Mechanisms of acetaminophen-induced hepatotoxicity: Role of mitochondrial permeability transition, reactive oxygen, and reactive nitrogen in freshly isolated mouse hepatocytes

Hepatic necrosis mediated by both acetaminophen (APAP) and chloroform occurs with formation of a reactive metabolites leading to GSH depletion and covalent binding. To separate metabolic events from toxic events, freshly isolated mouse hepatocytes were preincubated with the hepatotoxins, washed to remove the drugs, and reincubated in media alone. At the end of the preincubation, toxicity was not different between control and treated cells; however, treated cells reincubated with media alone resulted in a marked increase in toxicity. Confocal microscopy studies utilizing the dyes TMRM, calcein AM, and JC-1 indicated that toxicity occurred with mitochondrial permeability transition (MPT) and loss of mitochondrial membrane potential. Addition of the MPT inhibitors cyclosporine A and/or trifluoperazine in the reincubation phase inhibited toxicity, MPT, and loss of mitochondrial membrane potential. Inclusion of the redox sensitive dye, dichlorodihydrofluorescin, in the reincubation phase showed a large increase in fluorescence indicating oxidative stress. These data suggested that MPT and oxidative stress are important mechanisms in the hepatocyte toxicity produced by APAP and chloroform. Hepatocytes were also incubated with APAP and chloroform to evaluate the role of the nitrating agent peroxynitrite as the toxic reactive oxygen species. Western blot and/or ELISA indicated nitration of tyrosine in treated hepatocytes, and the amount of nitration correlated with development of toxicity. Inclusion of constitutive nitric oxide synthase inhibitors and peroxynitrite scavengers in the reincubation media resulted in inhibition of toxicity, dichlorodihydrofluorescin oxidation, and protein nitration; however, inducible nitric oxide synthase inhibitors had no effect. These data are consistent with a hypothesis where APAP- and chloroform-induced cell death occurs in two phases, a metabolic phase and a toxic phase. The toxic phase occurs with increased oxidative stress, loss of mitochondrial membrane potential, MPT, protein nitration, and toxicity. These data suggest that the toxic reactive oxygen species is peroxynitrite, and it is formed by a constitutive nitric oxide synthase.