Vascular and hepatocellular peroxynitrite formation during acetaminophen toxicity: Role of mitochondrial oxidant stress

Peroxynitrite may be involved in acetaminophen-induced liver damage. Howeve r, it is unclear if peroxynitrite is generated in hepatocytes or in the vas culature. To address this question, we treated C3Heb/FeJ mice with 300 mg/k g acetaminophen and assessed nitrotyrosine protein adducts as indicator for peroxynitrite formation. Vascular nitrotyrosine staining was evident befor e liver injury between 0.5 and 2 h after acetaminophen treatment. However, liver injury developed parallel to hepatocellular nitrotyrosine staining be tween 2 and 6 h after acetaminophen. The mitochondrial content of glutathio ne disulfide, as indicator of reactive oxygen formation determined 6 h afte r acetaminophen, increased from 2.8 +/- 0.6% in controls to 23.5 +/- 5.1%. A high dose of allopurinol (100 mg/kg) strongly attenuated acetaminophen pr otein-adduct formation and prevented the mitochondrial oxidant stress and l iver injury after acetaminophen. Lower doses of allopurinol, which are equa lly effective in inhibiting xanthine oxidase, were not protective and had n o effect on nitrotyrosine staining and acetaminophen protein adduct formati on. In vitro experiments showed that allopurinol is not a direct scavenger of peroxynitrite. We conclude that there is vascular peroxynitrite formatio n during the first 2 h after acetaminophen treatment. On the other hand, re active metabolites of acetaminophen bind to intracellular proteins and caus e mitochondrial dysfunction and superoxide formation. Mitochondrial superox ide reacts with nitric oxide to form peroxynitrite, which is responsible fo r intracellular protein nitration. The pathophysiological relevance of vasc ular peroxynitrite for hepatocellular peroxynitrite formation and liver inj ury remains to be established.