Western blot analysis for nitrotyrosine protein adducts in livers of saline-treated and acetaminophen-treated mice.

Abstract

The hepatic centrilobular necrosis produced by the analgesic/antipyretic acetaminophen correlates with metabolic activation of the drug leading to its covalent binding to protein. However, the molecular mechanism of the toxicity is not known. Recent immunohistochemical analyses using an antinitrotyrosine antiserum indicated that nitrotyrosine protein adducts co-localized with the acetaminophen-protein adducts in the centrilobular cells of the liver. Nitration of proteins is believed to occur by peroxynitrite, a substance formed by the rapid reaction of superoxide with nitric oxide. Nitric oxide and superoxide may be formed by activated Kupffer cells or by other cells. Because we were unable to successfully utilize the commercial antiserum in Western blot analyses of liver fractions, we developed a new antiserum. With our antiserum, liver fractions from saline-treated control and acetaminophen-treated mice were successfully analyzed for nitrated proteins. The immunogen for this new antiserum was synthesized by coupling 3-nitro-4-hydroxybenzoic acid to keyhole limpet hemocyanin. A rabbit immunized with this adduct yielded a high titer of an antiserum that recognized BSA nitrated with peroxynitrite. Immunoblot analysis of nitrated BSA indicated that nitrotyrosine present in a protein sample could be easily detected at levels of 20 pmoles. Immunohistochemical analyses indicated that nitrotyrosine protein adducts were detectable in the centrilobular areas of the liver. Immunoblot analysis of liver homogenates from both saline-treated and acetaminophen-treated mice (300 mg/kg) indicate that the major nitrotyrosine protein adducts produced have molecular weights of 36 kDa, 44 kDa, and 85 kDa. The 85-kDa protein stained with the most intensity. The hepatic homogenates of the acetaminophen- treated mice showed significantly increased levels of all protein adducts.