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Chem. Res. Toxicol. 2003, 16, 56-65
Stereochemical Aspects in the 4-Vinylcyclohexene Biotransformation with Rat Liver Microsomes and Purified Cytochrome P450s: Diepoxide Formation and Hydrolysis Cinzia Chiappe,*,† Antonietta De Rubertis,‡ Giandomenico Piegari,† Giada Amato,‡ and Pier Giovanni Gervasi*,‡ Dipartimento di Chimica Bioorganica e Biofarmacia, Universita` di Pisa, via Bonanno 33, 56126 Pisa, Italy, and Istituto di Fisiologia Clinica, area della Ricerca CNR, Via G. Moruzzi, 56100 Pisa, Italy Received June 12, 2002
The stereochemical course of the biotransformation of 1,2-monoepoxides of 4-vinylcyclohexene (2 and 3) by liver microsomes from control and induced rats and by purified P4502B1 and P4502E1 has been determined. The epoxidation of monoepoxides cis-4-vinylcyclohexene 1,2epoxide (2) and trans-4-vinylcyclohexene 1,2-epoxide (3) gives the corresponding eight isomeric diepoxides cis-4-vinylcyclohexene diepoxide (9) and trans-4-vinylcyclohexene diepoxide (10). The stereoselectivity of this process is affected by P450 induction. Phenobarbital is able to enhance the yield of epoxidation to give preferentially diepoxide (1R,2S,4R,7R)-trans-10b. This enantiomer is also formed as nearly the sole product by P450-catalyzed epoxidation of (1R,2S,4R)-trans-3b, the monoepoxide that, as a consequence of the selective formation from 4-vinylcyclohexene and/or reduced elimination by epoxide hydrolase, tends to accumulate in rat. Also, the P4502B1 but not 2E1, in a reconstituted system, is able to perform the epoxidation of (1R,2S,4R)-trans-3b to produce selectively the same diepoxide. Diepoxides cis-9 and trans10 are biotransformed by mEH catalyzed hydrolysis. Although the hydrolysis of diepoxides 9 is characterized by a lower substrate enantioselection, the reaction of diepoxides 10 occurs with a good substrate enantioselectivity favoring the hydrolysis of the epoxides (1R,2S,4R,7S)trans-10b and (1S,2R,4S,7S)-trans-10a. Diepoxide (1R,2S,4R,7R)-trans-10b is therefore the isomer primarily formed by P450-catalyzed oxidation of monoepoxide trans-3, and it is also the compound showing the lower propensity to undergo mEH-catalyzed hydrolysis. On the basis of this result, the ovotoxicity of 4-vinylcyclohexene is expected to be due to the stereoisomer diepoxide (1R,2S,4R,7R)-trans-10b, whose biological reactivity, via cross-linking, may be strongly different to the other isomer diepoxides, being dependent by its specific conformation.
Introduction 4-Vinylcyclohexene (VCH) is a colorless liquid formed by spontaneous dimerization of 1,3-butadiene during the rubber process (1). VCH is also an intermediate in the epoxide resin formation and in polymer manufacturing (1). Various studies have shown that a chronic exposure to VCH causes in rodents toxic effects, especially in the ovary (2, 3). Mice are reported to be the most susceptible species in VCH toxicity. A prolonged exposure of VCH provokes in mice, but not in rats, a premature ovarian failure, and ovarian neoplasms (3). These differences are likely to be related to biotransformation of VCH to epoxide metabolites, VCH-1,2-epoxide and VCH-7,8epoxide, and in particular to the mutagenic and carcinogenic 4-vinylcyclohexene diepoxide, thought to be the ultimate ovitoxic metabolite (2). The activities of the main enzymatic systems engaged in the metabolism of VCH were studied in various species (4-7). Mice have a greater capacity than rats to oxidize VCH by P450 * To whom correspondence should be addressed. E-mail: cinziac@ farm.unipi.it. † Universita ` di Pisa. ‡ Istituto di Fisiologia Clinica.
