450
Chem. Res. Toxicol. 1997, 10, 450-456
Stereochemical Aspects of 1,3-Butadiene Metabolism and Toxicity in Rat and Mouse Liver Microsomes and Freshly Isolated Rat Hepatocytes Joe L. Nieusma, David J. Claffey, Chris Maniglier-Poulet, Tomasz Imiolczyk, David Ross, and James A. Ruth* Molecular Toxicology and Environmental Health Sciences Program, Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, Denver, Colorado 80262 Received December 6, 1996X
1,3-Butadiene (BD) is a gas used heavily in the rubber and plastics industry. BD and its epoxide metabolites have been shown to be carcinogenic and mutagenic in rodents, and BD has been classified by IARC as a group 2A carcinogen. We have examined the role of stereochemistry in species-dependent metabolism and toxicity of BD. Diastereo- and enantioselective synthetic routes to butadiene monoxide (BMO), butadiene bisoxide (BBO), and 3,4epoxybutane-1,2-diol isomers have been developed. These routes have allowed the development of chiral gas chromatographic and GC/MS analytical procedures for quantitation of these metabolites in biological experiments. We have utilized hepatic microsomes from male B6C3F1 mice and hepatic microsomes and intact hepatocytes from male Sprague-Dawley rats as experimental systems. At 30 min, BMO production from BD was two times higher in mouse hepatic microsomes than in rats, and stereoselective analysis was used to determine the relative formation of (R)- and (S)-BMO. Formation of BBO from both (R)- and (S)-BMO was characterized in rat and mouse microsomal systems. As expected, more BBO was formed in mouse hepatic microsomes (3-4-fold) than in rat hepatic microsomes. No difference in total BBO formed from either isomer was observed in rat microsomes, but in mouse microsomes significantly more BBO was produced from (S)-BMO than from (R)-BMO. The cytotoxicity of each BMO and BBO enantiomer was examined in freshly isolated rat hepatocytes. (R)-BMO showed greater cytotoxicity than (S)-BMO. Stereospecific cytotoxicity was also observed using BBO enantiomers and (meso)-BBO was more cytotoxic than either the (R:R) or the (S:S)-BBO. The results show that stereochemistry plays an important role in BD metabolism and cytotoxicity and for the purposes of risk assessment needs to be compared across species.
Introduction 1
1,3-Butadiene (BD) is a gas used heavily in the rubber and plastics industry as an intermediate in production processes that could potentially result in occupational exposure (1). BD has also been detected in automobile exhaust and urban air (2). An increased risk for hematopoietic cancers has been suggested for workers involved in the manufacture and use of BD (3), although the interpretation of these studies has been questioned (4, 5). BD is classified by IARC as a group 2A carcinogen (probably carcinogenic to humans) (3). Carcinogenicity studies in rats and mice have shown BD to be carcinogenic (6-9), and reactive epoxide metabolites formed from BD have been shown to be mutagenic (10-13). BD requires metabolic activation to produce toxicity, and several groups have extensively characterized metabolism of BD in mouse, rat, and human microsomal systems from liver and lungs (14-18), and metabolism of inhaled BD has been examined in monkeys (19). Comparative stereochemical aspects of butadiene metabolism between species, however, have not been reported. This paper examines the stereospecificity of sequential metabolic * Address correspondence to this author at the Department of Pharmaceutical Sciences, School of Pharmacy, UCHSC Box C-238, 4200 East Ninth Ave, Denver, CO 80262. Telephone: (303) 315-7569. X Abstract published in Advance ACS Abstracts, April 1, 1997. 1 Abbreviations: BD, 1,3-butadiene; BMO, butadiene monoxide; BBO, butane-1,2:3,4-bisoxide; TCPO, trichloropropylene oxide.
