Hepatocyte-like HepaRG Cells Research Articles

Development of a strategy to identify and evaluate direct and indirect activators of constitutive androstane receptor in rats.

Constitutive androstane receptor (CAR) is a nuclear receptor that plays a key role in drug metabolism and disposition and in the development of liver tumors in rodents. CAR is activated by ligands and indirect activators, which do not bind to the receptor but activate it through cellular signaling. In this study, we sought to identify direct and indirect activators of rat CAR (rCAR). Assessment of the influence of mutations on the transcriptional activity of rCAR identified a mutant termed rCAR-3A-G354Q that displays low constitutive activity and high ligand responsiveness. Reporter assays using the mutant were performed with compounds that increased the mRNA levels of Cyp2b1, a CAR target gene, in rat primary hepatocytes. Several compounds activated rCAR-3A-G354Q and were implicated as rCAR ligands. Since indirect CAR activators are considered to display little species differences, we then determined CYP2B6 mRNA levels in human hepatocyte-like HepaRG cells after treatment with compounds that increased Cyp2b1 mRNA levels in rat hepatocytes but did not activate rCAR-3A-G354Q. The results demonstrated six compounds as possible rCAR indirect activators. Taken together, the combined measurement of Cyp2b1 mRNA levels in rat primary hepatocytes and rCAR-3A-G354Q activation in reporter assays can be useful for evaluating rCAR activation by chemicals.

Plasma Concentration Profiles for Hepatotoxic Pyrrolizidine Alkaloid Senkirkine in Humans Extrapolated from Rat Data Sets Using a Simplified Physiologically Based Pharmacokinetic Model.

The main aim of the current study was to obtain forward dosimetry assessments of pyrrolizidine alkaloid senkirkine plasma and liver concentrations by setting up a human physiologically based pharmacokinetic (PBPK) model based on the limited information available. The risks associated with plant-derived pyrrolizidine alkaloids as natural toxins have been assessed. The pyrrolizidine alkaloid senkirkine was investigated because it was analyzed in a European transcriptomics study of natural hepatotoxins and in a study of the alkaloidal constituents of traditional Japanese food plants Petasites japonicus. The in silico human plasma and liver concentrations of senkirkine were modeled using doses reported for acute-term toxicity in humans. Using a simplified PBPK model established using rat pharmacokinetic data, forward dosimetry was conducted. Since in vitro rat and human intrinsic hepatic clearances were similar; an allometric scaling approach was applied to rat parameters to create a human PBPK model. After oral administration of 1.0 mg/kg in rats in vivo, water-soluble senkirkine was absorbed and cleared from plasma to two orders of magnitude below the maximum concentration in 8 h. Human in silico senkirkine plasma concentration curves were generated after virtual daily oral administrations of 3.0 mg/kg senkirkine (the dose involved in an acute fatal hepatotoxicity case). A high concentration of senkirkine in the culture medium caused in vitro hepatotoxicity as evidenced by lactate dehydrogenase leakage from human hepatocyte-like HepaRG cells. Higher virtual concentrations of senkirkine in human liver and plasma than those in rat plasma were estimated using the current rat and human PBPK models. Current simulations suggest that if P. japonicus (a water-soluble pyrrolizidine alkaloid-producing plant) is ingested daily as food, hepatotoxic senkirkine could be continuously present in human plasma and liver.

PXR Suppresses PPARα-Dependent HMGCS2 Gene Transcription by Inhibiting the Interaction between PPARα and PGC1α.

