Abstract
Acetaminophen (AAP) overdose causes formation of nitrotyrosine, a footprint of peroxynitrite, in centrilobular hepatocytes. The importance of peroxynitrite for the pathophysiology, however, is unclear. C3Heb/FeJ mice were treated with 300 mg/kg AAP. To accelerate the restoration of hepatic glutathione (GSH) levels as potential endogenous scavengers of peroxynitrite, some groups of animals received 200 mg of GSH/kg i.v. at different time points after AAP. AAP induced severe liver cell damage at 6 h. Total liver and mitochondrial glutathione levels decreased by >90% at 1 h but recovered to 75 and 45%, respectively, of untreated values at 6 h after AAP. In addition, the hepatic and mitochondrial glutathione disulfide (GSSG) content was significantly increased over baseline, suggesting a mitochondrial oxidant stress. Moreover, centrilobular hepatocytes stained for nitrotyrosine. Treatment with GSH at t= 0 restored hepatic GSH levels and completely prevented the mitochondrial oxidant stress, peroxynitrite formation, and liver cell injury. In contrast, treatment at 1.5 and 2.25 h restored hepatic and mitochondrial GSH levels but did not prevent the increase in GSSG formation. Nitrotyrosine adduct formation and liver injury, however, was substantially reduced. GSH treatment at 3 h after AAP was ineffective. Similar results were obtained when these experiments were repeated with glutathione peroxidase-deficient animals. Our data suggest that early GSH treatment (t = 0) prevented cell injury by improving the detoxification of the reactive metabolite of AAP. Delayed GSH treatment enhanced hepatic GSH levels, which scavenged peroxynitrite in a spontaneous reaction. Thus, peroxynitrite is an important mediator of AAP-induced liver cell necrosis.
Footnotes
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This work was supported in part by National Institutes of Health Grant AA 12916.
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DOI: 10.1124/jpet.102.038968
- Abbreviations:
- AAP
- acetaminophen
- NAPQI
- N-acetyl-p-benzoquinone imine
- GSH
- reduced glutathione
- iNOS
- inducible nitric-oxide synthase
- Gpx1
- glutathione peroxidase-1
- ALT
- alanine aminotransferase
- NEM
- N-ethylmaleimide
- GSSG
- glutathione disulfide
- KPP
- potassium phosphate buffer
- BSA
- bovine serum albumin
- NT
- nitrotyrosine
- Received May 15, 2002.
- Accepted July 2, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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