infection and replication using the HepaRG and HepG2.2.2.15 cell lines. Of the series tested, four peptides showed 40-75% inhibition of HBV infection in HepaRG cells, HLP1-23 , containing the GRRRR cationic cluster, being the most potent. Interestingly, this cluster is one of the two glycosaminoglycan binding sites of the native HLf involved in its antiviral activity; however, the mechanism of the HLP1-23 action was different from that of the full-length protein, the peptide inhibiting HBV infection when pre-incubated with the virus, while no effect was observed on the target cells. It is suggested that the cationic cluster is sufficient for the peptide to interact stably with negatively charged residues on the virion envelope, while the absence of the second glycosaminoglycan binding site prevents its efficient attachment to the cells. In conclusion, this peptide may constitute a non-toxic approach for potential clinical applications in inhibiting HBV entry by neutralizing the viral particles.
• Dual roles of nuclear receptor liver X receptor &alpha; (LXR&alpha;) in the CYP3A4 expression in human hepatocytes as a positive and negative regulator.Watanabe K, Sakurai K, Tsushiya Y, Yamazoe Y, Yoshinari K
  Biochem Pharmacol, May 2013
  Abstract : CYP3A4 is a major drug-metabolizing enzyme in humans, whose expression levels show large inter-individual variations and are associated with several factors such as genetic polymorphism, physiological and disease status, diet and xenobiotic exposure. Nuclear receptor pregnane X receptor (PXR) is a key transcription factor for the xenobiotic-mediated transcription of CYP3A4. In this study, we have investigated a possible involvement of liver X receptor α (LXRα), a critical regulator of cholesterol homeostasis, in the hepatic CYP3A4 expression since several recent reports suggest the involvement of CYP3A enzymes in the cholesterol metabolism in humans and mice. Reporter assays using wild-type and mutated CYP3A4 luciferase reporter plasmids and electrophoretic mobility shift assays revealed that LXRα up-regulated CYP3A4 through the known DNA elements critical for the PXR-dependent CYP3A4 transcription, suggesting LXRα as a positive regulator for the CYP3A4 expression and a crosstalk between PXR and LXRα in the expression. In fact, reporter assays showed that LXRα activation attenuated the PXR-dependent CYP3A4 transcription. Moreover, a PXR agonist treatment-dependent increase in CYP3A4 mRNA levels was suppressed by co-treatment with an LXRα agonist in human primary hepatocytes and HepaRG cells. The suppression was not observed when LXRα expression was knocked-down in HepaRG cells. In conclusion, the present results suggest that sterol-sensitive LXRα positively regulates the basal expression of CYP3A4 but suppresses the xenobiotic/PXR-dependent CYP3A4 expression in human hepatocytes. Therefore, nutritional, physiological and disease conditions affecting LXRα might be one of the determinants for the basal and xenobiotic-responsive expression of CYP3A4 in human livers.
• Replication-competent infectious hepatitis B virus vectors carrying substantially sized transgenes by redesigned viral polymerase translation.Wang z, Wu L, Cheng X, Liu S, Li B, Li H, Kang F, Wang J, Xia H, Ping C, Nassal M, Sun D
  PLoS One, Apr 2013
  Abstract : Viral vectors are engineered virus variants able to deliver nonviral genetic information into cells, usually by the same routes as the parental viruses. For several virus families, replication-competent vectors carrying reporter genes have become invaluable tools for easy and quantitative monitoring of replication and infection, and thus also for identifying antivirals and virus susceptible cells. For hepatitis B virus (HBV), a small enveloped DNA virus causing B-type hepatitis, such vectors are not available because insertions into its tiny 3.2 kb genome almost inevitably affect essential replication elements. HBV replicates by reverse transcription of the pregenomic (pg) RNA which is also required as bicistronic mRNA for the capsid (core) protein and the reverse transcriptase (Pol); their open reading frames (ORFs) overlap by some 150 basepairs. Translation of the downstream Pol ORF does not involve a conventional internal ribosome entry site (IRES). We reasoned that duplicating the overlap region and providing artificial IRES control for translation of both Pol and an in-between inserted transgene might yield a functional tricistronic pgRNA, without interfering with envelope protein expression. As IRESs we used a 22 nucleotide element termed Rbm3 IRES to minimize genome size increase. Model plasmids confirmed its activity even in tricistronic arrangements. Analogous plasmids for complete HBV genomes carrying 399 bp and 720 bp transgenes for blasticidin resistance (BsdR) and humanized Renilla green fluorescent protein (hrGFP) produced core and envelope proteins like wild-type HBV; while the hrGFP vector replicated poorly, the BsdR vector generated around 40% as much replicative DNA as wild-type HBV. Both vectors, however, formed enveloped virions which were infectious for HBV-susceptible HepaRG cells. Because numerous reporter and effector genes with sizes of around 500 bp or less are available, the new HBV vectors should become highly useful tools to better understand, and combat, this important pathogen.
• Exploring the zebrafish embryo as an alternative model for the evaluation of liver toxicity by histopathology and expression profiling.Driessen M, Kienhuis AS, Pennings JL, Pronk TE, van de Brandhof EJ, Roodbergen M, Spaink HP, van de Water B, van der Ven LT
  Arch Toxicol, Apr 2013
  Abstract : The whole zebrafish embryo model (ZFE) has proven its applicability in developmental toxicity testing. Since functional hepatocytes are already present from 36 h post fertilization onwards, whole ZFE have been proposed as an attractive alternative to mammalian in vivo models in hepatotoxicity testing. The goal of the present study is to further underpin the applicability of whole ZFE for hepatotoxicity testing by combining histopathology and next-generation sequencing-based gene expression profiling. To this aim, whole ZFE and adult zebrafish were exposed to a set of hepatotoxic reference compounds. Histopathology revealed compound and life-stage-specific effects indicative of toxic injury in livers of whole ZFE and adult zebrafish. Next-generation sequencing (NGS) was used to compare transcript profiles in pooled individual RNA samples of whole ZFE and livers of adult zebrafish. This revealed that hepatotoxicity-associated expression can be detected beyond the overall transcription noise in the whole embryo. In situ hybridization verified liver specificity of selected highly expressed markers in whole ZFE. Finally, cyclosporine A (CsA) was used as an illustrative case to support applicability of ZFE in hepatotoxicity testing by comparing CsA-induced gene expression between ZFE, in vivo mouse liver and HepaRG cells on the levels of single genes, pathways and transcription factors. While there was no clear overlap on single gene level between the whole ZFE and in vivo mouse liver, strong similarities were observed between whole ZFE and in vivo mouse liver in regulated pathways related to hepatotoxicity, as well as in relevant overrepresented transcription factors. In conclusion, both the use of NGS of pooled RNA extracts analysis combined with histopathology and traditional microarray in single case showed the potential to detect liver-related genes and processes within the transcriptome of a whole zebrafish embryo. This supports the applicability of the whole ZFE model for compound-induced hepatotoxicity screening.
• Comparison of human Hepatoma hepaRG Cells with Human and Rat Hepatocytes in Uptake transport assays in order to Predict a risk of Drug Induced Hepatotoxicity.Szabo M, Veres Z, Baranyai Z, Kjakab F, Jemnitz K
  PloS One, Mar 2013
  Abstract : Human hepatocytes are the gold standard for toxicological studies but they have several drawbacks, like scarce availability, high inter-individual variability, a short lifetime, which limits their applicability. The aim of our investigations was to determine, whether HepaRG cells could replace human hepatocytes in uptake experiments for toxicity studies. HepaRG is a hepatoma cell line with most hepatic functions, including a considerable expression of uptake transporters in contrast to other hepatic immortalized cell lines. We compared the effect of cholestatic drugs (bosentan, cyclosporinA, troglitazone,) and bromosulfophthalein on the uptake of taurocholate and estrone-3-sulfate in human and rat hepatocytes and HepaRG cells. The substrate uptake was significantly slower in HepaRG cells than in human hepatocytes, still, in the presence of drugs we observed a concentration dependent decrease in uptake. In all cell types, the culture time had a significant impact not only on the uptake process but on the inhibitory effect of drugs too. The most significant drug effect was measured at 4 h after seeding. Our report is among the first concerning interactions of the uptake transporters in the HepaRG, at the functional level. Results of the present study clearly show that concerning the inhibition of taurocholate uptake by cholestatic drugs, HepaRG cells are closer to human hepatocytes than rat hepatocytes. In conclusion, we demonstrated that HepaRG cells may provide a suitable tool for hepatic uptake studies.
• Farnesoid X receptor inhibits the transcriptional activity of carbohydrate response element binding protein in human hepatocytes.Caron S, Samanez CH, Dehondt H, Ploton M, Briand O, Lien F, Dorchies E, Dumont J, Postic C, Cariou B, Lefebvre P, Staels B
  Mol cell biol, Mar 2013
  Abstract : The glucose-activated transcription factor carbohydrate response element binding protein (ChREBP) induces the expression of hepatic glycolytic and lipogenic genes. The farnesoid X receptor (FXR) is a nuclear bile acid receptor controlling bile acid, lipid, and glucose homeostasis. FXR negatively regulates hepatic glycolysis and lipogenesis in mouse liver. The aim of this study was to determine whether FXR regulates the transcriptional activity of ChREBP in human hepatocytes and to unravel the underlying molecular mechanisms. Agonist-activated FXR inhibits glucose-induced transcription of several glycolytic genes, including the liver-type pyruvate kinase gene (L-PK), in the immortalized human hepatocyte (IHH) and HepaRG cell lines. This inhibition requires the L4L3 region of the L-PK promoter, known to bind the transcription factors ChREBP and hepatocyte nuclear factor 4α (HNF4α). FXR interacts directly with ChREBP and HNF4α proteins. Analysis of the protein complex bound to the L4L3 region reveals the presence of ChREBP, HNF4α, FXR, and the transcriptional coactivators p300 and CBP at high glucose concentrations. FXR activation does not affect either FXR or HNF4α binding to the L4L3 region but does result in the concomitant release of ChREBP, p300, and CBP and in the recruitment of the transcriptional corepressor SMRT. Thus, FXR transrepresses the expression of genes involved in glycolysis in human hepatocytes.
• Metabolomic signatures in lipid-loaded HepaRGs reveal pathways involved in steatotic progression.Brown M, Compton S, Milburn M, Lawton K, Cheatham B
  Obesity , Mar 2013
  Abstract : "OBJECTIVES: A spectrum of disorders including simple steatosis, nonalcoholic steatohepatitis, fibrosis, and cirrhosis is described by nonalcoholic fatty liver disease (NAFLD). With the increased prevalence of obesity, and consequently NAFLD, there is a need for novel therapeutics in this area. To facilitate this effort, a cellular model of hepatic steatosis was developed using HepaRG cells and the resulting biochemical alterations were determined. DESIGN AND METHODS: Using global metabolomic profiling, by means of a novel metabolite extraction procedure, the metabolic profiles in response to the saturated fatty acid palmitate, and a mixture of saturated and unsaturated fatty acids, palmitate and oleate (1:2) were examined. RESULTS: We observed elevated levels of the branched chain amino acids, tricarboxylic acid cycle intermediates, sphingosine and acylcarnitines, and reduced levels of carnitine in the steatotic HepaRG model with both palmitate and palmitate:oleate treatments. In addition, elevated levels of diacylglycerols and monoacylglycerols as well as altered bile acid metabolism were selectively displayed by palmitate-induced steatotic cells. CONCLUSIONS: Biochemical changes in pathways important in the transition to hepatic steatosis including insulin resistance, altered mitochondrial metabolism, and oxidative stress are revealed by this global metabolomic approach. Moreover, the utility of this in vitro model for investigating the mechanisms of steatotic progression, insulin resistance, and lipotoxicity in NAFLD was demonstrated. "
• Proteoglycans Act as Cellular Hepatitis Delta Virus Attachment Receptors.lamas longarela O, Schmidt TT, Schoneweis K, Romeo R, Wedemeyer H, Urban S, Schulze A
  PloS One, Mar 2013
  Abstract : The hepatitis delta virus (HDV) is a small, defective RNA virus that requires the presence of the hepatitis B virus (HBV) for its life cycle. Worldwide more than 15 million people are co-infected with HBV and HDV. Although much effort has been made, the early steps of the HBV/HDV entry process, including hepatocyte attachment and receptor interaction are still not fully understood. Numerous possible cellular HBV/HDV binding partners have been described over the last years; however, so far only heparan sulfate proteoglycans have been functionally confirmed as cell-associated HBV attachment factors. Recently, it has been suggested that ionotrophic purinergic receptors (P2XR) participate as receptors in HBV/HDV entry. Using the HBV/HDV susceptible HepaRG cell line and primary human hepatocytes (PHH), we here demonstrate that HDV entry into hepatocytes depends on the interaction with the glycosaminoglycan (GAG) side chains of cellular heparan sulfate proteoglycans. We furthermore provide evidence that P2XR are not involved in HBV/HDV entry and that effects observed with inhibitors for these receptors are a consequence of their negative charge. HDV infection was abrogated by soluble GAGs and other highly sulfated compounds. Enzymatic removal of defined carbohydrate structures from the cell surface using heparinase III or the obstruction of GAG synthesis by sodium chlorate inhibited HDV infection of HepaRG cells. Highly sulfated P2XR antagonists blocked HBV/HDV infection of HepaRG cells and PHH. In contrast, no effect on HBV/HDV infection was found when uncharged P2XR antagonists or agonists were applied. In summary, HDV infection, comparable to HBV infection, requires binding to the carbohydrate side chains of hepatocyte-associated heparan sulfate proteoglycans as attachment receptors, while P2XR are not actively involved.
• Ezetimibe blocks hepatitis B virus infection after virus uptake into hepatocytes.Lucifora J, Esser K, Protzer U
  Antiviral Res., Feb 2013
  Abstract : Current treatment of chronic hepatitis B virus (HBV) infection mainly targets viral replication in hepatocytes and leads to curing only in exceptional cases. Despite their potential to improve therapeutic success, no drugs interfering with early infection steps of the hepatotropic pathogen HBV are available to date. Recently, entry of the hepatitis C virus (HCV) has been shown to occur along hepatic cholesterol uptake pathways and ezetimibe, a drug which blocks this lipid transport, has been shown to inhibit HCV infection. We here investigated the effect of ezetimibe on HBV infection using differentiated HepaRG cells as a cell-culture infection model. Treatment with ezetimibe inhibited establishment of intrahepatic cccDNA and expression of viral replication markers when cells were infected with HBV virions, while we observed no effect when the HBV viral genome was transduced via an adenoviral vector. Our data suggest that modulating hepatic cholesterol uptake by ezetimibe inhibits early HBV infection and that ezetimibe sensitive lipid transport pathways represent new targets for antiviral therapy in HBV infection.
• Transcriptomic responses generated by hepatocarcinogens in a battery of liver-based in vitro models.Doktorova TY, Yildirimman R, Vinken M, Vilardell M, Vanhaecke T, Gmuender H, Bort R, Brolen G, Holmgren G, Li R, Vhesne C, Van Deft J, Kleinjans J
  Carcinogenis, Feb 2013
  Abstract : As the conventional approach to assess the potential of a chemical to cause cancer in humans still includes the 2-year rodent carcinogenicity bioassay, development of alternative methodologies is needed. In the present study, the transcriptomics responses following exposure to genotoxic (GTX) and non-genotoxic (NGTX) hepatocarcinogens and non-carcinogens (NC) in five liver-based in vitro models, namely conventional and epigenetically stabilized cultures of primary rat hepatocytes, the human hepatoma-derived cell lines HepaRG and HepG2 and human embryonic stem cell-derived hepatocyte-like cells, are examined. For full characterization of the systems, several bioinformatics approaches are employed including gene-based, ConsensusPathDB-based and classification analysis. They provide convincingly similar outcomes, namely that upon exposure to carcinogens, the HepaRG generates a gene classifier (a gene classifier is defined as a selected set of characteristic gene signatures capable of distinguishing GTX, NGTX carcinogens and NC) able to discriminate the GTX carcinogens from the NGTX carcinogens and NC. The other in vitro models also yield cancer-relevant characteristic gene groups for the GTX exposure, but some genes are also deregulated by the NGTX carcinogens and NC. Irrespective of the tested in vitro model, the most uniformly expressed pathways following GTX exposure are the p53 and those that are subsequently induced. The NGTX carcinogens triggered no characteristic cancer-relevant gene profiles in all liver-based in vitro systems. In conclusion, liver-based in vitro models coupled with transcriptomics techniques, especially in the case when the HepaRG cell line is used, represent valuable tools for obtaining insight into the mechanism of action and identification of GTX carcinogens.
• Induction of Hepatic CYP3A Enzymes by Pregnancy-Related Hormones: Studies in Human Hepatocytes and Hepatic Cell Lines.Papageorgiou I, Grepper S, Unadkat Jd
  Drug metab Dispos, Feb 2013
  Abstract : CYP3A activity is induced by approximately 2-fold during the third trimester of human pregnancy. Placental growth hormone (PGH), estrogens (primarily 17β-estradiol), cortisol, and progesterone have the potential to modulate CYP3A activity. Therefore, we determined whether the elevated plasma concentrations of these hormones during pregnancy induce hepatic CYP3A expression. We incubated sandwich-cultured human hepatocytes (SCHH) from premenopausal female donors (n = 2) with the physiologic (unbound, 1× total) and the 10× total third trimester hormone plasma concentrations (individually and in combination) and determined their effect on CYP3A activity and the transcripts of CYP3A4, CYP3A5, and the respective hormone receptors (growth hormone receptor, glucocorticoid receptor, and estrogen receptor alpha). Of all the hormones, cortisol was the most potent inducer of CYP3A activity and CYP3A4, CYP3A5 mRNA expression. The combination of PGH/growth hormone and cortisol induced CYP3A activity and expression significantly more than did cortisol alone. When incubated with the unbound or total plasma concentration of all the hormones, CYP3A activity in SCHH was induced to an extent comparable to that observed in vivo during the third trimester. These hormones had only a modest effect on the mRNA expression of the hormone receptors. The pattern of induction observed in SCHH was reproduced in HepaRG cells but not in HuH7/HepG2 cells. SCHH or HepaRG cells could be used to determine the mechanistic basis of CYP3A induction during pregnancy and to predict the magnitude of induction likely to be observed during the first and second trimesters, when phenotyping studies to measure in vivo CYP3A activity are logistically difficult to perform.
• 3D organotypic cultures of human HepaRG cells: a tool for in vitro toxicity studies.Gunness P, Mueller D, Shevshenko V, Heinzle E, Ingelmann-Sundberg M, Noor F
  Toxicological Sciences, Feb 2013
  Abstract : Drug-induced human hepatotoxicity is difficult to predict using the current in vitro systems. In this study, long-term 3D organotypic cultures of the human hepatoma HepaRG cell line were prepared using a high-throughput hanging drop method. The organotypic cultures were maintained for 3 weeks and assessed for (1) liver specific functions, including phase I enzyme and transporter activities, (2) expression of liver-specific proteins, and (3) responses to three drugs (acetaminophen, troglitazone, and rosiglitazone). Our results show that the organotypic cultures maintain high liver-specific functionality during 3 weeks of culture. The immunohistochemistry analyses illustrate that the organotypic cultures express liver-specific markers such as albumin, CYP3A4, CYP2E1, and MRP-2 throughout the cultivation period. Accordingly, the production rates of albumin and glucose, as well as CYP2E1 activity, were significantly higher in the 3D versus the 2D cultures. Toxicity studies show that the organotypic cultures are more sensitive to acetaminophen- and rosiglitazone-induced toxicity but less sensitive to troglitazone-induced toxicity than the 2D cultures. Furthermore, the EC50 value (2.7mM) for acetaminophen on the 3D cultures was similar to in vivo toxicity. In summary, the results from our study suggest that the 3D organotypic HepaRG culture is a promising in vitro tool for more accurate assessment of acute and also possibly for chronic drug-induced hepatotoxicity.
