title: Studies on the metabolic activation of disulfiram in rat. Evidence for electrophilic S-oxygenated metabolites as inhibitors of aldehyde dehydrogenase and precursors of urinary N-acetylcysteine conjugates.
abstract: Recent studies on the mechanism by which disulfiram inhibits aldehyde dehydrogenase have provided evidence for the formation of reactive intermediates that are thought to carbamoylate, and thereby inactivate the enzyme. In our study, rats were dosed with either disulfiram (0.25 mmol kg-1 i.p.) or its reduced metabolite diethyldithiocarbamate (DDTC; 0.5 mmol kg-1 i.p.) and urine was collected for the analysis of metabolites derived from putative reactive intermediates. By means of ionspray LC-MS/MS, two novel N-acetylcysteine (NAC) conjugates, i.e., N-acetyl-S-(N, N-diethylcarbamoyl)cysteine and N-acetyl-S-(N, N-diethylthiocarbamoyl)cysteine, were identified in urine specimens. Quantitative analyses indicated that, over the 0- to 24-hr period after drug administration, urinary excretion of N-acetyl-S-(N, N-diethylcarbamoyl)cysteine accounted for 7.5 +/- 4.0 and 6.2 +/- 1.0%, respectively, of the dose of disulfiram and diethyldithiocarbamate, while the corresponding thiocarbamoyl conjugate, N-acetyl-S-(N, N-diethylthiocarbamoyl)cysteine, accounted for a further 0.5 +/- 0.3 and 0.3 +/- 0.1%, respectively, of the dose. These conjugates are believed to derive from reactive sulfoxide and sulfone metabolites of disulfiram, namely S-methyl-N, N-diethylthiocarbamate sulfoxide (DETC-MeSO), S-methyl-N, N-diethylthiocarbamate sulfone (DETC-MeSO2), S-methyl-N, N-diethyldithiocarbamate sulfoxide (DDTC-MeSO) and S-methyl-N, N-diethyldithiocarbamate sulfone (DDTC-MeSO2), which were found to carbamoylate N-acetylcysteine in vitro with the following rank order of reactivity: DDTC-MeSO2 > DETC-MeSO2 > DDTC-MeSO > DETC-MeSO. In vitro experiments with aldehyde dehydrogenase showed that all four S-oxygenated metabolites inhibited the enzyme effectively. Furthermore, inclusion of NAC in incubation media attenuated significantly the inhibition by DDTC-MeSO2, DETC-MeSO2 and DDTC-MeSO, but had little effect on that by DETC-MeSO. Our results are consistent with the hypothesis that disulfiram and diethyldithiocarbamate undergo activation by a sequence of metabolic reactions leading to the formation of electrophilic S-methyl sulfoxides and sulfones that carbamoylate, and thereby inhibit, aldehyde dehydrogenase and possibly other enzymes.
authors.affiliation: Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Seattle, USA.
authors.forename: P
authors.initials: P
authors.lastname: Hu
authors.forename: L
authors.initials: L
authors.lastname: Jin
authors.forename: T A
authors.initials: TA
authors.lastname: Baillie
mesh_terms: D000111:Acetylcysteine / Q000737:chemistry / Q000652:urine*; D000427:Alcohol Deterrents / Q000493:pharmacokinetics*; D000444:Aldehyde Dehydrogenase / Q000037:antagonists & inhibitors*; D000818:Animals; D001711:Biotransformation; D002853:Chromatography, Liquid / Q000379:methods; D004221:Disulfiram / Q000493:pharmacokinetics*; D008297:Male; D013058:Mass Spectrometry / Q000379:methods; D010100:Oxygen / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D000427:Alcohol Deterrents; D000444:Aldehyde Dehydrogenase; D010100:Oxygen; D004221:Disulfiram; D000111:Acetylcysteine