Metabolism of the Chemoprotective Agent Diallyl Sulfide to

The chemoprotective effects of diallyl sulfide (DAS), a flavor component of garlic, have been .... facilitate the development of novel chemoprotective...
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Chem. Res. Toxicol. 1997, 10, 318-327

Metabolism of the Chemoprotective Agent Diallyl Sulfide to Glutathione Conjugates in Rats Lixia Jin† and Thomas A. Baillie*,† Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Seattle, Washington 98195 Received October 16, 1996X

The chemoprotective effects of diallyl sulfide (DAS), a flavor component of garlic, have been attributed to its inhibitory effects on CYP2E1-mediated bioactivation of certain carcinogenic chemicals. In addition to being a competitive inhibitor of CYP2E1 in vitro, DAS is known to cause irreversible inhibition of CYP2E1 in rats in vivo. The latter property is believed to be mediated by the DAS metabolite diallyl sulfone (DASO2), which is thought to be a mechanismbased inhibitor of CYP2E1, although the underlying mechanism remains unknown. In order to investigate the nature of the reactive intermediate(s) responsible for the inactivation of CYP2E1 by DAS and its immediate metabolites, the present studies were carried out to detect and identify potential glutathione (GSH) conjugates of DAS and its metabolites diallyl sulfoxide (DASO) and DASO2. By means of ionspray LC-MS/MS, ten GSH conjugates were identified in bile collected from rats dosed with DAS, namely: S-[3-(S′-allyl-S′-oxomercapto)-2-hydroxypropyl]glutathione (M1, M2; diastereomers), S-[3-(S′-allyl-S′-dioxomercapto)-2-hydroxypropyl]glutathione (M5), S-[2-(S′-allyl-S′-dioxomercapto)-1-(hydroxymethyl)ethyl]glutathione (M3, M4; diastereomers), S-[3-(S′-allylmercapto)-2-hydroxypropyl]glutathione (M6), S-(3-hydroxypropyl)glutathione (M7), S-(2-carboxyethyl)glutathione (M8), allyl glutathionyl disulfide (M9), and S-allylglutathione (M10). With the exception of M6, all of the above GSH conjugates were detected in the bile of rats treated with DASO, while only M3, M4, M5, M7, M8, and M10 were found in the bile of rats treated with DASO2. Experiments conducted in vitro showed that GSH reacted spontaneously with DASO to form conjugates M9 and M10, and with DASO2 to form M10. In the presence of NADPH and GSH, incubation of DAS with cDNA-expressed rat CYP2E1 resulted in the formation of metabolites M6, M9, and M10, while incubation with DASO led to the formation of M3, M4, M5, M9, and M10. When DASO2 acted as substrate, CYP2E1 generated only conjugates M3, M4, M5, and M10. These results indicate that while DAS and DASO undergo extensive oxidation in vivo at the sulfur atom, the allylic carbon, and the terminal double bonds, CYP2E1 preferentially catalyzes oxidation of the sulfur atom to form the sulfoxide and the sulfone (DASO and DASO2). However, it appears that the end product of this sequence, namely, DASO2, undergoes further CYP2E1-mediated activation of the olefinic π-bond, a reaction which transforms many terminal olefins to potent mechanismbased P450 inhibitors. We hypothesize, therefore, that it is this final metabolic event with DASO2 which leads to autocatalytic destruction of CYP2E1 and which is mainly responsible for the chemoprotective effects of DAS in vivo.

Introduction A variety of naturally occurring chemicals found in certain fruits and vegetables have the ability to inhibit the initiation and promotion of cancers in animal models (1, 2). Among these compounds is diallyl sulfide (DAS),1 a flavor component of garlic. DAS has been shown to protect rodents from carcinogenesis induced by various chemicals, including aflatoxin B1 (3), benzo[a]pyrene (4), 7,12-dimethylbenz[a]anthracene (5-7), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (8), 1,2-dimethylhydrazine (DMH) (9-12), azoxymethane (13), and a * Corresponding author. † Present address: Department of Drug Metabolism, Merck Research Laboratories, WP26A-2044, West Point, PA 19486. X Abstract published in Advance ACS Abstracts, February 15, 1997. 1 Abbreviations: DAS, diallyl sulfide; DASO, diallyl sulfoxide; DASO2, diallyl sulfone; NDMA, N-nitrosodimethylamine; DMH, 1,2-dimethylhydrazine; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1butanone; BSTFA, N,O-bis(trimethylsilyl)trifluoroacetamide; TFA, trifluoroacetic acid; TMS, trimethylsilyl; GC-EIMS, gas chromatography-electron impact mass spectrometry; GC-CIMS, gas chromatography-chemical ionization mass spectrometry; LC-MS/MS, liquid chromatography-tandem mass spectrometry; CID, collisionallyinduced dissociation.

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number of nitrosamines (14-17). Mechanisms proposed to explain this chemoprotective activity include inhibition of the bioactivation of procarcinogens (14, 18-21), induction of phase II detoxification enzymes (4, 11, 22, 23), and scavenging of ultimate electrophilic carcinogenic species by the sulfur atom of DAS (24). Although it is not clear how individual mechanisms interplay for a given carcinogenic chemical, it is believed that, at least in the case of DMH, azoxymethane, and N-nitrosodimethylamine (NDMA), all of which require metabolic activation by CYP2E1, inhibition of the initial metabolic event represents the primary anti-carcinogenic action of DAS (25). Inhibition of CYP2E1-mediated bioactivation of carcinogenic agents is a complex process. Thus, DAS, which has been reported to be a competitive inhibitor of CYP2E1 (18, 26), undergoes sequential metabolism in rats to diallyl sulfoxide (DASO) and diallyl sulfone (DASO2) (26). While DASO also is a competitive inhibitor of CYP2E1 (26), DASO2 appears to be a mechanism-based inhibitor of the enzyme (26). Administration of DAS to © 1997 American Chemical Society

Glutathione Conjugates of Diallyl Sulfide

rats led to time- and dose-dependent decreases in CYP2E1 activity which were irreversible in nature, and was accompanied by suppression of CYP2E1 protein levels (18, 19). It was suggested that this in vivo enzyme inactivation was the result of DASO2-mediated mechanism-based inhibition of CYP2E1 (26). Consistent with this view, administration of DASO2 to animals led to a more rapid reduction of CYP2E1 activity and protein level than was observed following dosing with DAS (19). Therefore, blockage by DAS of the bioactivation of procarcinogenic substrates of CYP2E1 may be due to the combined effects of competitive inhibition by DAS, DASO, and DASO2, and inactivation of the enzyme by DASO2. The mechanism by which DASO2 destroys CYP2E1 remains unclear. Theoretically, at least two potential reactive intermediates may be generated through cytochrome P450-mediated metabolism of DASO2, either or both of which may play a role in the process. On the one hand, oxidation of the terminal double bonds may lead to the formation of rective intermediates which could alkylate the prosthetic heme moiety of cytochrome P450, and thereby cause destruction of the enzyme (27, 28). On the other hand, oxidation of DASO2 at the allylic position and subsequent S-dealkylation would yield acrolein, a highly reactive aldehyde, which could alkylate a critical nucleophilic residue on the cytochrome P450, resulting in enzyme inactivation (29). In order to gain a better understanding of the mechanism by which DAS exerts its chemoprotective effects, the present study was designed to investigate whether either of the above types of reactive intermediate was formed from DAS in the rat in vivo. Since it was anticipated that electrophilic metabolites of DAS would be short-lived species in vivo, the experimental approach adopted in this investigation was to examine specimens of bile from rats dosed with the compounds of interest for the presence of the corresponding S-linked adducts with GSH. The specific objectives of the present studies, therefore, were: (i) to detect and identify drug-related GSH conjugate(s) in the bile of rats treated with DAS, DASO, or DASO2, and (ii) to detect and identify GSH adducts from incubations of DAS, DASO, or DASO2 with cDNAexpressed rat CYP2E1 in order to establish the role of this isoform of cytochrome P450 in mediating the formation of the corresponding biliary GSH conjugates. It was hoped that the results of the above experiments would provide valuable information on the mechanism by which DAS inhibits CYP2E1 in vivo, which, in turn, may facilitate the development of novel chemoprotective agents.