S0893-228x(96)00199-3 CCC: $14.00
reactions in the biotransformation of BD and the cytotoxicity of stereoisomers of BD metabolites. Stereochemical considerations arise following the initial oxidation of BD to butadiene monoxide (BMO) because a stereocenter is generated by this oxidation step and the chirality of this metabolite may then influence subsequent biotransformation pathways. Current studies in our laboratory are addressing which metabolic pathway predominates for each enantiomer of BMO and butadiene bisoxide (BBO). The use of isolated hepatocytes provides an intact cellular system with integrated metabolic pathways to study the fate of individual enantiomers of BD metabolites. In this work we have examined the influence of stereochemistry on BD metabolism and toxicity. We have examined stereoselectivity in the production of BMO, the subsequent hydrolysis of BMO to 1,2-butene-3,4-diol, and BBO production using rat and mouse hepatic microsomal systems. Additionally, cytotoxicity induced by enantiomers of BMO and BBO was measured in freshly isolated rat hepatocytes.
Materials and Methods Caution: The following chemicals are hazardous potential carcinogens and should be handled carefully: BD, BMO, and
© 1997 American Chemical Society
Stereochemical Aspects of 1,3-Butadiene Metabolism BBO should be used in a 100% vented biosafety hood at all times. BD gas, racemic BMO and BBO, calcium hydride, and sodium hydroxide were obtained from Aldrich Chemical Co. (Milwaukee, WI). Ethyl acetate, pyridine, acetic anhydride, and dimethyl sulfoxide were obtained from Mallinckrodt Chemical Co. (St. Louis, MO). The enantiomeric (R)- and (S)-1-tosyloxy-3buten-2-ols and (R)- and (S)-3-butene-1,2-diols were obtained from Eastman Fine Chemicals (Rochester, NY). 18O-enriched water (98% isotopic purity) was obtained from Cambridge Laboratories (Andover, MA). All other chemicals employed were reagent grade. Buffers were prepared with deionized water. Ethyl acetate was freshly distilled from calcium hydride under an argon atmosphere for use in the hydrolysis experiments. Male Sprague-Dawley rats (150-200 g) were purchased from Sasco (Lincoln, NE), and male B6C3F1 mice (6-8 weeks of age) were obtained from The Jackson Laboratory (Bar Harbor, ME). Oxidation of BD to BMO, an Analysis of BMO Enantiomers. Microsomes were prepared according to standard methods (20). Assays for the initial oxidation of BD to BMO contained hepatic microsomes (4 mg of protein/mL) in isotonic buffer (100 mM NaPO4, pH 7.42) containing MgCl2 (5 mM), trichloropropylene oxide (TCPO), an epoxide hydrolase inhibitor (100 µM), and NAD(P)H (1 mM). The final incubation volume was 2 mL, incubation temperature was 37 °C, and incubates were performed for 3, 10, 15, and 30 min. Controls included incubations without TCPO, without NAD(P)H, without BD, and without microsomes. The samples were split into two 1-mL aliquots for either headspace analysis on an HP-5 GC column (cross-linked 5% phenylmethyl silicone, 0.2 mm i.d., 0.33 µm film thickness, 25 m length) to quantitate total BMO production (21) or for analysis following extraction by n-pentane on a nickel camphorate column (Supelco, 0.55 mm i.d., 0.5 µm film thickness, and 60 m length) in order to distinguish the S/R ratio for BMO isomer formation as described by Schurig (22). 2-Butanol (250 µM) was used as internal standard for each headspace sample. GC conditions for the HP-5 column were oven (30 °C), injection port (100 °C), 25 psi head pressure, and flame ionization detector (250 °C). GC conditions for the nickel camphorate column were oven (90 °C), injector port (150 °C), 15 psi head pressure, and flame ionization detector (250 °C). Helium was the carrier gas for both columns. Standard curves were prepared daily for quantitation. Hydrolysis of BMO Enantiomers to 1,2-Butene-3,4-diols. A recent report (23) described a method for the generation of BMO enantiomers in which the commercial hydroxy tosylate enantiomers were treated with sodium hydroxide to generate an alkoxide that displaced the tosylate in an intramolecular fashion generating an epoxide. In this manner, the stereocenter at C2 was preserved and