Background: PXR is a xenobiotic-responsive nuclear receptor that controls the expression of drug-metabolizing enzymes. Drug-induced activation of PXR sometimes causes drug–drug interactions due to the induced metabolism of co-administered drugs. Our group recently reported a possible drug–drug interaction mechanism via an interaction between the nuclear receptors CAR and PPARα. As CAR and PXR are structurally and functionally related receptors, we investigated possible crosstalk between PXR and PPARα. Methods: Human hepatocyte-like HepaRG cells were treated with various PXR ligands, and mRNA levels were determined by quantitative reverse transcription PCR. Reporter assays using the HMGCS2 promoter containing a PPARα-binding motif and mammalian two-hybrid assays were performed in HepG2 or COS-1 cells. Results: Treatment with PXR activators reduced the mRNA levels of PPARα target genes in HepaRG cells. In reporter assays, PXR suppressed PPARα-dependent gene expression in HepG2 cells. In COS-1 cells, co-expression of PGC1α, a common coactivator of PPARα and PXR, enhanced PPARα-dependent gene transcription, which was clearly suppressed by PXR. Consistently, in mammalian two-hybrid assays, the interaction between PGC1α and PPARα was attenuated by ligand-activated PXR. Conclusion: The present results suggest that ligand-activated PXR suppresses PPARα-dependent gene expression by inhibiting PGC1α recruitment.

In vitro profiling of toxic effects of environmental polycyclic aromatic hydrocarbons on nuclear receptor signaling, disruption of endogenous metabolism and induction of cellular stress

Polycyclic aromatic hydrocarbons (PAHs) may interact with multiple intracellular receptors and related signaling pathways. We comprehensively evaluated the toxicity profiles of six environmentally relevant PAHs differing in structure, genotoxicity and their ability to activate the aryl hydrocarbon receptor (AhR). We focused particularly on their impact on intracellular hormone-, xenobiotic- and lipid-sensing receptors, as well as on cellular stress markers, combining a battery of human reporter gene assays and qRT-PCR evaluation of endogenous gene expression in human hepatocyte-like HepaRG cells, with LC/MS-MS analysis of cellular sphingolipids. The effects of PAHs included: activation of estrogen receptor α (in case of fluoranthene (Fla), pyrene (Pyr), benz[a]anthracene (BaA), benzo[a]pyrene (BaP)), suppression of androgen receptor activity (Fla, BaA, BaP and benzo[k]fluoranthene (BkF)), enhancement of dexamethasone-induced glucocorticoid receptor activity (chrysene (Chry), BaA, and BaP), and potentiation of triiodothyronine-induced thyroid receptor α activity (all tested PAHs). PAHs also induced transcription of endogenous gene targets of constitutive androstane receptor (Fla, Pyr), or repression of target genes of pregnane X receptor and peroxisome proliferator-activated receptor α (in case of the AhR-activating PAHs - Chry, BaA, BaP, and BkF) in HepaRG cells. In the same cell model, the AhR agonists reduced the expression of glucose metabolism genes (PCK1, G6PC and PDK4), and they up-regulated levels of glucosylceramides, together with a concomitant induction of expression of UGCG, glucosylceramide synthesis enzyme. Finally, both BaP and BkF were found to induce expression of early stress and genotoxicity markers: ATF3, EGR1, GDF15, CDKN1A/p21, and GADD45A mRNAs, while BaP alone increased levels of IL-6 mRNA. Overall, whereas low-molecular-weight PAHs exerted significant effects on nuclear receptors (with CYP2B6 induction observed already at nanomolar concentrations), the AhR activation by 4-ring and 5-ring PAHs appeared to be a key mechanism underlying their impact on nuclear receptor signaling, endogenous metabolism and induction of early stress and genotoxicity markers.

Drug-Drug Interaction Potential, Cytotoxicity, and Reactive Oxygen Species Production of Salix Cortex Extracts Using Human Hepatocyte-Like HepaRG Cells.

