Introduction

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Disulfiram [bis(diethylthiocarbamoyl) disulfide, Antabuse] (fig. 1), that is used widely in the clinical treatment of alcoholism, elicits its pharmacological effects via inhibition of liver mitochondrial low K_m ALDH (Brien and Loomis, 1985). Although itself a potent and reversible inhibitor of ALDH in vitro, disulfiram in vivo acts as an irreversible inhibitor of the enzyme, and recent evidence indicates that this inhibition is mediated by a reactive metabolite(s) of the drug (Johansson, 1989; Yourick and Faiman, 1991).

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Structures of compounds referred to in the text. SG denotes the glutathion-S-yl moiety and SNAC denotes the N-acetyl-cystein-S-yl moiety.

As depicted in figure 2, disulfiram undergoes rapid enzymatic reduction in the blood to DDTC by glutathione reductase (Strömme, 1963; Cobby et al., 1977) and also is reduced non-enzymatically by albumin (Agarwal et al., 1983). Subsequently, DDTC is metabolized by both thiol methyltransferase and thiopurine methyltransferase to DDTC-Me (Gessner and Jakubowski, 1972; Glauser et al., 1993; Lill et al., 1996) which, in turn, is oxidized primarily by cytochrome P-450 with a minor contribution from FMO to DETC-Me (Johansson et al., 1989; Hart et al., 1990) and DETC-MeSO (Hart and Faiman, 1992; Madan et al., 1993; Madan et al., 1995). DETC-MeSO has been found to be a potent inhibitor of ALDH both in vitro and in vivo, and it has been suggested that this sulfoxide is the active species responsible for the inhibitory effects of disulfiram in vivo (Hart and Faiman, 1992, 1994). Specifically, it has been proposed that DETC-MeSO inactivates ALDH through carbamoylation of a critical thiol group at the active site of the enzyme. Consistent with this hypothesis, SDEG, the adduct generated by carbamoylation of the endogenous thiol glutathione (GSH) by DETC-MeSO, was identified as a major conjugate in the bile of rats treated with either disulfiram or DDTC (Jin et al., 1994).

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Proposed metabolic pathways for disulfiram leading to the formation of the electrophilic intermediates DETC-MeSO, DETC-MeSO2, DDTC-MeSO and DDTC-MeSO2, each of which may carbamoylate, and thereby inhibit, aldehyde dehydrogenase or give rise to the GSH conjugates SDEG or SDETG.

Recently, DDTC-MeSO, the dithio analog of DETC-MeSO, was identified in incubations of DDTC-Me with rat liver microsomal preparations (Madan and Faiman, 1994a). The formation of DDTC-MeSO was shown to be catalyzed primarily by cytochrome P-450 with a minor contribution from FMO. Although DDTC-MeSO was shown to inhibit ALDH both in vitro and in vivo, this sulfoxide was not detected in plasma from rats treated with either disulfiram, DDTC-Me or DDTC-MeSO (Madan and Faiman, 1994b). However, SDETG, the corresponding GSH conjugate, was found to be a prominent component in the bile of rats dosed with either disulfiram or DDTC, consistent with the formation of DDTC-MeSO as a transient intermediate in vivo (Jin et al., 1994). Therefore, this dithiocarbamate sulfoxide (and/or the corresponding sulfone, DDTC-MeSO₂) also may play a role in mediating the inhibition of ALDH by disulfiram in vivo.

Studies on the thiocarbamate herbicide eptam (EPTC) (fig. 1), a structural analog of DETC-Me, have shown that successive oxidation reactions occur when the compound is incubated with mouse or fish liver microsomes, leading to the formation of EPTC sulfoxide and sulfone (Chen and Casida, 1978; Cashman et al., 1989). It has been proposed that DETC-Me undergoes similar reactions to form the corresponding sulfone (DETC-MeSO₂), a potent inhibitor of ALDH both in vitro and in vivo (Nagendra et al., 1994; Mays et al., 1995). However, attempts to detect DETC-MeSO₂ either in incubations of DETC-Me or DETC-MeSO with rat liver microsomes or in the plasma of rats treated with DETC-MeSO or DETC-MeSO₂ were unsuccessful (Nagendra et al., 1994), and it was suggested that the high reactivity of DETC-MeSO₂ toward endogenous nucleophiles may preclude its detection in biological matrices (Mays et al., 1995).