Experimental Procedures Materials. DAS was purchased from the Aldrich Chemical Co. (Milwaukee, WI) and purified by distillation under reduced pressure (bp ) 58 °C at 20 mmHg). DASO and DASO2 were synthesized as described previously (19, 30). S-[3-(S′Allylmercapto)-2-hydroxypropyl]glutathione, S-[3-(S′-allyl-S′oxomercapto)-2-hydroxypropyl]glutathione, S-[3-(S′-allyl-S′dioxomercapto)-2-hydroxypropyl]glutathione, S-[2-(S′-allyl-S′dioxomercapto)-1-(hydroxymethyl)ethyl]glutathione, S-(3hydroxypropyl)glutathione, S-(2-carboxyethyl)glutathione, S-allylglutathione, and allyl glutathionyl disulfide were synthesized as outlined below. All other chemicals were obtained from commercial sources and were of analytical grade. cDNA-expressed rat CYP2E1, recombinant rat NADPHcytochrome P450 oxidoreductase and purified rat liver cytochrome b5 were generous gifts from Mr. Weiqiao Chen, Dr. Raimund Peter, and Dr. Sidney D. Nelson (University of Washington).

Chem. Res. Toxicol., Vol. 10, No. 3, 1997 319 Instrumentation and Analytical Methods. 1H NMR spectra were recorded at 400 Hz on a Varian Unity 400 spectrometer (Varian Associates, Palo Alto, CA). For samples dissolved in CDCl3 or CD3OD, chemical shifts are expressed in parts per million (δ) downfield from tetramethylsilane, while for samples dissolved in D2O, sodium 3-(trimethylsilyl)-[2,2,3,32H ]propionate was used as the internal reference. Signal 4 multiplicities are reported as follows: s, d, t, dd (doublet of doublets), ddd (doublet of doublet of doublets), dt (doublet of triplets), tm (triplet of multiplets), and m. Gas chromatography-electron impact mass spectrometry (GC-EIMS) was performed on a VG 7070H double-focusing mass spectrometer interfaced to a Hewlett Packard 5710A gas chromatograph. Analyses were carried out at an electron energy of 70 eV using a DB-1 (J & W Scientific, Folsom, CA) capillary GC column (30 m × 0.32 mm i.d., 0.25 µm film thickness). The ion source, GC interface, and injection port temperatures were maintained at 200, 250, and 250 °C, respectively. Helium was employed as carrier gas (head pressure 15 psi), and samples of trimethylsilyl ether derivatives of synthetic diols were injected in the splitless mode onto the column at 40 °C. After 0.5 min, the oven temperature was raised to 80 °C at a rate of 40 °C min-1, and then at 8 °C min-1 to 250 °C. The trimethylsilyl ether derivatives were prepared by treating individual diols (10 µmol) with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA, 100 µL) and heating at 90 °C for 30 min. Gas chromatography-chemical ionization mass spectrometry (GC-CIMS) was carried out using a Fisons Trio 2 mass spectrometer equipped with a Hewlett Packard 5890 gas chromatograph. Analyses were performed in the negative ion mode with an electron energy of 70 eV. The ion source, GC interface, and injection port temperatures were maintained at 200, 250, and 190 °C, respectively. Helium was employed as carrier gas (head pressure 15 psi), and ammonia served as CI reagent gas. A DB-5 capillary GC column (30 m × 0.32 mm i.d., 0.25 µm film thickness) was used, and samples were injected onto the column at 40 °C with a split ratio of 1:20. The GC temperature program was identical to that used in GC-EIMS analyses. Liquid chromatography-tandem mass spectrometry (LC-MS/ MS) was carried out on a Perkin-Elmer Sciex API III Plus triplequadrupole mass spectrometer equipped with an atmospheric pressure ion source and an IonSpray interface. Analyses were 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. Collisionally-induced dissociation (CID) of selected precursor ions was performed in the rf-only quadrupole region where argon was employed as target gas at a thickness of 1.9 × 1014 molecules cm-1. Specimens of filtered bile (20 µL) or incubation mixture (100 µL) were injected directly onto a Beckman Ultrasphere narrow-bore C18 column (150 mm × 2.0 mm i.d.) coupled to a splitter so that one-fourth of the column effluent entered the mass spectrometer. The mobile phase, which consisted of a mixture of solvent A (0.06% aqueous trifluoroacetic acid (TFA)) and solvent B (acetonitrile containing 0.06% TFA), was delivered by a Hewlett Packard 1050 liquid chromatographic system at a constant flow rate of 200 µL min-1. The gradient started at 100% solvent A, followed by a linear increase in solvent B to 10% B in 30 min, then at 5% B min-1 to 90% B. Initially, bile samples were analyzed by the constant neutral loss scanning (loss of 129 Da) technique in order to screen for the presence of unknown GSH conjugates (31). Once candidate GSH conjugates had been detected by this approach, bile samples were reanalyzed to record the product ion mass spectra of individual conjugates by CID of their respective MH+ species. 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 the metabolite was demonstrated to possess LC and MS/MS properties identical to those of the corresponding reference standard. Synthesis. S-[3-(S′-Allyl-S′-oxomercapto)-2-hydroxypropyl]glutathione (M1, M2). To a solution of 3-mercapto-1,2propanediol (10 mmol) in ethanol (50 mL) was added solid NaOH (10 mmol). The resulting solution was cooled to 0 °C

