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Institut National de la Santé et de la Recherche Médicale U620; Université de Rennes 1, Rennes, France (R.J., C.A., J.D., F.M., A.G.); Institut National de la Santé et de la Recherche Médicale U522, Hôpital Pontchaillou, Rennes, France (D.G., C.G.-G.); and Agence Française de Sécurité Sanitaire des Aliments, Laboratoire d'Etudes et de Recherches sur les Médicaments Vétérinaires et les Désinfectants, Fougères, France (V.F., J.-M.P.)
(Received November 28, 2007; accepted March 13, 2008)
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). It is also a major cause of attrition in drug development, indicating that preclinical evaluation of new drugs is of critical importance. A number of drugs and other xenobiotics are potentially hepatotoxic, either directly or more frequently after bioactivation, leading to the formation of chemically reactive metabolites or generation of reactive oxygen species. However, it must be borne in mind that around half of the drugs found to be responsible for liver injury during clinical trials did not cause any liver damage in animal experiments (Olson et al., 2000). Similarly, although they are known to be hepatocarcinogens in animals, especially in rodents, a number of chemicals, with few exceptions such as aflatoxin B1 (AFB1), are only suspected to induce liver tumors in humans. Indeed, their carcinogenic activity is usually based on epidemiological studies showing a higher frequency of cancers with occupational exposure or certain alimentary habits. Such species differences can be explained, at least in many cases, by the frequent major variations in the routes and the rates of chemical metabolism between laboratory animals and humans. Xenobiotic biotransformation is also one of the major determinants of genotoxic effects of chemicals. In in vitro mutagenicity assays, tested compounds are routinely bioactivated using exogenous rodent subcellular systems (generally the liver S9 fraction) containing drug-metabolizing enzymes (Paolini et al., 1997). However, doubts persist regarding the real validity of these in vitro models in representing both in vivo conditions and species differences.
Obviously, there is a need for more accurate prediction of chemical hepatic cytotoxicity and genotoxicity in humans. In vitro human liver preparations appear to be the most pertinent models; they include both cellular (tissue slices, suspensions and primary cultures of hepatocytes, hepatic cell lines) and subcellular (liver microsomes, recombinant enzymes) systems (Guillouzo, 1998). Only living cells are potentially able to express the whole metabolic equipment and consequently to mimic the diverse mechanisms of toxicity occurring in the liver. Primary human hepatocytes and human immortalized liver cell lines have been widely used but both models have limitations (Guillouzo, 1998). Indeed, primary hepatocytes have scarce and unpredictable availability, limited growth activity and life span, and undergo early and variable changes in various functions, including the major cytochromes P450 (P450) involved in drug metabolism. However, this model remains the gold standard model for xenobiotic metabolism and toxicity studies (Guillouzo, 1998; Hewitt et al., 2007). Indeed, liver cell lines obtained either from tumors or by oncogenic immortalization lack a variable and substantial set of liver-specific functions, making them unsuitable for mimicking in vivo hepatocytes (Sassa et al., 1987; Wilkening et al., 2003; Majer et al., 2004). In particular, they exhibit only quite low, if any, P450 activities. However, we have recently reported that a new human hepatoma cell line, named HepaRG, has retained the major P450 activities, as well as various liver-specific functions, at levels comparable with those found in primary human hepatocytes (Aninat et al., 2006, 2008).
Various mechanisms can contribute to drug-induced liver injury, and only a few compounds induce hepatotoxicity within a few days. Moreover, most drugs cause liver injury infrequently, and only a few exert dose-dependent effects (Lee, 2003). In most cases, liver injury is related to long-term treatments, thereby supporting the idea that there is a need to predict not only acute toxicity and genotoxic effects but also chronic toxicity due to repeated exposures to low doses or even a mixture of compounds in human liver (Guillouzo et al., 2006). This led us to question as to whether HepaRG cells could remain functionally stable at confluence for several weeks or more and consequently could represent a suitable tool for such purposes. We demonstrate that differentiated HepaRG cells retained various liver-specific markers at relatively stable levels for several weeks at confluence and that AFB1, a potent hepatotoxicant and carcinogen, induced in these cells dose-dependent and cumulative cytotoxicity as well as DNA damage as evidenced by the Comet assay.
