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.

The Role of Catecholamines in Pathophysiological Liver Processes.

Over the last few years, the number of research publications about the role of catecholamines (epinephrine, norepinephrine, and dopamine) in the development of liver diseases such as liver fibrosis, fatty liver diseases, or liver cancers is constantly increasing. However, the mechanisms involved in these effects are not well understood. In this review, we first recapitulate the way the liver is in contact with catecholamines and consider liver implications in their metabolism. A focus on the expression of the adrenergic and dopaminergic receptors by the liver cells is also discussed. Involvement of catecholamines in physiological (glucose metabolism, lipids metabolism, and liver regeneration) and pathophysiological (impact on drug-metabolizing enzymes expression, liver dysfunction during sepsis, fibrosis development, or liver fatty diseases and liver cancers) processes are then discussed. This review highlights the importance of understanding the mechanisms through which catecholamines influence liver functions in order to draw benefit from the adrenergic and dopaminergic antagonists currently marketed. Indeed, as these molecules are well-known drugs, their use as therapies or adjuvant treatments in several liver diseases could be facilitated.

Retrodifferentiation of Human Tumor Hepatocytes to Stem Cells Leads to Metabolic Reprogramming and Chemoresistance.

Human hepatocellular carcinoma (HCC) heterogeneity promotes recurrence and therapeutic resistance. We recently demonstrated that inflammation favors hepatocyte retrodifferentiation into progenitor cells. Here, we identify the molecular effectors that induce metabolic reprogramming, chemoresistance, and invasiveness of retrodifferentiated HCC stem cells. Spheroid cultures of human HepaRG progenitors (HepaRG-Spheres), HBG-BC2, HepG2, and HuH7 cells and isolation of side population (SP) from HepaRG cells (HepaRG-SP) were analyzed by transcriptomics, signaling pathway analysis, and evaluation of chemotherapies. Gene expression profiling of HepaRG-SP and HepaRG-Spheres revealed enriched signatures related to cancer stem cells, metastasis, and recurrence and showed that HepaRG progenitors could retrodifferentiate into an immature state. The transcriptome from these stem cells matched that of proliferative bad outcome HCCs in a cohort of 457 patients. These HCC stem cells expressed high levels of cytokines triggering retrodifferentiation and displayed high migration and invasion potential. They also showed changes in mitochondrial activity with reduced membrane potential, low ATP production, and high lactate production. These changes were, in part, related to angiopoietin-like 4 (ANGPTL4)-induced upregulation of pyruvate dehydrogenase kinase 4 (PDK4), an inhibitor of mitochondrial pyruvate dehydrogenase. Upregulation of ANGPTL4 and PDK4 paralleled that of stem cells markers in human HCC specimens. Moreover, the PDK4 inhibitor dichloroacetate reversed chemoresistance to sorafenib or cisplatin in HCC stem cells derived from four HCC cell lines. In conclusion, retrodifferentiated cancer cells develop enhanced invasion and therapeutic resistance through ANGPTL4 and PDK4. Therefore, restoration of mitochondrial activity in combination with chemotherapy represents an attractive therapeutic approach in HCC. SIGNIFICANCE: Restoring mitochondrial function in human hepatocellular carcinomas overcomes cancer resistance.

Dioxin-mediated up-regulation of aryl hydrocarbon receptor target genes is dependent on the calcium/calmodulin/CaMKIalpha pathway.