BMO enantiomers were generated. This enabled the enantiomeric distribution of the hydrolysis products to be characterized from each individual BMO isomer. Hepatic microsomal hydrolysis of BMO enantiomers to 1,2butene-3,4-diol utilized 3 mg of protein/mL in order to ensure product formation following the addition of substrate in 5 mL of headspace (23). Incubations were performed at 37 °C for 30 min. NAD(P)H was omitted from the 3-mL incubations in potassium phosphate buffer (25 mM, pH 7.4) so that further oxidation of BMO could not occur. Catalytic hydrolysis was corrected for non-catalytic rates determined in the absence of microsomes. For assessment of the stereochemical course of hydrolysis, the incubation samples were extracted with 3 mL of ethyl acetate as described previously (21). The concentrated extract was dissolved in 50 µL of pyridine and 50 µL of acetic anhydride and heated for 10 min at 70 °C to ensure complete acetylation of the hydroxyl groups. The sample was then subjected to GC/MS analysis using a Hewlett-Packard 5890 GC with flame ionization detection or in line with a HewlettPackard 5988 mass spectrometer (electron impact mode). A 25 m × 0.3 mm × 0.3m 10% persilated β-cyclodextrin column (Supelco, Inc., Bellefont, PA) was employed with helium as the carrier gas (1 mL/min). Other GC conditions included injection port temperature at 250 °C; oven temperature at 100 °C for 5 min, then reduced to 80 °C at 20 °C/min, and held for the
Chem. Res. Toxicol., Vol. 10, No. 4, 1997 451 duration of the analysis. A sharper 1,2-butene-3,4-diol peak resulted from starting the oven at 100 °C, and better peak separation was obtained by lowering the oven temperature to 80 °C. Calibrations were performed by extracting, derivatizing, and analyzing known mixtures of commercial enantiomeric butenediols (S:R 100, 70, 50, 30, 0%). Oxidation of (R)- and (S)-BMO to BBO. Oxidation of (R)or (S)-BMO to BBO were performed using the same conditions as described for BD oxidation to BMO except that hepatic microsomes were used at a concentration of 2 mg of protein/ mL. Samples were extracted into ethyl acetate as described previously (21) at 5, 10, 15, 20, and 30 min following addition of either (R)- or (S)-BMO. BBO formation was monitored by GC/MS (Hewlett-Packard 5890 Series II GC/ Hewlett-Packard 5972 Series quadrapole mass selective detector) analysis using selected-ion monitoring at m/z ) 29, 55, and 57 for BBO and at m/z ) 56, 69 and 84 for hexanol. Hexanol (150 µM) was used as an internal standard. Other GC conditions included injection port temperature at 200 °C, oven temperature at 50 °C for 3.2 min and then increasing at 30 °C/min to 120 °C for 1.5 min. Helium was the carrier gas, and the injection was splitless for 1 min. BBO levels were quantitated against a standard curve. Cytotoxicity in Freshly Isolated Rat Hepatocytes. Hepatocytes were isolated from Sprague-Dawley rats by a method utilizing collagenase perfusion originally described by Moldeus (24). Viability and hepatocyte density were determined by counting cells diluted 50 times with trypan blue dye (1 mL of 0.4% trypan blue stain, 5.6 mL of 0.9% NaCl) using a hemocytometer. Freshly isolated hepatocytes were added to KrebsHepes buffer (pH 7.4) in 25-mL round-bottom flasks for a final cell density of one million cells/mL. The flasks were attached to a five spot glass apparatus, which allows the cells to be maintained under a 95% O2/5% CO2 atmosphere while rotating the flasks through a 37 °C water bath. At various timepoints, 10 µL of the hepatocyte suspension was combined with 10 µL of trypan blue dye, and viability was determined by counting 100 cells. Statistical Analysis. Statistical analyses were performed using Statview 512+ (BrainPower, Inc. Calabasas, CA) and Excel (Microsoft, Seattle, WA). Differences in the cytotoxicity of various treatments in isolated hepatocytes were determined by ANOVA followed by a Dunnett’s t-test for comparison of multiple parameters to a single control. Differences in 1,2butene-3,4-diol composition and BBO formation in microsomal systems were determined by student’s t-test. The level of significance was set at p < 0.05.
