Identification of Epoxide- and Quinone-Derived Bromobenzene Adducts to Protein Sulfur Nucleophiles

Abstract

Bromobenzene (BB) hepatotoxicity is widely attributed to the alkylation of cellular proteins by chemically reactive metabolites, particularly BB-3,4-oxide. This laboratory recently reported the first conclusive evidence that BB epoxides actually do alkylate proteins; i.e., acid hydrolysates of hepatic proteins from phenobarbital-(PB-) induced BB-treated rats contain S-(o-, S-(m-, and S-(p-bromophenyl)cysteine [Weller, P. E., and Hanzlik, R. P. (1991) Chem. Res. Toxicol. 4, 17–20]. However, these three compounds account for Ã0.5% of total protein covalent binding. Bro-moquinone metabolites of BB are also suspected of alkylating proteins. To search for such adducts to protein cysteinyl or methionyl residues, we heated hepatic proteins from PB-induced BB-treated rats with a two-phase mixture of 16 N KOH and CH3I (“alkaline permethylation”). Under these conditions S-alkylated residues are cleaved via elimination and the phenoxide and thiophenoxide groups on the fragments are methylated. Product analysis by 14C HPLC and GC/MS revealed o-, m-, and p-bromothioanisoles in amounts comparable to the content of S-(bromophenyl)cysteines found by acid hydrolysis (para ≫ meta, ortho). This, too, clearly implicates protein-SH alkylation by BB-2,3- and 3,4-oxides. In addition, 2,3-dimethoxy-5-bromothioanisole and another unidentified isomer were observed. However, by far the major adduct (5–6% of total covalent binding) was 2,5-dimethoxythioanisole (i.e., a debrominated adduct). When BB-d5 was administered, the latter contained mostly 3 deuterium atoms/mol. These latter results clearly show that alkylation of protein sulfur nucleophiles in vivo by quinone metabolites is 10–15 times more extensive than their alkylation by BB epoxides. After BB-d5 was administered, the bromothioanisoles and dimethoxybromothioanisoles contained 4 and 2 deuterium atoms/mol, respectively. A weighted average calculation of deuterium retention across the six major sulfur adducts agreed well with 3H/14C retention ratios determined earlier for total liver protein covalent binding of dual-labeled [3H/14C]BB, indicating that the overall pattern of BB metabolite binding to all protein nucleophiles may closely parallel that seen here specifically for protein sulfhydryl groups. The identification of a variety of specific BB-derived adducts to protein now affords the opportunity to investigate their relative contributions to the toxicity of bromobenzene. © 1991, American Chemical Society. All rights reserved.