• The HepaRG cell line : a valuable in vitro tool hepatitis virus infection studies.Ceelen L, Lieveld M, Forsyth R, vinken M
  Hepatol Int, Jan 2013
  Abstract : Hepatitis virus infections, mainly hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, constitute a major problem for public health since they have a worldwide distribution and because they are associated with hepatocellular carcinoma and death. Current anti- HBV vaccines seem to be effective in the majority of people. However, an important issue waiting to be tackled nowadays is how to cure patients with chronic hepatitis B. Moreover, no vaccine is available today for the prevention of HCV infection. Therefore, the use of adequate in vitro infection systems is a prerequisite for the molecular understanding of the infection events of these viruses, which could result in the development of novel powerful therapeutics. In this respect, the HepaRG cell line exhibits a hepatocyte-like morphology and displays drug metabolism capacity similar to that of primary hepatocytes. HepaRG cells have yet been proven to be a useful tool in the study of viral infections, particularly for deciphering the mechanism of HBV entry into hepatocytes.
• In vitro biokinetics of chlorpromazine and the influence of different dose metrics on effect concentrations for cytotoxicity in Balb/c 3T3, Caco-2 and HepaRG cell cultures.Broeders JJ, Blaauboer BJ, Hermens JL
  Toxicological Sciences, Jan 2013
  Abstract : The extrapolation of in vitro to in vivo toxicity data is a challenge. Differences in sensitivity between cell systems may be due to intrinsic properties of the cell but also because of differences in exposure. In this study, the cytotoxicity and biokinetics of the antipsychotic chlorpromazine (CPZ) were studied in in vitro assays using different cell types and exposure conditions. Different dose metrics were assessed to express the sensitivity to CPZ. The biokinetics of CPZ were measured in cell cultures of Balb/c 3T3, Caco-2 and HepaRG cells. Cytotoxicity was measured by Alamar Blue and expressed using different dose metrics, including the nominal, measured total and measured free CPZ medium concentrations. CPZ was taken up by the cells; the highest amounts in the cell compartments were found in the Caco-2 and HepaRG cells. CPZ was highly protein-bound in the Caco-2 cell medium containing 10% fetal bovine serum, resulting in lower bioavailable exposure concentrations. Moreover, also uptake into the cells strongly influenced the concentration in the medium. The Balb/c 3T3 cells were the most sensitive to the toxic effect of CPZ. The use of different dose metrics influenced the cytotoxicity results found in the three cell types. The data show that in comparing the sensitivity of the tested cell systems, the freely dissolved concentration is a more appropriate dose metric than total concentration in the medium. The ranking in sensitivity of the three cell types for CPZ was dependent on the dose metric used.
• Truncated Active Human Matrix Metalloproteinase-8 Delivered by a Chimeric Adenovirus-Hepatitis B Virus Vector Ameliorates Rat Liver CirrhosisLiu J, Cheng X, Guo Z, Wang Z, Li D, Kang F, Li H, Li B, Cao Z, Nassal M, Sun D
  PLOS one, Jan 2013
  Abstract : BACKGROUND: Liver cirrhosis is a potentially life-threatening disease caused by progressive displacement of functional hepatocytes by fibrous tissue. The underlying fibrosis is often driven by chronic infection with hepatitis B virus (HBV). Matrix metalloproteinases including MMP-8 are crucial for excess collagen degradation. In a rat model of liver cirrhosis, MMP-8 delivery by an adenovirus (Ad) vector achieved significant amelioration of fibrosis but application of Ad vectors in humans is subject to various issues, including a lack of intrinsic liver specificity. METHODS: HBV is highly liver-specific and its principal suitability as liver-specific gene transfer vector is established. HBV vectors have a limited insertion capacity and are replication-defective. Conversely, in an HBV infected cell vector replication may be rescued in trans by the resident virus, allowing conditional vector amplification and spreading. Capitalizing on a resident pathogen to help in its elimination and/or in treating its pathogenic consequences would provide a novel strategy. However, resident HBV may also reduce susceptibility to HBV vector superinfection. Thus a size-compatible truncated MMP-8 (tMMP8) gene was cloned into an HBV vector which was then used to generate a chimeric Ad-HBV shuttle vector that is not subject to superinfection exclusion. Rats with thioacetamide-induced liver cirrhosis were injected with the chimera to evaluate therapeutic efficacy. RESULTS: Our data demonstrate that infectious HBV vector particles can be obtained via trans-complementation by wild-type virus, and that the tMMP8 HBV vector can efficiently be shuttled by an Ad vector into cirrhotic rat livers. There it exerted a comparable beneficial effect on fibrosis and hepatocyte proliferation markers as a conventional full-length MMP-8Ad vector. CONCLUSIONS: Though the rat cirrhosis model does not allow assessing in vivo HBV vector amplification these results advocate the further development of Ad-HBV vectors for liver-specific gene therapy, including and perhaps particularly for HBV-related disease.
• Suppression of AKT Anti-Apoptotic Signaling by a Novel Drug Candidate Results in Growth Arrest and Apoptosis of Hepatocellular Carcinoma Cells.Cuconati A, Mills C, Goddard C, Zhang X, Yu W, Guo H, Xu X, Block TM
  PloS One, Jan 2013
  Abstract : Hepatocellular carcinoma (HCC) is the third most common cause of cancer fatalities worldwide, with limited treatment options and five year survival rates of between <5 and 15%. To address this medical need, we conducted a screen of a drug-like small molecule library for HCC-selective cytotoxins. We report here the identification of a disubstituted aminothiazole termed HBF-0079, with remarkable selective toxicity for HCC-derived cell lines versus non-HCC liver lines and most other cancer lines. HBF-0079 caused irreversible growth arrest and apoptosis of the HCC lines Huh7, Hep3B, HepaRG as well as the hepatoblastoma line HepG2, with CC₅₀ values from ∼0.7-7.7 µM, while more than 45 µM was needed to achieve CC₅₀ values for the immortalized normal hepatocyte lines THLE-2 and PH5CH. Of the sixty cancer lines from the National Cancer Institute panel, only five exhibited >50% growth inhibition by HBF-0079. In Huh7 cells, HBF-0079 induced cell cycle arrest in G1 and concomitant apoptosis, and its effects were irreversible after removal of the compound. These observations corroborate a loss of AKT phosphorylation at the mTORC2-targeted residue S473, with concurrent loss of phosphorylation of the mTORC1 targets SK6 and 4EBP1 in Huh7 but not PH5CH cells. Finally, growth of Hep3B-derived tumors in a murine xenograft model was significantly repressed by the compound through either systemic or intratumoral administration of formulated HBF-0079. The potential for development of this drug candidate is discussed.
• Differential regulation of drug transporter expression by all-trans retinoic acid in hepatoma HepaRG cells and human hepatocytes.Le Vee M, Jouan E, Stieger B, Fardel O
  Eur J Pharm Sci., Jan 2013
  Abstract : All-trans retinoic acid (atRA) is the active form of vitamin A, known to activate retinoid receptors, especially the heterodimer retinoid X receptor (RXR):retinoic acid receptor (RAR) that otherwise may play a role in regulation of some drug transporters. The present study was designed to characterize the nature of human hepatic transporters that may be targeted by atRA and the heterodimer RXR:RAR. Exposure of human hepatoma HepaRG cells and primary human hepatocytes to 5 μM atRA down-regulated mRNA levels of various sinusoidal solute carrier (SLC) influx transporters, including organic anion transporting polypeptide (OATP) 2B1, OATP1B1, organic cation transporter (OCT) 1 and organic anion transporter (OAT) 2, and induced those of the canalicular breast cancer resistance protein (BCRP). The retinoid concomitantly reduced protein expression of OATP2B1 and OATP1B1 and activity of OATPs and OCT1 and induced BCRP protein expression in HepaRG cells. Some transporters such as OATP1B3 and the bile salt export pump (BSEP) were however down-regulated by atRA in primary human hepatocytes, but induced in HepaRG cells, thus pointing out discrepancies between these two liver cell models in terms of detoxifying protein regulation. atRA-mediated repressions of OATP2B1, OATP1B1, OAT2 and OCT1 mRNA expression were finally shown to be counteracted by knocking-down expression of RARα and RXRα through siRNA transfection in HepaRG cells. atRA thus differentially regulated human hepatic drug transporters, mainly in a RXR:RAR-dependent manner, therefore establishing retinoids and retinoid receptors as modulators of liver drug transporter expression.

2012

• Assessment of an automated in vitro basal cytotoxicity test system based on metabolically-competent cells.Rodrigues RM, Bouhifd M, Bories G, Sacco MG, Gribaldo L, Fabbri M, Coecke S, Whelan MP
  Toxicol In vitro, Dec 2012
  Abstract : When in vitro test systems are evaluated for assessment of the toxicity of chemical compounds, particular efforts are made to mimic the in vivo reality as close as possible. Cellular models with appropriate metabolic competence, i.e. with the potency to biotransform chemical compounds, are considered crucial since some metabolites have a different toxicity than their parent compounds. In this study a cell based in vitro test system is proposed to investigate the basal cytotoxicity of several reference chemicals. Both metabolic competent HepaRG cells and cells with no or low hepatic enzyme activity (undifferentiated HepaRG and proliferating HepG2) were used. The classic Neutral Red Uptake (NRU) assay proved to be robust and reliable to be applied as viability assay. The test was performed on a robotic platform, which enabled fully automated and simultaneous screening of the compounds. The outcome of these tests grouped the tested compounds in three categories following their detoxification effect (benzo(a)pyrene, valproic acid), their bio-activation effect (aflatoxin B1) and their specific effect on inhibition of cell proliferation (cycloheximide, sodium lauryl sulphate, atropine sulphate monohydrate, acetylsalicylic acid).
• Substantial Phase 1 and Phase 2 Drug Metabolism and Bile Acid Production of HepaRG Cells in a Bioartificial Liver in Absence of Dimethyl Sulfoxide.Hoekstra R, Nibourg Ga, Van der Hoeven TV, Plomer G, Sppen J, Ackermans MT, CamusS, Kulik W, van Gulik TM, Oude Elferink RP, Chalumeau RA
  Drug metab Dispos, Dec 2012
  Abstract : The human liver cell line HepaRG has been recognized as a promising source for in vitro testing of metabolism and toxicity of compounds. However, currently the hepatic differentiation of these cells relies on exposure to dimethylsulfoxide (DMSO), which, as a side effect, has a cytotoxic effect and represses an all-round hepatic functionality. The AMC-bioartificial liver (AMC-BAL) is a three-dimensional bioreactor that has previously been shown to upregulate various liver functions of cultured cells. We therefore cultured HepaRG cells in the AMC-BAL without DMSO and characterized the drug metabolism. Within 14 days of culture, the HepaRG-AMC-BALs contained highly polarized viable liver-like tissue with heterogeneous expression of CYP3A4. We found a substantial metabolism of the tested substrates, ranging from 26% (UDP-glucuronosyltransferase 1A1), 47% (CYP3A4), to 240% (CYP2C9) of primary human hepatocytes. The CYP3A4 activity could be induced 2-fold by rifampicin, whereas CYP2C9 activity remained equally high. The HepaRG-AMC-BAL secreted bile acids at 43% the rate of primary human hepatocytes and demonstrated hydroxylation, conjugation, and transport of bile salts. Concluding, culturing HepaRG cells in the AMC-BAL yields substantial phase 1 and phase 2 drug metabolism, while maintaining high viability, rendering DMSO addition superfluous for the promotion of drug metabolism. Therefore, AMC-BAL culturing makes the HepaRG cells more suitable for testing metabolism and toxicity of drugs.
• Effects of acute-liver-failure-plasma exposure on hepatic functionality of HepaRG-AMC-Bioartificial Liver.Nibourg GA, Hoekstra R, Van der Hoeven TV, Ackermans MT, Hakvoort TB, Van Gulik TM, Chamuleau RA
  Liver Int., Dec 2012
  Abstract : "BACKGROUND & AIMS: The AMC-bioartificial liver loaded with the human hepatoma cell line HepaRG as biocomponent (HepaRG-AMC-BAL) has recently proven efficacious in rats with acute liver failure (ALF). However, its efficacy may be affected by cytotoxic components of ALF plasma during treatment. In this study, we investigated the effects of ALF-plasma on the HepaRG-AMC-BAL. METHODS: HepaRG-AMC-BALs were connected to the blood circulation of rats with total liver ischaemia, either during the first 5 h after induction of ischaemia (mild ALF group), or during the following 10 h (severe ALF group). After disconnection, the BALs were assessed for cell leakage, gene transcript levels, ammonia elimination, urea production, cytochrome P450 3A4 activity, apolipoprotein A 1 production, glucose and amino acid metabolism. RESULTS: Cell leakage increased 2.5-fold in the severe ALF group, but remained limited in all groups. Hepatic gene transcript levels decreased (max 40-fold) or remained stable. In contrast, hepatic functions increased slightly or remained stable. Particularly, urea production increased 1.5-fold, with a concurrent increase in arginase 2 transcription and arginine consumption, with a trend towards reduced conversion of ammonia into urea. The amino acid consumption increased, however, the net glucose consumption remained stable. CONCLUSIONS: The HepaRG-AMC-BAL retains functionality after both mild and severe exposure to ALF plasma, but urea production may be increasingly derived from arginase 2 activity instead of urea cycle activity. Nevertheless, the increase in cell leakage and decrease in various hepatic transcript levels suggest that a decrease in hepatic functionality may follow upon extended exposure to ALF plasma. "
• Myristoylated preS1-domain of the hepatitis B virus L-protein mediates specific binding todifferentiated hepatocytes.Meier A, Mehrle S, Weiss Ts, Mier W, Urban S
  Hepatology , Dec 2012
  Abstract : Chronic infection with the human hepatitis B virus (HBV) is a global health problem and a main cause of progressive liver diseases. HBV exhibits a narrow host range, replicating primarily in hepatocytes. Both host and hepatocyte specificity presumably involve specific receptor interactions on the target cell; however, direct evidence for this hypothesis is missing. Following the observation that HBV entry is specifically blocked by L-protein-derived preS1-lipopeptides, we visualized specific HBV receptor/ligand complexes on hepatic cells and quantified the turnover kinetics. Using fluorescein isothiocyanate-labeled, myristoylated HBV preS1-peptides we demonstrate (1) the presence of a highly specific HBV receptor on the plasma membrane of HBV-susceptible primary human and tupaia hepatocytes and HepaRG cells but also on hepatocytes from the nonsusceptible species mouse, rat, rabbit and dog; (2) the requirement of a differentiated state of the hepatocyte for specific preS1-binding; (3) the lack of detectable amounts of the receptor on HepG2 and HuH7 cells; (4) a slow receptor turnover at the hepatocyte membrane; and (5) an association of the receptor with actin microfilaments. The presence of the preS1-receptor in primary hepatocytes from some non-HBV-susceptible species indicates that the lack of susceptibility of these cells is owed to a postbinding step. Conclusion: These findings suggest that HBV hepatotropism is mediated by the highly selective expression of a yet unknown receptor* on differentiated hepatocytes, while species specificity of the HBV infection requires selective downstream events, e.g., the presence of host dependency or the absence of host restriction factors. The criteria defined here will allow narrowing down reasonable receptor candidates and provide a binding assay for HBV-receptor expression screens in hepatic cells.
• Inhibition of p53 attenuates steatosis and liver injury in a mouse model of non-alcoholic fatty liver disease.Derdak Z, Villegas KA, Harb R, Wu AM, Sousa A, Wands JR
  J Hepatol, Dec 2012
  Abstract : BACKGROUND & AIMS: p53 and its transcriptional target miRNA34a have been implicated in the pathogenesis of fatty liver. We tested the efficacy of a p53 inhibitor, pifithrin-α p-nitro (PFT) in attenuating steatosis, associated oxidative stress and apoptosis in a murine model of non-alcoholic fatty liver disease (NAFLD).METHODS: C57BL/6 mice were fed a high-fat (HFD) or control diet for 8 weeks; PFT or DMSO (vehicle) was administered three times per week. Markers of oxidative stress and apoptosis as well as mediators of hepatic fatty acid metabolism were assessed by immunohistochemistry, Western blot, real-time PCR, and biochemical assays.RESULTS: PFT administration suppressed HFD-induced weight gain, ALT elevation, steatosis, oxidative stress, and apoptosis. PFT treatment blunted the HFD-induced upregulation of miRNA34a and increased SIRT1 expression. In the livers of HFD-fed, PFT-treated mice, activation of the SIRT1/PGC1α/PPARα axis increased the expression of malonyl-CoA decarboxylase (MLYCD), an enzyme responsible for malonyl-CoA (mCoA) degradation. Additionally, the SIRT1/LKB1/AMPK pathway (upstream activator of MLYCD) was promoted by PFT. Thus, induction of these two pathways by PFT diminished the hepatic mCoA content by enhancing MLYCD expression and function. Since mCoA inhibits carnitine palmitoyltransferase 1 (CPT1), the decrease of hepatic mCoA in the PFT-treated, HFD-fed mice increased CPT1 activity, favored fatty acid oxidation, and decreased steatosis. Additionally, we demonstrated that PFT abrogated steatosis and promoted MLYCD expression in palmitoleic acid-treated human HepaRG cells.CONCLUSIONS: The p53 inhibitor PFT diminished hepatic triglyceride accumulation and lipotoxicity in mice fed a HFD, by depleting mCoA and favoring the β-oxidation of fatty acids.
• Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus.Yan H, Zhong G, Xu G, He W, jing Z, Gao Z, Huang Y, Qi Y, Peng B, Wang H, Fu l, Song M, Chen P
  eLife, Nov 2012
  Abstract : Human hepatitis B virus (HBV) infection and HBV-related diseases remain a major public health problem. Individuals coinfected with its satellite hepatitis D virus (HDV) have more severe disease. Cellular entry of both viruses is mediated by HBV envelope proteins. The pre-S1 domain of the large envelope protein is a key determinant for receptor(s) binding. However, the identity of the receptor(s) is unknown. Here, by using near zero distance photo-cross-linking and tandem affinity purification, we revealed that the receptor-binding region of pre-S1 specifically interacts with sodium taurocholate cotransporting polypeptide (NTCP), a multiple transmembrane transporter predominantly expressed in the liver. Silencing NTCP inhibited HBV and HDV infection, while exogenous NTCP expression rendered nonsusceptible hepatocarcinoma cells susceptible to these viral infections. Moreover, replacing amino acids 157-165 of nonfunctional monkey NTCP with the human counterpart conferred its ability in supporting both viral infections. Our results demonstrate that NTCP is a functional receptor for HBV and HDV.
• Oxidative stress plays a major role in chlorpromazine-induced cholestasis in human heparg cells.Anthérieu S, Azzi PB, Dumont J, Abdel-Razzak Z, Guguen-Guillouzo C, Fromenty B, Robin MA, Guillouzo A
  Hepatology, Nov 2012
  Abstract : "Drugs induce cholestasis by diverse and still poorly understood mechanisms in humans. Early hepatic effects of chlorpromazine (CPZ), a neuroleptic drug known for years to induce intrahepatic cholestasis, were investigated using the differentiated human hepatoma HepaRG cells. Generation of reactive oxygen species (ROS) was detected as early as 15 minutes after CPZ treatment and was associated with an altered mitochondrial membrane potential and disruption of the pericanalicular distribution of F-actin. Inhibition of [3H]-taurocholic acid efflux was observed after 30 minutes and was mostly prevented by N-acetyl cysteine (NAC) cotreatment, indicating a major role of oxidative stress in CPZ-induced bile acid (BA) accumulation. Moreover, 24-hour treatment with CPZ decreased messenger RNA (mRNA) expression of the two main canalicular bile transporters, bile salt export pump (BSEP) and multidrug resistance protein 3 (MDR3). Additional CPZ effects included inhibition of Na+ -dependent taurocholic cotransporting polypeptide (NTCP) expression and activity, multidrug resistance-associated protein 4 (MRP4) overexpression and CYP8B1 inhibition that are involved in BA uptake, basolateral transport, and BA synthesis, respectively. These latter events likely represent hepatoprotective responses which aim to reduce intrahepatic accumulation of toxic BA. Compared to CPZ effects, overloading of HepaRG cells with high concentrations of cholic and chenodeoxycholic acids induced a delayed oxidative stress and, similarly, after 24 hours it down-regulated BSEP and MDR3 in parallel to a decrease of NTCP and CYP8B1 and an increase of MRP4. By contrast, low BA concentrations up-regulated BSEP and MDR3 in the absence of oxidative stress. CONCLUSION: These data provide evidence that, among other mechanisms, oxidative stress plays a major role as both a primary causal and an aggravating factor in the early CPZ-induced intrahepatic cholestasis in human hepatocytes."
