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Within the EuroMix project, we have previously developed an adverse outcome pathway (AOP)-based in vitro assay toolbox to investigate the combined effects of liver steatosis-inducing compounds in human HepaRG hepatocarcinoma cells. In this study, we applied the toolbox to further investigate mixture effects of combinations, featuring either similarly acting or dissimilarly acting substances. The valproic acid structural analogs 2-propylheptanoic acid (PHP) and 2-propylhexanoic acid (PHX) were chosen for establishing mixtures of similarly acting substances, while a combination with the pesticidal active substance clothianidin (CTD) was chosen for establishing mixtures of dissimilarly acting compounds. We first determined relative potency factors (RPFs) for each compound based on triglyceride accumulation results. Thereafter, equipotent mixtures were tested for nuclear receptor activation in transfected HepG2 cells, while gene expression and triglyceride accumulation were investigated in HepaRG cells, following the proposed AOP for liver steatosis. Dose addition was observed for all combinations and endpoints tested, indicating the validity of the additivity assumption also in the case of the tested mixtures of dissimilarly acting substances. Gene expression results indicate that the existing steatosis AOP can still be refined with respect to the early key event (KE) of gene expression, in order to reflect the diversity of molecular mechanisms underlying the adverse outcome. Supplementary Information The online version contains supplementary material available at 10.1007/s00204-021-03182-1.

Spodoptera frugiperda, a highly polyphagous insect pest from America, has recently invaded and widely spread throughout Africa and Asia. Effective and environmentally safe tools are needed for successful pest management of this invasive species. Natural molecules extracted from plants offer this possibility. Our study aimed to determine the insecticidal efficacy of a new molecule extracted from Alpinia galanga rhizome, the 1&prime, S-1&prime, acetoxychavicol acetate (ACA). The toxicity of ACA was assessed by topical application on early third-instar larvae of S. frugiperda. Results showed that ACA caused significant larval growth inhibition and larval developmental abnormalities. In order to further explore the effects of this molecule, experiments have been performed at the cellular level using Sf9 model cells. ACA exhibited higher toxicity on Sf9 cells as compared to azadirachtin and was 38-fold less toxic on HepG2 cells. Inhibition of cell proliferation was observed at sublethal concentrations of ACA and was associated with cellular morphological changes and nuclear condensation. In addition, ACA induced caspase-3 activity. RT-qPCR experiments reveal that ACA induces the expression of several caspase genes. This first study on the effects of ACA on S. frugiperda larvae and cells provides evidence that ACA may have potential as a botanical insecticide for the control of S. frugiperda.

International audience; Physiologically-based toxicokinetic (PBTK) models are important tools for in vitro to in vivo or inter-species extrapolations in health risk assessment of foodborne and non-foodborne chemicals. Here we present a generic PBTK model implemented in the EuroMix toolbox, MCRA 9 and predict internal kinetics of nine chemicals (three endocrine disrupters, three liver steatosis inducers, and three developmental toxicants), in data-rich and data-poor conditions, when increasingly complex levels of parametrization are applied. At the first stage, only QSAR models were used to determine substance-specific parameters, then some parameter values were refined by estimates from substance-specific or high-throughput in vitro experiments. At the last stage, elimination or absorption parameters were calibrated based on available in vivo kinetic data. The results illustrate that parametrization plays a capital role in the output of the PBTK model, as it can change how chemicals are prioritized based on internal concentration factors. In data-poor situations, estimates can be far from observed values. In many cases of chronic exposure, the PBTK model can be summarized by an external to internal dose factor, and interspecies concentration factors can be used to perform interspecies extrapolation. We finally discuss the implementation and use of the model in the MCRA risk assessment platform.

Exposure to complex chemical mixtures requires a tiered strategy for efficient mixture risk assessment. As a part of the EuroMix project we developed an adverse outcome pathway (AOP)-based assay toolbox to investigate the combined effects of the liver steatosis-inducing compounds imazalil, thiacloprid, and clothianidin in human HepaRG hepatocarcinoma cells. Compound-specific relative potency factors were determined using a benchmark dose approach. Equipotent mixtures were tested for nuclear receptor activation, gene and protein expression, and triglyceride accumulation, according to the molecular initiating events and key events proposed in the steatosis AOP. All three compounds affected the activity of nuclear receptors, but not key genes/proteins as proposed. Triglyceride accumulation was observed with three different methods. Mixture effects were in agreement with the assumption of dose additivity for all the combinations and endpoints tested. Compound-specific RPFs remained similar over the different endpoints studied downstream the AOP. Therefore, it might be possible to reduce testing to a smaller battery of key tests. The results demonstrate the suitability of our in vitro assay toolbox, integrated within an AOP framework and combined with the RPF approach, for the analysis of steatotic effects of chemical mixtures. However, mRNA results suggest that the steatosis AOP still needs improvement.

Adverse outcome pathways (AOPs) describe causal relationships between molecular perturbation and adverse cellular effects and are being increasingly adopted for linking in vitro mechanistic toxicology to in vivo data from regulatory toxicity studies. In this work, a case study was performed by developing a bioassay toolbox to assess key events in the recently proposed AOP for chemically induced liver steatosis. The toolbox is comprised of in vitro assays to measure nuclear receptor activation, gene and protein expression, lipid accumulation, mitochondrial respiration, and formation of fatty liver cells. Assay evaluation was performed in human HepaRG hepatocarcinoma cells exposed to the model compound cyproconazole, a fungicide inducing steatosis in rodents. Cyproconazole dose-dependently activated RARα and PXR, two molecular initiating events in the steatosis AOP. Moreover, cyproconazole provoked a disruption of mitochondrial functions and induced triglyceride accumulation and the formation of fatty liver cells as described in the AOP. Gene and protein expression analysis, however, showed expression changes different from those proposed in the AOP, thus suggesting that the current version of the AOP might not fully reflect the complex mechanisms linking nuclear receptor activation and liver steatosis. Our study shows that cyproconazole induces steatosis in human liver cells in vitro and demonstrates the utility of systems-based approaches in the mechanistic assessment of molecular and cellular key events in an AOP. AOP-driven in vitro testing as demonstrated can further improve existing AOPs, provide insight regarding molecular mechanisms of toxicity, and inform predictive risk assessment

International audience; Tea is an interesting source of antioxidants capable of counteracting the oxidative stress implicated in liver diseases. We investigated the impact of antioxidant molecules provided by a mixture of teas' leaves (green, oolong, pu-erh) after different infusion durations in the prevention of oxidative stress in isolated rat hepatocytes, by comparison with pure epigallocatechin-3-gallate (EGCG), the main representative of tea catechins. Dried aqueous tea extracts (ATE) obtained after 5, 15 and 30 min infusion time were characterized for total polyphenols (gallic acid equivalent), catechins, gallic acid and caffeine (HPLC-DAD/ESI-MS) contents, and for scavenging ability against 2,2-diphenyl-1-picrylhydrazyl free radical. Hepatoprotection was evaluated through hepatocyte viability tests using tert-butyl hydroperoxide as a stress inducer, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, neutral red uptake, real-time cellular impedance) and mitochondrial function tests. We showed that a 5-min incubation time is sufficient for an optimal bioaccessibility of tea compounds with the highest antioxidative ability, which decreases for longer durations. A 4-h pretreatment of cells with ATE significantly prevented cell death by regulating reactive oxygen species production and maintaining mitochondrial integrity. Pure EGCG, at doses similar in ATE (5-12 µM), was inefficient, suggesting a plausible synergy of several water-soluble tea compounds to explain the ATE beneficial effects.