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Cell Cultures. HepaRG cells were obtained from a liver tumor of a female patient suffering from hepatocarcinoma and cultured as previously described (Gripon et al., 2002). Briefly, liver pieces were digested with 0.025% collagenase D in Hepes buffer supplemented with 0.075% CaCl2, and the resulting cell population was suspended in Williams' E medium and then maintained in culture by serial passages. The cells reached maximum differentiation when maintained at confluency for 2 weeks in the presence of 2% DMSO and 5x 10–5 M hydrocortisone hemisuccinate. For the present studies, HepaRG cells were seeded at a density of 2.6 x 104 cells/cm2 in the growth medium composed of Williams' E medium supplemented with 10% FCS, 100 units/ml penicillin, 100 µg/ml streptomycin, 5 µg/ml insulin, 2 mM glutamine, and 5 x 10–5 M hydrocortisone hemisuccinate. At such a low-density seeding, HepaRG cells took a morphology of elongated undifferentiated cells characterized by a clear cytoplasm and actively divided until they reached confluence. After 2 weeks, they were shifted to the same culture medium supplemented with 2% DMSO for 2 more weeks to obtain differentiated HepaRG cell cultures (Aninat et al., 2006; Cerec et al., 2007).
Human hepatocytes from adult donors undergoing resection for primary and secondary tumors were obtained by collagenase perfusion of histologically normal liver fragments (Guguen-Guillouzo et al., 1982) and were used for the preparation of RNA samples and primary cultures. Hepatocyte cultures were obtained by seeding 170,000 cells/cm2 in 96-well plates in a Williams' E medium supplemented with 10% FCS, 100 units/µl penicillin, 100 µg/ml streptomycin, 1 µg/ml insulin, 2 mM glutamine, and 1 µg/ml bovine serum albumin. The medium was discarded 12 h after seeding, and cells were thereafter maintained in serum-free medium supplemented with 5 x 10–5 M hydrocortisone hemisuccinate. The medium was renewed every day. Human hepatocytes were used 24 h after seeding.
HepG2 cells obtained from the American Type Culture Collection (HB-8065; American Type Culture Collection (Manassas, VA) were passaged using a split ratio of 1:3 at 5-day intervals and were used at the time of confluency. The growth medium was composed of minimum essential medium 
supplemented with 10% FCS, nonessential amino acids, 100 units/ml penicillin, and 100 µg/ml streptomycin. All experimental procedures complied with French laws and were approved by the National Ethics Committee.
Isolation of RNA and RT-qPCR Analysis. Total RNA was extracted from 106 HepaRG cells or 106 human hepatocytes with the SV total RNA isolation system (Promega, Madison, WI), which directly included a DNase treatment step. RNAs were reversed-transcribed into cDNA using the High-Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA). Real-time quantitative PCR was performed by the fluorescent dye SYBR Green methodology using the SYBR Green PCR Master Mix (Applied Biosystems) and the ABI Prism 7000 (Applied Biosystems). Table 1 shows primer pairs for each transcript chosen with Primers 3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). The amplification curves were read with the ABI Prism 7000 SDS Software using the comparative cycle threshold method. The relative quantification of the steady-state mRNA levels was calculated after normalization of the total amount of cDNA tested by an active reference, 18S RNA. Furthermore, a dissociation curve was performed after the PCR reaction to verify the specificity of the amplification. Results were expressed as percentages of mRNA levels measured at day 30 after seeding, arbitrarily set at 100%. For induction studies, HepaRG cells were first exposed to either 50 µM rifampicin or 5 µM 3-MC for 24 h before cell harvesting for RNA extraction.
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Determination of Drug-Metabolizing Enzyme Activities. For the determination of CYP3A4- and 1A2-related activities, HepaRG cells were incubated with 200 µM testosterone for 2 h and 200 µM phenacetin for 20 h, respectively, in phenol red-free medium deprived of both FCS and DMSO. For induction studies, the cells were first exposed to either 50 µM rifampicin or 5 µM 3-MC for 48 h before incubation with the specific substrates. Culture media were then collected and centrifuged for 2 min at 10,000 rpm; 50 µlof sample was injected in the high-performance liquid chromatography system for quantification of the specific metabolites, 6β-hydroxytestosterone and acetaminophen, resulting from 6β-hydroxylation of testosterone and phenacetin deethylation activities, respectively. A nucleosil C18 column (150 x 4.6 mm, 5 µm) was used for chromatographic separation of testosterone metabolites at 254 nm. The mobile phase consisted of two solvents, A (0.1% acetic acid) and B (acetonitrile), with the following gradient: 0 min, 20% B; and 20 min, 33% B. Chromatographic separation of phenacetin metabolites was carried out with an Interchrom C18 column (250 x 4.0 mm, 5 µm) at 250 nm. The mobile phase consisted of two solvents, A (2 mM ammonium acetate, pH 2.6) and B (acetonitrile) with a linear gradient (20 min, 3% B).
