Although safe at therapeutic doses, overdoses of acetaminophen produce a centrilobular hepatic necrosis that can be fatal. Each year, more than 9,000 individuals in the US sustain liver damage due to acetaminophen, with 53 deaths reported in 1996. The rationale for this proposal is based on key findings from our preliminary work that challenge the currently accepted paradigm of toxicity. It is generally accepted that acetaminophen is metabolized by CYP-450 to the reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI), which reacts with GSH, leading to its depletion (and thus decreased peroxide detoxification), and subsequently forming acetaminophen-protein adducts. Our findings indicate that metabolism of acetaminophen to NAPQI may not be the sole determinant of cell lysis and death. We find that the metabolic stress activates resident cells, leading to increase synthesis of nitric oxide (NO) and superoxide, which combine to form peroxynitrite. This entity reacts to form nitrotyrosine-protein adducts and has hydroxyl radical like activity. We detect nitrotyrosine-protein adducts in the hepatic centrilobular cells of acetaminophen-treated mice, the site of the toxicity. Thus, based on this and other preliminary data, we propose a paradigm of acetaminophen hepatotoxicity whereby peroxynitrite generation, coupled with acetaminophen-protein adduct formation, act synergistically to cause cell lysis and death. We hypothesize that peroxynitrite generated during or as a result of acetaminophen metabolic activation is a major determinant of acetaminophen hepatotoxicity. To test this hypothesis, we plan to SA1) Determine the time and dose relationships between acetaminophen metabolism, NO formation, and development of toxicity; SA2) Investigate the roles of NO, superoxide, and peroxynitrite generation in acetaminophen hepatotoxicity by inhibiting NO formation and by using NO, superoxide, and peroxynitrite scavengers; and SA3) Identify the liver cells responsible for NO and superoxide generation during acetaminophen hepatotoxicity. By understanding the role of peroxynitrite in acetaminophen hepatotoxicity, new treatment paradigms may be developed for hepatotoxicity, and this mechanism may be important with other toxins.
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