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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; 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.

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.

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.

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.

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.

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.