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Benzophenone-2 (BP2) is widely used as a UV screen in both industrial products and cosmetic formulations, where it is frequently found associated with fragrance compounds, such as isoeugenol and coumarin. BP2 is now recognized as an endocrine disruptor, but to date, no information has been reported on its fate in humans. The intrinsic clearance (Clint) and metabolic interactions of BP2 were explored using cryopreserved human hepatocytes in primary cultures. In vitro kinetic experiments were performed to estimate the Michaelis-Menten parameters. The substrate depletion method demonstrated that isoeugenol was cleared more rapidly than BP2 or coumarin (Clint = 259, 94.7 and 0.40 μl/min/10(6) cells respectively). This vitro model was also used to study the metabolic interactions between BP2 and isoeugenol and coumarin. Coumarin exerted no effects on either isoeugenol or BP2 metabolism, because of its independent metabolic pathway (CYP2A6). Isoeugenol appeared to be a potent competitive substrate inhibitor of BP2 metabolism, equivalent to the specific UGT1A1 substrate: estradiol. Despite the fact that inhibition of UGT by xenobiotics is not usually considered to be a major concern, the involvement of UGT1A1 in BP2 metabolism may have pharmacokinetic and pharmacological consequences, due to the its polymorphisms in humans and its pure estrogenic effect., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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.

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., (© The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., (Copyright © 2014. Published by Elsevier Ltd.)

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.

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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 I and 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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2012 John Wiley & Sons, Ltd.)

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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.

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.

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.

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.

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.

Background: Organophosphorus insecticides are well known to induce hepatotoxicity. One among this is chlorpyrifos (CPF), which is an insecticide inducing various toxicities including in liver. Objective: This investigation focused on CPF-induced oxidative damage in rat hepatocytes primary culture and the protective effect of Cactus cladode aqueous extract. Materials and Methods: Hepatocytes were treated with CPF (50, 75, and 150 μM) and cactus aqueous extract. On treatment for 48 h, mortality within these cells was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide test, reactive oxygen species (ROS) levels were measured by H2DCFDA test. Furthermore, heat shock protein 70 (HSP70) and CYP1A1/2 levels were determined using western blot analysis. Annexin V and 4',6-diamidino-2-phenylindole analysis was run to determine the level of cell death and cytoprotection exerted by CPF and extracts, respectively. Results: The results showed that CPF increases the levels of H2O2 and HSP70 and induces CYP1A1/2 and mortality within these cells. In the other side of this study, the co-treatment of these cells with CPF and Cactus cladode aqueous extract showed a recovery of these parameters. It also has been found that the Cactus cladode aqueous extract has the potential to do cytoprotective effect by preventing necrosis induced by CPF. Conclusion: Taken together, these findings suggest that the toxicity exerted by CPF in hepatocytes are involved with the generation of ROS and the regulation of well-controlled programmed cell death, which could be well protected by the Cactus cladode extract pretreatment. [ABSTRACT FROM AUTHOR]

Induction of both CYP1A1 and metallothioneins (MTs) in fish liver is increasingly being used in ecotoxicological studies. The interaction of heavy metals (Cd, Cu, Zn, Pb) with the CYP1A induction response and MT levels was studied in trout (Oncorhynchus mykiss) hepatocyte cultures. Cells were exposed to 3-methylcholanthrene (3-MC) or to increasing heavy metal concentrations or to a mixture of both (3-MC and one heavy metal). Metal cytotoxicity was assessed by the neutral red test. Ranking of toxicity was Cd(II) < Cu(II) < Zn(II) < Pb(II) (EC50: 45, 222, 873, and 945 µM, respectively). CYP1A1 expression was monitored by ethoxyresorufin-O-deethylase (EROD) activity as well as by Western and Northern blots. As expected, 3-MC induced EROD activity in a time- and dose-dependent manner (maximal induction 5 times that of the control at 0.5 µM and after a 72-h exposure period). These data were confirmed by Western blot (intense band of 55-60 KDa) and Northern blot analyses. Induction caused by 0.5 µM 3-MC was reduced to less than 50% of control by the concomitant exposure to Cd, Cu, Pb, or Zn (EC50: from 1 µM for Cd(II) to 18 µM for Pb(II)). The MTs were significantly induced in hepatocytes exposed to heavy metals for 24 h. In the presence of 3-MC (0.5 µM), MT levels were significantly lower than those found in cells treated with metals alone at 24 h only. Our results lead to the conclusion that heavy metals significantly affect CYP expression and that a CYP1A1 inducer (3-MC) can modulate the induction of MTs. These data have to be taken into consideration in biomarker monitoring. [ABSTRACT FROM AUTHOR]