Identification of Novel Glutathione Conjugates of Disulfiram and

Mar 8, 1994 - Disulfiram and Diethyldithiocarbamate in Rat Bile by ... (GSH) conjugates in the bileof rats dosed ip with either disulfiram (75 mg kg-*...
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Chem. Res. Toxicol. 1994, 7, 526-533

526

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 Lixia Jin, M a r g a r e t R. Davis, Pei Hu, a n d T h o m a s A. Baillie' Department of Medicinal Chemistry, School of Pharmacy, BG-20, University of Washington, Seattle, Washington 98195 Received March 8, 1994"

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-K, form of aldehyde dehydrogenase (ALDH) may be mediated by a reactive metabolite@.) 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-l) 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-diethylcarbamoy1)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-ethyland S-[N-(carboxymethyl)-Ncarbamoyl)glutathione, S-(N-ethylthiocarbamoyl)glutathione, ethylcarbamoyl]glutathione, the structures of which indicate that metabolic oxidation takes place a t the thiono sulfur group and a t each of the carbon atoms of disulfiram and DDTC. The results of this study are consistent with the hypothesis that the title compounds undergo metabolism to methylN,N-diethylthiocarbamoylsulfoxide (DETC-MeSO)and that this reactive sulfoxide (and/or the corresponding sulfone, DETC-MeS02) carbamoylates, and thereby inhibits, a key residue a t the active site of liver ALDH.

Introduction Disulfiram [bis(diethylthiocarbamoyl) disulfide, Antabuse; Figure 11 has been employed therapeutically in the treatment of alcoholism for over 40 years (1). The basis of the clinical use of this compound as an alcohol deterrent is that coingestion of disulfiram and ethanol produces an unpleasant reaction characterized by intense vasodilation of the face and neck, tachycardia, and tachypnea followed by nausea, vomiting, pallor, and hypotension (2). It is well established that disulfiram acts through its inhibitory effectson liver aldehyde dehydrogenase (ALDH)' (31,such that when ethanol is consumed together with disulfiram, acetaldehyde (the primary metabolite of ethanol and a highly toxic compound) accumulates in the systemic circulationdue to ALDH inhibition and produces the above Abstract published in Aduance ACS Abstracts, June 1, 1994. ALDH, aldehydedehydrogeme(EC 1.2.1.3); DDTC, diethyldithiocarbamic acid; DDTC-Me,methyl diethyldithiocarbamate; DETC-Me, S-methyldiethylthiocarbamate;DETC-MeSO,methylN,Ndiethylthiocarbamoyl sulfoxide; DETC-MeSOz, methyl N,N-diethylthiocarbamoyl sulfone; EPTC, S-ethyl dipropylthiocarbamate (eptam); SBG,S-(N-benzylcarbamoy1)glutathione;SDEG, S-(N,N-diethylcarbamoy1)glutathione;SDETG,S-(NJV-diethylthiocarbamoy1)glutathione; SEG, S-(N-ethylcarbamoy1)glutathione;SETG, S-(N-ethylthiocarbamoy1)glutathione;SCEG, S- [N(carboxymethyl)-N-ethylcarbamoyllglutathione;SDEC,S-(N,N-diethylcarbamoyl)cysteine;TFA, trfiuoroacetic acid; LC-MS/MS, liquid chromatography-tandem mass spectrometry; CID,collisionally-induced dissociation; SRM, selected reaction monitoring. @

1 Abbreviations:

S Et Et

\N-c

II

/

S

-S-S-C-

II

E ,t N

'Et Disulfiram

/N-c-R3 R2 DDTC: DDTC-Me: SDETG: SETG:

R, = R2 = Et; R3 = SH R1 = R2 = Et; R3 = SMe R, = R2 = Et; R3 = SG (metabolite 'B) R, = Et; R2 = H; ,R3 = SG (metabolite 'E')

0 Rl\

II

/N-c-R3

R2

DETC-Me: DETC-Mew: DETC-MeS02: SDEG: SEG: SCEG: SDEC: SBG:

Rl = R2= Et; R3 = SMe R, = R2 = Et: R3 = S(0)Me R 1 = R2 = Et; R3 = S 0 2 W R1 = R2 = Et; R3 = SG (metabolite '4') R, = Et; R2 = H: R3 = SG (metabolite'C') R1 = Et; R2 = CH-H; R3 = SG (metabolite 'D) R,=RZ=Et: R3=SCyS R1= CH2Ph; R2 = H; R3 = SG

Figure 1. Structures of compounds referred to in the text.

adverse reactions. However, although disulfiram itself has been shown to be a potent, reversible inhibitor of ALDH in vitro (4) and was believed for many years to be the 0 1994 American Chemical Society

