Papers by André Guillouzo
Toxicology in Vitro, Dec 1, 2015
In vitro experiments have a high potential to improve current chemical safety assessment and redu... more 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 14 days. 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 14 days 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.
Toxicology, Mar 1, 2010
Better prediction of safety risk and understanding of mechanism of action of drug candidates rema... more Better prediction of safety risk and understanding of mechanism of action of drug candidates remain a major challenge in order to prevent late stage attrition. Continuous efforts are made to improve and develop new models, especially in some areas such as hepatotoxicity. Besides primary hepatocytes and transformed liver cell lines, stem cells either isolated from embryos or adult tissues or obtained by reprogramming somatic cells are emerging as a new potential source of unlimited numbers of hepatocytes. Presently, only hepatocyte-like cells expressing low levels of liver-specific markers, especially drug metabolizing and detoxifying enzymes, are usually obtained, making them still unsuitable as metabolically competent cells for toxicity studies. The only exceptions are some hepatoma cell lines, particularly the HepaRG cell line that can differentiate from a bipotent progenitor stage to attain the functional capacity of normal adult hepatocytes in primary culture without losing the indefinite growth property of transformed cells. Since the research field on stem cells is growing fast marked advances might be expected in the next future.
Drug Metabolism and Disposition, Jul 7, 2014
Several factors are thought to be implicated in the occurrence of idiosyncratic adverse drug reac... more 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 downregulation 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 proinflammatory 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.
Journal of Biological Chemistry, Jul 1, 2002
Oltipraz, a synthetic derivative of the cruciferous vegetable product 1,2-dithiole-3-thione, is c... more 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 M oltipraz treatment increased CYP1A1 mRNA (ϳ30-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.
The human hepatoma HepaRG cells are able to differentiate in vitro into hepatocyte-like cells and... more The human hepatoma HepaRG cells are able to differentiate in vitro into hepatocyte-like cells and to express various liver-specific functions, including the major cytochromes P450. This study was aimed to determine whether differentiated HepaRG cells retained their specific functional capacities for a long time period at confluence. We show that expression of transcripts encoding CYP1A2, 2B6, 3A4 and 2E1, several phase II and antioxidant enzymes, membrane transporters, including OCT1 and BSEP, the nuclear receptors CAR and PXR and aldolase B remained relatively stable for at least the 4-week confluence period tested. Similarly, activities of CYP3A4 and CYP1A2 and their responsiveness to prototypical inducers were well preserved. Aflatoxin B1, a potent hepatotoxicant and carcinogen, induced a dose-dependent and cumulative cytotoxicity. Furthermore, at a concentration as low as 0.1µM, this mycotoxin caused a decrease in both CYP3A4 activity and intracellular ATP associated with morphological alterations, after 14 days following every two days exposure. Moreover, using the comet assay a dose-dependent DNA damage was observed after a 3h treatment of differentiated HepaRG cells with 1 to 5µM aflatoxin B1, in the absence of any cell damage and this DNA damaging effect was strongly reduced in the presence of ketoconazole, a CYP3A4 inhibitor. These results bring the first demonstration of long-term stable expression of liver-specific markers in HepaRG hepatocyte cultures maintained at confluence and show that these cells represent a suitable in vitro liver cell model for analysis of acute and chronic toxicity as well as genotoxicity of chemicals in human liver. This article has not been copyedited and formatted. The final version may differ from this version.
