Stereochemical and Kinetic Comparisons of Mono-and Diepoxide

Chem. Res. Toxicol. 2003, 16, 933-944

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Stereochemical and Kinetic Comparisons of Mono- and Diepoxide Formation in the In Vitro Metabolism of Isoprene by Liver Microsomes from Rats, Mice, and Humans Bernard T. Golding,*,† Lisa Cottrell,‡ Daniel Mackay,‡ Daping Zhang,† and William P. Watson*,‡ School of Natural SciencessChemistry, Bedson Building, University of Newcastle upon Tyne, Newcastle upon Tyne, NE1 7RU, United Kingdom, and Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, SK10 4TJ, United Kingdom Received March 25, 2003

Isoprene (2-methylbuta-1,3-diene) is a large scale petrochemical used principally in the manufacture of synthetic rubbers. It is also produced by plants and trees and is formed endogenously in mammals as a major endogenous hydrocarbon. Mammalian metabolism of isoprene involves cytochrome P450-dependent monooxygenases to give the regioisomeric monoepoxides, prop-2-enyloxirane and 2-ethenyl-2-methyloxirane. The isoprene monoepoxides are further oxidized to the mutagenic diepoxides, 2-methyl-2,2′-bioxiranes. The present studies have investigated the stereochemistry and comparative rates of the metabolic epoxidation in vitro of isoprene to mono- and diepoxides by liver microsomes from rat, mouse, and human in order to identify stereochemical and kinetic differences between species in the formation of these epoxide metabolites, which are key to understanding the toxicology of isoprene. The assignments of stereochemistry were based on comparisons with synthetic standards, the syntheses for which are described. Comparative enzyme kinetic parameters (apparent Km and apparent Vmax values) for the in vitro formation of all of the monoepoxide and diepoxide stereoisomers have been obtained. The rates of formation of both mono- and diepoxides were greater in the rodent systems as compared with the human in vitro system. The results provide comparative kinetic data that have potential for modeling and assessing the relevance of the animal carcinogenicity data for man. The possibility of human interindividual variation was also investigated with liver preparations from several individual humans, but significant differences between individuals were not observed in the formation of the monoepoxides from isoprene.

Introduction

Chart 1

Isoprene (2-methylbuta-1,3-diene) (1) (Chart 1) is a large scale petrochemical obtained principally from the cracking of naphtha or gas oil. The world production level per annum is over 1.3 × 109 kg, of which about 95% is used in the manufacture of synthetic rubbers. Isoprene is also a natural product formed by plants and trees (1) at a level of about 4 × 1011 kg per annum (2). This gives rise to concentrations of isoprene of about 1 ppb in rural areas. Isoprene is also formed in combustion processes and is present in engine exhausts and tobacco smoke (3). Isoprene is the major endogenous hydrocarbon produced in humans and other mammals. It is probably derived from mevalonate (4-6) because dietary and other factors that affect cholesterol biosynthesis have an effect on the amount of exhaled isoprene (5). In humans, the rate of production of isoprene, which comprises 30-70% of the exhaled hydrocarbons (7), is ca. 0.15 µmol/kg/h (8). This * To whom correspondence should be addressed. [email protected] or [email protected]. † University of Newcastle upon Tyne. ‡ Syngenta Central Toxicology Laboratory.

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10.1021/tx034061x CCC: $25.00 © 2003 American Chemical Society Published on Web 07/01/2003

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Chem. Res. Toxicol., Vol. 16, No. 7, 2003

corresponds to 2-4 mg of exhaled isoprene per day per individual (7). It seems therefore that human exposure to isoprene is unavoidable. 1,3-Butadiene, which has been classified as a category 2A (probable human) carcinogen by the International Agency for Research on Cancer (9) and as a category 1 (human) carcinogen by the U.S. National Toxicology Program (NTP) (10) and the European Commission (11), is the parent diene of isoprene. The toxicological properties discovered for 1,3-butadiene have been a major stimulus for studies on the mammalian toxicology of isoprene. The animal carcinogenicity of isoprene has been evaluated in several studies in B6C3F1 mice (12-14) and also with Fischer F344 rats (13, 15). In a 26 week inhalation exposure study at doses ranging from 70 to 7000 ppm for 6 h/day, 5 days/week, followed by a 26 week recovery period, isoprene was shown to have carcinogenic activity in male B6C3F1 mice (12, 13). A similar study in male F344/N rats gave inconclusive results; the only effect observed was a marginal increase in benign testicular interstitial cell tumors at the highest dose (13). The profile of tumors observed in mice exposed to isoprene was similar to those reported for 1,3-butadiene with the exception of the early onset of T-cell lymphoma caused by 1,3-butadiene (14). The carcinogenic potency of isoprene to mice has been estimated to be about 1 order of magnitude less than that of 1,3-butadiene (12). Male and female F344/N rats were exposed to isoprene by whole body inhalation for 2 years in a U.S. NTP carcinogenesis study (15). In this study, there was evidence of carcinogenic activity in both male and female animals based on increased incidences of mammary gland neoplasms, renal tubule adenoma, and testicular adenoma (15). Increased incidences of renal tubule hyperplasia and splenic fibrosis were also observed in the male rats. There was also some evidence of carcinogenic activity in female F344/N rats based on increased incidences of mammary gland fibroadenoma. A low incidence of rare brain neoplasms in exposed female rats may also have been due to exposure to isoprene. On the basis of a review of the available published data, the International Agency for Research on Cancer has classified isoprene as a group 2B carcinogen, possibly carcinogenic to humans (16). There is currently no epidemiological evidence for cancer in humans following occupational exposure to isoprene (17). As with 1,3-butadiene, oxidative metabolism is the major contributor to the toxicological behavior of isoprene. Nevertheless, and in contrast to the mutagenic butadiene monoepoxide, the monoepoxides of isoprene, 2-ethenyl-2-methyloxirane (2) and prop-2-enyloxirane (3), have been reported to be nonmutagenic (18). However, the diepoxides of butadiene and isoprene, bioxirane and 2-methyl-2,2′-bioxirane (4), respectively, are mutagenic (18-20). From studies with rodents, the tissue concentrations of the total isoprene metabolites formed in rats and mice have been shown to be higher than the total butadiene metabolites, after comparable exposures (2123). Although the blood levels of total metabolites were similar when both rats and mice were exposed to isoprene, the internal dose of isoprene was greater in mice than rats after exposure to the same concentration (24, 25). While the resulting higher internal dose could contribute to the greater sensitivity of mice to isoprene, the concentration of reactive metabolites, presumably of the diepoxide in particular, is the more relevant dose.

Golding et al.

The difference in carcinogenic potencies between butadiene and isoprene might be explained by differences in reactivities of the metabolites of butadiene and isoprene, particularly the diepoxides (26, 27). The stereochemistry and rate of formation of the isoprene diepoxides could have toxicological relevance. The present studies have therefore investigated stereochemical and kinetic differences between species in the formation of the reactive metabolites, the isoprene monoand diepoxides, to provide comparative kinetic data for modeling and to aid in assessing the relevance of the animal carcinogenicity data for man. Studies on the stereochemistry and rates of the metabolic epoxidation in vitro of isoprene to mono- and diepoxides by liver microsomes from rat, mouse, and human were therefore carried out. In particular, comparative enzyme kinetic parameters (apparent Km and Vmax) for the in vitro formation of all of the monoepoxide and diepoxide stereoisomers have been obtained. Human interindividual variation has also been investigated with liver preparations from several individual humans. The absolute stereochemistry of all of the possible mono- and diepoxide isomers is shown in Chart 1. Chemical syntheses of appropriate reference standards of isoprene epoxides were carried out to identify the nature and stereochemistry of the metabolites formed in the metabolism studies.