Herbal preparations of willow bark (Salix cortex) are available in many countries as non-prescription medicines for pain and inflammation, and also as dietary supplements. Currently only little information on toxicity and drug interaction potential of the extracts is available. This study now evaluated the effects of two Salix cortex extracts on human hepatocyte-like HepaRG cells, in view of clinically relevant CYP450 enzyme activity modulation, cytotoxicity and production of reactive oxygen species (ROS). Drug metabolism via the CYP450 enzyme system is considered an important parameter for the occurrence of drug-drug interactions, which can lead to toxicity, decreased pharmacological activity, and adverse drug reactions. We evaluated two different bark extracts standardized to 10 mg/ml phenolic content. Herein, extract S6 (S. pentandra, containing 8.15 mg/ml total salicylates and 0.08 mg/ml salicin) and extract B (industrial reference, containing 5.35 mg/ml total salicylates and 2.26 mg/ml salicin) were tested. Both Salix cortex extracts showed no relevant reduction in cell viability or increase in ROS production in hepatocyte-like HepaRG cells. However, they reduced CYP1A2 and CYP3A4 enzyme activity after 48 h at ≥25 μg/ml, this was statistically significant only for S6. CYP2C19 activity inhibition (0.5 h) was also observed at ≥25 μg/ml, mRNA expression inhibition by 48 h treatment with S6 at 25 μg/ml. In conclusion, at higher concentrations, the tested Salix cortex extracts showed a drug interaction potential, but with different potency. Given the high prevalence of polypharmacy, particularly in the elderly with chronic pain, further systematic studies of Salix species of medical interest should be conducted in the future to more accurately determine the risk of potential drug interactions.

Curcumin inhibited hepatitis B viral entry through NTCP binding

Hepatitis B virus (HBV) has been implicated in hepatitis and hepatocellular carcinoma. Current agents (nucleos(t)ide analogs and interferons) could only attenuate HBV infection. A combination of agents targeting different stages of viral life cycle (e.g., entry, replication, and cccDNA stability) was expected to eradicate the infection. Curcumin (CCM) was investigated for inhibitory action toward HBV attachment and internalization. Immortalized hepatocyte-like cells (imHCs), HepaRG and non-hepatic cells served as host cells for binding study with CCM. CCM decreased viral load, HBeAg, HBcAg (infectivity), intracellular HBV DNA, and cccDNA levels. The CCM-induced suppression of HBV entry was directly correlated with the density of sodium-taurocholate co-transporting polypeptide (NTCP), a known host receptor for HBV entry. The site of action of CCM was confirmed using TCA uptake assay. The affinity between CCM and NTCP was measured using isothermal titration calorimetry (ITC). These results demonstrated that CCM interrupted HBV entry and would therefore suppress HBV re-infection.

Cytochrome P450 1A1/2, 2B6 and 3A4 HepaRG Cell-Based Biosensors to Monitor Hepatocyte Differentiation, Drug Metabolism and Toxicity.

Human hepatoma HepaRG cells express most drug metabolizing enzymes and constitute a pertinent in vitro alternative cell system to primary cultures of human hepatocytes in order to determine drug metabolism and evaluate the toxicity of xenobiotics. In this work, we established novel transgenic HepaRG cells transduced with lentiviruses encoding the reporter green fluorescent protein (GFP) transcriptionally regulated by promoter sequences of cytochromes P450 (CYP) 1A1/2, 2B6 and 3A4 genes. Here, we demonstrated that GFP-biosensor transgenes shared similar expression patterns with the corresponding endogenous CYP genes during proliferation and differentiation in HepaRG cells. Interestingly, differentiated hepatocyte-like HepaRG cells expressed GFP at higher levels than cholangiocyte-like cells. Despite weaker inductions of GFP expression compared to the strong increases in mRNA levels of endogenous genes, we also demonstrated that the biosensor transgenes were induced by prototypical drug inducers benzo(a)pyrene and phenobarbital. In addition, we used the differentiated biosensor HepaRG cells to evidence that pesticide mancozeb triggered selective cytotoxicity of hepatocyte-like cells. Our data demonstrate that these new biosensor HepaRG cells have potential applications in the field of chemicals safety evaluation and the assessment of drug hepatotoxicity.

Bulk Organocatalytic Synthetic Access to Statistical Copolyesters from l-Lactide and ε-Caprolactone Using Benzoic Acid.