320 Chem. Res. Toxicol., Vol. 10, No. 3, 1997 and treated with allyl bromide (12 mmol) with stirring. After 10 min, the solvent was removed under reduced pressure and the residue was dissolved in H2O (20 mL) and extracted with ethyl acetate (3 × 100 mL). The combined organic extracts were dried over MgSO4, filtered, and evaporated to give a viscous liquid, which was purified by flash column chromatography (CH2Cl2/MeOH, 95:5 v/v) to afford 3-(S-allylmercapto)1,2-propanediol (yield ) 82%). 1H NMR (CD3OD): δ 2.50 (dd, J ) 6.8 and 13.5 Hz, 1H, -SCH2CH(OH)-), 2.62 (dd, J ) 5.7 and 13.5 Hz, 1H, -SCH2CH(OH)-), 3.17 (dm, J ) 7.2 Hz, 2H, CH2dCHCH2-), 3.51 (dd, J ) 5.90 and 11.3 Hz, 1H, -CH2OH), 3.58 (dd, J ) 4.55 and 11.3 Hz, 1H, -CH2OH), 3.71 (m, 1H, -CH(OH)-), 5.10 (m, 2H, CH2dCH-), and 5.79 (m, 1H, CH2dCH-). GC-EIMS (TMS derivative): m/z 292 (M•+, 0.5), 277 ([M - CH3]+, 0.8), 205 ([TMSO-CH2CH)OTMS]+, 6), 189 ([M - CH2-OTMS]+, 4), 147 ([Me2SidOTMS]+, 37), 129 ([TMSOdCHCHdCH2]+, 27), 117 ([TMSO-CH2CH2]+, 15.5), 87 ([M - 205]+, 19), 73 ([TMS]+, 100), and 59 ([Me2SiH]+, 10). A solution of 3-(S-allylmercapto)-1,2-propanediol (5 mmol) in pyridine (10 mL) was cooled to 0 °C and treated with ptoluensulfonyl chloride (5 mmol). The solution was kept in a refrigerator for 2 days and then poured into ice water (100 mL) with stirring. The resulting tosylate derivative was taken up into diethyl ether (3 × 50 mL), and the combined ethereal extracts were washed with cold 50% HCl (2 × 20 mL) and water (2 × 20 mL), dried over MgSO4, and evaporated to give crude 3-(S-allylmercapto)-1,2-propanediol mono-tosylate (yield 75%) which was used in the next step without further purification. A solution of 3-(S-allylmercapto)-1,2-propanediol mono-tosylate (1 mmol) in acetic acid (5 mL) was treated with 35% hydrogen peroxide (1 mmol) and stirred at room temperature for 3 h. GSH (2 mmol) dissolved in water (15 mL) was added to the above solution, and the resulting mixture was adjusted to pH 8 with 10 M NaOH. After stirring under N2 overnight, the reaction mixture was subjected to analysis by HPLC (C18, 250 mm × 10 mm i.d.; mobile phase 4% aqueous acetonitrile containing 0.06% TFA) to give the two stereoisomers of S-[3(S′-allyl-S′-oxomercapto)-2-hydroxypropyl]glutathione (yield ∼60%). The individual stereoisomers (denoted M1 and M2) were characterized by 1H NMR and MS/MS analysis. M1: 1H NMR (D2O): δ 2.25 (m, 2H, Glu-β,β′), 2.61 (m, 2H, Glu-γ,γ′), 2.99 (m, 4H, -CH2CH(OH)CH2- and Cys-β), 3.17 (m, 2H, -CH2CH(OH)- and Cys-β′), 3.62 (dd, 1H, J ) 7.6 and 13.3 Hz, CH2dCHCH2-), 3.82 (dd, 1H, J ) 7.0 and 13.3 Hz, CH2dCHCH2-), 4.00 (t, 1H, J ) 6.4 Hz, Glu-R), 4.05 (s, 2H, GlyR,R′), 4.28 (m, 1H, -CH(OH)-), 4.64 (m, 1H, Cys-R), 5.54 (m, 2H, CH2dCH-), and 5.95 (m, 1H, CH2dCH-). MS/MS (CID of MH+ at m/z 454): m/z 413 ([MH - CH2dCHCH2]•+), 379 ([MH Gly]+), 338 ([413 - Gly]•+), 325 (MH - pyroglutamic acid]+), 284 ([325 - CH2dCHCH2]•+), 274 ([GSH2 - H2S]+), 179 ([CysGly + H]+), 145 ([179 - H2S]+), and 130 ([pyroglutamic acid + H]+). M2: 1H NMR (D2O): δ 2.22 (m, 2H, Glu-β,β′), 2.59 (m, 2H, Glu-γ,γ′), 2.92 (m, 3H, -CH(OH)CH2- and Cys-β), 3.15 (m, 2H, -CH2CH(OH)- and Cys-β′), 3.28 (m, 1H, -CH2CH(OH)-), 3.68 (dd, 1H, J ) 8.2 and 13.3 Hz, CH2dCHCH2-), 3.82 (dd, 1H, J ) 6.0 and 13.3 Hz, CH2dCHCH2-), 3.88 (t, 1H, J ) 6.0 Hz, Glu-R), 4.02 (s, 2H, Gly-R,R′), 4.32 (m, 1H, -CH(OH)-), 4.65 (m, 1H, CysR), 5.55 (m, 2H, CH2dCH-), and 5.97 (m, 1H, CH2dCH-). MS/ MS: CID of the MH+ ion (m/z 454) gave a product ion spectrum which was qualitatively similar to that of M1. S-[2-(S′-Allyl-S′-dioxomercapto)-1-(hydroxymethyl)ethyl]glutathione (M3, M4). To a stirred solution of 3-(Sallylmercapto)-1,2-propanediol (25 mmol) in CH2Cl2 (50 mL) at 0 °C was added m-chloroperoxybenzoic acid (55 mmol) in CH2Cl2 (200 mL). The resulting mixture was brought to room temperature and stirred for 8 h. The product was extracted with water (3 × 50 mL), and the combined extracts were dried under reduced pressure. The residue then was purified by flash column chromatography (CH2Cl2/MeOH; 9:1 v/v) to afford 3-(S-allyl-S-dioxomercapto)-1,2-propanediol as a waxy solid (yield ) 53%). 1H NMR (CD3OD): δ 3.11 (m, 1H, -SO2CH2CH(OH)-), 3.26 (dd, J ) 9.1 and 14.8 Hz, 1H, -SO2CH2CH(OH)-),

