4A-C). Similar to rodent hepatocytes,28 rhodamine fluorescence peaked in HepaRG cells (12 hours) after the peak of Mitosox Red fluorescence (Fig. 4A-C). HepaRG cells are bipotent progenitors that differentiate into two morphologically distinct cell populations.23, 24 The hepatocyte-like cells have a characteristic granular appearance and grow in small clusters or “hepatocyte islands” (Figs. 3, 4D). Surrounding these islands are flatter, clearer biliary epithelial-like cells (Figs. 3, 4D). To assess the contribution of each cell type to our data showing APAP toxicity, check details APAP-treated cells were exposed to PI, which stains nuclei of necrotic cells red (Fig. 4E,F). At 24 hours

the majority of the PI staining was seen in the hepatocyte-like cells, with very little among the biliary epithelial-like cells (Fig. 4E,F). The distribution was similar at 48 hours (data not shown). This suggests that APAP mainly affects the hepatocytes. Together, these data indicate that—similar to rodent hepatocytes—cell death in human HepaRG cells is preceded by GSH depletion, protein binding, formation of reactive

click here oxygen and peroxynitrite, and mitochondrial dysfunction. To compare HepaRG cells with other hepatoma cell lines, APAP toxicity was evaluated in HepG2 cells. HepG2 cells treated with 20 mM APAP for 24 hours showed no evidence of GSH depletion, mitochondrial DNA ligase dysfunction (JC-1 assay), or cell injury (LDH release) in response to the toxic dose of APAP (Table 1). However, low levels of protein adducts were identified despite the absence of toxicity (Table 1). Thus, the near absence of drug-metabolizing enzymes drastically reduced the metabolic activation of APAP and prevented any toxicity in HepG2

cells. Loss of mitochondrial membrane integrity can result in the release of proapoptotic proteins, including the caspase activator cytochrome c, into the cytosol. To determine whether or not APAP toxicity in HepaRG cells involves apoptosis, caspase-3 activity was measured in lysates of cells treated for 24 hours with APAP. There was no significant increase in caspase activity over control with 20 (Fig. 5A), 5, or 10 mM APAP (data not shown). In addition, the potent pan-caspase inhibitor Z-VD-fmk had no effect on APAP-induced LDH release at 24 hours (Fig. 5B), suggesting that APAP did not cause apoptosis in HepaRG cells. In contrast, caspase activity was significantly increased when cells were exposed to 100 ng/mL human TNF alpha (rhTNFα) and 5 mM galactosamine for 16.5 hours as a positive control (Fig. 5A). The caspase inhibitor prevented the increase in caspase activity after G/TNF. This indicates that HepaRG cells do have the capacity to undergo apoptotic cell death in response to an appropriate stimulus.