Material and Methods Caution: All work involving isoprene epoxides and their derivatives should be performed with protective clothing and in a well-ventilated fume cupboard. Chemicals. Racemic 2 (containing 95% 2 and 5% 3) and m-CPBA1 were purchased from Aldrich Chemical Co. (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 (28). The iodometric assay indicated 99+% purity. Petrol was redistilled (bp range 40-60 °C). Chromatographic Methods for Synthetic Procedures. GC analyses were performed with a Pye Unicam GCD gas chromatograph equipped with a 1.5 m × 4 mm i.d. 10% poly(ethyleneglycol) PEG-terephthalate column and a flame ionization detector. Preparative GC was performed with a Varian series 2700 gas chromatograph containing a 2.5 m × 4 mm i.d. 10% PEG 20 M column and a thermal conductivity detector. Nitrogen was used as the carrier gas for both instruments. Purification of rac-2. The commercial epoxide was purified to 99.8% purity by preparative GC (conditions: 80-140 °C at a gas flow rate of 50 mL/min and a temperature gradient of 8 °C/min; retention time, 2.26 min). General Procedure A for the Reaction of the Enolate of (6R,7R,15S)-15-Methyl-1,8,13,16-tetraoxadispiro[5.0.5.4]hexadecan-14-one (9) with an Aldehyde. To a stirred solution of diisopropylamine (0.2-0.4 M) in THF at -78 °C under nitrogen atmosphere was added 1 mol equiv of nbutyllithium (2.5 M in hexane) and DMPU (8 equiv). The mixture was stirred for 15 min before the addition of a solution of 9 (29) in THF via cannula. The mixture was stirred for 15 min, and then, a second portion of n-butyllithium was added. After a further 15 min, the enolate was quenched with 1.5-5 mol equiv of the aldehyde and the reaction was stirred until complete as judged by TLC (around 15 min). The solution was poured into an excess of saturated 2/1 (v/v) ammonium chloride/ water, and the product was extracted with ethyl acetate. The 1 Abbreviations: DBU, 1,8-diazbicyclo[5.4.0]undec-7-ene; m-CPBA, 3-chloroperoxybenzoic acid; DMPU, 1,3-dimethyl-3,4,5,6-tetrahydro2(1H)-pyrimidinone; LDA, lithium diisopropylamide; pmb, p-methoxybenzyl; triflate, trifluoromethane sulphonate.

Metabolism of Isoprene combined organic extracts were dried (MgSO4) and filtered, and the solvent was removed. The residue was purified by medium pressure chromatography (silica, elution with 1:4 ethyl acetate/ petrol). General Procedure B for Preparation of rac- or (S)-2Methylbut-3-ene-1,2-diol. To LiAlH4 in dry ether (either diethyl ether or THF) was added dropwise racemic or (S)-2hydroxy-2-methylbut-3-enoate in dry ether at -5 to 0 °C. After the reaction was complete, the mixture was quenched with H2O/ NaOH (15%, w/v)/H2O (1/1/3). The mixture was filtered, and the filter pad was washed with diethyl ether. The remaining solid was continuously extracted with diethyl ether overnight in a Soxhlet apparatus. The combined ether extracts were dried (MgSO4), and the solvent was removed to give a crude product that was purified by medium pressure chromatography (silica, elution with 2.5% methanol in dichloromethane). General Procedure C for Preparation of rac- or (S)-1O-Tosylate of 2-Methylbut-3-ene-1,2-diol. To 2-methylbut3-ene-1,2-diol (racemic or S isomer) in dry pyridine under nitrogen was added freshly recrystallized p-toluenesulfonyl chloride over 10 min. The mixture was stored at ∼0 °C for 18 h. After ice-cold water was added, the product was extracted into diethyl ether. The combined ether extracts were washed with ice-cold 1 M HCl, followed by saturated sodium bicarbonate solution, and then dried (MgSO4). The solvent was removed, and the residue was purified by medium pressure chromatography (silica, elution with dichloromethane). General Procedure D for Preparation of Monoepoxide of Isoprene. Racemic or (S)-tosylate of 2-methylbut-3-ene-1,2diol contained in a glass bulb attached to a ground glass joint was added, by rotating the bulb, to sodium ethane-1,2-diolate in ethylene glycol (0.9 M, 1.0 mol equiv) under high vacuum (0.05 mmHg). Compound 2 distilled from the reaction mixture (30) and was condensed in a trap immersed in solid CO2/acetone. Preparation of rac-Ethyl-2-hydroxy-2-methylbut-3-enoate (6). A solution of vinylmagnesium bromide (100 mL, 100 mmol, 1 M in THF) was added dropwise during 1 h to freshly redistilled ethyl pyruvate (5) (11.6 g, 11.0 mL, 81 mmol) in THF (100 mL) under nitrogen at -78 °C. The resulting mixture was stirred for an additional 15 min before the temperature was allowed to rise to ca. 20 °C. The reaction mixture was poured slowly into saturated aqueous ammonium chloride (100 mL). The resulting mixture was extracted with diethyl ether (3 × 50 mL). The combined ether extracts were dried over anhydrous magnesium sulfate. The ether was removed, and the residue was purified by medium pressure chromatography (silica, elution with 1:3 ether/petrol) to give a clear oil (5.86 g, 50%). 1H NMR (200 MHz, CD CN): 6.10 (1H, dd, J 17, 10 Hz, dCH), 3 5.43 (1H, dd, J 1, 17 Hz, dCHH), 5.19 (1H, dd, J 1, 10 Hz, dCHH), 4.23 (2H, q, J 7 Hz, CH3CH2), 3.76 (1H, s, OH), 1.47 (3H, s, CH3), 1.30 (3H, t, J 7 Hz, CH3CH2). 13C (50 MHz, CDCl3): 175.5 (CdO), 139.7 (dCH), 114.5 (CH2d), 74.7 (C), 62.2 (CH2), 25.8 (CH3C), 14.1 (CH3CH2). IR (film): 3505 (O-H), 3097, (dC-H), 2985 (C-H), 1730 (CdO), 1644 (CdC), 1127 (C-O) cm-1. MS (EI): m/z 145 (MH+), 132, 83, 65. Accurate mass calcd for C7H12O3 (MH+), 145.0864; found, 145.0855. Preparation of rac-2-Methylbut-3-ene-1,2-diol (7). Compound 6 (5.86 g, 41 mmol) in diethyl ether was reacted with LiAlH4 (0.85 g, 22 mmol) by general procedure B to give a clear oil (4.17 g, 100%). 1H NMR (500 MHz, CDCl3): 5.81 (1H, dd, J 17, 10 Hz, dCH), 5.24 (1H, dd, J 1, 17 Hz, dCHH), 5.08 (1H, dd, J 1, 10 Hz, dCHH), 3.59 (1H, s, OH), 3.51 (1H, s, OH), 3.39 (2H, m, CH2), 1.18 (3H, s, CH3). 13C NMR (125 MHz, CDCl3): 141.8 (dCH), 114.1 (CH2d), 73.7 (C), 69.4 (CH2), 23.7 (CH3). IR (film): 3375 (O-H), 3092 (dC-H), 2976 (C-H), 1647 (Cd C), 1127 (C-O) cm-1. MS (EI): m/z 102 (M+), 91, 71, 69, 57, 55, 53. Accurate mass calcd for C5H10O2 (M+1), 102.0680; found, 102.0678. Anal. calcd for C5H10O2: C, 58.80; H, 9.87. Found: C, 58.30; H, 10.33. Preparation of the 1-O-Tosylate of rac-2-Methylbut-3ene-1,2-diol (8). Compound 7 (4.17 g, 41 mmol) was reacted with p-toluenesulfonyl chloride (7.9 g, 41 mmol) using general