system, whereas the microsomal epoxide hydrolase (mEH)1-catalyzed hydrolysis of the epoxide intermediates is less efficient in mice than in rats (3, 8). Furthermore, previous studies have shown that the P4502A and 2B isozymes, which are the major enzymes implied in the epoxidation of VCH (9, 10), are inducible by VCH exposure in mice but not in rats (7). Thus, the higher activity of the P450 monooxygenase involved in the metabolic activation of VCH in mice along with the lower activity involved in the detoxication of epoxide intermediates may account for the high susceptibility of this species. VCH is a chiral compound, and its epoxidation by P450 system may form eight monoepoxides, which, in turn, may be further oxidized to eight diepoxides. Recently, stereochemical aspects of VCH biotransformation and regioselectivity in the formation of endocyclic cis- and trans-VCH-1,2-epoxides in rodents and human have been reported (11, 12). It was demonstrated that in rats both endocyclic and exocyclic monoepoxides undergo hydrolization by mEH with diastereo- and 1 Abbreviations: mEH, microsomal epoxide hydrolase; PB, phenobarbital; DEX, dexamethasone; PYR, pyrazole; TCPO, trichloropropene oxide; GC, gas chromatography.
10.1021/tx025573z CCC: $25.00 © 2003 American Chemical Society Published on Web 12/06/2002
Stereochemistry of 4-Vinylcyclohexene Biotransformation
enantioselectivity (12). It was observed that the endocyclic epoxides, (1R,2S,4R)-trans-3b and (1S,2R,4R)-cis2b, whose formation was favored in phenobarbitalinduced microsomes, had also a major resistance to the enzymatic hydrolysis. Therefore, these epoxides are expected to accumulate in the liver and undergo further oxidation in a stereochemical manner to specific reactive diepoxides. In the present work, we have investigated the stereochemical and mechanistic aspect of the second step of VCH metabolism. By using liver microsomes from rats pretreated with phenobarbital (PB), pyrazole (PYR), dexamethasone (DEX), and purificated rat P4502B1 or P4502E1, we have examined the stereochemistry of the oxidation of endocyclic cis- and trans-VCH-1,2-epoxides to the isomeric diepoxides. Subsequently, we have studied the stereoselectivity of the mEH-catalyzed hydrolysis of the racemic diepoxides to epoxydiols.
Materials and Methods Caution! The following chemicals are hazardous and should be handled carefully: 4-vinyl cyclohexene, 4-vinylcyclohexene 1,2epoxide, 4-vinylcyclohexene 7,8-epoxide, 4-vinylcyclohexene diepoxide. Materials. DEX, PYR, and β-NF were obtained from common commercial sources. PB is a control drug and was sold by Fluka (Buchs, Switzerland) only for research purpose. 4-Vinyl cyclohexene (1), 4-vinylcyclohexene 1,2-epoxide (1:1 mixture of cis-2 and trans-3) and 4-vinylcyclohexene diepoxide (mixture of four cis-9 isomers and four trans-10 isomers) were purchased from Aldrich Chemical Co (Milwaukee, WI). TCPO was obtained from EGA-Chemie (Steinheim-Albuch, Germany). (S)-4-Vinylcyclohexene (1a), (()-4-vinylcyclohexene 7,8-epoxide (4), (()-4-vinylcyclohexene 7,8-epoxide (5), (()-4-vinylcyclohexene 1,2-diol (6), (()-4-vinylcyclohexene 7,8-diol (7) and (()-4-vinylcyclohexene 7,8-diol (8), (4S,7S)-4-vinylcyclohexene 7,8-diol (8), (4S,7R)-4vinylcyclohexene 7,8-diol (7), (4S,7S)-4-vinylcyclohexene 7,8epoxide (5), and (4R,7S)-4-vinylcyclohexene 7,8-epoxide (4) were prepared as previously reported (12). 