Identification of the human and rat P450 enzymes responsible for the sulfoxidation of S-methyl N,N-diethylthiolcarbamate (DETC-ME). The terminal step in the bioactivation of disulfiram.
The present study investigated the role of rat and human cytochrome P450 enzymes in the sulfoxidation of S-methyl N,N-diethylthiolcarbamate (DETC-Me) to S-methyl N,N-diathylthiolcarbamate sulfoxide (DETC-Me sulfoxide), the putative active metabolite of disulfiram. DETC-Me sulfoxidation by microsomes from male and female rats treated with various cytochrome P450-enzyme inducers suggested that multiple enzymes can catalyze this reaction, and these include, CYP1A1/2, CYP2B1/2, and CYP3A1/2. All cDNA-expressed human cytochrome P450 enzymes examined catalyzed the sulfoxidation of DETC-Me. The turnover rates (min-1) of DETC-Me sulfoxidation by the cDNA-expressed cytochrome P450 enzymes ranked as follows: CYP3A4 > CYP2A6 = CYP2C9 > CYP1A2 > CYP2B6 = CYP2E1 > CYP1A1 > CYP2D6. Interestingly, CYP3A4 ranked first or last, depending on whether or not additional NADPH-cytochrome P450 reductase was coexpressed in the lymphoblastoid cells. This complicated estimates of the contribution of CYP3A4 to DETC-Me sulfoxidation by human liver microsomes. The sample-to-sample variation in DETC-Me sulfoxidation by bank of human liver microsomes (N=13) correlated highly with coumarin 7-hydroxylation (r=0.88) and testosterone 6beta-hydroxylation (r=0.90), suggesting that CYP2A6 and CYP3A4/5 contribute to the sulfoxidation of DETC-Me by human liver microsomes. Although, chlorzoxazone 6-hydroxylation (a marker for CYP2E1) correlated poorly with DETC-Me sulfoxidation, the correlation improved from r=0.07 to r=0.44 when DETC-Me sulfoxidation was studied in the presence of the CYP2A6 inhibitor, coumarin. Similarly, when DETC-Me sulfoxidation was studied in the presence of diethyldithiocarbamate (DDTC), the inhibited DETC-Me sulfoxidase activity correlated better (r=0.50) with chlorzoxazone 6-hydroxylase, compared with DETC-Me sulfoxidase activity in the absence of DDTC (r=0.09). Polyclonal antibodies against CYP2E1 caused a modest inhibition (30%) of DETC-Me sulfoxidation by human liver microsomes. Anti-CYP3A1 antibodies completely inhibited DETC-Me sulfoxidation by cDNA-expressed CYP3A4. Under similar conditions, DETC-Me sulfoxidation by human liver microsomes was only partially inhibited by anti-CYP3A1 antibodies. Although studies with the rat and cDNA-expressed cytochrome P450 enzymes suggested that CYP1A2 contributed to DETC-Me sulfoxidation, this reaction was not inhibited by either furafylline ( a mechanism-based inhibitor of CYP1A2) or antibodies against CYP1A1/2. A significant role for CYP2C9 was excluded by the inability of sulfaphenazole to inhibit the sulfoxidation of DETC-Me by human liver microsomes. Collectively, these data suggest that multiple cytochrome P450 enzymes can catalyze the sulfoxidation of DETC-Me. In human liver microsomes the CYP2A6, CYP2E1, and CYP3A4/5 all contribute significantly to the sulfoxidation of DETC-Me. It is interesting to note that DDTC, the reduced metabolite of disulfiram, is known to inhibit these same enzymes. The ability of DDTC to block the formation of DETC-Me sulfoxide may explain why the dose of disulfiram required to produce a disulfiram-ethanol reaction in alcoholics is so variable and often inadequate.
Madan
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Department of Pharmacology and Toxicology, University of Kansas, Lawrence 66045, USA.
Parkinson
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Faiman
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MD
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001711:Biotransformation; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000502:physiology*; D004221:Disulfiram / Q000493:pharmacokinetics*; D004050:Ditiocarb / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D013859:Thiocarbamates / Q000378:metabolism*
D013859:Thiocarbamates; C073220:S-methyl N,N-diethylthiolcarbamate sulfoxide; C070631:S-methyl diethylthiocarbamate; D003577:Cytochrome P-450 Enzyme System; D004050:Ditiocarb; D001189:Aryl Hydrocarbon Hydroxylases; C465138:Cyp3a23-3a1 protein, rat; D051544:Cytochrome P-450 CYP3A; D004221:Disulfiram