Cell Biology and Toxicology
Administration of tacrine (THA) for the treatment of Alzheimer's disease results in a reversible ... more Administration of tacrine (THA) for the treatment of Alzheimer's disease results in a reversible hepatotoxicity in 30^50% of patients, as indicated by an increase in transaminase levels. However, the intracellular mechanisms underlying such a toxicity have not yet been elucidated. In this study, we performed short-term and long-term in vitro treatments on primary human and rat hepatocyte cultures as well as on nonparenchymal rat liver epithelial cells (RLEC), known as CYP1A-de¢cient cells. Cell ultrastructure was analyzed under di¡erent conditions and the release of lactate dehydrogenase (LDH) was used to evaluate cytotoxicity. The e¡ects of THA on protein synthesis, intermediary metabolism and reduced glutathione (GSH) level were also determined in rat hepatocytes. THA induced dose-dependent toxic e¡ects in liver parenchymal and nonparenchymal cells, with human hepatocytes being less sensitive. This toxicity appeared to be unrelated to metabolism of THA since similar e¡ects were observed in rat hepatocytes and RLEC, in which THA metabolism was found negligible. Ribosome aggregation appeared only at high concentrations (41 mmol/L) and was not speci¢c to hepatocytes. Therefore, the THA-induced decrease in protein synthesis observed at lower concentrations was likely not related to this alteration. ATP and glycogen levels as well as GSH content were reduced upon THA. However, while glycogen level decreased at THA doses similar to those inducing an increase in LDH release, the fall in ATP and GSH contents occurred at higher doses. Thus, glycogen level in hepatocytes appeared to be a more sensitive indicator of THA toxicity than were ATP and GSH levels. We also found that protein synthesis started to decrease at THA doses that were still ine¡ective on LDH release. This might suggest that the decrease in synthesis of one or several proteins upon THA treatment represents the early signal leading cells to death.
Cell Biology and Toxicology, 2000
Cytochrome P4502E1 (CYP2E1) plays a key role in the metabolism of numerous drug substrates, mostl... more Cytochrome P4502E1 (CYP2E1) plays a key role in the metabolism of numerous drug substrates, mostly in mammalian liver. Both the apoprotein and mRNA levels are increased in response to interleukin 4 (IL-4) in primary human hepatocyte cultures. We developed a human hepatoma cell model that faithfully reproduces the responsiveness of the CYP2E1 gene to IL-4 at least in part through
Cell Biology and Toxicology, 2000
We investigated the involvement of diverse protein kinases and phosphatases in the transduction p... more We investigated the involvement of diverse protein kinases and phosphatases in the transduction pathways elicited by phenobarbital (PB), a well-known inducer of some hepatic cytochromes P450 (CYP). Different inhibitors or activators of protein kinases or phosphatases were assessed for their ability to modulate PB-induction of CYP2B and CYP3A mRNA expression. Rat hepatocytes in primary culture were treated with the test
Toxicological Sciences, 2015
Alteration of bile acid (BA) profiles and secretion by cholestatic drugs represents a major clini... more Alteration of bile acid (BA) profiles and secretion by cholestatic drugs represents a major clinical issue. Species differences exist in BA composition, synthesis, and regulation; however presently, there is no in vitro reproducible cell model to perform studies on BAs in humans. We have evaluated the capacity of the human HepaRG cell line to synthesize, conjugate, and secrete BAs, and analyzed changes in BA content and profile after cyclosporine A (CsA) treatment. Our data show that HepaRG cells produced normal BAs at daily levels comparable, though in different proportions, to those measured in primary human hepatocytes. A 4-h treatment with CsA led to BA accumulation and profile changes associated with occurrence of cholestatic features, while after 24 h BAs were decreased in cell layers and increased in media. The latter effects resulted from reduced function of BA uptake transporter (Na þ-taurocholate cotransporting polypeptide), reduced expression of BA metabolizing enzymes, including cytochrome P4507A1, cytochrome P4508B1, and cytochrome P45027A1, and induction of alternative basolateral transporters. Noteworthy, HepaRG cells incubated in a 2% serum-supplemented medium showed dose-dependent accumulation of the cytotoxic BA lithocholic acid in a nonsulfoconjugated form associated with early inhibition of the canalicular transporter MRP2 and sulfotransferase 2A1. In summary, our data bring the first demonstration that an in vitro human liver cell line is able to produce and secrete conjugated BAs, and to accumulate endogenous BAs transiently, concomitantly to occurrence of various other cholestatic features following CsA treatment. Retention of the hydrophobic lithocholic acid supports its toxic role in drug-induced cholestasis. Overall, our results argue on the suitability of HepaRG cells for investigating mechanisms involved in the development of the disease.