• New models of hepatitis E virus replication in human and porcine hepatocyte cell lines.Rogee S, Talbot N, Caperna T, Bouquet J, Barnaud E, Pavio N
  J Gen Virol., Nov 2012
  Abstract : Hepatitis E virus (HEV) causes acute, enterically transmitted hepatitis in human. It is associated with large epidemics in tropical and subtropical regions where it is endemic or with sporadic cases in non-endemic regions. Unlike other hepatitis viruses, HEV has several animal reservoirs. Phylogenetic studies on HEV human and animal sequences, and the identification of cases of direct transmission from animal to human strongly suggest that HEV is a zoonotic agent. The lack of efficient cell culture models limits studies on molecular and cellular aspects of HEV infection and species barrier crossing. The present study reports on the development of two new in vitro models of HEV replication using a human hepatoma-derived cell line, HepaRG, and a porcine embryonic stem cell-derived cell line, PICM-19. These two cell lines have morphological and functional properties similar to primary hepatocytes. These in vitro culture systems support HEV replication and release of encapsidated RNA. These new models represent a powerful tool for studying the viral replication cycle, species barrier crossing and virulence factors.
• Modulation of ethanol effect on hepatocyte proliferation by polyamines.Do TH, Gaboriau F, Morel I, Lepage S, Cannie I, Loréal O, Lescoat G
  Amino Acids, Oct 2012
  Abstract : An occurrence and a magnitude of alcoholic liver diseases depend on the balance between ethanol-induced injury and liver regeneration. Like ethanol, polyamines including putrescine, spermidine, and spermine modulate cell proliferation. Thus, the purpose of this study was to evaluate the relationship between effect of ethanol on hepatocyte (HC) proliferation and polyamine metabolism using the HepaRG cell model. Results showed that ethanol effect in proliferating HepaRG cells was associated with a decrease in intracellular polyamine levels and ornithine decarboxylase (ODC) activity. Ethanol also induced disorders in expression of genes coding for polyamine-metabolizing enzymes. The α-difluoromethyl ornithine, an irreversible inhibitor of ODC, amplified ethanol toxicity on cell viability, protein level, and DNA synthesis through accentuation of polyamine depletion in proliferating HepaRG cells. Conversely, putrescine reversed ethanol effect on cell proliferation parameters. In conclusion, this study suggested that ethanol effect on HC proliferation was closely related to polyamine metabolism and that manipulation of this metabolism by putrescine could protect against the anti-proliferative activity of ethanol.
• Functional consequences of WNT3-Frizzled7- mediated signaling in non-transformed hepatic cells.Nambotin SB, Tomimaru Y, Merle P, Wands JR, Kim M
  Oncogenesis, Oct 2012
  Abstract : We have previously demonstrated that WNT3 and Frizzled7 (FZD7) expression levelswere upregulated in hepatocellular carcinoma (HCC) and that they directly interact to activate the canonical Wnt/β-catenin pathway in HCC cell lines. In this study, we investigated the functional consequences of WNT3 and FZD7 expression levels in non-transformed hepatic cells to address the question of whether WNT3/FZD7-mediated signal transduction could be involved in cellular transformation. After stable transfection of WNT3 and FZD7, the activation of the Wnt/β-catenin pathway was confirmed by western blot, immunostaining and quantitative real-time reverse transcriptase-PCR (qRT-PCR) analysis in two non-transformed hepatocyte-derived cell lines. In vitro characteristics of the malignant phenotype were measured, including cell proliferation, migration, invasion and anchorage-independent growth in soft agar. Stable expression of WNT3 and FZD7 in the two cell lines led to cellular accumulation of β-catenin and expression of downstream target genes activated by this pathway. In the stable WNT3/FZD7-expressing clones, hepatic cell proliferation, migration, invasion as well as soft agar colony formation were enhanced compared with the non-transformed control cells. The epithelial-mesenchymal transition (EMT) factors, Twist, Snail and Vimentin, were increased in cells expressing WNT3 and FZD7. However, the WNT3/FZD7-expressing cells did not form tumors in vivo. We conclude that activation of the WNT3/FZD7 canonical pathway has a role in the early stages of hepatocarcinogenesis by promoting the acquisition of a malignant phenotype with features of EMT.
• The effect of rat acute-liver-failure plasma on HepaRG cells.Hoekstra R, Nibourg GA, Hoeven TV, Ackermans MT, Hakvoort TB, Gulik TM, Elferink RP, Chamuleau RA
  Int J Artif Organs, Sep 2012
  Abstract : "PURPOSE: We recently demonstrated the high liver functionality of the human liver cell line HepaRG, including ammonia eliminating capacity, making it a valuable biocomponent of a bioartificial liver (BAL) to support patients with acute liver failure (ALF). This cell line further gains detoxification properties when cultured with dimethyl sulfoxide (DMSO). In this paper we describe whether its functionality is compromised by the toxic effects of ALF plasma, as has been shown for primary hepatocytes. METHODS: We exposed -DMSO and +DMSO HepaRG cultures during 16 hours to healthy plasma and ALF-rat plasma. The cultures were analyzed for lipid accumulation, cell leakage, apolipoprotein A-1 production, nitrogen metabolism and transcript levels of hepatic genes. RESULTS: The -DMSO cultures showed increased cell leakage after healthy and ALF plasma exposure in contrast to +DMSO cultures, but otherwise the -DMSO and +DMSO cultures were equally affected by exposure to the plasmas. Exposure to both plasmas caused lipid accumulation and decreased transcript levels of various hepatic genes. ALF plasma decreased urea cycle activity, but increased urea production from arginine by upregulated arginase 2. However, total ammonia elimination was not affected by exposure to either plasma, indicating its predominant elimination by fixation into amino acids. In addition, apolipoprotein A-1 production remained constant. CONCLUSIONS: HepaRG cells are negatively affected by rat plasma, even of healthy origin. However, their ammonia eliminating capacity is relatively resistant, underlining their suitability for BAL application. DMSO pre-treatment may increase their viability in plasma. "
• Preservation, induction or incorporation of metabolism into the in vitro cellular system.Pelkonen O, Turpeinen M, Hakkola J, Abass K, Pasanen M, Raunio H, Vähäkangas K
  Tox In Vitro, Aug 2012
  Abstract : Metabolism plays a major role in the toxicokinetics of a vast majority of substances, although other dispositional processes have to be considered as well. There are currently a large repertoire of primary or permanent cells/cell lines with variable metabolic capacities and a number of experimental approaches to preserve, induce or incorporate biotransformation enzymes for the development of metabolically competent cells. Many of these cell lines possess also other important dispositional characteristics mimicking the in vivo situation. Such cell models can be employed in studies targeted for estimating metabolic disposition of a substance or the production of active metabolites and ensuing toxic end points. There are also ways to collect metabolic information by using a large number of non-cellular systems and build a coherent view on metabolism, although not really replacing the actual cellular system. Early consideration of metabolic competence is a necessary prerequisite for the validation and use of cellular systems for toxicity studies and in vitro-in vivo extrapolation.
• Expression and transport function of drug uptake transporters in differentiated HepaRG cells.Kotani N, Maeda K, Debori Y, Camus S, Li R, Chesne C, Sugiyama Y
  Mol Pharmacol., Aug 2012
  Abstract : HepaRG cells have the ability to differentiate into hepatocyte-like cells. Many papers have shown that these hepatocyte-like cells share several functional properties with intact human hepatocytes. However, although previous studies have indicated the partial maintenance of mRNA expression of drug transporters, their expression and function have not been quantitatively characterized. In the present study, the mRNA and protein expression levels and transport activities of hepatic uptake transporters, organic anion transporting polypeptides (OATPs) and Na(+)-taurocholate cotransporting polypeptide (NTCP) in HepaRG cells were compared with those in cryopreserved human hepatocytes. The mRNA expression levels of OATP1B1, OATP1B3, OATP2B1, and NTCP in HepaRG cells were 22-38%, 2-15%, 82-113%, and 191-247% of those in human hepatocytes, respectively. The relative protein expression of these transporters was comparable with their mRNA expression. We observed saturable uptake of typical substrates of NTCP and OATPs except for cholecystokinin octapeptide (OATP1B3-selective substrate), and Na(+)-dependent uptake of taurocholate was confirmed. Their relative uptake clearances were well explained by their mRNA and protein expression levels. Additionally, inhibition potencies of 12 OATP1B1 inhibitors were investigated both in HepaRG cells and in OATP1B1-expressing HEK293 cells to demonstrate the usefulness of HepaRG cells for the characterization of OATP1B1-mediated drug-drug interactions. The K(i) values in both cell lines were comparable and showed significant correlation. These results suggest that the hepatic uptake transport function of OATP and NTCP transporters was relatively well maintained in HepaRG, although OATP1B3 function was too low to be detected.
• Investigation of ifosfamide nephrotoxicity induced in a liver kidney co-culture biochip.Choucha-Snouber L, Aninat C, Grsicom L, Madalinski G, Brochot C, Poleni PE, Razan F, Guillouzo CG, Legallais C, Corlu A, Leclerc E
  Biotechnol Bioeng., Aug 2012
  Abstract : In this article, we present a liver-kidney co-culture model in a micro fluidic biochip. The liver was modeled using HepG2/C3a and HepaRG cell lines and the kidney using MDCK cell lines. To demonstrate the synergic interaction between both organs, we investigated the effect of ifosfamide, an anticancerous drug. Ifosfamide is a prodrug which is metabolized by the liver to isophosforamide mustard, an active metabolite. This metabolism process also leads to the formation of chloroacetaldehyde, a nephrotoxic metabolite and acrolein a urotoxic one. In the biochips of MDCK cultures, we did not detect any nephrotoxic effects after 72 h of 50 µM ifosfamide exposure. However, in the liver-kidney biochips, the same 72 h exposure leads to a nephrotoxicity illustrated by a reduction of the number of MDCK cells (up to 30% in the HepaRG-MDCK) when compared to untreated co-cultures or treated MDCK monocultures. The reduction of the MDCK cell number was not related to a modification of the cell cycle repartition in ifosfamide treated cases when compared to controls. The ifosfamide biotransformation into 3-dechloroethylifosfamide, an equimolar byproduct of the chloroacetaldehyde production, was detected by mass spectrometry at a rate of apparition of 0.3 ± 0.1 and 1.1 ± 0.3 pg/h/biochips in HepaRG monocultures and HepaRG-MDCK co-cultures respectively. Any metabolite was detected in HepG2/C3a cultures. Furthermore, the ifosfamide treatment in HepaRG-MDCK co-culture system triggered an increase in the intracellular calcium release in MDCK cells on contrary to the treatment on MDCK monocultures. As 3-dechloroethylifosfamide is not toxic, we have tested the effect of equimolar choloroacetaldehyde concentration onto the MDCK cells. At this concentration, we found a quite similar calcium perturbation and MDCK nephrotoxicity via a reduction of 30% of final cell numbers such as in the ifosfamide HepaRG-MDCK co-culture experiments. Our results suggest that ifosfamide nephrotoxicity in a liver-kidney micro fluidic co-culture model using HepaRG-MDCK cells is induced by the metabolism of ifosfamide into chloroacetaldehyde whereas this pathway is not functional in HepG2/C3a-MDCK model. This study demonstrates the interest in the development of systemic organ-organ interactions using micro fluidic biochips. It also illustrated their potential in future predictive toxicity model using in vitro models as alternative methods.
• Proteomic analysis of plasma membranes isolated from undifferentiated and differentiated HepaRG cells.Sokolowska I, Dorobantu C, Woods AG, Macovei A, Branza-Nichita N, Darie CC
  Proteome Sci., Aug 2012
  Abstract : Liver infection with hepatitis B virus (HBV), a DNA virus of the Hepadnaviridae family, leads to severe disease, such as fibrosis, cirrhosis and hepatocellular carcinoma. The early steps of the viral life cycle are largely obscure and the host cell plasma membrane receptors are not known. HepaRG is the only proliferating cell line supporting HBV infection in vitro, following specific differentiation, allowing for investigation of new host host-cell factors involved in viral entry, within a more robust and reproducible environment. Viral infection generally begins with receptor recognition at the host cell surface, following highly specific cell-virus interactions. Most of these interactions are expected to take place at the plasma membrane of the HepaRG cells. In the present study, we used this cell line to explore changes between the plasma membrane of undifferentiated (-) and differentiated (+) cells and to identify differentially-regulated proteins or signaling networks that might potentially be involved in HBV entry. Our initial study identified a series of proteins that are differentially expressed in the plasma membrane of (-) and (+) cells and are good candidates for potential cell-virus interactions. To our knowledge, this is the first study using functional proteomics to study plasma membrane proteins from HepaRG cells, providing a platform for future experiments that will allow us to understand the cell-virus interaction and mechanism of HBV viral infection.
• Analysis of the epitope and neutralizing capacity of human monoclonal antibodies.Tajiri K, Ozawa T, Jin A, Tokimitsu Y, Minemura M, Kishi H, Sugiyama Y, Muraguchi A
  Antiviral Research, Jul 2012
  Abstract : Hepatitis B virus (HBV) is an infectious agent that is a significant worldwide public health issue. However, the mechanism by which vaccination-induced antibodies prevent HBV infection remains unclear. To investigate the mechanism by which antibodies induced by hepatitis B surface Ag (HBsAg)-vaccination prevent HBV infection in humans, we prepared human monoclonal antibodies (mAbs) against HBsAg using a novel cell-microarray system from peripheral blood B-lymphocytes from vaccinated individuals. We then characterized the IgG subclass, L-chain subtype, and V-gene repertoire of the H/L-chain, as well as affinities of each of these mAbs. We also determined the epitopes of the individual mAbs using synthesized peptides, and the HBV-neutralizing activities of mAbs using the hepatocyte cell line HepaRG. Consequently, IgG1 and kappa chain was mainly used as the mAbs for HBsAg. Seventy percent of the mAbs bound to the loop domain of the small-HBsAg and showed greater neutralizing activities. There were no relationships between their affinities and neutralization activities. A combination of mAbs recognizing the first loop domain showed a synergistic effect on HBV-neutralizing activity that surpassed conventional hepatitis B-Ig (HBIG) in the HepaRG cell line assay. These results may contribute to the development of effective mAb treatment against HBV infection replacing conventional HBIG administration.
• Characterization of the Host Factors Required for Hepadnavirus Covalently Closed Circular (ccc) DNA Formation.Guo H, Xu C, Zhou T, Block TM, Guo JT
  Plos One, Jul 2012
  Abstract : Synthesis of the covalently closed circular (ccc) DNA is a critical, but not well-understood step in the life cycle of hepadnaviruses. Our previous studies favor a model that removal of genome-linked viral DNA polymerase occurs in the cytoplasm and the resulting deproteinized relaxed circular DNA (DP-rcDNA) is subsequently transported into the nucleus and converted into cccDNA. In support of this model, our current study showed that deproteinization of viral double-stranded linear (dsl) DNA also took place in the cytoplasm. Furthermore, we demonstrated that Ku80, a component of non-homologous end joining DNA repair pathway, was essential for synthesis of cccDNA from dslDNA, but not rcDNA. In an attempt to identify additional host factors regulating cccDNA biosynthesis, we found that the DP-rcDNA was produced in all tested cell lines that supported DHBV DNA replication, but cccDNA was only synthesized in the cell lines that accumulated high levels of DP-rcDNA, except for NCI-H322M and MDBK cells, which failed to synthesize cccDNA despite of the existence of nuclear DP-rcDNA. The results thus imply that while removal of the genome-linked viral DNA polymerase is most likely catalyzed by viral or ubiquitous host function(s), nuclear factors required for the conversion of DP-rcDNA into cccDNA and/or its maintenance are deficient in the above two cell lines, which could be useful tools for identification of the elusive host factors essential for cccDNA biosynthesis or maintenance.
• 3D HepaRG Model as an attractive tool for toxicity testing.Leite SB, Wilk-Zasadna I, Zaldivar Comenges JM, Airola E, Reis-Fernandes MA, Mennecozzi M, Guguen-Guillouzo C, Chesne C, Gouillou C, Alves PM, Coecke S
  Toxicol Sci., Jul 2012
  Abstract : The culture of HepaRG cells as three dimensional (3D) structures in the spinner-bioreactor may represent added value as a hepatic system for toxicological purposes. The use of a cost-effective commercially available bioreactor, which is compatible with high-throughput cell analysis, constitutes an attractive approach for routine use in the drug testing industry. In order to assess specific aspects of the biotransformation capacity of the bioreactor-based HepaRG system, the induction of CYP450 enzymes (i.e., CYP1A2, 2B6, 2C9, and 3A4) and the activity of the phase II enzyme, uridine diphosphate glucuronoltransferase (UGT), were tested. The long-term functionality of the system was demonstrated by 7-week stable profiles of albumin secretion, CYP3A4 induction, and UGT activities. Immunofluorescence-based staining showed formation of tissue-like arrangements including bile canaliculi-like structures and polar distribution of transporters. The use of in silico models to analyze the in vitro data related to hepatotoxic activity of acetaminophen (APAP) demonstrated the advantage of the integration of kinetic and dynamic aspects for a better understanding of the in vitro cell behavior. The bioactivation of APAP and its related cytotoxicity was assessed in a system compatible to high-throughput screening. The approach also proved to be a good strategy to reduce the time necessary to obtain fully differentiated cell cultures. In conclusion, HepaRG cells cultured in 3D spinner-bioreactors are an attractive tool for toxicological studies, showing a liver-like performance and demonstrating a practical applicability for toxicodynamic approaches.
• Evaluation of Organic Anion-Transporting Polypstide 1B1 and CYP3A4 activities in primary human hepatocytes and HepaRGcells cultured in a dynamic three-dimensional bioreactor system.Ulvestad M, Darnell M, Molden E, Ellis E, Asberg A, Andersson TB
  J Pharmacol Exp Ther., Jul 2012
  Abstract : The long-term stability of liver cell functions is a major challenge when studying hepatic drug transport, metabolism, and toxicity in vitro. The aim of the present study was to investigate organic anion-transporting polypeptide (OATP) 1B1 and CYP3A4 activities in fresh primary human hepatocytes and differentiated cryopreserved HepaRG cells when cultured in a three-dimensional (3D) bioreactor system. OATP1B1 activity was determined by loss from media experiments of [(3)H]estradiol-17β-D-glucuronide and atorvastatin acid (ATA) for up to 7 days in culture. ATA metabolite formation was determined at days 3 to 4 to evaluate CYP3A4 activity. Overall, the results showed that freshly isolated human hepatocytes inoculated in the bioreactor retained OATP1B1 activity for at least 7 days, whereas in HepaRG cells no OATP1B1 activity was observed beyond day 2. The activity data were in agreement with immunohistochemical stainings, which showed that OATP1B1 protein expression was preserved for at least 9 days in fresh human hepatocytes, whereas OATP1B1 was expressed markedly lower in HepaRG cells after 9 days in culture. Fresh human hepatocytes and HepaRG cells exhibited similar CYP3A4 activity in bioreactor culture, and immunohistochemical stainings supported these findings. Activity and mRNA expression of OATP1B1 and CYP3A4 in primary human hepatocytes compared with HepaRG cells in fresh suspensions were in agreement with data obtained in bioreactor culture. In conclusion, freshly isolated human hepatocytes cultured in a 3D bioreactor system preserve both OATP1B1 and CYP3A4 activities, allowing long-term in vitro studies on drug disposition and toxicity.
• In vitro evaluation of major in vivo drug metabolic pathways using primary human hepatocytes and HepaRG cells in suspension and in a dynamic three-dimensional bioreactor system.Darnell MH, Ulvestad M, Ellis E, Weidolf L, Andersson TB
  J Pharmacol Exp Ther., Jul 2012
  Abstract : Major human specific metabolites, not detected during in vivo and in vitro preclinical studies, may cause unexpected drug interactions and toxicity in human and delays in clinical programs. Thus, reliable preclinical tools for the detection of major human metabolites are of high importance. The aim of this study was to compare major drug metabolic pathways in HepaRG cells, a human hepatoma cell line, to fresh human hepatocytes, cryopreserved human hepatocytes, and human in vivo data. Furthermore, the maintenance of cytochrome P450 (P450) and UDP-glucuronosyltransferase (UGT) activities in a dynamic three-dimensional (3D) bioreactor were evaluated over time by using HepaRG cells and human hepatocytes. (14)C-diclofenac and a candidate from AstraZeneca's drug development program, (14)C-AZD6610, which are metabolized by P450 and UGT in vivo, were used as model substrates. The proportion of relevant biotransformation pathways of the investigated drug was clearly different in the various cell systems. The hydroxylation route was favored in primary human hepatocytes, whereas the glucuronidation route was favored in HepaRG cells. The human in vivo metabolite profile of AZD6610 was best represented by human hepatocytes, whereas all major diclofenac metabolites were detected in HepaRG cells. Moreover, the metabolite profiles in cryopreserved and fresh human hepatocytes were essentially the same. The liver bioreactor using both fresh human hepatocytes and HepaRG cells retained biotransformation capacity over 1 week. Thus, the incubation time can be increased from a few hours in suspension to several days in 3D cultures, which opens up for detection of metabolites from slowly metabolized drugs.
