Investigating the in vitro steatotic mixture effects of similarly and dissimilarly acting test compounds using an adverse outcome pathway-based approach.

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.

The Anti-Cancer Drug Dabrafenib Is a Potent Activator of the Human Pregnane X Receptor.

The human pregnane X receptor (hPXR) is activated by a large set of endogenous and exogenous compounds and plays a critical role in the control of detoxifying enzymes and transporters regulating liver and gastrointestinal drug metabolism and clearance. hPXR is also involved in both the development of multidrug resistance and enhanced cancer cells aggressiveness. Moreover, its unintentional activation by pharmaceutical drugs can mediate drug-drug interactions and cause severe adverse events. In that context, the potential of the anticancer BRAF inhibitor dabrafenib suspected to activate hPXR and the human constitutive androstane receptor (hCAR) has not been thoroughly investigated yet. Using different reporter cellular assays, we demonstrate that dabrafenib can activate hPXR as efficiently as its reference agonist SR12813, whereas it does not activate mouse or zebrafish PXR nor hCAR. We also showed that dabrafenib binds to recombinant hPXR, induces the expression of hPXR responsive genes in colon LS174T-hPXR cancer cells and human hepatocytes and finally increases the proliferation in LS174T-hPXR cells. Our study reveals that by using a panel of different cellular techniques it is possible to improve the assessment of hPXR agonist activity for new developed drugs.

An adverse outcome pathway-based approach to assess steatotic mixture effects of hepatotoxic pesticides in vitro.

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 Pathway-Driven Analysis of Liver Steatosis in Vitro: A Case Study with Cyproconazole.

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.

Antioxidant properties of tea blunt ROS-dependent lipogenesis: beneficial effect on hepatic steatosis in a high fat-high sucrose diet NAFLD obese rat model.

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.

Trans-nonachlor decreases miR-141-3p levels in human melanocytes in vitro promoting melanoma cell characteristics and shows a multigenerational impact on miR-8 levels in Drosophila.

Epidemiological association studies have revealed a role for pesticides in cancer occurrence, while a growing number of reports have highlighted the deleterious epigenetic modifications that can be produced by environmental factors. However, epidemiological data currently lack molecular support to unravel the epigenetic impact of pesticides on carcinogenesis. Based on epidemiological studies of melanoma, our data show for the first time that trans-nonachlor (TNC), a component of the pesticide chlordane, modulates the microRNA miR-141-3p in human melanocytic cells in vitro, with effects on melanomagenesis parameters. TNC downregulates the level of miR-141-3p in normal melanocytes to levels found endogenously in several melanoma cell lines. Ectopic expression of either a synthetic miRNA mimic or inhibitor in human melanocytic cells revealed that TNC counteracts the inhibitory effects of miR-141-3p on melanoma cell anchorage-independent growth ability, their invasive potential, and expression of a multipotent, embryonic-like, aggressive cancer phenotype (termed vasculogenic mimicry), involving VE-Cadherin. In addition, the data suggest that miR-141-3p regulates vasculogenic mimicry through extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphatidyl inositol-3-kinase (PI3K). Notably, in the Drosophila animal model, TNC also decreased the level of miR-8, the sole miR-141-3p fly ortholog. Importantly, the downregulation of miR-8 levels induced by TNC in ancestors was transmitted through multigenerations, with a progressive reversion over time. Such a decrease in miR-8 levels translated to a loss-of-weight phenotype in offspring. Providing support to epidemiological data, these results altogether suggest that TNC may favor melanomagenesis by lowering the levels of miR-141-3p, thereby activating melanoma cell processes. Although any such conclusions in humans are yet to be determined, these experiments in Drosophila demonstrate that TNC can promote an epigenetic multigenerational inheritance of the miR-141-3p/miR-8 defect. This study therefore justifies the development of molecular investigations to decipher the toxic epigenetic heritable impact of pesticides on cancer occurrence.

Synergistic activation of human pregnane X receptor by binary cocktails of pharmaceutical and environmental compounds.

Humans are chronically exposed to multiple exogenous substances, including environmental pollutants, drugs and dietary components. Many of these compounds are suspected to impact human health, and their combination in complex mixtures could exacerbate their harmful effects. Here we demonstrate that a pharmaceutical oestrogen and a persistent organochlorine pesticide, both exhibiting low efficacy when studied separately, cooperatively bind to the pregnane X receptor, leading to synergistic activation. Biophysical analysis shows that each ligand enhances the binding affinity of the other, so the binary mixture induces a substantial biological response at doses at which each chemical individually is inactive. High-resolution crystal structures reveal the structural basis for the observed cooperativity. Our results suggest that the formation of 'supramolecular ligands' within the ligand-binding pocket of nuclear receptors contributes to the synergistic toxic effect of chemical mixtures, which may have broad implications for the fields of endocrine disruption, toxicology and chemical risk assessment.

High-content screening imaging and real-time cellular impedance monitoring for the assessment of chemical's bio-activation with regards hepatotoxicity.

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 (HCS) 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.

Effect of Brewing Duration on the Antioxidant and Hepatoprotective Abilities of Tea Phenolic and Alkaloid Compounds in a t-BHP Oxidative Stress-Induced Rat Hepatocyte Model.

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.

Is bisphenol S a safe substitute for bisphenol A in terms of metabolic function? An in vitro study.