The development of synthetic strategies to produce statistical copolymers based on l-lactide (l-LA) and ε-caprolactone (CL), denoted as P(LA- stat-CL), remains highly challenging in polymer chemistry. This is due to the differing reactivity of the two monomers during their ring-opening copolymerization (ROcP). Yet, P(LA- stat-CL) materials are highly sought after as they combine the properties of both polylactide (PLA) and poly(ε-caprolactone) (PCL). Here, benzoic acid (BA), a naturally occurring, cheap, readily recyclable, and thermally stable weak acid, is shown to trigger the organocatalyzed ring-opening copolymerization (OROcP) of l-LA and CL under solvent-free conditions at 155 °C, in presence of various alcohols as initiators, with good control over molar masses and dispersities (1.11 < Đ < 1.35) of the resulting copolyesters. Various compositions can be achieved, and the formation of statistical compounds is shown through characterization by 1H, 13C, and diffusion ordered spectroscopy NMR spectroscopies and by differential scanning calorimetry, as well as through the determination of reactivity ratios ( rLA = 0.86, rCL = 0.86), using the visualization of the sum of squared residuals space method. Furthermore, this BA-OROcP process can be exploited to access metal-free PLA- b-P(LA- stat-CL)- b-PLA triblock copolymers, using a diol as an initiator. Finally, residual traces of BA remaining in P(LA- stat-CL) copolymers (<0.125 mol %) do not show any cytotoxicity toward hepatocyte-like HepaRG cells, demonstrating the safety of this organic catalyst.

Farnesol induces fatty acid oxidation and decreases triglyceride accumulation in steatotic HepaRG cells

Non-alcoholic fatty liver disease is manifested by hepatic accumulation of triglycerides (TG) and is commonly associated with metabolic syndrome. The isoprenoid farnesol (FOH) modulates lipid metabolism and reduces hepatic TG content in rodents. This effect involves activation of at least two nuclear receptors, peroxisome proliferator-activated receptor α (PPARα) and farnesoid X receptor. We evaluated the effects of FOH (100 μM) in a cellular model of human hepatic steatosis by loading hepatocyte-like HepaRG cells with oleic acid (OA, 0.66 mM). FOH treatment decreased OA-induced TG accumulation by ~25%. Using PCR arrays, we found that FOH treatment modulated the mRNA levels of several lipid-metabolizing enzymes, both alone and when cells were loaded with OA. While FOH activated PPARα and the constitutive androstane receptor (CAR), most of the FOH-mediated effects on lipid-metabolizing genes could be attributed to activation of PPARα. In OA-loaded HepaRG cells, FOH increased fatty acid oxidation, which was accompanied by up-regulation of PPARα target genes involved in mitochondrial fatty acid oxidation, including hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase and acetyl-coenzyme A acyltransferase 2. These effects on gene expression were lost when the cells were co-treated with the PPARα antagonist, GW6471. OA treatment alone decreased the mRNA levels of the drug-metabolizing enzymes, cytochrome P450 (CYP)1A2, 2B6, and 3A4, and increased CYP2E1 expression, all of which were attenuated by FOH co-treatment. These findings show that FOH treatment increases fatty acid oxidation and decreases TG accumulation in steatotic HepaRG cells, which is likely attributable to PPARα-mediated induction of mitochondrial fatty acid oxidation.

Cellular and Molecular Effects of Prolonged Low-Level Sodium Arsenite Exposure on Human Hepatic HepaRG Cells.

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 noncytotoxic 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, overexpression 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.

Initial sites of hepadnavirus integration into host genome in human hepatocytes and in the woodchuck model of hepatitis B-associated hepatocellular carcinoma

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.

Efficient transfection of Xenobiotic Responsive Element-biosensor plasmid using diether lipid and phosphatidylcholine liposomes in differentiated HepaRG cells

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.

Activation of nuclear receptor CAR by an environmental pollutant perfluorooctanoic acid.

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.