To gain a better understanding of the mechanism by which disulfiram undergoes metabolic activation and inhibits ALDH in vivo, our studies were carried out to investigate the role of DETC-MeSO, DETC-MeSO₂, DDTC-MeSO and DDTC-MeSO₂ as reactive intermediates and as potential mediators of the inhibitory effects. Specifically, we proposed that each of the above S-oxygenated metabolites of disulfiram may carbamoylate, and thereby inhibit, ALDH or give rise to the GSH conjugates SDEG or SDETG which may be further metabolized to the corresponding NAC conjugates ADECC and ADETCC, respectively. To test the hypothesis, in one set of experiments, ionspray LC-MS/MS was used to identify and quantify putative NAC conjugates of these carbamoylating intermediates in the urine of rats treated with disulfiram and DDTC, while in a parallel series of studies, experiments were conducted in vitro to compare the carbamoylating activity and the corresponding ALDH inhibitory potency of these sulfoxide and sulfone metabolites of disulfiram and DDTC.

	Experimental Procedures

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Materials

Disulfiram and sodium DDTC·3H₂O were purchased from the Aldrich Chemical Co. (Milwaukee, WI). DDTC-MeSO₂, ADECC and ADETCC were obtained by synthesis, as described below. DETC-MeSO₂ (Jin et al., 1994), DDTC-MeSO (Madan and Faiman, 1994a) and DETC-MeSO (Hart and Faiman, 1992) were prepared as outlined previously. APrCC (ca. 95% pure) was a gift from Dr. Xiangming Guan (University of Washington). All the other synthetic compounds were greater than 99.5% pure as determined by HPLC and NMR analysis. Other chemicals were obtained from commercial sources and were of analytical grade. Potassium-activated aldehyde dehydrogenase from bakers yeast [Aldehyde:NAD(P)⁺ oxidoreductase; EC1.2.1.5] was purchased from the Sigma Chemical Co. (St. Louis, MO).

Instrumentation and Analytical Methods

¹H NMR spectra were recorded on a Varian VXR 300 spectrometer (Varian Associates, Palo Alto, CA). For samples dissolved in CDCl₃ or Me₂SO-d₆, chemical shifts are expressed in parts per million () downfield from tetramethylsilane, while for samples dissolved in D₂O, sodium 3-(trimethylsilyl)-[2,2,3,3-d₄]propionate was used as internal reference. Signal multiplicities are reported as follows: s, d, t, dd (doublet of doublets) and m.

LC-MS/MS was carried out on a Perkin-Elmer Sciex API III Plus triple-quadrupole mass spectrometer equipped with an atmospheric pressure ion source and an IonSpray interface. Analysis was performed with an ionizing voltage of 4.6 kV, and high-purity air was used as the nebulizing gas at an operating pressure of 40 psi. CID of selected precursor ions was performed in the radio frequency only quadrupole region where argon was used as target gas at a thickness of 1.9 × 10¹⁴ molecules cm¹. Samples of filtered urine (20 µl) were injected onto a Beckman (Beckman Instruments, San Ramon, CA) Ultrasphere narrow-bore C₁₈ column (150 × 2.0 mm i.d., 5 µm) coupled to a splitter so that one-fourth of the effluent entered the mass spectrometer. The mobile phase, which consisted of a mixture of solvent "A" (0.1% aqueous formic acid) and solvent "B" (acetonitrile containing 0.1% formic acid), was delivered by an Hewlett Packard (Hewlett-Packard Co., Palo Alto, CA) 1050 liquid chromatography system at a constant flow rate of 200 µl min¹. The gradient started with 10% solvent "B," followed by a linear increase in solvent "B" at a rate of 1% min¹. Both NAC conjugates of interest, i.e,. ADECC and ADETCC, were detected directly using the product ion scanning technique in which ions with the appropriate m/z values for the predicted MH⁺ species were subjected to CID. Confirmation of the identities of the urinary NAC conjugates was obtained when the metabolites were shown to possess LC and MS/MS properties identical to those of the corresponding reference compounds prepared by synthesis.