Results Oxidation of BD to BMO occurs by 3 min in both rat and mouse microsomes and plateaus at 15 min in each species (Figure 1A,B). More total BMO is produced by mouse liver microsomes than by rat liver microsomes. The monoepoxide level declines in rat microsomes while in mouse microsomes BMO levels remain constant between 15 and 30 min of incubation. Initially, rat microsomes produce three times more (S)-BMO than (R)BMO and over time the S/R ratio falls below 1 (Figure 1C). Mouse microsomes consistently produce slightly greater amounts of (S)-BMO at early time points, and this ratio remains essentially constant over 30 min (Figure 1D). The presence of TCPO (100 µM), an inhibitor of epoxide hydrolase, significantly increased the amount of BMO produced from BD (25 K ppm, 30 min, at 37 °C) by rat liver microsomes (2 mg of protein/mL) from 27.8 ( 5.7 to 49.7 ( 7.0 nmol/mL. Conversely, TCPO did not affect the production of BMO from BD in mouse liver microsomes (BMO production ) 93.6 ( 18 and 108 ( 23 nmol/mL, n ) 3, in the presence and absence of TCPO, respectively). Enantiomeric ratios of BMO were not significantly affected by TCPO (100 µM) in either species (data not shown).
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Figure 1. BMO production from 25 000 ppm BD in the presence of 100 µM TCPO: (A) rat liver microsomes, (B) mouse liver microsomes, (C) enantiomeric characterization in rat liver microsomes, (D) enantiomeric characterization in mouse liver microsomes. Table 1. Enantiomeric Distribution of 3-Butene-1,2-diol Formed upon Incubation of Rat and Mouse Liver Microsomes with (R)- and (S)-Butadiene Monoxidea starting isomer rat mouse
diol composition (% (S) and (R)) (R)-monoxide (S)-monoxide racemic monoxide (S) 3.3 ( 1.3 (R) 94.7 ( 1.3* (S) 24.0 ( 3.8 (R) 76.0 ( 3.8*
(S) 98.4 ( 0.2 (R) 0.6 ( 0.2* (S) 84.3 ( 0.5 (R) 15.7 ( 0.5*
(S) 58.3 ( 0.9 (R) 41.7 ( 0.9* (S) 51.3 (0.3 (R) 48.7 ( 0.3
a Values represent the mean ( SEM of 3-7 independent determinations. (*) Significantly different from percentage of (S) at p < 0.05 (Student’s t-test). Diol formed upon hydrolysis of BMO enantiomers by rat or mouse liver microsomes was extracted with ethyl acetate as described in the methods section. The 1,2-butene3,4-diol was derivitized to a bisacetate ester with pyridine and acetic anhydride. The acetates were subjected to gas chromatographic analysis on a 10% β-cyclodex chiral column (persilated cyclodextrin) as described. The enantiomeric composition was determined by calibration with known mixtures of acetates prepared from commercial enantiomeric 1,2-butene-3,4-diol standards.
Once generated, two of the major metabolic fates of BMO enantiomers are represented by hydrolysis to 1,2butene-3,4-diol catalyzed by epoxide hydrolase or a second cytochrome P450-mediated oxidation to BBO. We first examined hydrolysis of BMO enantiomers catalyzed by microsomal epoxide hydrolase. It has been shown previously that the contribution of cytosolic epoxide hydrolase to BMO ring opening is minimal (14). The enantiomeric distributions of 1,2-butene-3,4-diols resulting from hepatic microsomal hydrolysis of BMO enantiomers are shown in Table 1. Incubation of rat liver microsomes with (S)-BMO resulted in 98% retention of configuration at C2. Treatment of rat liver microsomes with (R)-BMO resulted in approximately 95% retention of configuration at C2. Interestingly, treatment of mouse liver microsomes with (S)-BMO resulted in approxi-
Nieusma et al.
