Identification of Three Protein Targets for Reactive Metabolites of Bromobenzene in Rat Liver Cytosol

Abstract

The hepatotoxicity of bromobenzene and many other simple organic chemicals is believed to be associated with covalent binding of chemically reactive metabolites to cellular proteins. Recently, a rat liver microsomal esterase was shown to be targeted by bromobenzene metabolites formed in vitro [Rombach, E. M., and Hanzlik, R. P. (1998) Chem. Res. Toxicol. 11, 178-184]. To identify protein targets for bromobenzene metabolites in cytosol, we incubated liver microsomes and glutathione-depleted liver cytosol from phenobarbital-treated rats with [(14)C]bromobenzene in vitro. In a separate experiment, we intraperitoneally injected a hepatotoxic dose of [(14)C]bromobenzene to phenobarbital-treated rats. The cytosol fractions from both experiments were recovered and analyzed for protein-bound radioactivity. Under the conditions that were used, 2.6 and 3.9 nmolar equiv of bromobenzene/mg of cytosolic protein was bound in vitro and in vivo, respectively. Denaturing polyacrylamide gel electrophoresis of these cytosolic proteins followed by phosphor imaging analysis revealed several radiolabeled protein bands over a broad molecular mass range, the patterns observed in vitro and in vivo being generally similar to each other. Cytosolic proteins labeled in vitro were separated by ion exchange chromatography and electrophoresis, and three major radioactive bands with estimated molecular masses of ca. 14, 25, and 30 kDa were in-gel digested with trypsin, followed by on-line HPLC electrospray ionization mass spectrometry of the resulting peptide mixtures. For the three protein bands, the observed peptide masses were found to match the predicted tryptic fragments of liver fatty acid binding protein, glutathione transferase subunit A1, and carbonic anhydrase isoform III, respectively, with 83, 45, and 59% coverage of the corresponding complete sequences. The possible relationship of the adduction of these proteins to the toxicological outcome is discussed.