European Journal of Biochemistry, 2000
Phenobarbital (PB) has long been known as an inducer of drug-metabolizing enzymes in liver, but t... more Phenobarbital (PB) has long been known as an inducer of drug-metabolizing enzymes in liver, but the molecular mechanism underlying this induction is still poorly understood. Using primary mouse hepatocyte culture, we have investigated the possible involvement of different regulatory pathways in PB action, by exposing PB-treated cells to various protein kinase/phosphatase modulators. Our results showed a negative role of the cAMP-dependent pathway, as treatment with cAMP-dependent protein kinase (PKA) activators (10 mm dibutyryl-cAMP and 50 mm forskolin) dramatically inhibited PB-induced Cyp2b9/10 mRNA accumulation, whereas PKA inhibitor potentiated the PB responsiveness of this gene. The cGMP-dependent protein kinase (PKG) seems to play a positive role as PKG inhibitor reduced the PB-induced level of Cyp2b9/10 mRNA. We also obtained two lines of evidence for the involvement of Ca 2+ in modulating PB action. Firstly, measurements of intracellular Fura-2 fluorescence ratio in murine hepatocytes showed that long-term PB incubation (24 and 48 h) led to a significant increase of [Ca 2+ ] i. Secondly, treatment with an intracellular Ca 2+ chelator (BAPTA-AM) nearly completely abolished PB-induced Cyp2b9/10 expression. Ca 2+ thus appeared to mediate PB action likely via Ca 2+ /calmodulin-dependent protein kinase II, as KN62, a specific inhibitor of this enzyme, also dramatically inhibited PB induction of the Cyp2b9/10 genes.
The Journal of pharmacology and experimental therapeutics, 2000
Tacrine (THA), used in the treatment of Alzheimer's disease, is known to induce hepatotoxicit... more Tacrine (THA), used in the treatment of Alzheimer's disease, is known to induce hepatotoxicity, the mechanisms of which remain to be fully established. We have previously shown that THA reduced intracellular glutathione concentration in rat hepatocytes in primary culture, thus pointing to a possible role for oxidative stress in THA toxicity. To test this, the effects of antioxidant molecules, namely, the flavonoids silibinin, silibinin dihydrogensuccinate, and silymarin, were evaluated on the toxicity of THA in cultured rat hepatocytes. This toxicity was investigated after a 24-h treatment over a concentration range from 0 to 1 mM, in the presence or absence of antioxidant (1 and 10 microM). We found that simultaneous treatment of hepatocytes with any of the antioxidants and THA remained ineffective on the lactate dehydrogenase release induced by THA. Then, the production of lipid-derived radicals (to estimate lipid peroxidation) was measured in THA (0.05-0.50 mM)-treated cells ...
The Journal of pharmacology and experimental therapeutics, 1999
Several of the hepatic microsomal cytochromes P-450 (CYP) including CYP3A are inducible by phenob... more Several of the hepatic microsomal cytochromes P-450 (CYP) including CYP3A are inducible by phenobarbital (PB). However, the intracellular pathways involved in the action of PB on CYP3A remain poorly known. With the aim to unravel some of the main aspects of PB signaling, we first devised a simple model of mouse cultured primary hepatocytes in which CYP3A mRNA and protein were strongly induced by PB in the absence of dexamethasone and were at maximum levels after a 48-h treatment with a 2-mM dose of PB. Under these culture conditions, we studied the effects of inhibitors and activators of different protein kinases or phosphatases on CYP3A mRNA and protein induction by PB. CYP3A-induced expression was inhibited by activators of cyclic AMP-dependent protein kinase (PKA) (dibutyryl-cyclic AMP and forskolin) whereas inhibition of PKA by PKA inhibitor enhanced induction. 8-br-cGMP produced effects similar to the activators of PKA, and so did the specific inhibitor of cGMP-dependent protei...
