Detoxication Pathways Involving Glutathione and Epoxide Hydrolase

Sep 6, 2003 - ... were obtained from Lancaster Synthesis (Newgate, Lancashire, U.K.). ...... In vitro metabolic fate of a novel structural class: Evid...
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Chem. Res. Toxicol. 2003, 16, 1287-1297

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Detoxication Pathways Involving Glutathione and Epoxide Hydrolase in the in Vitro Metabolism of Chloroprene Tony Munter,† Lisa Cottrell,† Bernard T. Golding,*,‡ and William P. Watson*,† Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, SK10 4TJ, United Kingdom, and School of Natural SciencessChemistry, Bedson Building, University of Newcastle upon Tyne, Newcastle upon Tyne, NE1 7RU, United Kingdom Received May 28, 2003

Chloroprene (2-chloro-1,3-butadiene, 1) is an important industrial chemical, which is carcinogenic in experimental animals and possibly in humans. It is metabolized to the monoepoxides, 2-chloro-2-ethenyloxirane (2a,b) and (1-chloroethenyl)oxirane (3a,b), together with electrophilic chlorinated aldehydes and ketones. This study has investigated the detoxication of these chloroprene metabolites in vitro by glutathione (GSH) and epoxide hydrolase (EH) in liver microsomes from Sprague-Dawley rats, B6C3F1 mice, and humans. In incubations of chloroprene with liver microsomes containing GSH, several GSH conjugates were identified. These were 1-hydroxy-4-(S-glutathionyl)butan-2-one (13), 1,4-bis-(S-glutathionyl)butan-2-one (15), and (Z)-2-(S-glutathionyl)but-2-en-1-al (16). A fourth GSH conjugate was identified as either 2-chloro-3-hydroxy-4-(S-glutathionyl)butene (12a,b) or 1-chloro-4-(Sglutathionyl)-butan-2-one (14), which were indistinguishable by LC/MS. Structural assignments of metabolites were based on chromatographic and spectroscopic comparisons with synthetic standards. There were significant differences between species in the amounts of 3a,b formed in microsomal incubations, the order being mouse > rat > human. Hydrolysis by microsomal EHs showed a distinct selectivity for S-(1-chloroethenyl)oxirane (3b) resulting in an accumulation of the R-enantiomer; the ratio of the amounts between species was 20:4:1 for mouse:rat: human, respectively.

Introduction

Scheme 1

Chloroprene (2-chloro-1,3-butadiene, 1) (Scheme 1) is used as a monomer in the production of polychloroprene, an elastomer for the manufacture of automotive rubber goods and personal protection clothing (1). The principal source of exposure to chloroprene is occupational (2). The International Agency for Research on Cancer has classified chloroprene as a group 2B carcinogen (possible human carcinogen) on the basis of sufficient evidence for carcinogenicity in experimental animals but inadequate evidence in humans (3). Chloroprene has been found to be a multisite carcinogen in F344/N rats and B6C3F1 mice following long-term inhalation exposure (4, 5). Cohort studies have suggested an increase in the risk of lung and liver cancers among workers exposed to chloroprene (6-9); however, the existing studies are considered limited due to poor exposure characterization, lack of control of potential confounding factors, incompleteness in cohort enumeration, short follow up periods, and small numbers of cancer cases (6). We have shown that chloroprene is metabolized in mouse, rat, and human liver microsomes to the epoxide metabolites, (1-chloroethenyl)oxirane (3a,b) and 2-chloro2-ethenyloxirane (2a,b), by cytochromes P450 (Scheme 1). In addition to the epoxides, a variety of multifunc* To whom correspondence should be addressed. (B.T.G.) E-mail: [email protected]. (W.P.W.) E-mail: [email protected]. † Syngenta Central Toxicology Laboratory. ‡ University of Newcastle upon Tyne.

10.1021/tx034107m CCC: $25.00 © 2003 American Chemical Society Published on Web 09/06/2003

1288 Chem. Res. Toxicol., Vol. 16, No. 10, 2003

tional aldehydes and ketones are also formed (10). The major metabolite (1-chloroethenyl)oxirane has been found to induce mutagenicity comparable to that of ethenyloxirane in various strains of Salmonella typhimurium but was found to be nonclastogenic in an in vitro study using Chinese hamster V79 cells (11). An additional metabolite, (Z)-2-chlorobut-2-en-1-al (8), found in microsomal oxidations of chloroprene, is a potent direct-acting bacterial mutagen inducing about 200 000 revertants/µmol in S. typhimurium strain TA100 (12). In a recent study, we demonstrated that (1-chloroethenyl)oxirane reacts with nucleosides and double-stranded DNA with a preferential alkylation at N7 of 2′-deoxyguanosine and N3 of 2′deoxycytidine (13). The modification of DNA by the chloroprene metabolites may play a fundamental role in the carcinogenesis of chloroprene. Detoxication of highly reactive electrophilic metabolites may occur by spontaneous conjugation with GSH or enzymatic conjugation by GSH transferases to form less reactive conjugates that can be readily excreted (14). Summer and Greim have reported that the metabolism of chloroprene in the rat leads to formation of GSH conjugates with concomitant depletion of hepatic GSH and increased excretion of thioethers (15). The mutagenic activities of 8 and other acrolein derivatives in bacteria were considerably reduced in the presence of a liver S9 mix due to inactivation by GSH (12, 16). One of the chloroprene metabolites, 1-hydroxybut-3-en-2-one (5), has also been postulated as a reactive metabolite of 1,3butadiene (17, 18). Recently, Krause et al. identified the GSH conjugate, 1-hydroxy-4-(S-glutathionyl)butan-2-one (13), in P450 oxidation of 3-butene-1,2-diol in mouse, rat, and human liver microsomes (19). Mammalian EHs1 are present in liver and other tissues to detoxicate epoxides. They catalyze the hydrolysis of epoxides to more soluble and easily excretable vicinal diols (14, 20). The reactive epoxides of chloroprene, (1chloroethenyl)oxirane and 2-chloro-2-ethenyloxirane, were effectively hydrolyzed by EH to 3-chlorobut-3-ene-1,2-diol (4a,b) and 5, respectively (10). The inactivation of the metabolites by EH and by GSH conjugation may prevent their covalent binding to DNA and proteins. The overall toxicity is determined by the balance of the oxidative activating reactions and hydrolytic and conjugative detoxication reactions. In this work, we present the results of studies on the detoxication pathways of metabolites by GSH and EH in the in vitro microsomal metabolism of chloroprene. The GSH conjugates and metabolites were identified by comparison with synthetic reference standards.

Experimental Procedures Caution: Care should be exercised in the handling of chloroprene and its metabolites; the work should be performed with protective clothing and in a well-ventilated fume hood. Chemicals. GSH, anhydrous DMSO, NADPH, LiCl, and m-CPBA were purchased from Sigma-Aldrich Company (Poole, Dorset, U.K.). The commercial m-CPBA was purified by washing with phosphate buffer (pH 7.5), filtering, and drying the residue in a vacuum desiccator (21). Dess-Martin periodinane, 2-butyne-1,4-diol, and sodium borohydride were obtained from 1 Abbreviations: EH, epoxide hydrolase; SIM, selected ion monitoring; ESI-MS, electrospray ionization mass spectrometry; HMBC, heteronuclear multiple bond connectivity NMR spectroscopy (longrange 1H-13C NMR spectroscopy); m-CPBA, 3-chloroperoxybenzoic acid.