Metabolic Activation of Disulfiram active inhibitory species (5), recent mechanistic studies have indicated that the inhibition in vivo differs from that in vitro (6) in that it is irreversible in nature and appears to be mediated by a reactive metabolite(,$ of disulfiram (7, 8). Disulfiram is reduced rapidly in vivo [enzymatically by glutathione reductase in erythrocytes (9,10) and nonenzymatically by plasma albumin (11)l to yield diethyldithiocarbamate (DDTC), which degrades slowly to carbon disulfide and diethylamine (12)and also forms mixed disulfides with proteins (5, 10, 11). In addition, DDTC undergoes metabolism in vivo to the corresponding S-linked glucuronide (13)and two S-methylated compounds, methyl diethyldithiocarbamate (DDTC-Me) (10, 14)and S-methyl diethylthiocarbamate (DETC-Me) (15, 16) (Figure 1). The observation that DDTC-Me and DETC-Me were potent inhibitors of ALDH in vivo, yet were ineffective in vitro, suggested that these compounds probably represented intermediates in the metabolic activation pathway, further biotransformation of which would be required to generate the ultimate enzyme inhibitor (8). Moreover, since pretreatment of rats with n-octylamine, an inhibitor of cytochrome P-450 enzymes (17), blocked the inhibitory effects of disulfiram, DDTCMe, and DETC-Me on the hepatic mitochondrial lOW-Km form of ALDH, it was proposed that metabolic activation of these compounds must entail one or more cytochrome P-450-dependent steps (8). Recently, Hart and Faiman (18) reported on the identification of methyl N,N-diethylthiocarbamoyl sulfoxide (DETC-MeSO; Figure 1) as a metabolite of disulfiram in rats and showed that this reactive thiocarbamate sulfoxide was a potent inhibitor of rat liver mitochondrial low-KmALDH both in vitro (IC50 = 0.75 pM) and in vivo. Indeed, the dose of DETC-MeSO which inhibited ALDH by 50 5% in rats in vivo was only 3.5 mg kg' ip, considerably lower than the doses of disulfiram, DDTC, DDTC-Me, or DETC-Me required to elicit a similar effect (16,18). On the basis of these findings, it was suggested that DETCMeSO may represent the ultimate inhibitory species formed by metabolic activation of both disulfiram and DDTC (18). In support of this hypothesis, pretreatment of rats with inhibitors of cytochrome P-450 prevented the formation of DETC-MeSO from DETC-Me, but did not block ALDH inhibition when DETC-MeSO itself was administered (18). However, Johansson has reported, in a preliminary communication, that DETC-MeSO is oxidized further in vivo to the corresponding sulfone, DETCMeSOz (Figure l),and that this species is an even more potent inhibitor of the rat liver mitochondrial low-K, ALDH in vitro and in vivo than is DETC-MeSO (5). It appears, therefore, that the inhibitory effects of disulfiram in vivo probably are mediated by both DETC-MeSO and DETC-MeS02, two reactive S-oxidized metabolites of the drug which are thought to carbamoylate a critical thiol group at the active site of the enzyme (5, 18). Many thiocarbamate herbicides with structures similar to that of DETC-Me also undergo metabolic S-oxidation, a process which may be catalyzed both by cytochrome P-450 and flavin-containingmonooxygenases (19,20).Such reactions typically result in the formation of electrophilic sulfoxides and sulfones which carbamoylate cellular nucleophiles, e.g., glutathione (GSH) and proteins (15c23). Also, thiocarbamate S-oxides can deplete GSH stores in vivo, and it is now recognized that S-oxidation of this

Chem. Res. Toxicol., Vol. 7,No.4, 1994 527 class of herbicides enhances their carbamoylating activity and their hepatotoxic potential (24-27). By analogy with these systems, it might be expected that the reactive disulfiram metabolites DETC-MeSO and DETC-MeS02 also would react with GSH and that biliary excretion of the resulting adduct, S-(N,N-diethylcarbamoy1)glutathione (SDEG; Figure l), could serve as a valuable indirect measure of metabolic activation of the parent drug in vivo. The primary objective of the present investigation, therefore, was to apply mass spectrometric-based screening techniques to determine whether SDEG indeed was present in the bile of rats dosed with disulfiram and, if so, to quantify the biliary excretion of this conjugate. Related goals were to establish whether DDTC (the reduced metabolite of disulfiram) also gave rise to SDEG in the rat, and whether other GSH adducts were generated during biotransformation of disulfiram and DDTC in vivo.

