Mechanism of Acetaminophen-Induced Hepatotoxicity: Covalent Binding versus Oxidative Stress

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• 作者：Jennifer D. Gibson ; Neil R. Pumford ; Victor M. Samokyszyn ; and Jack A. Hinson
• 刊名：Chemical Research in Toxicology
• 出版年：1996
• 出版时间：April 1996
• 年：1996
• 卷：9
• 期：3
• 页码：580 - 585
• 全文大小：143K
• 年卷期：v.9,no.3(April 1996)
• ISSN：1520-5010

文摘

The hepatotoxicity of acetaminophen is believed to be mediated bythe reactive metaboliteN-acetyl-p-benzoquinone imine; however, themechanism by which this metabolite producesthe toxicity is unknown. The metabolite, which is both anelectrophile and an oxidizing agent,may covalently bind to critical proteins, or it may initiate oxidativedamage. We have previouslydeveloped a Western blot assay for detection of acetaminophencovalently bound to proteinand have reported the relationship between covalent binding and thedevelopment ofhepatotoxicity. Recently, we developed a Western blot assay forprotein aldehyde formation,which may occur via the reactive oxygen species, the hydroxyl radical.In this paper, we havecompared covalent binding to protein aldehyde formation. Toxicdoses of acetaminophen (400mg/kg) were administered to mice, and the mice were subsequently killedat 0, 1, 2, 4, and 6h. Since the oxidizing agent FeSO₄ has been reportedto potentiate lipid peroxidation whenadministered with acetaminophen, other mice received FeSO₄(100 mg/kg) plus acetaminophen.Compared to saline-treated control mice, acetaminophen treatmentsignificantly increasedserum alanine aminotransferase levels, an index of hepatotoxicity, at 4and 6 h, but not at 1or 2 h. Acetaminophen plus FeSO₄ treatment of micesignificantly increased serum alanineaminotransferase levels at 2, 4, and 6 h compared to controls.Levels of alanine aminotransferase in serum of acetaminophen plus ferrous sulfate-treated mice werehigher at 4 and 6 hthan those of acetaminophen-treated mice, but not significantlydifferent. FeSO₄ alone didnot increase alanine aminotransferase levels. Western blot assaysrevealed that acetaminophendid not cause an increase in protein aldehydes over control at anytime, nor did acetaminophenplus FeSO₄; however, FeSO₄ alone increased theintensity of staining of the immunoblot forprotein aldehydes over control at all times after 0 time.Acetaminophen-protein adducts weredetected in acetaminophen- and acetaminophen plusFeSO₄-treated mice. In vitroexperimentsindicated that FeSO₄ plus tert-butylhydroperoxide in the presence of bovine serum albuminincreased protein aldehyde formation. Inclusion of acetaminophenin the incubation mixtureinhibited protein oxidation of bovine serum albumin in a concentrationdependent manner.The data indicate that acetaminophen quenches protein oxidation,presumably by reactingwith the hydroxyl radical. These data are consistent with thetheory that acetaminophencovalent binding is the primary mechanism of toxicity and argue againsta role for proteinoxidation in acetaminophen hepatotoxicity.