• Nanofibrillar cellulose hydrogel promotes three-dimensional liver cell culture.Bhattacharya M, Malinen MM, Lauren P, Lou YR, Kuisma SW, Kanninen L, Lille M, Corlu A, Guguen-Guillouzo C, Ikkala O, Laukkanen A, Urtti A, Yliperttula M
  J Control Release, Jul 2012
  Abstract : Over the recent years, various materials have been introduced as potential 3D cell culture scaffolds. These include protein extracts, peptide amphiphiles, and synthetic polymers. Hydrogel scaffolds without human or animal borne components or added bioactive components are preferred from the immunological point of view. Here we demonstrate that native nanofibrillar cellulose (NFC) hydrogels derived from the abundant plant sources provide the desired functionalities. We show 1) rheological properties that allow formation of a 3D scaffold in-situ after facile injection, 2) cellular biocompatibility without added growth factors, 3) cellular polarization, and 4) differentiation of human hepatic cell lines HepaRG and HepG2. At high shear stress, the aqueous NFC has small viscosity that supports injectability, whereas at low shear stress conditions the material is converted to an elastic gel. Due to the inherent biocompatibility without any additives, we conclude that NFC generates a feasible and sustained microenvironment for 3D cell culture for potential applications, such as drug and chemical testing, tissue engineering, and cell therapy.
• The HepaRG cell line: a unique in vitro tool for understanding drug metabolism and toxicology in human.Andersson TB, Kanebratt KP, Kenna JG
  Expert Opin Drug Metab Toxicol, Jul 2012
  Abstract : "INTRODUCTION: HepaRG is a unique cell line showing a great plasticity, which differentiates to both canaliculae-like and hepatocyte-like cells. The long-term stability of key cell functions, for example, the drug-metabolizing cytochrome P450 (CYP) enzyme activities, in culture is especially useful in drug metabolism, disposition and toxicity studies. AREAS COVERED: This review describes features of the HepaRG cells focusing on drug-metabolizing enzymes and drug transporters, their functionality and regulation. Several applications in drug discovery studies are discussed and the use of HepaRG, as a human relevant predictive in vitro CYP induction model, is described. In addition, promising studies using HepaRG cells for understanding liver toxicity mechanisms by drug compounds are also discussed. EXPERT OPINION: HepaRG cells exhibit features which make them useful as an in vitro model for drug metabolism, disposition and toxicity studies, and could, for many studies, replace the requirement for primary human hepatocytes. Care should be taken since HepaRG cells are of a specific genotype which is reflected in the expression of drug processing proteins. The finding that HepaRG cells form tight junctions provides the basis for formation of functional canalicular structures and this should be investigated further to aid development of human relevant hepatic in vitro 2D and 3D models."
• Proliferative human cell sources applied as biocomponent in bioartificial livers: a review.Nibourg GA, Chamuleau RA, van Gulik TM, Hoekstra R
  Expert Opin Biol Ther., Jul 2012
  Abstract : "INTRODUCTION: Bioartificial livers (BALs) are urgently needed to bridge severe liver failure patients to liver transplantation or liver regeneration. When based on primary hepatocytes, their efficacy has been shown in animal experiments and their safety was confirmed in clinical trials. However, a proliferative human cell source with therapeutic functionality is needed to secure availability and move BAL application forward. AREAS COVERED: This review compares the performance of BALs based on proliferative human biocomponents and primary hepatocytes. This review evaluates relevant studies identified by searching the MEDLINE database until July 2011 and some of our own unpublished data. EXPERT OPINION: All the discussed hepatocyte-like biocomponents show deficiencies in their hepatic functionality compared with primary hepatocytes, particularly functions occurring late in liver development. Nonetheless, the HepaRG, HepG2-GS-CYP3A4, and mesenchymal stem cells show efficacy in a statistically well-powered animal model of acute liver failure, when applied in a BAL device. Various methods to gain higher functionality of BALs, including genetic modification, the usage of combinatory cell sources, and improvement of culture methods, have scarcely been applied, but may further pave the path for BAL application. Clinical implementation of a BAL based on a human proliferative biocomponent is still several years away. "
• Time-Dependent Inhibition and Estimation of CYP3A Clinical Pharmacokinetic Drug-Drug Interactions Using Plated Human Cell Systems.Albaugh DR, Fullenwider CL, Fisher MB, Hutzler JM
  Drug Metab Dispos., Jul 2012
  Abstract : The current studies assessed the utility of freshly plated hepatocytes, cryopreserved plated hepatocytes, and cryopreserved plated HepaRG cells for the estimation of inactivation parameters k(inact) and K(I) for CYP3A. This was achieved using a subset of CYP3A time-dependent inhibitors (fluoxetine, verapamil, clarithromycin, troleandomycin, and mibefradil) representing a range of potencies. The estimated k(inact) and K(I) values for each time-dependent inhibitor were compared with those obtained using human liver microsomes and used to estimate the magnitude of clinical pharmacokinetic drug-drug interaction (DDI). The inactivation kinetic parameter, k(inact), was most consistent across systems tested for clarithromycin, verapamil, and troleandomycin, with a high k(inact) of 0.91 min(-1) observed for mibefradil in HepaRG cells. The apparent K(I) estimates derived from the various systems displayed a range of variability from 3-fold for clarithromycin (5.4-17.7 μM) to 6-fold for verapamil (1.9-12.6 μM). In general, the inactivation kinetic parameters derived from the cell systems tested fairly replicated what was observed in time-dependent inhibition studies using human liver microsomes. Despite some of the observed differences in inactivation kinetic parameters, the estimated DDIs derived from each of the tested systems generally agreed with the clinically reported DDI within approximately 2-fold. In addition, a plated cell approach offered the ability to conduct longer primary incubations (greater than 30 min), which afforded improved ability to identify the weak time-dependent inhibitor fluoxetine. Overall, results from these studies suggest that in vitro inactivation parameters generated from plated cell systems may be a practical approach for identifying time-dependent inhibitors and for estimating the magnitude of clinical DDIs.
• Perfusion flow rate substantially contributes to the performance of the HepaRG-AMC-bioartificial liver.Nibourg GA, Boer JD, van der Hoeven TV, Ackermans MT, van Gulik TM,, Chamuleau RA, Hoekstra R
  Biotechnol Bioeng., Jun 2012
  Abstract : Bioartificial livers (BALs) are bioreactors containing liver cells that provide extracorporeal liver support to liver-failure patients. Theoretically, the plasma perfusion flow rate through a BAL is an important determinant of its functionality. Low flow rates can limit functionality due to limited substrate availability, and high flow rates can induce cell damage. This hypothesis was tested by perfusing the AMC-BAL loaded with the liver cell line HepaRG at four different medium flow rates (0.3, 1.5, 5, and 10 mL/min). Hepatic functions ammonia elimination, urea production, lactate consumption, and 6β-hydroxylation of testosterone showed 2-20-fold higher rates at 5 mL/min compared to 0.3 mL/min, while cell damage remained stable. However, at 10 mL/min cell damage was twofold higher, and maximal hepatic functionality was not changed, except for an increase in lactate elimination. On the other hand, only a low flow rate of 0.3 mL/min allowed for an accurate measurement of the ammonia and lactate mass balance across the bioreactor, which is useful for monitoring the BAL's condition during treatment. These results show that (1) the functionality of a BAL highly depends on the perfusion rate; (2) there is a universal optimal flow rate based on various function and cell damage parameters (5 mL/min for HepaRG-BAL); and (3) in the current set-up the mass balance of substrate, metabolite, or cell damage markers between in-and out-flow of the bioreactor can only be determined at a suboptimal, low, perfusion rate (0.3 mL/min for HepaRG-BAL).
• Liver Progenitor Cell Line HepaRG Differentiated in a Bioartificial Liver Effectively Supplies Liver Support to Rats with Acute Liver Failure.Nibourg GA, Chamuleau RA, van der Hoeven TV, Maas MA, Ruiter AF, Lamers WH, Oude Elferink RP, van Gulik TM, Hoekstra R
  PLoS One, Jun 2012
  Abstract : A major roadblock to the application of bioartificial livers is the need for a human liver cell line that displays a high and broad level of hepatic functionality. The human bipotent liver progenitor cell line HepaRG is a promising candidate in this respect, for its potential to differentiate into hepatocytes and bile duct cells. Metabolism and synthesis of HepaRG monolayer cultures is relatively high and their drug metabolism can be enhanced upon treatment with 2% dimethyl sulfoxide (DMSO). However, their potential for bioartificial liver application has not been assessed so far. Therefore, HepaRG cells were cultured in the Academic Medical Center bioartificial liver (AMC-BAL) with and without DMSO and assessed for their hepatic functionality in vitro and in a rat model of acute liver failure. HepaRG-AMC-BALs cultured without DMSO eliminated ammonia and lactate, and produced apolipoprotein A-1 at rates comparable to freshly isolated hepatocytes. Cytochrome P450 3A4 transcript levels and activity were high with 88% and 37%, respectively, of the level of hepatocytes. DMSO treatment of HepaRG-AMC-BALs reduced the cell population and the abovementioned functions drastically. Therefore, solely HepaRG-AMC-BALs cultured without DMSO were tested for efficacy in rats with acute liver failure (n = 6). HepaRG-AMC-BAL treatment increased survival time of acute liver failure rats ∼50% compared to acellular-BAL treatment. Moreover, HepaRG-AMC-BAL treatment decreased the progression of hepatic encephalopathy, kidney failure, and ammonia accumulation. These results demonstrate that the HepaRG-AMC-BAL is a promising bioartificial liver for clinical application.
• Phosphonium-Containing Diblock Copolymers for Enhanced Colloidal Stability and Efficient Nucleic Acid Delivery.Hemp ST, Smith AE, Bryson JM, Allen MH Jr, Long TE
  Biomacromolecules, Jun 2012
  Abstract : RAFT polymerization successfully controlled the synthesis of phosphonium-based AB diblock copolymers for nonviral gene delivery. A stabilizing block of either oligo(ethylene glycol(9)) methyl ether methacrylate or 2-(methacryloxy)ethyl phosphorylcholine provided colloidal stability, and the phosphonium-containing cationic block of 4-vinylbenzyltributylphosphonium chloride induced electrostatic nucleic acid complexation. RAFT polymerization generated well-defined stabilizing blocks (M(n) = 25000 g/mol) and subsequent chain extension synthesized diblock copolymers with DPs of 25, 50, and 75 for the phosphonium-containing block. All diblock copolymers bound DNA efficiently at ± ratios of 1.0 in H(2)O, and polyplexes generated at ± ratios of 2.0 displayed hydrodynamic diameters between 100 and 200 nm. The resulting polyplexes exhibited excellent colloidal stability under physiological salt or serum conditions, and they maintained constant hydrodynamic diameters over 24 h. Cellular uptake studies using Cy5-labeled DNA confirmed reduced cellular uptake in COS-7 and HeLa cells and, consequently, resulted in low transfection in these cell lines. Serum transfection in HepaRG cells, which are a predictive cell line for in vivo transfection studies, showed successful transfection using all diblock copolymers with luciferase expression on the same order of magnitude as Jet-PEI. All diblock copolymers exhibited low cytotoxicity (>80% cell viability). Promising in vitro transfection and cytotoxicity results suggest future studies involving the in vivo applicability of these phosphonium-based diblock copolymer delivery vehicles.
• Decreased infectivity of nucleoside analogs-resistant hepatitis B virus mutants.Billioud G, Pichoud C, Parent R, Zoulim F
  J Hepatol, Jun 2012
  Abstract : "BACKGROUND & AIMS: To understand the mechanisms of emergence and selection of HBV polymerase variants, which may also harbor mutations in the overlapping envelope protein, we analyzed the in vitro virus production and infectivity of the main viral mutants resistant to lamivudine and adefovir. METHODS: HBV-resistant mutants (rtL180M+M204V, rtV173L+L180M+M204V, rtM204I, rtL180M+M204I, rtN236T, rtA181V, rtA181V+rtN236T, rtA181T+N236T, and rtA181T) were produced in HepG2 cells permanently expressing the respective viral genomes. Viral protein expression, secretion, and viral particle production were studied by ELISA, Western blot, and transmission electron microscopy. To study only the effect of surface gene mutants on virus infectivity, HepaRG cells were inoculated with HDV pseudo-particles coated with the mutant HBV envelopes. To evaluate infectivity and replication in a global fashion, HepaRG cells were inoculated with HBV mutants. RESULTS: HBeAg was expressed and secreted in cell supernatants in all mutant-expressing cell lines. As expected, mutants harboring a sW196Stop mutation in the surface gene did not express small envelope proteins. All mutants expressing HBsAg were able to produce viral particles. HDV particles coated with mutant envelopes were less infectious than WT in HepaRG cells. Finally, we found that resistant mutants exhibit lower infectivity and replication ability than WT virus. CONCLUSIONS: Based on this study, we found that envelope substitutions modulate viral protein expression, HDV coating, and viral infectivity. These envelope modifications provide novel insights into the features of emerging HBV variants during antiviral therapies and suggest that such mutants are less prone to transmission than their WT counterpart. "
• Characterization of human cytochrome P450 induction by pesticides.Abass K, Lämsä V, Reponen P, Küblbeck J, Honkakoski P, Mattila S, Pelkonen O, Hakkola J
  Toxicology, May 2012
  Abstract : Pesticides are a large group of structurally diverse toxic chemicals. The toxicity may be modified by cytochrome P450 (CYP) enzyme activity. In the current study, we have investigated effects and mechanisms of 24 structurally varying pesticides on human CYP expression. Many pesticides were found to efficiently activate human pregnane X receptor (PXR) and/or constitutive androstane receptor (CAR). Out of the 24 compounds tested, 14 increased PXR- and 15 CAR-mediated luciferase activities at least 2-fold. While PXR was predominantly activated by pyrethroids, CAR was, in addition to pyrethroids, well activated by organophosphates and several carbamates. Induction of CYP mRNAs and catalytic activities was studied in the metabolically competent, human derived HepaRG cell line. CYP3A4 mRNA was induced most powerfully by pyrethroids; 50 μM cypermethrin increased CYP3A4 mRNA 35-fold. CYP2B6 was induced fairly equally by organophosphate, carbamate and pyrethroid compounds. Induction of CYP3A4 and CYP2B6 by these compound classes paralleled their effects on PXR and CAR. The urea herbicide diuron and the triazine herbicide atrazine induced CYP2B6 mRNA more than 10-fold, but did not activate CAR indicating that some pesticides may induce CYP2B6 via CAR-independent mechanisms. CYP catalyzed activities were induced much less than the corresponding mRNAs. At least in some cases, this is probably due to significant inhibition of CYP enzymes by the studied pesticides. Compared with human CAR activation and CYP2B6 expression, pesticides had much less effect on mouse CAR and CYP2B10 mRNA. Altogether, pesticides were found to be powerful human CYP inducers acting through both PXR and CAR.
• Statins increase cytochrome P450 4F3-mediated eicosanoids production in human liver cells: A PXR dependent mechanism.Plée-Gautier E, Antoun J, Goulitquer S, Le Jossic-Corcos C, Simon B, Amet Y, Salaün JP, Corcos L
  Biochem Pharmacol., May 2012
  Abstract : In the present study, the ability of lovastatin, a competitive inhibitor of HMG-CoA reductase, to regulate the gene expression and function of Cytochrome P450 4F3B (CYP4F3B) was examined in the well differentiated HepaRG human hepatoma cell line. Statins induced CYP4F3B mRNA, protein and the production of 20-hydroxyeicosatetraenoic acid (20-HETE), a product of arachidonic acid metabolism and a peroxisome proliferator activated receptor (PPAR) ligand. This response was not dependent on cholesterol shortage or on sterol regulatory element binding protein activation. By both a pharmacological and a siRNA approaches, we demonstrated that recruitment of the Pregnane X Receptor (PXR) was required to mediate CYP4F3 induction by lovastatin. Furthermore, the CYP4F3 gene promoter was transcriptionally activated by PXR, and responded to lovastatin. Finally, the expression of fatty acid-responsive genes was increased in response to the statin or 20-HETE in a CYP4F3-dependent way. We propose that metabolites produced by CYP4F3 could modulate lipid metabolism in response to lovastatin. These results suggest the existence of a novel pathway, operating in liver cells, through which statins could lower lipid levels.
• Optimization of the HepaRG cell model for drug metabolism and toxicity studies.Anthérieu S
  Toxicology in vitro, May 2012
  Abstract : The HepaRG cell line is the first human cell line able to differentiate in vitro into mature hepatocyte-like cells. Our main objective within the framework of the EEC-LIINTOP project was to optimize the use of this cell line for drug metabolism and toxicity studies, especially after repeat treatments. The main results showed that differentiated HepaRG cells: (i) retained their drug metabolism capacity (major CYPs, phase 2 enzymes, transporters and nuclear receptors) and responsiveness to prototypical inducers at relatively stable levels for several weeks at confluence. The levels of several functions, including some CYPs such as CYP3A4, were dependent on the addition of dimethyl sulfoxide in the culture medium; (ii) sustained the different types of chemical-induced hepatotoxicity, including steatosis, phospholipidosis and cholestasis, after acute and/or repeat treatment with reference drugs. In particular, drug-induced vesicular steatosis was demonstrated in vitro for the first time. In conclusion, our results from the LIINTOP project, together with other studies reported concomitantly or more recently in the literature, support the conclusion that the metabolically competent human HepaRG cells represent a surrogate to primary human hepatocytes for investigating drug metabolism parameters and both acute and chronic effects of xenobiotics in human liver.
• The human hepatocyte cell lines IHH and HepaRG: models to study glucose, lipid and lipoprotein metabolism.Samanez CH
  Arch Physiol Biochem., May 2012
  Abstract : Metabolic diseases reach epidemic proportions. A better knowledge of the associated alterations in the metabolic pathways in the liver is necessary. These studies need in vitro human cell models. Several human hepatoma models are used, but the response of many metabolic pathways to physiological stimuli is often lost. Here, we characterize two human hepatocyte cell lines, IHH and HepaRG, by analysing the expression and regulation of genes involved in glucose and lipid metabolism. Our results show that the glycolysis pathway is activated by glucose and insulin in both lines. Gluconeogenesis gene expression is induced by forskolin in IHH cells and inhibited by insulin in both cell lines. The lipogenic pathway is regulated by insulin in IHH cells. Finally, both cell lines secrete apolipoprotein B-containing lipoproteins, an effect promoted by increasing glucose concentrations. These two human cell lines are thus interesting models to study the regulation of glucose and lipid metabolism.
