Acetaminophen (APAP)‐Induced Hepatotoxicity and Protein Nitration in neuronal Nitric Oxide Synthase (nNOS) Knockout Mice

Abstract

In overdose APAP is hepatotoxic. Toxicity occurs by metabolism to N‐acetyl‐p‐benzoquinone imine which covalently binds to proteins followed by protein nitration. Nitration is by peroxynitrite, formed from superoxide and nitric oxide (NO). In freshly isolated hepatocytes we reported that the NO is from nNOS which is believed to be in mitochondria. MnSOD is nitrated by 1 hr in APAP toxicity in mice resulting in MnSOD inactivation. This leads to increased peroxynitrite. To understand the role of nNOS in APAP toxicity and MnSOD nitration, nNOS knockout mice (KO) and wildtype mice (WT) were administered APAP (300 mg/kg). Serum ALT significantly increased at 6 and 8 h in WT and at 8 h in KO. ALT levels in WT were significantly higher than in KO at 6 h but not 8 h. There were no significant differences in hepatic GSH depletion, APAP protein covalent binding, or histopathological evaluation of hepatic necrosis. Hepatic MnSOD activity was significantly lower at 1–4 h in WT and 8 h in KO. MnSOD activity in WT was significantly higher than in KO at 8 h. Western blot analysis for MnSOD nitration indicated a significant increase at 1–8 h in WT and at 6 and 8 h in KO. There was dramatically more nitration of MnSOD in WT than in KO. Nitration in WT was significantly different from KO at 1–6 h. These data indicate that nNOS is important in protein nitration but the role of nNOS and nitration in in vivo toxicity is unclear. Supported by 1 R01 DK079008.