Munter et al. Lancaster Synthesis (Newgate, Lancashire, U.K.). [35S]GSH at a specific activity of 30 mCi (1.11 TBq)/mmol was purchased from PerkinElmer NEN Life Sciences (Cambridge, U.K.) and was diluted with unlabeled GSH and used at a specific activity of 5.2 KBq/µmol. 1-Tosyloxy-but-3-en-2-ol was prepared according to the method of Crawford et al. (22) and purified by chromatography on silica (eluent petrol:ether, 1:1 v/v). 2,6Dimethoxybenzenethiol (20) was prepared according to a literature procedure (23). Chloroprene, racemic and (S)-(1chloroethenyl)oxirane, 8, and 4a,b were prepared as described previously (10). Petrol refers to the solvent with a boiling range of 40-60 °C. HPLC and LC/MS. LC/MS analyses were performed on a Finnigan LCQ quadrupole ion trap mass spectrometer (Thermo Finnigan, Hemel Hempstead, Herts, U.K.) equipped with an electrospray source and operated in the positive ion mode. The conditions applied to the electrospray source were as follows: spray voltage, 4.5 kV; capillary temperature, 200 °C; and capillary voltage, 33 V. Nitrogen was used as the sheath gas (80 arbitrary units) and as an auxiliary gas (10 arbitrary units). Scanning from m/z 50-800 was applied for the scanning of the full scan mass spectra. The HPLC separations were performed on a Hewlett-Packard 1100 system (Agilent Technologies UK Ltd., West Lothian, U.K.) consisting of a quaternary pump, a vacuum degasser, an autosampler, a thermostated column oven, a UV variable wavelength detector, and a Canberra Radiomatic flo-ae radiochemical detector (Canberra Harwell, Didcot, Oxfordshire, U.K.). The GSH reaction mixtures were chromatographed on a 5 µm, 4.6 mm × 250 mm, reversed-phase C18 analytical column (Luna, Phenomenex, Macclesfield, Cheshire, U.K.). The column was eluted with a gradient of methanol (eluent A) in water (eluent B). Formic acid was added to both eluents (0.1% v/v). The gradient applied was as follows: for 5 min eluent A 0.1%, at 11 min A 1%, at 20 min A 20%, at 25 min A 50%, at 26 min A 80%, and at 30 min A 80%. The flow rate was 1 mL/min, and the injection volume was 20 µL. For the analysis of the reaction mixture of 20 with 1-chlorobut-3-en-2one (6), a linear gradient starting from 40% A and ending after 30 min at 80% A was applied. Preparative isolation of the conjugates from the reaction mixtures was performed by HPLC on a semipreparative 5 µm, 10 mm × 250 mm, reversed-phase C18 column (Spherisorb ODS2, Hichrom, Theale, Berkshire, U.K.). The column was coupled to a Gilson HPLC system, which consisted of a Gilson 305 pump, a 303 pump, a 811 dynamic mixer, a 802 manometric module, and a Holocrome variable wavelength UV spectrophotometric detector. The GSH conjugate, (Z)-2-(S-glutathionyl)but-2-en-1-al (16), was desalted by column chromatograpy on a 3 cm × 8 cm column of reversed-phase C18 silica gel 100 (particle size, 40-63 µm; Sigma-Aldrich Company, Gillingham Dorset, U.K.). GC/MS Analysis of 3a,b and 4a,b. Compounds 3a,b and 4a,b were quantified by a GC/MS system comprising of a Hewlett-Packard 5973 mass selective detector and HewlettPackard 6890 GC equipped with a split/splitless injector. The compounds were separated using a Chiraldex G-PN (γ-cyclodextrin propionyl) capillary column (30 m × 0.25 mm i.d.) with a helium flow of 1 mL/min and an injector temperature of 200 °C with a split ratio of 5:1. For the analysis of the enantiomers of 3a,b, the oven temperature was held at 40 °C for 1 min and then increased at 10 °C/min to 60 °C for 4 min. The MS was operated in SIM mode, and ions were selected at m/z 39, 69, and 104. For the analysis of 4a,b, the oven temperature was held at 40 °C for 10 min and then increased at 10 °C/min for the following program: 60 (4 min), 100 (10 min), and 150 °C (1 min). The MS was operated in SIM mode, and the ions selected were m/z 91 and 104. The compounds were quantified by reference to standard curves based on measurement of a range of concentrations of 3a,b and 4a,b in the test medium in the absence of enzymes. MS Excel 2000 was used for the preparation of plots and trend lines.

Detoxication of Chloroprene Metabolites NMR. The 1H and 13C NMR spectra were recorded with either a 500 MHz JEOL-JNM-LA 500 spectrometer (JEOL, Tokyo, Japan) or a 300 MHz Bruker AVANCE spectrometer (Bruker, Rheinstetten, Germany) operating at frequencies given with the spectral data for each compound. The samples were dissolved in CDCl3 or D2O, and the solvent was used as the internal reference standard. The 1H NMR signal assignments were based on chemical shifts and 1H-1H and 13C-1H correlation data. Assignments of carbon signals were based on chemical shifts and 13C-1H correlations. Animals. Male Sprague-Dawley rats (8-9 weeks old) and B6C3F1 mice (5-7 weeks old) were supplied by Charles River (Manston, Kent, U.K.) and were acclimatized for at least 4 days before use. Human, Rat, and Mouse Liver Microsomes. Pooled microsomes from male human liver were obtained from In Vitro Technologies (Baltimore, MD). The pools were prepared from 15 donors. Rat and mouse liver microsomes were prepared as previously described (10). Metabolism of Chloroprene to 3a,b and 4a,b. Experiments were carried out using rat, mouse, or human liver microsomes at varying concentrations of chloroprene (0.01-10 mM). Microsomes (1.5 mg of protein) were incubated with chloroprene in acetonitrile (5 µL) and 10 mM NADPH in 0.1 M potassium phosphate buffer (pH 7.4) in a total volume of 1 mL. The samples were incubated in 10 mL gastight vials at 37 °C for 30 min with shaking. No EH inhibitor (10) was added to the samples. Controls were prepared where no substrate or no NADPH was added. Headspace samples were removed after 30 min and analyzed by GC/MS to quantify the amount of 3a,b. The reaction was then stopped by addition of 2 vol of ice cold ethyl acetate. This solvent was then used to extract the 4a,b from the samples. Conjugation of Chloroprene Metabolites with GSH. Experiments were carried out using rat, mouse, or human liver microsomes at varying concentrations of chloroprene (0.01-10 mM). Microsomes (1.5 mg of protein) were incubated with chloroprene in acetonitrile (5 µL), [35S]GSH (0.01-10 mM), and 10 mM NADPH in 0.1 M potassium phosphate buffer (pH 7.4) in a total volume of 1 mL. The samples were incubated in 10 mL gastight vials at 37 °C for 30 min with shaking. The reaction was stopped by the addition of 2 vol of acetonitrile, the samples were thoroughly mixed and centrifuged at 13 000g for 10 min, and the supernatant was removed. The samples were concentrated and analyzed by radiochemical HPLC using the analytical HPLC column and the same gradient program as described for the LC/MS method. Epoxidation of 3a,b with m-CPBA. (1-Chloroethenyl)oxirane was oxidized by m-CPBA to a mixture of diastereoisomers of chloro-2,2′-bioxiranes (9) as previously described (10). After a reaction time of 160 h, D2O (100 µL) was added and the two phase mixture was stirred for 32 h. The CDCl3 phase was monitored by NMR and showed the formation of 2-chloro-1oxiran-2-ylethanone (10). 1H NMR (500 MHz, CDCl3): δ 4.09 (AB, 2 H, 2 × H-2′, J ) 16.1 Hz), 3.60 (dd, 1 H, H-2, J ) 4.6, 2.4 Hz), 3.04 (dd, 1 H, H-3a, J ) 5.5 and 4.9 Hz), 2.89 (dd, 1 H, H-3b, J ) 5.5, 2.4 Hz). 13C NMR (125.65 MHz, CDCl3): δ 198.6 (CO), 52.4 (C-2), 46.9 (C-3), 44.6 (C-2′). EI-MS m/z 122/120 (5/ 30, M+), 85 (30, M+ - Cl), 79/77 (10/31, M+ - C2H3O), 71 (100, M+ - CH2Cl). Epoxidation of Chloroprene to 9. Chloroprene (15 mg, 0.17 mmol) was added to a solution of m-CPBA (60 mg, 0.35 mmol) in CDCl3 (0.8 mL). The mixture was held at room temperature for 44 h and was monitored by GC/MS. After 24 h, there was an appearance of a peak in the GC/MS with a mass ion at m/z 120/122 and fragment ions at m/z 85, 79, 77, and 71. In Vitro Detection of 9. Various experimental methods were employed to determine whether the product of oxidation of chloroprene with m-CPBA could be detected in vitro. Rat and mouse liver microsomes (2 mg) were incubated with 20 mM chloroprene, 10 mM NADPH, and 1 mM cyclohexene oxide for 30, 60, 150, and 240 min. The samples were extracted with ice