The high-performance liquid chromatography apparatus consisted of an Agilent 1100 Series high-performance liquid chromatograph equipped with an autosampler and Agilent 1100 Series fluorescence and UV detectors (Agilent Technologies, Palo Alto, CA). A computer running Agilent 1100 software (ChemStation) was used to integrate and calculate the separated peak area and to plot metabolite patterns. Metabolites were identified by comparison of retention times, and quantifications were estimated from calibration.
Evaluation of AFB1 Cytotoxicity. The effects of varying concentrations of AFB1 were evaluated on primary human hepatocytes, HepaRG cells, and HepG2 cells after various times of exposure. Incubations were performed with a medium supplemented with both 2% DMSO and 10% FCS. For long time treatments of HepaRG cells, the mycotoxin was added every 2 days with each medium renewal. The cultures were examined every day by phase-contrast microscopy using an Olympus 1 x 70 (Olympus, Tokyo, Japan). Cytotoxicity was estimated by intracellular ATP measurements using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). The test was performed according to the manufacturer's instructions. ATP concentrations were expressed as chemoluminescence units and converted into percentages by the ratio of the values measured in treated and corresponding control samples. In addition, functional capacity of remaining living hepatocytes was selectively evaluated by determination of CYP3A4 activity at different time points.
Comet Assay. Undifferentiated (5 days after seeding) and differentiated (30 days after seeding) HepaRG cells were exposed to 1, 2.5, or 5 µM AFB1 dissolved in DMSO (final concentration, 0.2%) or 5 µg/ml MMS (positive control) in an FCS-free medium for 3 h. Then, they were trypsinized, centrifuged (2 min, 1000 rpm), and cell pellets were suspended in a prewarmed low-melting point agarose (0.5% in PBS) and deposited on conventional microscope slides (initially dipped in 1% agarose and dried) precoated with normal agarose (0.8% in PBS). The slides were then put in a lysis solution composed of 2.5 M NaCl, 0.1 M EDTA, and 10 mM Tris-HCl, pH 10, extemporarily added with 10% DMSO and 1% Triton X-100, for 1 h at around 5°C. DNA was allowed to unwind for 40 min in fresh electrophoresis buffer (0.3 M NaOH, 1 mM EDTA, pH 13), and the electrophoretic migration was then performed at room temperature (24 min, 0.7 V/cm, 300 mA). The slides were bathed twice for 5 min in neutralizing solution (0.4 M Tris-HCl, pH 7.5) and dried for conservation with 95% ethanol for 5 min. DNA was stained with propidium iodide (2.5 µg/ml in PBS) just before slide examination with a fluorescence microscope (Leica DMR; Leica, Wetzlar, Germany) equipped with a CCD-200E video camera. At least 100 images per dose and treatment time were analyzed using the comet assay IV software (Perceptive Instruments, Haverhill, UK). Two parameters, the Olive tail moment (OTM), defined as the product of the distance between the barycenters of the head and tail by the proportion of DNA in the tail, and the percentage of DNA in the comet tail (percentage DNA) were used to evaluate the extent of DNA damage in individual cells.
Statistical Analysis. Each value corresponded to the mean ± S.E.M. of three independent experiments. The Kruskal-Wallis nonparametric test was used to compare mRNA levels and P450 activities between different time points. The Mann-Whitney U test was applied to compare cytotoxicity between treated and corresponding control samples and CYP3A4 activity in cultures treated with 0.5 or 1 µM compared with 0.1 µM AFB1 for 24 h as well as in cultures treated for 7 or 14 days compared with 1 day. For the Comet assay, DNA damage was expressed as the median OTM or percent DNA in the tail of 100 cells. Comparisons between cell cultures submitted to different treatment conditions were made using analysis of variance followed by pair-wise comparison using the Tukey's test. Data were considered significantly different when p < 0.05.