Experimental Procedures Materials. Disulfiram and sodium DDTCe3HzO were purchased from the Aldrich Chemical Co. (Milwaukee, WI). The sulfone DETC-MeSOz, the glutathione conjugates SDEG, [S(N,N-diethylcarbamoyl)glutathionel, SDETG [S- (N,N-diethylthiocarbamoyl)glutathione], SCEG [S- [N-(carboxymethyl)N-ethylcarbamoyl]glutathionel, and SETG [S- (Nethylthiocarbamoyl)glutathione], and the cysteine conjugate SDEC [S- (N,N-diethylcarbamoyl)cysteine] were obtained by synthesis, as outlined below. SEG [S-(N-ethylcarbamoy1)and SBG [S- (N-benzylcarbamoyl)glutathione] glutathione] (28) (29)were prepared as described previously. All conjugates were >95% pure as determined by HPLC analysis. Other chemicals were obtained from commercial sources and were of analytical grade. Porcine liver esterase was purchased from the Sigma Chemical Co. (St. Louis, MO). According to the manufacturer, 1 unit of this enzyme is defined as the amount required to hydrolyze 1.0 pmol of methyl butyrate to butyric acid min-1 at pH 8.0 and 25 OC. Instrumentation and Analytical Methods. Proton NMR spectra were recorded at 300 MHz on a Varian VXR 300 spectrometer (Varian Associates, Palo Alto, CA). For samples dissolved in CDC13 or MezSO-ds, chemical shifts are expressed in parts per million (6) downfield from tetramethylsilane, while for samples dissolved in DzO, sodium 3-(trimethylsilyl)propionate served as reference. Signal multiplicities are reported as follows: s, d, t, q (quartet), dd (doublet of doublets), and m. Mass spectrometry and combined liquid chromatographytandem mass spectrometry (LC-MS/MS) were carried out on a Perkin-Elmer SciexAPI I11triple-quadrupole mass spectrometer equipped with an atmospheric pressure ion source and an IonSpray interface. Analyses were performed with an ionizing voltage of 5 kV, and high-purity air was used as the nebulizing gas at operating pressures of 40 psi for direct introduction and flow injection analyses and 50 psi for LC-MS/MS experiments. Collisionally-induced dissociation (CID) of selected precursor ions was performed in the rf-only quadrupole region and employed argon as target gas at a thickness of 3.4 X 1014 molecules cm-1. Synthetic standards were dissolved in MeOH/1 % aqueous formic acid (1:lv/v) at a concentration of 10 pg mL-1, and the resulting solutions were introduced directly into the mass spectrometer at a flow rate of 5 p L min-1. For the detection of unknown GSH conjugates in rat bile, specimens of filtered bile (20 p L ) were analyzed by LC-MS/MS using the constant neutral loss scanning technique (lossof 129Da) (30).A narrow-bore Cle HPLC column (150 mm X 2.0 mm i.d.1 was used for LC separations, and was coupled via a splitting tee to both the mass spectrometer and an Applied Biosystems Model 785A UV-visible detector set to monitor absorbance at 214 nm. The mobile phase, which consisted of a mixture of solvent A 10.06% aqueous trifluoroacetic

528 Chem. Res. Toricol., Vol. 7, No. 4, 1994

acid (TFA)] and solvent B (0.06% TFA inCH&N), was delivered by an Applied Biosystems Model 140B pump at a constant flow rate of 200 pL min-l, of which 50 pL min-l entered the mass spectrometer and the remainder was directed to the UV monitor. LC-MS/MS analyses employed the following gradient: 10% solvent B for 10 min, followed by a linear increase in solvent B at a rate of 1% min-l. Once candidate GSH conjugates had been detected by this approach, bile samples were diluted (X50) with MeOH/l% aqueous formic acid (1:1v/v) and reanalyzed by direct introduction MS/MS. By this approach, product ion spectra were obtained directly for the MH+ species of the more abundant conjugates (SDEG and SDETG). For the less abundant adducts, it was necessary to first fractionate bile by HPLC in order to obtain more concentrated specimens for MS/MS analysis. Finally, as authentic samples of the conjugates of interest became available, the identity of each biliary GSH adduct was verified by LC-MS/MS analysis when specimens of bile (or HPLC fractions thereof) were coinjected with the corresponding synthetic materials in order to demonstrate identity of both LC retention time and fragmentation behavior under MS/MS conditions. Quantitative analysis of SDEG in bile was carried out by selected reaction monitoring (SRM) MS/MS. Specimens of filtered bile (20 pL) were treated with internal standard (SBG, 2.0 pg) and diluted (to 1.0 mL) with MeOH/l% aqueous formic acid (1:lv/v). Aliquots (20 pL) of these samples were introduced into the mass spectrometer by the flow injection technique, using a mobile phase consisting of equal proportions of solvent A and solvent B (flow rate 50 pL min-1). The analyte and internal standard (SBG) were detected by monitoring the transitions mlz 407 100 and 441 209, respectively, and the ratios of the resulting ion currents were employed to calculate the amount of SDEG in the bile sample by reference to a calibration curve. The latter was prepared by adding varying amounts of SDEG (0-4.0 jig) and a fixed amount of SBG (2.0 pg) to specimens of drug-free bile (20 pL), which then were treated as above and analyzed together with the unknowns. The standard curve was linear over the range of SDEG concentrations of interest (0-200 pg mL-l bile; r2 = 0.999). Synthesis. (a)Met hyl-N,N-Diet hylthiocarbamoyl Sulfone (DETC-MeS02). To a solution of DETC-Me [prepared according to Kitson (31);3 mmol] in CHzCl2 (5.0 mL) at 0 OC was added dropwise with stirring m-chloroperbenzoic acid (6 mmol) in CHzClz (10 mL). The reaction mixture was stirred for 30 min at 0 OC and for a further 4 h at ambient temperature before being filtered. The filtrate was washed with saturated NaHCOasolution (3 X 15 mL), dried (MgS04), and evaporated to give DETCMeSOz (yield = 90%). 1H NMR (CDC13): 6 1.23 (t,J =7.14 Hz, 3H, CH3CHz), 1.30 (t, J = 6.95 Hz, 3H, CHsCHz), 3.14 (9, 3H, SO2CH3),3.42 (4,J = 7.16 Hz, 2H, CHsCHz) and 3.75 (9,J = 7.05 Hz, 2H, CH3CHz). MS/MS (CID of MH+ at mlz 180): mlz 100 ([CzH&NCO+], 72 [(CzH&N+ or (C2H5)NHCO+l, and 29 (C2H6+)* (b) S-(NJV-Diethylcarbamoy1)glutathione (SDEG).Glutathione (3 mmol) was dissolved in H2O (10 mL), and the pH of the solution was adjusted to 7.8 with 20 M NaOH. A solution of DETC-MeSO2(3 mmol) in MeOH (10 mL) was added, and the resulting mixture was stirred under Nz at ambient temperature for 2 h. The product was filtered, washed with MeOH/HZO (1:l v/v), and dried under vacuum. Yield = 52%. ‘H NMR (DzO): 6 1.11-1.16 [m, 6H, (CZ&CHz)zN], 2.10-2.18 (m, 2H, Glu-@,@’), J=8.28and 14.52Hz, lH, 2.48-2.53 (m,2H,Glu-y,y’),3.20(dd, Cys-P), 3.37-3.47 [m, 5H, (CH~CHZ)~N and Cys-81, 3.79 (t,J = 6.35 Hz, IH, Glu-a),3.95 (s,2H, Gly-a,a’), and 4.62 (dd, J = 5.05 and 8.25 Hz, lH, Cys-a). MS/MS (CID of MH+ at mlz 407): see Figure 3. (c)S-(N,N-Diethylthiocarbamoy1)glutathione(SDETG). To a stirred solution of DDTC (3 mmol) in THF (45 mL) were added triethylamine (3 mmol) and ethyl chloroformate (3mmol). Stirring was continued for 30 min, following which this mixture was added dropwise, via a funnel packed with glass wool, to a solution containing GSH (3 mmol) in HzO (30 mL) and THF