Chem. Res. Toxicol., Vol. 16, No. 10, 2003 1289 cold ethyl acetate and analyzed by GC/MS using the same GC/ MS conditions as described for 4a,b. The MS was operated in SIM mode with ions selected at m/z 71, 85, and 120/122. This procedure was repeated with 3a,b as the substrate. Rat and mouse liver microsomes were also incubated with 5 mM 6 and 10 mM NADPH at 37 °C for 15, 30, and 60 min. The samples were extracted and analyzed as above. Preparation of 1,4-Dihydroxybutan-2-one. This compound was essentially prepared according to a published method (24). HgSO4 (0.9 g, 3 mmol) and concentrated sulfuric acid (2.0 g) were added to a solution of 2-butyne-1,4-diol (17.2 g, 0.2 mol) in water (152 mL). The reaction mixture was stirred at 30 °C for 20 h, carefully neutralized by barium carbonate, filtered through Celite, and concentrated by evaporation under reduced pressure to give 1,4-dihydroxybutan-2-one as a pale yellow oil (15.2 g, 73%). 1H NMR (300 MHz, D2O): δ 4.26 (s, 2 H, 2 × H-1), 3.74 (t, 2 H, 2 × H-4, J ) 6.0 Hz), 2.59 (t, 2 H, 2 × H-3, J ) 6.0 Hz). Preparation of 5. Concentrated H3PO4 (1.5 g) was added to 1,4-dihydroxybutan-2-one (15 g, 0.14 mol), and the mixture was heated to 110-120 °C at reduced pressure (about 10 mmHg). The distillate was collected and cooled in a bath of dry ice/ acetone. The distillation gave 4.2 g of a colorless liquid that was further purified by column chromatography on silica (eluent CH2Cl2/diethyl ether; 9:1 v/v). The solvent was carefully removed by evaporation under reduced pressure to give pure 5 as a colorless liquid (1.85 g, 15%). 1H NMR (300 MHz, CDCl3): δ 6.37 (dd, 1 H, H-3, J ) 17.8, 9.7 Hz), 6.27 (dd, 1 H, H-4Z, J ) 17.8, 1.9 Hz), 5.91 (dd, 1 H, H-4E, J ) 9.7, 1.9 Hz), 4.41 (s, 2 H, 2 × H-1), 3.20 (br, 1 H, OH). 13C NMR (75.5 MHz, CDCl3): δ 199.0 (C-2), 132.6 (C-3 or C-4), 130.4 (C-3 or C-4), 66.8 (C-1). EI-MS: m/z 86 (7, M+), 55 (100, M+ - CH2OH), 31 (20, M+ C3H3O), 27 (30, M+ - C2H3O2). Preparation of 1-Chlorobut-3-en-2-ol. LiCl (1.3 g, 31 mmol) was added to a solution of 1-tosyloxy-but-3-en-2-ol (6 g, 25 mmol) in anhydrous DMSO (30 mL), and the solution was stirred for 20 h at 60 °C under nitrogen. The mixture was poured into ice water (about 200 mL) and extracted with ether (3 × 80 mL). The combined ether extracts were washed with water (5 × 80 mL) and dried (Na2SO4), and the solvent was removed by evaporation under reduced pressure to yield 1-chlorobut-3-en2-ol as a colorless liquid (2.0 g, 75%). 1H NMR (500 MHz, CDCl3): δ 5.80 (1 H, ddd, H-3, J ) 17.1, 10.6, 5.5 Hz), 5.34 (dt, 1 H, H-4Z, J ) 17.1, 1.4 Hz), 5.21 (dt, 1 H, H-4E, J ) 10.7, 1.4 Hz), 4.29 (br, 1 H, H-2), 3.58 (dd, 1 H, H-1a, J ) 11.2, 3.9 Hz), 3.45 (dd, 1 H, H-1b, J ) 11.2, 7.2 Hz), 2.40 (br, 1 H, OH). 13C NMR (125.65 MHz, CDCl3): δ 136.3 (C-3), 117.5 (C-4), 72.3 (C2), 49.4 (C-1). EI-MS: m/z 106 (2, M+), 71 (8, M+ - Cl), 57 (100, M+ - CH2Cl). Preparation of 6. A solution of 1-chlorobut-3-en-2-ol (1.6 g, 15 mmol) in dry dichloromethane (10 mL) was added to a stirred solution of Dess-Martin periodinane (7.6 g, 18 mmol) in dry dichloromethane (45 mL) under nitrogen. The mixture was stirred for 3 h at room temperature in the dark. The mixture was chromatographed on silica (eluent dichloromethane), and the solvent was carefully distilled off using a Vigreux fractionating column to yield 6 as a colorless liquid residue (1.2 g, 77%). 1H NMR (500 MHz, CDCl ): δ 6.52 (dd, 1 H, H-3, J ) 17.4, 3 10.7 Hz), 6.32 (d, 1 H, H-4Z, J ) 17.4 Hz), 5.90 (d, 1 H, H-4E, J ) 10.7 Hz), 4.21 (s, 2 H, 2 × H-1). 13C NMR (125.65 MHz, CDCl3): δ 191.4 (C-2), 132.5 (C-3), 130.8 (C-4), 46.7 (C-1). EIMS: m/z 106/104 (5/14, M+), 79/77 (1/3, M+ - C2H3), 55 (100, M+ - CH2Cl), 51/49 (4/12, M+ - C3H3O), 27 (30, M+ - C2H2ClO). Analytical-Scale Reactions of GSH with Chloroprene Metabolites. Compound 5 (42 mg, 0.49 mmol) was reacted with 0.49 and 0.049 mmol of GSH. Compound 6 (17 mg, 0.16 mmol) was reacted with 0.33 and 0.08 mmol of GSH. Compounds 3a,b (17 mg, 0.16 mmol) and 8 (17 mg, 0.16 mmol) were reacted with 0.08 mmol of GSH. The reactions were performed in 5 mL of 0.1 M phosphate buffer (pH 7.4) and incubated at 37 °C on a roller mixer. Aliquots of the reaction mixtures were analyzed