Toxicology, 2010
Better prediction of safety risk and understanding of mechanism of action of drug candidates rema... more Better prediction of safety risk and understanding of mechanism of action of drug candidates remain a major challenge in order to prevent late stage attrition. Continuous efforts are made to improve and develop new models, especially in some areas such as hepatotoxicity. Besides primary hepatocytes and transformed liver cell lines, stem cells either isolated from embryos or adult tissues or obtained by reprogramming somatic cells are emerging as a new potential source of unlimited numbers of hepatocytes. Presently, only hepatocyte-like cells expressing low levels of liver-specific markers, especially drug metabolizing and detoxifying enzymes, are usually obtained, making them still unsuitable as metabolically competent cells for toxicity studies. The only exceptions are some hepatoma cell lines, particularly the HepaRG cell line that can differentiate from a bipotent progenitor stage to attain the functional capacity of normal adult hepatocytes in primary culture without losing the indefinite growth property of transformed cells. Since the research field on stem cells is growing fast marked advances might be expected in the next future.
Mutagenesis, 2011
The in vitro micronucleus test is considered as an attractive tool for genotoxicity testing of ch... more 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 Cooperation 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.
Journal of Biological Chemistry, 2002
Oltipraz, a synthetic derivative of the cruciferous vegetable product 1,2-dithiole-3-thione, is c... more 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 M oltipraz treatment increased CYP1A1 mRNA (ϳ30-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.
Drug Metabolism and Disposition, 2008
The human hepatoma HepaRG cells are able to differentiate in vitro into hepatocyte-like cells and... more The human hepatoma HepaRG cells are able to differentiate in vitro into hepatocyte-like cells and to express various liver-specific functions, including the major cytochromes P450. This study was aimed to determine whether differentiated HepaRG cells retained their specific functional capacities for a long time period at confluence. We show that expression of transcripts encoding CYP1A2, 2B6, 3A4 and 2E1, several phase II and antioxidant enzymes, membrane transporters, including OCT1 and BSEP, the nuclear receptors CAR and PXR and aldolase B remained relatively stable for at least the 4-week confluence period tested. Similarly, activities of CYP3A4 and CYP1A2 and their responsiveness to prototypical inducers were well preserved. Aflatoxin B1, a potent hepatotoxicant and carcinogen, induced a dose-dependent and cumulative cytotoxicity. Furthermore, at a concentration as low as 0.1µM, this mycotoxin caused a decrease in both CYP3A4 activity and intracellular ATP associated with morphological alterations, after 14 days following every two days exposure. Moreover, using the comet assay a dose-dependent DNA damage was observed after a 3h treatment of differentiated HepaRG cells with 1 to 5µM aflatoxin B1, in the absence of any cell damage and this DNA damaging effect was strongly reduced in the presence of ketoconazole, a CYP3A4 inhibitor. These results bring the first demonstration of long-term stable expression of liver-specific markers in HepaRG hepatocyte cultures maintained at confluence and show that these cells represent a suitable in vitro liver cell model for analysis of acute and chronic toxicity as well as genotoxicity of chemicals in human liver. This article has not been copyedited and formatted. The final version may differ from this version.
British Journal of Pharmacology, 1999
The effects of tacrine (THA) on intracellular pH (pHi) were examined in a rat liver biliary epith... more The effects of tacrine (THA) on intracellular pH (pHi) were examined in a rat liver biliary epithelial cell line (RLEC) in HEPES‐buffered medium. pHi was recorded using the pH‐sensitive fluoroprobe, carboxy‐SNARF‐1 (carboxy‐seminaphtorhodafluor). In the steady state, short‐term exposures to THA resulted in alkalinization and re‐acidification at 0.1 and 0.25 mM. Following a 24 h‐treatment, no significant difference in pHi could be detected at 0.1 and 0.25 mM THA, whereas at 0.05 mM, pHi was slightly more acid (7.17±0.02, n=16 versus 7.21±0.02, n=24 [control]). In control and short‐term treated cells, intracellular intrinsic buffering power (βi) increased roughly linearly as pHi decreased. This dependence was not seen following long‐term treatment. In all cases, βi was increased by THA (by 1.6 to 3.5 fold). Following an acid load (induced by 20 mM NH4Cl removal), pHi recovery in RLEC relied upon Na+/H+ exchange. A short‐term treatment (0.25 mM THA) did not affect total acid extrusion....
Impact of inflammation on chlorpromazine-induced cytotoxicity and cholestatic features
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Papers by André Guillouzo