mately 16% inversion of configuration at C2, while incubation with the (R)-BMO resulted in 24% inversion of configuration at C2. Incubation of microsomes with racemic BMO for 10 min, to assess initial stereochemical substrate preferences, was also performed, and the results are listed in Table 1. No preference for one enantiomer was observed in hydrolysis by mouse liver microsomes, but rat liver microsomes displayed a small but significant preference for hydrolysis of (S)-BMO (58%: 42%). In order to determine if the inversion observed in the hydrolysis of (R)-BMO by mouse liver microsomes involved any racemization, experiments were conducted in 18O-enriched water. Figure 2 illustrates a representative separation of acetylated (R)- and (S)-1,2-butene-3,4-diols arising from this incubation with mouse liver microsomes. Baseline separation of the enantiomers is attained in this system with a selectivity factor (a) of 1.05. A mass spectrum of the (R)-enantiomer is shown in Figure 2A. As is frequently the case with bis esters, no molecular ion is observed (25). The fragment of m/z 131 represents the loss of acetyl from the molecule containing 1 atom of 18O (MW 174). The ion at m/z 99 arises from the loss of the C1 portion of the molecule as acetoxymethyl, leaving an acetoxypropenyl fragment. If the fragment contained 18O, the ion would be shifted to m/z 101. Figure 3 illustrates the selected ion traces for ion m/z 99 and 101 for the two enantiomers formed by mouse liver microsomes. As can be seen, the (R)-enantiomer contains no 18O at C2 and therefore must originate from the addition of water to the terminal (C1) carbon of the epoxide. The addition of 18O to carbon 2 is observed only in the (S)-enantiomer and represents an inversion of configuration. A small amount of (S)-enantiomer containing 16O at C2 is observed. The small amount of inversion with [16O]water most likely results from uncatalyzed epoxide (SN2) opening by enzyme in which bound water had not equilibrated with isotopic medium (26). These data demonstrate a regiochemical difference in hydrolysis catalyzed by mouse microsomal epoxide hydrolase with epoxide ring opening occurring at C2 of the (S)-BMO enantiomer but not at C2 of the (R)-BMO enantiomer. The hydrolysis of either enantiomer of BMO with epoxide ring opening at C2 was not apparent using rat hepatic microsomes. Stereospecific conversion of BMO to BBO was next examined using rat and mouse hepatic microsomes. Figure 4 illustrates the formation of BBO by rat and mouse liver microsomes. The level of BBO formed is 3-4-fold higher in mouse liver microsomes (Figure 4B) than the level of BBO formed by rat liver microsomes (Figure 4A). In mice, BBO formation from (S)-BMO is significantly greater than BBO formation from (R)-BMO. No significant differences between BBO formation from (R)- or (S)-BMO could be detected using rat liver microsomes, and if anything the stereochemical preference was reversed from that observed in mouse microsomes with a trend toward greater formation of BBO from (R)BMO. In addition to metabolism being affected by stereochemistry, we also examined a potential stereochemical influence on cytotoxicity. Cytotoxicity, as measured by trypan blue dye exclusion, induced by (R)- or (S)-BMO in freshly isolated rat hepatocytes is shown in Figure 5 as the mean and standard error for three separate experiments. Hepatocytes were exposed to either (R)- or (S)-BMO over 6 h. Time-dependent cytotoxicity was
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A
B
Figure 2. (A) Electron-impact mass spectrum of (R)-1,2-bisacetoxy-3-butene. (B) GC separation of (R)- and (S)-1,2-bisacetoxy-3butenes, formed by mouse liver microsomes and acetylated as described in the Materials and Methods, using a 10% β-cyclodex (persilated cyclodextrin) column. The (S)-enantiomer elutes first. The selectivity factor R ) 1.07.