1290 Chem. Res. Toxicol., Vol. 16, No. 10, 2003 by LC/MS. In the positive ion ESI-MS, ions were observed as follows [m/z (relative abundance, formation)]: 13: m/z 394 (100, MH+), 319 (2, MH+ - C2H4O3 + H), 247 (7, MH+ - C4H8N2O4 + H); 15: m/z 683 (100, MH+), 554 (8, MH+ - C5H8NO3 + H). Reaction of 3a,b with GSH. To a solution of GSH (150 mg, 0.49 mmol) in 10 mL of 0.1 M phosphate buffer solution (pH 7.4) was added 3a,b (102 mg, 0.98 mmol), and the mixture was stirred for 4 h at 37 °C. The mixture was extracted with ether (5 mL), and the conjugates were isolated by semipreparative HPLC of the aqueous solution. The column was eluted isocratically with 3% acetonitrile in 0.01 M HCOONH4 for 3 min and then with a gradient from 3 to 40% acetonitrile over the course of 27 min. The collected fraction was evaporated to dryness under reduced pressure to yield 176 mg of a 10:1 mixture of 2-chloro-3-hydroxy-4-(S-glutathionyl)butene (12a,b) and 2-chloro4-hydroxy-3-(S-glutathionyl)butene (11a,b) as a colorless oil. Conjugates 12a,b: 1H NMR (500 MHz, D2O): δ 5.44 (s, 1 H, H-1a), 5.32 (s, 1 H, H-1b), 4.45 (m, 1 H, H-3), 4.30 (dd, 1 H, H-7, J ) 9.4, 6.1 Hz), 3.63 (s, 2 H, 2 × H-16), 3.62 (m, 1 H, H-12), 2.98 (dd, 1 H, H-4a, J ) 14.3, 4.8 Hz), 2.80-2.68 (m, 3 H, 2 × H-6, H-4b), 2.39 (m, 2 H, 2 × H-10), 2.01 (q, 2 H, 2 × H-11, J ) 7 Hz). 13C NMR (125.65 MHz, D2O): δ 177.0, 175.75 and 175.62, 174.8, 172.7, 142.25 and 142.23 (C-2), 116.22 and 116.18 (C-1), 74.19 and 74.11 (C-3), 55.1 (C-12), 54.25 and 54.12 (C-7), 44.4 (C-16), 36.59 and 36.44 (C-6), 34.4 (C-4), 32.4 (C10), 27.2 (C-11). Conjugates 11a,b: 13C NMR (125.65 MHz, D2O): δ 140.0 and 139.5 (C-2), 118.4 and 118.2 (C-1), 62.4 (C4), 54.7 (C-3 or C-7), 33.4 and 33.1 (C-10). The other carbon signals from the GSH moiety were not observed due to overlap by the signals of 12a,b. ESI-MS: 12a,b: m/z 414/412 (100, MH+), 285/283 (2/6, MH+ - C5H8NO3 + H), 163 (1, MH+ C9H15ClN2O4 + H); 11a: m/z 414/412 (100, MH+), 285/283 (7/2, MH+ - C5H8NO3 + H), 163 (8, MH+ - C9H15ClN2O4 + H). Reaction of 8 with GSH. To a solution of GSH (200 mg, 0.65 mmol) in 13 mL of 0.1 M phosphate buffer solution (pH 7.4) was added 8 (136 mg, 1.3 mmol), and the mixture was stirred for 2 h at 37 °C. The mixture was extracted with ether (3 × 10 mL) to remove unreacted 8, and the phosphate buffer solution containing the GSH conjugate was desalted by use of reversed phase C18 column chromatography. The desalted solution was evaporated to dryness under reduced pressure to give 162 mg of 16 as a dark yellow solid. 1H NMR (500 MHz, D2O): δ 9.21 (s, 1 H, CHO), 7.38 (q, 1 H, H-3, J ) 6.7 Hz), 4.23 (dd, 1 H, H-7, J ) 9.0, 4.9 Hz), 3.77 (s, 2 H, H-16), 3.69 (t, 1 H, H-12, J ) 6.4 Hz), 3.10 (dd, 1 H, H-6a, J ) 14.3, 4.8 Hz), 2.89 (dd, 1 H, H-6b, J ) 14.4, 8.9 Hz), 2.39 (m, 2 H, 2 × H-10), 2.022.08 (m, 5 H, 2 × H-11, CH3). 13C NMR (125.65 MHz, D2O): δ 195.5 (CHO), 175.5, 174.5, 173.0, 164.7 (C-3), 136.9 (C-2), 54.7 (C-7), 54.3 (C-12), 42.6 (C-16), 33.8 (C-6), 32.2 (C-10), 26.9 (C11), 17.9 (CH3). ESI-MS: m/z 376 (100, MH+), 229 (11, MH+ C4H8N2O4 + H). Reaction of 6 with GSH. To a solution of GSH (300 mg, 0.98 mmol) in 15 mL of 0.1 M phosphate buffer solution (pH 7.4) was added 6 (203 mg, 1.95 mmol), and the mixture was stirred for 2 h at 37 °C. The major GSH conjugate was isolated by semipreparative HPLC. The column was eluted isocratically with 2% acetonitrile in 0.01 M HCOONH4 for 3 min and then with a gradient from 2 to 40% acetonitrile over the course of 27 min. The fraction containing the purified GSH conjugate was concentrated by evaporation under reduced pressure to about 25 mL and freeze-dried overnight to yield 1-chloro-4-(S-glutathionyl)butan-2-one (14) (340 mg) as a white solid. 1H NMR (500 MHz, D2O): δ 4.45 (dd, 1 H, H-7, J ) 8.8, 4.9 Hz), 4.35 (s, 2 H, 2 × H-1), 3.62-3.70 (m, 3 H, H-12 and 2 × H-16), 2.96 (dd, 1 H, H-6a, J ) 14.0, 4.4 Hz), 2.83 (t, 2 H, 2 × H-4, J ) 6.7 Hz), 2.75 (dd, 1 H, H-6b, J ) 13.9, 9.3 Hz), 2.70 (m, 2 H, 2 × H-3), 2.40 (m, 2 H, 2 × H-10), 2.04 (dd, 2 H, 2 × H-11, J ) 14.1, 6.9 Hz). 13C NMR (125.65 MHz, D2O): δ 206.2 (C-2), 176.8, 175.8, 174.9, 172.9, 55.1 (C-12), 54.0 (C-7), 50.1 (C-1), 44.2 (C-16), 40.5 (C-4), 34.2 (C-6), 32.4 (C-10), 27.2 (C-11), 26.3 (C-3). ESI-MS: m/z 414/412 (36/100, MH+), 285/283 (1/3, MH+ - C5H8NO3 + H), 267/265 (2/5, MH+ - C4H8N2O4 + H).

Munter et al. Reduction of 14 with Sodium Borohydride. To a solution of 14 (60 mg, 0.14 mmol) in water (3 mL) was added sodium borohydride (8 mg, 0.21 mmol), and the mixture was stirred at room temperature for 23 h. The major product was isolated and purified by semipreparative HPLC. The column was eluted isocratically with 2% acetonitrile in 0.01 M HCOONH4 for 3 min and then with a gradient from 2 to 40% acetonitrile over the course of 17 min. The isolated fraction was evaporated to dryness under reduced pressure to yield 50 mg of N5-(1{[carboxymethylamino]carbonyl}vinyl)glutamine (dehydroglutathione) (19) as colorless oil. 1H NMR (500 MHz, D2O): δ 5.62 (d, 1 H, H-1a, J ) 1.0 Hz), 5.59 (d, 1 H, H-1b, J ) 1.1 Hz), 3.70 (s, 2 H, 2 × H-11), 3.66 (t, 1 H, H-7, J ) 6.4 Hz), 2.45 (m, 2 H, H-5), 2.05 (dd, 2 H, 2 × H-6, J ) 13.4, 6.7 Hz).13C NMR (125.65 MHz, D2O): δ 174.9, 169.4, 164.8, 133.7 (C-2), 110.8 (C-1), 52.4 (C-7), 41.9 (C-11), 30.0 (C-5), 24.2 (C-6). ESI-MS: m/z 274 (100, MH+), 145 (10, MH+ - C5H8NO3 + H). Reaction of 6 with 20. Compound 6 (170 mg, 1.63 mmol) was added to a suspension of 20 (140 mg, 0.82 mmol) in a mixture of methanol (20 mL) and water (5 mL). The mixture was stirred for 1.5 h at 37 °C and monitored by LC/MS. The solution was concentrated by evaporation under reduced pressure to about 15 mL and cooled overnight at - 20 °C. The solid precipitate (100 mg) was filtered off, dissolved in acetonitrile, and purified by semipreparative HPLC. The column was eluted isocratically with 48% acetonitrile in water for 4 min and then with a gradient from 48% acetonitrile to 70% acetonitrile over the course of 16 min. The purified fraction was concentrated by evaporation under reduced pressure to remove acetonitrile, extracted with ether (3 × 30 mL), and dried (MgSO4), and the solvent was evaporated under reduced pressure to give 1-chloro4[(2,6-dimethoxyphenyl)sulfanyl]butan-2-one (21) (48 mg, 21%) as a colorless oil. 1H NMR (500 MHz, CDCl3): δ 7.20 (t, 1 H, ArH, J ) 8.4 Hz), 6.51 (d, 2 H, ArH, J ) 8.6 Hz), 4.00 (s, 2 H, 2 × H-1), 3.81 (s, 6 H, 2 × OCH3), 2.99 (t, 2 H, 2 × H-4, J ) 7.0 Hz), 2.68 (t, 2 H, 2 × H-3, J ) 7.0 Hz). 13C NMR (125.65 MHz, CDCl3): δ 201.1 (C-2), 161.1 (C-2′ and C-6′), 130.1 (C-4′), 108.8 (C-1′), 104.1 (C-3′ and C-5′), 56.1 (OCH3), 48.4 (C-1), 39.9 (C-4), 27.8 (C-3). ESI-MS: m/z 299/297 (15/47, MNa+), 277/275 (36/ 100, MH+), 239 (12, MH+ - HCl), 183 (75, MH+ - C3H5ClO), 171 (46, MH+ - C4H6ClO + H).