-

-

J i n et al. (45 mL). (The latter solution was prepared by dissolving GSH in HzO and adjusting the pH to 7.8 with 20 M NaOH before adding the THF.) After stirring for 1.5h a t ambient temperature, the reaction mixture was acidified (topH 3) with concentrated HCI and the THF was removed under reduced pressure. The product, which was isolated from the resulting aqueous solution by HPLC (CIS column; mobile phase 20% aqueous CH3CN containing 0.06% TFA), was obtained in an overall yield of 1% . MSIMS (CID of MH+ at mlz 423): see Figure 4. (d) S[ N-(Carboxymethy 1)-N-e hylcarbamoyl]glutathione t (SCEG).Glycine methyl ester hydrochloride (25 mmol) was dissolved in MeOH (20 mL), and an equimolar amount of solid NaOH was added. The solution was stirred at ambient temperature until all the NaOH had dissolved (ca. 30 min) and a fine white precipitate of NaCl formed. Iodoethane (10 mmol) was added, and the reaction mixture was stirred at ambient temperature overnight and then filtered. After removing the MeOH under reduced pressure, the residue was taken up in HsO (20 mL) andextractedwithdiethylether (3 X 30mL). The combined organic extracts were dried (MgSOI), filtered, and evaporated under reduced pressure to give a mixture of mono- and bis-Nethylated glycine methyl ester, together with some starting material. This mixture was used directly to prepare, in turn, S-methyl N-(carboxymethyl)-N-ethylthiocarbamate and the corresponding sulfone, as described above for the synthesis of DETC-Me and DETC-MeS02. The sulfone then was treated with GSH, following which the reaction mixture was subjected to HPLC (CIS column; mobile phase 20% aqueous CH&N containing 0.06 % TFA),which afforded pure SCEG methyl ester in 50% yield. lH NMR (DzO): 6 1.11and 1.18 (t,J = 6.95 and 7.22 Hz, 3H, CH~CHZN, two rotational isomers), 2.17-2.24 (m, 2H, Glu-@,@’), 2.52-2.58 (m, 2H, Glu-y,y’), 3.25 (dd,J = 8.03 and 14.43 Hz, lH, Cys-P), 3.42-3.52 (m, 3H, CH3CHzN and Cys-p), 3.77 and 3.79 (s,3H,COOCH3, two isomers), 3.97 (t,J = 6.56 Hz, lH, Glu-a), 4.01 (9, 2H, Gly-a,a’), 4.22 and 4.32 (8, 2H, NCHzCOOCHs, two isomers), and 4.64 (dd, J = 5.07 and 7.95 Hz, lH, Cys-a). MS: mlz 451 (MH+). A specimen of the free acid form of SCEG was obtained by incubating SCEG methyl ester (300 mg, 0.67 mmol) with porcine liver esterase (300 pL; 840 units of enzyme) in 0.1 M potassium phosphate buffer (pH 7.8,5 mL) at 25 OC for 4 h. The product was purified by HPLC (CIS column; mobile phase 13% aqueous CH&N containing 0.06% TFA), which afforded a compound whose NMR spectrum (DzO)was essentially identical to that of SCEG methyl ester, the only difference being the absence of the COOCH, resonance at 3.79 ppm. MS/MS (CID of MH+ at mlz 437): 362 (MH+- Gly), 308 (MH+- pyroglutamic acid or GSHz+), 291 (308 - NH3), and 205 (HO~CCH~N(CZH~)COSCHZCH= NHz+). (e)S-(N-Et hylthiocarbamoy1)glutathione(SETG).GSH (1mmol) was dissolved in HzO (15 mL), and the pH was adjusted to 8 with 20 M NaOH. To this solution was added ethyl isothiocyanate (3 mmol) in CH3CN (15 mL), and the reaction mixture was stirred at ambient temperature for 30 min under a Nz atmosphere. The product, SETG, which precipitated from solution as a white solid, was collected and washed with CH3CN/H20 (1:l v/v) and dried under vacuum (yield = 90%). 1H NMR (MezSO-ds): 6 1.13 (t, 3H, J =7.22 Hz, CHsCHzNH), 1.851.95 (m, 2H, Glu-j3,@’),2.28-2.33 (m, 2H, Glu-y,y’), 3.22-3.37 (m, 2H, Glu-a and Cys-p),3.53-3.58 (m, 2H, CHsCHzNH), 3.66-3.77 (m,3H,Cys-@andGly-a,af),4.49(dd,1H, J=4.67and9.77Hz, Cys-a), 8.48-8.54 (m, 2H, CONH), and 10.06 (b, lH, SCSNH). MS/MS (CIDof MH+at mlz 395): 320 (MH+- Gly),308 (GSHz+), 266 (MH+ - pyroglutamic acid), 249 (266 - NH3), 233 ([GSHz Gly]+), 199 (233 - HzS), 179 (Cys-Gly + H+), 163 (CzHaNHCSSCH2CH=NHz+), 162 (179 - NH3), 130 (pyroglutamic acid + H+), 88 ([C2HbNH=C==S]+)and 76 (Gly + H+). (f) S-(N,N-Diethylcarbamoy1)cysteine(SDEC). This compound was prepared by a method similar to that described above for the synthesis of SDEG, the only difference being that cysteine was used in place of GSH. The crude product was subjected to HPLC (Cle column;mobile phase 25% aqueous CH3-