Figure 3. Electron-impact ion chromatograms of m/z 99 (A) and 101 (B) for the (S)- and (R)-enantiomers of 1,2-bisacetoxy-3butene following formation by mouse liver microsomes and acetylation as described in Materials and Methods.
observed following treatment of rat hepatocytes with BMO enantiomers, but greater cytotoxicity was observed using (R)-BMO than (S)-BMO. Stereochemical effects were also observed when the cytotoxicity of BBO was examined. BBO has three possible stereochemical orientations: (R:R), (S:S), and (meso). Figure 6 shows significant time-dependent cytotoxicity following exposure to BBO enantiomers (1 mM). Cytotoxicity assays in freshly isolated rat hepatocytes indicated that (meso)BBO produced significantly greater cytotoxicity at 1 mM
than either the (S:S)- or the (R:R)-enantiomers of BBO. The (R:R)-enantiomer consistently produced the lowest level of cytotoxicity. This result was consistent with data obtained after exposure of rat hepatocytes to BBO enantiomers at concentrations of both 750 and 500 µM (data not shown).
Discussion BD oxidation to BMO in rat and mouse liver microsomes has been extensively characterized (14-17),
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Nieusma et al.
Figure 6. Time-dependent cytotoxicity induced by 1 mM BBO enantiomers. Open triangles represent control values, filled squares represent (R:R)-BBO-induced cytotoxicity, filled circles represent (S:S)-BBO-induced cytotoxicity, and filled triangles represent (meso)-BBO-induced cytotoxicity. Three asterisks (***) indicate a significant difference (p > 0.05) from all other treatments. Two asterisks (**) indicate a significant difference (p > 0.05) from control and (RR)-BBO treatments. An asterisk (*) indicates a significant difference (p > 0.05) from control. Error bars represent the mean ( SEM for three separate determinations.
Figure 4. BBO production from 500 µM (R)- and (S)-BMO: (A) rat liver microsomes, (B) mouse liver microsomes. Squares represent BBO produced from (R)-BMO and circles represent BBO produced from (S)-BMO. An asterisk (*) indicates a significant difference (p > 0.05) from (R)-BMO. Error bars represent the mean ( SEM for three separate determinations.
Figure 5. Time-dependent cytotoxicity induced by 1 mM (R)or (S)-BMO in freshly isolated rat hepatocytes. Open triangles represent control values, filled squares represent (R)-BMO, and filled circles represent (S)-BMO. Two asterisks (**) indicate a significant difference (p > 0.05) from control and other treatments. An asterisk (*) indicates a significant difference (p > 0.05) from control. Error bars represent the mean ( SEM for three separate determinations.
and enantiomeric distributions have been reported for rat (22) but not for mouse. Mouse hepatic microsomes produce a constant level of BMO between 15 and 30 min following the addition of BD gas, whereas rat hepatic microsomal incubations produce less total BMO than mice and the level decreases after peaking at 15 min. Mice are known to produce more BMO from BD than rats, and this could potentially contribute to the greater toxicity of BD observed in mice relative to rats. The data in this paper demonstrate that the enantiomeric distribution of BMO generated from BD is also quite different between species. Initially, rats produce three times more
(S)-BMO than (R)-BMO in the early minutes following exposure to BD gas. This may reflect either selective production of (S)-BMO or selective removal of (R)-BMO, and studies are currently under way to distinguish between these possibilities. Our results in rat hepatic microsomes are in contrast to results obtained by Schurig (22), who observed an enantiomeric ratio at 30 min of 30% (R)-BMO and 70% (S)-BMO. Slightly greater levels of (S)-BMO than (R)-BMO are produced in mouse hepatic microsomes after exposure to BD gas. Stereochemical influences are also apparent in the second oxidation step whereby BMO is converted to BBO by cytochrome P450. Our data demonstrate that mice produce 3-4-fold higher levels of BBO, the most cytotoxic BD metabolite, than rat microsomes. Interestingly, there is a stereochemical difference between species. Mice produce significantly greater amounts of BBO from (S)BMO than from (R)-BMO. This was not observed using rat microsomes, and in fact a trend toward greater production