Chem. Res. Toxicol., Vol. 16, No. 7, 2003 935 procedure C to give the title compound as a clear oil (4.37 g, 44%). 1H NMR (200 MHz, CDCl3): 7.72 (2H, d, J 8 Hz, H2,6), 7.29 (2H, d, J 8 Hz, H3,5), 5.74 (1H dd, J 17, 10 Hz, CHd), 5.29 (1H, dd, J 17, 1 Hz, CHH), 5.07 (1H, dd, J 10, 1 Hz, CHH), 3.82 (2H, s, CH2), 2.39 (3H, s, ArCH3), 1.21 (3H, s, Me). 13C NMR (125 MHz, CDCl3): 145.1 (C-3), 140.0 (C-1′), 132.6 (C-3′,C-5′), 129.9 (C-2′,C-6′), 128.0 (C-4′), 115.2 (C-4), 75.8 (C-1), 71.9 (C2), 24.2 (ArCH3), 21.6 (CH3). MS (EI): m/z 256 (M+), 241, 227, 220, 172, 155, 139, 107, 91, 71, 65. Accurate mass calcd for C12H16O4S (M+1), 256.0503; found, 256.0502. Preparation of Compound 2. The 1-O-tosylate of 8 (2.0 g, 80 mmol) was reacted with sodium ethane-1,2-diolate in ethylene glycol (9.0 mL) using general procedure D to give a colorless oil (0.5 g, 80% based on tosylate). 1H NMR (200 MHz, CDCl3): 5.61 (1H dd, J 17, 10 Hz, CHd), 5.30 (1H, dd, J 17, 1 Hz, CHH), 5.20 (1H, dd, J 10, 1 Hz, CHH), 2.71 (2H, 2 × d, J 5 Hz, CH2), 1.35 (3H, s, Me). 13C NMR (125 MHz, CDCl3): 140.1 (C-3), 117.8 (C-4), 55.8 (C-1 or C2), 55.7 (C-1 or C-2), 19.2 (CH3). Preparation of (S)-Prop-2-enyloxirane (3b). Compound 3b with an 88% ee was prepared according to the method described by Wistuba et al. (31). 1H NMR (200 MHz, CD3CN): 5.10 (2H, m, CH2d), 3.22 (1H, m, CH), 2.70 (2H, m, CH2), 1.50 (3H, s, Me). 13C (50 MHz, CD3CN): 142.0 (C-3), 113.5 (C-4), 53.5 (C-2), 45.8 (C-1), 14.9 (CH3). Preparation of 3. This compound was prepared by the procedure of Harwood (32). The spectroscopic data were identical to that of the S isomer. Preparation of 4, Mixture of Diastereoisomers. To a solution of m-chloroperoxybenzoic acid (16.8 g, 79 mmol) in dry dichloromethane (200 mL) was added 2 (5.9 g, 6.9 mL). The mixture was stirred at room temperature while the disappearance of rac-2 was monitored by TLC (20:1 CH2Cl2/MeOH). After 18 h, the reaction was complete (the solution changed from clear to milky). The mixture was filtered, and the filtrate was washed twice with 5% sodium sulfite (w/v) and saturated sodium carbonate and dried (Na2SO4). The solvent was removed by atmospheric distillation through a 12 cm Vigreux column to give the crude product as a pale yellow oil, which was further purified by distillation under water pump pressure to afford 4 as a colorless oil (4.4 g, 62%); bp 72-73 °C/12 mmHg; lit. 75-77 °C/ 70 mmHg (33). 1H NMR (500 MHz, CD3CN): 2.81 (1H, m, CH), 2.60 (2H, m, CH2CH), 2.50 (2H, m, CH2C), 1.20, 1.09 (3H, 2 × s, Me). 13C NMR (125 MHz, CD3CN): 56.2, 55.5 (C), 54.1, 53.7 (CH), 51.7, 51.7 (CH2CH), 44.9, 44.8 (CH2C), 17.8, 16.9 (CH3). Preparation of (6R,7R,15S,(S))-15-(1-Hydroxyethyl)-15methyl-1,8,13,16-tetraoxadispiro[5.0.5.4]hexadecan-14one (11) (29). Compound 9 (29) (1.3 g, 5.0 mmol) in THF (25 mL) was reacted with n-butyllithium followed by acetaldehyde (1.1 g, 25 mmol, 1.40 mL) and more n-butyllithium using general procedure A to give 11 (1.3 g, 86%, Rf ) 0.28) and (6R,7R,15S,(R))-15-(1-hydroxyethyl)-15-methyl-1,8,13,16-tetraoxadispiro[5.0.5.4]hexadecan-14-one (0.10 g, 4%, TLC Rf ) 0.38, ethyl acetate/petrol bp range 40-60 °C, 1:4) as white solids. 1H and 13C NMR and IR data for compound 11 were similar to those reported (29); mp 102-104 °C; lit. 110-113 °C (29). MS (EI): m/z 301 (MH+1), 256, 201, 185, 168, 111, 101. Accurate mass calcd for C15H25O6 (MH+), 301.1651; found, 301.1642. Anal. calcd for C15H24O6: C, 60.00; H, 8.05. Found: C, 60.29; H, 8.08. [R]20D -68° (c ) 4.6 g/100 mL, CHCl3); lit. [R]28D -88° (c ) 1.0 g/100 mL, CHCl3) (29). Data for the minor diastereoisomer (6R,7R,15S,(R)) were also similar to those reported (29). Preparation of the Triflate of (6R,7R,15S,(S))-15-(1Hydroxyethyl)-15-methyl-1,8,13,16-tetraoxadispiro[5.0.5.4]hexadecan-14-one (12). Trifluoromethanesulfonic anhydride (5.5 mmol, 1.6 g, 0.9 mL) was added dropwise to a precooled solution of 11 (1.5 g, 5.0 mmol) in pyridine (5.5 mmol, 0.40 g, 0.5 mL) and dichloromethane (20 mL) at ∼ -5 °C under N2. Stirring was continued for an additional 30 min. The mixture was filtered through a glass-sintered funnel that was subsequently washed with dichloromethane. The solvent was removed to give a yellow solid (3.4 g, crude product), which was pure enough to be used directly for the next step. 1H NMR (200 MHz,

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CDCl3): 5.13 (1H, q, J 6.4 Hz, H1′), 3.92-3.82 (2H, m, 2 × H-9), 3.79-3.61 (2H, m, 2 × H2), 2.11-1.44 (12H, m, 6 × CH2), 1.57 (3H, s, Me), 1.48 (3H, d, J 6.3 Hz, MeCHOTf). Preparation of (6R,7R,15S)-15-Methyl-15-vinyl-1,8,13,16-tetraoxadispiro[5.0.5.4]hexadecan-14-one (13). A solution of DBU (2.3 mL, 2.4 g, 15.5 mmol) was added dropwise, under a nitrogen atmosphere, to a solution of the crude triflate of 12 (∼2.16 g) in dichloromethane (50 mL). The yellow solution was refluxed for 48 h, and the solvent was removed. The residue was purified by medium pressure chromatography (silica, elution with 5:1 ethyl acetate/petrol) to give a white solid (1.0 g, 71%); mp 50-51°C. 1H NMR (200 MHz, CDCl3): 5.90 (1H, dd, J 17, 10 Hz, CHd), 5.52 (1H, dd, J 17, 1 Hz, CHH), 5.11 (1H, dd, J 10, 1 Hz, CHH), 3.89-3.76 (2H, m, 2 × H9), 3.733.62 (2H, m, 2 × H2), 1.98-1.47 (12H, m, 6 × CH2), 1.56 (3H, s, Me). 13C (50 MHz, CD3CN): 171.0, 139.7, 115.1, 104.0, 96.0, 76.21, 62.13, 62.08, 29.00, 28.76 (4 CH2), 27.15 (Me), 25.10, 24.37 (2 × CH2), 18.26, 17.51 (2 × CH2). IR (film): 3024 (dC-H), 2951 (C-H), 1743 (CdO), 1640 (CdC), 1128 (C-O) cm-1. MS (EI): m/z 283 (MH+1), 203, 183, 168, 111, 98, 82, 55. Accurate mass calcd for C15H23O5 (MH+1), 283.1545; found, 283.1545. Anal. calcd for C15H22O5: C, 63.81; H, 7.86. Found: C, 63.79; H, 8.29. [R]20D -91° (c ) 4.8 g/100 mL, CHCl3). Preparation of (S)-2-Hydroxy-2-methylbut-3-enoic Acid (10). Compound 13 (0.50 g, 1.7 mmol) in 95% trifluoroacetic acid (TFA) (5 mL, ∼30 equiv) was stirred at room temperature until the starting material had disappeared (∼5 h, monitored by TLC). The excess of TFA was removed in vacuo. The residue was taken up in ethyl acetate (5 mL), and the acid was extracted into saturated sodium carbonate (4 × 5 mL). The combined extracts were acidified with 2 M sulfuric acid until pH ∼ 2, and the acid was extracted with ethyl acetate (4 × 5 mL). The combined extracts were dried (MgSO4), and the solvent was removed to give a white solid (0.20 g, 88%). An analytical sample was obtained by recrystallization from diethyl ether/acetone; mp 86-87 °C. 1H NMR (200 MHz, CD3CN): 6.12 (1H, dd, J 17, 10 Hz, CHd), 5.45 (1H, dd, J 17, 1 Hz, CHH), 5.22 (1H, dd, J 10, 1 Hz, CHH), 1.50 (3H, s, Me). 13C (50 MHz, CD3CN): 175.1 (Cd O), 139.9 (C3), 113.3 (C4), 74.2 (C2), 25.0 (CH3). IR (KBr): 3439 (O-H), 3006 (dC-H), 2993 (C-H), 1733 (CdO), 1638 (CdC), 1130 (C-O) cm-1. MS (EI): m/z 101 ((M - Me)+), 98, 71, 55, 53. Accurate mass calcd for C4H5O3 ((M - Me)+1), 101.0239; found, 101.0241. Anal. calcd for C5H8O3: C, 51.72; H, 6.89. Found: C, 51.52; H, 6.88. [R]19D -30° (c ) 4.0 g/100 mL, methanol). Preparation of (S)-2-Methylbut-3-ene-1,2-diol (7a). Compound 10 (0.60 g, 5.2 mmol) in THF (5 mL) was reacted with LiAlH4 (0.30 g, 7.8 mmol) by general procedure B to give a colorless oil (0.50 g, 96%). The spectroscopic data were identical to that for the rac compound. [R]20D -10° (c ) 3.5 g/100 mL, MeOH). Preparation of the 1-O-Tosylate of (S)-2-Methylbut-3ene-1,2-diol (8a). Compound 7a (0.40 g, 4.1 mmol) was reacted with p-toluenesulfonyl chloride (0.80 g, 4.1 mmol) using general procedure C to give the title compound as white crystals (0.50 g, 51%); mp 89-90 °C. The spectroscopic data were identical to that of the racemate. Preparation of (S)-2-Ethenyl-2-methyloxirane (2b) (34). The 1-O-tosylate of 8a (0.20 g, 0.80 mmol) was reacted with sodium ethane-1,2-diolate in ethylene glycol (0.90 mL) using general procedure D to give a colorless oil (0.04 g, 80% based on tosylate). 1H NMR (200 MHz, CDCl3): 5.58 (1H, dd, J 17, 10 Hz, CHd), 5.29 (1H, dd, J 17, 1 Hz, CHH), 5.17 (1H, dd, J 10, 1 Hz, CHH), 2.72 (2H, 2 × dd, J 5 Hz, CH2), 1.39 (3H, s, Me). 13C NMR (125 MHz, CDCl ): 139.4 (C-3), 116.9 (C-4), 55.7 3 (C-1), 55.6 (C-2), 18.9 (CH3). [R]19D +18° (c ) 6.2 g/100 mL, ethyl acetate). Preparation of (6R,7R,15S,(S))-15-(1-Hydroxy-2-(4-methoxybenzyloxy)ethyl)-15-methyl-1,8,13,16-tetraoxadispiro[5.0.5.4]hexadecan-14-one (14). Using general procedure A, 9 (2.6 g, 10.0 mmol) was reacted with 4-methoxybenzylacetaldehyde (2.7 g, 15 mmol) to give the title compound as a colorless