Regulation of genes targeted by the ligand-activated aryl hydrocarbon receptor (AhR) has been shown to be controlled by calcium (Ca(2+)) changes induced by AhR agonists such as the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The present study was designed to characterize this link between Ca(2+) and the AhR pathway. We report that fast elevation of intracellular Ca(2+) in TCDD-exposed mammary MCF-7 cells was associated with transient enhanced activity of the Ca(2+)/calmodulin (CaM)-dependent protein kinase (CaMK) pathway. Chemical inhibition of this pathway using the CaM antagonist W7 or the CaMK inhibitor KN-93 strongly reduced TCDD-mediated induction of the AhR target gene CYP1A1. Small interfering RNA (siRNA)-mediated knockdown expression of CaMKIalpha, one of the CaMK isoforms, similarly prevented CYP1A1 up-regulation. Both KN-93 and siRNA targeting CaMKIalpha were found to abolish TCDD-mediated activation of CYP1A1 promoter and TCDD-triggered nuclear import of AhR, a crucial step of the AhR signaling pathway. TCDD-mediated inductions of various AhR targets, such as the drug metabolizing CYP1B1, the cytokine interleukin-1beta, the chemokines interleukin-8 and CCL1, the adhesion molecule beta7 integrin, and the AhR repressor, were also prevented by KN-93 in human macrophages. Taken together, these data identified the Ca(2+)/CaM/CaMKIalpha pathway as an important contributing factor to AhR-mediated genomic response.

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.

Opsonisation of nanoparticles prepared from poly(ß-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.

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(ß-malic acid)-b-poly(ß-hydroxybutyrate) (PMLA-b-PHB) and poly(ß-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.

The human glutathione transferase alpha locus: genomic organization of the gene cluster and functional characterization of the genetic polymorphism in the hGSTA1 promoter.

By searching the human genome sequence database with human hGSTA1 and hGSTA4 cDNA sequences, we identified three PAC and one BAC clones covering more than 400 kilobases and containing the entire GST alpha gene cluster. The cluster consists of five genes: hGSTA1, hGSTA2, hGSTA3, hGSTA4 and hGSTA5, and seven pseudogenes that are distinguished as such by single-base and/or complete exon deletions. Using gene-specific probes we demonstrated that hGSTA1, hGSTA2 and hGSTA4 mRNAs are widely expressed in human tissues, whereas hGSTA3 mRNA appears to be a rare message subject to splicing defects. Although examination of the hGSTA5 gene sequence suggests that it is a functional gene, hGSTA5 mRNA could not be detected in human tissues we studied. hGSTA1 expression has been shown to be influenced by a genetic polymorphism, that consists of two alleles hGSTA1*A and hGSTA1*B, containing three linked base substitutions in the proximal promoter, at positions -567, -69 and -52. Constructs consisting of the luciferase gene controlled by variant hGSTA1 promoters showed differential expression when transfected into HepG2, GLC4 and Caco-2 cells: hGSTA1*A > hGSTA1*B. Directed mutagenesis for each base substitution indicated that the base change -52G>A was responsible for the differential promoter activity of hGSTA1*A and hGSTA1*B. The base at position -52 also altered binding of the ubiquitous transcription factor Sp1, as determined by gel shift analysis. Thus it may be postulated that hGSTA1 genotyping will be of importance to determine individual susceptibility to certain cancers or the efficacy of chemotherapeutics via its effect on hGSTA1 expression.

Regulation of signal transduction by glutathione transferases.

Glutathione transferases (GST) are essentially known as enzymes that catalyse the conjugation of glutathione to various electrophilic compounds such as chemical carcinogens, environmental pollutants, and antitumor agents. However, this protein family is also involved in the metabolism of endogenous compounds which play critical roles in the regulation of signaling pathways. For example, the lipid peroxidation product 4-hydroxynonenal (4-HNE) and the prostaglandin 15-deoxy-Δ12,14-prostaglandin J(2) (15d-PGJ(2)) are metabolized by GSTs and these compounds are known to influence the activity of transcription factors and protein kinases involved in stress response, proliferation, differentiation, or apoptosis. Furthermore, several studies have demonstrated that GSTs are able to interact with different protein partners such as mitogen activated protein kinases (i.e., c-jun N-terminal kinase (JNK) and apoptosis signal-regulating kinase 1 (ASK1)) which are also involved in cell signaling. New functions of GSTs, including S-glutathionylation of proteins by GSTs and ability to be a nitric oxide (NO) carrier have also been described. Taken together, these observations strongly suggest that GST might play a crucial role during normal or cancer cells proliferation or apoptosis.