4-Vinylcyclohexene 1,2-Epoxide-7,8-diols (11). To a solution of m-chloroperbenzoic acid (9 mmol) in chloroform (8 mL) a 1:1 mixture of diols 7 and 8 (3 mmol) was added, and the mixture was allowed to stay for 16 h at room temperature under stirring and 4 h at 4 °C. The solution was filtered, and 400 mg of KF, previously activated by heating at 120 °C in vacuo (1 mmHg) for 2 h, was added. The mixture was stirred at room temperature for 1 h; then the insoluble complexes were filtered off, and the solvent was removed under reduced pressure to give a mixture of eight diastereoisomeric epoxydiols 11. The crude product was analyzed by NMR. 1H NMR (CDCl ) δ (ppm): 1.4-2.0 (m, 28H); 2.1-2.5 (m, 8H, 3 epoxide CH-CH); 3.1-3.7 (m, 12H, CHOH and CH2OH). 13C NMR and DEPT (CDCl3) δ (ppm): 20.5 (CH2); 21.5(CH2); 23.1 (CH2); 23.1(CH2); 23.3 (CH2); 24.3 (CH2); 24.3 (CH2); 24.5 (CH2); 26.0 (CH2); 26.5 (CH2); 27.2 (CH2); 28.0 (CH2); 33.3 (CH); 33.9 (CH); 36.2 (CH); 36.8 (CH); 50.8 (epoxide CH); 51.4 (epoxide CH); 51.6 (epoxide CH); 51.7 (epoxide CH); 51.8 (epoxide CH); 52.7 (epoxide CH); 52.8(epoxide CH); 53.0 (epoxide CH); 67.8-68.2 (4CH2OH); 79.5 (CHOH); 79.6 (CHOH); 80.1 (CHOH); 80.2(CHOH). 4-Vinylcyclohexene 1,2-Diol-7,8-epoxides (12). The mixture of four diastereoisomeric epoxydiols 12 was synthesized by epoxidation with m-chloroperbenzoic acid from (()-4-vinylcyclohexene 1,2-diol (6), according to the above-reported procedure. The crude product was analyzed by NMR. 1H NMR (CDCl ) δ (ppm): 1.4-2.0 (m, 14H); 2.5 (m, 2H, 3 epoxide CH); 2.7 (m, 2H, epoxide CH); 2.9 (m, 2H, epoxide CH); 3.4 (m, 2H, CHOH); 3.6 (m, 2H, CHOH). 13C NMR and DEPT (CDCl3) δ (ppm): 24.5 (CH2); 25.1 (CH2); 28.4 (CH2); 28.7 (CH2); 30.9 (CH2); 33.2 (CH2); 36.5 (CH); 36.7 (CH); 46.5 (epoxide CH2);
Chem. Res. Toxicol., Vol. 16, No. 1, 2003 57
Figure 1. Gas chromatographic separation of a mixture (ca. 2:8) of monepoxides (1S,2R,4R)-cis-2 and (1R,2S,4R)-trans-3 arising from enzymatic resolution of the commercial mixture of (()-cis-2 and (()-trans-3. Analysis conditions are outlined in the Materials and Methods.
46.7 (epoxide CH2); 46.8 (epoxide CH); 47.0 (epoxide CH); 71.2 (CHOH); 71.5 (CHOH); 73.8(CHOH); 74.0 (CHOH). (1R,2S,4R)-trans-4-Vinylcyclohexene 1,2-Epoxide (3b) was prepared by enzymatic kinetic resolution of a 1:1 mixture of monoepoxides cis-2 and trans-3 using a bovine liver microsomal preparation. An ethanolic stock solution (0.2 mL) of the mixture of (()-cis-2 and (()-trans-3 (160 mg) was added to 6 mL of bovine liver microsomal preparation, containing 5 mg of protein/mL, at pH 7.4 (Tris-HCl), and the mixture was incubated with shaking at 37 °C. After 6 h, a saturating amount of NaCl was added to precipitate the microsomal proteins and the incubation mixture was extracted with hexane (3 × 10 mL) to remove the unreacted epoxide. The organic phase was dried (MgSO4) evaporated under reduced pressure to give a 2:8 mixture of the two monepoxides (1S,2R,4R)-cis-2b and (1R,2S,4R)-trans-3b or pure (1R,2S,4R)-trans-3b. As shown in Figure 1, GC analysis was conducted on a 50 m chiral CPCyclodex B (CHROMPACK) column (helium flow of 50 kPa, and with an evaporator and detector set at 200 °C, at 90 °C). (1S,2R,4S,7S)-, (1S,2R,4S,7R)-, (1R,2S,4S,7S)-, and (1R,2S,4S,7R)-4-Vinylcyclohexene Diepoxide (cis-9a, trans10a). To a solution of m-chloroperbenzoic acid (5 mmol) in chloroform (15 mL) (S)-4-vinylcyclohexene (1a, 2 mmol) was added and the mixture was allowed to stay for 2 days at room temperature and 4 h at 4 °C. The solution was filtered, and 650 mg of KF, previously activated by heating at 120 °C in vacuo (1 mmHg) for 2 h, was added. The mixture was stirred at room temperature for 1 h; then, the insoluble complexes were filtered off, and the solvent was removed under reduced pressure to give a mixture of (1S, 2R, 4S, 7S)-, (1S, 2R, 4S,7R)-, (1R,2S,4S,7S)-, and (1R,2S,4S,7R)-4-vinylcyclohexene diepoxides (cis-9a and trans-10a). The crude product was analyzed by GC (Figure 2; trace b) on a 25 m Chiraldex G-TA (ASTEC) chiral column at 98 °C (helium flow of 50 kPa, with an evaporator and detector set at 200 °C). Mixture of (1R,2S,4S,7R)-cis-4-Vinylcyclohexene Diepoxide (9a) and (1S,2R,4S,7R)-trans-4-Vinylcyclohexene Diepoxide (10a). The mixture of (1R,2S,4S,7R)-cis-9a and (1S,2R,4S,7R)-trans-10a was synthesized by epoxidation with m-chloroperbenzoic acid from (4S,7R)-4-vinylcyclohexene 7,8epoxide (4a), prepared as previously reported (12), according to the above-reported procedure. The crude product was analyzed by GC under conditions reported above. Figure 2, trace d. Mixture of (1R,2S,4S,7S)-cis-4-Vinylcyclohexene Diepoxide (9a) and (1S,2R,4S,7S)-trans-4-Vinylcyclohexene Diepoxide (10a). To a solution of m-chloroperbenzoic acid (2.5 mmol) in chloroform (10 mL) was added (4S,7S)-4-vinylcyclohexene 7,8-epoxide (5a, 1 mmol), prepared as previously reported (12), and the mixture was allowed to stay for 2 days at room temperature and 4 h at 4 °C. The solution was filtered, and 300 mg of KF, previously activated by heating at 120 °C in vacuo (1 mmHg) for 2 h, was added. The mixture was stirred at room temperature for 1 h; then the insoluble complexes were filtered off, and the solvent was removed under reduced pressure to give a ca. 1:1 mixture of (1R,2S,4S,7S)-cis-9a and
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Figure 2. Gas chromatographic separation of diepoxides cis-9 and trans-10. (a) commercial mixture of diepoxides cis-9 and trans-10. The continuous line connects the enantiomer couples determined on the basis of the peaks areas (1.6; 2.3; 4.7; 5.8). (b) mixture of four diepoxides having configuration (4S), arising from epoxidation of 1a. (c) Mixture of the two diepoxides having configuration (4S,7S), arising from epoxidation of (4S,7S)-5a. (d) Mixture of the two diepoxides having configuration (4S,7R), arising from epoxidation of (4S,7R)-5a. (e) Mixture of the four diepoxides arising from epoxidation of a 2:8 mixture of monepoxides (1S,2R,4R)-trans-3 and (1R,2S,4R)-trans-3. Peaks 1 and 5 correspond to diepoxides having configuration (1R,2S,4R), peaks 3 and 7 correspond to diepoxides having configuration (1S,2R,4R). On the basis of the information arising from these analyses the following configurations may be attributed to the eight diepoxides: (1R,2S,4R,7R)-trans-10b (1); (1R,2S,4S,7S)cis-9a (2); (1S,2R,4R,7R)-cis-9b (3); (1R,2S,4S,7R)-cis-9a (4); (1R,2S,4R,7S)-trans-10b (5); (1S,2R,4S,7S)-trans-10a (6); (1S,2R,4R,7S)-cis-9b (7); (1S,2R,4S,7R)-trans-10a (8). Analysis conditions are outlined in the Materials and Methods. (1S,2R,4S,7S)-trans-10a. The crude product was analyzed by GC under conditions reported above. Figure 2, trace c. Mixture of (1R,2S,4R,7R)-trans-4-Vinylcyclohexene Diepoxide (10b) and (1R,2S,4R,7S)-trans-4-Vinylcyclohexene Diepoxide (10b). The mixture of (1R,2S,4R,7R)-trans-10b and (1R,2S,4R,7S)-10b was synthesized by epoxidation with mchloroperbenzoic acid from (1R,2S,4R)-trans-4-vinylcyclohexene 1,2-epoxide (3b) according to the above-reported procedure. The crude product was analyzed by GC under conditions reported above. Animal Microsomal Preparations and Enzymes. Male Sprague-Dawley rats were purchased from Charles River (Calco, Como, Italy). Male rats were treated ip for 3 days with PB (80 mg/kg daily), DEX (50 mg/kg daily), or PYR (200 mg/kg daily), and microsomes were obtained from the liver as previously described (13). Microsomal protein concentrations were assayed by using the method of Lowry et al. (14); the total P450 concentration was measured according to Omura and Sato (15). P4502B1 and P4502E1 and NADPH cytochrome P450 reductase