As bisphenol A (BPA) has been shown to induce adverse effects on human health, especially through the activation of endocrine pathways, it is about to be withdrawn from the European market and replaced by analogues such as bisphenol S (BPS). However, toxicological data on BPS is scarce, and so it is necessary to evaluate the possible effects of this compound on human health. We compared the effect of BPA and BPS on obesity and hepatic steatosis processes using low doses in the same range as those found in the environment. Two in vitro models were used, the adipose cell line 3T3-L1 and HepG2 cells, representative of hepatic functions. We analyzed different parameters such as lipid and glucose uptakes, lipolysis, leptin production and the modulation of genes involved in lipid metabolism and energy balance. BPA and BPS induced an increase in the lipid content in the 3T3-L1 cell line and more moderately in the hepatic cells. We also observed a decrease in lipolysis after bisphenol treatment of adipocytes, but only BPS was involved in the increase in glucose uptake and leptin production. These latter effects could be linked to the modulation of SREBP-1c, PPARγ, aP2 and ERRα and γ genes after exposure to BPA, whereas BPS seems to target the PGC1α and the ERRγ genes. The findings suggest that both BPA and BPS could be involved in obesity and steatosis processes, but through two different metabolic pathways.

A concentration addition model to assess activation of the pregnane X receptor (PXR) by pesticide mixtures found in the French diet.

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.

Potential involvement of chemicals in liver cancer progression: an alternative toxicological approach combining biomarkers and innovative technologies.

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'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.

Comparative study of bisphenol A and its analogue bisphenol S on human hepatic cells: a focus on their potential involvement in nonalcoholic fatty liver disease.

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, PLIN). 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.

Atrazine represses S100A4 gene expression and TPA-induced motility in HepG2 cells.

Atrazine (ATZ) is probably the most widely used herbicide in the world. However there are still many controversies regarding its impacts on human health. Our investigations on the role of pesticides in liver dysfunctions have led us to detect an inhibition of FSP1 expression of 70% at 50µm and around 95% at 500µM of ATZ (p<0.01). This gene encodes the protein S100a4 and is a clinical biomarker of epithelial-mesenchymal transition (EMT), a key step in the metastatic process. Here we investigated the possible effect of ATZ on cell migration and noticed that it prevents the EMT and motility of the HepG2 cells induced by the phorbol ester TPA. ATZ decreases Fak pathway activation but has no effect on the Erk1/2 pathway known to be involved in metastasis in this cell line. These results suggest that ATZ could be involved in cell homeostasis perturbation, potentially through a S100a4-dependant mechanism.

Crosstalk between beta-catenin and snail in the induction of epithelial to mesenchymal transition in hepatocarcinoma: role of the ERK1/2 pathway.

Epithelial to mesenchymal transition (EMT) is an integral process in the progression of many epithelial tumors. It involves a coordinated series of events, leading to the loss of epithelial features and the acquisition of a mesenchymal phenotype, resulting in invasion and metastasis. The EMT of hepatocellular carcinoma (HCC) cells is thought to be a key event in intrahepatic dissemination and distal metastasis. In this study, we used 12-O-tet-radecanoylphorbol-13-acetate (TPA) to dissect the signaling pathways involved in the EMT of HepG2 hepatocarcinoma cells. The spectacular change in phenotype induced by TPA, leading to a pronounced spindle-shaped fibroblast-like cell morphology, required ERK1/2 activation. This ERK1/2-dependent EMT process was characterized by a loss of E-cadherin function, modification of the cytoskeleton, the acquisition of mesenchymal markers and profound changes to extracellular matrix composition and mobility. Snail was essential for E-cadherin repression, but was not sufficient for full commitment of the TPA-triggered EMT. We found that TPA triggered the formation of a complex between Snail and ß-catenin that activated the Wnt pathway. This study thus provides the first evidence for the existence of a complex network governed by the ERK1/2 signaling pathway, converging on the coregulation of Snail and the Wnt/ß-catenin pathway and responsible for the onset and the progression of EMT in hepatocellular carcinoma cells.

Organochlorine pesticides induce epithelial to mesenchymal transition of human primary cultured hepatocytes.

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.

Effects of endosulfan on hepatoma cell adhesion: Epithelial-mesenchymal transition and anoikis resistance.

Endosulfan is an organochlorine pesticide commonly used in agriculture yet classified by the Stockholm Convention in 2011 as a persistent organic pollutant (POP). Its potential toxicity makes its continued use a major public health concern. Despite studies in laboratory animals, the molecular mechanisms underlying the carcinogenic effects of endosulfan in human liver remain poorly understood. In this study, we investigated the phenotypical effects of endosulfan on HepG2 liver cells. First, we found that endosulfan disrupted the anoikis process. Indeed, cells exposed to endosulfan were initially sensitized to anoikis and thereafter recovered their resistance to this process. This phenomenon occurred in parallel to the induction of the epithelial to mesenchymal (EMT) process, as demonstrated by: (1) reorganization of the actin cytoskeleton together with activation of the FAK signaling pathway; (2) repression of E-cadherin expression; (3) induction of Snail and Slug; (4) activation of the WNT/ß-catenin pathway; and (5) induction and reorganization of mesenchymal markers (S100a4, vimentin, fibronectin, MMP-7). Secondly, despite the acquisition of mesenchymal characteristics, HepG2 cells exposed to endosulfan failed to migrate. This incapacity to acquire a motile phenotype could be attributed to a disruption of the interaction between the ECM and the cells. Taken together, these results indicate that endosulfan profoundly alters the phenotype of liver cells by inducing cell detachment and partial EMT as well as disrupting the anoikis process. All these events account, at least in part, for the carcinogenic potential of endosulfan in liver.

Molecular investigation of the effects of lindane in rat hepatocytes: microarray and mechanistic studies.

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.

Bis(hydroxyphenyl)methane-bisphenol F-metabolism by the HepG2 human hepatoma cell line and cryopreserved human hepatocytes.

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.

Lindane and cell death: at the crossroads between apoptosis, necrosis and autophagy.

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.