Jin and Baillie 3.50 (dd, J ) 5.60 and 11.20 Hz, 1H, -CH2OH), 3.56 (dd, J ) 5.40 and 11.20 Hz, 1H, -CH2OH), 3.87 (ddd, J ) 1.0, 7.0, and 14.0 Hz, 1H, CH2dCHCH2-), 4.02 (dd, J ) 7.8 and 14.0 Hz, 1H, CH2dCHCH2-), 4.16 (m, 1H, -CH(OH)-), 5.49 (m, 2H, CH2dCH-), and 5.94 (m, 1H, CH2dCH-). GC-EIMS (TMS derivative): m/z 309 ([M - CH3]+, 28), 251 ([M - TMS]+, 4), 147 ([Me2Si)OTMS]+, 63), 129 ([TMSOdCHCHdCH2]+, 20), 103 ([CH2)OTMS]+, 87), 75 ([TMSOH]+, 13), 73 ([TMS]+, 100), and 59 ([Me2SiH]+, 7). 3-(S-Allyl-S-dioxomercapto)-1,2-propanediol was tosylated by the method described above for the preparation of 3-(Sallylmercapto)-1,2-propanediol mono-tosylate. The crude product thus obtained was purified by flash column chromatography (CH2Cl2/MeOH; 98:2 v/v) to yield 1-(p-toluenesulfonyloxy)-3(S-allyl-S-dioxomercapto)-2-propanol (yield ) 75%). 1H NMR (CD3OD): δ 2.46 (s, 3H, CH3), 3.01 (ddd, J ) 1.1, 3.1, and 14.8 Hz, 1H, -SO2CH2CH(OH)-), 3.26 (dd, J ) 8.8 and 14.8 Hz, 1H, -SO2CH2CH(OH)-), 3.84 (m, 1H, -CH(OH)CH2O-), 3.96 (m, 1H, -CH(OH)CH2O-), 4.01 (dd, J ) 5.7 and 10.3 Hz, 1H, CH2dCHCH2-), 4.05 (dd, J ) 4.4 and 10.3 Hz, 1H, CH2dCHCH2-), 4.30 (m, 1H, -SO2CH2CH(OH)-), 5.45 (m, 2H, CH2dCH-), 5.89 (m, 1H, CH2dCH-), 7.45 (m, 2H, C6H4-), and 7.81 (m, 2H, C6H4-). MS/MS (CID of MH+ at m/z 335): m/z 163 ([MH - p-toluenesulfonyloxy]+), 121 ([163 - CH2dCHCH3]+), and 107 ([CH2dCHCH2SO2H2]+). 1-(p-Toluenesulfonyloxy)-3-(S-allyl-S-dioxomercapto)-2-propanol (3 mmol) was dissolved in anhydrous methanol (20 mL), treated with K2CO3 (1 mmol), and stirred at room temperature for 1.5 h. The solvent was removed under reduced pressure, and the residue was dissolved in water (25 mL) and extracted with CH2Cl2 (3 × 25 mL). The combined extracts were dried and evaporated to give 3-(S-allyl-S-dioxomercapto)-2-propen-1ol (yield ) 82%). 1H NMR (CD3OD): δ 3.83 (d, J ) 7.6 Hz, 2H, CH2dCHCH2-), 4.31 (dd, J ) 2.2 and 3.2 Hz, 2H, -CH2OH), 5.41 (m, 2H, CH2dCH-), 5.85 (m, 1H, CH2dCH-), 6.58 (dt, J ) 15.0 and 2.2 Hz, 1H, -SO2CHdCH-), and 6.95 (dt, J ) 15.0 and 3.2 Hz, 1H, -SO2CHdCH-). GSH (1.2 mmol) was dissolved in water (10 mL), and the pH of the solution was adjusted to 7.8 with 10 M NaOH. A solution of 3-(S-allyl-S-dioxomercapto)-2-propen-1-ol (1 mmol) in methanol (10 mL) was added, and the resulting mixture was stirred at room temperature for 4 h. The product was subjected to HPLC (C18, 250 mm × 10 mm i.d.; mobile phase 5% aqueous methanol containing 0.06% TFA), which afforded the two diastereomers of S-[2-(S′-allyl-S′-dioxomercapto)-1-(hydroxymethyl)ethyl]glutathione (denoted M3, M4). These products were characterized by 1H NMR and MS/MS analysis. M3: 1H NMR (D2O): δ 2.25 (m, 2H, Glu-β,β′), 2.61 (m, 2H, Glu-γ,γ′), 3.02 (dd, J ) 8.4 and 14.0 Hz, 1H, Cys-β), 3.21 (dd, J ) 5.0 and 14.0 Hz, 1H, Cys-β′), 3.44 (m, 2H, -SO2CH2CH(SG)-), 3.61 (dd, J ) 5.0 and 8.4 Hz, 1H, -SO2CH2CH(SG)-), 3.80 (m, 2H, -CH2OH), 4.04 (s, 2H, Gly-R,R′), 4.08 (m, 3H, CH2dCHCH2and Glu-R), 4.65 (dd, J ) 5.0 and 8.4 Hz, 1H, Cys-R), 5.60 (m, 2H, CH2dCH), and 5.93 (m, 1H, CH2dCH-). MS/MS (CID of MH+ at m/z 470): m/z 395 ([MH - Gly]+), 341 ([MH pyroglutamic acid]+), 324 ([341 - NH3]+), 306 ([GS]+), 238 ([CH2dCHCH2SO2CH2CH(CH2OH)SCH2CHdNH2]+), 221 ([238 - NH3]+), 177 ([GS - pyroglutamic acid]+), 160 ([177 - NH3]+), 145 ([Cys-Gly + H - H2S]+), and 130 ([pyroglutamic acid + H]+). M4: 1H NMR (D2O): δ 2.21 (m, 2H, Glu-β,β′), 2.59 (m, 2H, Glu-γ,γ′), 3.07 (dd, J ) 8.4 and 14.0 Hz, 1H, Cys-β), 3.22 (dd, J ) 5.2 and 14.0 Hz, 1H, Cys-β′), 3.44 (m, 1H, -CH(SG)-), 3.53 (m, 1H, -SO2CH2CH(SG)-), 3.65 (dd, J ) 5.4 and 14.6 Hz, 1H, -SO2CH2CH(SG)-), 3.82 (m, 3H, -CH2OH and Glu-R), 4.01 (s, 2H, Gly-R,R′), 4.12 (m, 2H, CH2dCHCH2-), 4.68 (dd, J ) 5.2 and 8.4 Hz, 1H, Cys-R), 5.62 (m, 2H, CH2dCH-), and 5.96 (m, 1H, CH2dCH-). MS/MS: CID of MH+ ion (m/z 470) gave a product ion spectrum which was qualitatively similar to that of M3. S-[3-(S′-Allyl-S′-dioxomercapto)-2-hydroxypropyl]glutathione (M5). p-Nitroperoxybenzoic acid (1 mmol), which was prepared according to the method of Silbert et al. (32), was added to a solution of DASO2 (1 mmol) in CHCl3 (150 mL). The

