Liver Protein Targets of Hepatotoxic 4-Bromophenol Metabolites

Abstract

The hepatotoxicity of bromobenzene (BB) is directly related to the covalent binding of both initially formed epoxide and secondary quinone metabolites to at least 45 different liver proteins. 4-Bromophenol (4BP) is a significant BB metabolite and a precursor to reactive quinone metabolites, yet when administered exogenously it has negligible hepatotoxicity compared to BB. The protein adducts of 4BP were thus labeled as non-toxic (Monks, T. J.; Hinson, J. A.; Gillette, J. R. (1982) Life Sci. 30, 841–848). To help identify which BB-derived adducts might be related to its cytotoxicity, we sought to identify the supposedly non-toxic adducts of 4BP and eliminate them from the BB target protein list. Administration of [14C]-4BP to phenobarbital-induced rats resulted in covalent binding of 0.25, 0.33 and 0.42 nmol-eq 4BP/mg protein in the mitochondrial, microsomal and cytosolic fractions, respectively. These values may be compared to published values of 3–6 nmol/mg protein from a comparable dose of [14C]-BB. After subcellular fractionation and 2D electrophoresis, 47 radioactive spots on 2D gels of the mitochondrial, microsomal and cytosolic fractions were excised, digested and analyzed by LC-MS/MS. Twenty nine of these spots contained apparently single proteins, of which 14 were non-redundant. Nine of the 14 are known BB targets. Incubating freshly-isolated rat hepatocytes with 4BP (0.1–0.5 mM) produced time- and concentration-dependent increases in lactate dehydrogenase release and changes in cellular morphology. LC-MS/MS analysis of the cell culture medium revealed rapid and extensive sulfation and glucuronidation of 4BP as well as formation of a quinone-derived glutathione conjugate. Studies with 7-hydroxycoumarin (7HC), (−)-borneol or D-(+)-galactosamine (DGN) showed that inhibiting the glucuronidation/sulfation of 4BP increased the formation of a GSH-bromoquinone adduct, increased covalent binding of 4BP to hepatocyte proteins and potentiated its cytotoxicity. Taken together, our data demonstrate that protein adduction by 4BP metabolites can be toxicologically consequential, and provide a mechanistic explanation for the failure of exogenously administered 4BP to cause hepatotoxicity. Thus the probable reason for the low toxicity of 4BP in vivo is that rapid conjugation limits its oxidation and covalent binding and thus its toxicity.