Bromobenzene (BB) is a model hepatotoxin believed to exert its harmful effects by undergoing biotransformation to one or more chemically reactive metabolites capable of reacting covalently with cellular nucleophiles, thereby disrupting vital cellular processes. While BB-3,4-oxide has long been regarded to be the critical reactive metabolite, recent work in several laboratories including our own suggests an important role for quinone metabolites in BG covalent binding. However, the toxicological significance of quinone metabolites of BB has ben questioned. We hope to clarify this confusing situation. One objective of the proposed study is to elucidate the chemistry of metabolic activation and covalent binding of BB and several of its hepatotoxic derivatives. For this we will determine the complete chemical structures of products from the chemical trapping of reactive metabolites in vitro, and the structures of amino acid-bromobenzene (aa-BB) adducts found in hydrolysates of proteins covalently labeled with BB metabolites. A second objective is to determine whether microsomal systems accurately reflect the covalent binding processes which occur in living cells and in vivo. For this we will compare HPLC profiles of aa-BB adducts formed in microsomes, isolated hepatocytes, and in liver tissue in vivo. We will also compare profiles of adducts formed from bromophenols to those formed from BB to determine whether any adducts arise uniquely from BB (e.g. via an epoxide metabolite). The complete chemical structures of major adducts, and those which appear uniquely from BB, will be determined. Finally, we will compare adduct profiles from control, phenobarbital- and 7,8-naphthoflavone-pretreated rats to determine if certain adducts are increased (or decreased) more than others by treatments which potentiate (or diminish) the hepatotoxicity of bromobenzene. In this way we hope to determine which adducts are toxicologically the most significant.
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