• An adaptation of the human HepaRG cells to the in vitro micronucleus assay.Jossé R, Rogue A, Lorge E, Guillouzo A
  Mutagenesis, May 2012
  Abstract : The in vitro micronucleus test is considered as an attractive tool for genotoxicity testing of chemicals because of its simplicity of scoring and wide applicability in different cell types. However, most of the cells currently in use are devoid of the enzyme equipment required for activation of promutagens in the genotoxic metabolites. We postulated that the human HepaRG cell line, which can express xenobiotic metabolising enzymes at levels close to those found in primary human hepatocytes and has retained the indefinite growth capacity of transformed cells, could represent a more suitable model for genotoxicity testing of chemicals requiring metabolic activation. Based on the recommendations of the Organisation for Economic Co-operation and Development test guideline TG 487 for testing of chemicals, HepaRG cell cultures containing >80% mature hepatocytes were treated in situ with various chemicals for 24 h followed by a 3-day mitogenic stimulation with epidermal growth factor without cytokinesis block. In such culture conditions, HepaRG cells underwent >1.5 cell cycle per cell during the mitogenic stimulation. While non-genotoxic compounds (mannitol and staurosporine) did not increase the rate of micronucleated mononucleated cells, all aneugens (colchicine, nocodazole and dichlorodiphenyldichloroethylene) as well as the direct acting clastogen methyl methanesulfonate and clastogens requiring metabolic activation (aflatoxin B1, benzo(a)pyrene and 2-nitrofluorene) induced a statistically significant concentration-related increase in the number of mono-micronucleated cells. The micronucleus test was also performed after 7-day repeat exposure of HepaRG cells to the chemicals. Noticeably, a time-dependent effect was obtained with the three clastogens requiring metabolic activation. In conclusion, our results obtained with HepaRG hepatocytes exposed to various genotoxic compounds requiring or not bioactivation, compared favorably with those reported in various other cell types. They support the view that metabolically competent HepaRG cells have unique potential benefits for testing genotoxic compounds using the in vitro micronucleus assay.
• Co-cultures of enterocytes and hepatocytes for retinoid transport and metabolism.Rossi C
  Toxicol In Vitro, Apr 2012
  Abstract : Dietary retinoid bioavailability involves the interplay of the intestine (transport and metabolism) and the liver (secondary metabolism). To reproduce these processes in vitro, differentiated human intestinal Caco-2/TC7 cells were co-cultured with two hepatocyte cell lines. Murine 3A cells and the more highly differentiated human HepaRG hepatocytes were both shown to respond to β-carotene (BC) and retinol (ROH) treatment by secreting Retinol Binding Protein 4 (RBP4). In co-culture experiments, Caco-2/TC7 were differentiated on filter inserts and transferred for the time of the experiment to culture wells containing confluent 3A or differentiated HepaRG cells. Functionality of the co-cultures was assayed using as endpoints the retinol-dependent secretion of RBP4 and the retinoic acid-dependent induction of CYP26A1 in hepatocytes. BC and ROH added to intestinal Caco-2/TC7 induced a reduction in intracellular RBP4 levels in the underlying hepatocytes and its secretion into the medium. HepaRG hepatocytes were also shown to up-regulate the expression of CYP26A1 mRNA in response to retinoid treatment. This in vitro model represents a useful tool to analyze the absorption and metabolism of retinoids and could be further developed to investigate other dietary compounds and molecules of pharmacological interest.
• Characterization of primary human hepatocytes, HepG2 cells, and HepaRG cells at the mRNA level and CYP activity in response to inducers and their predictivity for the detection of human hepatotoxins.Gerets HH, Tilmant K, Gerin B, Chanteux H, Depelchin BO, Dhalluin S, Atienzar FA
  Cell Biol Toxicol., Apr 2012
  Abstract : In the pharmaceutical industry, improving the early detection of drug-induced hepatotoxicity is essential as it is one of the most important reasons for attrition of candidate drugs during the later stages of drug development. The first objective of this study was to better characterize different cellular models (i.e., HepG2, HepaRG cells, and fresh primary human hepatocytes) at the gene expression level and analyze their metabolic cytochrome P450 capabilities. The cellular models were exposed to three different CYP450 inducers; beta-naphthoflavone (BNF), phenobarbital (PB), and rifampicin (RIF). HepG2 cells responded very weakly to the different inducers at the gene expression level, and this translated generally into low CYP450 activities in the induced cells compared with the control cells. On the contrary, HepaRG cells and the three human donors were inducible after exposure to BNF, PB, and RIF according to gene expression responses and CYP450 activities. Consequently, HepaRG cells could be used in screening as a substitute and/or in complement to primary hepatocytes for CYP induction studies. The second objective was to investigate the predictivity of the different cellular models to detect hepatotoxins (16 hepatotoxic and 5 nonhepatotoxic compounds). Specificity was 100% with the different cellular models tested. Cryopreserved human hepatocytes gave the highest sensitivity, ranging from 31% to 44% (depending on the donor), followed by lower sensitivity (13%) for HepaRG and HepG2 cells (6.3%). Overall, none of the models under study gave desirable sensitivities (80-100%). Consequently, a high metabolic capacity and CYP inducibility in cell lines does not necessarily correlate with a high sensitivity for the detection of hepatotoxic drugs. Further investigations are necessary to compare different cellular models and determine those that are best suited for the detection of hepatotoxic compounds.
• Interactions between hepatitis B virus and aflatoxin B(1): effects on p53 induction in HepaRGcells.Lereau M, Gouas D, Villar S, Besaratinia A, Hautefeuille A, Berthillon P, Martel-Planche G, Nogueira da Costa A, Ortiz-Cuaran S, Hantz O, Pfeifer GP, Hainaut P, Chemin I
  J Gen Virol., Mar 2012
  Abstract : Infection by hepatitis B virus (HBV) and dietary exposure to aflatoxin B(1) (AFB(1)) are the main risk factors for the development of chronic liver disease and hepatocellular carcinoma (HCC). How these factors cooperate is still largely unknown. AFB(1) activation leads to DNA adduction and mutagenesis, with a specific mutation at codon 249 in TP53 (p.R249S). So far, only limited studies have addressed the effects of AFB(1) on HBV replication. We have analysed the effects of both risk factors on p53 induction during HBV infection in HepaRG, a cell line with hepatocyte-like morphology that metabolizes AFB(1) and supports HBV infection. Exposure to AFB(1) up to 5 µM induced a downregulation of HBV replication after 48 h, as measured by a decrease in viral antigens in the culture medium (HBsAg, HBeAg and large envelope protein) and in intracellular levels of HBV transcripts, DNA and HBsAg. Conversely, HBV infection did not significantly modify AFB(1)-DNA adduct formation or repair as assessed by immunodot-blot assay, and the induction of p53 in response to AFB(1) was similar in infected and non-infected HepaRG cells. Overall, our results suggest that AFB(1) exposure decreases HBV replication, whereas DNA damage by AFB(1) and subsequent p53 induction is not affected by the presence of the virus. Thus, in HepaRG cell line, AFB(1) and HBV do not cooperate to increase DNA damage by AFB(1). Further studies on the effects of both factors in a context of chronicity are needed to better understand synergistic effects.
• Hepatocyte polarization is essential for the productive entry of the hepatitis B virus.Schulze A, Mills K, Weiss TS, Urban S
  Hepatology, Feb 2012
  Abstract : "Human hepatitis B virus (HBV) is characterized by a high species specificity and a distinct liver tropism. Within the liver, HBV replication occurs in differentiated and polarized hepatocytes. Accordingly, the in vitro HBV infection of primary human hepatocytes (PHHs) and the human hepatoma cell line, HepaRG, is restricted to differentiated, hepatocyte-like cells. Though preparations of PHH contain up to 100% hepatic cells, cultures of differentiated HepaRG cells are a mixture of hepatocyte-like and biliary-like epithelial cells. We used PHH and HepaRG cells and compared the influence of virus inoculation dose, cell differentiation, and polarization on productive HBV infection. At multiplicities of genome equivalents (mge) >8,000, almost 100% of PHHs could be infected. In contrast, only a subset of HepaRG cells stained positive for HBcAg at comparable or even higher mge. Infection predominantly occurred at the edges of islands of hepatocyte-like HepaRG cells. This indicates a limited accessibility of the HBV receptor, possibly as a result of its polar sorting. Multidrug resistance protein 2 (MRP2), a marker selectively transported to the apical (i.e., canalicular) cell membrane, revealed two polarization phenotypes of HepaRG cells. HBV infection within the islands of hepatocyte-like HepaRG cells preferentially occurred in cells that resemble PHH, exhibiting canalicular structures. However, disruption of cell-cell junctions allowed the additional infection of cells that do not display a PHH-like polarization. CONCLUSION: HBV enters hepatocytes via the basolateral membrane. This model, at least partially, explains the difference of PHH and HepaRG cells in infection efficacy, provides insights into natural HBV infection, and establishes a basis for optimization of the HepaRG infection system. "
• Antiproliferative and iron chelating efficiency of the new bis-8-hydroxyquinoline benzylamine chelator S1 in hepatocyte cultures.lescoat G, Leonce S, Perré A, Gouffier L, Gaboriau F
  Chem Biol Interact., Jan 2012
  Abstract : If a new generation of iron chelators specifically devoted for cancer chemotherapy emerged these last years, any of them has not yet been approved at this time. Accordingly, there is a need to optimize new chelating molecules for iron chelation therapy and cancer treatment. So, the objective of the present investigation was to characterize the antiproliferative activity and the iron chelating capacity of the iron chelator S1 [bis-N-(8-hydroxyquinoline-5-ylmethyl)benzylamine]. Its effects were compared to O-trensox which binds ferric iron with a very high affinity (pFe(3+)=29.5). For this purpose, primary rat hepatocyte stimulated by EGF and human hepatoma HepaRG cell cultures were used. In these models, the anti-proliferative effect, the inhibition of DNA synthesis and the iron-chelating efficiency of increasing concentrations of S1 and O-trensox (0 up to 200 μM) were investigated. In the two cell culture models, we observed that S1 was about 100 times more efficient than O-trensox and the antiproliferative effect of S1 in HepaRG cells appeared at concentrations as low as 0.1 μM without cytotoxicity. Moreover, the stoichiometry of S1 for iron seemed to be in the range S1/Fe(3+)=1. Using the calcein fluorescence assay, we demonstrated that the affinity of S1 for iron was better than that of O-trensox since it was at least two times more effective to restore the fluorescence of calcein previously quenched by iron. So, the iron chelating efficiency of S1 could explain at least partially its higher anti-proliferative effect compared to O-trensox. Finally, these results suggest that molecules such as S1 may constitute a promising starting point to improve cancer treatment.
• Identification of early target genes of aflatoxin B1 in human hepatocytes, inter-individual variability and comparison with other genotoxic compounds.Josse R, Dumont J, Fautrel A, Robin MA, Guillouzo A
  Toxicol Appl Pharmacol., Jan 2012
  Abstract : Gene expression profiling has recently emerged as a promising approach to identify early target genes and discriminate genotoxic carcinogens from non-genotoxic carcinogens and non-carcinogens. However, early gene changes induced by genotoxic compounds in human liver remain largely unknown. Primary human hepatocytes and differentiated HepaRG cells were exposed to aflatoxin B1 (AFB1) that induces DNA damage following enzyme-mediated bioactivation. Gene expression profile changes induced by a 24h exposure of these hepatocyte models to 0.05 and 0.25μM AFB1 were analyzed by using oligonucleotide pangenomic microarrays. The main altered signaling pathway was the p53 pathway and related functions such as cell cycle, apoptosis and DNA repair. Direct involvement of the p53 protein in response to AFB1 was verified by using siRNA directed against p53. Among the 83 well-annotated genes commonly modulated in two pools of three human hepatocyte populations and HepaRG cells, several genes were identified as altered by AFB1 for the first time. In addition, a subset of 10 AFB1-altered genes, selected upon basis of their function or tumor suppressor role, was tested in four human hepatocyte populations and in response to other chemicals. Although they exhibited large variable inter-donor fold-changes, several of these genes, particularly FHIT, BCAS3 and SMYD3, were found to be altered by various direct and other indirect genotoxic compounds and unaffected by non-genotoxic compounds. Overall, this comprehensive analysis of early gene expression changes induced by AFB1 in human hepatocytes identified a gene subset that included several genes representing potential biomarkers of genotoxic compounds.
• The role of CYP3A4 mRNA transcript with shortened 3'-untranslated region in hepatocyte differentiation, liver development, and response to drug induction.Li D, Gaedigk R, Hart SN, Leeder JS, Zhong XB
  Mol Pharmacol., Jan 2012
  Abstract : Cytochrome P450 3A4 (CYP3A4) metabolizes more than 50% of prescribed drugs. The expression of CYP3A4 changes during liver development and may be affected by the administration of some drugs. Alternative mRNA transcripts occur in more than 90% of human genes and are frequently observed in cells responding to developmental and environmental signals. Different mRNA transcripts may encode functionally distinct proteins or contribute to variability of mRNA stability or protein translation efficiency. The purpose of this study was to examine expression of alternative CYP3A4 mRNA transcripts in hepatocytes in response to developmental signals and drugs. cDNA cloning and RNA sequencing (RNA-Seq) were used to identify CYP3A4 mRNA transcripts. Three transcripts were found in HepaRG cells and liver tissues: one represented a canonical mRNA with full-length 3'-untranslated region (UTR), one had a shorter 3'-UTR, and one contained partial intron-6 retention. The alternative mRNA transcripts were validated by either rapid amplification of cDNA 3'-end or endpoint polymerase chain reaction (PCR). Quantification of the transcripts by RNA-Seq and real time quantitative PCR revealed that the CYP3A4 transcript with shorter 3'-UTR was preferentially expressed in developed livers, differentiated hepatocytes, and in rifampicin- and phenobarbital-induced hepatocytes. The CYP3A4 transcript with shorter 3'-UTR was more stable and produced more protein compared with the CYP3A4 transcript with canonical 3'-UTR. We conclude that the 3'-end processing of CYP3A4 contributes to the quantitative regulation of CYP3A4 gene expression through alternative polyadenylation, which may serve as a regulatory mechanism explaining changes of CYP3A4 expression and activity during hepatocyte differentiation and liver development and in response to drug induction.
• Interindividual variability in gene expression profiles in human hepatocytes and comparison with HepaRG cells.Rogue A, Lambert C, Spire C, Claude N, Guillouzo A
  Drug Metab Dispos, Jan 2012
  Abstract : Interindividual variations in functions other than drug metabolism activity, remain poorly elucidated in human liver. In the present study, the whole transcriptome of several human hepatocyte populations and the differentiated human HepaRG cell line have been analyzed and compared, using oligonucleotide pangenomic microarrays. We show that, although the variation in the percentages of expressed genes did not exceed 14% among the primary human hepatocyte populations, huge interindividual differences in the transcript levels of many genes were observed. These genes were related to various functions; in addition to drug metabolism, they mainly concerned carbohydrate, amino acid, and lipid metabolism. HepaRG cells expressed from 81 to 92% of the genes active in human hepatocytes and, in addition, a specific gene subset mainly related to their transformed status, some chromosomal abnormalities, and the presence of primitive biliary epithelial cells. Of interest, a relationship was evidenced between abnormal basal expression levels of some target genes and their corresponding previously reported fold changes in one of four human hepatocyte populations treated with the hepatotoxic drug troglitazone and not with other nonhepatotoxic peroxisome proliferator-activated receptor agonists (PLoS One 6:e18816, 2011). Taken together, our results support the view that HepaRG cells express most of the genes active in primary human hepatocytes and show that expression of most human hepatic genes can quantitatively greatly vary among individuals, thereby contributing to explain the huge interindividual variability in susceptibility to drugs and other environmental factors.
• Comparison of cryopreserved HepaRG cells with cryopreserved human hepatocytes for prediction of clearance for 26 drugs .Zanelli U, Pasquale Caradonna N, Hallifax D, Turlizzi E, Houston JB
  DMD Fast Forward, Jan 2012
  Abstract : Prediction of clearance in drug discovery currently relies on human primary hepatocytes, which can vary widely in drug-metabolizing enzyme activity. Potential alternative in vitro models include the HepaRG cell (from immortalized hepatoma cells), which in culture can express drug-metabolizing enzymes to an extent comparable to that of primary hepatocytes. Utility of the HepaRG cell will depend on robust performance, relative to that of primary hepatocytes, in routine high-throughput analysis. In this study, we compared intrinsic clearance (CL(int)) in the recently developed cryopreserved HepaRG cell system with CL(int) in human cryopreserved pooled hepatocytes and with CL(int) in vivo for 26 cytochrome P450 substrate drugs. There was quantitative agreement between CL(int) in HepaRG cells and human hepatocytes, which was linear throughout the range of CL(int) (1-2000 ml · min(-1) · kg(-1)) and not dependent on particular cytochrome P450 involvement. Prediction of CL(int) in HepaRG cells was on average within 2-fold of in vivo CL(int) (using the well stirred liver model), but average fold error was clearance-dependent with greater underprediction (up to at least 5-fold) for the more highly cleared drugs. Recent reporting of this phenomenon in human hepatocytes was therefore confirmed with the hepatocytes used in this study, and hence the HepaRG cell system appears to share an apparently general tendency of clearance-limited CL(int) in cell models. This study shows the cryopreserved HepaRG cell system to be quantitatively comparable to human hepatocytes for prediction of clearance of drug cytochrome P450 substrates and to represent a promising alternative in vitro tool.

2011

• Lactate is an ideal non-invasive marker for evaluating temporal alterations in cell stress and toxicity in repeat dose testing regimes.Limonciel A, Aschauer L, Wilmes A, Prajczer S, Leonard MO, Pfaller W, Jennings P
  Toxicol In Vitro, Dec 2011
  Abstract : Technological developments are driving in vitro methods towards integrated "omic" strategies. However, there is still an over reliance on classical viability assays for dose range finding. Such assays are not readily suited to the investigation of subtle alterations in cell function and most require termination of the experiment, which makes it difficult to monitor temporal alterations in repeat-dose long term exposure experiments. To this end, we investigated the use of lactate production as a marker of cell stress in long term repeat dose experiments. We conducted daily exposures to eight compounds at five concentrations for 14 days on human renal proximal tubular cells (RPTEC/TERT1), human hepatoma cells (HepaRG) and mouse fibroblasts (BALB-3T3) cells. Compounds were chosen from a training set used in the 7th EU Framework project Predict-IV and consisted of amiodarone, diclofenac, troglitazone, cadmium chloride, cephaloridine, cidofovir, cyclosporine A and buflomedil. At days 1, 3, 7 and 14, lactate was measured in the supernatant medium. At day 14, cells were assayed for resazurin reduction capability and subsequently lysed in methanol for ATP determination. Compound-induced loss of viability was comparable across all cell lines. For all cell types, when cell viability was compromised at day 14, lactate production was induced during the treatment period. In some situations, lactate also fell below control values, indicating cell death. Thus, temporal alterations in supernatant lactate provides information on the time and concentration of stress induction and the time and concentration where cell death becomes the dominant factor. Supernatant lactate production is a simple, cheap and non-invasive parameter. Since many molecular pathways converge on the glycolytic pathway, enhanced lactate production may be considered as a global marker of sub-lethal injury and thus an ideal marker for investigating temporal alterations in long term repeat dose testing in vitro regimes. Copyright © 2011 Elsevier Ltd. All rights reserved.
• Hepatitis B virus X protein is essential to initiate and maintain virus replication after infection.Lucifora J, Arzberger S, Durantel D, Belloni L, Strubin M, Levrero M, Zoulim F, Hantz O
  J Hepatol, Nov 2011
  Abstract : "BACKGROUND & AIMS: The molecular biology of hepatitis B virus (HBV) has been extensively studied but the exact role of the hepatitis B X protein (HBx) in the context of natural HBV infections remains unknown. METHODS: Primary human hepatocytes and differentiated HepaRG cells allowing conditional trans complementation of HBx were infected with wild type (HBV(wt)) or HBx deficient (HBV(x-)) HBV particles and establishment of HBV replication was followed. RESULTS: We observed that cells inoculated with HBx-deficient HBV particles (HBV(x-)) did not lead to productive HBV infection contrary to cells inoculated with wild type HBV particles (HBV(wt)). Although equal amounts of nuclear covalently closed circular HBV-DNA (cccDNA) demonstrated comparable uptake and nuclear import, active transcription was only observed from HBV(wt) genomes. Trans-complementation of HBx was able to rescue transcription from the HBV(x-) genome and led to antigen and virion secretion, even weeks after infection. Constant expression of HBx was necessary to maintain HBV antigen expression and replication. Finally, we demonstrated that HBx is not packaged into virions during assembly but is expressed after infection within the new host cell to allow epigenetic control of HBV transcription from cccDNA. CONCLUSIONS: Our results demonstrate that HBx is required to initiate and maintain HBV replication and highlight HBx as the key regulator during the natural infection process. "
• CYP4F3B Expression Is Associated with Differentiation of HepaRG Human Hepatocytes and Unaffected by Fatty Acid Overload.Madec S, Cerec V, Plée-Gautier E, Antoun J, Glaise D, Salaun JP, Guguen-Guillouzo C, Corlu A
  Drug Metab Dispos, Oct 2011
  Abstract : Fatty acid microsomal ω-oxidation involves cytochrome P450 enzymes. Some of them belonging to the CYP4F3 family are mainly expressed in the liver, making this organ a major player in energy homeostasis and lipid metabolism. To study this important regulation pathway, we used HepaRG cells, which gradually undergo a complete differentiation process. Even at the early stage of the differentiation process, CYP4F3B generated by alternative splicing of the CYP4F3 gene represented the prevalent isoform in HepaRG cells as in the liver. Its increasing expression associated with hepatocyte differentiation status suggested a hepatic-specific control of this isoform. As in liver microsomes, the catalytic hydroxylation of the CYP4F3B substrate [1-¹⁴C]Z9(10)-epoxystearic acid led to major production of 18-hydroxy-9(10)-epoxystearic acid. When treated with saturated, monounsaturated, or polyunsaturated fatty acids, CYP4F3B and CYP4A11 expression remained unchanged whereas CYP4F2 and CYP4F12 expression was transiently up-regulated. A 24-h exposure of differentiated HepaRG cells to various polyunsaturated fatty acids and derivatives induced microvesicular steatosis; down-regulation of lipid metabolism gene regulators such as sterol regulatory element-binding protein-1c, fatty acid synthase, peroxisome proliferator-activated receptor γ (PPARγ), PPARα, and decreased expression of glucose-dependent metabolism genes, which could limit de novo lipogenesis. Docosahexaenoic acid seemed to be the most effective compound. These results suggest that a PPARα-independent pathway could participate to limit lipogenesis and emphasize the role of hepatocytes in the fatty acid ω-hydroxylation pathway. They also give insights on the use of HepaRG hepatocytes to open new avenues of investigations on factors mediating the lipid metabolic pathway and finding new hypolipidemic molecules.
