Mechanism of disulfiram inhibition of P450 2E1 in human liver microsomes

Numerous environmental contaminants and some therapeutic drugs can cause hepatotoxicity when metabolized by cytochrome P450 2E1. Disulfiram and its primary metabolite diethydithiocarbamate (DDTC) inhibit P450 2E1 in vitro and in vivo, and disulfiram may prevent metabolism-based toxicity. Disulfiram and DDTC are mechanism-based inhibitors requiring P450 2E1-mediated activation to the ultimate inhibitory species. Disulfiram is extensively metabolized in vivo, however, the identity of the most inhibitory disulfiram metabolite is unknown. This project tests the hypothesis that disulfiram metabolites is/are the ultimate species responsible for P450 2E1 inhibition. Human liver microsomal P450 2E1 activity was determined by chlorzoxazone 6-hydroxylation, in the presence and absence (controls) of the disulfiram metabolites: DDTC, S-methyl-N, N-diethyldithio-carbamate (DDTC-Me) S-methyl-N, N-diethylthiocarbamate (DETC-Me), S-methyl-N, N-diethylthiocarbamate sulfoxide (DETC-MeSO) S-methyl-N, N-diethyldithiocarbamate sulfoxide (DDTC-MeSO), S-methyl-N, N-diethylthiocarbamate sulfone (DETC-MeSO2), S-methyl-N, N-diethyldithiocarbamate sulfone (DDTC-MeSO2), and carbon disulfide (CS2). Initially, reaction mixtures contained human liver microsomes (0.15mg), chlorzoxazone (62μM), and inhibitor (0 or 0.3-1,000μM) in 100mM KPi buffer. NADPH (1mM final) was added to start the reaction (10 min., 37°C). 6-hydroxychlorzoxazone was extracted and concentrations determined by HPLC. To investigate if inhibitors required metabolic activation, experiments were repeated, however, inhibitor was incubated with microsomes and NADPH for 15 minutes before adding substrate. Data were analyzed by non-linear regression analysis of sigmoidal log concentration-rate curves. All the compounds showed inhibitory activity. Compounds containing a diethyldithiol or dithiol moiety were better inhibitors than those containing a diethylthiol group. The most effective inhibitors were the dithiol containing compounds. Furthermore, pre-incubation of the diethyldithiol compounds significantly lowered the IC50. In conclusion, diethyldithiol compounds showed evidence of metabolic activation. Furthermore, the diethyldithiol metabolites of disulfiram are likely to be the actual inhibitors of P450 2E1. This knowledge could be useful to enhance the prevention of toxicity caused by anesthetics, other drugs or carcinogens. Inhibitor No Pre- Pre-incubation, incubation, IC50 (μM) IC50(μM) DDTC 5 1 DDTC-Me 51 7 DDTC-MeSO 24 2 DDTC-MeSO2 3 2 DETC-ME 211 172 DETC-MeSO 77 243 DETC-MeSO2 249 201 CS2 11 5.

Duke Scholars

Author Evan Kharasch Anesthesiology, General, Vascular, High Risk Transplant & Cr ...