Roles of FMO and CYP450 in the metabolism in human liver microsomes of S-methyl-N,N-diethyldithiocarbamate, a disulfiram metabolite

Background: The conversion of S-methyl-N,N-diethyldithiocarbamate (MeDDC) t o MeDDC sulfine is the first step after methylation in the metabolic pathwa y of disulfiram, an alcohol deterrent, to its ultimate active metabolite. V arious isoforms of CYP450 have recently been shown to catalyze this reactio n, but the involvement of flavin monooxygenase (FMO) in this metabolism in humans has not been evaluated. In this study we examined the ability of rec ombinant human FMO3 in insect microsomes to metabolize MeDDC, and investiga ted the relative roles of FMO and CYP450 in the metabolism of MeDDC in huma n liver microsomes. Methods: HPLC-mass spectrometry was used to identify the products of MeDDC formed by human liver microsomes and by recombinant human FMO3. MeDDC metab olism in human liver microsomes was studied by using either heat inactivati on to inhibit FMO, or N-benzylimidazole (NBI) or antibodies to the CYP450 N ADPH reductase to inhibit CYP450. Results: We confirmed by HPLC-mass spectrometry that MeDDC sulfine was the major product of MeDDC formed by human liver microsomes and by FMO3. Recomb inant FMO3 was an efficient catalyst for the formation of MeDDC sulfine (5. 3 +/- 0.2 nmol/min/mg, mean +/- SEM, n = 6). Inhibition studies showed MeDD C was metabolized primarily by CYP450 in human liver microsomes at pH 7.4, with a 10% contribution from FMO (total microsomal activity 3.1 +/- 0.2, n = 17). In the course of this work, methyl p-tolyl sulfide (MTS), sulfoxidat ion of which is used by some investigators as a specific probe for FMO acti vity, was found to be a substrate for both FMO and CYP450 in human liver mi crosomes. Conclusions: Our results prove that MeDDC sulfine is the major product of M eDDC oxidation in human liver microsomes, MeDDC is a good substrate for hum an FMO3, and MeDDC is metabolized in human liver microsomes primarily by CY P450. We also showed that use of MTS sulfoxidation as an indicator of FMO a ctivity in microsomes is valid only in the presence of a CYP450 inhibitor, such as NBI.