Golding et al. oil (3.2 g, 73%). 1H NMR (200 MHz, CDCl3): 7.21 (2H, d, J 9 Hz, H-2, H-6), 6.80 (2H, d, J 9 Hz, H-3, H-6), 4.44 (2H, m, ArCH2), 3.93-3.45 (10H, m, inc. 3.73, 3H, CH3OAr; 2 × CH2; 1H, H-1′; 2H, H-2′), 1.98-1.41 (15H, inc. 1.47, 3H, Me; 6 × CH2). 13C NMR (125 MHz, CDCl ): 169.9, 159.1, 130.3, 129.3, 113.7, 3 103.8, 95.8, 78.69, 72.68, 70.24, 62.82, 62.33, 55.23, 28.85, 28.58, 24.27, 24.13, 23.45, 18.13, 17.25. IR (film): 3476 (O-H), 3059 (Ar-H), 2952 (C-H), 1744 (CdO), 1158 (C-O) cm-1. MS (EI): m/z 436 (M+), 335, 269, 255, 235, 220, 204, 185, 168, 137, 121, 111, 101. Accurate mass calcd for C23H32O8 (M+1), 436.2097; found, 436.2114. Anal. calcd for C23H32O8: C, 63.28; H, 7.41. Found: C, 63.69; H, 7.91. [R]20D -37° (c ) 5.1 g/100 mL, MeOH). Preparation of (3S,4S)-3,4-Dihydroxy-3-methyl-butyrolactone (15). Compound 14 (2.3 g, 5.2 mmol) was dissolved in 95% TFA (20 mL, ∼30 equiv), and the resulting solution was stirred at room temperature for 5 h until the starting material had disappeared (monitored by TLC). The excess of TFA was removed by rotary evaporation. To the residue was added methanol (∼20 mL), and the mixture was stirred for 0.5 h. The methanol was removed, and another portion of methanol (∼20 mL) was added. After the reaction was left overnight, filtration and removal of the methanol gave a residue that was purified by medium pressure chromatography (silica, elution with ether) to give the title compound as a colorless oil (0.60 g, 78%). 1H NMR (200 MHz, CDCl3): 4.32 (1H, dd, J 10, 3 Hz, CHH), 4.26 (1H, dd, J 10, 1 Hz, CHH), 4.11 (1H, m, CH), 1.40 (3H, s, Me). 13C NMR (125 MHz, CDCl ): 178.58 (CdO), 73.74 (C), 73.24 3 (CH2), 72.20 (CH), 21.31 (CH3). IR (KBr disk): 3414 (O-H), 2983 (C-H), 1778 (CdO), 1106 (C-O) cm-1. MS (EI): m/z 133 (MH+), 101, 98, 88, 83, 74, 70, 61, 55. Accurate mass calcd for C5H9O4 (MH+1), 133.0501; found, 133.0496. [R]17D +26° (c ) 3.8 g/100 mL, methanol). Preparation of (2R,3S)-2-Methylbutane-1,2,3,4-tetraol (16). Compound 15 (0.20 g, 1.5 mmol) in dry methanol (1.0 mL) was added to sodium borohydride (0.30 g, 7.4 mmol) in methanol (2.0 mL). The reaction was monitored by TLC until complete (overnight). The reaction mixture was worked up by addition of ethereal HCl (1 M in diethyl ether, 7.4 mL). After the solvent was removed, the resulting pale yellow oily solid was purified by medium pressure chromatography on silica with methanol/ CH2Cl2 (1/4) as eluent to give the title compound as a colorless oil (0.20 g, 100%). 1H NMR (200 MHz, D2O): 3.92 (1H, m, CH), 3.65 (2H, m, HOCH2CH), 3.43 (2H, s, HOCH2C), 1.20 (3H, s, Me). 13C NMR (125 MHz, D2O): 76.3 (C), 75.2 (CH), 66.3 (HOCH2CH), 62.6 (HOCH2C), 19.9 (CH3). MS (EI): m/z 105 (M - CH2OH)+, 87, 75, 61, 57. Accurate mass calcd for C4H9O3 (M - CH2OH)+1, 105.0552; found, 105.0554. [R]20D -8° (c ) 4.2 g/100 mL, MeOH). Preparation of the 1,4-Di-O-mesylate of (2R,3S)-2-Methylbutane-1,2,3,4-tetraol (17). To 16 (0.2 g, 1.5 mmol) in dry pyridine (1.0 mL) under a nitrogen atmosphere was added methanesulfonyl chloride (0.3 g, 0.2 mL, 3.0 mmol) over 10 min. After the reaction was finished (ca. 10 min), the mixture was directly purified by medium pressure chromatography on silica (twice) with methanol/dichloromethane (1/20) as eluent to give a white solid (0.2 g, 37%); mp 90-91°C. 1H NMR (500 MHz, CD3OD): 4.27 (2H, m, MsOCH2CH), 4.07 (2H, 2 × dd, J 10, 10 Hz, MsOCH2C), 3.77 (1H, dd, J 2, 8 Hz, CH), 2.87 (6H, s, 2 × MeSO2). 13C NMR (125 MHz, CD3OD): 75.3, 73.4, 72.8, 72.4, 37.2, 37.1, 18.5. MS (EI): m/z 292 (M+), 265, 234, 220, 184, 165, 154, 98, 88, 79, 57. Anal. calcd for C7H16O8S2: C, 28.77; H, 5.48. Found: C, 28.40; H, 5.40. [R]20D -13° (c ) 5.7 g/100 mL, MeOH). Preparation of (2R,2′S)-2-Methylbioxirane (4d) (6). The dimesylate of 17 (0.10 g, 0.40 mmol) in a glass bulb attached to a ground glass joint was added, by rotating the bulb, to sodium ethane-1,2-diolate in ethylene glycol (0.9 M, 0.9 mL) under vacuum (0.04 mmHg). (2R,2′S)-2-Methylbioxirane (0.02 g, 50%) was distilled from the reaction mixture and condensed in a trap immersed in liquid nitrogen. 1H NMR (500 MHz, CD3CN): 3.11 (1H, dd, J 4, 2 Hz, CH), 2.80 (1H, dd, J 5, 4 Hz, CHHCH), 2.76 (1H, d, J 5 Hz, CHHC), 2.68 (1H, dd, J 5, 2 Hz, CHHCH), 2.67 (1H, d, J 5 Hz, CHHC), 1.41 (3H, s, Me). 13C (125 MHz, CD3-

