Roles of FMO and CYP450 in the Metabolism in Human Liver Microsomes of S‐Methyl‐N, N‐Diethyldithiocarbamate, a Disulfiram Metabolite

Abstract

The conversion of S-methyl-N,N-diethyldithiocarbamate (MeDDC) to MeDDC sulfine is the first step after methylation in the metabolic pathway of disulfiram, an alcohol deterrent, to its ultimate active metabolite. Various isoforms of CYP450 have recently been shown to catalyze this reaction, but the involvement of flavin monooxygenase (FMO) in this metabolism in humans has not been evaluated. In this study we examined the ability of recombinant human FMO3 in insect microsomes to metabolize MeDDC, and investigated the relative roles of FMO and CYP450 in the metabolism of MeDDC in human liver microsomes. HPLC-mass spectrometry was used to identify the products of MeDDC formed by human liver microsomes and by recombinant human FMO3. MeDDC metabolism in human liver microsomes was studied by using either heat inactivation to inhibit FMO, or N-benzylimidazole (NBI) or antibodies to the CYP450 NADPH reductase to inhibit CYP450. We confirmed by HPLC-mass spectrometry that MeDDC sulfine was the major product of MeDDC formed by human liver microsomes and by FMO3. Recombinant 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 MeDDC 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), sulfoxidation of which is used by some investigators as a specific probe for FMO activity, was found to be a substrate for both FMO and CYP450 in human liver microsomes. Our results prove that MeDDC sulfine is the major product of MeDDC oxidation in human liver microsomes, MeDDC is a good substrate for human FMO3, and MeDDC is metabolized in human liver microsomes primarily by CYP450. We also showed that use of MTS sulfoxidation as an indicator of FMO activity in microsomes is valid only in the presence of a CYP450 inhibitor, such as NBI.