Quantitation of drug-related conjugates in urine was carried out by SRM LC-MS/MS. Specimens of urine (20 µl) were treated with internal standard (APrCC, 1 µg) and diluted (to a final volume of 1 ml) with 0.1% aqueous formic acid. Aliquots (20 µl) of these samples were injected onto the narrow bore C₁₈ column with an isocratic mobile phase consisting of 75% solvent "A" and 25% solvent "B." The analytes and internal standard were detected by monitoring the transitions m/z 263  100 for ADECC, 279  116 for ADETCC and 249  164 for APrCC. The ratios of the ion currents of metabolites to internal standard were used to determine the amounts of conjugates in urine samples with reference to calibration curves which were prepared by adding varying amounts of reference compounds together with a fixed amount of APrCC to specimens of drug-free rat urine.

Synthesis

DDTC-MeSO₂. DDTC-MeSO₂ was prepared according to a modification of a published method (Nilsson et al., 1972). Briefly, to a solution of Na₂SO₃ (2 mol) in water (800 ml), was slowly added CH₃SO₂Cl (5 g) and 50% aqueous NaOH in alternating portions so that the pH of the solution was kept around 8 to 9. The reaction mixture was stirred for 30 min at 45 to 55°C and then lyophilized to dryness. The dry residue was pulverized and extracted with 1.5 l warm (60-70°C) absolute ethanol. The extract was filtered and dried under reduced pressure to give CH₃SO₂Na. CH₃SO₂Na (0.15 mol) then was added together with diethylthiocarbamoyl chloride (0.15 mol) to a mixture of benzene and water (400 ml, 1:1 v/v), and stirred for 3 hr at 40°C. The organic phase was dried over CaCl₂ and evaporated to give DDTC-MeSO₂ (yield = 34%). ¹H NMR (CDCl₃):  1.27 (t, J = 7.10 Hz, 3H, CH₃CH₂-), 1.35 (t, J = 7.05 Hz, 3H, CH₃CH₂-), 3.43 (s, 3H, -SO₂CH₃), 3.84 (q, J = 7.10 Hz, 2H, CH₃CH₂-), 4.06 (q, J = 7.05 Hz, 2H, CH₃CH₂-). MS/MS (CID of MH⁺ at m/z 196): m/z 116 ([Et₂N=C=S]⁺), 88 ([EtNH=C=S]⁺), 60 ([NH₂= C=S]⁺).

ADECC. NAC (6 mmol) was dissolved in water (25 ml) and the pH was adjusted to 7.8 with 1 M aqueous NaOH. A solution of DETC-MeSO₂ (7 mmol) in MeOH (25 ml) was added, and the reaction mixture was stirred under N₂ overnight at room temperature. The crude product was purified by HPLC (C₁₈ column; mobile phase 20% aqueous CH₃CN containing 0.06% TFA) to give ADECC (yield = 52%). ¹H NMR (D₂O):  1.13 (m, 6H, (CH₃CH₂)₂N-), 2.02 (s, 3H, -COCH₃), 3.24 (dd, J = 14.52 and 8.06 Hz, 1H, Cys-), 3.40 (q, J = 7.16 Hz, 4H, NCH₂CH₃), 3.49 (dd, J = 14.52 and 4.49 Hz, 1H, Cys-'), 4.63 (dd, J = 8.06 and 4.49 Hz, 1H, Cys-). MS/MS (CID of MH⁺ at m/z 263): m/z 221 ([MH -NCH₂CO]⁺), 175 ([Et₂N-CO-S-CH₂-CH=NH₂]⁺), 146 ([Et₂N-CO-S-CH₂]⁺), 134 ([Et₂N-CO-SH + H]⁺), 130 ([CH₂-CH(COOH)-NH-CO-CH₃]⁺), 118 ([EtNH-CO-S-CH₂]⁺), 112 ([130 -NH₂O]⁺), 100 ([Et₂N=C=O]⁺), 88 ([CH₂-CH(COOH)-NH₂]⁺), 76 ([NH₃-CH₂-COOH]⁺) and 72 ([EtNH=C=O]⁺).