Metabolism of Isoprene CN): 56.3 (C), 53.7 (CH), 51.7 (CH2CH), 44.9 (CH2C), 17.8 (CH3). [R]19D -0.4° (c ) 5.8 g/100 mL, ethyl acetate). Preparation of Rat and Mouse Liver Microsomes. Animals were supplied by Charles River (Manston, Kent, U.K.) and were acclimatized for at least 4 days before use. Liver microsomes were prepared from animals sacrificed in a slowrising concentration of CO2 followed by cardiac puncture using previously developed and described methods (35). The prepared microsomes were stored at -80 °C. This procedure was used for the preparation of microsomes from male Sprague-Dawley rats (n ) 12, 220-260 g body weight) and male B6C3F1 mice (n ) 30, 16-20 g body weight). The concentration of microsomal protein was determined by assay with Coomassie blue (Biorad, Pierce Chemicals). Human Liver Microsomes. Liver microsomes prepared from individual donors were obtained from the either the Queen Elizabeth Hospital (Birmingham, U.K.; human 9 and 10) or the U.K. Human Tissue Bank at De Montfort University (Leicester, U.K.; human 12). Details of the individual donors were as follows: human 9, male, age 43, death from head injury following a fall; human 10, male, age 2, death by head injury in a road traffic accident; human 12, age 47, death by cardiac arrest. All of the individual donors were recorded as nonsmokers with no history of drug or alcohol use. Pooled human liver microsomes (lot no. 1035) prepared from livers of 10 male donors and representing, according to the supplier, an average male were obtained from In Vitro Technologies (Baltimore, MD). The composition and history, as provided by the supplier, of the donor pool were as follows: eight Caucasian, age range 15-53, average age 32; one African American, age 36; one Asian, age 57. Five of the donors were recorded as smokers, five used alcohol, and two used drugs. GC-MS Analysis of Reference Compounds. Reference standards of 2-4 were analyzed by a GC-MS system comprised of a Hewlett-Packard 5973 mass selective detector and HewlettPackard 6890 GC with a split/splitless injector. Compounds 2 and 3 were separated into enantiomer pairs (2a/2b and 3a/3b) using a Chiraldex G-PN (γ-cyclodextrin propionyl) capillary column (30 m × 0.25 mm i.d.) with a helium flow of 1.1 mL/ min and an injector temperature of 200 °C (split ratio 100:1). The oven temperature was held at 30 °C for 10 min. The MS was operated in scan mode with a GC-MS interface temperature of 280 °C. These conditions gave retention times of 4.3 and 4.5 min for the R and S enantiomers of 2 and for the R and S enantiomers of 3, 6.0 and 5.8 min, respectively. The reference standard of 4 was analyzed using a Chiraldex G-TA (γcyclodextrin trifluoroacetyl) capillary column (30 m × 0.25 mm i.d.) with a helium flow of 0.8 mL/min and an injector temperature of 200 °C (split ratio of 100:1.) The oven temperature was held at 70 °C for 10 min and then increased at 10 °C/min for the following program: 75 (1 min), 100 (10 min), and 70 °C (10 min). These conditions gave retention times of 12.90, 13.05, 13.17, and 13.27 min for the isomers of 4. Metabolism of 1 to 2 and 3. The experiments to determine reaction kinetics were carried out in 2 mL vials with a total incubation volume of 600 µL. Each incubation contained 4.2 mg of NADPH (5 µmol), 0.1 M potassium phosphate buffer (pH 7.4), 1 mM cyclohexene oxide, 2 mg of microsomal protein, and an added substrate concentration ranging from 0.1 to 40 mM. The reaction mixture was incubated for 5 min at 37 °C to allow inhibition of epoxide hydrolase prior to addition of substrate. The samples were prepared in triplicate and incubated at 37 °C for 2.5-60 min. Reactions were terminated by addition of 400 µL of ice-cold methanol containing 1 mM butadiene monoepoxide as an internal standard. The termination of the reaction and the addition of the internal standard were necessary to ensure reproducibility. Control samples were each run in the absence of either substrate or NADPH. The formation of isoprene monoepoxide metabolites was quantified using calibration curves prepared using reference standards of 2 and 3. Samples were prepared in 600 µL of 0.1 M potassium phosphate buffer (pH 7.4) with 400 µL of 1 mM butadiene monoepoxide added as

Chem. Res. Toxicol., Vol. 16, No. 7, 2003 937 an internal standard. Samples and standard were kept on ice prior to analysis to minimize hydrolysis of 2 to the corresponding diol. For analyses, samples and reference standards were heated at 37 °C for 10 min to allow equilibration of the internal standard and the isoprene monoepoxides between the gas and the liquid phases. The headspace was then sampled using a 1 mL pressure-lock gas tight syringe and analyzed by GC-MS in selective ion-monitoring mode using the GC conditions described in the above section, GC-MS Analysis of Reference Compounds, with a split ratio of 10:1. The MS was operated in the selective ion-monitoring mode with monitoring of fragment ions of the monoepoxides at m/z 53 and 55. Metabolism of 2 and 3 to 4. The experiments for kinetic studies were carried out in 2 mL vials with a total incubation volume of 1 mL. Each incubation contained 4.2 mg of NADPH (5 µmol), 0.1 M potassium phosphate buffer (pH 7.4), 10 mM cyclohexene oxide, 2 mg of microsomal protein, and an added substrate concentration ranging from 0.2 to 40 mM. The incubation mixture was maintained for 5 min at room temperature to allow inhibition of epoxide hydrolase prior to the addition of substrate. The reactions were started by the addition of substrate and incubated at 37 °C for 2.5-60 min. Samples were run in triplicate, and control incubations were run in the absence of either NADPH or substrate. The reactions were terminated by the addition of ice-cold diethyl ether (1 mL) and cycloheptanone as the internal standard. Samples were then extracted with diethyl ether (3 × 1 mL), and the organic extracts were pooled, dried, and concentrated to a volume of ca. 100 µL. Samples were quantified by correlation with a standard curve of 4 in 0.1 M potassium phosphate buffer (pH 7.4). Reference standard samples used for preparing the standard curve were extracted, dried, and concentrated in an identical manner to the samples for determination. GC-MS Analysis of 4 from Microsomal Incubations. The diethyl ether extracts of 4 were analyzed by GC-MS using one of the methods described below. In cases where sufficient analyte was present, a Chiraldex G-TA capillary column (30 m × 0.25 mm i.d.) was used, with a helium flow of 0.8 mL/ min and an injector temperature of 200 °C (split ratio of 50:1). The oven temperature conditions were held at 70 °C for 10 min and then increased at 10 °C/min for the following program: 75 (1 min), 150 (10 min), and 70 °C (1 min). Because of the lower sensitivity of detection when using the chiral GC column, some samples were analyzed using a CP WAX 57 capillary column (50 m × 0.32 mm i.d.). This gave two peaks for the diastereoisomers of 4 under the following conditions: helium pressure of 23 Kpa, injector temperature of 200 °C, split ratio of 1:1. The oven temperature was held at 60 °C for 5 min and then increased at 10°C/min for the following program: 70 (12 min), 150 (2 min), and 60 °C (1 min). For both columns, the MS was operated in the selective ion-monitoring mode with monitoring of the fragment ion at m/z 55. This corresponded to the loss of C2H5O from the molecular ion and was the base peak in the scan spectrum of the diepoxide. Measurement of Partition Coefficients of 1-3. The method of measurement of the partition coefficients for 1-3 was based on the procedure described by Gargas et al. (36). The method was adapted so that the vial size and headspace correlated with the conditions used in the microsomal incubations. Boiled microsomes in 0.1 M potassium phosphate buffer were used as the aqueous medium. Estimation of Enzyme Kinetic Data. The parameters Km and Vmax were estimated with Table Curve 2D version 4 (AISN Software Inc.). Chemical Oxidation of 3b. Compound 3b (10 µL) was added to a solution of m-CPBA (10 mg) in CDCl3 (0.25 mL). The mixture was stirred at room temperature for 1 h prior to direct analysis by GC-MS using the method described for the diepoxide standard (4). The GC analysis of the mixture showed two major product peaks with retention times of 13.05 and 13.27 min, corresponding to the 2S,2′R (4c) and 2R,2′R (4b) isomers of 4.

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Chem. Res. Toxicol., Vol. 16, No. 7, 2003 Scheme 1a

a Reagents and conditions: (i) Vinylmagnesium bromide, THF, 50%. (ii) LiAlH4, ether, 99.7%. (iii) p-Toluenesulfonyl chloride, pyridine, 44%. (iv) NaOCH2CH2OH, HOCH2CH2OH, 80%.