Results and Discussion Chemical Reactions of Chloroprene Metabolites with GSH. Reactions of the chloroprene metabolites with GSH were performed at 37 °C in aqueous phosphate buffer solution at pH 7.4. The reactions were monitored by LC/MS analyses, and the chromatograms of the reaction mixtures are shown in Figures 1 and 2. The major GSH conjugates were isolated by semipreparative HPLC and characterized by 1H and 13C NMR spectroscopy and ESI-MS. The reaction of racemic 3a,b with GSH resulted in the formation of three conjugates as detected by LC/MS analysis (Figure 1A). The two major conjugates 12a,b were identified as a diastereoisomeric pair of 2-chloro3-hydroxy-4-(S-glutathionyl)butene (Scheme 2). The minor conjugate 11a was tentatively identified as one of the diastereoisomers of 2-chloro-4-hydroxy-3-(S-glutathionyl)butene (11a,b) on the basis of 13C NMR and mass spectral data. The peak corresponding to the other diastereoisomer could not be distinguished in the chromatogram probably due to overlapping by the major peaks of 12a,b. The GSH conjugates were isolated from the reaction mixture by semipreparative HPLC. It was not possible to separate the diastereoisomers or regioisomers by the semipreparative column, and they eluted as a single peak. The 13C NMR spectrum of the collected fraction showed five signals in an abundance of about

Detoxication of Chloroprene Metabolites

Chem. Res. Toxicol., Vol. 16, No. 10, 2003 1291 Scheme 2

Figure 1. C18 analytical column HPLC chromatogram of the reaction mixture (A) of 2 molar equiv of (1-chloroethenyl)oxirane with GSH and (B) 2 molar equiv of 8 with GSH held at 37 °C and pH 7.4 for 1 h. For analysis conditions, see Experimental Procedures.

Figure 2. C18 analytical column HPLC chromatogram of the reaction mixture (A) of 5 with equimolar GSH and (B) 2 molar equiv of 6 with GSH held at 37 °C and pH 7.4 for 0.5 h. For analysis conditions, see Experimental Procedures.

10% of the major signals from 12a,b. Three of these signals appeared as doublets in the spectrum with very small differences in their chemical shifts indicating a diastereoisomeric pair of 11a,b. The signal at δ ) 62.4 ppm indicated a primary hydroxyl group in the molecule

and was assigned as C-4. The C-2 and C-1 carbon signals were observed at δ ) 140.0 and 139.5 (C-2) and at δ ) 118.4 and 118.2 ppm (C-1). The mass spectrum showed the protonated molecular ion at m/z 414/412 in a ratio of 1:3, indicating the presence of one chlorine atom in 11a,b. The 1H NMR spectrum of 12a,b showed in addition to the signals from the GSH protons, one proton signals at δ ) 5.44, 5.32, 4.45, and 2.98 ppm and a multiplet signal at 2.80-2.68 ppm overlapped by the signals of the two H-6 protons. The signals at δ ) 5.44 and 5.32 ppm were assigned as the two vinyl protons H-1a and H-1b. The signal at δ ) 4.45 ppm was assigned to H-3 on the basis of the observed 1H-1H correlations with the signal at δ ) 2.98 ppm (H-4a) and with the overlapped multiplet at δ ) 2.80-2.68 ppm (H-4b). In the 13C NMR spectrum, some of the carbon signals appeared as doublets with small differences in their chemical shifts due to the

1292 Chem. Res. Toxicol., Vol. 16, No. 10, 2003

Figure 3. Mass spectra of the mono-GSH conjugate (16) of 8 formed from (A) incubation of chloroprene with rat liver microsomes in the presence of GSH and (B) authentic 8.

presence of two diastereoisomers. The 13C NMR spectrum showed carbon signals at δ ) 142.25 and 142.23, 116.22 and 116.18, 74.19 and 74.11, and at δ ) 34.4 ppm in addition to the signals from GSH. In the long-range C-H correlation spectrum (HMBC), connectivities were observed between C-2 at δ ) 142.25 and 142.23 ppm and the H-1a and H-1b protons. The signal at δ ) 74.19 and 74.11 was assigned to C-3 on the basis of the observed C-H long-range correlations with the H-1b and H-4b proton signals. The C-4 carbon signal at δ ) 34.4 ppm exhibited long-range C-H correlation with H-3. The mass spectrum showed the protonated molecular ion at m/z 414/412 in a ratio of 1:3. Reaction of GSH with 8 gave one major conjugate, which was identified as 16 (Figure 1B). The 1H NMR spectrum exhibited three signals at δ ) 9.21, 7.28, and 2.05 ppm, besides the signals from GSH. The signal at δ ) 9.21 ppm was assigned to the formyl proton on the basis of the downfield shift and the one bond C-H correlation with the carbon signal at δ ) 195.5 ppm. The quartet at δ ) 7.38 ppm was assigned to H-3, and it showed a one bond C-H correlation with the carbon signal at δ ) 164.7 ppm. The three proton signal at δ ) 2.05 ppm displayed a one bond C-H correlation with the carbon at δ ) 17.9 ppm and was assigned as the methyl protons. A Z-configuration was assigned on the basis of the chemical shift of the methyl protons in comparison with that reported for 8 (10). The mass spectrum showed the protonated molecular ion peak at m/z 376. The fragment ion at m/z 229 corresponded to the loss of the glycine unit together with the carbonyl group of cysteine and the carboxylic acid group of glutamate (Figure 3B). The conjugate appears to be formed by sulfur displacement of the C-2 chlorine atom as previously shown for GSH conjugation of mucochloric acid (25). The mechanism of formation of the conjugate could be by Michael addition of the sulfhydryl group to the β-carbon of the

Munter et al.

Figure 4. Mass spectra of the mono-GSH conjugate (13) of 5 formed from (A) incubation of chloroprene with rat liver microsomes in the presence of GSH and (B) authentic 5.

double bond followed by an intramolecular attack of sulfur on C-2 with displacement of chloride to form an episulfonium intermediate. This could be captured by chloride to give 3-chloro-2-glutathionylbutanal, which could undergo β-elimination of chloride to give 16. Previously, a variety of R,β-unsaturated carbonyl compounds have been shown to react by Michael addition with GSH (26, 27) and GSH-derived episulfonium ion intermediates have been suggested as intermediates in the biotransformation of 1,2-dibromo-3-chloropropane and 1,2-dibromoethane (28, 29). If an electrophilic episulfonium ion intermediate is formed, it could have the potential to react with nucleophilic sites in DNA. GSHderived episulfonium intermediates from biotransformation of 1,2-dibromoethane and 1,2-dibromo-3-chloropropane have been shown to alkylate N7 of guanine in DNA (29, 30). The reaction of GSH with 5 was carried out both with the reactants in equimolar amounts and with a 10-fold excess of 5 relative to GSH. The LC/MS analyses of the reaction mixture of equimolar amounts of GSH and 5 showed that one major conjugate was formed (Figure 2A). The conjugate was identified on the basis of its mass spectrum as 13. The protonated molecular ion peak was observed at m/z 394, and the fragment ion peak at m/z 247 was formed by loss of the glycine unit together with the carbonyl group of cysteine and the carboxylic acid group of glutamate from MH+ (Figure 4B). The mass spectrum was in all essential features identical to that of 13 reported previously by Krause et al. (19). When the reaction was carried out with a 10-fold excess of 5, the conjugate 13 was a minor product and three later eluting peaks with retention times of 17.4, 18.0, and 18.4 min were formed. The conjugate eluting at 17.4 min showed the protonated molecular ion at m/z 480. The two other conjugates both showed MH+ at m/z 566. Most likely,

Detoxication of Chloroprene Metabolites

Chem. Res. Toxicol., Vol. 16, No. 10, 2003 1293 Scheme 3

Scheme 4

Figure 5. Mass spectra of the bis-GSH conjugate (15) of 6 formed from (A) incubation of chloroprene with rat liver microsomes in the presence of GSH and (B) authentic 6.

these products are GSH conjugates of a dimer and a trimer of 1-hydroxybut-3-en-2-one. The reaction of GSH with 6 was performed both with a 2-fold excess of 6 relative to GSH and with a 2-fold excess of GSH relative to 6. When 6 was in excess, the major conjugate detected was 14 formed by a Michael addition of GSH to the double bond of 6 (Figure 2B). The minor product was identified on the basis of its mass spectrum as the diglutathionyl conjugate 1,4-bis-(Sglutathionyl)butan-2-one (15). The bis-conjugate showed the protonated molecular ion at m/z 683, and the fragment peak at m/z 554 was formed by cleavage of one of the glutamate units from MH+ (Figure 5B). When the reaction was carried out with an excess of GSH, formation of 15 predominated. The 1H NMR spectrum of 14 showed three two proton signals at δ ) 4.35, 2.83, and 2.70 ppm besides the signals from GSH. The signal at δ ) 4.35 ppm was assigned to the two methylene protons of the chloromethyl group. The H-4 protons at δ ) 2.83 ppm displayed a one bond C-H correlation with the carbon signal at δ ) 40.5 ppm and a H-H correlation with the H-3 protons at δ ) 2.70 ppm. In the 13C spectrum, four signals at δ ) 206.2, 50.1, 40.5, and 26.3 ppm were observed in addition to the 10 carbon signals of GSH. The signal observed at δ ) 206.2 was assigned to the carbonyl carbon. The signal at δ ) 26.3 ppm displayed a one bond C-H correlation with the H-3 methylene protons at δ ) 2.70 ppm. The signals at δ ) 50.1 and 40.5 ppm were assigned to the chloromethyl carbon C-1 and the methylene carbon C-4, respectively. The mass spectrum showed the protonated molecular ion peaks at m/z 414/412 in a 1:3 ratio. The fragment peaks at m/z 285/283 resulted from cleavage of the glutamate group from MH+. The fragment ions observed at m/z 267/ 265 corresponded to the loss of the glycine unit together with the carbonyl group of cysteine and the carboxylic acid group of glutamate.