Chem. Res. Toxicol., Vol. 7, No. 4, 1994 529

Metabolic Activation of Disulfiram CN containing 0.06 % TFA), which afforded pure SDEC in 55 % yield. 1H NMR (D2O): 6 1.13 (t, 3H, J = 6.73 Hz, CHs), 1.21 (t, 3 H , J = 6.71 Hz,CH~),3.36-3.47 [m,5H, (CHaCH2)2N and Cysb], 3.58 (dd, lH, J = 3.91 and 15.20 Hz, CYS-~), and 4.18 (dd, lH, J = 3.99 and 6.59 Hz, Cys-a). MS/MS (CID of MH+ a t m/z 221): 100 ([C2H6]2N=CxO+) and 72 ([C2HaNH=C=Ol+). Biological Studies. Male Sprague-Dawleyrata (240-280 g), obtained from Charles River Laboratories (Wilmington, MA), were anesthetized with an injection of urethane (1.1g k g l ip). A bile duct cannula then was inserted into each rat by making an incision on the common bile duct between the liver and the duodenum into which was inserted a length of PE-10 tubing. Disulfiram (75 mg kgl) suspended in 0.5% methylcellulose or sodium DDTCe3H20 (114 mg kgl) dissolved in isotonic saline was administered to the rats by ip injection, and bile was collected over ascorbic acid (200 mg) for 4 h. These bile specimens then were taken for analysis by mass spectrometry,as describedabove. Drug-freebile for construction of the calibration curve for SDEG was collected from a rat which received an ip injection of 0.5% methylcellulose only. Thiol Exchange Experiments with SDEG. Synthetic SDEG was incubated (at 0.5mM) with cysteine (2.5 mM) in 0.1 M aqueous phosphate buffer (37 "C, 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 8 h, mixed with internal standard (5-glutathion-S-yl-3-oxovalproic acid (32); 10 pL of a freshly-prepared 2.5 mM aqueous solution], and frozen immediatelywith liquid N2. Samples were stored at -80 OC until analyzed by HPLC for the corresponding cysteine conjugate, SDEC. This was carried out using a Shimadzu LC-600 liquid chromatograph equipped with a Beckman ODS column (25 cm x 4.6 mm i.d., 5 pm). The mobile phase was CH&N/H20 (15:85 v/v) containing 0.06% TFA. The flow rate was 1.0 mL min-I, and compounds eluting from the column were detected by monitoring the UV absorbance at 214 nm (Shimadzu SPD-6A spectrophotometer). Under these conditions,theretention times of SDEC, SDEG, and the internal standard (SBG) were 20.5, 23.4, and 25.7 min, respectively. Inhibition of Aldehyde DehydrogenaseActivity in Vitro. The ability of SDEG to inhibit yeast aldehyde dehydrogenasein vitro was determined by a published method (33) in which the enzyme is exposed to the inhibitor for 15min prior to the addition of NAD+to initiate the reaction. The activity of the ALDH then is determined 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 SDEG examined were 0.3, 1.5, and 3.0 mM.

