Acute ethanol intoxication induces oxidative stress and apoptosis in primary cultured hepatocytes. Oxidative stress can trigger mitochondrial cytochrome c release initiating the mitochondrial pathway of apoptosis. Based on this information, we formulated the hypothesis that ethanol-induced oxidative stress causes mitochondrial dysfunction resulting in apoptosis. In the present study, we found that the mitochondrial membrane permeability transition (MPT) is essential for induction of mitochondrial cytochrome c release and caspase activation of ethanol. The short-term incubation with ethanol (50 mmol/L) induced the MPT, cytochrome c release, caspase activation, and apoptosis of cultured rat hepatocytes. Hepatocyte apoptosis was prevented by caspase inhibitors (i.e., Z-VAD-fmk, DEVD-cho, and DMQD-cho). An MPT inhibitor, cyclosporin A, also prevented ethanol-induced cytochrome c release, caspase activation, and apoptosis, suggesting that acute ethanol-induced apoptosis is MPT dependent. Ethanol-induced MPT was also attenuated by N′N′-dimethylthiourea (DMTU, a scavenger of hydrogen peroxide, 10 mmol/L) and N-acetyl-cysteine (NAC, an antioxidant, 5 mmol/L). Preventing hepatocyte MPT by DMTU or NAC attenuated cytochrome c release as well as caspase activation, suggesting that ethanol-induced oxidative stress mediates the MPT. Thus, acute ethanol induces MPT via oxidative stress, and the MPT mediates mitochondrial pathway of apoptosis in hepatocytes exposed to acute ethanol.
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