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) were in the range of the levels measured in 1- to 3-day primary human hepatocyte cultures.
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Drug-Metabolizing Enzyme Activities. 6β-Hydroxylation of testosterone and deethylation of phenacetin activities catalyzed by CYP3A4 and CYP1A2, respectively, were also analyzed at different time points and as observed for the corresponding transcripts were found to be relatively stable over the 4-week studied period. Whatever the time point considered, the values were not statistically different (Fig. 2). For 6β-hydroxylation of testosterone, they ranged between 600 and 800 pmol/min/mg proteins, being similar to those previously reported in HepaRG cells and comparable with those usually found in primary human hepatocyte cultures (Aninat et al., 2006). Similarly, during the whole confluent period studied, HepaRG cells retained their responsiveness to rifampicin and 3-MC (Fig. 3). At each time point analyzed, at least a 2-fold induction was observed with either inducer.
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Dose-Dependent and Cumulative Cytotoxic Effects of AFB1. In a first series of experiments, 24-h primary human hepatocytes, differentiated HepaRG cells, and confluent HepG2 cells were exposed to varying concentrations of AFB1 for 3, 24, and 72 h. Cytotoxic effects were estimated by measurement of intracellular ATP content. As shown in Fig. 4, AFB1 did not cause any damage to the 3 cell types after a 3-h treatment, whereas after 24 h, cytotoxicity was evidenced with 5 and even 1 µM in both primary human hepatocytes and HepaRG cells. The effects were much more pronounced after 72 h. However, the values of intracellular ATP did not drop under 60%; this was explained by the fact that, as evidenced by light microscopic examination, morphological alterations of HepaRG cells were restricted to hepatocyte-like cells and were characterized by cell rounding and cytoplasmic granular appearance (Fig. 1, B–F). An exception was with the highest mycotoxin concentration of 5 µM, which caused both a stronger decrease in ATP content and some damage to biliary-like cells (Fig. 1B). Furthermore, variations in the sensitivity to the mycotoxin were noticed among the three exposed human hepatocyte populations, probably related to differences in the levels of the enzymes involved in its metabolism. No damage was observed in HepG2 cells over the 72-h exposure, even with 5 µM.
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In another series of experiments, differentiated HepaRG cells were exposed to 0.01, 0.1, and 0.5 µM AFB1 for up to 14 days. The mycotoxin was added every 2 days with each medium renewal. Morphological changes and limited decrease in both intracellular ATP content and CYP3A4 activity occurred after 7 days with 0.1 µM AFB1 (Figs. 1D and 5). No morphological changes were visualized in biliary-like cells after a 7-day exposure to AFB1 at concentrations of 0.01 µM (Fig. 1F). Consistent with a cumulative cytotoxic effect of AFB1, CYP3A4 activity was significantly decreased after 14 days with 0.1 µM(p < 0.001 compared with day 1).
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Genotoxic Effects of AFB1 Identified by the Comet Assay. A positive relationship was obtained between the extent of DNA damage, estimated by the OTM or the percent DNA in the tail, and AFB1 concentrations after a 3-h exposure of differentiated HepaRG cells to the mycotoxin (Fig. 6). A 9- and 4-fold increase in OTM and percentage DNA in the tail, respectively, was recorded in 5 µM AFB1-treated versus corresponding untreated cultures. DNA damage induced by the mycotoxin occurred in the absence of increased cytotoxicity estimated by intracellular ATP content (Fig. 4) and trypan blue exclusion and annexin V tests (data not shown) when compared with untreated controls. In contrast, undifferentiated HepaRG cells that did not express CYP3A4 and CYP1A2 were not sensitive to AFB1. A 1-h prior and concomitant treatment of the cells with 10 µM ketoconazole, a CYP3A4 inhibitor, was associated with a statistically significant reduction of DNA damage in 5 µM AFB1-treated cultures. No effect was evidenced with 10 µM ketoconazole alone (Table 3). As expected, MMS, a direct alkylating compound, was highly genotoxic to both undifferentiated and differentiated HepaRG cells, being slightly more DNA damaging in the latter (Fig. 6).