Further proof for the structure of 14 was obtained by reduction of the carbonyl group using sodium borohydride in water (Scheme 3). One major product 19 was isolated by semipreparative HPLC. The 13C spectrum showed the two vinyl carbons C-1 and C-2 at δ ) 110.8 and 133.7 ppm, respectively. The carbon signal C-11 from the glycine moiety was observed at δ ) 41.9 ppm and the three carbon signals from glutamate at δ ) 52.4 (C-7), 30.0 (C-5), and 24.2 ppm (C-6). In the 1H NMR spectrum, the two vinyl protons H-1a and H-1b were observed at δ ) 5.62 and 5.59 ppm, respectively. These assignments are in agreement with data previously published for dehydroglutathione (31, 32). The mass spectrum showed the protonated molecular ion at m/z 274 and a fragment peak at m/z 145 corresponding to the loss of the glutamyl group. In the reaction, the chloride of chlorohydrin 17 is displaced by an intramolecular attack by sulfur and 3-hydroxytetrahydrothiophene is cleaved from the sulfonium intermediate 18 to give 19. Alternatively, under the basic conditions of the reduction of 14 to 17, the chlorohydrin may cyclize to an epoxide intermediate, which undergoes cyclization to 18 in an analogous manner to that described for the reaction of isoprene diepoxide with N-acetylcysteine methyl ester (33). The reaction of 6 with the sulfur-containing nucleophile, 20, was studied in order to compare the reaction with that of GSH and the spectral data with 14 (Scheme 4). The reaction was performed at 37 °C in methanol containing 20% water. The reaction gave one major product, 21, which was isolated by semipreparative HPLC. The protons of the two methylene groups appeared as triplets at δ ) 2.99 (H-4) and 2.68 ppm (H-3) in the 1H NMR spectrum. The two protons of the

1294 Chem. Res. Toxicol., Vol. 16, No. 10, 2003

Munter et al.

Scheme 5

chloromethyl group were observed as a singlet at δ ) 4.00 ppm. In the 13C spectrum, four carbon signals at δ ) 201.1 (C-2), 48.4 (C-1), 39.9 (C-4), and 27.8 ppm (C-3) were observed in addition to the five signals of the dimethoxybenzenethiol moiety. In the mass spectrum, the protonated molecular ions were observed at m/z 277/ 275 in a 1:3 ratio confirming the presence of one chlorine atom in the structure. Chemical Oxidation of 3a,b. The epoxidation of rac3a,b with m-CPBA was followed by 1H NMR and showed the formation of 9 as a mixture of diastereoisomers (10). The chloro-2,2′-bioxiranes were relatively stable in CDCl3, but addition of D2O and stirring at room temperature for a further 32 h caused rearrangement to 10 (Scheme 5). The 13C NMR spectrum showed four carbon signals. The signal at δ ) 46.9 ppm showed C-H correlation with the two AB protons of H-2′ and was assigned as the chloromethyl carbon C-2′. The carbonyl carbon was observed at δ ) 198.6 ppm, and the oxirane carbons C-2 and C-3 were observed at δ ) 52.4 and 46.9 ppm, respectively. In the mass spectrum, the molecular ion peaks were observed at m/z 122/120 in a 1:3 ratio indicating the presence of one chlorine atom. Cleavage of the chloromethyl group from M+ produced the fragment ion at m/z 71. Studies on Possible Diepoxide Formation in Microsomal Incubations. The reaction of chloroprene with m-CPBA in chloroform showed that the chloro-2,2′bioxiranes could be formed by chemical oxidation of chloroprene and that they were relatively stable in the low polarity solvent used. The mixture of these diepoxides was however very unstable under aqueous conditions and underwent hydrolysis to give 10, as was also shown in the chemical oxidation of (1-chloroethenyl)oxirane. Compound 10 was identified in mixtures by its mass spectrum, which showed molecular ions at m/z 120/122, a major ion at m/z 71 corresponding to a loss of CH2Cl from M+, and a smaller characteristic ion at m/z 85 representing loss of the chlorine atom from M+. These ions were used to monitor by GC/MS the formation of this compound as a metabolite in incubations with liver microsomes. Incubations of male rat or mouse liver microsomes with either chloroprene or (1-chloroethenyl)oxirane as a substrate in the presence of EH inhibitor (cyclohexene oxide) failed to provide any evidence for the formation of 10. The only detectable metabolite of (1-chloroethenyl)oxirane under these microsomal oxidation conditions was 3-chlorobut-3-ene-1,2-diol indicating that the aqueous hydrolysis of (1-chloroethenyl)oxirane was faster than any further metabolic oxidation to diepoxides. However, when 6 was incubated as the substrate with liver microsomes, a minor product was detected, which had

Figure 6. Mass spectra of 10 formed from (A) chemical oxidation of (1-chloroethenyl)oxirane by m-CPBA and (B) oxidation of 6 by rat liver microsomes.

an identical mass spectrum and gas chromatographic retention time as compared with 10 (Figure 6). This product was detected as a minor metabolite in incubations using liver microsomes from each of the three species, with slightly higher levels being formed by mouse liver microsomes. The absence of metabolic formation of 10 from (1chloroethenyl)oxirane and the formation of only a very low level of this compound from 6, together with the observation that 6 itself rapidly conjugated with GSH, suggests that systemic levels of 10 following exposure to chloroprene in vivo would be very low or possibly undetectable. The in vitro metabolism of chloroprene therefore appears to differ from that of isoprene (33, 34) and butadiene (35, 36) on the basis that a stable diepoxide, or a possible rearrangement product, was not detected in the in vitro metabolism of the monoepoxides of chloroprene. Formation of GSH Conjugates in Microsomal Incubations. Initial studies on the conjugation of chloroprene metabolites with GSH were carried out with rat liver microsomes, which were supplemented with GSH either pre- or postincubation. The rates and amount of conjugation were determined by monitoring the levels of the metabolites in the microsomal incubations by GC/ MS. The effect of GSH transferases on the conjugation reactions was investigated by addition of liver cytosol to the incubations. It was found from these studies that even in the absence of GSH transferase enzymes each of the metabolites 5, 6, and 8 was rapidly conjugated by the added GSH. The metabolites formed by the rearrangement of 2-chloro-2-ethenyloxirane (2a,b) thus reacted very readily with GSH. Incubations with added GSH showed this potential detoxication pathway to be so rapid that unconjugated metabolites could not be detected. The other chloroprene metabolites that included

Detoxication of Chloroprene Metabolites

Chem. Res. Toxicol., Vol. 16, No. 10, 2003 1295

Figure 7. Radio-HPLC profile of GSH conjugates identified from the incubation of chloroprene with rat liver microsomes in the presence of [35S]GSH.

