23 Then they were

scraped in 0.1 N NaOH; the remaining radiolabeled substrate was measured through scintillation counting to determinate TA efflux. To discriminate between basolateral and canalicular efflux, cells were incubated in parallel in either standard MG-132 in vivo or Ca2+ and Mg2+-free buffer for 30 minutes after TA uptake before measuring the remaining radiolabeled TA. Canalicular efflux was calculated using this equation: Canalicular efflux = Radioactivity in efflux mediumCa2+ and Mg2+ free buffer − Radioactivity in efflux mediumStandard buffer.23 6β-Hydroxylation of testosterone by CYP3A4 was measured as described previously.18 The Mann-Whitney U test was applied to compare data between treated cells and corresponding control cultures. Data were considered significantly different when P < 0.05. The MTT test was first used to evaluate cytotoxic effects of CPZ in HepaRG cells after 6- and 24-hour exposure. Whereas no cytotoxicity was observed after 6 hours (data

not shown), CPZ induced dose-dependent toxic effects after a 24-hour treatment, with a median inhibitory concentration (IC50) value equal to 80 μM (Fig. 1A). Based on these data, nontoxic (20 and 35 μM) and subtoxic Osimertinib supplier (50 μM, corresponding to 80% cell viability) concentrations of CPZ were used to investigate early events leading to the toxic response. At these concentrations, CPZ induced formation of intracytoplasmic vesicles. These vesicles appeared as lamellar bodies under electron microscopy and, accordingly, expression of target genes of phospholipidosis was found to be modulated (Supporting data). ROS generation was determined by DHE staining in 50 μM CPZ-treated HepaRG cells (Fig. 1B). Superoxide anions were detected in

hepatocyte-like cells as early as 15 minutes after CPZ exposure. Superoxide anions formation was totally prevented up to 6 hours and only partially after 24 hours by coincubation with the antioxidant NAC. Moreover, expression of three oxidative stress-related genes was analyzed 6 and 24 hours after addition of 50 μM CPZ (Fig. 1C). The NF-E2-related factor 2 (Nrf2) that regulates antioxidant-responsive element-mediated induction of cytoprotective genes and its target gene heme oxygenase 1 (HO-1) were significantly up-regulated at both timepoints, whereas the expression of the antioxidant enzyme, manganese superoxide 上海皓元医药股份有限公司 dismutase (MnSOD), was enhanced only after a 24-hour CPZ treatment. As expected, fold-induction of the three genes was reduced in the presence of NAC. ROS production is known to generate mitochondrial injury and to disrupt F-actin distribution. Mitochondrial membrane potential was followed by JC-1 staining. CPZ seemed to alter the inner mitochondrial membrane potential, as the green fluorescence associated with monomer forms of JC-1 was more pronounced in CPZ-treated cells compared to untreated cells in which the red fluorescence associated with JC-1 dimers was predominant (Fig. 2A).