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; Le Vee et al., 2006) can be further maintained functionally stable for several weeks and used for chronic toxicity studies using AFB1 as a reference hepatotoxic compound. No obvious morphological changes occurred in HepaRG cell cultures during such a long-term confluence period. The cells still remained able to transdifferentiate when seeded at low density and to sustain typical cultures composed of both hepatocyte- and biliary-like cells, each representing around 50% of the total cell population (Cerec et al., 2007) (data not shown). It may be therefore assumed that the 4-week functional stability of differentiated HepaRG cells at confluence can certainly be greatly extended.
Transcripts of genes involved in various pathways of xenobiotic metabolism and/or recognized as liver-specific markers were analyzed in differentiated HepaRG cells by RT-qPCR at varying time points over a 4-week period. Compared with the levels measured in freshly isolated hepatocytes those encoding liver-specific functions were lower and more comparable with the corresponding ones quantified in 1- to 3-day pure human hepatocyte cultures; they included aldolase B, a marker of adult hepatocytes, CYP3A4, the nuclear receptor CAR, as well as the transporters OCT1 and BSEP. OCT1 is located in the basolateral membrane of the hepatocyte and transports diverse organic cations including toxins and endogenous compounds (Jonker et al., 2004), whereas BSEP plays a central role in bile acid secretion and bile flow (Alrefai et al., 2007). However, it must be emphasized that hepatocyte-like HepaRG cells represented only around 50% of the total cell population, thereby meaning that the levels of liver-specific transcripts should be nearly doubled to be correctly compared with those measured in pure freshly isolated hepatocyte populations. In contrast, other genes were expressed at levels much closer to those found in freshly isolated hepatocytes; they include MnSOD and catalase, two enzymes involved in cellular defense against toxic oxygen species during endogenous metabolism and biotransformation of xenobiotics. In agreement with gene transcription, stability of enzyme activities and their responsiveness to prototypical inducers were also well maintained during the 4-week period as evidenced with CYP3A4 and CYP1A2 activities and their induction by rifampicin and 3-MC, respectively.
As stressed for their corresponding transcripts, CYP3A4 and CYP1A2 activities would be more elevated by considering only hepatocyte-like cells in HepaRG cell cultures. On this basis, when compared with the levels determined in primary human hepatocyte cultures, those expressed in HepaRG cells would even be higher for CYP3A4 (Aninat et al., 2006) and close to the lowest values for CYP1A2. Indeed, as previously reported (Guillouzo, 1998), CYP1A2 activity, using phenacetin as a substrate, greatly varied in primary human hepatocytes from different donors (0.1 to 2.5 nmol/min/mg protein, n = 25, after 18–24 h of culture). The -fold induction with prototypical inducers was also in agreement with previously reported results from primary human hepatocyte cultures (Diaz et al., 1990; Morel et al., 1990). However, it is quite important to keep in mind that a -fold induction is related not only to the inducer and its concentration but also to the substrate and the basal activity; the lower the basal activity, the higher the induction level. Indeed, the results are not the same when ethoxyresorufin is used instead of phenacetin as a substrate for CYP1A2 determinations. Activities of CYP1A are quite low with ethoxyresorufin; consequently, the -fold change after induction is quite high (Diaz et al., 1990; Aninat et al., 2006). In contrast, when phenacetin is used as a substrate, basal activity is higher, and -fold induction is quite lower, the extent of CYP1A2 induction being only around 2- to 3-fold in both primary hepatocytes and HepaRG cells. This is supported by in vivo observations; after a 4-day treatment with omeprazole, the levels of CYP1A2 were increased by around 2- to 3-fold, except in one patient having a low basal activity who showed a 15-fold induction (Diaz et al., 1990). The same conclusion could be drawn from previously reported data for CYP3A4 in HepaRG cells (Aninat et al., 2006).