General population exposure to pesticides mainly occurs via food and water consumption. However, their risk assessment for regulatory purposes does not currently consider the actual co-exposure to multiple substances. To address this concern, relevant experimental studies are needed to fill the lack of data concerning effects of mixture on human health. For the first time, the present work evaluated on human microsomes and liver cells the combined metabolic effects of, chlorfenvinphos, ethion and linuron, three pesticides usually found in vegetables of the European Union. Concentrations of these substances were measured during combined incubation experiments, thanks to a new analytical methodology previously developed. The collected data allowed for calculation and comparison of the intrinsic hepatic clearance of each pesticide from different combinations. Finally, the results showed clear inhibitory effects, depending on the association of the chemicals at stake. The major metabolic inhibitor observed was chlorfenvinphos. During co-incubation, it was able to decrease the intrinsic clearance of both linuron and ethion. These latter also showed a potential for metabolic inhibition mainly cytochrome P450-mediated in all cases. Here we demonstrated that human detoxification from a pesticide may be severely hampered in case of co-occurrence of other pesticides, as it is the case for drugs interactions, thus increasing the risk of adverse health effects. These results could contribute to improve the current challenging risk assessment of human and animal dietary to environmental chemical mixtures.

International audience; French consumers are exposed to mixtures of pesticide residues in part through food consumption. As a xenosensor, the pregnane X receptor (hPXR) is activated by numerous pesticides, the combined effect of which is currently unknown. We examined the activation of hPXR by seven pesticide mixtures most likely found in the French diet and their individual components. The mixture's effect was estimated using the concentration addition (CA) model. PXR transactivation was measured by monitoring luciferase activity in hPXR/HepG2 cells and CYP3A4 expression in human hepatocytes. The three mixtures with the highest potency were evaluated using the CA model, at equimolar concentrations and at their relative proportion in the diet. The seven mixtures significantly activated hPXR and induced the expression of CYP3A4 in human hepatocytes. Of the 14 pesticides which constitute the three most active mixtures, four were found to be strong hPXR agonists, four medium, and six weak. Depending on the mixture and pesticide proportions, additive, greater than additive or less than additive effects between compounds were demonstrated. Predictions of the combined effects were obtained with both real-life and equimolar proportions at low concentrations. Pesticides act mostly additively to activate hPXR, when present in a mixture. Modulation of hPXR activation and its target genes induction may represent a risk factor contributing to exacerbate the physiological response of the hPXR signaling pathways and to explain some adverse effects in humans.

International audience; Chronic exposure to low doses of pesticides present in the environment is increasingly suspected to cause major health issues to humans. Toxicological evaluations become more complex when the exposure concerns chemical combinations. Atrazine, chlorpyrifos, and endosulfan are pesticides used worldwide in agriculture and are therefore currently found at residual levels in food and the environment, even in countries in which they are now banned. Our study aimed to use Real-Time Cell Impedance Analyzer to investigate changes in phenotypical status of primary human hepatocytes and differentiated HepaRG cells induced by short and chronic exposures to these three chemicals. In contrast to the traditionally used endpoint cytotoxicity test, this technology allows kinetic measurements in real-time throughout the entire experiment. Our data show significantly higher cytotoxic effects of mixtures as compared to individual pesticides and a greater susceptibility of human hepatocytes as compared to HepaRG to short-term exposure (24 h). Repeated exposure over 2 weeks to endosulfan and endosulfan-containing mixture induced HepaRG cell death in a time- and dose-dependent manner. Of the typical genes involved in metabolism and cell-response to xenobiotics, we found an exposure time- and condition-dependent deregulation of the expression of CYP3A4 and UGT1A in HepaRG cells exposed to low doses of pesticides and mixtures. Our data demonstrate the usefulness of real-time cell monitoring in long-term toxicological evaluations of co-exposure to xenobiotics. In addition, they support but at the same time highlight certain limitations in the use of HepaRG cells as the gold standard liver cell model in toxicity studies.

International audience; Oxidative stress could trigger lipid accumulation in liver and thus hepatic steatosis. Tea is able to prevent liver disorders, but a direct link between antioxidant capacities and prevention of steatosis has not been reported yet. We aimed to investigate such relationship in a rat model of high fat-high sucrose diet (HFS)-induced obesity and to explore more deeply the mechanisms in isolated hepatocytes. Wistar rats were divided into a control group (standard diet), an HFS group (high fat-sucrose diet) and an HFS+tea group (HFS diet with ad-libitum access to tea drink). Body weight, fat mass, glycemic parameters in blood, lipid and oxidative stress parameters in blood and liver were measured in each group after 14 weeks. Isolated hepatocytes were treated with the reactive oxygen species (ROS) inducer t-BHP in the presence or not of antioxidants (tempol or tea), and superoxide anion production and lipid accumulation were measured using specific fluorescent probes. We reported that the HFS diet highly increased hepatic lipids content, while tea consumption attenuated steatosis and improved the oxidative status (decrease in hepatic oxidative stress, increase in plasma total antioxidant capacity). The role of antioxidant properties of tea in such phenomenon was confirmed in primary cultured rat hepatocytes. Indeed, the increase of mitochondrial ROS production with t-BHP resulted in lipid accumulation in hepatocytes (positive linear regression), and antioxidants (tempol or tea) normalized both. We reported that the antioxidant properties of tea protect rats from an obesogenic HFS diet-induced hepatic steatosis by counteracting the ROS-dependent lipogenesis.