Results Identification of GSH Conjugates in Bile. Bile samples collected over 0-4 h from rats dosed with either disulfiram (75 mg kg' ip) or sodium DDTC (114 mg kg' ip) were analyzed directly by LC-MS/MS, when candidate GSH adducts were detected on t h e basis of the characteristic loss from the parent (MH+) ion of 129 Da [elimination of the elements of pyroglutamic acid (3013 which is observed with this class of conjugate under CID conditions. Closely similar results were obtained from the disulfiram- and DDTC-treated animals, and a representative reconstructed ion current chromatogram for the 129-Da constant neutral loss LC-MS/MS experiment (derived from a DDTC-dosed rat) is reproduced in Figure 2. By this approach, one major GSH adduct (A) was detected, together with three minor conjugates (B-D). Subsequent studies revealed the presence of trace amounts of a fifth conjugate (E) which is scarcely visible in the chromatogram shown in Figure 2 but whose elution time is indicated. Analysis of the data from this constant neutral loss scanning experiment revealed that the mlz

Rsvniion Time (mi")

Figure 2. Detection of GSH conjugates in the bile of a rat which had been treated with DDTC (591 pmol k g l ip). The chromatogram was obtained from constant neutral loss scanning LC-MS/ MS analysis of a bile specimen collected between 0 and 4 h postdose and depicts all constituents of the sample which eliminated 129 Da upon CID. Retention times of the conjugates referred to as metabolites A-E are indicated. The peaks eluting between 35 and 37 min also were present in drug-free bile and therefore were ascribed to endogenous biliary metabolites.

"1

nyz

Figure 3. Spectrum of product ions obtained by CID of the MH+ ion (m/z 407) of SDEG (metabolite A). The origins of the structurally-diagnostic product ions at m/z 278 ([MH - 129]+) and 100 ([EhNCO]+) are as indicated, while the identities of other fragments are discussed in the text. values for the MH+ions of these candidateGSH conjugates were as follows: A, 407; B, 423; C, 379; D, 437; and E, 395. Following detection of the above metabolites by LCMS/MS, specimens of crude bile (or concentrated HPLC fractions from bile) were introduced directly into the mass spectrometer and t h e MH+ ion of each conjugate was subjected t o CID in order t o obtain a spectrum of product ions. In t h e case of the most abundant conjugate (metabolite A), the product ion spectrum from mlz 407 exhibited a number of structurally-informative fragments (Figure 3). These included ions characteristic of the glutathionyl moiety, e.g., mlz 332 (MH+- Gly), 278 (MH+ - pyroglutamic acid), 199 ([GluNHC(CH2)COI+), 145 ([CH2CH(NH2)CONHCH2COzHI+), and 130 ([pyroglutamic acid HI+),together with fragments indicative of the N,N-diethylcarbamoyl residue derived from disulfiram, e.g., mlz 175 ([Et2NCOSCH2CH=NH21+) (301, 100 ([EtzN=C=Ol+), and 72 ([EtNH=C=OI+ or EhN+). On the basis of this product ion spectrum, metabolite A appeared t o be S-(N,N-diethylcarbamoy1)glutathione (SDEG), a conclusion which was verified when it was found

+

530 Chem. Res. Toxicol., Vol. 7,No.4,1994 2

3

lld

I

A Im

1yI

Figure 4. Spectrum of product ions obtained by CID of the MH*ion (m/z423)of SDETG (metaboliteB). The origins of the structurally-diagnosticproduct ions at m/z 294 ([MH- 129]+) and 116 ([EhNCS]+)are as indicated, while the identities of other fragments are discussed in the text.

that the corresponding synthetic material exhibited identical HPLC properties and afforded the same product ion spectrum as the biological compound. The product ion spectrum of metabolite B (Figure 4) was similar in many respects to that of conjugate A, although both the parent ion (MH+ at mlz 423) and fragments which retained the disulfiram residue were shifted 16 Da to higher mass. This suggested that metabolite B corresponded to the dithio analog of metabolite A, namely, S-(N,N-diethylthiocarbamoy1)glutathione (SDETG), a conclusion which was verified when synthetic SDETG was found to exhibit identical LC-MS/ MS properties to those of the biliary compound. Metabolite C, whose MH+ ion appeared at mlz 379, proved to be the known (28) GSH conjugate, S-(N-ethylcarbamoy1)glutathione (SEG), while metabolite E (MH+ at mlz 395) gave a product ion spectrum in which all of the fragments retaining the drug moiety were shifted 16 Da to higher mass compared with the spectrum of C. It was suspected, therefore, that metabolite E corresponded to the dithio analog of C, namely, S- (N-ethylthiocarbamoy1)glutathione (SETG), and this proposal was found to be correct when asynthetic sample of SETG became available. The final GSH conjugate, metabolite D, afforded an MH+ ion at mlz 437, CID of which yielded abundant fragments at mlz 308 ([MH+ - pyroglutamic acid] and/or GSH2+) and 205. The fact that both the parent ion and the fragment at mlz 205 were 30 Da higher in mass than the corresponding ions in the spectrum of metabolite A suggested that the xenobiotic moiety of conjugate E differed from that of A by the addition of two oxygen atoms and the loss of two hydrogens. One possible structure for D which is compatible with these mass shifts is the acetic acid derivative, S- [ N -(carboxymethyl)-Nethylcarbamoyl] glutathione (SCEG), formed by oxidation of one of the terminal carbons of the diethylamino group to a carboxylic acid. In order to test this hypothesis, a dried specimen of bile was treated with anhydrous methanolic HC1 in order to convert all carboxylate groups to the corresponding methyl esters. When this methylated bile sample was reanalyzed by LC-MS/MS, the increase in the retention time of metabolite D was found to be proportionately greater than for the other GSH conjugates