ADETCC. NAC (2 mmol) and diethylthiocarbamoyl chloride (2 mmol) were dissolved in tetrahydrofuran (15 ml) and stirred under N₂ for 4 hr at ambient temperature. The solvent was removed under reduced pressure, and the residue was taken up in CH₃CN (5 ml). The crude product was subjected to semi-preparative HPLC (C₁₈ column; mobile phase 25% aqueous CH₃CN containing 0.06% TFA) to afford ADETCC (yield = 44%). ¹H NMR (DMSO):  1.18 (t, J = 6.57 Hz, 3H, CH₃CH₂-), 1.22 (t, J = 6.08 Hz, 3H, CH₃CH₂-), 1.84 (s, 3H, -COCH₃), 3.38 (dd, J = 13.51 and 8.91 Hz, 1H, Cys-), 3.74 (m, 2H, CH₃CH₂-), 3.79 (dd, J = 4.83 and 13.51 Hz, 1H, Cys-'), 3.96 (m, 2H, CH₃CH₂-), 4.46 (m, 1H, Cys-), 8.35 (d, J = 8.19 Hz, 1H, -CONH-). MS/MS (CID of MH⁺ at m/z 279): m/z 150 ([Et₂NHCS-SH + H]⁺), 130 ([CH₂-CH(COOH)-NH-CO-CH₃]⁺), 116 ([Et₂N=C= S]⁺), 88 ([EtNH=C=S]⁺).

Biological Experiments

In vivo metabolism study. Adult male Sprague-Dawley rats (240-270 g), obtained from Charles River Laboratories (Wilmington, MA), were housed in Nalgene metabolic cages and had free access to food and water. One group of five animals was given an i.p. dose of disulfiram (0.25 mmol kg¹) suspended in 0.5% methylcellulose, although a second group was dosed ip with sodium DDTC·3H₂O (0.5 mmol kg¹) dissolved in isotonic saline. Urine was collected over ascorbic acid (0.5 g) for 24 hr and stored at -20°C until analyzed.

Carbamoylating activity in vitro. DETC-MeSO, DETC-MeSO₂, DDTC-MeSO, or DDTC-MeSO₂ (0.5 mM) was incubated with NAC (2.5 mM) in aqueous phosphate buffer (50 mM, pH 7.4; total volume = 3.0 ml) in a shaking water bath. Aliquots (0.1 ml) of the reaction mixture were withdrawn over intervals up to 4 hr, mixed with internal standard (4-nitrophenylacetic acid; 15 µl of a 10 mM aqueous solution) and frozen immediately with liquid N₂. Samples were stored at -80°C until analyzed by HPLC for the corresponding NAC adducts, i.e., ADECC or ADETCC. This was carried out using a Shimadzu LC-600 liquid chromatograph equipped with a Beckman ODS column (25 cm × 4.6 mm i.d., 5 µm). The mobile phase was CH₃CN/H₂O (15:85, v/v) containing 0.06% TFA. The flow rate was 1.0 ml min¹ and compounds eluting from the column were detected by monitoring the UV absorbance at 214 nm (Shimadzu SPD-6A spectrophotometer; Shimadzu Corp., Kyoto, Japan). Under these conditions, the retention times of ADECC, ADETCC and the internal standard were 10.6, 14.8 and 19.2 min, respectively. The second order rate constants for carbamoylation reactions were obtained from plots of product concentrations versus time and a least squares fitting method.