Recent studies suggested that oxidative stress could trigger lipid accumulation in liver and thus subsequent development of hepatic steatosis which contributes to alter blood lipid level leading to increase cardiovascular risk factors. Tea has been described to prevent liver disorders and decrease cardiovascular risk. However, whether tea decreases oxidative stress and thus prevents hepatic steatosis has never been investigated. Therefore we aimed to investigate the effects of tea on oxidative stress and lipid accumulation in a rat model of high fatsucrose diet (HFS) induced metabolic syndrome (fed during 14 weeks) and in isolated rat hepatocytes.Wistar rats were randomly divided into three groups: Ctrl, HFS and HFS+Tea rats which had free access to tea infusion drink instead of water (n=15). Lipid profile of the liver, lipogenesis gene expression and oxidative stress were measured in each group of rat. Isolated hepatocytes were treated with the ROS inducer t-BHP in the presence or not of antioxidant tempol or tea. Then, superoxide anion production and lipid accumulation were measured using specific fluorescent probes. We reported that HFS diet-induced elevated hepatic lipid content by enhancing lipogenic gene expression in HFS rats compared to Ctrl ones. HFS diet-induced hepatic steatosis was attenuated by tea which was mainly associated with decrease hepatic oxidative stress and increased plasma total antioxidant capacity. The key role of antioxidant properties of tea in such phenomenon was confirmed in primary culture of rat hepatocytes. Indeed, we reported that increased ROS production with t-BHP resulted in lipid accumulation in hepatocytes, which was normalized by both tempol and tea. To conclude, we clearly reported that the antioxidant properties of tea protect rats from HFS diet-induced hepatic steatosis via its anti-lipogenesis effects. The consequence of such nutritional strategy on cardiovascular risk factor would constitute the next step of this work. The author declares a conflict of interest : Les travaux ont ete realises dans le cadre d’une bourse CIFRE Thes de la Pagode-Universite de Toulon

Background/Aims: Bile acids damage the liver, essentially by inducing hepatocyte apoptosis. Clinical studies have shown that several activators of the pregnane X receptor (PXR) may induce the remission of cholestasis. However, the molecular mechanisms involved in this beneficial effect remain unclear. We analysed the effect of an activator of PXR, clotrimazole (CLO), on the apoptosis induced by bile acids in primary cultures of rat hepatocytes. Methods: Rat hepatocytes were isolated by collagenase perfusion of the liver. Then, cells were pretreated with CLO for 24 h, after which they were exposed to deoxycholic and glycochenodeoxycholic acids (DCA, GCDCA). Apoptosis and necrosis were monitored morphologically and biochemically using cytotoxicity assays, phase-contrast microscopy, Annexin V/propidium iodide staining and evaluations of lactate dehydrogenase release. The activation of caspases and the proteolysis of their substrates were analysed by enzyme assays and Western blot. The signal transductions involved in the protective effect of the PXR activation were analysed by assessing the phosphorylation status of kinases belonging to the ERK, Akt and p38 pathways and by analysing pro- and anti-apoptotic proteins. Results: CLO protected rat hepatocytes against DCA- and GCDCA-induced apoptosis, preventing morphological aspects of this process (membrane blebbing, nuclear and chromatin condensation and DNA breakdown). This effect was attributable, at least partly, to caspases inhibition, Bcl-xL induction, the activation of ERK and Akt signalling and p38 inhibition. Conclusion: This study provides the description of the cytoprotective effect of PXR activation against bile acid-induced apoptosis and highlights molecular pathways that could be targeted in the treatment of cholestasis.

Lindane, a persistent organochlorine pesticide, is recognized as a major public health concern because of its potential toxic effects on human health. Despite observations pointing to the toxicity of lindane, mechanisms underlying its deleterious effects in liver have yet to be understood. In this study, we investigated the effects of lindane on autophagic, apoptotic and necrotic cell death in primary cultured rat hepatocytes. We found that lindane deregulated the autophagic process as demonstrated by (1) the formation of enlarged acidic vesicles labeled with LC3, Rab7 and LAMP1 (specific markers of autophagic vacuole maturation), (2) the conversion of LC3-I (the cytosolic form) into LC3-II (membrane bound), (3) the deregulation of the Beclin 1 protein expression and (4) the enhanced formation of the Bcl-xL/Beclin 1 complex. Lindane induced vacuolization together with the inhibition of spontaneous and intrinsic apoptosis. This disruption of cell suicide was linked to Bcl-xL up-regulation, Bax down-expression, prevention of cytochrome c release, and inhibition of caspase-9 and -3 activities. Lindane-induced disruption of apoptosis and autophagy occurred in parallel with necrosis induction in rat hepatocytes. In consequence, we proposed that lindane toxicity in primary rat hepatocytes could be jointly attributed to the disruption of autophagic process, the inhibition of apoptotic cell death and the induction of necrosis. These events account, at least in part, for the involvement of both cytotoxic and carcinogenic signaling pathways in the action of lindane in the liver.

Species differences and metabolism are the most crucial factors in considering the effects of genistein. The aim of this study was to have a better knowledge of the metabolic fate of genistein in humans as compared with rats. For this purpose, radiolabeled genistein was incubated with human and rat liver microsomes and with cryopreserved hepatocytes from both species. Incubations were performed using a wide range of genistein concentrations to analyze the kinetics of formation of the metabolites. Metabolite profiling was obtained using an HPLC system connected to a radioactivity detector. Identification of the metabolites was based on their retention times as compared with those of authentic standards and on LC-MS (ESI-MS/MS) or NMR analyses. In both species, liver microsomes produced the same three hydroxylated metabolites (8-OH, 6-OH and 3'-OH-genistein) whereas cryopreserved hepatocytes produced the same glucurono- and sulfo-conjugates (genistein 4'-O-sulfate 7-O-glucuronide, genistein 7-O-glucuronide, genistein 4'-O-glucuronide, genistein 7-O-sulfate and genistein 4'-O-sulfate). The rate of metabolism varied with species. 3'-Hydroxygenistein was the predominant metabolite produced by rat liver microsomes, whereas in humans 3'-hydroxy and 8-hydroxygenistein were produced in the same range. In both human and rat hepatocyte incubations, genistein 7-O-glucuronide represented more than 50% of the incubated dose. Our results on hepatocytes confirmed the predominance of conjugation reaction compared to oxidative reaction observed in vivo.

Oxidative stress is recognized to be implicated in the pathogenesis of cardiovascular and non-alcoholic fatty liver diseases. It is well know that tea is a rich source of phenolic compounds known for their antioxidant activity. Consequently, the antioxidant and protective effects of phenolic compounds from a pu-erh tea extract (PTE) was evaluated on primary culture of rat hepatocytes. PTE was quantified for its composition in catechins and polyphenol content by HPLC analysis. The antioxidant capacity of tea products was determined using TAC and DPPH assay methods. Then, antioxidant and hepatoprotective effects were determined by pretreating hepatocytes during 4h with various concentrations of PTE (25, 50 and 100μg/ml), epigallocatechin-3-gallate (EGCG) as major catechin of PTE (12μM corresponding to 100μg/ml PTE) and N-acetylcystein (NAC) (0.1 and 1mM) as an antioxidant reference. Then, cells were stressed for 1h with 150μM tert-Butyl hydroperoxide (TBHP). Viability was determined by real time cellular impedance and MTT assays. Oxidative stress was measured by CellRox, MitoSox and TMRE stainings and evaluated by fluorescence microscopy on an ArrayScanXTI high Content Analysis Reader (Cellomics Inc.). We found that TBHP induced oxidative stress (+1.5 fold increase vs control) was prevented by PTE pretreatment (+1.07 fold increase vs ctrl) and EGCG (+1.1 fold increase vs ctrl). We also demonstrated that PTE pretreatment protected rat hepatocytes (–28% mortality relative to TBHP) against TBHP induced mortality (+23% mortality relative to ctrl). However, EGCG did not prevented death in the same proportion than PTE (–9% mortality relative to TBHP). In this study, we reported that PTE pre-exposure prevented oxidative stress and mortality induced by TBHP. Moreover, we reported here that PTE has higher antioxidative and protective effects than EGCG alone, well known for its antioxidant effects, which means that EGCG may act in synergy with other PTE components.

