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 modification 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), which is the reactive metabolite thought to be most significant toxicologically, also arylates protein side chains, although to a lesser extent. To facilitate the detection, isolation, and identification of rat liver proteins specifically adducted by BBO, we raised polyclonal antibodies capable of recognizing S-(p-bromophenyl)cysteine moieties (anti-BP) by immunizing rabbits with p-bromophenylmercapturic acid conjugated to keyhole limpet hemocyanin. The antiserum had a high titer, showed a high specificity for hapten in competitive ELISA with hapten analogues, and performed well in Western blot experiments using synthetically haptenized control proteins. When used for Western analysis of protein fractions from in vitro incubations of rat liver microsomes with [14C]BB, affinity-purified anti-BP recognized a limited number of bands, each of which also contained 14C. One of these bands corresponds to hydrolase B, a nonspecific esterase known to contain one free sulfhydryl group and previously shown to be a target protein for [14C]BB metabolites.

Identification of a rat liver microsomal esterase as a target protein for bromobenzene metabolites.

The hepatotoxicity of bromobenzene and many other simple organic molecules has been associated with their biotransformation to chemically reactive metabolites and the subsequent covalent binding of those metabolites to cellular macromolecules. To identify proteins targeted by bromobenzene metabolites, we incubated [14C]bromobenzene in vitro with liver microsomes from phenobarbital-induced rats under conditions which typically led to covalent binding of 2-4 nmol equiv of bromobenzene/mg of protein. Microsomal proteins were solubilized with detergent, separated by chromatography and electrophoresis, and analyzed for 14C by phosphorimaging of stained blots. Much of the radioactivity was associated with several bands of proteins of ca. 50-60 kDa, plus another prominent band around 70 kDa, but labeling density appeared to vary considerably overall. A major radiolabeled protein was purified by preparative electrophoresis and submitted to automated Edman microsequencing. Its N-terminal sequence was found to correspond to that of a known rat liver microsomal carboxylesterase (E.C. 3.1.1.1) previously identified as a target for reactive metabolites of halothane. The extent to which covalent modification of this protein by reactive metabolites contributes to the production of hepatotoxic effects remains to be determined.

Detection of benzoquinone adducts to rat liver protein sulfhydryl groups using specific antibodies.

Benzoquinone is an electrophilic metabolite of bromobenzene and other simple aromatic compounds of toxicological interest including benzene, phenol, hydroquinone, and acetaminophen. In reacting with proteins benzoquinone shows great selectivity for Michael addition to sulfhydryl groups and formation of S-(2,5-dihydroxyphenyl) protein adducts. To facilitate the specific detection and eventual isolation and identification of such adducted proteins, we prepared an antiserum capable of recognizing hydroquinone moieties by immunizing rabbits with keyhole limpet hemocyanin modified with 3-[2,5-dihydroxyphenyl)thio]propanoyl groups as haptens. The antiserum had a high titer and showed high specificity for hapten in competitive ELISA with hapten analogues. In Western blot experiments the antiserum detected not only synthetically haptenized control proteins but also several proteins from rat liver microsomes that had been incubated in vitro with [14C]bromobenzene. This binding was completely blocked by free hapten, showing that it was hapten-specific. Each of the microsomal protein bands detected in the Western blots also contained radioactivity, but not all radioactive protein bands reacted with antibody. This antiserum should prove useful in exploring the role of protein arylation by benzoquinone in cytotoxic responses to its metabolic precursors.