• Predictive Utility of In Vitro Rifampin Induction Data Generated in Fresh and Cryopreserved Human Hepatocytes, Fa2N-4, and HepaRG Cells.Templeton IE, Houston JB, Galetin A
  Drug Metab Dispos, Oct 2011
  Abstract : Rifampin is a potent inducer of CYP3A4 in vitro and precipitates numerous drug-drug interactions (DDIs) when coadministered with CYP3A4 substrates. In the current study, we have critically assessed reported rifampin in vitro CYP3A4 induction data in Fa2N-4, HepaRG, and cryopreserved or primary human hepatocytes, using either CYP3A4 mRNA or probe substrate metabolism as induction endpoints. An in vivo data base of intravenously administered victim drugs (assuming hepatic induction only) was collated (n = 18) to assess the predictive utility of these in vitro systems and to optimize rifampin in vivo E(max). In addition, the effect of substrate hepatic extraction ratio on prediction accuracy was investigated using prediction boundaries proposed recently (Drug Metab Dispos 39:170-173). Incorporation of hepatic extraction ratio in the prediction model resulted in accurate prediction of 89% of intravenous induction DDIs (n = 18), regardless of the in vitro system or induction endpoint (mRNA or CYP3A4 activity). Effects of in vitro parameters from different cellular systems, and optimized in vivo E(max), on the prediction of 21 oral DDIs were assessed. Use of mRNA data resulted in pronounced overprediction across all systems, with 86 to 100% of DDIs outside the acceptable prediction limits; in contrast, CYP3A4 activity predicted up to 62% of the oral DDIs within limits. Although prediction accuracy of oral DDIs was improved when using intravenous optimized rifampin E(max), >35% of DDIs were incorrectly assigned, suggesting potential differential E(max) between intestine and liver. Implications of the findings and recommendations for prediction of rifampin DDIs are discussed.
• The HepaRG cell line is suitable for bioartificial liver application.Hoekstra R, Nibourg GA, van der Hoeven TV, Ackermans MT, Hakvoort TB, van Gulik TM, Lamers WH
  Int J Biochem Cell Biol, Oct 2011
  Abstract : For bioartificial liver application, cells should meet the following minimal requirements: ammonia elimination, drug metabolism and blood protein synthesis. Here we explore the suitability of HepaRG cells, a human cell line reported to differentiate into hepatocyte clusters and surrounding biliary epithelial-like cells at high density and after exposure to dimethyl sulfoxide (DMSO). The effect of carbamoyl-glutamate (CG), an activator of urea cycle enzyme carbamoylphosphate synthetase (CPS) was studied additionally. The effects of DMSO and/or CG were assessed in presence of (15)NH(4)Cl on HepaRG cells in monolayer. We tested hepatocyte-specific functions at transcript and biochemical level, cell damage parameters and performed immunostainings. Ureagenesis, ammonia/galactose elimination and albumin, glutamine synthetase and CPS transcript levels were higher in -DMSO than +DMSO cultures, probably due to a higher cell content and/or cluster-neighbouring regions contributing to their functionality. DMSO treatment increased cytochrome P450 (CYP) transcript levels and CYP3A4 activity, but also cell damage and repressed hepatic functionality in cluster-neighbouring regions. The levels of ammonia elimination, apolipoprotein A-1 production, and transcription of CYP3A4, CYP2B6 and albumin reached those of primary hepatocytes in either the + or -DMSO cultures. Preconditioning with CG increased conversion of (15)NH(4)Cl into (15)N-urea 4-fold only in -DMSO cultures. Hence, HepaRG cells show high metabolic and synthetic functionality in the absence of DMSO, however, their drug metabolism is only high in the presence of DMSO. An unparalleled broad hepatic functionality, suitable for bioartificial liver application, can be accomplished by combining CG treated -DMSO cultures with +DMSO cultures. Copyright © 2011 Elsevier Ltd. All rights reserved.
• Interaction of sirolimus and everolimus with hepatic and intestinal organic anion-transporting polypeptide transporters.Picard N, Levoir L, Lamoureux F, Yee SW, Giacomini KM, Marquet P
  Xenobiotica, Sep 2011
  Abstract : The goal of this study was to assess the interaction of the mTOR inhibitors (ImTORs) sirolimus and everolimus with the human organic anion-transporting polypeptides (OATPs) expressed in hepatocytes and enterocytes by conducting uptake experiments using (i) transfected HEK293T cells, (ii) the hepatocyte-like HepaRG cell line and (iii) the enterocyte-like Caco-2 cell line. Sirolimus and everolimus inhibited in a dose-dependent manner the uptake of [³H]-estrone sulphate by OATP1A2 and OATP1B1 and that of mycophenolic acid 7-O-glucuronide (MPAG) by OATP1B3. ImTOR apparent 50% inhibitory concentrations (IC₅₀) for OATPs were 11.9 µM (OATP1A2), 9.8 µM (OATP1B1) and 1.3 µM (OATP1B3) for sirolimus and 4.2 µM (OATP1A2), 4.1 µM (OATP1B1) and 4.3 µM (OATP1B3) for everolimus. No transport of sirolimus or everolimus by OATP1A2, OATP1B1 or OATP1B3 was observed in HEK-transfected cells and the OAT/OATP/MRP chemical inhibitor probenecid did not significantly decrease the uptake of sirolimus and everolimus in HepaRG and Caco-2 cells, but tended to increase their intracellular accumulation presumably through efflux inhibition. In conclusion, our data suggest that the major OATP transporters expressed in the liver and the intestine do not contribute to the pharmacokinetics of sirolimus and everolimus. However, ImTORs are inhibitors of these transporters.
• Automated detection of hepatotoxic compounds in human hepatocytes using HepaRG cells and image-based analysis of mitochondrial dysfunction with JC-1 dye.Pernelle K, Le Guevel R, Glaise D, Stasio CG, Le Charpentier T, Bouaita B, Corlu A, Guguen-Guillouzo C
  Toxicol Appl Pharmacol., Aug 2011
  Abstract : In this study, our goal was to develop an efficient in situ test adapted to screen hepatotoxicity of various chemicals, a process which remains challenging during the early phase of drug development. The test was based on functional human hepatocytes using the HepaRG cell line, and automation of quantitative fluorescence microscopy coupled with automated imaging analysis. Differentiated HepaRG cells express most of the specific liver functions at levels close to those found in primary human hepatocytes, including detoxifying enzymes and drug transporters. A triparametric analysis was first used to evaluate hepatocyte purity and differentiation status, mainly detoxication capacity of cells before toxicity testing. We demonstrated that culturing HepaRG cells at high density maintained high hepatocyte purity and differentiation level. Moreover, evidence was found that isolating hepatocytes from 2-week-old confluent cultures limited variations associated with an ageing process occurring over time in confluent cells. Then, we designed a toxicity test based on detection of early mitochondrial depolarisation associated with permeability transition (MPT) pore opening, using JC-1 as a metachromatic fluorescent dye. Maximal dye dimerization that would have been strongly hampered by efficient efflux due to the active, multidrug-resistant (MDR) pump was overcome by coupling JC-1 with the MDR inhibitor verapamil. Specificity of this test was demonstrated and its usefulness appeared directly dependent on conditions supporting hepatic cell competence. This new hepatotoxicity test adapted to automated, image-based detection should be useful to evaluate the early MPT event common to cell apoptosis and necrosis and simultaneously to detect involvement of the multidrug resistant pump with target drugs in a human hepatocyte environment.
• Long-term propagation of serum hepatitis C virus (HCV) with production of enveloped HCV particles in human HepaRG hepatocytes.Ndongo-Thiam N, Berthillon P, Errazuriz E, Bordes I, De Sequeira S, Trépo C, Petit MA
  Hepatology, Aug 2011
  Abstract : HepaRG human liver progenitor cells exhibit morphology and functionality of adult hepatocytes. We investigated the susceptibility of HepaRG hepatocytes to in vitro infection with serum-derived hepatitis C virus (HCV) particles (HCVsp) and the potential neutralizing activity of the E1E2-specific monoclonal antibody (mAb) D32.10. The infection was performed using HCVsp when the cells actively divided at day 3 postplating. HCV RNA, E1E2, and core antigens were quantified in HCV particles recovered from culture supernatants of differentiated cells for up to 66 days. The density distributions of particles were analyzed on iodixanol or sucrose gradients. Electron microscopy (EM) and immune-EM studies were performed for ultrastructural analysis of cells and localization of HCV E1E2 proteins in thin sections. HCV infection of HepaRG cells was documented by increasing production of E1E2-core-RNA(+) HCV particles from day 21 to day 63. Infectious particles sedimented between 1.06 and 1.12 g/mL in iodixanol gradients. E1E2 and core antigens were expressed in 50% of HCV-infected cells at day 31. The D32.10 mAb strongly inhibited HCV RNA production in HepaRG culture supernatants. Infected HepaRG cells frozen at day 56 were reseeded at low density. After only 1-3 subcultures and induction of a cell differentiation process the HepaRG cells produced high titer HCV RNA and thus showed to be sustainably infected. Apolipoprotein B-associated empty E1E2 and complete HCV particles were secreted. Characteristic virus-induced intracellular membrane changes and E1E2 protein-association to vesicles were observed. CONCLUSION: HepaRG progenitor cells permit HCVsp infection. Differentiated HepaRG cells support long-term production of infectious lipoprotein-associated enveloped HCV particles. The E1E2-specific D32.10 mAb neutralizes the infection and this cellular model could be used as a surrogate infection system for the screening of entry inhibitors. Copyright © 2011 American Association for the Study of Liver Diseases.
• In vitro genotoxicity test approaches with better predictivity: summary of an IWGT workshop.Pfuhler S, Fellows M, van Benthem J, Corvi R, Curren R, Dearfield K, Fowler P, Frötschl R
  Mutat Res., Aug 2011
  Abstract : Improving current in vitro genotoxicity tests is an ongoing task for genetic toxicologists. Further, the question on how to deal with positive in vitro results that are demonstrated to not predict genotoxicity or carcinogenicity potential in rodents or humans is a challenge. These two aspects were addressed at the 5th International Workshop on Genotoxicity Testing (IWGT) held in Basel, Switzerland, on August 17-19, 2009. The objectives of the working group (WG) were to make recommendations on the use of cell types or lines, if possible, and to provide evaluations of promising new approaches. Results obtained in rodent cell lines with impaired p53 function (L5178Y, V79, CHL and CHO cells) and human p53-competent cells (peripheral blood lymphocytes, TK6 and HepG2 cells) suggest that a reduction in the percentage of non-relevant positive results for carcinogenicity prediction can be achieved by careful selection of cells used without decreasing the sensitivity of the assays. Therefore, the WG suggested using p53- competent - preferably human - cells in in vitro micronucleus or chromosomal aberration tests. The use of the hepatoma cell line HepaRG for genotoxicity testing was considered promising since these cells possess better phase I and II metabolizing potential compared to cell lines commonly used in this area and may overcome the need for the addition of S9. For dermally applied compounds, the WG agreed that in vitro reconstructed skin models, once validated, will be useful to follow up on positive results from standard in vitro assays as they resemble the properties of human skin (barrier function, metabolism). While the reconstructed skin micronucleus assay has been shown to be further advanced, there was also consensus that the Comet assay should be further evaluated due to its independence from cell proliferation and coverage of a wider spectrum of DNA damage. Copyright © 2011 Elsevier B.V. All rights reserved.
• The promoter of human telomerase reverse transcriptase is activated during liver regeneration and hepatocyte proliferation.Sirma H, Kumar M, Meena JK, Witt B, Weise JM, Lechel A, Ande S, Guguen-Guillouzo C
  Gastroenterology, Jul 2011
  Abstract : BACKGROUND & AIMS: Telomerase activity has not been detected in healthy human liver biopsy samples, but it is up-regulated in most human liver tumors. It is not clear whether telomerase is activated in response to acute or chronic liver injury. Telomerase activity is closely associated with expression of its catalytic subunit, telomerase reverse transcriptase (TERT). We analyzed the activity of the human TERT (hTERT) promoter during liver regeneration in vivo and hepatocyte proliferation in vitro. METHODS: We used hTERTp-lacZ transgenic mice, which contain an 8.0-kilobase pair fragment of the hTERT gene promoter, to study the role of TERT in liver regeneration following partial hepatectomy. As an in vitro model, we used the HepaRG cell line as a new model system for human hepatocyte proliferation and differentiation. RESULTS: Activity of the hTERT promoter increased significantly after partial hepatectomy; it was also induced in hepatocytes, based on immunohistologic analysis. Similar to the in vivo results, telomerase activity and hTERT expression were up-regulated in proliferating HepaRG cells and repressed in response to growth arrest and differentiation. Promoter mapping revealed that a proximal 0.3-kilobase pair fragment contains all elements necessary for regulation of hTERT in HepaRG cells. We identified E2F2 and E2F7 as transcription factors that control the differential expression of hTERT in proliferating hepatocytes, in vitro and in vivo. CONCLUSIONS: hTERT is induced in hepatocytes during liver regeneration, indicating a functional role for telomerase in human liver. Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.
• Cytochrome P450-dependent metabolism in HepaRG cells cultured in a dynamic three-dimensional bioreactor.Darnell M, Schreiter T, Zeilinger K, Urbaniak T, Söderdahl T, Rossberg I, Dillnér B, Berg AL
  Drug Metab Dispos, Jul 2011
  Abstract : Reliable and stable in vitro cellular systems maintaining specific liver functions important for drug metabolism and disposition are urgently needed in preclinical drug discovery and development research. The cell line HepaRG exhibits promising properties such as expression and function of drug-metabolizing enzymes and transporter proteins, which resemble those found in freshly isolated human hepatocytes. In this study, HepaRG cells were cultured up to 68 days in a three-dimensional multicompartment capillary membrane bioreactor, which enables high-density cell culture under dynamic conditions. The activity of drug-metabolizing cytochrome P450 (P450) enzymes was investigated by a cocktail of substrates for CYP1A1/2 (phenacetin), CYP2C9 (diclofenac), CYP2B6 (bupropion), and CYP3A4 (midazolam). The model P450 substrates, which were introduced to the bioreactor system mimicking in vivo bolus doses, showed stable metabolism over the entire experimental period of several weeks with the exception of bupropion hydroxylase, which increased over time. Ketoconazole treatment decreased the CYP3A4 activity by 69%, and rifampicin induced the CYP3A4- and CYP2B6-dependent activity 6-fold, which predicts well the magnitude of changes observed in vivo. Moreover, polarity of transporter expression and formation of tissue-like structures including bile canaliculi were demonstrated by immune histochemistry. The long-lasting bioreactor system using HepaRG cells thus provides a promising and stable liver-like in vitro model for continuous investigations of the hepatic kinetics of drugs and of drug-drug interactions, which well predict the situation in vivo in humans
• Expression systems of cytochrome P450 proteins in studies of drug metabolism in vitroPawlowska M, Augustin E
  Postepy Hig Med Dosw (Online), Jun 2011
  Abstract : Cytochrome P450 proteins are the most important enzymes involved in metabolic activation or detoxification of various drugs used in clinical practice. However, some drug metabolism pathways may be responsible for their increased toxicity. New expression systems of cytochrome P450 proteins in mammalian cells, including human, are designed to explore the influence of metabolism on the cellular and molecular mechanisms of action of potential drugs and those used therapeutically. They can also be used to study the effect of tested compounds on activity and expression of metabolizing enzymes. Human tumor cell lines with overexpression of cytochrome P450 isoenzymes are of particular importance, especially in studies of potential chemotherapeutics. The HepG2 cell line, derived from human liver cancer, is the most commonly used in studies on drug metabolism and toxicity. However, due to the low level of metabolizing enzymes in these cells, the Hep3A4 cell line with overexpression of CYP3A4 isoenzyme was developed. The stable overexpression of cytochrome P450 isoenzymes was also obtained in other human cancer cell lines, including hepatoma HepaRG cells, ovarian cancer IGROV-1 cells, colon cancer Caco-2, and LS180 cells. This review describes currently developed bacterial, yeast, insect and mammalian (including human) cytochrome P450 protein expression systems, in terms of their advantages and disadvantages in the context of their suitability for basic research and use on a commercial scale.
• Induction of vesicular steatosis by amiodarone and tetracycline is associated with up-regulation of lipogenic genes in HepaRG cells.Anthérieu S, Rogue A, Fromenty B, Guillouzo A, Robin MA
  Hepatology, Jun 2011
  Abstract : Drug-induced liver injury occurs in general after several weeks and is often unpredictable. It is characterized by a large spectrum of lesions that includes steatosis and phospholipidosis. Many drugs such as amiodarone and tetracycline have been reported to cause phospholipidosis and/or steatosis. In this study, acute and chronic hepatic effects of these two drugs were investigated using well-differentiated human hepatoma HepaRG cells. Accumulation of typical lipid droplets, labeled with Oil Red O, was observed in hepatocyte-like HepaRG cells after repeat exposure to either drug. Amiodarone caused the formation of additional intracytoplasmic vesicles that did not stain in all HepaRG cells. At the electron microscopic level, these vesicles appeared as typical lamellar bodies and were associated with an increase of phosphatidylethanolamine and phosphatidylcholine. A dose-dependent induction of triglycerides (TG) was observed after repeat exposure to either amiodarone or tetracycline. Several genes known to be related to lipogenesis were induced after treatment by these two drugs. By contrast, opposite deregulation of some of these genes (FASN, SCD1, and THSRP) was observed in fat HepaRG cells induced by oleic acid overload, supporting the conclusion that different mechanisms were involved in the induction of steatosis by drugs and oleic acid. Moreover, several genes related to lipid droplet formation (ADFP, PLIN4) were up-regulated after exposure to both drugs and oleic acid. CONCLUSION: Our results show that amiodarone causes phospholipidosis after short-term treatment and, like tetracycline, induces vesicular steatosis after repeat exposure in HepaRG cells. These data represent the first demonstration that drugs can induce vesicular steatosis in vitro and show a direct relationship between TG accumulation and enhanced expression of lipogenic genes. Copyright © 2011 American Association for the Study of Liver Diseases.