Inhibition of aldehyde dehydrogenase activity in vitro. The ability of DETC-MeSO, DETC-MeSO₂, DDTC-MeSO and DDTC-MeSO₂ to inhibit ALDH in vitro was determined by a published method (Nagasawa et al., 1989) in which the enzyme is exposed to inhibitors for 10 min before the addition of benzaldehyde and NAD⁺ to initiate the reaction. The activity of the ALDH then is calculated from the rate of formation of NADH over 2 min by measuring the increase in absorbance at 340 nm on a Cary 3E UV-visible spectrophotometer. The concentrations of the inhibitors examined were 10, 25, 50, 100, 200, 300, 400 and 500 µM. To investigate the effects of NAC on the inhibition of ALDH by these carbamoylating agents, the enzyme was preincubated with NAC (at 1 or 2 mM) for 5 min before exposure to the inhibitors (500 µM).

	Results

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Identification of N-acetylcysteine conjugates in urine. Based on our previous studies on the biotransformation of disulfiram in the rat (Jin et al., 1994), it was expected that further metabolism of the disulfiram GSH conjugates SDEG and SDETG in vivo would lead to the formation of ADECC and ADETCC, respectively. Therefore, specimens of urine collected from rats treated with either disulfiram or DDTC were analyzed directly by product ion scanning LC-MS/MS. By this approach, ADECC and ADETCC were detected in urine (fig. 3), and their identities were verified with authentic samples of ADECC and ADETCC that were shown to possess identical LC-MS/MS properties to those of the respective metabolites. Thus, under the LC conditions employed in this study, ADECC eluted at 18.8 min and CID of its [MH]⁺ ion at m/z 263 afforded the product ion spectrum reproduced in figure 4. This spectrum contained a number of ions indicative of the N, N-diethylcarbamoyl residue, e.g., m/z 175 ([Et₂N-CO-S-CH₂-CH=NH₂]⁺), 146 ([Et₂N-CO-S-CH₂]⁺), 134 ([Et₂N-CO-SH + H]⁺), 118 ([EtNH-CO-S-CH₂]⁺), 100 ([Et₂N=C=O]⁺) and 72 ([EtNH=C=O]⁺). Fragmentation of the N-acetylcysteinyl moiety of this conjugate, on the other hand, yielded ions at m/z 221 ([MH -NCH₂CO]⁺), 130 ([CH₂-CH(COOH)-NH-CO-CH₃]⁺), 112 ([130 -NH₂O]⁺), 88 ([CH₂-CH(COOH)-NH₂]⁺) and 76 ([NH₃-CH₂-COOH]⁺). ADETCC exhibited a retention time of 24.6 min and afforded the product ion spectrum shown in figure 5. The spectrum consisted of structurally-informative ions at m/z 150 ([Et₂N-CS-SH + H]⁺), 130 ([CH₂-CH(COOH)-NH-CO-CH₃]⁺), 116 ([Et₂N=C=S]⁺) and 88 ([EtNH=C=S]⁺ or [CH₂-CH(COOH)-NH₂]⁺). The fragments which retained the drug residue (i.e., ions at m/z 150 and 116) exhibited m/z ratios 16 Da higher than the corresponding ions in the spectrum of ADECC, reflecting the fact that ADETCC is the thiocarbamoyl analog of ADECC.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Detection of ADECC and ADETCC in the urine of a rat that had been treated with disulfiram (0.25 mmol kg1). The upper and lower chromatograms were obtained by product ion scanning LC-MS/MS analysis of a urine specimen collected between 0 and 24 hr post-dose in which the predicted [MH]+ ions of ADECC (m/z 263) and ADETCC (m/z 279) were subjected to CID, respectively.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Spectrum of product ions obtained by CID of the [MH]+ ion (m/z 263) of N-acetyl-S-(N, N-diethylcarbamoyl)cysteine (ADECC). The origins of characteristic product ions are discussed in the text.