International audience; Testing hepatotoxicity is a crucial step in the development and toxicological assessment of drugs and chemicals. Bio-activation can lead to the formation of metabolites which may present toxicity for the organism. Classical cytotoxic tests are not always appropriate and are often insufficient, particularly when non metabolically-competent cells are used as the model system, leading to false-positive or false-negative results. We tested over 24 h the effects of eight reference compounds on two different cell models: primary cultures of rat hepatocytes and FAO hepatoma cells that lack metabolic properties. We performed inter-assay validation between three classical cytotoxicity assays and real-time cell impedance data. We then complemented these experiments with high-content screening (HC5) to determine the cell function disorders responsible for the observed effects. Among the different assays used, the neutral red test seemed to be well suited to our two cell models, coupled with real-time cellular impedance which proved useful in the detection of bio-activation. Indeed, impedance monitoring showed a high sensitivity with interesting curve profiles yet seemed unsuitable for evaluation of viability on primary culture. Finally, HCS in the evaluation of hepatotoxicity is likely to become an essential tool for use in parallel to a classical cytotoxic assay in the assessment of drugs and environmental chemicals.

International audience; In vivo distribution of the neonicotinoid insecticide, imidacloprid, was followed during 72 h in six biological compartments of Apis mellifera L: head, thorax, abdomen, haemolymph, midgut and rectum. Honeybees were treated orally with 100 µg of 14 C-imidacloprid per kg of bee, a dose close to the median lethal dose. Elimination half-life of total radioactivity in honeybee was 25 h. Haemolymph was the compartment with the lowest and rectum that with the highest level of total radioactivity during the whole study, with a maximum 24 h after treatment. Elimination half-life of imidacloprid in whole honeybee was 5 h. Imidacloprid was readily distributed and metabolised only by Phase I enzymes into five metabolites: 4/5-hydroxy-imidacloprid, 4,5-dihydroxy-imidacloprid, 6-chloronicotinic acid, and olefin and urea derivatives. The guanidine derivative was not detected. The urea derivative and 6-chloronicotinic acid were the main metabolites and appeared particularly in midgut and rectum. The olefin derivative and 4/5-hydroxy-imidacloprid preferentially occurred in head, thorax and abdomen, which are nicotinic acetylcholine receptor-rich tissues. Moreover, they presented a peak value around 4 h after imidacloprid ingestion. These results explain the prolongation of imidacloprid action in bees, and particularly the differences between rapid intoxication symptoms and late mortality.

Microcystin-LR (MCLR) is a potent inhibitor of protein phosphatases 1 and 2A and causes alterations in cytoskeletal filaments and morphological changes that underlie apoptosis in rat hepatocytes. It has also been reported that it caused several cases of human deaths and illness. As no study on the effect of microcystins on human hepatocytes was done, yet, the aim of the study is to evaluate the toxicity of MCLR on primary human hepatocytes. The hepatocytes were incubated in 12.5-50 nM MCLR for 3, 6 and 9 h, fixed and stained with fluorescent probes for actin filaments and nuclei. Spectral laser-scanning confocal microscopy revealed that in the MCLR-treated primary human hepatocytes the actin mesh collapsed into the center of the cell, similarly as it has been described for rat hepatocytes. Cells were blebbing, fragmenting, and separated from each other. The nuclei in the affected cells condensed. In conclusion, this study confirms that MCLR is toxic to primary human hepatocytes, and it may be responsible for the liver failure cases observed after acute cyanobacterial poisoning.

Permethrin, an insecticide of the pyrethroid family, is suspected to induce hormonal and neuronal disorders. Permethrin is usually sold and found in the environment as a mixture of two isomers cis/trans. In a previous study, Scollon et al. (2009) characterized the metabolism of permethrin in human hepatic microsomes and observed that the metabolism of isomers was altered when incubated together. In this study, we propose to determine the metabolism of isomers of permethrin alone and as a mixture in primary human hepatocytes, recognized as the most suitable in vitro model for such studies. The formation of two metabolites, 3-phenoxybenzoic acid (3-PBA) and cis/trans 3-(2,2-dichlorovinyl)-2,2-dimethyl-(1-cyclopropane) carboxylic acid (DCCA), was also assessed. Platted primary human hepatocytes were incubated with either cis or trans-permethrin at 7 concentrations (5–125 μM) for 10 min, 30 min, 1 h and 3 h. The depletion of permethrin and the formation of two metabolites were analysed using the Michaelis Menten model. Metabolism was also evaluated at low concentrations below Km to estimate the intrinsic clearance when cells are exposed to both isomers. As observed in previous studies, the metabolism of cis-permethrin is slower than trans-permethrin one. Michaelis–Menten parameters were estimated for all compounds except DCCA because the saturation phase was not reached at the highest exposure concentration. No significant differences for the metabolic rates of parent compounds and metabolites were observed for cis- and trans-isomers incubated alone or in mixture. Our results could be integrated in physiologically based pharmacokinetic models.

International audience; Pesticides as well as many other environmental pollutants are considered as risk factors for the initiation and the progression of cancer. In order to evaluate the in vitro effects of chemicals present in the diet, we began by combining viability, real-time cellular impedance and high throughput screening data to identify a concentration "zone of interest" for the six xenobiotics selected: endosulfan, dioxin, carbaryl, carbendazim, p ' p ' DDE and hydroquinone. We identified a single concentration of each pollutant allowing a modulation of the impedance in the absence of vital changes (nuclear integrity, mitochondrial membrane potential, cell death). Based on the number of observed modulations known to be involved in hepatic homeostasis dysfunction that may lead to cancer progression such as cell cycle and apoptosis regulators, EMT biomarkers and signal transduction pathways, we then ranked the pollutants in terms of their toxicity. Endosulfan, was able to strongly modulate all the studied cellular processes in HepG2 cells, followed by dioxin, then carbendazim. While p,p,p ' DDE, carbaryl and hydroquinone seemed to affect fewer functions, their effects nevertheless warrant close scrutiny. Our in vitro data indicate that these xenobiotics may contribute to the evolution and worsening of hepatocarcinoma, whether via the induction of the EMT process and/or via the deregulation of liver key processes such as cell cycle and resistance to apoptosis.