Chiappe et al. were purified from rat liver as previously described (16). Enzymatic Incubations. (1) Determination of the P450 Activity and of the Enantiomeric Excesses of the Products. Incubation mixtures (10 mL) containing 100 mM potassium phosphate buffer (pH 7.4), 3 mg/mL of hepatic microsomal proteins, NADPH-regenerating system, consisting of 0.5 mM NADP+, 5 mM glucose 6-phosphate, and 0.5 units/mL glucose 6-phosphate dehydrogenase and trichloropropene oxide (TCPO, 1 mM) to inhibit the mEH, were preincubated at 37 °C for 5 min, and the reaction were initiated by the addition of a proper amount of the 1:1 mixture of epoxides 2 and 3 or of the enantiomerically pure epoxide (1R,2S,4R)-trans-3b. The substrate concentration was 10 mM to saturate the P450 enzymes (8). After 60 min, a saturating amount of NaCl was added to precipitate the microsomal proteins. The reaction products were extracted with dichloromethane (2 × 5 mL) and analyzed by GC after addition of appropriate amounts of 2-cyclohexen-1-one as an internal standard. The enantiomeric composition of diepoxides was determined by GC on a chiral 30 m Chiraldex G-TA (ASTEC) column (helium flow of 50 kPa and with an evaporator and detector set at 200 °C, and column at 98 °C). The absolute configurations of the excess enantiomers of diepoxides cis-9 and trans-10 were determined by comparison of the retention times of each enantiomer with those of enantiomerically pure compounds obtained by epoxidation of chiral monopoxides prepared by enzymatic resolution or asymmetric synthesis. The epoxidations of the enantiomerically pure epoxide (1R,2S,4R)-trans-3b were also measured in a reconstituted system (0.5 mL) containing 0.1 nmol of purified rat P4502B1 or 2E1, 0.3 nmol of P450 reductase, 30 µg of dilauroylphosphatidylcholine and the substrate at concentration 10 mM. The reactions were carried out at 37 °C for 60 min after the addition of 1 mM NADPH and the products were extracted and analyzed as described above. (2) Determination of the Substrate Enantioselectivity of the mEH-Catalyzed Hydrolysis of Diepoxides cis-9 and trans-10. Aliquots (20 µL) of an ethanolic stock solution of the commercial mixture of diepoxides cis-9 and trans-10 were added to 2 mL of a microsomal preparation (Control) containing 3 mg of protein/mL in a such way to obtain a 20 mM substrate concentration, and the reaction mixtures were incubated at 37 °C. At prefixed times (between 1 and 5 h), a saturating amount of NaCl was added to precipitate the microsomal proteins and the incubation mixtures were extracted with dichloromethane (3 × 1 mL) to remove the unreacted epoxides followed by brief centrifugation to separate the emulsion. 2-cyclohexen-1-one was added as a standard to the combined extracts, which were analyzed directly by GC on 30 m Chiraldex G-TA (ASTEC) column (helium flow of 50 kPa, and with an evaporator and detector set at 200 °C, and column at 98 °C). To determine the nature of the products the commercial mixture of epoxides cis-9 and trans-10 as neat liquid (40-60 mg) was added to 20 mL of microsomal preparation and the reaction mixtures were incubated with shaking at 37 ° C for the time necessary to obtain a practically complete conversion (6 h). The incubations were then extracted with ethyl acetate (3 × 10 mL), and the organic phase was dried and evaporated under reduced pressure. The product mixture was analyzed by NMR. Blank experiments carried out with boiled microsomal preparations show that the spontaneous hydrolysis does not contribute to the diols formation under the incubation conditions.
