Mitochondrial permeability transition and peroxynitrite in acetaminophen toxicity in freshly isolated mouse hepatocytes

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The role of metabolism in the hepatotoxicity of the analgesic acetaminophen (APAP) is well described. At therapeutic doses the CYP metabolite N-acetyl-p-benzoquinone imine is detoxified by conjugation with glutathione (GSH); however, at high doses hepatic GSH is depleted and the metabolite covalently binds to protein. We and others have shown that covalent binding correlates with hepatic necrosis under a variety of conditions. However, biochemical mechanisms of toxicity are poorly understood. We have examined the role of mitochondrial permeability transition (MPT) and peroxynitrite in APAP toxicity in freshly isolated mouse hepatocytes. These hepatocytes have high levels of CYP enzymes necessary for development of toxicity. MPT occurs as a result of oxidative stress and leads to increased oxidative stress. It results in decreased ATP synthesis and is a lethal event. As previously reported we found that incubation of hepatocytes with APAP (1 mM) for 2 hrs results in minimal toxicity (ALT release). Subsequently hepatocytes were washed twice to remove APAP. Reincubation in media alone resulted in significant toxicity in the reincubation phase (3-5 hr) with toxicity occurring in approximately 75% of the hepatocytes by 5 hrs. The amount of toxicity was not significantly different if APAP was added back during the reincubation phase. Confocal microscopy studies utilizing the dye calcein indicated MPT.