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resulting solution was heated under reflux for 10 h, then cooled to room temperature, and filtered. The filtrate was washed with saturated aqueous NaHCO3 (2 × 50 mL) and water (50 mL), dried over MgSO4, and concentrated under reduced pressure. The product was subjected to flash column chromatography (hexane/ethyl acetate, 1:1 v/v) to afford 3-(S-allyl-S-dioxomercapto)-1,2-epoxypropane (yield ) 21%). 1H NMR (CDCl3): δ 2.67 (dd, J ) 2.6 and 4.4 Hz, 1H, -CH(O)CH2), 2.97 (t, J ) 4.4 Hz, 1H, -CH(O)CH2), 3.05 (dd, J ) 8.4 and 14.8 Hz, 1H, -CH2CH(O)CH2), 3.23 (dm, J ) 14.8 Hz, 1H, -CH2CH(O)CH2), 3.41 (m, 1H, -CH(O)CH2), 3.78 (m, 1H, CH2dCHCH2-), 3.91 (dd, J ) 8.2 and 14.2 Hz, 1H, CH2dCHCH2-), 5.51 (m, 2H, CH2dCH-), and 5.93 (m, 1H, CH2dCH-). GC-CI MS: m/z 161 ([M - H]-), 145 ([M - H - O]-), 121 ([M - CH2dCHCH2]-), 105 ([CH2dCHCH2SO2]-), and 64 ([SO2]-). 3-(S-Allyl-S-dioxomercapto)-1,2-epoxypropane (1 mmol) was dissolved in methanol (7.5 mL) and added to a solution of GSH in water (7.5 mL). The resulting solution was adjusted to pH 8 using 10 M NaOH and stirred at room temperature overnight. The crude product was purified by HPLC (C18, 250 mm × 10 mm i.d.; mobile phase 3% aqueous acetonitrile containing 0.06% TFA) to afford S-[3-(S′-allyl-S′-dioxomercapto)2-hydroxypropyl]glutathione (M5, yield ) 30%). 1H NMR (D2O): δ 2.24 (m, 2H, Glu-β,β′), 2.59 (m, 2H, Glu-γ,γ′), 2.80 (m, 2H, -CH2SG), 2.93 (m, 1H, Cys-β), 3.08 (m, 1H, Cys-β′), 3.44 (m, 2H, -SO2CH2CH(OH)-), 4.00 (s, 2H, Gly-R,R′), 4.04 (m, 3H, CH2dCHCH2- and Glu-R), 4.35 (m, 1H, -CH(OH)-), 4.58 (m, 1H, Cys-R), 5.54 (m, 2H, CH2dCH-), and 5.89 (m, 1H, CH2dCH-). MS/MS (CID of MH+ at m/z 470): m/z 395 ([MH - Gly]+), 341 ([MH - pyroglutamic acid]+), 324 ([341 - NH3]+), 306 ([GS]+), 238 ([CH2dCHCH2SO2CH2CH(OH)CH2SCH2CHdNH2]+), 221 ([238 - NH3]+), 177 ([GS - pyroglutamic acid]+), 160 ([177 NH3]+), 145 ([Cys-Gly + H - H2S]+), and 130 ([pyroglutamic acid + H]+). S-[3-(S′-Allylmercapto)-2-hydroxypropyl]glutathione (M6). 3-(S-Allylmercapto)-1,2-propanediol mono-tosylate (1 mmol) was dissolved in anhydrous methanol (15 mL), treated with K2CO3 (2 mmol), and stirred at room temperature for 3 h. The reaction mixture was filtered, and the filtrate was added to a solution of GSH (2 mmol) in water (15 mL). The resulting solution was stirred under N2 overnight, and the product, S-[3(S′-allylmercapto)-2-hydroxypropyl]glutathione (M6), was isolated by HPLC (C18, 250 mm × 10 mm i.d.; mobile phase 13% aqueous acetonitrile containing 0.06% TFA) (yield ) 60%). 1H NMR (D2O): δ 2.21 (m, 2H, Glu-β,β′), 2.59 (m, 2H, Glu-γ,γ′), 2.65-2.81 (m, 3H, -CH2CH(OH)CH2SG), 2.90 (m, 1H, -CH2SG), 2.97 (dd, J ) 7.2 and 14.3 Hz, 1H, Cys-β), 3.14 (dd, J ) 5.0 and 14.3 Hz, 1H, Cys-β′), 3.26 (d, J ) 6.8 Hz, 2H, CH2dCHCH2-), 3.86 (t, 1H, J ) 6.4 Hz, Glu-R), 3.97 (m, 1H, 4.04, -CH(OH)-), 4.02 (s, 2H, Gly-R,R′), 4.63 (m, 1H, Cys-R), 5.21 (m, 2H, CH2dCH-), and 5.88 (m, 1H, CH2dCH-). MS/MS (CID of MH+ at m/z 438): m/z 363 ([MH - Gly]+), 309 (MH - pyroglutamic acid]+), 291 ([309 - H2O]+), 217 ([309 - Gly - NH3]+), 188 ([291 - Gly - CO]+), and 163 ([CH2dCHCH2SCH2CH(OH)CH2S]+). S-(3-Hydroxypropyl)glutathione (M7). GSH (5 mmol) was dissolved in water (35 mL), and the pH of the solution was adjusted to 8.2 with 10 M NaOH. The solution was cooled to 0 °C and 3-bromo-1-propanol (5 mmol) in methanol (35 mL) was added with stirring. Stirring was continued for 3 h, following which the reaction mixture was concentrated under reduced pressure. The product was purified by HPLC (C18 column; mobile phase 15% aqueous methanol containing 0.06% TFA) to afford S-(3-hydroxypropyl)glutathione (M7; yield ) 42%). 1H NMR: δ 1.88 (m, 2H, Glu-β,β′), 2.21 (m, 2H, Glu-γ,γ′), 2.59 (m, 4H, HOCH2CH2CH2S-), 2.84 (dd, J ) 8.8 and 14.0 Hz, 1H, Cysβ), 3.01 (dd, J ) 5.2 and 14.0 Hz, 1H, Cys-β′), 3.62 (m, 2H, HOCH2CH2CH2S-), 3.98 (s, 3H, Gly-R,R′), 4.06 (m, 1H, Glu-R), and 4.54 (dd, J ) 5.2 and 8.8 Hz, 1H, Cys-R). MS/MS (CID of MH+ at m/z 366): m/z 291 ([MH - Gly]+), 237 ([MH pyroglutamic acid]+), 220 ([237 - NH3]+), 202 ([220 - H2O]+), 134 ([HOCH2CH2CH2SCH2CHdNH2]+), 130 ([pyroglutamic acid + H]+), and 117 ([134 - NH3]+).

Figure 1. Detection of GSH conjugates in the bile of a rat which had been treated with: (A) DAS, (B) DASO, or (C) DASO2. The chromatograms were obtained from constant neutral loss scanning LC-MS/MS analyses of bile specimens collected between 0 and 4 h post-dose, and depict all constituents of the samples which eliminated 129 Da upon CID. S-(2-Carboxyethyl)glutathione (M8). To a stirred aqueous solution of GSH (5 mmol; 40 mL) at 0 °C was added solid KOH (5 mmol). After all the KOH had dissolved, the solution was treated with 3-bromopropionic acid (5 mmol) in methanol (40 mL). The mixture was stirred for 4 h and concentrated under reduced pressure. The product was isolated by HPLC (C18 column; mobile phase 15% aqueous methanol containing 0.06% TFA) to afford M8 (yield ) 65%). 1H NMR: δ 2.25 (m, 2H, Glu-β,β′), 2.61 (m, 2H, Glu-γ,γ′), 2.73 (t, J ) 6.8 Hz, 2H, HO2CCH2CH2S-), 2.86 (m, 2H, HO2CCH2CH2S-), 2.93 (dd, J ) 8.8 and 14.0 Hz, 1H, Cys-β), 3.11 (dd, J ) 5.2 and 14.0 Hz, 1H, Cys-β′), 4.04 (m, 3H, Gly-R,R′ and Glu-R), and 4.62 (dd, J ) 5.2 and 8.8 Hz, 1H, Cys-R). MS/MS (CID of MH+ at m/z 380): m/z 305 ([MH - Gly]+), 251 ([MH - pyroglutamic acid]+), 234 ([251 - NH3]+), 216 ([234 - H2O]+), 148 ([HO2CCH2CH2SCH2CHdCH2]+), and 130 ([pyroglutamic acid + H]+).

