Identification of the human P-450 enzymes responsible for the sulfoxidation and thiono-oxidation of diethyldithiocarbamate methyl ester: role of P-450 enzymes in disulfiram bioactivation.
Diethyldithiocarbamate methyl ester (DDTC-Me) is a precursorto the formation of S-methyl-N,N-diethylthiolcarbamate sulfoxide, the active metabolite proposed to be responsible for the alcohol deterrent effects of disulfiram. The present study investigated the role of human cytochrome P-450 (CYP) enzymes in sulfoxidation and thiono-oxidation of DDTC-Me, intermediary steps in the activation of disulfiram. Several approaches were used in an attempt to delineate the particular P-450 enzyme(s) involved in the sulfoxidation and thiono-oxidation of DDTC-Me. These approaches included the use of cDNA-expressed human P-450 enzymes, correlation analysis with sample-to-sample variation in human P-450 enzymes in a bank of human liver microsomes, and chemical and antibody inhibition studies. Multiple human P-450 enzymes (CYP3A4, CYP1A2, CYP2A6, and CYP2D6) catalyzed the sulfoxidation of DDTC-Me, as determined with cDNA-expressed enzymes. Several lines of evidence suggest that the sulfoxidation of DDTC-Me by human liver microsomes is primarily catalyzed by CYP3A4/5, including (1) a high correlation between DDTC-Me sulfoxidation and testosterone 6beta-hydroxylation; (2) increased DDTC-Me sulfoxidation in the presence of alpha-naphthoflavone, an activator of CYP3A enzymes; (3) inhibition of this reaction by inhibitors of CYP3A4/5 enzymes, such as troleandomycin and ketoconazole; and (4) inhibition of DDTC-Me sulfoxidation by antibodies against CYP3A enzymes. On the other hand, several lines of evidence suggested that the thiono-oxidation of DDTC-Me by human liver microsomes is catalyzed in part by CYP1A2, CYP2B6, CYP2E1, and CYP3A4/5, including (1) these human P450 enzymes among others have the capacity to catalyze this reaction, as determined with cDNA-expressed enzymes; (2) a high correlation between DDTC-Me thiono-oxidation and testosterone 6beta-hydroxylation, weak inhibition by ketoconazole, troleandomycin, and anti-CYP3A antibodies suggested a minor role for CYP3A4; (3) a high correlation with immunoreactive CYP2B6 suggested involvement of this enzyme; (4) weak inhibition of DDTC-Me thiono-oxidation by furafylline and anti-CYP1A antibody suggested involvement of CYP1A2; and (5) inhibition of DDTC-Me thiono-oxidation by DDTC and anti-CYP2E antibodies suggested a role for CYP2E1. Collectively, these data suggested CYP3A4/5 enzymes are the major contributors to the sulfoxidation of DDTC-Me by human liver microsomes, and CYP1A2, CYP2B6, CYP2E1, and CYP3A4/5 contribute toward DDTC-Me thiono-oxidation by human liver microsomes. This study, in conjunction with others (Madan et al., Drug Metab. Dispos. 23:1153-1162, 1995), may help explain the variability in disulfiram's effectiveness as an alcohol deterrent.
Department of Pharmacology and Toxicology, University of Kansas, Lawrence 66045, USA.
A
A
Madan
A
A
Parkinson
M D
MD
Faiman
D000427:Alcohol Deterrents / Q000493:pharmacokinetics*; D000444:Aldehyde Dehydrogenase / Q000037:antagonists & inhibitors*; D001711:Biotransformation; D046508:Culture Techniques; D003577:Cytochrome P-450 Enzyme System / Q000502:physiology*; D004221:Disulfiram / Q000493:pharmacokinetics*; D004050:Ditiocarb / Q000031:analogs & derivatives* / Q000493:pharmacokinetics; D004791:Enzyme Inhibitors / Q000493:pharmacokinetics*; D006801:Humans; D008862:Microsomes, Liver / Q000201:enzymology; D010084:Oxidation-Reduction
D000427:Alcohol Deterrents; D004791:Enzyme Inhibitors; C112317:S-methyl-N,N-diethyldithiocarbamate sulfine; C016571:methyl diethyldithiocarbamate; D003577:Cytochrome P-450 Enzyme System; D004050:Ditiocarb; D000444:Aldehyde Dehydrogenase; D004221:Disulfiram