Involvement of pregnane X receptor in the regulation of CYP2B6 gene expression by oltipraz in human hepatocytes.

Oltipraz, a synthetic derivative of the cruciferous vegetable product 1,2-dithiole-3-thione, is considered as a potent chemoprotectant. Previously, we have demonstrated that CYP2B6 expression is induced in cultured human hepatocytes by a 24h treatment with oltipraz. The aim of this study was to further determine mechanisms involved in the regulation of CYP2B6 by this compound. An increase of CYP2B6 mRNA is observed after a 4h exposure and maximum induction is reached after 24h. The rapid induction of CYP2B6 mRNA in oltipraz-treated cells suggests a transcriptional activation of corresponding gene. To test this hypothesis, we performed transient transfections with constructs containing the CYP2B6 gene 5'-flanking region upstream of the luciferase gene in order to measure the transcriptional activity of CYP2B6 gene in human hepatoma HepG2 cells, in absence or presence of oltipraz. The results demonstrate that transcriptional activation of CYP2B6 gene is mediated mainly by the pregnane X receptor (PXR) and the Phenobarbital Responsive Element Module (PBREM). The nuclear factor-erythroid 2-related factor 2 (Nrf2) and an antioxidant responsive element (ARE), located upstream the PBREM, might also have a role in this activation but their involvement remains unclear. Despite increasing CYP2B6 apoprotein levels in human hepatocytes, oltipraz has little effect, if any, on testosterone 16beta-hydroxylation which is catalyzed by CYP2B6. This can be explained by a dose-dependent inhibition of CYP2B6 activity in presence of oltipraz as demonstrated with human hepatocyte microsomes. Altogether, this study provides the first demonstration of PXR involvement in oltipraz transcriptional activation of CYP2B6 gene and of the inhibitory effect of oltipraz on CYP2B6 activity.

Involvement of Nrf2 activation in resistance to 5-fluorouracil in human colon cancer HT-29 cells.

Acquisition of drug resistance by cancer cells is attributed to various factors including alterations in apoptotic pathways, enhanced expression of multidrug resistance-associated proteins, altered drug metabolism or uptake and/or overexpression of cytoprotective genes. Thus, potential induction of defence pathways by anticancer drugs might have a marked incidence on cancer cell resistance. 5-Fluorouracil (5-FU) remains the most commonly used anticancer drug for the treatment of colorectal cancer, although objective response rates are as low as 20%. The aim of our study was to investigate the effects of 5-FU on cytoprotective systems in human colon HT-29 cells. Our results demonstrate that 5-FU induced the expression of mRNAs encoding glutathione transferases and antioxidant enzymes. To further determine the mechanisms involved in 5-FU effects, we investigated whether it activates the Nrf2/antioxidant response element pathway which is implicated in the regulation of several genes involved in cytoprotection. Translocation of Nrf2 into the nucleus after 5-FU exposure was demonstrated by immunocytochemistry and western blotting. Using an ARE-driven reporter gene assay, activation of the luciferase activity by 5-FU was also evidenced. Moreover, transfection of HT-29 cells with siRNA directed against Nrf2 inhibited induction of Nrf2 target genes and increased 5-FU cytotoxicity. In conclusion, we demonstrate for the first time that 5-FU activates the Nrf2/ARE pathway which in turn induces cytoprotective genes and modulates chemosensitivity of HT-29 colon cancer cells. Therefore, we postulate that Nrf2 might represent a potential therapeutic target in 5-FU treatment of colon cancer.

Glutathione transferases kappa 1 and kappa 2 localize in peroxisomes and mitochondria, respectively, and are involved in lipid metabolism and respiration in Caenorhabditis elegans.

