Reactive chemicals re often cytotoxic because they covalently modify (i.e. denature) important cellular macromolecules. Chemically reactive metabolites of otherwise innocuous compounds also covalently modify cellular macromolecules, in most cases with toxic consequences. Bromobenzene (BB) exemplifies the latter situation, and is an important model compound for studying biochemical mechanisms of chemically-induced hepatotoxicity. The applicant's laboratory recently identified the structure of ten protein-S-adducts and one protein-histidine adduct; these derived from two epoxide- and four quinone metabolites of BB and represented ca. 10% of total covalent binding. The applicant now proposes to utilize this background to pursue two major objectives. One is the elucidation of structures which account for the majority of radioactivity covalently bound to liver proteins of rats treated with [14C]-BB. Although adducts have been released by acid hydrolysis, the use of milder enzymatic hydrolysis will be explored, and systematic separation and elucidation of adduct structures will be carried out. The second major objective is to isolate and identify specific rat hepatocyte target proteins adducted by reactive metabolites of BB, to determine the nature of the adduct(s) formed to each, to evaluate whether metabolite adduction of a given protein contributes to the hepatotoxicity of BB, and to determine (via inter-laboratory collaboration) whether the same proteins are targeted by reactive metabolites of other hepatotoxic chemicals (e.g. from acetaminophen or halothane). Polyclonal anti-hepten antibodies capable of selective recognition of protein adducts of BB- epoxide vs. -quinone metabolites in ELISA assays have been raised. However, in Western blotting applications, these antibodies cross-reacted with some non-adducted liver proteins was detected. Therefore, monoclonal (i.e. monospecific) anti-hapten antibodies will also be raised and evaluated. In addition, autoradiography has been used successfully to detect proteins adducted with [14C]-BB on PAGE gels. These methods will be used to detect and monitor the purification of BB-adducted proteins formed in vitro and in vivo. Target proteins will be analyzed using combinations of peptide mapping, N-terminal and internal peptide sequencing and electrospray LC-MS/MS. It is anticipated that identifying target proteins will help bridge a major knowledge-gap between early cellular events which specifically involve bromobenzene or its metabolites, and late-phase events which may be common cytotoxic mechanisms activated by many different cytotoxic chemicals. Conceivably, better understanding of toxic mechanisms could lead to more effective ways of managing clinical toxicities resulting from chemicals and/or drugs which are bioactivated to chemically reactive metabolites.
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