Identification of Novel Glutathione Conjugates of Disulfiram and Diethyldithiocarbamate in Rat Bile by Liquid Chromatography-Tandem Mass Spectrometry. Evidence for Metabolic Activation of Disulfiram in vivo

Abstract

Recent studies have shown that the inhibitory effects of disulfiram and diethyldithiocarbamate (DDTC) (to which disulfiram is rapidly reduced in vivo) on the liver mitochondrial low-Km form of aldehyde dehydrogenase (ALDH) may be mediated by a reactive metabolite(s) of these compounds. In order to investigate the nature of such electrophilic intermediates in vivo, the present study was carried out with the goal of detecting and identifying their respective glutathione (GSH) conjugates in the bile of rats dosed ip with either disulfiram (75 mg kg-1) or sodium DDTC (114 mg kg-1). By means of highly selective screening strategies based on coupled liquid chromatography-tandem mass spectrometry techniques, one major and four minor GSH adducts were identified as common biliary metabolites of disulfiram and DDTC. The major conjugate, whose excretion into bile over 4 h accounted for ca. 1% of the dose of either precursor, was identified as S-(N,N-diethylcarbamoyl)glutathione (SDEG). In vitro experiments with synthetic SDEG demonstrated that this carbamate thioester derivative is chemically stable in aqueous media under physiological conditions and does not carbamoylate nucleophiles such as cysteine. Consistent with these findings, SDEG failed to inhibit yeast ALDH in vitro. The minor GSH conjugates in bile were identified as S-(N,N-diethylthiocarbamoyl)glutathione, S-(N-ethyl-carbamoyl)glutathione, S-(N-ethylthiocarbamoyl)glutathione, and S-[N-(carboxymethyl)-N- ethylcarbamoyl]glutathione, the structures of which indicate that metabolic oxidation takes place at the thiono sulfur group and at each of the carbon atoms of disulfiram and DDTC.(ABSTRACT TRUNCATED AT 250 WORDS)