To elucidate the function of kappa class glutathione transferases (GSTs) in multicellular organisms, their expression and silencing were investigated in Caenorhabditis elegans. In contrast with most vertebrates, which possess only one GST kappa gene, two distinct genes encoding GSTK-1 and GSTK-2 are present in the C. elegans genome. The amino acid sequences of GSTK-1 and GSTK-2 share around 30% similarity with the human hGSTK1 sequence and, like the human transferase, GSTK-1 contains a C-terminal peroxisomal targeting sequence. gstk-1 and gstk-2 genes show distinct developmental and tissue expression patterns. We show that GSTK-2 is localized in the mitochondria and expressed mainly in the pharynx, muscles and epidermis, whereas GSTK-1 is restricted to peroxisomes and expressed in the intestine, body wall muscles and epidermis. In order to determine the potential role(s) of GST kappa genes in C. elegans, specific silencing of the gstk-1 and gstk-2 genes was performed by an RNA interference approach. Knockdown of gstk-1 or gstk-2 had no apparent effect on C. elegans reproduction, development, locomotion or lifespan. By contrast, when biological functions (oxygen consumption and lipid metabolism) related to peroxisomes and/or mitochondria were investigated, we observed a significant decrease in respiration rate and a lower concentration of the monounsaturated fatty acid cis-vaccenic acid (18:1omega7) when worms were fed on bacteria expressing RNA interference targeting both gstk-1 and gstk-2. These results demonstrate that GST kappa, although not essential for the worm's life, may be involved in energetic and lipid metabolism, two functions related to mitochondria and peroxisomes.

Aryl hydrocarbon receptor- and calcium-dependent induction of the chemokine CCL1 by the environmental contaminant benzo[a]pyrene.

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed immunotoxic environmental contaminants well known to regulate expression of pro-inflammatory cytokines such as interleukine-1beta and tumor necrosis factor-alpha. In the present study, we demonstrated that the chemokine CCL1, notably involved in cardiovascular diseases and inflammatory or allergic processes, constitutes a new molecular target for PAHs. Indeed, exposure to PAHs such as benzo[a]pyrene (BP) markedly increased mRNA expression and secretion of CCL1 in primary human macrophage cultures. Moreover, intranasal administration of BP to mice enhanced mRNA levels of TCA3, the mouse orthologue of CCL1, in lung. CCL1 induction in cultured human macrophages was fully prevented by targeting the aryl hydrocarbon receptor (AhR) through chemical inhibition or small interfering RNA-mediated down-modulation of its expression. In addition, BP and the potent AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin were found to enhance activity of a CCL1 promoter sequence containing a consensus xenobiotic-responsive element known to specifically interact with AhR. Moreover, 2,3,7,8-tetrachlorodibenzo-p-dioxin triggered AhR binding to this CCL1 promoter element as revealed by chromatin immunoprecipitation experiments and electrophoretic mobility shift assays. In an attempt to further characterize the mechanism of CCL1 induction, we demonstrated that BP was able to induce an early and transient increase of intracellular calcium concentration in human macrophages. Inhibition of this calcium increase, using the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester or the calcium store-operated channel inhibitor 2-aminoethoxydiphenyl borate, fully blocked CCL1 up-regulation. Taken together, these results bring the first demonstration that PAHs induce expression of the chemokine CCL1 in an AhR- and calcium-dependent manner.

Oltipraz regulates different categories of genes relevant to chemoprevention in human hepatocytes.

Numerous chemical compounds are cytotoxic or carcinogenic to human beings and attention is now focusing on preventative strategies. One agent, oltipraz (OPZ), regarded as one of the most promising chemoprotectors, has been shown to be a potent inducer of phase II enzymes involved in the detoxification of carcinogens, including aflatoxins. However, little is known about its effects on global gene expression in human cells. Thus, we used microarrays and reverse transcription-quantitative polymerase chain reaction to test the effects of OPZ on the overall pattern of mRNA expression of multiple metabolic pathways in human hepatocytes in primary culture. Our results show for the first time that OPZ significantly alters the expression of human genes within different functional categories (detoxification of xenobiotics, antioxidant defences, xenobiotic transport, cell cycle and stress responses), at both the mRNA and protein levels, some of which are highly relevant to chemoprevention. Amongst these genes, several have never been described as being regulated by OPZ before. We also demonstrate variations in response to OPZ, depending on the individual from whom the cells were derived, that might potentially contribute to differences in efficacy of chemopreventive treatments between individuals. Moreover, comparison of our results with those obtained in rodents demonstrates species differences in response to OPZ for some genes, underlying the importance of studies on human cells to predict the effects of chemopreventive agents.