Results and Discussion The stereochemical course of formation of 4-vinylcyclohexene diepoxides by epoxidation of the exocyclic double bond of a mixture of monoepoxides 2 and 3 or of the pure (1R,2S,4R)-3b was investigated using various microsomal preparations from male rats and in reconstituted monooxygenase systems containing either
Stereochemistry of 4-Vinylcyclohexene Biotransformation
P4502B1 or 2E1. This study was performed with male rats since level of overall VCH metabolism was found higher in male than in female rat liver microsomes according to our previous published paper (12). Microsomal preparations from male rats were also used to study the stereochemistry of hydrolysis of 4-vinylcyclohexene diepoxides. In both cases, as a convenient analytical tool, the inclusion gas chromatography, which enables a time-dependent enantiomer screening of diepoxides in the nmol range, was employed for the determination of the enantiomeric ratios (Figure 2) and for the absolute configurations. To use this method to determine the absolute configuration of the formed or residual diepoxides, the synthesis of reference substances with unequivocal stereochemistries was necessary. Synthesis of Reference Diepoxides and GC Analysis on a Chiral Column. The four diastereoisomeric diepoxides cis-9a and trans-10a, having (S) configuration at C-4, were prepared by the epoxidation of (S)-4vinylcyclohexene 1a (12) with m-chloroperbenzoic acid. Analogously, mixtures of two diasteroisomeric epoxides, (1S,2R,4S,7R)-trans-10a + (1R,2S,4S,7R)-cis-9a, (1S,2R,4S,7S)-trans-10a + (1R,2S,4S,7S)-cis-9a, (1R,2S,4R,7S)-trans-10b + (1R,2S,4R,7R)-trans-10b, were prepared by epoxidation with peracid of the corresponding monoepoxides (4S,7R)-4, (4S,7S)-5, and (1R,2S,4R)-trans3b. The oxidation of a 2:8 mixture of the diastereosiomeric epoxides (1S,2R,4R)-cis-2b and (1R,2S,4R)-trans-3b, obtained by partial enzymatic kinetic resolution, gave a mixture of four diepoxides, (1S,2R,4R,7R)-cis-9b, (1S,2R,4R,7S)-cis-9b, (1R,2S,4R,7S)-trans-10b, and (1R,2S,4R,7R)-trans-10b, characterized by a ca. 2:8 ratio between the isomers 9 and 10. The commercial mixture of four couples of enantiomers of diepoxides cis-9 and trans-10 give, when analyzed by GC on a chiral column, as expected eight peaks (Figure 2, trace a). On the basis of the areas of the peaks four couples of enantiomers can be evidenced. In particular, peaks 1 and 6 having the same area correspond to a pair of enantiomer. Analogously, peaks 2 and 3, 4 and 7, and finally 5 and 8 correspond to the other three couples of enantiomers. On the basis of the retention times of the four diepoxides arising by exhaustive oxidation of (4S)1a (Figure 2, trace b), it was possible to attribute the configuration (4S) to diastereoisomers corresponding to the peaks 2, 4, 6, and 8. Analogously, considering the starting material, configuration (4S,7S) was attributed to peaks 2 and 6 (Figure 2, trace c), (4S,7R) to peaks 4 and 8 (Figure 2, trace d), (1R,2S,4R) to peaks 1 and 5, and (1S,2R, 4R) to peaks 3 and 7 (Figure 2, trace e). On the basis of this information, taking into account the enantiomeric couples (Figure 2, traces a), the absolute configuration was assigned to each peak, as reported in Figure 2. Epoxidation of 1,2-Monoepoxides of 4-Vinylcyclohexene 2 and 3 by Rat Liver Microsomes. The oxidation of the exocyclic double bond of monoepoxides cis-2 and trans-3 can give four pairs of enantiomers. cis- and trans-4-vinylcyclohexene 1,2-epoxides possess a prochiral carbon-carbon double bond and three chiral centers. The enzymatic epoxidation of these compounds is therefore a competitive process which can occur with substrate and/or product enantio- and diastereoselectivity. The correct determination of stereoselectivity in the epoxidation requires the complete inhibition of
Chem. Res. Toxicol., Vol. 16, No. 1, 2003 59 Table 1. Stereochemical Course for the Oxidation on the Exocyclic Double Bond of a 1:1 Mixture of 2 and 3 by Microsomes from Male Control and PB-Treated Rats diepoxides (1R,2S,4R,7S)-10b (1R,2S,4R,7R)-10b (1S,2R,4S,7R)-10a (1S,2R,4S,7S)-10a (1S,2R,4R,7R)-9b (1S,2R,4R,7S)-9b (1R,2S,4S,7R)-9a (1R,2S,4S,7S)-9a
a b h g c d e f
CTR nmol (%)
PB nmol (%)d
3.3 ( 0.6 (4.5) 27 ( 3 (36.9) 16 ( 2 (21.9) 3.6 ( 1 (4.9) 11 ( 2 (15.0) 3.2 ( 0.8 (4.4) ndc 9 ( 2 (12.3)
10 ( 2* (4.9) 66 ( 7* (32.7) 27 ( 3* (13.3) 14 ( 2* (6.9) 39 ( 4* (19.3) 8 ( 1* (3.9) 5 ( 0.8* (2.47) 33 ( 5* (16.3)
a Each value represents the mean ((SD) of at least three determinations. b a, b, c, d, e, f, g, and h refer pathways reported in Scheme 5. c nd ) not detected. d The asterisk (*) indicates values significantly different from control (p < 0.01).