AFB1 is a reference hepatotoxic compound, acting mainly by binding to DNA and proteins through its exo-8,9 epoxide metabolite formed mainly by CYP3A4 (Guengerich et al., 1998). We previously found that differentiated HepaRG cells were much more sensitive to AFB1 than HepG2 cells that do not express both CYP3A4 and CYP1A2 (Aninat et al., 2006). Our present results confirm this observation and show that HepaRG cells are as sensitive as primary human hepatocytes to this mycotoxin, on the basis of cytotoxicity evaluation by determination of intracellular ATP content that has been reported to be more relevant than classical viability tests. However, comparable results were observed by using the neutral red assay (data not shown). Assuming that cytotoxicity induced by the mycotoxin is concentration-dependent and cumulative, differentiated HepaRG cells were exposed every 2 days for 14 days to varying concentrations of AFB1. As expected, in addition to a concentration-dependent toxicity following a unique exposure, a cumulative toxicity was observed affecting primarily hepatocyte-like cells. A concentration as low as 0.1 µM caused a decrease in CYP3A4 activity starting after 1 week of treatment, i.e., corresponding to three additions of the mycotoxin. The cytotoxic effects were amplified after 14 days of treatment. These observations support the idea that hepatocyte-like cells mixed with biliary-like cells that do not express the major cytochromes P450 represent an appropriate model to identify compounds that require oxidative metabolism to be hepatotoxic. More precise characterization of phenotypic changes in the two cell populations could be conducted by their selective enzymatic detachment prior to functional analyses.
In vitro genotoxic effects of AFB1 were studied using the single-cell gel electrophoresis (comet) assay, which is a sensitive method for the detection of DNA damage and repair induced by genotoxic compounds in individual cells and is extensively used in genotoxicity studies. The alkaline version, used in this study, detects single- and double-strand DNA breaks as well as abasic sites (Collins, 2004; Witte et al., 2007). The exo-8,9 metabolite of AFB1 binds to the N-7 position of guanine residues in DNA and can depurinate to give an abasic site or to be repaired by the nucleotide excision repair pathway (Bedard et al., 2006). Using the comet assay, dose-dependent DNA damage induced by AFB1 was demonstrated in differentiated HepaRG cells in the absence of any sign of cytotoxicity, indicating that DNA alterations evidenced by this test were specific of AFB1-induced genotoxic lesions. In agreement with a genotoxic effect related to the epoxide, DNA alterations were not observed in AFB1-treated undifferentiated cultures that do not express CYP3A4 and were greatly decreased in cells treated with the CYP3A inhibitor ketoconazole. Interestingly, similar dose-dependent effects of AFB1 were obtained with differentiated HepaRG cells using the micronucleus assay (unpublished observations). However, DNA-damaging effects of AFB1 were much lower than those caused by the alkylating agent MMS. These differences might have several nonexclusive explanations; indeed, it is likely that during a 3-h exposition period, AFB1 metabolism and formation of DNA-reactive metabolites were not complete and that a large proportion of the epoxides was inactivated by epoxide hydratase and possibly GST enzymes. Such a difference between direct and indirect genotoxic agents has already been observed in the comet assay with other human cells (Uhl et al., 1999). Moreover, although both cell types were damaged by MMS, only hepatocyte-like cells were likely to be sensitive to the mycotoxin. Further experiments will allow the definition of the lowest AFB1 concentration inducing detectable DNA lesions under well defined experimental conditions that could be further applied for investigating specific DNA lesions induced by a wide variety of xenobiotics.
Taken together, our results clearly demonstrate that HepaRG cells not only can fully differentiate but in addition have the unique property to retain their differentiation state for weeks at confluence. We bring here the proof of concept that metabolically competent HepaRG cells represent a suitable model to study chronic toxicity and genotoxicity of chemicals in human liver and to identify early and late target genes of any potential hepatotoxic compound.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS: AFB1, aflatoxin B1; P450, cytochrome P450; 3-MC, 3-methylcholanthrene; DMSO, dimethylsulfoxide; MMS, methyl methane sulfonate; FCS, fetal calf serum; RT-qPCR, reverse transcriptase-quantitative polymerase chain reaction; PBS, phosphate-buffered saline; OTM, Olive tail moment; UGT, UDP-glucuronosyl transferase; GST, glutathione transferase; mEH, microsomal epoxide hydrolase; MDR, multidrug resistance protein; MRP, multidrug resistance-associated protein; OCT, organic cation transporter; BSEP, bile salt export pump; PXR, pregnane X receptor; CAR, constitutive androstane receptor; MnSOD, manganese superoxide dismutase.
Address correspondence to: Guillouzo André, Institut National de la Santéet de la Recherche Médicale U620, Détoxication et Réparation Tissulaire, Faculté des Sciences Pharmaceutiques et Biologiques, Université de Rennes I, 35043 Rennes cedex, France. E-mail: andre.guillouzo{at}univ-rennes1.fr
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