View larger version (11K): [in this window] [in a new window]   Fig. 5.   Spectrum of product ions obtained by CID of the [MH]+ ion (m/z 279) of N-acetyl-S-(N, N-diethylthiocarbamoyl)cysteine (ADETCC). The origins of characteristic product ions are discussed in the text.

Quantitative analysis of ADECC and ADETCC in urine. SRM LC-MS/MS was used to determine the fraction of disulfiram (75 mg kg¹; 0.25 mmol kg¹) or DDTC (114 mg kg¹; 0.5 mmol kg¹) excreted in urine over a 24-hr period as ADECC and ADETCC. Because disulfiram is reduced rapidly and quantitatively to DDTC in vivo (Cobby et al., 1977), a dose of 0.25 mmol kg¹ of disulfiram may be considered equivalent to a dose of 0.5 mmol kg¹ of the monomer DDTC. Consistent with this view, the urinary excretion of ADECC was found to account for 7.5 ± 4.0 and 6.2 ± 1.0% (mean ± SD, n = 5), respectively, of the administered dose of disulfiram and DDTC. ADETCC accounted for additional 0.5 ± 0.3 and 0.3 ± 0.1%, respectively, of the administered dose of disulfiram and DDTC.

Carbamoylation of NAC by S-oxygenated metabolites of disulfiram. Under physiological conditions of temperature and pH, DETC-MeSO, DETC-MeSO₂, DDTC-MeSO and DDTC-MeSO₂ were found to carbamoylate NAC readily, resulting in the formation of S-linked adducts of NAC. Among the four compounds, DDTC-MeSO₂ demonstrated the highest level of reactivity toward NAC, followed by DETC-MeSO₂, DDTC-MeSO and DETC-MeSO.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Carbamoylation of NAC by (a) DDTC-MeSO2 (at 4°C), (b) DETC-MeSO2 (at 4°C), (c) DDTC-MeSO (at 37°C) and (d) DETC-MeSO (at 37°C). The carbamoylating agents were incubated (at 0.5 mM) with NAC (2.5 mM) in aqueous phosphate buffer (50 mM, pH 7.4; total volume = 3.0 ml) in a shaking water bath. All values represent means ± SD (n = 3) except in the case of DDTC-MeSO2 where a single measurement was used for each data point. Note the differences in the time axis in different experiments.

Inhibition of ALDH in vitro by S-oxygenated metabolites of disulfiram. As illustrated in figure 7, 10-min incubation of ALDH with DDTC-MeSO, DDTC-MeSO₂, DETC-MeSO or DETC-MeSO₂ (0-500 µM) led to concentration-dependent losses of enzyme activity. All four compounds caused marked inhibition of ALDH, with DETC-SO₂ being the most potent member of the group. When NAC was included in incubation media, it was found that this thiol attenuated significantly, in a concentration-dependent fashion, the loss of enzyme activity caused by DDTC-MeSO, DDTC-MeSO₂ and DETC-MeSO₂. However, NAC failed to protect against the ALDH-inhibitory properties of DETC-MeSO, which was the least reactive member of the group (table 1).

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Inhibition of yeast aldehyde dehydrogenase (ALDH) in vitro by S-oxygenated metabolites of disulfiram. Yeast ALDH was incubated for 10 min with DETC-MeSO, DETC-MeSO2, DDTC-MeSO or DDTC-MeSO2 (10-500 µM) at 37°C, after which ALDH activity was determined as described in "Experimental Procedures." The results (mean ± S.D., n = 3) are expressed as % ALDH activity remaining compared to control incubations which did not contain inhibitor (control activity = 13.9 nmol unit1 min1).