International audience; For several decades, people have been in contact with bisphenol A (BPA) primarily through their diet. Nowadays it is gradually replaced by an analogue, bisphenol S (BPS). In this study, we compared the effects of these two bisphenols in parallel with the positive control diethylstilbestrol (DES) on different hepatocyte cell lines. Using a cellular impedance system we have shown that BPS is less cytotoxic than BPA in acute and chronic conditions. We have also demonstrated that, contrary to BPA, BPS is not able to induce an increase in intracellular lipid and does not activate the PXR receptor which is known to be involved in part, in this process. In parallel, it failed to modulate the expression of CYP3A4 and CYP2B6, the drug transporter ABCB1 and other lipid metabolism genes (FASN, PUN). However, it appears to have a weak effect on GSTA4 protein expression and on the Erk1/2 pathway. In conclusion, in contrast to BPA, BPS does not appear to induce the metabolic syndrome that may lead to non-alcoholic fatty liver disease (NAFLD), in vitro. Although we have to pay special attention to BPS, its use could be less dangerous concerning this toxicological endpoint for human health. (C) 2014 Published by Elsevier Ltd.

In hepatocytes, the amount of the Spot 14 (S14) protein is closely related to the full expression of enzymes involved in the glycolytic and lipogenic pathways. In the present study we address the role played by this protein in the control of transcription of the L-type pyruvate kinase (L-PK) gene in primary hepatocytes. We show that human S14, which by itself does not bind to the L-PK promoter, physically interacts with the human chicken ovalbumin upstream promoter-transcription factor 1 (COUP-TF1) and induces the switch of this factor from a repressor to an activator. However, the enhancing activity of S14 and COUP-TF1 depends on the presence of a proximal GC-rich box (the L0 element) that specifically binds nuclear proteins from the livers of rats fed a glucose-rich diet. Moreover, the L0 element, which strongly binds dephosphorylated specificity protein 1 (Sp1), loses all affinity when this factor is phosphorylated by cAMP-dependent protein kinase. Mutations that affect binding of Sp1 and nuclear proteins to the L0 box also decrease basal transcription and impair glucose responsiveness of the promoter. These results therefore shed light on the mechanism by which the S14 protein, whose concentration rapidly rises after glucose intake, contributes to the full activity of the L-PK promoter.

Malaria remains the most prevalent infectious disease of tropical and subtropical areas of the world. It represents a crucial problem in public health care, affecting 750 million people annually, of whom at least two million die. Various antimalarials currently used were studied for their capability to induce expression of the cytochrome P450 1A1 (CYP1A1) gene, an enzyme that plays an important role in the activation of xenobiotics to genotoxic derivatives. Studies on human hepatocytes and HepG2 cell lines showed that primaquine was capable of dose dependently increasing both the ethoxyresorufin-O-deethylase activity and CYP1A1 mRNAs, suggesting a transcriptional activation of this gene. Moreover, alpha-naphthoflavone, a partial aryl hydrocarbon receptor (AhR) antagonist, and 8-methoxypsoralen, which interferes with the binding of activated AhR to the xenobiotic responsive element, were shown to suppress CYP1A1 induction when added to the cultures. However, neither primaquine nor its metabolites were able to displace [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin from AhR in competitive binding studies using 9S-enriched fractions of human cytosol. These data, together with the induction of CYP1A1 promoter-directed chloramphenicol acetyl transferase gene expression, suggest that CYP1A1 induction involves the participation of the AhR but not a direct primaquine-receptor interaction. This supports the notion that an alternative ligand-independent mechanism has to be considered. Given the pharmaco-toxicological significance of CYP1A1 induction, these findings may have important implications in the treatment of malaria with primaquine and new analogs.

Persistent organic pollutants (POPs) are a group of organic or chemicals that adversely affect human health and are persistent in the environment. These highly toxic compounds include industrial chemicals, pesticides such as organochlorines, and unwanted wastes such as dioxins. Although studies have described the general toxicity effects of organochlorine pesticides, the mechanisms underlying its potential carcinogenic effects in the liver are not well understood. In this study, we analyzed the effect of three organochlorine pesticides (dichlorodiphenyltrichloroethane, heptachlore and endosulfan) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the epithelial to mesenchymal transition (EMT) in primary cultured human hepatocytes. We found that these compounds modified the hepatocyte phenotype, inducing cell spread, formation of lamellipodia structures and reorganization of the actin cytoskeleton in stress fibers. These morphological alterations were accompanied by disruption of cell-cell junctions, E-cadherin repression and albumin down-regulation. Interestingly, these characteristic features of dedifferentiating hepatocytes were correlated with the gain of expression of various mesenchymal genes, including vimentin, fibronectin and its receptor ITGA5. These various results show that organochlorines and TCDD accelerate cultured human hepatocyte dedifferentiation and EMT processes. These events could account, at least in part, for the carcionogenic and/or fibrogenic activities of these POPs.

International audience; Low amounts of residual pesticides are present in the environment, often as mixtures of chemicals which contaminate drinking water and food, being a source of chronic exposure for humans and a growing matter of concern in public health policy. Despite of the needs and growing investigation, little is known about the impact of low doses and mixtures of these chemicals on human health. The purpose of this study was to enlighten if modifications of liver cell metabolic- and/or defence-related capacities could occur under such exposures. In vitro perturbations of several metabolic, stress and survival pathways in human and mice cultured hepatocytes and liver cells were evaluated under exposure to low doses of single molecules or equimolecular combinations of the three pesticides, atrazine, chlorpyrifos and endosulfan. Mainly phases land II enzymes of detoxification were found modulated, together with apoptotic process deregulation. Hence, CYP3A4 and CYP3A11 were upregulated in primary cultured human and mouse hepatocytes, respectively. These inductions were correlated to an anti-apoptotic process (increased Bcl-xL/Bax ratio, inhibition of the PARP protein cleavage). Such disturbances in pathways involved in cell protection may possibly account for initiation of pathologies or decrease in drugs efficiency in humans exposed to multiple environmental contaminants.