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Figure 2. Spectrum of product ions obtained by CID of the MH+ ion (m/z 454) of metabolite M1. The proposed origins of characteristic product ions are as indicated. CID of the MH+ ion (m/z 454) of metabolite M2 afforded a product ion spectrum almost identical to that of M1. Allyl Glutathionyl Disulfide (M9). To a stirred solution of GSH (5 mmol) in water (40 mL) at 0 °C was added allyl mercaptan (5 mmol) in methanol (40 mL). The mixture was treated with 35% hydrogen peroxide (5 mmol) and stirred for 2 h. The product was isolated by HPLC (C18 column; mobile phase 45% aqueous methanol containing 0.06% TFA) to afford M9 (yield ) 45%). 1H NMR: δ 2.26 (m, 2H, Glu-β,β′), 2.62 (m, 2H, Glu-γ,γ′), 3.00 (dd, J ) 9.4 and 14.3 Hz, 1H, Cys-β), 3.26 (dd, J ) 4.4 and 14.3 Hz, 1H, Cys-β′), 3.42 (d, J ) 7.6 Hz, 2H, CH2dCHCH2), 4.04 (s, 2H, Gly-R,R′), 4.10 (t, J ) 6.4 Hz, 1H, Glu-R), 4.75 (dd, J ) 4.4 and 9.4 Hz, 1H, Cys-R), 5.25 (m, 2H, CH2dCH-), and 5.92 (m, 1H, CH2dCH-). MS/MS (CID of MH+ at m/z 380): m/z 305 ([MH - Gly]+), 251 ([MH - pyroglutamic acid]+), 234 ([251 - NH3]+), 209 ([Gly-Cys-S]+), 177 ([GS pyroglutamic acid]+), 160 ([177 - NH3]+), 148 ([CH2dCHSSCH2CHdNH2]+), 130 ([pyroglutamic acid + H]+), 112 ([130 - H2O]+), 105 ([CH2dCHCH2SS]+), and 73 ([CH2dCHCH2S]+). S-Allylglutathione (M10). GSH (5 mmol) was dissolved in water (40 mL), and the solution was adjusted to pH 8 with 10 mM NaOH. Allyl bromide (5 mmol) was added with stirring, following which methanol (40 mL) was added to render the mixture homogeneous. The reaction mixture was stirred for 4 h, concentrated under reduced pressure, and subjected to HPLC (C18 column; mobile phase 45% aqueous methanol containing 0.06% TFA) to afford S-allylglutathione (M10; yield ) 58%). 1H NMR: δ 2.56 (m, 2H, Glu-β,β′), 2.61 (m, 2H, Glu-γ,γ′), 2.86 (dd, J ) 8.4 and 14.0 Hz, 1H, Cys-β), 3.03 (dd, J ) 5.2 and 14.0 Hz, 1H, Cys-β′), 3.24 (d, J ) 6.8 Hz, 2H, CH2dCHCH2-), 4.04 (s, 2H, Gly-R,R′), 4.06 (t, J ) 6.8 Hz, 1H, Glu-R), 4.59 (dd, J ) 5.2 and 8.4 Hz, 1H, Cys-R), 5.22 (m, 2H, CH2dCH-), and 5.86 (m, 1H, CH2dCH-). MS/MS (CID of MH+ at m/z 348): m/z 273 ([MH - Gly]+), 219 ([MH - pyroglutamic acid]+), 202 ([219 NH3]+), 130 ([pyroglutamic acid + H]+), and 116 ([CH2dCHSCH2CHdNH2]+). In Vivo Metabolism Studies. Male Sprague-Dawley rats (250-280 g), obtained from Taconic Farm (Germantown, NY), were anesthetized with an ip injection of a mixture of ketamine (68.2 mg kg-1) and xylazine (4.4 mg kg-1) (diluted with saline to a final volume of 2 mL). A bile duct cannula made of PE-10 tubing (1 cm) connected to silastic tubing (50 cm) was inserted into the common bile duct between the liver and the duodenum of each rat. The cannula was introduced under the skin with an eye probe and externalized on the upper back between the shoulder blades where the cannula was secured to a stainless steel button tether assembly (Instech Laboratories, PA). Fol-

lowing recovery from anesthesia, the animal was given an ip dose of one of the following compounds: DAS (200 mg kg-1) dissolved in corn oil (0.6 mL), DASO (226 mg kg-1) or DASO2 (256 mg kg-1) dissolved in isotonic saline. Bile was collected over ascorbic acid for 4 h post-dose, then filtered, and analyzed directly by LC-MS/MS, as described above. In Vitro Metabolism Studies. Homogenate of Tricoplusia ni TN5 cells, which had been infected with a recombinant baculovirus containing a full-length rat CYP2E1 cDNA, was reconstituted with recombinant rat NADPH-cytochrome P450 oxidoreductase and purified rat liver cytochrome b5 at a molar ratio of 1:2:2, respectively. The reconstituted CYP2E1 was dialyzed against 1000 volumes of 0.1 M potassium phosphate buffer (pH 7.4) for 2 h at room temperature to remove glycerol. DAS, DASO, or DASO2 (1 mM) was incubated at 37 °C with the above reconstituted rat CYP2E1 (0.15 nmol) in the presence of GSH (5 mM), NADPH (1 mM), EDTA (1 mM), deferoxamine mesylate (1 mM), and potassium phosphate buffer (100 mM, pH 7.4) in a final volume of 1 mL. The incubations were carried out for 5 h and stopped by adding acetonitrile (3 mL). The mixtures were centrifuged to sediment the protein, and the supernatant was dried under N2, reconstituted with water (300 µL), and subjected to LC-MS/MS analysis.

Results In Vivo Studies. Following administration to rats of DAS, DASO, or DASO2, bile was collected over a period of 4 h and analyzed directly by LC-MS/MS. By means of the constant neutral loss scanning (loss of 129 Da) technique (31), ten GSH conjugates (denoted M1-M10, Figure 1A) were detected in the bile of rats dosed with DAS, none of which was present in bile from control animals which were dosed with vehicle (corn oil) only. With the exception of M6, each of these GSH conjugates also was detected in bile collected from DASO-treated rats (Figure 1B), while conjugates M3, M4, M5, M7, M8, and M10 were found in bile collected from rats dosed with DASO2 (Figure 1C). Metabolites M1 and M2 both exhibited an MH+ ion at m/z 454, suggesting that these two compounds may be isomers. Consistent with this proposal, the product ion mass spectra of M1 and M2, obtained by on-line LC-MS/

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Figure 3. Spectra of product ions obtained by CID of the MH+ ion (m/z 470) of M3 (A) and M5 (B). The proposed origins of characteristic product ions are as indicated. CID of the MH+ ion (m/z 470) of M4 afforded a product ion spectrum almost identical to that of M3.