Results and Discussion The present studies have investigated the stereochemical and kinetic differences between species in the formation of all of the possible isoprene epoxides. The overall aim was to generate comparative kinetic data that could be used for estimating in vivo concentrations of the reactive metabolites and could also assist in judging the relevance of the animal carcinogenicity data for man. The stereochemical features and relative rates of the metabolic epoxidation in vitro of isoprene to its mono- and diepoxides by liver microsomes from male and female Sprague-Dawley rats, B6C3F1 mice, and humans have therefore been studied. The identities and stereochemistry of metabolites were established by comparisons with synthetic standards. Syntheses of Chemical Reference Standards. 1. Synthesis of rac-2 (Scheme 1). Commercial 2 contains 5% 3, and it was necessary to develop an efficient method for obtaining compound 2 in a higher state of purity. Two methods were developed for this purpose. One method was by preparative GC, which was suitable for purifying up to 1 g quantities of 2. The other method was synthesis from ethyl pyruvate, which was suitable for obtaining relatively large amounts of 2. Vinylmagnesium bromide was reacted with 5 in dry THF at -78 °C to give 6 (Scheme 1). Reduction of 6 using lithium aluminum hydride in diethyl ether gave 7 in quantitative yield. Diol 7 was converted into the corresponding 1-O-tosylate (8). Treatment of 8 with base followed by direct distillation of the product from the reaction mixture (30) gave 2,

Golding et al.

essentially 100% pure, and containing none of the isomeric 3. 2. Synthesis of 2b (Scheme 2). A key intermediate for the preparation of 2b is 10, which was prepared from the “dispoke-protected lactate” (9) (Scheme 2). Ley and co-workers (29) have shown that condensation with acetaldehyde of the carbanion derived by deprotonation of 9 with LDA gives predominately diastereoisomer 11. We converted 11 into its triflate (12), which underwent DBU-induced elimination to 13. Deprotection of 13 by treatment with 95% TFA gave 10. The synthesis of 2b was completed by reduction of 10 to 7a, tosylation of 7a to 8a, treatment of 8a with base, and direct distillation of 2b from the reaction mixture. 3. Synthesis of rac-3 and 3b. The racemic compound was synthesized according to a procedure described by Harwood et al. (32), while the S isomer (88% ee) was obtained according to Wistuba et al. (31). 4. Synthesis of 4, Mixture of Diastereoisomers. A mixture of 4a-d was obtained by the oxidation of rac-2 by m-CPBA. The ratio of the isomers 2R,2′S:2S,2′R: 2S,2′S:2R,2′R was 3:3:2:2 as judged by GC-MS analysis with monitoring on fragment ions at m/z 55, which represented the base peak (M - C2H5O) in the scan spectra. 5. Synthesis of 4d (Scheme 3). Compound 4d was also prepared from the dispoke-protected lactate (9) (29) (Scheme 3). Condensation of 2-(4-methoxybenzyloxy)acetaldehyde with the carbanion derived by deprotonation of 9 with LDA was assumed to give predominately diastereoisomer 14 by analogy with the steric course observed with acetaldehyde and other aldehydes (29). Compound 14 was converted into lactone 15 by treatment with 95% TFA. Reduction of 15 to tetraol 16 was followed by selective mesylation of the primary hydroxy groups to give 17 and finally base-induced ring closure to diepoxide 4d. Chromatographic Separation and Analytical Detection of Isoprene Monoepoxides. We have previously developed separations using GC equipped with a chiral stationary phase to separate enantiomers of each of the isoprene monoepoxides: (R)-2 and (S)-2 (2a,b) and (R)-3 (3a) and (S)-3 (3b) (27). A Chiraldex G-PN chiral GC column completely resolved all of the enantiomers of

Scheme 2. Synthesis of 2ba

a Reagents and conditions: (i) LDA, DMPU, n-BuLi, THF, and then CH CHO, 93%. (ii) Trifluoromethanesulfonic anhydride, pyridine, 3 CH2Cl2, 100%. (iii) DBU, CH2Cl2, 71%. (iv) 95% TFA, 88%. (v) LiAlH4, ether, 100%. (vi) p-Toluenesulfonyl chloride, pyridine, 51%. (vii) NaOCH2CH2OH in HOCH2CH2OH, 80%.

Metabolism of Isoprene

Chem. Res. Toxicol., Vol. 16, No. 7, 2003 939 Scheme 3. Synthesis of (2R,2′S)-2-Methylbioxirane (4d)a

a Reagents and conditions: (i) LDA, DMPU, n-BuLi, THF, and then pmbOCH CHO, 73%. (ii) 95% TFA, 78%. (iii) NaBH , MeOH, 2 4 100%. (iv) Methanesulfonyl chloride in pyridine, 37%. (v) NaOCH2CH2OH in HOCH2CH2OH, 49%.

Figure 1. Gas chromatographic chiral separation of 2-methyl-2,2′-bioxirane isomers: (1) (2R,2′S), (2) (2S,2′R), (3) (2S,2′S), and (4) (2R,2′R) using a Chiraldex G-TA capillary column (30 m × 0.25 mm i.d.); helium flow, 0.8 mL/min; injector temperature, 200 °C (split ratio of 50:1). The temperature conditions were as follows: 70 °C for 10 min and then changed at 10 °C/min to the following temperatures: 75 (1 min), 150 (10 min), and 70 °C (1 min).

the monoepoxides. Selective ion monitoring GC-MS for ions m/z 53 and 55 was used for detection. These ions correspond to the loss of CH3O+ and CHO, respectively, from each of the molecular ions. The molecular ions at m/z 84 were not used for monitoring the monoepoxides because a coeluting peak gave rise to an ion with the same mass. These analytical conditions permitted a quantitative comparison of the formation of each pair of enantiomers of prop-2-yloxirane and 2-ethenyl-2-methyloxirane in incubations of isoprene with liver microsomes from rat, mouse, and human. The assignments for peak identities were made by chromatographic and mass spectral comparisons with the synthetic reference standards described. Separation and Detection of Isoprene Diepoxides. The analysis conditions using the Chiraldex G-PN column employed for separation of the isoprene monoepoxides were unsuitable for the complete resolution of all of the stereoisomers of the isoprene diepoxides. Effective resolution of the diepoxides, 4a-d, was however achieved using a Chiraldex G-TA column. Selective ionmonitoring of the fragment ion (base peak) at m/z 55, corresponding to the loss of C2H5O from the molecular ion, was used for detection and quantification of each of the diepoxides. The assignment of the identities of peaks to each of the enantiomers of the diastereoisomers was achieved by comparison of the single peak for the synthetic 2R,2′S isomer and the two peaks from the oxidation of 3b by m-CPBA with the peaks for the

diepoxide mixture. Under the analysis conditions, the order of elution of individual isomers of 4 from the Chiraldex G-TA column was (2R,2′S)-, (2S,2′R)-, (2S,2′S)-, and (2R,2′R)-2-methyl-2,2′-bioxirane (Figure 1). Kinetics of the Metabolism of 1 to 2 and 3. There have been a number of previous studies in vitro that have investigated kinetic parameters for the cytochrome P450mediated oxidation of isoprene (31, 37, 38), and values for the apparent Km and Vmax have been reported. The most detailed study is that reported by Bogaards et al. (39) in which the oxidation of isoprene to its epoxides and their hydrolysis with epoxide hydrolase or conjugation with GSH were described. To date, however, there have been no studies that have comprehensively investigated and compared kinetic parameters for the formation of all of the stereoisomers of the mono- and diepoxides of isoprene. The present study therefore describes the first reported comparisons of values for apparent Km, the Michaelis-Menten constant, and Vmax, the maximum initial velocity, for the formation of each single enantiomer of the two isoprene monoepoxides, 2 and 3, and each enantiomer of the pairs of diastereomers for the four isoprene diepoxides, 4a-d. The oxidations exhibited Michaelis-Menten kinetics, and typical MichaelisMenten plots are shown (Figure 2) for the formation of (R)-2 and (S)-2 from isoprene by rat liver microsomes. Quantification of epoxides in microsomal incubations was based on comparisons with standard curves obtained from reference standards maintained under similar

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Golding et al.