Polybrominated diphenyl ethers (PBDEs) are flame retardants widely used in electronic and domestic goods. These persistent pollutants are present in the environment and in humans, and their toxicological properties are of growing concern. PBDEs can be metabolised into compounds suspected to be responsible for their toxicity. These metabolites have been characterised quite well in rodents and fish, but available information in humans remains scarce. For their identification, an efficient method for the simultaneous analysis of PBDEs, hydroxylated PBDEs (OH-PBDEs), and other PBDE metabolites in a single run was needed and has been developed in this work. Atmospheric pressure ionisation modes were compared, and Atmospheric Pressure Photo-Ionization (APPI) was selected. After careful setting of APPI parameters such as dopant and operating temperature, the optimised method was based on APPI ionization coupled to High-Resolution Mass Spectrometry operating in the full scan mode at a resolution of 60 000. This provided excellent sensitivity and specificity, allowing the discrimination of signals which could not be resolved on a triple quadrupole used as a reference. The full-scan high-resolution acquisition mode allowed monitoring of both parent PBDEs and their metabolites, including hydroxylated PBDEs, with detection limits ranging from 0.1 pg to 4.5 pg injected on-column based on the investigated standard compounds. The method was applied to the study of BDE-47 metabolism after incubation with human primary cultures of hepatocytes, and proved to be efficient not only for monitoring the parent compound and expected hydroxylated metabolites, but also for the identification of other non-targeted metabolites. In addition to hydroxy-BDE-47, several conjugated metabolites could be located, and the formation of a dihydrodiol derivative was evidenced for the first time in the case of PBDEs in this work.

The authors received Public Institutional Funding from INRA and French; International audience; Although many studies of lindane toxicity have been carried out, we still know little about the underlying molecular mechanisms. We used a microarray specifically designed for studies of the hepatotoxic effects of xenobiotics to evaluate the effects of lindane on specific gene expression in primary cultured rat hepatocytes. These genes were assigned to detoxication processes (CYP3A4, Gsta2, CYP4A1), cell signalling pathways and apoptosis (Eif2b3, Eif2b4, PKC). In this study, we demonstrate that lindane up-regulates PKC by increasing oxidative stress. TEMPO (a well known free radical scavenger) and Ro 31-8220 (an inhibitor of classical PKCs) prevented the inhibition of spontaneous and intrinsic apoptosis pathway (characterised by Bcl-xL induction, Bax down-regulation, caspases inhibition) and the induction of necrosis by lindane in rat hepatocytes. Thus, these findings indicate that several dependent key signalling pathways, including detoxification, apoptosis, PKC activity and redox status maintenance, contribute to lindane-induced toxicity in primary cultured rat hepatocytes. This may account more clearly for the acute and chronic effects of lindane in vivo, with the induction of cell death and tumour promotion, respectively.

author cannot archive publisher's version/PDF; International audience; Bisphenol F (BPF) is present in the environment and as a contaminant of food. Humans may, therefore, be exposed to BPF, and an assessment of this risk is required. BPF has been shown to have genotoxic and endocrine-disruptor properties in a human hepatoma cell line (HepG2), which is a model system for studies of xenobiotic toxicity. In this study, we investigated the ability of HepG2 cells to biotransform BPF, because metabolism may affect the observed effects of BPF, and we compared this metabolic capacity with that of human hepatocytes. Cells were incubated for 24 hours with [(3)H]-BPF. The culture medium was then concentrated and its metabolites were isolated by high-performance liquid chromatography and identified by mass spectrometry. BPF was largely metabolized into the corresponding sulfate by the HepG2 cell line. BPF was metabolized into both sulfate and glucuronide by human hepatocytes, but with differences between individuals. The metabolism of BPF in both HepG2 cells and human hepatocytes suggests the existence of a detoxification pathway. Thus, these two cell models differ in metabolic capacity. It is, therefore, very important, when assessing the toxic effects of substances in vitro, to determine, in parallel, the biotransformation capacities of the model used to extrapolate in vivo.

A multicentre validation study of the acute in vitro cytotoxicities of 31 liquid or solid chemicals was carried out by six laboratories, using primary rat hepatocyte cultures as a model system. We report here a comparison of neutral red uptake IC50 and LD50 values. Oral, i.p. and i.v. LD50 values were available for 27, 24 and 18 chemicals, respectively, and an IC50 value was obtained for 15, 14 and 11 of these compounds, respectively. A significant correlation was found only between IC50 and i.v. LD50 values.

Metabolism of mitoxantrone was studied in primary cultures of hepatocytes freshly obtained from rat, rabbit and humans. Metabolic pattern was evaluated by a high performance liquid Chromatographic method which specifically resolved mitoxantrone from its mono- and dicarboxylic acid derivatives. Studies were carried out over 48 hr and at [ 14 C]mitoxantrone concentrations ranging between 1 and 20 μM. In all species studied, metabolism occurred: extracellular unchanged mitoxantrone concentrations represented around 50, 25 and 20% of total extracellular radiolabel at 48 hr in rat, rabbit and humans, respectively. Although minor interspecies variability was observed in total amount of drug biotransformed by hepatocytes, large variability in the metabolic pattern occurred between the different species: hence, in rats the main derivatives were two polar compounds and only trace amounts of the mono- and dicarboxylic acid metabolites were present. In both rabbits and humans however, these polar derivatives represented minor metabolic pathways and the main metabolites were the mono- and dicarboxylic acid derivatives. While the percentage of total biotransformation was similar in these two latter species, the monocarboxylic acid derivative was the main metabolite in rabbits while the dicarboxylic acid was predominant in humans. Only small interindividual differences (N = 4) were observed in the metabolism of mitoxantrone by human hepatocytes in primary culture. These data demonstated that: (i) in all species, mitoxantrone was biotransformed into metabolites which rapidly effluxed in the extracellular compartment, (ii) there were low interspecies differences between rat, rabbit and humans in terms of total biotransformed drug, but (iii) large interspecies variability was demonstrated in the qualitative (rat versus both rabbit and human) and relative (rabbit versus man) patterns of the metabolites. Furthermore, the metabolism of mitoxantrone was linear over a wide range of concentrations (i.e. 1–20 μM). In conclusion, rabbit appears to be the animal species most closely related to humans in terms of mitoxantrone metabolism.

The pregnane X receptor (PXR) plays a major role in the protection of the body by regulating the genes involved in the metabolism and elimination of potentially toxic xeno- and endobiotics. We previously described that PXR activator dexamethasone protects hepatocytes from spontaneous apoptosis. We hypothesise a PXR-dependent co-regulation process between detoxication and programmed cell death. Using primary cultured human and rat hepatocytes, we investigated to determine if PXR is implicated in the regulation of Bcl-2 and Bcl-xL, two crucial apoptosis inhibitors. In the present study we demonstrated that the treatment of primary cultured hepatocytes with PXR agonists increased hepatocyte viability and protects them from staurosporine-induced apoptosis. The anti-apoptotic capacity of PXR activation was correlated with Bcl-2 and Bcl-xL induction at both the transcriptional and protein levels in man and rats, respectively. The inhibition of PXR expression by antisense oligonucleotide abolished PXR activators Bcl-xL induction. Accordingly, PXR overexpression in HepG2 cells led to bcl-2 induction upon clotrimazole treatment and protects cells against Fas-induced apoptosis. Our results demonstrate that PXR expression is required for Bcl-2 and Bcl-xL up-regulation upon PXR activators treatment in human and rat hepatocytes. They also suggest that PXR may protect the liver against chemicals by simultaneously regulating detoxication and the apoptotic pathway.