Jin et al. (which were converted to bis-methyl esters), and the MH+ ion shifted to mlz 479, a gain of 42 Da. On the basis of these results, it was concluded that metabolite D contained three carboxylic acid functionalities, and therefore, the compound most likely was SCEG. This proposal was shown to be correct when an authentic sample of SCEG was prepared by synthesis and found to exhibit HPLC and MS/MS characteristics identical to those of the biological conjugate. Quantitative Analysis of SDEG in Rat Bile. On the basis of the results of the above qualitative studies, it was evident that SDEG (metabolite A) was by far the most abundant GSH conjugate in the bile of rats dosed with either disulfiram or DDTC. In order to assess the quantitative importance of this metabolite in bile, a SRM MS/MS assay was developed for SDEG in which both analyte and internal standard (a related GSH conjugate) were determined in specimens of crude bile. By this technique, it was found that the amounts of SDEG excreted in 0-4 h bile specimens of rats dosed with disulfiram (75 mg kg’; 253 pmol kg-l; 506 pmol of DDTC equivalents k g l ) and sodium DDTC.3H20 (114 mg kg’; 506 pmol k g l ) corresponded to 1.03 f 0.13% (mean f SD, N = 4) and 0.85 f 0.15% ( N = 5 ) of the dose, respectively. No attempt was made to quantify the biliary excretion of the minor conjugates in bile. In Vitro Stability and ALDH Inhibitory Properties of SDEG. In order to determine whether SDEG served as a carbamoylating agent invitro, a sample of the synthetic conjugate was incubated with a 5-fold molar excess of cysteine in aqueous phosphate buffer at pH 7.4 and the reaction mixture was analyzed periodically for the appearance of the corresponding cysteine conjugate, SDEC. Over a period of 8 h, no evidence was obtained for the formation of SDEC, and no loss of SDEG from the solution was noted during this interval. It was concluded, therefore, that SDEG was chemically stable under these conditions. Similarly, followingincubation of SDEG with yeast ALDH in vitro, loss of enzyme activity was found not to be statistically different from that in control incubations from which SDEG had been omitted. Therefore, on the basis of these in vitro studies, it was concluded that SDEG is not a carbamoylating agent and does not inhibit ALDH.

Discussion The present studies, which were made possible by the use of highly selective LC-MS/MS “screening”approaches for GSH conjugates (30, 321, demonstrated that when either disulfiram or DDTC is administered to rats by ip injection, one major and four minor GSH conjugates, accounting collectively for ca. 1% of the dose, are eliminated into bile collected over 4 h. From both a qualitative and quantitative standpoint, administration of disulfiram was equivalent to dosing with 2 mol of DDTC, consistent with early reports that disulfiram undergoes rapid and essentially complete reduction to DDTC in vivo (10). Formation of the above GSH adducts, none of which has been identified previously as a metabolite of disulfiram or DDTC, indicates that both title compounds must undergo biotransformation to several chemically-reactive intermediates which are then “trapped” by GSH in the form of S-linked conjugates. This indirect evidence for reactive metabolite formation is pertinent to recent hypotheses on the mechanism of action of disulfiram, which contend that disulfiram must undergo metabolic activation

Chem. Res. Toxicol., Vol. 7, No. 4, 1994 531

Metabolic Activation of Disulfiram

1 CH,-CH,

\ ?

CH,-Cy/N-C

-S-H

DDTC

. . DETC.MeS0

CH,-CHI

CH

CH. I

.

\ N -C-S P

'I

Inhibition of Aldehyde

/C-NH-CU2-COeH

-CH,-CH

,C%H

\

'NH-C-CHZ-CH~

A

-CH

"%

SDEG (metabclhe'N)

Figure 5. Proposed metabolic pathway for disulfiram leading to the formation of the electrophilicintermediatesDETC-MeSO and DETC-MeSO2, each of which would be expected to inhibit aldehyde dehydrogenase and to give rise to the conjugate SDEG (metabolite A). The precursors of some of the minor GSH conjugatesidentified in this study (notshown)also may contribute to enzyme inhibition. Adapted from Johannson (5) and Yourick and Faiman (8).

for the expressionof ita ALDH-inhibitory properties. Thus, it has been proposed that a sequence of metabolic reactions transforms the parent drug to the sulfoxide DETC-MeSO and/or the sulfone DETC-MeSOz, both of which are electrophilic species which carbamoylate the active site thiol of ALDH and thereby inhibit the enzyme (5,8,18). This view is supported by the fact that the maximum inhibitory effect of disulfiram on ALDH in vivo is manifest only after a certain lag time (141, presumably reflecting the period required for metabolic activation of the drug, and also by the observation that inhibitors of cytochrome P-450 enzymes protect animals against the ALDHinhibitory properties of disulfiram (8). It should be noted, however, that the metabolic activation hypothesis is not universally accepted, and arguments have been made in favor of disulfiram itself as the active inhibitory species (31). The most abundant of the GSH adducts present in the bile of rats dosed with disulfiram or DDTC was the tertiary carbamate thioester derivative SDEG (metabolite A), which is the product expected from reaction of GSH with DETC-MeSO and/or DETC-MeS02 (Figure 5). The identification of SDEG as a metabolite of disulfiram therefore lends support to the above hypothesis that one or both of the S-oxidized derivatives of DETC-Me serves as a carbamoylating agent which inactivates ALDH by covalent modification of an active site thiol (5, 18). Conversion of DETC-MeSO (or DETC-MeSOZ) to SDEG