Figure 2. Michaelis-Menten plots of the metabolism of isoprene to (A) 3a and (B) 3b by liver microsomes from male SpragueDawley rats.

conditions to the incubations taking account of the possible spontaneous hydrolysis of the epoxides (t1/2 2, 1.25 h; 3, 73 h). Because of the short incubation times of microsomal incubations, values obtained were not corrected for possible small differences in spontaneous hydrolysis between the incubations and the reference standards. Km values measured in vitro do not necessarily represent exactly the Km values that occur in vivo. There is a straightforward and obvious explanation for this. In vitro studies of the type described here employ homogenates of liver tissues from which the microsomal fraction containing the membrane associated cytochrome P450s is isolated. The isolation procedure involves cellular disruption and centrifugation. The membrane conditions employed in vitro therefore do not mimic precisely the conditions in vivo due to such differences as the degree of oxygenation and the concentration of cofactors. Relatively high concentrations of substrate are often required in order to observe the formation of the products of interest. The Km values from in vitro studies are calculated from the substrate concentration in the incubations and are therefore higher than would occur in vivo and are referred to as apparent values. This situation is not necessarily a disadvantage, particularly for comparative

studies between species, where identical conditions in vitro are used thus allowing direct and valid comparisons. The availability of kinetic data in vivo for a given compound and species permits a direct comparison with the values obtained in vitro and hence allows extrapolation to other species, especially man. This is the so-called parallelogram approach for interspecies in vitro-in vivo extrapolations (40-42). In the case of isoprene, in vivo kinetic data have already been generated for mice from gas uptake studies (43), which can be used as the in vivo reference and the basis for extrapolation to other species. In the microsomal oxidation studies described here, the kinetic measurements were performed with nominal added concentrations of isoprene in the range of 0.1-40 mM and 2 and 3 in the range of 0.2-20 mM. These nominal values were corrected to actual concentrations in the microsomal mixture by determining the partition coefficient for isoprene and the monoepoxides in the microsomal incubation mixture according to the method of Gargas et al. (36). When corrected for the partition coefficient, the actual concentration range for isoprene in the microsomal fraction was 0.02-5.62 mM and for the monoepoxides the range was 0.16-16 mM. Because of the relatively low metabolic conversion of isoprene and monoepoxides in vitro, substrate concentrations in this


Chem. Res. Toxicol., Vol. 16, No. 7, 2003 941

Table 1. Kinetic Data for Oxidation of Isoprene to the Enantiomers of 2 and 3 by Liver Microsomes from Sprague-Dawley Rats, B6C3F1 Mice, and Human (Pooled and Individual)a compound 2 R

a

compound 3 S

S

R

1.17 ( 0.05 0.43 ( 0.08 2.72 0.97

0.68 ( 0.03 0.23 ( 0.04 2.96 0.96

Vmax (nmol/min/mg) Km (mM) Vmax/Km R2

1.65 ( 0.09 0.27 ( 0.06 6.11 0.94

SD rat 2.04 ( 0.11 0.23 ( 0.05 8.87 0.94


2.07 ( 0.13 0.30 ( 0.07 6.90 0.94

B6C3F1 mouse 1.97 ( 0.17 0.23 ( 0.05 8.57 0.93

0.60 ( 0.05 0.41 ( 0.11 1.46 0.92

0.60 ( 0.04 0.30 ( 0.07 2.00 0.93


0.60 ( 0.037 0.17 ( 0.042 3.64 0.92

human pooled 0.84 ( 0.07 0.20 ( 0.06 4.22 0.89

0.24 ( 0.02 0.28 ( 0.08 0.87 0.92

0.23 ( 0.02 0.28 ( 0.12 0.83 0.87


0.59 ( 0.05 0.10 ( 0.04 5.97 0.72

human 9 0.66 ( 0.06 0.09 ( 0.04 6.74 0.65

0.27 ( 0.02 0.21 ( 0.06 1.31 0.92

0.31 ( 0.02 0.24 ( 0.07 1.30 0.91


0.61 ( 0.04 0.12 ( 0.03 4.92 0.87

human 10 0.69 ( 0.04 0.10 ( 0.03 6.61 0.86

0.30 ( 0.02 0.26 ( 0.09 1.15 0.86

0.32 ( 0.13 0.25 ( 0.04 1.27 0.97


0.58 ( 0.05 0.14 ( 0.05 4.27 0.83

human 12 0.78 ( 0.07 0.16 ( 0.7 4.96 0.80

0.28 ( 0.02 0.27 ( 0.10 1.06 0.89

0.32 ( 0.04 0.47 ( 0.24 0.69 0.89

Data given as follows: mean ( SD, n ) 3.

range were necessary to observe and detect the formation of the isoprene epoxides and achieve satisfactory quantification. The complete kinetic data for the oxidation of isoprene by cytochrome P450 to each enantiomer of the two monoepoxides, 3 and 2, in liver microsomes from male SD rat, male B6C3F1 mice, and male human are shown in Table 1. The apparent kinetic parameters for monoepoxidation of isoprene by rat or mouse liver microsomes were very similar. Although somewhat lower values were observed using human liver microsomes, overall, only marginal differences between species were found for the oxidation of isoprene to its monoepoxides. This is consistent with the findings by Bogaards et al. (39). Levels of cytochrome P450 2E1, the cytochrome principally responsible for isoprene oxidation, were lower in human microsomes as compared to microsomes from rat and mouse. The levels of cytochrome P450 2E1 in liver microsomes from rat and mouse, determined according to methods previously used (35), were found to be 3.5 and 4.8 nmol/mg protein, respectively. The value for the human pooled microsomes was 1.9 nmol/mg protein. Significant differences between individuals in respect of the formation of the isoprene monoepoxides were not observed with liver microsomes from three separate human subjects. The results from the studies with liver microsomes from the individual human subjects were similar to those obtained using pooled microsomal material, which represented the average response from 10 individuals. Incubations of isoprene with samples of either pooled or individual male human liver microsomes gave both lower Vmax and Km values as compared with rat or mouse microsomes. This indicates that human liver

has a lower rate of conversion of isoprene to oxidative metabolites as compared with liver of rat or mouse. The Vmax values obtained for oxidations using mouse liver microsomes were comparable with the results of Bogaards et al. (39) and were in the same range as the in vivo kinetic constants reported by Filser et al. (44). The Km values found in this present study were also similar to those determined by Bogaards et al. (39). The apparent Km values determined in vitro were considerably higher than values determined for rats and mice in vivo (43) for the reasons outlined above. Appropriate in vitro-in vivo scaling is therefore necessary for the use of these values in physiologically based pharmaco toxico kinetic modeling. Kinetic parameters for the metabolism of 1,3-butadiene have also been determined in vitro in studies with microsomes from rat, mouse, and human by gas depletion and formation of the monoepoxide (45). The apparent Km values in those studies were determined to be in the micromolar range. The use of glycerol in the storage procedure for microsomes has been reported (46) to give rise to false increases in Km values, but this reagent was not used in any of the current studies. While it is recognized that the overall toxicology of isoprene is determined by the balance of oxidative and hydrolysis reactions, in order to make comparisons across the species for the oxidative metabolism, it was necessary to inhibit the enzymatic hydrolysis reactions. In the present study on the regio- and stereochemical aspects of enzymatic oxidation of isoprene, a main feature of interest found in the oxidative metabolism of isoprene to its monoepoxides was the stereoselectivity in the formation of 3b by rat liver microsomes: the ratio of the

942


Golding et al.

Table 2. Kinetic Data for Oxidation of 3 to the Isomers of 2-Methyl-2,2′-bioxirane by Liver Microsomes from Sprague-Dawley Rats, B6C3F1 Mice, and Human (Pooled)a 2-methyl-2,2′-bioxirane 2R,2′S

a

2S,2′R

2S,2′S

2R,2′R

0.81 ( 0.13 1.83 ( 0.96 0.44 0.77

0.97 ( 0.10 1.71 ( 0.61 0.57 0.87


1.68 ( 0.13 1.75 ( 0.45 0.96 0.93

SD rat 1.06 ( 0.08 1.76 ( 0.45 0.60 0.93


2.03 ( 0.26 1.24 ( 0.58 1.63 0.79

B6C3F1 mouse 2.14 ( 0.30 1.12 ( 0.58 1.91 0.72

1.76 ( 0.23 1.40 ( 0.66 1.26 0.80

1.82 ( 0.22 1.58 ( 0.68 1.15 0.83


0.65 ( 0.13 4.54 ( 2.50 0.14 0.80

human pooled 0.45 ( 0.06 2.88 ( 1.28 0.16 0.84

0.46 ( 0.10 3.30 ( 2.10 0.14 0.70

0.46 ( 0.08 3.08 ( 1.56 0.15 0.80

Data given as follows: mean ( SD, n ) 3).