of BBO from (R)-BMO was observed in rat liver microsomes. The combined effect of greater (S)BMO generated from BD in mice and greater production of the highly cytotoxic BBO from (S)-BMO may contribute to the increased toxicity of BD observed in mice. Although mice produce greater amounts of BMO from BD than rats, the role of hydrolysis appears to be greater in rat liver microsomal suspensions than in mouse liver microsomal samples. TCPO, an inhibitor of epoxide hydrolase, significantly increased the amount of BMO produced in rat liver microsomes but not in mouse liver microsomes. This result suggests that the contribution of epoxide hydrolase is greater in rat liver than in mouse liver, and this may contribute to the resistance to BD toxicity seen in rats relative to mice. This is consistent with previous studies that have compared the role of epoxide hydrolase in BD metabolism in rats and mice (14). Wistuba and Schurig (27) followed the disappearance of BMO enantiomers from incubations with rat liver microsomes and reported a regiochemical selectivity for hydrolytic opening at C1. Regioselectivity was also
Stereochemical Aspects of 1,3-Butadiene Metabolism Scheme 1. Possible Hydrolytic Pathways for Conversion of BMO to 1,2-Butene-3,4-diol
Chem. Res. Toxicol., Vol. 10, No. 4, 1997 455
tion of BD and its epoxide metabolites. Mouse hepatic microsomes produced more total BMO than rats and more (S)-BMO than (R)-BMO. Higher BBO production occurred in mice with significantly more BBO generation from (S)-BMO than from (R)-BMO. Our data also demonstrate stereochemical differences in cytotoxicity induced by BMO and BBO enantiomers in rat hepatocytes. These data demonstrate the importance of stereochemistry in BD biotransformation and cytotoxicity and emphasize the need for these studies to be extended to human systems for use in BD risk assessment.
Acknowledgment. This work was supported by Grant ES06440 from the National Institute of Environmental Health Sciences.
References
observed with some disubstituted epoxides that were opened by inversion of the stereocenter (27). Addition of water to the terminal (C1) carbon would not involve the stereocenter and would result in retention of configuration at that center. Our data are consistent with previous studies (26) and indicate retention of configuration of (R)- and (S)-BMO following epoxide hydrolysis in rat liver microsomes. With mouse microsomes, however, particularly upon hydrolysis of the (R)-monoxide, a 24% inversion of configuration is observed. This could result from SN2 type addition of water to cleanly invert the stereocenter or could result from the generation of a planar cationic intermediate that undergoes racemization (Scheme 1). If the latter pathway were operative, the enantiomeric distribution would reflect 48% of the BMO undergoing racemization. The results of the hydrolysis experiment in [18O]water clearly show that no racemization occurred. The results suggest regiochemical possibilities of hydrolysis by mouse microsomes that do not occur in rat microsomes, at least for this substrate. The significance of this difference must await the further elucidation of stereochemical differences in BD metabolism and toxicity in rat and mouse. We examined stereochemical effects on cytotoxicity using freshly isolated rat hepatocytes. This intact cellular system provides an integrated metabolic system that includes both activation and deactivation mechanisms for BD metabolites. Stereochemical differences in cytotoxicity were observed with enantiomers of both the monoxide and the bisoxide of BD. (R)-BMO was more toxic to rat hepatocytes than (S)-BMO. This could reflect interactions with other chiral enzymes in the cell such as epoxide hydrolase and glutathione-S-transferase. The (meso)-BBO enantiomer was more toxic to rat hepatocytes than either the (S:S) or (R:R) configurations of BBO, while (R:R)-BBO was consistently the least toxic enantiomer in rat hepatocytes. Although we have characterized species differences in metabolism, whether species differences in the cytotoxicity of BD metabolites exist between mouse and rat hepatocytes remains to be elucidated. To summarize, we have shown multiple stereochemical differences between species involving the biotransforma-
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