Many simple organic molecules containing phenyl substituents or benzene rings become cytotoxic upon biotransformation to reactive electrophilic metabolites. Prime examples include halothane, acetaminophen and bromobenzene (BB). Their hepatotoxicity is correlated with covalent binding of reactive metabolites to cellular proteins. As a start toward elucidating the biochemical mechanism(s) of their cytotoxicity we identified the structures of ten adducts of BB metabolites to protein-SH groups; most arose via quinone metabolites, but we also found that BB-3,4-oxide (BBO), thought to be the primary """"""""toxic"""""""" metabolite of BB, alkylates histidine and lysine as well as cysteine residues of rat liver proteins. Key questions concerning the mechanism of cell injury by reactive metabolites include the identity of the proteins they target and the functional consequences of their covalent modification. We recently identified several rat liver proteins targeted by BB metabolites. One was a nonspecific esterase also known to be a target for metabolites of halothane and molinate. Another, surprisingly, was epoxide hydrolase, which is supposed to detoxify BBO. To address the mechanism of BB-induced cytotoxicity it is essential to expand this list by identifying other liver proteins targeted by BB metabolites. In doing so we will emphasize mitochondrial proteins but will continue to explore cytosolic and microsomal proteins. To facilitate recognition of BBO adducts, we raised antibodies to p-bromophenyl-cysteine and demonstrated their utility for western blotting; we will now develop antibodies to p-bromophenyl-histidine and p-bromophenyl-lysine as well. These antibodies, coupled with [C14]-BB, will give us a broad and powerful means for finding and identifying those proteins of greatest toxicological interest. Very little is known about the chemistry and consequences of protein adduction by reactive metabolites. Thus we will elucidate in detail the specific site(s), metabolite(s) and linkage(s) involved in adduct formation for select BB target proteins. For those target proteins which are enzymes, we will evaluate the effect of adduction on catalytic activity to assess its potential contribution to cell injury. Comparing the proteins modified by bromobenzene to those modified by other small bioactivated toxins may reveal the existence, or the lack of, a """"""""common pathway"""""""" for chemically-induced cytotoxic responses.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Special Emphasis Panel (ZRG1-ALTX-1 (01))
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University of Kansas Lawrence
Schools of Pharmacy
United States
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