In vitro Toxicity Evaluation and in vivo Biodistribution of Polymeric Micelles Derived from Poly(ethylene glycol)-b-poly(benzyl malate) Copolyme

Background: The development of polymeric micelles for site-specific drug delivery is an exponentially growing field of research. In this context, we have designed two degradable amphiphilic copolymers, the poly(ethylene glycol)-b-poly(benzyl malate) (PEG42-b-PMLABe73) and the biotin-poly(ethylene glycol)- b-poly(benzyl malate) (Biot-PEG62-b-PMLABe73). The copolymer bearing biotin residue was synthesized in order to formulate micelles for grafting biotinylated cyclic RGD peptide onto their surface via the bridging streptavidin. Objective: Our study aimed at investigating the in vitro and in vivo toxicity of such micelles and to evaluate the potential of these nanovectors for hepatocyte-targeted drug delivery. Methods: The toxicity of micelles obtained by the nanoprecipitation method and characterized by dynamic light scattering and zeta potential measurement was evaluated in vitro using the differentiated hepatocyte-like HepaRG cells and in vivo in mice. Results: The micelles derived from PEG42-b-PMLABe73, Biot-PEG62-b-PMLABe73 and RGDBiot-Strept- Biot-PEG62-b-PMLABe73 did not affect the cell proliferation and apoptotic indexes in HepaRG hepatoma cells and were well tolerated in mice following systemic injection. The hydrophobic fluorescent probes DiD oil and DiR were efficiently encapsulated in PEG-b-PMLABe-derived micelles allowing the visualization of their uptake into HepaRG cells. Furthermore, the addition of RGD peptide onto micelles strongly enhanced the cell uptake in vitro and liver targeting in vivo. Conclusion: These data demonstrate the low toxicity of poly(benzyl malate) derived copolymers towards hepatocyte- like cells and emphasize their potential use for the design of liver targeting nanovectors

Aberrant DNA methylation of imprinted loci in hepatocellular carcinoma and after in vitro exposure to common risk factors.

BackgroundHepatocellular 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.ResultsAnalysis 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.ConclusionThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-015-0053-9) contains supplementary material, which is available to authorized users.

Expression and characterization of myristoylated preS1-conjugated nanocages for targeted cell delivery

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.

Reduction of the mitochondrial content in cardiolipins decreases O2 consumption and increases ATP synthesis efficiency in human hepatocyte-like HepaRG cells

Reduction of the mitochondrial content in cardiolipins decreases O2 consumption and increases ATP synthesis efficiency in human hepatocyte-like HepaRG cells

Hepatocyte Polarization Is Essential for the Productive Entry of the Hepatitis B Virus

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. 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.

Induction of vesicular steatosis by amiodarone and tetracycline is associated with up-regulation of lipogenic genes in heparg cells

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. 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.

CYP4F3B is induced by PGA1 in human liver cells: a regulation of the 20-HETE synthesis

The regulation of the human liver-specific cytochrome P450 4F3B (CYP4F3B) isoform, a splice variant of the CYP4F3 gene with strong substrate specificity for long chain fatty acids, is yet an unsolved question. This report provides the first evidence that CYP4F3B is uniquely induced by prostaglandin A1 (PGA1) in human hepatocyte-like HepaRG cells and leads to the synthesis of 20-hydroxy-eicosatetraenoic acids (HETEs). Real time PCR, immunoblot analysis with a specific antipeptide antibody, and determination of fatty acid omega-hydroxylase activity demonstrate that PGA1 treatment strongly increases expression of CYP4F3B. This induction drives the production of 20-HETE (19-fold increase). SiRNA-mediated-silencing of CYP4F3 suppresses both 20-HETE synthesis and PGA1 induced 20-HETE production. Taken together, these results provide evidence that CYP4F3B is the key enzyme to produce 20-HETE by omega-hydroxylation of arachidonic acid in liver cells. Since 20-HETE is a potent activator of PPARalpha and an important inflammatory mediator, CYP4F3B may exert important functions in lipid homeostasis and in inflammatory diseases.