(1-chloroethenyl)oxirane either did not react with GSH or the conjugation was very slow under these conditions. In separate studies where liver cytosol was added to incubations containing (1-chloroethenyl)oxirane and GSH, this had only a marginal effect on the amount of GSH conjugate formed. For quantitative and diagnostic studies, liver microsomes were incubated with chloroprene in the presence of [35S]GSH and the reaction course was monitored by HPLC with radiochemical detection and LC/MS. LC/ MS analysis and cochromatography with synthetic reference standards permitted the identification of three of the radiolabeled peaks (Figure 7) in incubations as the mono-GSH conjugate (13) of 1-hydroybut-3-en-2-one (peak 1), the bis-GSH conjugate (15) of 1-chlorobut-3-en-one (peak 2), and the mono-GSH conjugate (16) of 8 (peak 3). The mass spectrum of each of these conjugates as compared with the spectrum for the corresponding authentic standard is shown in Figures 3-5, respectively. A fourth radiolabeled conjugate (peak 4, Figure 7) was also detected. This product was either the mono-GSH conjugate (12a,b) of (1-chloroethenyl)oxirane or the mono-GSH conjugate (14) of 6, but it was not possible to establish unambiguously its identity since these two compounds coincidentally had similar chromatographic and mass spectral properties. At all concentrations of chloroprene in the range of 10 µM to 10 mM, the total amounts of metabolites that were conjugated with GSH after incubation of chloroprene with liver microsomes were in the order mouse > rat > human, reflecting the greater amounts of reactive metabolites formed by the mouse liver microsomal system as compared with the rat and human systems (Figure 8). Hydrolysis of Epoxide Metabolites by EH. The initial oxidative metabolism of chloroprene by liver microsomes gives the two monoepoxide metabolites 2-chloro-2-ethenyloxirane and (1-chloroethenyl)oxirane. The latter metabolite did not react readily with GSH in microsomal incubations in the absence of GSH transferase and preferentially hydrolyzed enzymatically to form 4a,b (10). The hydrolysis of (1-chloroethenyl)oxirane by EH is therefore of particular significance for the toxicology and detoxication of this metabolite since we have shown that this compound is reactive toward DNA in vitro with the formation of nucleoside adducts at basepairing sites (13). To investigate the enzymatic hydrolysis of (1-chloroethenyl)oxirane and the associated stereoselectivity by EHs in microsomes from rat, mouse, and human, a sensitive method was developed to detect each enanti-

Figure 8. Total amounts of GSH conjugates identified in 30 min incubations of chloroprene with microsomes from mouse (9), rat (]), or human (2) liver in the presence of [35S]GSH.

omer of (1-chloroethenyl)oxirane formed in the metabolism of low concentrations of chloroprene. This entailed analysis of a large volume of the headspace of the incubation samples by GC/MS with a chiral stationary phase and quantification of each enantiomer of (1chloroethenyl)oxirane as compared with a calibration curve. Under these conditions using liver microsomes containing EH, the (R)-(1-chloroethenyl)oxirane could be quantified in incubations carried out at substrate concentrations of chloroprene in the range of 10 µM to 10 mM. There was a significant difference between species in the amounts of each enantiomer of (1-chloroethenyl)oxirane present after incubation of the microsomes with chloroprene. For the R-enantiomer, the relative ratio of the amount of (1-chloroethenyl)oxirane present after 30 min in incubations with 10 µM chloroprene in mouse, rat, or human liver microsomes was approximately 20:4:1, respectively. This difference between species was also observed at substrate concentrations of chloroprene between 100 µM and 10 mM. The S-enantiomer of (1-chloroethenyl)oxirane could not be detected after 30 min in incubations of rat and human liver microsomes with 10 µM chloroprene. At this concentration of chloroprene, (S)-(1-chloroethenyl)oxirane was however detected with mouse liver microsomes. At substrate concentrations of chloroprene in the range of 100 µM to 10 mM, (S)-(1-chloroethenyl)oxirane could be detected in incubations with rat liver microsomes but at levels approximately 10 times less than those observed with mouse liver microsomes. (S)-(1-Chloroethenyl)oxirane could not be detected in incubations with human liver microsomes even at the highest concentration of chloroprene. An overall comparison of the amounts of Rvs S-enantiomer formed by liver microsomes from the different species is shown in Figure 9. These results demonstrate that microsomal oxidation of chloroprene in the presence of EH to (1-chloroethenyl)oxirane is most effective with microsomes from mouse liver. When EH was inhibited, there was a speciesdependent selectivity in the P450-catalyzed oxidative formation of either the R- or S-enantiomer (10). The results of the present studies show that the enzymatic hydrolysis of (1-chloroethenyl)oxirane by microsomal EH is enantioselective for the S-enantiomer resulting in an accumulation of (R)-(1-chloroethenyl)oxirane. This stereoselectivity could have some toxicological significance with respect to the greater sensitivity of the mouse

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Munter et al.

Figure 9. Amounts of R- and S-(1-chloroethenyl)oxirane formed in 30 min incubations of chloroprene with uninhibited microsomes from mouse (4 R, × S), rat ([ R, 2 S), or human (0 R) liver. Table 1. Amounts of 3a,b and 4a,b Formed in 30 min Incubations of Chloroprene with Uninhibited Microsomes from Rat, Mouse, or Human Livera chloroprene (mM)

rat

0.01 0.1 1 10

0.75 1.63 4.48 9.66

4a,b (nmol) mouse human 0.74 3.34 11.72 14.58

0.30 3.23 6.73 10.24

rat 0.11 0.60 0.91 1.41

3a,b (nmol) mouse human 0.49 4.08 13.57 16.73

0.02 0.36 0.60 1.02

a Limits of detection: 3-chlorobut-3-ene-1,2-diol, 0.02 nmol; (1chloroethenyl)oxirane, 0.01 nmol.

toward chloroprene-induced cancers (4, 5). It is also evident, however, that the overall balance between oxidative metabolism and enzymatic hydrolysis results in much lower amounts of (1-chloroethenyl)oxirane remaining in the human model system as compared to the rat and mouse systems (Table 1). 3-Chlorobut-3-ene-1,2-diol, the enzymatic hydrolysis product of (1-chloroethenyl)oxirane (10), was also measured in incubations of chloroprene with rat, mouse, and human microsomes. For each concentration of chloroprene substrate, more 3-chlorobut-3-ene-1,2-diol was detected with mouse liver microsomes as compared with rat or human microsomes. The amounts of 3-chlorobut3-ene-1,2-diol formed via the EH pathway for the different species at different concentrations of chloroprene together with the amounts of residual (1-chloroethenyl)oxirane detected are shown in Table 1.

Conclusions Chloroprene is metabolized in vitro by mammalian P450 oxidative enzymes to a range of metabolites, some of which are reactive toward DNA in vitro and are also mutagenic. In the case of the stable epoxide metabolite (1-chloroethenyl)oxirane, there were considerable differences between species in the amounts formed of this metabolite, with much less being formed by the human liver model system as compared with the rat and mouse models. This epoxide metabolite was a substrate for the detoxicating mammalian microsomal EHs, which showed a pronounced selectivity for the preferential hydrolysis of the S-(1-chloroethenyl)oxirane. The unstable epoxide metabolite 2-chloro-2-ethenyloxirane rapidly hydrolyzed to hydroxyketone, chloroketone, and chloroaldehyde metabolites, which were effectively conjugated by GSH both

in the absence or in the presence of GSH transferases in the model systems for all three species. In addition, no evidence was found to indicate the formation of a chloroprene diepoxide metabolite in any of the model systems. The chlorine atom thus leads to significantly different intoxication and detoxication profiles as compared with those identified for butadiene and isoprene. The overall toxicological profile of chloroprene will depend on the balance between the activating P450 metabolism and the detoxicating hydrolytic and conjugation reactions. The former oxidative process appears to be more quantitatively relevant in the rodent systems and the latter hydrolytic process more pronounced in the human model. It is possible that the stereoselective and quantitative differences in the formation of (1-chloroethenyl)oxirane may have some toxicological significance and could contribute to the species difference in response observed with rodents exposed to chloroprene. While a degree of care is necessary in interpreting the results from in vitro model studies, direct comparisons of metabolic activities and profiles across species can certainly be made. This would indicate that lower levels of (1-chloroethenyl)oxirane would be formed in humans in vivo as compared with rodents following exposure to similar concentrations of chloroprene, and therefore, that there is likely to be a marked difference in responses in vivo between rodents and humans. Mechanistic studies coupled with valid extrapolative human models, which do not suffer from the drawbacks of epidemiological studies, may assist in assessing the risk for humans.

Acknowledgment. We thank the Long-Range Research Initiative (LRI) of the European Chemical Industry Council (CEFIC) for financial support.

