Acetaminophen (APAP) poisoning is the most common cause of acute liver failure in the U.S. The early metabolic events in APAP-mediated toxicity are well described; however, the subsequent steps in development of toxicity are unknown. Using freshly isolated mouse hepatocytes treated with APAP, we recently showed that toxicity involves mitochondrial permeability transition (MPT), a mechanism mediated by oxidative stress and leads to a large increase in oxidative stress. We also demonstrated that protein tyrosine nitration, indicative of peroxynitrite, coincided with toxicity at later stages of treatment (2-5hr) following washing cells free of APAP. More recent studies have examined the mechanism leading to induction of MPT. Hence, addition of inhibitors of MPT, oxidant stress, and nitric oxide synthase (NOS1) inhibited mitochondrial injury, nitration, and development of APAP-mediated toxicity. Interestingly, GSH depletion was maximal by 0.5hr, but incubation of hepatocytes with APAP for 1hr followed by washing to remove APAP and reincubation did not result in toxicity. Therefore, we hypothesize that there are 3 stages of APAP toxicity: STAGE 1: the metabolic stage which occurs by 1hr and leads to STAGE 2: the oxidative stage which leads to mitochondrial injury and oxidant production prior to MPT; and finally STAGE 3: the toxicity stage which occurs with a large increase in protein nitration, MPT, and toxicity. Three specific aims are proposed. Specific Aim 1: Determine the early events (before 2 hr) important in the oxidative stage (Stage 2) of APAP-induced toxicity leading to MPT in hepatocytes. We hypothesize that oxidative stress develops in Stage 2 and is critical to the initiation of Stage 3. Specific Aim 2: Determine the molecular events which occur during and following MPT (Stage 3) in APAP-induced toxicity using in vitro and in vivo models. We hypothesize that mitochondrial superoxide and NOS1 activation play a fundamental role in MPT mediated toxicity. Specific Aim 3: Identify the hepatic proteins that are tyrosine nitrated in APAP toxicity. We hypothesize that a different subset of proteins are nitrated during the early onset of Stage 2 versus the later onset of Stage 3. The overall goal of the proposal is to test the hypothesis that mitochondrial protein nitration plays a fundamental role in induction and during MPT and toxicity of APAP. PUBLIC HEALTH RELEVANCE: Acetaminophen poisoning is the single most common cause of acute liver failure in the United States. An overdose of the analgesic can produce fatal liver necrosis. It is well recognized that metabolism to a reactive metabolite that depletes glutathione and covalently binds to protein is important. However, the subsequent events are unknown. Recent preliminary data in the PI's laboratory indicate that acetaminophen toxicity occurs with mitochondrial dysfunction and production of reactive oxygen/nitrogen species. The goal of the project will be to understand the role of reactive oxygen/nitrogen species and mitochondrial dysfunction in acetaminophen toxicity in mice. The ultimate goal is to develop new approaches to treat acetaminophen poisoning in humans.