Figure 3. Enantiomeric composition of diepoxides 9 and 10 arising by oxidation of a 1:1 mixture of monoepoxides cis-2 and trans-3 catalyzed by microsomes from PB-treated rats. Peaks: 1 (1R,2S,4R,7R)-trans-10b; 2, (1R,2S,4S,7S)-cis-9a; 3, (1S,2R,4R,7R)-cis-9b; 4, (1R,2S,4S,7R)-cis-9a; 5, (1R,2S,4R,7S)trans-10b; 6, (1S,2R,4S,7S)-trans-10a; 7, (1S,2R,4R,7S)-cis-9b; 8, (1S,2R,4S,7R)-trans-10a.
epoxide hydrolase, since the enzymatic hydrolysis of the starting monoepoxides or formed diepoxides may represent an efficient competing enantioselective process. The stereochemical course of oxidation of a 1:1 mixture of monoepoxides cis-2 and trans-3 to the corresponding diepoxides cis-9 and trans-10 was investigated by incubating the substrate (10 mM) in the presence of TCPO (1 mM), an efficient inhibitor of epoxide hydrolase (17), by using liver microsomes obtained from untreated rats or rats pretreated with PB, PYR, and DEX, classical inducers of 2B1/2, 2E1, and 3A1/2, respectively (16, 18). At prefixed times (60 min), the reactions were stopped by adding a saturating amount of NaCl, and the incubation mixtures were extracted with dichloromethane and analyzed by GC on a chiral column, after addition of 2-cyclohexen-1-one as an internal standard, to evaluate the chemical yields. An incubation time of 60 min was necessary to obtain a quantity of products enough to be reproducibly detected by GC analysis. The use of the chiral column allowed the determination of the diastereoisomeric product ratios among the formed diepoxides and, for each diastereoisomer, the enantiomeric ratio and the absolute configuration of the enantiomer in excess, by the comparison of the retention time of the formed products with those of samples of known configuration prepared as reported above. Table 1 reports the diastereo- and enantiomeric composition of diepoxides cis-9 and trans-10 obtained by oxidation of the mixture of monoepoxides cis-2 and trans-3 by liver microsomes from male control and PBinduced rats. Figure 3 shows the GC trace of the products obtained by oxidation using microsomes from PB-induced
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Chiappe et al. Scheme 1
Scheme 2
rats. It must be remarked that both the chemical yield of epoxidation and the stereoselection of the process strictly depend on the induction of the P450 isoformes involved in these reactions. The use of PB-induced microsomes compared to control microsomes considerably increases the yields, indicating the involvement of the 2B1 isoform. On the other hand, when DEX- or PYRinduced microsomes are used, the stereochemical course of epoxidation was qualitatively the same of that obtained with control microsomes for the formation of the diepoxides (1S,2R,4S,7R)-10a, (1R,2S,4R,7R)-10b, (1S,2R,4R,7R)-9b, and (1S,2S,4S,7S)-9a whereas the quantities of the other formed diepoxides were lower of the detection limit (