A stable hepatoma cell line expressing the human pregnane X receptor (hPXR) and the cytochrome P4503A4 (CYP3A4) distal and proximal promoters plus the luciferase reporter gene was developed to assess the ability of several xenobiotic agents to induce CYP3A4 and CYP2B6. After selection for neomycin resistance, one clone, displaying high luciferase activity in response to rifampicin (RIF), was isolated and the stable expression of hPXR was confirmed by reverse transcription polymerase chain reaction (RT-PCR). Dose-response curves were generated by treating these cells with increasing concentrations of RIF, phenobarbital (PB), clotrimazole (CLOT) or 5beta-pregnane-3,20-dione (5beta-PREGN). The effective concentrations for half maximal response (EC50) were determined for each of these compounds. RIF was the most effective compound, with maximal luciferase activity induced at 10 microM. The agonist activities of PXR-specific inducers measured using our stable model were consistent with those measured in transient transfectants. The abilities of organochlorine (OC), organophosphate (OP) and pyrethroid pesticides (PY) to activate hPXR were also assessed and found to be consistent with the abilities of these compounds to induce CYP3A4 and CYP2B6 in primary culture of human hepatocytes. These results suggest that CYP3A4 and CYP2B6 regulation through PXR activation by persistent pesticides may have an impact on the metabolism of xenobiotic agents and endogenous steroid hormones. Our model provides a useful tool for studying hPXR activation and for identifying agents capable of inducing CYP3A4 and CYP2B6.

Aim Tea is one of the most consumed beverages in the world and its healthpromoting effects have been widely investigated.Lipid-lowering effects of pu-erh tea have attracted growing interest.The importance of liver in lipid metabolism prompted us to investigate the lipid-lowering properties of pu-erh tea in rat primary culture hepatocytes.We tested the effect of a Hao Ling pu-erh tea extract (HLPT) and one of the major components of tea as Epigallocatechin-3-gallate (EGCG) which is largely recognized as a hypolipidemic molecule. Methods HLPT: an infusion of Hao Ling pu-erh tea was lyophilized and quantified for its composition in catechins by LC-MS. 24h after seeding on collagen, rat hepatocytes in primary culture were treated for 24h with various concentrations of HLPT(100,200,400,600 μg/ml) and EGCG(30,100 uM) and compared to CyclosporinA (hyperlipidemic reference) and Clofibrate (hypolipidemic drug used in human)(n=3 in triplicate).Lipid droplet accumulation was measured by LipidTox staining and evaluated by fluorescence microscopy on an ArrayScanXTI high Content Analysis Reader(Cellomics Inc.). Results We found that HLPT significantly prevented hepatocyte lipid accumulation (−56%) and in the same proportion to Clofibrate.EGCG also significantly attenuated lipid accumulation (−19%) but less than HLPT. Conclusion The main result of this study was to point out the major implication of liver cells in the hypolipidemic effects of HLPT.Moreover, we have shown here that this effect was partly due to the EGCG, well known for its antioxidant effects.However as we reported here that HLPT has a higher hypolipidemic effect than EGCG alone, which means that EGCG acts in synergy with other HLPT components such as theaflavins but this hypothesis has to be confirmed in further experiments.In future, the variation of expression of genes involved in lipid metabolism (THRSP, LXR, PPar-α) by qPCR induced by HLPT will allow us to improve the understanding of the effect induced by HLPT.

The effect of sodium bicarbonate on intracellular pH under conditions close to those in vivo, with both bicarbonate and nonbicarbonate buffering systems, is unknown. We postulated that this effect depends on the nonbicarbonate buffering capacity because the alkali-induced back-titration of these buffers results in a concentration-dependent release of CO2 in the extracellular space, leading to a decrease in intracellular pH.The study was conducted in two stages. First, human hepatocytes were perfused with pH 7 bicarbonate-buffered medium (5 mM HCO3-, 20 torr Pco2) containing no nonbicarbonate buffer or small amounts (5 mM 4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid [HEPES]) or large amounts (20 mM HEPES) of nonbicarbonate buffer. Second, the changes in intracellular pH of hepatocytes placed in acidotic human blood (pH 7, 5 mM HCO3-, 20 torr Pco2) at three hematocrits (40%, 20%, and 5%) were measured.Research laboratory at a medical university.Cryopreserved human hepatocytes thawed the day before the experiments.Sodium bicarbonate was infused for 10 mins to increase the HCO3- concentration from 5 to 30 mM. In the second part, 20 mM sodium bicarbonate was added directly to the blood bathing the cells.The intracellular pH was measured with the pH-sensitive fluorescent dye bis-carboxyethyl carboxy-fluorescein in its esterified form, acetoxy-methyl ester, by using a single-cell imaging technique. Gas analyses were performed before and during the sodium bicarbonate load. Sodium bicarbonate caused a decrease in intracellular pH with all media except the artificial medium containing no HEPES. This decrease was small in media with low nonbicarbonate buffering capacity (5 mM HEPES and 5% hematocrit blood) and large in media with high nonbicarbonate buffering capacity (20 mM HEPES and 40% hematocrit blood). The change in intracellular pH was linked closely to the change in Pco2 caused by the sodium bicarbonate.The effect of sodium bicarbonate on intracellular pH depends on changes in Pco2 in the medium bathing the cells. The increase in Pco2 is correlated with the extracellular nonbicarbonate buffering capacity because of the release of H+ ions coming from the back-titration of these buffers. We conclude that sodium bicarbonate may exacerbate cell acidosis under buffering conditions close to those in vivo and that the initial changes in cell pH caused by sodium bicarbonate depend on the extracellular nonbicarbonate buffering capacity.

Ethical, economic and technical reasons hinder regular supply of freshly isolated hepatocytes from higher mammals such as monkey for preclinical evaluation of drugs. Hence, we aimed at developing optimal and reproducible protocols to cryopreserve and thaw parenchymal liver cells from this major toxicological species. Before the routine use of these protocols, we validated them through a multi-laboratory study. Dissociation of the whole animal liver resulted in obtaining 1-5 billion parenchymal cells with a viability of about 86%. An appropriate fraction (around 20%) of the freshly isolated cells was immediately set in primary culture and various hepato-specific tests were performed to examine their metabolic, biochemical and toxicological functions as well as their ultrastructural characteristics. The major part of the hepatocytes was frozen and their functionality checked using the same parameters after thawing. The characterization of fresh and thawed monkey hepatocytes demonstrated the maintenance of various hepato-specific functions. Indeed, cryopreserved hepatocytes were able to survive and to function in culture as well as their fresh counterparts. The ability for synthesis (proteins, ATP, GSH) and conjugation and secretion of biliary acids was preserved after deep freeze storage. A better stability of drug metabolizing activities than in rodent hepatocytes was observed in monkey. After thawing, Phase I and Phase II activities (cytochrome P450, ethoxycoumarin-O-deethylase, aldrin epoxidase, epoxide hydrolase, glutathione transferase, glutathione reductase and glutathione peroxidase) were well preserved. The metabolic patterns of several drugs were qualitatively and quantitatively similar before and after cryopreservation. Lastly, cytotoxicity tests suggested that the freezing/thawing steps did not change cell sensitivity to toxic compounds.

