NITROTYROSINE-PROTEIN ADDUCTS IN HEPATIC CENTRILOBULAR AREAS FOLLOWING TOXIC DOSES OF ACETAMINOPHEN IN MICE

Treatment of mice with a toxic dose of acetaminophen (300 mg/kg, ip) s ignificantly increased hepatotoxicity at 4 h, as evidenced by histolog ical necrosis in the centrilobular areas of the liver, and increased s erum levels of alanine aminotransferase (ALT) (from 8 +/- 1 IU/L in sa line-treated mice to 3226 +/- 892 IU/L in the acetaminophen-treated mi ce). Serum levels of nitrate plus nitrite (a marker of nitric oxide sy nthesis) were also increased from 62 +/- 8 mu M in saline-treated mice to 110 +/- 14 mu M in acetaminophen-treated mice (P < 0.05). Regressi on analysis of serum ALT levels to serum nitrate plus nitrite levels i n individual mice revealed a positive, linear relationship between ser um ALT levels and serum nitrate plus nitrite levels with a correlation coefficient of 0.9 (P < 0.05). The y intercept value (nitrate plus ni trite level) was 63 +/- 15 mu M. Immunohistochemical analysis of liver sections from acetaminophen-intoxicated mice using an anti-3-nitrotyr osine antibody indicated tyrosine nitration in the proteins of the cen trilobular cells. Tyrosine nitration has been shown to occur by peroxy nitrite, a reactive intermediate formed by an extremely rapid reaction of nitric oxide and superoxide and a species which also has hydroxyl radical-like activity. Analysis of liver sections using an anti-acetam inophen antiserum indicated the centrilobular cells also contained ace taminophen-protein adducts, a reaction of the metabolite N-acetyl-p-be nzoquinone imine with cysteine residues on proteins. These data are co nsistent with acetaminophen metabolic activation leading to increased synthesis of nitric oxide and superoxide and to peroxynitrite as an im portant intermediate in the toxicity.