COMPARISON OF COVALENT BINDING OF ACETAMINOPHEN AND THE REGIOISOMER 3'-HYDROXYACETANILIDE TO MOUSE-LIVER PROTEIN

The hepatotoxicity of the analgesic acetaminophen has been previously attributed to metabolic activation by cytochrome P450 to the reactive intermediate N-acetyl-p-benzoquinone imine. At therapeutic doses this species is detoxified by reaction with glutathione; however, following a hepatotoxic dose, liver glutathione levels are depleted and the met abolite covalently binds primarily to cysteine groups on proteins as 3 -(cystein-S-yl)acetaminophen adducts. Altered function of critical pro teins has been postulated to be the mechanism of hepatotoxicity. Coval ent binding has been studied by both radiochemical methods and immunoc hemical methods. Utilizing Western blot analysis with an antiserum whi ch recognizes acetaminophen we have previously shown that covalent bin ding occurs on a number of proteins in various hepatic fractions. In a n effort to better understand the role of covalent binding in the toxi city, others have studied the non-hepatotoxic isomer 3'-hydroxyacetani lide. Administration of large doses of radiolabeled acetaminophen or 3 '-hydroxyacetanilide resulted in similar levels of covalent binding to proteins. To better understand the role of covalent binding in toxici ty we have administered mice 3'-hydroxyacetanilide and acetaminophen, and analyzed liver fractions for protein adducts using anti-3-(cystein -S-yl)acetaminophen and anti-arylacetamide antisera in Western blot as says. Analysis of liver fractions from acetaminophen-treated mice, wit h both antisera showed, as has been previously reported, that acetamin ophen covalently binds to a number of hepatic proteins. In liver from 3'-hydroxyacetanilide-treated mice, covalent adducts were detected wit h an anti-arylacetamide antiserum only. A major 3'-hydroxyacetanilide protein adduct was observed in microsomes at 50 kDa. Minor adducts wer e observed at 47 kDa in microsomes and 56 kDa in cytosol. 3'-Hydroxyac etanilide protein adducts were not observed in the 10 000 x g pellet. Densitometric analysis of a time course of 3'-hydroxyacetanilide prote in adducts indicated that peak levels of the 50 kDa microsomal protein adduct occurred at 1 h and subsequently decreased. Copyright (C) 1997 Elsevier Science Ireland Ltd.