• Critical selection of reliable reference genes for gene expression study in the HepaRG cell line.Ceelen L, De Spiegelaere W, David M, De Craene J, Vinken M, Vanhaecke T, Rogiers V
  Biochem Pharmacol., May 2011
  Abstract : The human HepaRG cell line has shown to be a valuable in vitro tool for repeated exposure to chemical compounds and to evaluate their potential toxic outcome. Seen the importance given by the actual EU legislation of cosmetics and chemical substances to the use of in vitro methods in human safety evaluation, one can expect that HepaRG cells will gain importance as human-relevant cell source. At the transcriptional level, RT-qPCR assays are often used to obtain quantitative results. The choice of internal control is important since it may affect the study outcome. Indeed, it is well-known that expression levels of traditional reference genes can vary across tissue types and across experimental settings within one specific tissue type. From a review of the scientific literature, it appears that, for HepaRG cells, S18 often is used as internal control, but without any evidence of its expression stability in this cell line. Therefore, we aimed to select the most optimal reference genes for gene expression studies in HepaRG cells and to check whether S18 is a suitable reference gene. Twelve candidate genes' expression stability level was analyzed by three algorithms (geNorm, BestKeeper, Normfinder), which identified the optimal single reference gene (TBP) and the most suitable set of reference genes (TBP, UBC, SDHA, RLP13, YHWAZ, HMBS, B2M and HPRT1) for HepaRG transcriptional profiling. This study provides a new set of reference genes that is suitable for testing whenever RT-qPCR data for HepaRG cells are generated. The most stable ones can then be selected for further normalization.
• Comparative gene expression profiles induced by PPAR&gamma; and PPAR&alpha;/&gamma; agonists in human hepatocytes.Rogue A, Lambert C, Jossé R, Antherieu S, Spire C, Claude N, Guillouzo A
  PLoS One, Apr 2011
  Abstract : BACKGROUND: Several glitazones (PPARγ agonists) and glitazars (dual PPARα/γ agonists) have been developed to treat hyperglycemia and, simultaneously, hyperglycemia and dyslipidemia, respectively. However, most have caused idiosyncratic hepatic or extrahepatic toxicities through mechanisms that remain largely unknown. Since the liver plays a key role in lipid metabolism, we analyzed changes in gene expression profiles induced by these two types of PPAR agonists in human hepatocytes.METHODOLOGY/PRINCIPAL FINDINGS: Primary human hepatocytes and the well-differentiated human hepatoma HepaRG cells were exposed to different concentrations of two PPARγ (troglitazone and rosiglitazone) and two PPARα/γ (muraglitazar and tesaglitazar) agonists for 24 h and their transcriptomes were analyzed using human pangenomic Agilent microarrays. Principal Component Analysis, hierarchical clustering and Ingenuity Pathway Analysis® revealed large inter-individual variability in the response of the human hepatocyte populations to the different compounds. Many genes involved in lipid, carbohydrate, xenobiotic and cholesterol metabolism, as well as inflammation and immunity, were regulated by both PPARγ and PPARα/γ agonists in at least a number of human hepatocyte populations and/or HepaRG cells. Only a few genes were selectively deregulated by glitazars when compared to glitazones, indicating that PPARγ and PPARα/γ agonists share most of their target genes. Moreover, some target genes thought to be regulated only in mouse or to be expressed in Kupffer cells were also found to be responsive in human hepatocytes and HepaRG cells.CONCLUSIONS/SIGNIFICANCE:This first comprehensive analysis of gene regulation by PPARγ and PPARα/γ agonists favor the conclusion that glitazones and glitazars share most of their target genes and induce large differential changes in gene profiles in human hepatocytes depending on hepatocyte donor, the compound class and/or individual compound, thereby supporting the occurrence of idiosyncratic toxicity in some patients.
• Antiproliferative effect on HepaRG cell cultures of new calix[4]arenes. Part II.Latxague L, Gaboriau F, Chassande O, Leger JM, Pires V, Rouge P, Dassonville-Klimpt A, Fardeau S
  Journal of Enzyme Inhibition, Apr 2011
  Abstract : Cell cycle progression is dependent on the intracellular iron level and chelators can lead to iron depletion and decrease cell proliferation. This antiproliferative effect can be inhibited by exogenous iron. In this work, we present the synthesis of some new synthetic calix[4]arene podands bearing diamino-tetraesters, diamino-tetraalcohols, diamino-tetraacid and tetraaryloxypentoxy groups at the lower rim, designed as potential iron chelators. We report their effect on cell proliferation, in comparison with the new oral chelator ICL670A (4-[3,5-bis-(2-hydroxyphenyl)-1,2,4-triazol-1-yl]-benzoic acid). The antiproliferative effect of these new compounds was studied in the human hepatocarcinoma HepaRG cell cultures using cell nuclei counting after staining with the DNA intercalating fluorescence dye, Hoechst 33342. Their cytotoxicity was evaluated by the extracellular LDH activity. Preliminary results indicated that their antiproliferative effect was mainly due to their cytotoxicity. The efficiency of these compounds, being comparable to that of ICL670, was independent of iron depletion. This effect remains to be further explored. Moreover, it also shows that the new substituted calix[4]arenes could open the way to valuable new approaches for medicinal chemistry scaffolding.
• HepaRG cells: a human model to study mechanisms of acetaminophen hepatotoxicity.McGill MR, Yan HM, Ramachandran A, Murray GJ, Rollins DE, Jaeschke H.
  Hepatology, Mar 2011
  Abstract : "Acetaminophen (APAP) overdose is the leading cause of acute liver failure in Western countries. In the last four decades much progress has been made in our understanding of APAP-induced liver injury through rodent studies. However, some differences exist in the time course of injury between rodents and humans. To study the mechanism of APAP hepatotoxicity in humans, a human-relevant in vitro system is needed. Here we present evidence that the cell line HepaRG is a useful human model for the study of APAP-induced liver injury. Exposure of HepaRG cells to APAP at several concentrations resulted in glutathione depletion, APAP-protein adduct formation, mitochondrial oxidant stress and peroxynitrite formation, mitochondrial dysfunction (assessed by JC-1 fluorescence), and lactate dehydrogenase (LDH) release. Importantly, the time course of LDH release resembled the increase in plasma aminotransferase activity seen in humans following APAP overdose. Based on propidium iodide uptake and cell morphology, the majority of the injury occurred within clusters of hepatocyte-like cells. The progression of injury in these cells involved mitochondrial reactive oxygen and reactive nitrogen formation. APAP did not increase caspase activity above untreated control values and a pancaspase inhibitor did not protect against APAP-induced cell injury. CONCLUSION: These data suggest that key mechanistic features of APAP-induc "
• Ethanol Effect on Cell Proliferation in the Human Hepatoma HepaRG Cell Line: Relationship With Iron Metabolism.Tuoi Do TH, Gaboriau F, Ropert M, Moirand R, Cannie I, Brissot P, Loréal O
  Alcohol Clin Exp res., Mar 2011
  Abstract : BACKGROUND: Alcoholism increases the risk of cirrhosis and/or hepatocellular carcinoma development. Iron, like ethanol, modulates the cell growth. However, the relationship between alcohol and iron toward hepatocyte proliferation has not been clearly elucidated. The purpose of this study was to evaluate, in the human HepaRG cell line model, the impact of ethanol on hepatocyte proliferation in relation to modulations of iron metabolism and the protective effect of iron metabolism manipulation by chelators in alcohol liver diseases. METHODS: The human hepatoma HepaRG cell line model was used. Cell viability was determined by measuring succinate dehydrogenase activity, total protein level by the Bradford method. DNA synthesis was evaluated by [(3)H]-methyl thymidine incorporation. Cytotoxicity was studied by release of lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT) in culture medium and apoptosis by measuring caspase 3/7 activity. Gene expression was analyzed by RT-qPCR. Total iron, soluble transferrin receptor, and ferritin levels were, respectively, measured by colorimetrical, immuno-nephelometrical, and immuno-turbidimetrical methods. Intracellular iron uptake and accumulation was examined by radionuclide (55)Fe (III) measurement and Perls staining. RESULTS: Results showed that ethanol decreased all the parameters associated with HepaRG cell proliferation (cell viability, total protein levels, and DNA synthesis) in a dose- and time-dependent manner. This effect was accompanied by cytotoxicity and apoptosis as evaluated by a significant increase in extracellular enzymes (LDH, AST, ALT) and caspase 3/7 activity, respectively. Ethanol exposure was accompanied by an increased cellular iron uptake, together with increased expression of genes involved in iron transport and storage such as l-ferritin, Divalent Metal transporter 1, transferrin, transferrin receptor 1, and ceruloplasmin. Ethanol impact was intensified by iron-citrate and decreased by iron chelators when added to the culture medium. CONCLUSIONS: The results indicated that (i) ethanol-induced iron metabolism dysfunction could be one of the underlying mechanisms of ethanol antiproliferative effect and (ii) exogenous iron may accentuate ethanol hepatoxicity. These data suggest that iron metabolism manipulation by chelators may be a useful therapeutic approach in alcohol-related liver diseases. Copyright © 2010 by the Research Society on Alcoholism.
• HepaRG human hepatic cell line utility as a surrogate for primary human hepatocytes in drug metabolism assessment in vitro.Lübberstedt M, Müller-Vieira U, Mayer M, Biemel KM, Knöspel F, Knobeloch D, Nüssler AK, Gerlach JC
  J Pharmacol Toxicol Methods, Feb 2011
  Abstract : INTRODUCTION: Primary human hepatocytes are considered as a highly predictive in vitro model for preclinical drug metabolism studies. Due to the limited availability of human liver tissue for cell isolation, there is a need of alternative cell sources for pharmaceutical research.METHODS: In this study, the metabolic activity and long-term stability of the human hepatoma cell line HepaRG were investigated in comparison to primary human hepatocytes (pHH). Hepatocyte-specific parameters (albumin and urea synthesis, galactose and sorbitol elimination) and the activity of human-relevant cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) were assayed in both groups over a period of 14 days subsequently to a two week culture period in differentiated state in case of the HepaRG cells, and compared with those of cryopreserved hepatocytes in suspension. In addition, the inducibility of CYP enzymes and the intrinsic clearances of eleven reference drugs were determined.RESULTS: The results show overall stable metabolic activity of HepaRG cells over the monitored time period. Higher albumin production and galactose/sorbitol elimination rates were observed compared with pHH, while urea production was not detected. CYP enzyme-dependent drug metabolic capacities were shown to be stable over the cultivation time in HepaRG cells and were comparable or even higher (CYP2C9, CYP2D6, CYP3A4) than in pHH, whereas commercially available hepatocytes showed a different pattern The intrinsic clearance rates of reference drugs and enzyme induction of most CYP enzymes were similar in HepaRG cells and pHH. CYP1A2 activity was highly inducible in HepaRG by β-naphthoflavone.DISCUSSION: In conclusion, the results from this study indicate that HepaRG cells could provide a suitable alternative to pHH in pharmaceutical research and development for metabolism studies such as CYP induction or sub-chronic to chronic hepatotoxicity studies.

2010

• The chinese Herbal Medicine Sophora flavescens Activates Pregnane X Receptor.Wang L, Li F, Lu J, Li G, Zhong XB, Guo G L, Ma X
  Drug Metab Dispos, Dec 2010
  Abstract : Sophora flavescens (SF) is an herbal medicine widely used for the treatment of viral hepatitis, cancer, viral myocarditis, gastrointestinal hemorrhage, and skin diseases. It was recently reported that SF up-regulates CYP3A expression. The mechanism of SF-induced CYP3A expression is unknown. In the current study, we tested the hypothesis that SF-induced CYP3A expression is mediated by the activation of pregnane X receptor (PXR). We used two cell lines, DPX2 and HepaRG, to investigate the role of PXR in SF-induced CYP3A expression. The DPX2 cell line is derived from HepG2 cells with the stable transfection of human PXR and a luciferase reporter gene linked with a human PXR response element identified in the CYP3A4 gene promoter. In DPX2 cells, SF activated PXR in a concentration-dependent manner. We used a metabolomic approach to identify the chemical constituents in SF, which were further analyzed for their effect on PXR activation and CYP3A regulation. One chemical in SF, N-methylcytisine, was identified as an individual chemical that activated PXR. HepaRG is a highly differentiated hepatoma cell line that mimics human hepatocytes. In HepaRG cells, N-methylcytisine significantly induced CYP3A4 expression, and this induction was suppressed by the PXR antagonist sulforaphane. These results suggest that SF induces CYP3A expression via the activation of PXR.
• The use of HepaRG and human hepatocyte data in predicting CYP induction drug-drug interactions via static equation and dynamic mechanistic modelling approaches.Grime K, Ferguson DD, Riley RJ.
  Curr Drug Metab, Dec 2010
  Abstract : he method of predicting CYP induction drug-drug interactions (DDIs) from a relative induction score (RIS) calibration has been developed to provide a novel model facilitating predictions for any CYP-inducer substrate combination by inclusion of parameters such as the fraction of hepatic clearance mediated by a specific CYP and fraction of the dose escaping intestinal extraction. In vitro HepaRG CYP3A4 induction data were used as a basis for the approach and a large number of DDIs were well predicted. Primary human hepatocyte data were also used to make predictions, using the HepaRG calibration as a foundation. Similar predictive accuracy suggests that HepaRG and primary hepatocyte data can be used inter-changeably within the same laboratory. A comparison of this 'indirect' calibration method with a direct in vitro-in vivo scaling approach was made and investigations undertaken to define the most appropriate in vivo inducer concentration to use. Additionally, a reasonably effective prediction model based on F(2) (the concentration of inducer taken to increase the CYP mRNA 2-fold above background) was established. An accurate prediction for the CYP1A2-dependent omeprazole-caffeine interaction was also made, demonstrating that the methods are useful for the evaluation of DDIs from induction involving mechanisms other than PXR activation. Finally, a dynamic mechanistic model accounting for the simultaneous influence of CYP induction and reversible and irreversible CYP inhibition in both the liver and intestine was written to provide a prediction of the overall DDI when several interactions occur concurrently. The rationale for using the various models described, alongside commercially available prediction tools, at various stages of the drug discovery process is described.
• Prediction of clinical CYP3A4 induction using cryopreserved human hepatocytes.Kaneko A, Kato M, Endo C, Nakano K, Ishigai M, Takeda K
  Xenobiotica, Dec 2010
  Abstract : The purpose of this study was to construct a method to predict CYP3A4 induction in the clinical setting from in vitro data using cryopreserved human hepatocytes. We recently developed an approach with in vitro assays of HepaRG cell lines for predicting CYP3A4 induction by using a novel value, termed the relative factor (RF), determined from the ratio of the concentration of an inducer to the reference standard. In this study, the applicability of the RF approach was expanded to cryopreserved human hepatocytes. Induction assays were performed in vitro using hepatocytes from four individual donors and eight typical inducers. The obtained RF values were related to the free plasma concentration of each inducer (expressed as Css,u/RF). A good relationship between the Css,u/RF values and the in vivo induction response was found for all donors. Inducers were classified by the Css,u/RF values into three categories for CYP3A4 induction risk (high, medium and low potency), and thereby the degree of CYP3A4 induction in vivo in humans could be predicted from the Css,u/RF values. The RF approach is applicable to human cryopreserved hepatocytes. Thus, a method to predict the potency of CYP3A4 inducers was constructed using cryopreserved human hepatocytes.
• Human hepatoma HepaRG cell line engraftment in severe combined immunodeficient × beige mice using mouse-specific anti-Fas antibody.Jiang L, Li JG, Lan L, Wang YM, Mao Q, You JP
  Transplant Proc, Nov 2010
  Abstract : Chimeric mice with repopulated human hepatocytes are widely used for drug development research. HepaRG cell line is a naturally immortalized human liver cell line with progenitor properties and inducible bipotent differentiation capability. It would be useful if HepaRG cells could repopulate damaged livers severe, combined immunodeficient × beige (SCID/bg) mice and exhibit special human hepatic features. After inducing mouse hepatocyte apoptosis with an antimouse agonistic Fas monoclonal antibody (Jo2 mAb), HepaRG cells with bipotent differentiation capability were repopulated in SCID/bg mouse livers. HepaRG cells were transplanted intrasplenically into SCID/bg mice treated with 0.2 mg/kg Jo2 mAb once a week for 10 weeks. Human hepatocyte repopulation was characterized by immunohistochemistry for human serum albumin (HSA), Hep Par1, and CK18 and by immunofluorescence staining for HSA. Human albumin mRNA was detected by reverse-transcription polymerase chain reactions. HSA levels were quantified by Western blotting and enzyme-linked immunosorbent assays. Our results showed that HepaRG cell engraftment protected mice from the effects of Jo2 mAb-mediated liver hemorrhage and hepatocyte apoptosis. At 2 weeks after transplantation, increase concentrations of HSA were detected in recipient blood and liver. At 12 weeks after transplantation, approximately 15%-20% of mice showed livers repopulated with human hepatocytes. In conclusion, normal SCID/bg mice were suitable recipients for HepaRG cell transplantation when induced with Jo2 mAb. This chimeric mouse model with HepaRG cells could serve as a useful model for in vivo studies of drug metabolism and hepatitis virus infections. Copyright © 2010 Elsevier Inc. All rights reserved.
• Preferential induction of the AhR gene battery in HepaRG cells after a single or repeated exposure to heterocyclic aromatic amines.Dumont J, Jossé R, Lambert C, Anthérieu S, Laurent V, Loyer P, Guillouzo A
  Toxico Appl Pharmacol, Nov 2010
  Abstract : 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) are two of the most common heterocyclic aromatic amines (HAA) produced during cooking of meat, fish and poultry. Both HAA produce different tumor profiles in rodents and are suspected to be carcinogenic in humans. In order to better understand the molecular basis of HAA toxicity, we have analyzed gene expression profiles in the metabolically competent human HepaRG cells using pangenomic oligonucleotide microarrays, after either a single (24-h) or a repeated (28-day) exposure to 10 μM PhIP or MeIQx. The most responsive genes to both HAA were downstream targets of the arylhydrocarbon receptor (AhR): CYP1A1 and CYP1A2 after both time points and CYP1B1 and ALDH3A1 after 28 days. Accordingly, CYP1A1/1A2 induction in HAA-treated HepaRG cells was prevented by chemical inhibition or small interference RNA-mediated down-regulation of the AhR. Consistently, HAA induced activity of the CYP1A1 promoter, which contains a consensus AhR-related xenobiotic-responsive element (XRE). In addition, several other genes exhibited both time-dependent and compound-specific expression changes with, however, a smaller magnitude than previously reported for the prototypical AhR target genes. These changes concerned genes mainly related to cell growth and proliferation, apoptosis, and cancer. In conclusion, these results identify the AhR gene battery as the preferential target of PhIP and MeIQx in HepaRG cells and further support the hypothesis that intake of HAA in diet might increase human cancer risk.
• Assessment of the genotoxic potential of indirect chemical mutagens in HepaRG cells by the comet and the cytokinesis-block micronucleus assays.Le Hegarat L, Dumont J, Josse R, Huet S, Lanceleur R, Guguen-Guillouzo C, Guillouzo A
  Mutagenesis, Nov 2010
  Abstract : Many chemical carcinogens require metabolic activation to form genotoxic compounds in human. Standard in vitro genotoxicity assays performed with activation systems, such as rat liver S9, are recognised to lead to a high number of false positives. The aim of this study was to evaluate the suitability of differentiated human hepatoma HepaRG cells as an in vitro model system for the detection of DNA damage induced by promutagens using the comet and the cytokinesis-block micronucleus assays. Several promutagens were tested, including aflatoxin B1 (AFB1), benzo[a]pyrene (B[a]P), acrylamide, N-nitrosodimethylamine (NDMA), cyclophosphamide (CPA), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). Cytotoxicity of these compounds was assessed by measuring lactate dehydrogenase leakage. A 24 h exposure was generally needed to obtain an obvious positive response in differentiated HepaRG cells in the comet and in the cytokinesis-block micronucleus assays. Comet formation was observed with all compounds except IQ. B[a]P, CPA and AFB1 showed a dose-dependent increase in micronucleated cells, whereas no increase was observed with PhIP, IQ and acrylamide. These preliminary data on genotoxicity in differentiated HepaRG cells are promising but more chemicals must be tested to determine the ability of HepaRG cells to assess genotoxicity of chemicals in humans.