may be viewed as a detoxification reaction, since SDEG proved to be chemically stable under simulated physiological conditions and was essentially devoid of carbamoylating activity toward cysteine, a representative sulfhydryl nucleophile. It may be inferred, therefore, that if inhibition of ALDH by disulfiram does indeed involve the proposed N,N-diethylcarbamoylation of an active site SH group, then a chemically stable adduct would result. The fact that inhibition of ALDH by disulfiram is known to be irreversible in vivo (reviewed in ref 5) agrees with this interpretation, while our observation that SDEG did not inhibit ALDH in vitro also would be consistent with this model. It should be pointed out that many N-monoeubstituted carbamate thioesters, such as the GSH conjugates of isocyanates, are chemically reactive species in their own right and can donate the elements of the parent isocyanate to sulfhydryl groups on peptides and proteins (34, 35). N,N-Disubstituted carbamate thioesters, on the other hand, must be activated (by S-oxidation) to carbamoylating species, and this would explain the ineffectiveness of DETC-Me as an inhibitor of ALDH in vitro compared to the corresponding electrophilic sulfoxide and sulfone derivatives which are potent inhibitors of the enzyme (5, 18). Thus, it would appear that S-oxidation of DETCMe represents a pivotal step in the metabolic activation of disulfiram to the ultimate inhibitory species. SOxidation plays a similar role in the metabolic activation of thiocarbamate herbicides such as eptam (EPTC),whose structure is closely similar to that of DETC-Me, inasmuch as conversion of eptam to the corresponding sulfoxide and sulfone serves to generate progressively more reactive carbamoylating agents (22, 23). In addition to SDEG, the bile from rats dosed with disulfiram or DDTC contained small amounts of the dithiocarbamate conjugate SDETG (metabolite B), together with the corresponding N-desethyl adducts (metabolites C and E). Also, a conjugate in which one of the ethyl groups of the disulfiram moiety had been oxidized to a carboxylic acid (metabolite D) was identified as a minor constituent. A scheme depicting the proposed metabolic origin of the major GSH conjugate is shown in Figure 5, which indicates that disulfiram is first reduced to DDTC which, in turn, undergoes S-methylation and oxidation at multiple sites. Once again, a comparison may be drawn with eptam since metabolism of this herbicide by liver microsomal preparations was found to result in mono- and dioxygenation at sulfur and hydroxylation at each alkyl carbon (25). Interestingly, while metabolic S-oxidation of thiocarbamate herbicides normally is viewed as a function of the flavin-dependent mixedfunction oxidases of mammalian liver, recent in vitro studies demonstrated that the conversion of DETC-Me to DETC-MeSO in male rat liver microsomes is mediated primarily by cytochrome P-450 enzymes and that CYP2Bl is the major catalyst in hepatic microsomes from phenobarbital-pretreated rata (36). Similarly, the conversion of DDTC-Me to DDTC-MeSO in rat liver microsomes was found to be catalyzed mainly by the cytochrome P-450 system, rather than by flavin-containing monooxygenases (37). These observations raise the intriguing possibility that reactive sulfoxides such as DETC-MeSO and DDTCMeSO might inhibit not only ALDH, but also those isoforms of cytochrome P-450 which participate in their formation. Indeed, it is well-known that both disulfiram and DDTC, which are known to serve as precursors of

532 Chem. Res. Toxicol., Vol. 7, No. 4, 1994

DETC-MeSO in vivo, are effective inhibitors of hepatic cytochromes P-450, although the underlying mechanism(s) remains obscure (38-40). In the interest of clarity, metabolic routes to the minor GSH conjugates identified in this study are not shown in Figure 5. However, S-oxidation of DDTC-Me followed by reaction with GSH would afford SDETG (metaboliteB),while N-dealkylation of DDTC-Me and DETC-Me would yield intermediates which could undergo S-oxidation and conjugation to give SETG (metabolite E) and SEG (metabolite C), respectively. The most likely origin of SCEG (metabolite D) involves w-oxidation of DETC-Me to the corresponding carboxylic acid, followed by S-oxidation and reaction with GSH. On the basis of the detection of the above group of GSH conjugates in the bile of rats dosed with disulfiram and DDTC, it is to be expected that the corresponding N-acetylcysteine adducts will be present in urine, where their cumulative excretion may provide a valuable index of metabolic activation of the parent drug in vivo. Currently, efforts are underway to detect and quantify these putative conjugates and to establish whether their excretion into urine could serve as a convenient, noninvasive approach to investigate the relationship between the metabolism of disulfiram to electrophilic intermediates and its ALDH-inhibitory effects in animals and human subjects.

Acknowledgment. We would like to thank Dr. Xiangming Guan (Department of Medicinal Chemistry, University of Washington) for assistance with the assay for ALDH activity. These studies were supported by Grants RR 05543 and ES 05500 from the National Institutes of Health, which are gratefully acknowledged.

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