COVALENT BINDING IN DRUG-INDUCED LIVER INJURY

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The objective of this project is to obtain information on the identities of reactive hepatotoxic metabolites of certain therapeutic agents and to investigate their interaction with proteins. The drugs to be studied include acetaminophen, isoniazid, iproniazid and valproic acid. The experimental approach to be adopted in this study is novel in that it takes advantage of the irreversible binding of reactive drug metabolites to protein in order to "trap" these short-lived toxic species. Both direct and indirect methods will be used to characterize the structures of the resulting covalent adducts which, in turn, will reveal the identities of the reactive intermediates themselves. Modern chromatographic and mass spectrometric techniques will play a central role in the proposed studies and the development of improved analytical methodology for the isolation and identification of drug-protein and drug-amino acid conjugates will be a key feature of this investigation. Such methods are likely to find widespread use in the field of biochemical toxicology and related disciplines. The specific aims of this project are: (1) to identify the covalent adducts formed in vitro between proteins and both hepatotoxic and non-hepatotoxic agents, (2) to compare the types of covalent adduct formed in vivo with those produced in vitro, (3) to study the different types of interaction which take place between proteins and both toxic and non-toxic metabolites, (4) to investigate species differences in the protective effect of ascorbate on acetaminophen-induced liver injury, (5) to identify hepatotoxic metabolites of valproic acid, and (6) to develop versatile and sensitive analytical methodology for the identification of trace amounts of drug-protein adducts in liver tissue and in hemoglobin. Information of the type sought in this project is necessary for the construction of a sound experimental basis from which to develop rational therapeutic and prophylactic treatments of drug-induced hepatitis and, ultimately, from which to predict the potential toxicity of new therapeutic agents.