MS analysis of bile, were almost identical to each other (Figure 2). In addition to ions indicative of the glutathionyl moiety, e.g., m/z 379 ([MH - Gly]+), 325 (MH pyroglutamic acid]+), 179 ([Cys-Gly + H]+), 145 ([179 H2S]+), and 130 ([pyroglutamic acid + H]+), the spectra were characterized by several radical cations resulting from the loss of the elements of the allyl radical (41 Da), e.g., m/z 413 ([MH - CH2dCHCH2]•+), 338 ([379 CH2dCHCH2]•+), and 284 ([325 - CH2dCHCH2]•+). This fragmentation behavior suggested that the conjugates contained an allylic group linked to a functionality which could stabilize the radical cations generated upon homolytic cleavage of the C-S bond in the drug moiety. Potential GSH conjugates which would have such properties, together with a molecular weight of 453 Da, were diastereomers of S-[3-(S′-allyl-S′-oxomercapto)-2-hydroxypropyl]glutathione which could be formed by oxidation of the double bond of DASO, with subsequent addition

of GSH to the resulting epoxide. Cleavage of the C-S bond upon CID of the MH+ ion would afford O-protonated sulfine radical cations (RS•+OH) which are stabilized by delocalization of both the unpaired electron and the positive charge between sulfur and oxygen atoms. This structural hypothesis was proven correct when authentic samples of two stereoisomers of S-[3-(S′-allyl-S′-oxomercapto)-2-hydroxypropyl]glutathione were obtained by synthesis and shown to possess LC-MS/MS characteristics identical to those of M1 and M2. (It should be noted that these conjugates may exist in several diastereomeric forms. No attempt was made in the present studies to establish the absolute stereochemistry of each metabolic adducts.) Metabolites M3, M4, and M5 all afforded an MH+ ion at m/z 470, which was 16 Da higher than that of metabolites M1 and M2, suggesting that M3, M4, and M5 were the sulfone analogs of M1 and M2, or regeoi-

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Figure 4. Spectrum of product ions obtained by CID of the MH+ ion (m/z 438) of metabolite M6. The proposed origins of characteristic product ions are as indicated.

somers thereof. In support of this view, MS/MS analysis of M3, M4, and M5 afforded closely similar product ion mass spectra (Figure 3). Finally, when the corresponding reference compounds became available through chemical synthesis, a comparison of the LC-MS/MS properties of M3, M4, and M5 with those of the standards led to identification of M3 and M4 as the diastereomers of S-[2(S′-allyl-S′-dioxomercapto)-1-(hydroxymethyl)ethyl]glutathione, and M5 as S-[3-(S′-allyl-S′-dioxomercapto)-2hydroxypropyl]glutathione (possibly a mixture of two diastereomers). Metabolite M6 exhibited a protonated molecular ion at m/z 438, 16 Da less than the MH+ ion of M1 and M2. This metabolite, therefore, appeared to be the sulfide analog of M1 and M2, namely, S-[3-(S′-allylmercapto)2-hydroxypropyl]glutathione (possibly a mixture of two diastereomers), a proposal which was shown to be correct when the corresponding synthetic material was found to exhibit identical HPLC properties and to afford the same product ion mass spectrum (Figure 4) as the metabolite. By a similar approach, M7, which exhibited an MH+ species at m/z 366, was identified as S-(3-hydroxypropyl)glutathione, while metabolite M8, exhibiting an MH+ ion at m/z 380, proved to be S-(2-carboxyethyl)glutathione. Metabolite M9, while also displaying an MH+ ion at m/z 380, was more lipophilic than M8. A potential structure for this metabolite was allyl glutathionyl disulfide. The product ion mass spectrum obtained by CID of the MH+ ion of M9 supported this view in that it contained a number of ions indicative of a disulfide bridge and an allyl moiety, e.g., m/z 209 ([SSCH2CH(NH2)COOH]+), 105 ([CH2dCHCH2SS]+), and 73 ([CH2dCHCH2S]+). This conclusion was verified when an authentic sample of allyl glutathionyl disulfide became available. Finally, metabolite M10, which displayed a molecular weight of 347 Da, proved to be S-allylglutathione. CID of the MH+ species (m/z 348) of M10 afforded ions at m/z 273 ([MH - Gly]+), 219 ([MH pyroglutamic acid]+), 202 ([219 - NH3]+), 130 ([pyroglutamic acid + H]+), and 116 ([CH2dCHCH2SCH2CHdNH2]+).

In Vitro Studies. To investigate the role of CYP2E1 in mediating the formation of the observed biliary GSH conjugates M1-M10, cDNA-expressed rat CYP2E1 was incubated with DAS, DASO, or DASO2 in the presence of GSH, and the products were analyzed by LC-MS/MS. First, when DAS was employed as substrate, conjugates M6, M9, and M10 were detected in the incubation mixture (Figure 5A), the formation of which was found to be CYP2E1- and NADPH-dependent (data not shown). Second, conjugates M3, M4, M5, M9, and M10 were identified in the incubation media when DASO was incubated with CYP2E1 (Figure 5B). While the formation of M3, M4, and M5 was CYP2E1- and NADPHdependent, M9 and M10 appeared to be generated chemically since incubation of DASO and GSH in the absence of both CYP2E1 and NADPH resulted in the production of the two adducts (data not shown). Finally, GSH adducts M3, M4, M5, and M10 were identified as metabolites of DASO2 (Figure 5C). The formation of M3, M4, and M5 was found to be CYP2E1- and NADPHdependent, but that of M10 appeared to be a chemical process (data not shown). Finally, it should be noted that two additional GSH conjugates, eluting from the HPLC column at 32 and 38 min (Figure 5C), were formed in the incubation with DASO2. Since these adducts appeared not to be formed in detectable amount from DAS in vivo, no attempt was made in the present studies to identify these conjugates rigorously. Nevertheless, their formation was found to be nonenzymatic, and both exhibited a protonated molecular ion at m/z 454 which is identical to that of conjugates M1 and M2. CID of the MH+ ions of these two conjugates, however, afforded product ion spectra which were quite distinct from those of M1 and M2, in that they consisted only of ions resulting from fragmentation of the glutathionyl moiety, e.g., m/z 379 ([MH Gly]+), 325 ([MH - pyroglutamic acid]+), 308 ([325 NH3]+), 222 ([RSCH2CHdNH2]+), and 179 ([Cys-Gly + H]+). The lack of such radical cations seen in the spectra of M1 and M2 (Figure 2) suggests that these two unidentified GSH adducts are not sulfoxide derivatives.

Glutathione Conjugates of Diallyl Sulfide

Figure 5. Detection of GSH conjugates in the incubation of cDNA-expressed rat CYP2E1 with: (A) DAS, (B) DASO, or (C) DASO2. The incubation mixtures contained reconstituted CYP2E1 (0.15 nmol), substrate (DAS, DASO, or DASO2; 1 mM), GSH (5 mM), NADPH (1 mM), EDTA (1 mM), deferoxamine mesylate (1 mM), and potassium phosphate buffer (100 mM, pH 7.4) in a final volume of 1 mL. The incubation was carried out for 5 h, and the incubation mixtures were analyzed by constant neutral loss scanning LC-MS/MS. The chromatograms depict all constituents of the samples which eliminated 129 Da upon CID.

Based on the electrophilic nature of DASO2, it is likely that these two conjugates may correspond to the products formed by addition of GSH to DASO2.

Discussion The present studies on the metabolic activation of the chemoprotective agent DAS focused on the detection and identification of GSH conjugates of DAS and its immediate metabolites DASO and DASO2 in vivo and in vitro. By means of ionspray LC-MS/MS, a total of ten GSH adducts (M1-M10) were detected in bile collected from rats dosed with DAS. The structures of M1-M10 indicate that DAS undergoes extensive oxidation in vivo at various positions in the molecule, as depicted in Figure 6.

