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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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

A method for the determination of chlorfenvinphos, ethion and linuron in liver samples by LC-MS/MS is described. Sample treatment was performed by using Sola™ polymeric reverse phase SPE cartridges after protein precipitation. Gradient elution using 10 mM ammonium formate in methanol (A) and 10 mM ammonium formate in water (B) was used for chromatographic separation of analytes on a Hypersil™ end-capped Gold PFP reverse phase column (100 mm × 2.1 mm, 3 μm). All analytes were quantified without interference, in positive ionization mode using multiple reaction monitoring (MRM) with chlorfenvinphos-d10 as internal standard. The whole procedure was validated according to the FDA guidelines for bioanalytical methods. The calibration curves for chlorfenvinphos, linuron and ethion compounds were linear over the concentration range of 0.005-2 μM (i.e. 0.0018-0.720 μg/mL, 0.0019-0.770 μg/mL and 0.0012-0.500 μg/mL respectively) with coefficients of determination higher than 0.998. A Lower limit of quantification of 0.005 μM was achieved for all analytes, i.e. 5.76, 6.08 and 3.84 μg/kg of liver for chlorfenvinphos, ethion and linuron respectively. Compounds extraction recovery rates ranged from 92.9 to 99.5% with a RSD of 2.3%. Intra- and inter-day accuracies were within 90.9 and 100%, and imprecision varied from 0.8 to 8.2%. Stability tests proved all analytes were stable in liver extracts during instrumental analysis (+12 °C in autosampler tray for 72 h) at the end of three successive freeze-thaw cycles and at -20 °C for up to 9 months. This accurate and robust analytical method is therefore suitable for contamination or metabolism studies., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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.)

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.)

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.)

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.)

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.