For APAP treatment, HepaRG or HepG2 cells were washed with phosphate-buffered saline (PBS) and changed to DMSO-free medium containing the desired concentration of APAP. For caspase inhibition, some cells were pretreated for 1 hour with medium containing 20 μM Z-VD-fmk see more (generous gift from Dr. S.X. Cai, Epicept, San Diego, CA), then changed to medium containing 20 μM Z-VD-fmk and 20 mM APAP. As a positive control for caspase activation, some cells were treated for 16.5 hours with 5 mM galactosamine and 100 ng/mL recombinant human tumor necrosis factor (rhTNFα) (Genzyme, Cambridge, MA). HepaRG cells were used at passages 18, 19, and 20. Within this range, no variation in glutathione (GSH)

depletion or in the kinetics of injury was observed after APAP exposure, suggesting no relevant

change in CYP expression or activity between these passages. After protease digestion, APAP-cysteine (APAP-CYS) adducts were measured in cells and in the culture medium by liquid chromatography dual mass spectrometry selleck screening library (LC-MS/MS) as described in detail in the Supporting Material. Cell death was assessed by lactate dehydrogenase (LDH) release, as described in detail.12 LDH release is a more sensitive parameter of cell death because HepaRG cells contain only low levels of alanine aminotransferase activity. The JC-1 Mitochondrial Membrane Potential Kit (Cell Technology, Mountain View, CA) was used according to the manufacturer’s instructions.12 Cellular glutathione was measured using a modified Tietze assay, as described.27 For measurement of mitochondrial ROS generation, HepaRG cells were seeded on glass bottom dishes and

ROS and peroxynitrite generation was measured using Mitosox Red and dihydrorhodamine, respectively, as described.28 Caspase activity based on Z-VAD-fmk inhibitable Ac-DEVD-AMC fluorescence was measured as described.29 Cells were seeded on glass bottom dishes and treated with APAP and 30 μM PI in DMSO-free, phenol red-free Williams’ E Medium with penicillin/streptomycin and 10% FBS. At various timepoints Sorafenib mouse the live cells were imaged on a Zeiss Axiovert inverted fluorescence microscope through a Texas Red filter to assess PI uptake. All fluorescence images were taken at the same exposure and later superimposed on phase contrast images of the same fields using ImageJ software. All results are expressed as mean ± standard error (SE). Comparisons between multiple groups were performed with one-way analysis of variance (ANOVA) followed by a post-hoc Bonferroni test. If the data were not normally distributed, we used the Kruskal-Wallis Test (nonparametric ANOVA) followed by Dunn’s Multiple Comparisons Test; P < 0.05 was considered significant. The first event in the pathogenesis of APAP hepatotoxicity in rodents is metabolism of the drug to the reactive intermediate NAPQI, which can bind to and deplete glutathione.