	Discussion

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The results from the present studies in rats have provided further evidence that disulfiram and its reduced metabolite DDTC undergo metabolic activation in vivo, based on the identification in urine of two N-acetylcysteine conjugates, ADECC and ADETCC. The detection of these S-linked conjugates is consistent with our previous findings that the bile of disulfiram-treated rats contained the corresponding glutathione adducts, SDEG and SDETG (Jin et al., 1994). In addition, the ratio of ADECC to ADETCC in urine (ca. 15:1) was similar to that of SDEG to SDETG in bile, suggesting that the urinary mercapturates are derived, as expected, from renal processing of the corresponding GSH adducts.

The formation of thio- and dithiocarbamate conjugates of disulfiram is indicative of the generation of reactive, carbamoylating metabolites of the drug, which most likely comprise the S-methyl sulfoxides DETC-MeSO and DDTC-MeSO, and the corresponding sulfones DETC-MeSO₂ and DDTC-MeSO₂. Currently, it is believed that one or more of these S-oxidized metabolites may be responsible for the inhibition of ALDH after administration of disulfiram, as a consequence of metabolite-mediated carbamoylation of a critical thiol group on the enzyme.

The above hypothesis for the mechanism of action of disulfiram is supported by three lines of evidence, as follows.

1) As demonstrated in the present study, both of the sulfoxides DETC-MeSO and DDTC-MeSO, and the corresponding sulfones DETC-MeSO₂ and DDTC-MeSO₂, serve as potent inhibitors of ALDH in vitro. For all but the least reactive member of the group (DETC-MeSO), the addition of NAC protected against this enzyme inhibition; such protection may have been due to competition between NAC and an active-site thiol moiety for reaction with the carbamoylating metabolite(s) (see below).

2) All four sulfoxide and sulfone metabolites of disulfiram reacted spontaneously in buffered aqueous solution (pH 7.4) with NAC to form the corresponding mercapturic acid derivatives ADECC and ADETCC, indicating that NAC (and probably other thiols, e.g., GSH, proteins) may act as scavengers of these reactive metabolites in vivo.

3) Recently, certain thio- and dithiocarbamate herbicides and pesticides, which are structural analogs of disulfiram and DDTC, and which also inhibit ALDH in vivo, have been shown to form reactive S-oxidized intermediates similar to those proposed for disulfiram (Quistad et al., 1994; Staub et al., 1995; Hart and Faiman, 1995).

Collectively, these observations are consistent with the proposed mechanism for inhibition of ALDH by disulfiram outlined in figure 2. According to this scheme, rapid reduction of disulfiram to DDTC is followed by S-methylation and oxidative desulfuration to generate DDTC-Me and DETC-Me, respectively. Both of these S-methyl derivatives then undergo successive oxidations on the thioether sulfur atom to generate the electrophilic sulfoxides DDTC-MeSO and DETC-MeSO, and sulfones DDTC-MeSO₂ and DETC-MeSO₂. One or more of the latter species are viewed as the ultimate carbamoylating agents that either bind covalently to proteins or are intercepted by GSH and form S-linked conjugates. It is proposed that inhibition of ALDH is a consequence of the carbamoylation of a critical thiol group, perhaps at the active site of the enzyme, by an S-oxidized metabolite of disulfiram. The fact that there did not appear to be a strict correlation between the carbamoylating activities and the ALDH inhibitory potencies of the S-oxygenated metabolites of disulfiram could be attributed to steric hindrance (Hart and Faiman, 1995). Thus, DDTC-MeSO₂, a dithiocarbamate derivative having a thiono group (C=S) that is bulkier than the corresponding carbonyl group (C=O) in the thiocarbamate analog DETC-MeSO₂, may not be able to gain access to the active site of ALDH as readily as DETC-MeSO₂. However, other sulfhydryl-dependent enzymes whose active sites are accessible to these metabolites should be expected to be subject to inhibition by this drug. Further studies will be required to investigate this intriguing possibility, which should provide a valuable test of the above mechanistic hypothesis.