Gene and protein characterization of the human glutathione S-transferase kappa and evidence for a peroxisomal localization.

Kappa class glutathione S-transferase (GST) cDNA sequences have been identified in rat, mouse, and human. In the present study, we determined the structure and chromosomal location of the human GST Kappa 1 (hGSTK1) gene, characterized the protein, and demonstrated its subcellular localization. The human gene spans approximately 5 kb, has 8 exons, and maps onto chromosome 7q34. The 5'-flanking region lacks TATA or CCAAT boxes, but there is an initiator element overlapping the transcription start site. hGSTK1 amino acid sequence showed homology to bacterial 2-hydroxychromene-2-carboxylate isomerase, an enzyme involved in naphthalene degradation pathway. hGSTK1 mRNA was expressed in all of the organs examined. Subcellular fractionation of HepG2 cells showed that the protein was located in peroxisomes and mitochondria and was not detectable in cytoplasm. The peroxisomal localization was confirmed by transfection of HepG2 cells with a plasmid coding a green fluorescent protein fused inframe to the N terminus of hGSTK1. The C terminus of hGSTK1 was essential for localization of the protein to peroxisomes, and the C-terminal sequence Ala-Arg-Leu represents a peroxisome targeting signal. This is the first time that a human GST has been found in peroxisomes, suggesting a new function for this family of enzymes.

Transcriptional induction of CYP1A1 by oltipraz in human Caco-2 cells is aryl hydrocarbon receptor- and calcium-dependent.

Oltipraz, a synthetic derivative of the cruciferous vegetable product 1,2-dithiole-3-thione, is considered as one of the most potent chemoprotectants. It modulates both cytochrome P-450 (CYP) and glutathione S-transferase expression and activities in rat tissues. Its effects, however, are variable according to the enzyme, tissue, and species. We show here that, as previously found in rat lung and kidney, CYP1A1 is inducible by oltipraz in both rat intestine and Caco-2 cells, a cell line originated from a human colon adenocarcinoma. In these cells, a 50 microm oltipraz treatment increased CYP1A1 mRNA ( approximately 30-fold), protein and activity. mRNA level was augmented as early as 2 h after the beginning of treatment, suggesting a transcriptional activation, and was maximal between 8 and 12 h. Transient transfection of Caco-2 cells with constructs containing different sizes of the 5'-flanking region of the CYP1A1 gene upstream of the luciferase reporter gene showed an increase in luciferase activity in oltipraz-treated cells, which correlates with the presence of the xenobiotic responsive element (XRE). Furthermore we demonstrated that resveratrol, an antagonist of the aryl hydrocarbon (Ah) receptor, inhibited the induction of both CYP1A1 promoter activity and mRNA by oltipraz, supporting the involvement of the Ah receptor in this induction. In an attempt to further characterize the mechanism of CYP1A1 induction, we showed a rapid increase in intracellular calcium concentration upon treatment of Caco-2 cells with oltipraz. Moreover, the effect of this compound on CYP1A1 was strongly abolished in the presence of BAPTA-AM, a well known chelator of intracellular calcium, and 2-aminoethyl diphenylborate, an inhibitor of store-operated calcium channels. These results bring the first demonstration that oltipraz activates transcription of the CYP1A1 gene through the Ah receptor-XRE pathway in Caco-2 cells and that CYP1A1 induction relies upon an increase of intracellular calcium concentration.