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

The hepatic centrilobular necrosis produced by the analgesic! antipyretic a cetaminophen correlates with metabolic activation of the drug leading to it s covalent binding to protein. However, the molecular mechanism of the toxi city is not known. Recent immunohistochemical analyses using an antinitroty rosine antiserum indicated that nitrotyrosine protein adducts co-localized with the acetaminophen-protein adducts in the centrilobular cells of the li ver. Nitration of proteins is believed to occur by peroxynitrite, a substan ce formed by the rapid reaction of superoxide with nitric oxide. Nitric oxi de and superoxide may be formed by activated Kupffer cells or by other cell s. 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 acetami nophen-treated mice were successfully analyzed for nitrated proteins. The i mmunogen for this new antiserum was synthesized by coupling 3-nitro-4-hydro xybenzoic acid to keyhole limpet hemocyanin. A rabbit immunized with this a dduct yielded a high titer of an antiserum that recognized BSA nitrated wit h peroxynitrite. Immunoblot analysis of nitrated BSA indicated that nitroty rosine 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-t reated mice (300 mg/kg) indicate that the major nitrotyrosine protein adduc ts produced have molecular weights of 36 kDa, 44 kDa, and 85 kDa, The 85-kD a protein stained with the most intensity. The hepatic homogenates of the a cetaminophen- treated mice showed significantly increased levels of all pro tein adducts.