References (1) Lynch, M. (2001) Manufacture and use of chloroprene monomer. Chem.-Biol. Interact. 135/136, 155-167. (2) Lynch, J. (2001) Occupational exposure to butadiene, isoprene, and chloroprene. Chem.-Biol. Interact. 135/136, 207-214. (3) International Agency for Research on Cancer (1999) Reevaluation of some organic chemicals, hydrazine and hydrogen peroxide. In IARC Monographs on the Evaluation of the Carcinogenic Risks to Humans, Vol. 71 (Part 1), pp 227-250, International Agency for Research on Cancer, Lyon, France. (4) Melnick, R. L., Sills, R. C., Portier, C. J., Roycroft, J. H., Chou, B.J., Grumbein, S. L., and Miller, R. A. (1999) Multiple organ carcinogenicity of inhaled chloroprene (2-chloro-1,3-butadiene) in F344/N rats and B6C3F1 mice and comparison of dose response with 1,3-butadiene in mice. Carcinogenesis 20, 867-878. (5) Melnick, R. L., and Sills, R. C. (2001) Comparative carcinogenicity of 1,3-butadiene, isoprene, and chloroprene in rats and mice. Chem.-Biol. Interact. 135/136, 27-42. (6) Acquavella, J. F., and Leonard, R. C. (2001) A review of the epidemiology of 1,3-butadiene and chloroprene. Chem.-Biol. Interact. 135/136, 43-52. (7) Colonna, M., and Laydevant, G. (2001) A cohort study of workers exposed to chloroprene in the department of Isere, France. Chem.Biol. Interact. 135/136, 505-514. (8) Bulbulyan, M. A., Margaryan, A. G., Ilychova, S. A., Astashevsky, S. V., Uloyan, S. M., Cogan, V. Y., Colin, D., Boffetta, P., and Zaridze, D. G. (1999) Cancer incidence and mortality of chloroprene workers from Armenia. Int. J. Cancer 81, 31-33. (9) Zaridze, D., Bulbulyan, M., Changuina, O., Margaryan, A., and Boffetta, P. (2001) Cohort studies of chloroprene-exposed workers in Russia. Chem.-Biol. Interact. 135/136, 487-503. (10) Cottrell, L., Golding, B. T., Munter, T., and Watson, W. P. (2001) In vitro metabolism of chloroprene: species differences, epoxide stereochemistry and a de-chlorination pathway. Chem. Res. Toxicol. 14, 1552-1562. (11) Himmelstein, M. W., Gladnick, N. L., Donner, E. M., Snyder, R. D., and Valentine, R. (2001) In vitro genotoxicity testing of (1-

Detoxication of Chloroprene Metabolites

(12)

(13)

(14) (15) (16)

(17)

(18)

(19)

(20)

(21) (22) (23)

chloroethenyl)oxirane, a metabolite of β-chloroprene. Chem.-Biol. Interact. 135/136, 703-713. Eder, E., Deininger, C., Deininger, D., and Weinfurtner, E. (1994) Genotoxicity of 2-halosubstituted enals and 2-chloroacrylonitrile in the Ames test and the SOS-chromotest. Mutat. Res. 322, 321328. Munter, T., Cottrell, L., Hill, S., Kronberg, L., Watson, W. P., and Golding, B. T. (2002) Identification of adducts derived from reactions of (1-chloroethenyl)oxirane with nucleosides and calf thymus DNA. Chem. Res. Toxicol. 15, 1549-1560. Seidega˚rd, J., and Ekstro¨m, G. (1997) The role of human glutathione transferases and epoxide hydrolases in the metabolism of xenobiotics. Environ. Health Perspect. 105 (Suppl. 4), 791-799. Summer, K.-H., and Greim, H. (1980) Detoxification of chloroprene (2-chloro-1,3-butadiene) with glutathione in the rat. Biochem. Biophys. Res. Commun. 96, 566-573. Neudecker, T., Eder, E., Deininger, C., and Henschler, D. (1991) Mutagenicity of 2-methylacrolein, 2-ethylacrolein and 2-propylacrolein in Salmonella typhimurium TA100. A comparative study. Mutat. Res. 264, 193-196. Bechtold, W. E., Strunk, M. R., Chang, I.-Y., Ward, J. B., and Henderson, R. F. (1994) Species differences in urinary butadiene metabolites: comparisons of metabolite ratios between mice, rats, and humans. Toxicol. Appl. Pharmacol. 127, 44-49. Richardson, K. A., Peters, M. M. C. G., Megens, R. H. J. J. J., van Elburg, P. A., Golding, B. T., Boogaard, P. J., Watson, W. P., and van Sittert, N. J. (1998) Identification of novel metabolites of butadiene monoepoxide in rats and mice. Chem. Res. Toxicol. 11, 1543-1555. Krause, R. J., Kemper, R. A., and Elfarra, A. A. (2001) Hydroxymethylvinyl ketone: a reactive Michael acceptor formed by the oxidation of 3-butene-1,2-diol by cDNA-expressed human cytochrome P450s and mouse, rat, and human liver microsomes. Chem. Res. Toxicol. 14, 1590-1595. Oesch, F. (1973) Mammalian epoxide hydrases: inducible enzymes catalyzing the inactivation of carcinogenic and cytotoxic metabolites derived from aromatic and olefinic compounds. Xenobiotica 3, 305-340. Schwartz, N. N., and Blumbergs, J. H. (1964) Epoxidations with m-chloroperbenzoic acid. J. Org. Chem. 29, 1976-1979. Crawford, R. J., Lutener, S. B., and Cockcroft, R. D. (1976) The thermally induced rearrangements of 2-vinyloxirane. Can. J. Chem. 54, 3364-3376. Trost, B. M., and Jungheim, L. N. (1980) 1-(Arylthio)cyclopropanecarboxaldehydes. Conjunctive reagents for secoalkylation. J. Am. Chem. Soc. 102, 7910-7925.

Chem. Res. Toxicol., Vol. 16, No. 10, 2003 1297 (24) Reppe, W., and Mitarbeiter (1955) A ¨ thinylierung. Justus Liebigs Ann. Chem. 596, 63-79. (25) LaLonde, R. T., and Xie, S. (1993) Glutathione and N-acetylcysteine inactivations of mutagenic 2(5H)-furanones from the chlorination of humics in water. Chem. Res. Toxicol. 6, 445-451. (26) Chien, C., Kirollos, K. S., Linderman, R. J., and Dauterman, W. C. (1994) R,β-Unsaturated carbonyl compounds: inhibition of rat liver glutathione S-transferase isozymes and chemical reaction with reduced glutathione. Biochim. Biophys. Acta 1204, 175-180. (27) Porthogese, P. S., Kedziora, G. S., Larson, D. L., Bernard, B. K., and Hall, R. L. (1989) Reactivity of glutathione with R,βunsaturated ketone flavouring substances. Food Chem. Toxicol. 27, 773-776. (28) Pearson, P. G., Soderlund, E. J., Dybing, E., and Nelson, S. D. (1990) Metabolic activation of 1,2-dibromo-3-chloropropane: evidence for the formation of reactive episulfonium ion intermediates. Biochemistry 29, 4971-4981. (29) Peterson, L. A., Harris, T. M., and Guengerich, F. P. (1988) Evidence for an episulfonium ion intermediate in the formation of S-[2-(N7-guanyl)ethyl]glutathione in DNA. J. Am. Chem. Soc. 110, 3284-3291. (30) Humphreys, W. G., Kim, D.-H., and Guengerich, F. P. (1991) Isolation and characterization of N7-guanyl adducts derived from 1,2-dibromo-3-chloropropane. Chem. Res. Toxicol. 4, 445-453. (31) Asquith, R. S., and Carthew, P. (1972) Synthesis and PMR properties of some dehydroalanine derivatives. Tetrahedron 28, 4769-4773. (32) Jones, A. J., Helmerhorst, E., and Stokes, G. B. (1983) The formation of dehydroalanine residues in alkali-treated insulin and oxidized glutathione. Biochem. J. 211, 499-502. (33) Watson, W. P., Cottrell, L., Zhang, D., and Golding, B. T. (2001) Metabolism and molecular toxicology of isoprene. Chem.-Biol. Interact. 135/136, 223-238. (34) Wistuba, D., Weigand, K., and Peter, H. (1994) Stereoselectivity of in vitro isoprene metabolism. Chem. Res. Toxicol. 7, 336-343. (35) Thornton-Manning, J. R., Dahl, A. R., Bechtold, W. E., and Henderson, R. F. (1995) Disposition of butadiene monoepoxide and butadiene diepoxide in various tissues of rats and mice following a low-level inhalation exposure to 1,3-butadiene. Carcinogenesis 16, 1723-1731. (36) Csana`dy, G. A., Guengerich, F. P., and Bond, J. A. (1992) Comparison of the biotransformation of 1,3-butadiene and its metabolite, butadiene monoepoxide, by hepatic and pulmonary tissues from humans rats and mice. Carcinogenesis 13, 1143-1153.

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