• Gene Expression Changes Induced by PPAR Gamma Agonists in Animal and Human Liver.Rogue A, Spire C, Brun M, Claude N, Guillouzo A
  PPAR research, Oct 2010
  Abstract : Thiazolidinediones are a class of Peroxisome Proliferator Activated Receptor γ (PPARγ) agonists that reduce insulin resistance in type 2 diabetic patients. Although no detectable hepatic toxicity has been evidenced in animal studies during preclinical trials, these molecules have nevertheless induced hepatic adverse effects in some treated patients. The mechanism(s) of hepatotoxicity remains equivocal. Several studies have been conducted using PCR analysis and microarray technology to identify possible target genes and here we review the data obtained from various in vivo and in vitro experimental models. Although PPARγ is expressed at a much lower level in liver than in adipose tissue, PPARγ agonists exert various PPARγ-dependent effects in liver in addition to PPARγ-independent effects. Differences in effects are dependent on the choice of agonist and experimental conditions in rodent animal studies and in rodent and human liver cell cultures. These effects are much more pronounced in obese and diabetic liver. Moreover, our own recent studies have shown major interindividual variability in the response of primary human hepatocyte populations to troglitazone treatment, supporting the occurrence of hepatotoxicity in only some individuals.
• Biotransformation pathway maps in WikiPathways enable direct visualization of drug metabolism related expression changes.Jennen DG, Gaj S., Giesbertz P.J
  Drug Discovery today, Oct 2010
  Abstract : In recent decades, our knowledge of the genetics and functional genomics of drug-metabolizing enzymes has increased and a wealth of data on drug-related 'omics' has become available. Despite the availability of large amounts of biological information on xenobiotic biotransformation, the number of available biotransformation pathway maps that can easily be used for visualization of multiple omics data is limited. Here, we created integrated biotransformation pathway maps suitable for multiple omics analysis using PathVisio. The ease of visualizing data on these maps was demonstrated by using published microarray data from human hepatocyte-like cell models, exemplifying - where a sufficient capacity for metabolizing chemicals is a prerequisite for a suited model - how the biotransformation pathway maps can be used for model selection.
• Involvement of aryl hydrocarbon receptor in basal and 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced expression of target genes in primary human hepatocytes.Le Vee M, Jouan E, Fardel O
  Toxicol In Vitro, Sep 2010
  Abstract : Aryl hydrocarbon receptor (AhR) is a drug-sensing receptor activated by environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and is known to drive regulation of target genes in various human cell types. Its involvement in TCDD-mediated regulation of target genes in human hepatocytes however remains to be formally demonstrated. To gain insights into this point, we have analyzed the effects of AhR silencing on the regulation of various genes targeted by TCDD in primary human hepatocytes and highly-differentiated human hepatoma HepaRG cells. Efficient AhR knocking-down was performed through dimethyl sulfoxide-based transfection of small-interfering RNAs targeting AhR (siAhR). SiAhR-transfected human hepatocytes or HepaRG cells, exposed to TCDD, were found to exhibit reduced mRNA expression of various TCDD-responsive genes, i.e. CYP1A1, CYP1A2, CYP1B1, ALDH3A1, IL17RB, FER1L3 and SLC7A5, when compared to TCDD-treated counterparts transfected with non-targeting small-interfering RNAs. AhR silencing was moreover shown to markedly counteract TCDD-mediated induction of CYP1A1/CYP1A2/CYP1B1-related ethoxyresorufin O-deethylase activity in both human hepatocytes and HepaRG cells. It also concomitantly decreased constitutive mRNA expression of some target genes such as CYP1A1, CYP1A2, CYP1B1 and ALDH3A1. Taken together, these data indicate that AhR plays a crucial role in both basal and TCDD-induced expression of target genes in human hepatocytes.
• A comparison of whole genome gene expression profiles of HepaRG cells and HepG2 cells to primary human hepatocytes and human liver tissues.Hart SN, Li Y, Nakamoto K, Subileau EA, Steen D, Zhong XB
  Drug Metab Dispos, Jun 2010
  Abstract : HepaRG cells, derived from a female hepatocarcinoma patient, are capable of differentiating into biliary epithelial cells and hepatocytes. More importantly, differentiated HepaRG cells are able to maintain activities of many xenobiotic-metabolizing enzymes, and expression of the metabolizing enzyme genes can be induced by xenobiotics. The ability of these cells to express and induce xenobiotic-metabolizing enzymes is in stark contrast to the frequently used HepG2 cells. The previous studies have mainly focused on a set of selected genes; therefore, it is of significant interest to know the extent of similarity of gene expression at whole genome levels in HepaRG cells and HepG2 cells compared with primary human hepatocytes and human liver tissues. To accomplish this objective, we used Affymetrix (Santa Clara, CA) U133 Plus 2.0 arrays to characterize the whole genome gene expression profiles in triplicate biological samples from HepG2 cells, HepaRG cells (undifferentiated and differentiated cells), freshly isolated primary human hepatocytes, and frozen liver tissues. After using similarity matrix, principal components, and hierarchical clustering methods, we found that HepaRG cells globally transcribe genes at levels more similar to human primary hepatocytes and human liver tissues than HepG2 cells. In particular, many genes encoding drug-processing proteins are transcribed at a more similar level in HepaRG cells than in HepG2 cells compared with primary human hepatocytes and liver samples. The transcriptomic similarity of HepaRG with primary human hepatocytes is encouraging for use of HepaRG cells in the study of xenobiotic metabolism, hepatotoxicology, and hepatocyte differentiation.
• Differential toxicity of heterocyclic aromatic amines and their mixture in metabolically competent HepaRG cells.Dumont J, Jossé R, Lambert C, Anthérieu S, Le Hegarat L, Aninat C, Robin MA, Guguen-Guillouzo C
  Toxicol Appl Pharmacol., Jun 2010
  Abstract : Human exposure to heterocyclic aromatic amines (HAA) usually occurs through mixtures rather than individual compounds. However, the toxic effects and related mechanisms of co-exposure to HAA in humans remain unknown. We compared the effects of two of the most common HAA, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), individually or in combination, in the metabolically competent human hepatoma HepaRG cells. Various endpoints were measured including cytotoxicity, apoptosis, oxidative stress and DNA damage by the comet assay. Moreover, the effects of PhIP and/or MeIQx on mRNA expression and activities of enzymes involved in their activation and detoxification pathways were evaluated. After a 24h treatment, PhIP and MeIQx, individually and in combination, exerted differential effects on apoptosis, oxidative stress, DNA damage and cytochrome P450 (CYP) activities. Only PhIP induced DNA damage. It was also a stronger inducer of CYP1A1 and CYP1B1 expression and activity than MeIQx. In contrast, only MeIQx exposure resulted in a significant induction of CYP1A2 activity. The combination of PhIP with MeIQx induced an oxidative stress and showed synergistic effects on apoptosis. However, PhIP-induced genotoxicity was abolished by a co-exposure with MeIQx. Such an inhibitory effect could be explained by a significant decrease in CYP1A2 activity which is responsible for PhIP genotoxicity. Our findings highlight the need to investigate interactions between HAA when assessing risks for human health and provide new insights in the mechanisms of interaction between PhIP and MeIQx.
• Comparison of HepG2 and HepaRG by whole-genome gene expression analysis for the purpose of chemical hazard identification.Jennen DG, Magkoufopoulou C, Ketelslegers HB, van Herwijnen MH, Kleinjans JC, van Delft JH.
  Toxicol Sci., May 2010
  Abstract : Direct comparison of the hepatoma cell lines HepG2 and HepaRG has previously been performed by only evaluating a limited set of genes or proteins. In this study, we examined the whole-genome gene expression of both cell lines before and after exposure to the genotoxic (GTX) carcinogens aflatoxin B1 and benzo[a]pyrene and the nongenotoxic (NGTX) carcinogens cyclosporin A, 17beta-estradiol, and 2,3,7,8-tetrachlorodibenzo-para-dioxin for 12 and 48 h. Before exposure, this analysis revealed an extensive network of genes and pathways, which were regulated differentially for each cell line. The comparison of the basal gene expression between HepG2, HepaRG, primary human hepatocytes (PHH), and liver clearly showed that HepaRG resembles PHH and liver the most. After exposure to the GTX and NGTX carcinogens, for both cell lines, common pathways were found that are important in carcinogenesis, for example, cell cycle regulation and apoptosis. However, also clear differences between exposed HepG2 and HepaRG were observed, and these are related to common metabolic processes, immune response, and transcription processes. Furthermore, HepG2 performs better in discriminating between GTX and NGTX carcinogens. In conclusion, these results have shown that HepaRG is a more suited in vitro liver model for biological interpretations of the effects of exposure to chemicals, whereas HepG2 is a more promising in vitro liver model for classification studies using the toxicogenomics approach. Although, it should be noted that only five carcinogens were used in this study.
• Entry of hepatitis B virus: mechanism and new therapeutic target.Xie Y, Zhai J, Deng Q, Tiollais P, Wang Y, Zhao M
  Pathol Biol, May 2010
  Abstract : Entry of hepatitis B virus (HBV) into human hepatocytes constitutes the initial step in viral infection. The study of HBV entry had long been hampered by the lack of efficient cell culture systems and small animal models. The situation was greatly improved in the last decade with the development of HBV-infectible HepaRG cell line and primary Tupaia hepatocyte culture. Armed with these new tools, marked progresses have been achieved in the elucidation of the mechanism of HBV entry. Plenty of evidences indicate that the viral large surface protein (LHBs) is essential for HBV entry. Several regions in the PreS1 domain of LHBs have been verified to contribute directly to the viral attachment. In addition, a myristate moiety linked to the N-terminal glycine of PreS1 appears critical for HBV infectivity. Recently, the cysteine-rich antigenic loop of the S domain was identified as another crucial determinant for HBV infectivity. On the other hand, several cellular proteins were implicated in HBV attachment to hepatic cells, though definitive proofs are required in support to their functional involvement in HBV infection. Aiming to blocking viral entry, a couple of approaches based on acylated PreS1-derived peptides and short PreS1-binding peptides are currently under investigation, which have the potential to become novel antiviral therapeutics.
• General review on in vitro hepatocyte models and their applications.Guguen-Guillouzo C, Guillouzo A
  Methods Mol Biol, May 2010
  Abstract : In vitro hepatocyte models represent very useful systems in both fundamental research and various application areas. Primary hepatocytes appear as the closest model for the liver in vivo. However, they are phenotypically unstable, have a limited life span and in addition, exhibit large interdonor variability when of human origin. Hepatoma cell lines appear as an alternative but only the HepaRG cell line exhibits various functions, including major cytochrome P450 activities, at levels close to those found in primary hepatocytes. In vitro hepatocyte models have brought a substantial contribution to the understanding of the biochemistry, physiology, and cell biology of the normal and diseased liver and in various application domains such as xenobiotic metabolism and toxicity, virology, parasitology, and more generally cell therapies. In the future, new well-differentiated hepatocyte cell lines derived from tumors or from either embryonic or adult stem cells might be expected and although hepatocytes will continue to be used in various fields, these in vitro liver models should allow marked advances, especially in cell-based therapies and predictive and mechanistic hepatotoxicity of new drugs and other chemicals. All models will benefit from new developments in throughput screening based on cell chips coupled with high-content imaging and in toxicogenomics technologies.
• The application of HepRG cells in evaluation of cytochrome P450 induction properties of drug compounds.Andersson TB
  Methods Mol Biol, May 2010
  Abstract : The liver is the major organ metabolising drugs. The hepatocyte contains a number of drug-metabolising enzyme systems, which most often generate a complex pattern of drug metabolites. Isolated primary hepatocytes would be an ideal in vitro model for drug metabolism research but erratic availability and poor stability of functions in culture limit their value. Recently a hepatoma cell line HepaRG was developed showing promising functions and stability. In the differentiated stage the cell line showed stable expression of mRNA coding for key proteins in drug metabolism and liver-specific functions for over 6 weeks. The cell line was found to reflect important hepatic functions and has been evaluated as a convenient model for evaluating cytochrome P450 induction properties of drug compounds. HepaRG cells could therefore be an alternative to human hepatocytes in investigating drug metabolism and induction of drug-metabolising enzymes.
• The HepaRG cell line: biological properties and relevance as a tool for cell biology, drug metabolism, and virology studies.Marion MJ,, Hantz O, Durantel D
  Methods Mol Biol, May 2010
  Abstract : Liver progenitor cells may play an important role in carcinogenesis in vivo and represent therefore useful cellular materials for in vitro studies. The HepaRG cell line, which is a human bipotent progenitor cell line capable to differentiate toward two different cell phenotypes (i.e., biliary-like and hepatocyte-like cells), has been established from a liver tumor associated with chronic hepatitis C. This cell line represents a valuable alternative to ex vivo cultivated primary human hepatocytes (PHH), as HepaRG cells share some features and properties with adult hepatocytes. The cell line is particularly useful to evaluate drugs and perform drug metabolism studies, as many detoxifying enzymes are expressed and functional. It is also an interesting tool to study some aspect of progenitor biology (e.g., differentiation process), carcinogenesis, and the infection by some pathogens for which the cell line is permissive (e.g., HBV infection). Overall, this chapter gives a concise overview of the biological properties and potential applications of this cell line.
• The use of hepatocytes to investigate HDV infection: the HDV/HepaRG model.Sureau C.
  Methods Mol Biol, May 2010
  Abstract : Worldwide, it is estimated that more than 350 million people are chronically infected with hepatitis B virus (HBV), approximately 15 million of whom are coinfected with hepatitis D virus (HDV), a satellite of HBV that uses the envelope proteins of the latter to assemble its infectious particles. For a long time after HBV discovery, research on the viral life cycle, viral entry in particular, has been hampered by the lack of practical tissue culture systems. To date, in vitro isolation and serial propagation of HBV are still problematic, but the examination of the entire HBV life cycle is possible using two separate systems: (i) permissive human hepatoma cell lines to study HBV DNA replication, viral transcription, translation, assembly, and release of viral particles and (ii) primary cultures of human or chimpanzee hepatocytes or the susceptible HepaRG cell line for viral entry examination. The experimental model described here for analyzing the function of HBV envelope proteins at viral entry is based on this dual tissue culture system, in which HDV is substituted to HBV for practical reasons.
• Antiproliferative effect on HepaRG cell cultures of new calix[4]arenes.Rouge P, Pires VS, Gaboriau F, Dassonville-Klimpt A, Guillon J, Nascimento SD, Leger JM, Lescoat G
  J Enzyme Inhib Med Chem., Apr 2010
  Abstract : Cell cycle progression is dependent on the intracellular iron level, and chelators lead to iron depletion and decrease cell proliferation. This antiproliferative effect can be inhibited by exogenous iron. In this work, we present the synthesis of new synthetic calix[4]arene podands bearing alkyl acid and alkyl ester groups at the lower rim, designed as potential iron chelators. We report their effect on cell proliferation, in comparison with the new oral chelator ICL670 (4-[3,5-bis-(2-hydroxyphenyl)-1,2,4-triazol-1-yl]-benzoic acid). The antiproliferative effect of these new compounds was studied in human hepatocarcinoma HepaRG cell cultures using the MTT assay. Their cytotoxicity was evaluated by extracellular LDH activity. Preliminary results indicate that their antiproliferative effect is due to their cytotoxicity. The efficiency of these compounds, comparable to that of ICL670, was independent of iron depletion. This effect remains to be further explored. Moreover, it also shows that novel substituted calix[4]arenes could open the way to new valuable medicinal chemistry scaffolding.
• Depletion of CoREST does not improve the replication of ICP0 null mutant herpes simplex virus type 1.Everett RD.
  J Virol, Apr 2010
  Abstract : It has been proposed that the cellular corepressor protein CoREST is involved in repressing herpes simplex virus type 1 (HSV-1) infection in the absence of the viral regulatory protein ICP0. This proposal predicts that depletion of CoREST should improve the plaque-forming efficiency and replication of ICP0 null mutant virus. To test this hypothesis, human HepaRG cells that were highly depleted of CoREST were isolated using RNA interference technology. Depletion of CoREST had no effect on the replication of ICP0 null mutant HSV-1, demonstrating that CoREST does not play an influential role in regulating HSV-1 infection in this cell type.
• Effects of deferasirox and deferiprone on cellular iron load in the human hepatoma cell line HepaRG.Gaboriau F, Leray AM, Ropert M, Gouffier L, Cannie I, Troadec MB, Loréal O, Brissot P
  Biometals, Apr 2010
  Abstract : Two oral chelators, CP20 (deferiprone) and ICL670 (deferasirox), have been synthesized for the purpose of treating iron overload diseases, especially thalassemias. Given their antiproliferative effects resulting from the essential role played by iron in cell processes, such compounds might also be useful as anticancer agents. In the present study, we tested the impact of these two iron chelators on iron metabolism, in the HepaRG cell line which allowed us to study proliferating and differentiated hepatocytes. ICL670 uptake was greater than the CP20 uptake. The iron depletion induced by ICL670 in differentiated cells increased soluble transferrin receptor expression, decreased intracellular ferritin expression, inhibited (55)Fe (III) uptake, and reduced the hepatocyte concentration of the labile iron pool. In contrast, CP20 induced an unexpected slight increase in intracellular ferritin, which was amplified by iron-treated chelator exposure. CP20 also promoted Fe(III) uptake in differentiated HepaRG cells, thus leading to an increase of both the labile pool and storage forms of iron evaluated by calcein fluorescence and Perls staining, respectively. In acellular conditions, compared to CP20, iron removing ability from the calcein-Fe(III) complex was 40 times higher for ICL670. On the whole, biological responses of HepaRG cells to ICL670 treatment were characteristic of expected iron depletion. In contrast, the effects of CP20 suggest the potential involvement of this compound in the iron uptake from the external medium into the hepatocytes from the HepaRG cell line, therefore acting like a siderophore in this cell model.
• Genotoxicity of a freshwater cyanotoxin, cylindrospermopsin, in two human cell lines: Caco-2 and HepaRG.Bazin E, Mourot A, Humpage AR, Fessard V
  Environ Mol Mutagen, Apr 2010
  Abstract : Cylindrospermopsin (CYN), a cyanotoxin produced by certain freshwater cyanobacteria, causes human intoxications and animal mortalities. CYN is a potent inhibitor of protein- and glutathione-synthesis. Preliminary evidence for in vivo tumor initiation has been found in mice but the mechanism remains unclear. Several in vitro and in vivo studies demonstrate that CYN is genotoxic and requires metabolic activation. In the present study, the genotoxicity of CYN was assessed in human hepatocyte and enterocyte cell lines, which are models for CYN target organs. The cytokinesis-block micronucleus assay was conducted on liver-derived HepaRG cells and colon-derived Caco-2 cells. Each cell-type was exposed to CYN in both the differentiated and the undifferentiated states, and both with and without the cytochrome P450 inhibitor, ketoconazole, to determine the involvement of metabolism in CYN genotoxicity. CYN increased the frequency of micronuclei in binucleated cells (MNBNC) in both Caco-2 and HepaRG cells. Moreover, ketoconazole reduced both the genotoxicity and cytotoxicity caused by CYN. Our results confirm the involvement of metabolic activation of CYN in mediating its toxicity and suggest that CYN is progenotoxic.
• The application of 3D micropatterning of agarose substrate for cell culture and in situ comet assays.Mercey E, Obeïd P, Glaise D, Calvo-Muñoz ML, Guguen-Guillouzo C, Fouqué B
  Biomaterials , Apr 2010
  Abstract : We report the fabrication of a 3D micropatterned agarose substrate that enables the culture of single or multiple cells. Patterning was performed on dried agarose using deep UV irradiation leading to 6-microm-deep micropatterns of 25-70 microm in diameter. Cell adhesion was facilitated by the specific grafting of ECM (extra cellular matrix) proteins such as fibronectin into the micropatterns. We show that the pattern size induced the adhesion of one or more cells, thus allowing precise control of the cell number used in the assay, and that cells proliferated similarly as in standard culture conditions. Moreover, cell polarity appeared well preserved on this substrate, so polarized cells like hepatoma HepaRG cells might maintain their differentiation status and act as primary human hepatocytes for hepatotoxicity testing. These 3D patterned culture slides have been successfully used for in situ comet assays and there is evidence that the genotoxic effects of sub-cytotoxic concentrations of drugs could be analyzed in a large number of single HeLa cells. Coupled with the parallel-based design of the 3D micropatterning, which allows automated image analysis, these results strongly indicate that this new cell array system is suitable for high-throughput cytotoxicity and genotoxicity screening applications.
• The pre-s2 domain of the hepatitis B virus is dispensable for infectivity but serves a spacer function for L-protein-connected virus assembly.Ni Y, Sonnabend J, Seitz S, Urban S