MECHANISM OF ACETAMINOPHEN-INDUCED HEPATOTOXICITY - COVALENT BINDING VERSUS OXIDATIVE STRESS

The hepatotoxicity of acetaminophen is believed to be mediated by the reactive metabolite N-acetyl-p-benzoquinone imine; however, the mechan ism by which this metabolite produces the toxicity is unknown. The met abolite, which is both an electrophile and an oxidizing agent, may cov alently bind to critical proteins, or it may initiate oxidative damage . We have previously developed a Western blot assay for detection of a cetaminophen covalently bound to protein and have reported the relatio nship between covalent binding and the development of hepatotoxicity. Recently, we developed a Western blot assay for protein aldehyde forma tion, which may occur via the reactive oxygen species, the hydroxyl ra dical. In this paper, we have compared covalent binding to protein ald ehyde formation. Toxic doses of acetaminophen (400 mg/kg) were adminis tered to mice, and the mice were subsequently killed at 0, 1, 2, 4, an d 6 h. Since the oxidizing agent FeSO4 has been reported to potentiate lipid peroxidation when administered with acetaminophen, other mice r eceived FeSO4 (100 mg/kg) plus acetaminophen. Compared to saline-treat ed control mice, acetaminophen treatment significantly increased serum alanine aminotransferase levels, an index of hepatotoxicity, at 4 and 6 h, but not at 1 or 2 h. Acetaminophen plus FeSO4 treatment of mice significantly increased serum alanine aminotransferase levels at 2, 4, and 6 h compared to controls. Levels of alanine aminotransferase in s erum of acetaminophen plus ferrous sulfate-treated mice were higher at 4 and 6 h than those of acetaminophen-treated mice, but not significa ntly different. FeSO4 alone did not increase alanine aminotransferase levels. Western blot assays revealed that acetaminophen did not cause an increase in protein aldehydes over control at any time, nor did ace taminophen plus FeSO4; however, FeSO4 alone increased the intensity of staining of the immunoblot for protein aldehydes over control at all times after 0 time. Acetaminophen-protein adducts were detected in ace taminophen- and acetaminophen plus FeSO4-treated mice. In vitro experi ments indicated that FeSO4 plus tert-butyl hydroperoxide in the presen ce of bovine serum albumin increased protein aldehyde formation. Inclu sion of acetaminophen in the incubation mixture inhibited protein oxid ation of bovine serum albumin in a concentration dependent manner. The data indicate that acetaminophen quenches protein oxidation, presumab ly by reacting with the hydroxyl radical. These data are consistent wi th the theory that acetaminophen covalent binding is the primary mecha nism of toxicity and argue against a role for protein oxidation in ace taminophen hepatotoxicity.