S to R enantiomer was 3:2. In contrast, there was no pronounced enantiomeric excess for the formation of either enantiomer of 3 by mouse or human liver microsomes. The enantioselectivity of this oxidation step cannot be measured satisfactorily in the absence of epoxide hydrolase inhibitor due to the competing enantioselective hydrolysis of the formed epoxides by epoxide hydrolases in the microsomal fraction. In all incubations of isoprene studied here with human, rat, or mouse liver microsomes, there was consistently a higher proportion of 2 formed as compared with 3. The ratio of these two regioisomers, 2 and 3, was ca. 2:1 for metabolic oxidations with human or rat liver microsomes and ca. 3:1 using mouse liver microsomes. Each of these monoepoxides has been shown to be substrates for microsomal epoxide hydrolase. In in vitro studies, Bogaards et al. (39) found that the enzyme-catalyzed rate of hydrolysis of 2 was considerably greater than that for 3. To make quantitative determinations for the epoxides formed in the microsomal oxidation, the epoxide hydrolase present in the microsomal mixture was therefore inhibited. The use of the epoxide hydrolase inhibitor was essential in the context of the stereochemical studies since the enzymatic hydrolysis is also stereoselective. In the present studies, this was effected principally by the use of the epoxide hydrolase inhibitor cyclohexene oxide. There was no difference in the results obtained using the inhibitor cyclohexene oxide at concentrations of either 1 or 10 mM. For some control incubations, epoxide hydrolase was inhibited with 3,3,3-trichloromethyloxirane at concentrations of 1 and 10 mM for comparison. Essentially, the same results were obtained with each of the inhibitors indicating that the monooxygenation step was not significantly affected by this procedure. Further oxidation of the monoepoxides can, in principle, also occur in the isoprene incubation system thus causing possible underestimation of the monoepoxides. However, in the isoprene oxidation system that we employed for the kinetic determinations, the formation of diepoxides could not be detected (see below). Kinetics of the Metabolism of 3 to 4. Because of the very low extent of formation of 4 from the microsomal oxidation of isoprene itself in this in vitro system, the rate of production of the isoprene diepoxide isomers could not be measured directly from isoprene. To study the formation of isoprene diepoxide isomers, rac-2 and 3 were

each incubated with microsomes from male SpragueDawley rats, B6C3F1 mice, and male humans to establish the rates of production of all of the isomers of 4. As described for studies on the formation of the isoprene monoepoxides, the investigations on isoprene diepoxide formation were also carried out in the presence of an epoxide hydrolase inhibitor. The results were essentially the same with the different inhibitors used. The data obtained for enzymatic oxidation of rac-3 by liver microsomes from male rat, mouse, and human liver are summarized in Table 2. The values for the apparent Km and Vmax were obtained for each enantiomer (4a-d) of the pair of diastereoisomers of 4. Overall, the highest rate of metabolism of 3 to isoprene diepoxides occurred with mouse liver microsomes. However, the rates of formation of diepoxides were not substantially different as compared with values obtained with rat liver microsomes. Pooled male human liver microsomes exhibited a lower rate of metabolism of monoepoxides to diepoxides than did microsomes from rat or mouse liver microsomes, a finding similar to that observed for the epoxidation of isoprene to its monoepoxides. Kinetics of the Metabolism of 2 to 4. In the studies using 2 as substrate, it was more difficult to quantify the individual isomers of the diepoxides using the chiral column. Therefore, to achieve satisfactory sensitivity for quantitative analyses of low level formation of diepoxides, the determinations for enzymatic oxidations of 2 were carried out with a nonchiral column (Chrompak CP WAX 57) to increase the sensitivity of detection of peaks corresponding to diepoxide. The results thus obtained for the kinetic measurements of formation for 4 from 2 (Table 3) were based on quantification of the diepoxide as a single peak representing the four isomers. For the formation of 4 from 2, there were no significant differences between the values for apparent Km and apparent Vmax for metabolism by rat or mouse liver microsomes. The main differences between species for the epoxidation of 2 were the lower Km and Vmax values observed with human liver microsomes as compared with liver microsomes from mouse and rat liver. Some of the incubations carried out with higher substrate concentrations (nominally 20 mM) of 2 were analyzed on the chiral G-TA column to determine the ratio of individual isomers. A summary of the ratio of the isomers of 4 (4a:4b:4c:4d) formed from the metabo-


Chem. Res. Toxicol., Vol. 16, No. 7, 2003 943

Table 3. Kinetic Data for Oxidation of 2 to 2-Methyl-2,2′-bioxirane by Liver Microsomes from Sprague-Dawley Rats, B6C3F1 Mice, and Human (Pooled)a 2-methyl-2,2′-bioxirane SD rat Vmax (nmol/min/mg) Km (mM) Vmax/Km R2

5.97 ( 0.77 2.70 ( 1.23 2.21 0.90

B6C3F1 mouse Vmax (nmol/min/mg) Km (mM) Vmax/Km R2

7.32 ( 0.33 3.79 ( 0.49 1.93 0.99

human pooled Vmax (nmol/min/mg) Km (mM) Vmax/Km R2 a

1.25 ( 0.17 0.96 ( 0.49 1.30 0.71

Data given as follows: mean ( SD, n ) 3).

Conclusions

Table 4. Isomeric Composition of 2-Methyl-2,2′-bioxiranes Formed by the Metabolism of Isoprene Monoepoxides by Liver Microsomes from Sprague-Dawley Rats, B6C3F1 Mice, and Human (Pooled) species

R and the S enantiomers. The oxidation of 3 by the human liver system showed a preference in the formation of 4d from attack on the re face of the R enantiomer (3a). Overall, while differences were observed in the stereoselectivity and the kinetics of formation of 4 by rat, mouse, and human liver microsomes that could have toxicological significance, the quantitative differences between rat and mouse are probably not sufficient to account for the large difference between species observed in vivo for the rodent carcinogenicity of isoprene. It has recently been shown that the isoprene diepoxides are also substrates for epoxide hydrolase (39, 47). These compounds are known to be mutagenic metabolites of isoprene, and the species differences in the carcinogenic response of rodents to isoprene could be due to differences in the effectiveness of detoxification of the diepoxide isomers or in the resulting cellular concentration of the hydrolysis products, i.e., the isoprene diol epoxides.

substrate

2R,2′S

2S,2′R

2S,2′S

2R,2′R

SD rat

compound 2 compound 3

0.23 0.37

0.30 0.24

0.19 0.18

0.28 0.21

B6C3F1 mouse


0.41 0.26

0.22 0.28

0.21 0.23

0.16 0.23

human


0.35 0.32

0.17 0.22

0.30 0.23

0.18 0.23

Scheme 4. Stereoselectivity in the Enzymatic Epoxidation of 3a

lism of the two regioisomeric isoprene monoepoxides is shown in Table 4. From analysis of the relative proportions of each diepoxide isomer formed, it appears that in the enzymatic oxidation of 3 with rat liver microsomes, the R enantiomer (3a) underwent preferential oxidative attack at the re face to give 4d (37%)2 (Scheme 4). In the oxidation of 2 by rat liver microsomes, a slight preference for attack on the re face of the R enantiomer (2a) to give 4c was observed. With mouse liver microsomes, the most significant selectivity resulted from oxidative attack on the si face of 2b to give 4d (41%) (11). In the case of 3 with mouse liver microsomes, there was a slight preference for oxidation at the si face of the S enantiomer (3b) to give 4c. The results found with liver microsomes from Sprague-Dawley rats and B6C3F1 mice were broadly consistent with those reported for Fischer 344 rats and B6C3F1 mice by Wistuba et al. (31). With microsomes from human liver, the epoxidation of 2 occurred with a significant preferential attack on the si face of both the 2 On the basis of IUPAC nomenclature, C-2 of 3 becomes C-2′ in the product 2-methyl-2,2′-bioxirane. From the Cahn, Ingold, and Prelog sequence rule, 2R stereochemistry of 3 becomes 2′S of the product 2-methyl-2,2′-bioxirane and 2S stereochemistry of 2 becomes 2R for the product 2-methyl-2,2′-bioxirane.

The data obtained from the present study showed that the qualitative profile of isoprene metabolites was similar in the in vitro metabolism of isoprene and its epoxides by liver microsomes from rodents and humans. Quantitatively, there were some differences in the relative amounts of metabolites and their rates of formation and also in the stereochemistry of the oxidative metabolism between species. Although these differences may have some toxicological significance, they may not be great enough to account entirely for the large difference in carcinogenic potency between rodent species. The overall observed trends in the stereochemistry of the P450catalyzed epoxidation were similar to those described for the structurally related dienes, butadiene (48) and chloroprene (34). Differences in the rates of detoxification of reactive metabolites between species may also be important in the overall carcinogenesis. As noted above, it is necessary to interpret in vitro studies with a degree of care because certain aspects of the corresponding in vivo processes may not be fully replicated by the in vitro conditions. For example, there may be limitation of metabolism by transport differences and differences in turnover and concentrations of cofactors. Nevertheless, data obtained from these in vitro studies are useful for comparing metabolic activities and specificities for different substrates and across species.

Acknowledgment. This work was supported at the Syngenta Central Toxicology Laboratory and at the University of Newcastle upon Tyne by the International Institute for Synthetic Rubber Producers (IISRP). We thank the members of the IISRP Scientific Committee for comments on the paper.

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