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Metabolite M6 was identified as S-[3-(S′-allylmercapto)-2-hydroxypropyl]glutathione, while M1-M2 and M3-M5 proved to be the sulfoxide and sulfone analogs of M6, respectively. These three groups of metabolites most likely represent the products of conjugation of GSH with the epoxides of DAS, DASO, and DASO2. It follows that DAS and its S-oxidized metabolites DASO and DASO2 undergo oxidative metabolism at the terminal double bonds, as shown in Figure 6, to yield electrophilic epoxides which, in turn, are “trapped” by GSH to afford the above S-linked adducts. Since 3-(S-allyl-S-dioxomercapto)-1,2-epoxypropane, the epoxide derivative of DASO2, rearranged spontaneously at room temperature to 3-(Sallyl-S-dioxomercapto)-2-propan-1-ol (data not shown), it is also possible that M3 and M4 are formed in vivo by the reaction of GSH with 3-(S-allyl-S-dioxomercapto)-2propan-1-ol (Figure 6). The fact that M1-M5 were detected in DASO-treated rat bile, while M3-M5 were present in DASO2-treated rat bile, lends further support to the operation of the metabolic pathways for the formation of M1-M6 outlined in Figure 6. The absence of M6 in DASO-treated bile, and that of M1, M2, and M6 in DASO2-treated bile, suggests that the in vivo reduction of DASO2 to DASO, and of DASO to DAS, does not occur to any appreciable extent, if at all. Metabolites M7 and M8 were identified as S-(3hydroxypropyl)glutathione and S-(2-carboxyethyl)glutathione, which correspond, respectively, to the products formed upon reduction and oxidation of S-(3-oxopropyl)glutathione. Note that S-(3-oxopropyl)glutathione is the GSH adduct of acrolein which has been shown to undergo rapid reduction or oxidation in vivo (33-35). Therefore, the presence of M7 and M8 in bile of rats dosed with DAS indicates that DAS undergoes oxidation at the allylic carbon followed by S-dealkylation to form the reactive aldehyde acrolein (Figure 6). Moreover, both M7 and M8 were found in bile collected from rats treated with either DASO or DASO2, suggesting that the sulfoxide and sulfone also undergo oxidation at the allylic positions. Metabolite M9 was identified as allyl glutathionyl disulfide and metabolite M10 as S-allylglutathione. M9 could be formed from conjugation of GSH with allyl sulfenic acid generated from either S-oxidation of allyl mercaptan (the product of S-dealkylation of DAS) or S-dealkylation of DASO. M9 also could be formed directly through chemical reaction of DASO with GSH, since incubation of DASO and GSH alone in vitro resulted in the formation of both M9 and M10. The latter may be attributed to the electrophilic nature of DASO, inasmuch as DASO may undergo an addition-elimination reaction with GSH to yield S-allylglutathione (M10) and allyl sulfenic acid; the latter species, in turn, would be expected to react with GSH to form M9. In a similar manner, the electrophilic DASO2 may react directly with GSH to form M10 and allyl sulfinic acid, consistent with the fact that M10, but not M9, was detected in bile of rats treated with DASO2 as well as in the incubation of DASO2 with GSH. Based on the above observations, it appears that metabolism of DAS in rats occurs by way of oxidation at the sulfur atom, the allylic carbon, and the terminal double bonds (Figure 6). Although no attempt was made to quantify individual GSH conjugates of DAS, inspection of the chromatograms obtained by LC-MS/MS analyses of bile collected from rats dosed with either DAS, DASO, or DASO2 suggests that oxidation at the sulfur atom is

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Figure 6. Proposed scheme for the metabolism of DAS to GSH conjugates M1-M10 in rats. Compounds shown in brackets have not been isolated, but represent proposed intermediates. The bold arrows indicate major metabolic pathways of DAS in rats. See text for details.

favored, followed by oxidation at the double bonds and at the allylic carbon. The in vitro experiments with cDNA-expressed rat CYP2E1 demonstrated that, in the presence of NADPH and GSH, CYP2E1 catalyzes the formation of M6, M9, and M10 from DAS, and of M3-M5 from both DASO and DASO2. Evidently, CYP2E1 mediates the oxidation of DAS to DASO and subsequently to DASO2, as well as the formation of epoxide derivatives of DAS and DASO2. In light of the previous finding that inactivation of CYP2E1 by DASO2 is a mechanism-based process (26), it appears most likely that CYP2E1-mediated oxidation of the terminal double bonds of DASO2 is the key event that leads to the autocatalytical destruction of the enzyme, while the formation of other electrophilic species such as allyl sulfenic acid and acrolein also may play a role in vivo. Although DAS itself undergoes CYP2E1-catalyzed oxidation at the terminal double bonds, this metabolic pathway represents a minor biotransformation process in rats. When DAS and DASO serve as substrates, CYP2E1 preferentially catalyzes oxidation of the sulfur atom, which thus explains the competitive CYP2E1inhibitory properties of DAS and DASO. With DASO2, on the other hand, CYP2E1-mediated oxidation occurs at the terminal π-bond. By analogy to many other terminal olefins, the latter metabolic event would lead to the formation of a reactive species which may either inactivate CYP2E1 by alkylating the prosthetic heme moiety of the cytochrome or collapse to an epoxide which is released from the enzyme active site (27, 28). As a result, both the oxidation status of the sulfur atom and the presence of the terminal double bonds must be critical factors in mediating the chemoprotective activity of DAS in rats. Indeed, DASO2 has been found to cause more rapid inactivation of CYP2E1 in rats than either DAS or DASO (19), consistent with the requirement for biotransformation of DAS and DASO to the sulfone prior to inactivation of the enzyme. Furthermore, dipropyl sul-

fide, the saturated analog of DAS, has been shown to have no effect in preventing DMH-induced colon cancer in mice (12), again consistent with the above mechanistic interpretation. The insight gained through the present in vivo and in vitro studies into the mechanism by which the naturally occurring compound DAS elicits its chemoprotective effects in rats may serve as a basis for further studies on the potential of DAS chemoprevention in humans, whose CYP2E1 has been shown to share most of the fundamental properties of the rat enzyme, in particular, that of catalyzing the bioactivation of many carcinogens and toxins (36). In order to provide a basis for future clinical studies with this interesting series of sulfurcontaining natural products, studies are underway to investigate whether DAS, DASO, and DASO2 interact with human CYP2E1 in a similar fashion to that with rat CYP2E1, and thereby inhibit the bioactiovation of carcinogenic chemicals.

Acknowledgment. We would like to thank Mr. Weiqiao Chen, Dr. Raimund Peter, and Dr. Sidney D. Nelson (Department of Medicinal Chemistry, University of Washington, Seattle, WA) for their generous gifts of cDNA-expressed rat CYP2E1, recombinant rat NADPHcytochrome P450 oxidoreductase, and purified rat liver cytochrome b5. We also thank Dr. George A. Doss (Merck Research Laboratories, Rahway, NJ) for obtaining 1H NMR spectra of 3-(S-allylmercapto)-1,2-propanediol and 3-(S-allyl-S-dioxomercapto)-1,2-propanediol, and Dr. Jianguo Zhao (Merck Research Laboratories, West Point, PA) for GC-CIMS analysis. These studies were supported by Grant ES05500 from the National Institutes of Health, which is gratefully acknowledged.

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