Alcoholism is a major health problem. Treatment modalities for alcoholism vary, and include psychological and behavioral interventions as well as pharmacotherapeutic deterrents. An example of one group of agents classified as therapeutic interventions is the aldehyde dehydrogenase (ALDH) inhibitors. Of these inhibitors, only disulfiram is used exclusively in the U.S. Clinically, disulfiram produces a disulfiram-ethanol reaction (DER) in those individuals ingesting alcohol after disulfiram administration. It is now apparent that disulfiram must be bioactivated to a chemical species which inhibits liver ALDH, and is thus responsible for initiating a DER. Studies will be carried out to test the hypothesis that: a) the active species responsible for liver mitochondrial low Km ALDH is S-methyl-N,N-diethylthiolcarbamate sulfoxide (DETC-MeSO); b) that DETC-MeSO is formed in the liver with cytochrome P-450 mediating this oxidation; and c) that DETC-MeSO is an irreversible active-site- directed inhibitor of rat liver ALDH. Both in vitro and in vivo studies will be employed to verify that DETC-MeSO is the obligatory intermediate which is responsible for disulfiram's action as an ALDH inhibitor. To verify the role of cytochrome P-450 in the formation of DETC-MeSO, P-450 inhibition (chemical inhibitors, antibodies) and purified reconstituted systems will be employed to identify the particular P-450 isozyme responsible for the oxidative reaction. To test the hypothesis that DETC-MeSO is an irreversible site-directed inhibitor of rat liver ALDH, experiments will be carried out to characterize the kinetics and mode of action of DETC-MeSO. These will include studies investigating such parameters as time dependence of the reaction, saturability, substrate protection, irreversibility and stoichiometry. In addition, the isolation and characterization of the covalent adduct of the mitochondrial low Km ALDH with DETC-MeSO will be investigated. Thus the overall objectives of the proposed studies are to definitively identify the chemical species which inhibits liver ALDH, to study the mechanism by which this active metabolite is formed, and to use this active chemical species in in vitro studies as a pharmacological tool and probe to better understand the mechanism by which ALDH is inhibited.
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