Microsomal cytochrome P450-dependent lauric acid hydroxylase activities were characterized in liver, kidney, and intestinal mucosa of the sea bass (Dicentrarchus labrax). Microsomes from these organs generated (omega-1)-hydroxylauric acid and a mixture of positional isomers including (omega)-, (omega-2)-, (omega-3)- and (omega-4)-hydroxylauric acids, which were identified by RP-HPLC and GC-MS analysis. Peroxisome proliferators, such as clofibrate and especially di(2-ethylhexyl) phthalate, increased kidney microsomal lauric acid hydroxylase activities. The synthesis of 11-hydroxylauric acid was enhanced 5.3-fold in kidney microsomes. Liver microsomal lauric acid hydroxylase activities were weakly affected and no significant induction was found in small intestine microsomes from clofibrate or di(2-ethylhexyl) phthalate-treated fish. The differences in lauric acid metabolisation and the tissue-specific induction by peroxisome proliferators suggest the involvement of several P450s in this reaction. Incubations of liver and kidney microsomes with lauric acid analogues (11- or 10-dodecynoic acids) resulted in a time- and concentration-dependent loss of lauric acid hydroxylase activities. The induction of these activities in fish by phthalates, which are widely-distributed environmental pollutants, may be taken into consideration for the development of new biomarkers.

The aim of this study was to evaluate rifabutin (RBT) and rifampicin (RIF) capabilities in inducing various xenobiotic metabolizing enzymes such as cytochromes P450 (CYPs) and UDP-glucuronosyl-transferases (UGTs) in cultured fresh and cryopreserved human hepatocytes. Enzyme induction was assessed through the use of several diagnostic markers, i.e. testosterone, midazolam (MDZ), diazepam (DZP) and 7-ethoxyresorufin for CYP-dependent enzyme reactions; and AZT for UGT-dependent enzyme reactions. RBT concentrations (0.118, 0.708 microM) were selected according to previously published pharmacokinetic data in patients. The known CYP3A4 inducer in humans, RIF, was used as a positive control. At the concentrations used, no sign of cytotoxicity was evidenced. Both compounds were able to dose-dependently induce the overall metabolism of testosterone (approximately 2-fold for RBT, 4-fold for RIF) and the formation of the 6beta-hydroxylated-derivative (up to approximately 4-fold over control for RBT and approximately 10-fold for RIF), which is CYP3A4 dependent. The other hydroxylated metabolites (16alpha-OH and 2alpha-OH) were also enhanced. The metabolism of MDZ, which is specifically metabolized by CYP3A4 in humans, was also investigated following drug's exposure to hepatocytes. DZP one, which is governed by various CYPs, including CYP3A, was also investigated. RBT was shown to increase the biotransformation of both benzodiazepines (approximately 1.9-fold over control). Moreover, the effects of both drugs on ethoxyresorufin O-deethylase activity (EROD), which is representative of CYPIA1/2 isoforms, were tested. Results showed only a moderate induction of this marker (approximately 2-fold over control) when compared to the high effect observed after hepatocyte exposure to 3-methylcholantene (approximately 14-fold over control). Finally, the action of RBT and RIF on UGTs expression was investigated by using AZT as diagnostic substrate: glucuronides formation was not significantly affected by the two rifamycin derivatives. On the whole, exposure of fresh or cryopreserved human hepatocytes to RBT dose-dependently affected the levels of drug metabolizing enzymes in a dose-dependent manner. However, as already demonstrated by in vivo pharmacokinetic studies, its inducing properties towards CYPs, CYP3A in particular, are less pronounced than RIF.

Successful liver transplantation depends on adequate preservation of cellular function. We therefore tested the effects of two currently used liver preservation fluids, Euro-Collins (EC) solution and University of Wisconsin (UW) solution, on the viability and some functional activities of hepatocytes isolated from human livers. Cells in primary culture were maintained under hypoxic (95% N2/5% CO2) and hypothermic (4 degrees C) conditions for 24 h, either in EC or UW solution. This treatment did not result in significant hepatocyte damage, as judged by phase contrast microscopy, intracellular LDH release, and the MTT mitochondrial test. However, neutral red uptake indicated that lysosomal functions were slightly affected (35% decrease) when compared to control conditions. At the end of the hypoxia/hypothermia period, hepatocyte monolayers were incubated at 37 degrees C under normoxic conditions for 24 h, in order to simulate the reperfusion of a transplanted liver. Three drugs--midazolam, diazepam, zidovudine--were used as diagnostic substrates to check the metabolic abilities of human hepatocytes replaced in normal conditions. Both phase I (hydroxylation, demethylation) and phase II (glucuronidation) metabolic reactions were affected by the hypoxia/hypothermia shock. Indeed, a 30%-50% decrease in these activities was observed as compared to values obtained in control hepatocytes. No difference could, however, be found at the cellular level regarding the solution used for cold storage. These results suggest that the superiority of UW over EC solution, already reported in clinical practice after transplantation of preserved human livers, was not due to a better preservation of the hepatocytes.

The level and number of CYP2E1 gene transcripts were investigated by northern blot analysis in various human adult tissues including liver, lung, placenta, skin and neurinoma. Three transcripts of 1.8, 2.6 and 4 Kb were expressed in a tissue-specific manner. The origin of the various transcripts was studied and showed that both 4 and 2.6 Kb mRNAs contained sequences from the 3' non-translated region of the gene and that the 4 Kb also contained region localized in the 5' non-translated region. Furthermore, it clearly appeared that a catalytically active CYP2E1 enzyme (as proved by NDMA demethylase activity) was only detected in tissues expressing the 1.8 Kb. The human CYP2E1 was also identified through immunohistochemical techniques. Finally, we observed a relation between the hypomethylation of the human CYP2E1 gene and the hypoexpression of the corresponding protein.

Un modèle de mesure de la clairance de la T4 à partir de culture d'hépatocytes a été développé afin de préciser le potentiel perturbateur thyroïdien relatif du fipronil et du fipronil sulfone par des approches de type data-réponse.