Detection of adducts of bromobenzene 3,4-oxide with rat liver microsomal protein sulfhydryl groups using specific antibodies

The hepatotoxicity of bromobenzene (BB) has been attributed to covalent mod ification of cellular proteins by reactive metabolites generated during its oxidative biotransformation. Much of the net covalent binding which occurs originates via quinone metabolites, but bromobenzene 3,4-oxide (BBO), whic h is the reactive metabolite thought to be most significant toxicologically , also arylates protein side chains, although to a lesser extent. To facili tate the detection, isolation, and identification of rat liver proteins spe cifically adducted by BBO, we raised polyclonal antibodies capable of recog nizing S-(p-bromophenyl)cysteine moieties (anti-BP) by immunizing rabbits w ith p-bromophenylmercapturic acid conjugated to keyhole limpet hemocyanin. The antiserum had a high titer, showed a high specificity for hapten in com petitive ELISA with hapten analogues, and performed well in Western blot ex periments using synthetically haptenized control proteins. When used for We stern analysis of protein fractions from in vitro incubations of rat liver microsomes with [C-14]BB, affinity-purified anti-BP recognized a limited nu mber of bands, each of which also contained C-14. One of these bands corres ponds to hydrolase B, a nonspecific esterase known to contain one free sulf hydryl group and previously shown to be a target protein for [C-14]BB metab olites.