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Chem. Res. Toxicol. 1997, 10, 672-676
Aflatoxin B1 8,9-Epoxide Hydrolysis in the Presence of Rat and Human Epoxide Hydrolase William W. Johnson,† Hiroshi Yamazaki,‡ Tsutomu Shimada,‡ Yune-Fang Ueng,†,§ and F. Peter Guengerich*,† Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University, School of Medicine, Nashville, Tennessee 37232-0146, and Osaka Prefectural Institute of Public Health, Osaka 537, Japan Received December 20, 1996X
Aflatoxin B1 (AFB1) must be activated to the electrophilic AFB1 exo-8,9-epoxide to be genotoxic and carcinogenic. A role for epoxide hydrolase in detoxication has been suggested but never directly addressed. In light of recent studies determining the instability of AFB1 exo-8,9-epoxide in H2O, a role for epoxide hydrolase appears dubious. Rat liver or recombinant rat epoxide hydrolase provided an enhancement to the already fast hydrolysis rate of up to 22%. Purified human epoxide hydrolase provided no detectable enhancement to the rate of chemical hydrolysis. Some reduction in the genotoxicity of AFB1 was observed when the ratio of rat epoxide hydrolase to cytochrome P450 was high (∼50-fold). An 80-fold excess of human epoxide hydrolase over cytochrome P450 only produced an effect of ∼25% inhibition. It appears, therefore, that there is little evidence to support a role for epoxide hydrolase in the detoxication of AFB1.
Introduction
Scheme 1. Hydrolysis of AFB1 8,9-Epoxide
The contribution of enzymes to the detoxication of carcinogens has been frequently and directly shown (1, 2). One such classic carcinogen-detoxicating enzyme is epoxide hydrolase, an efficient catalyst with a broad specificity for epoxides of lipophilic xenobiotics (3, 4). Hence, the electrophilic center is rendered inocuous. Variations in quantities of xenobiotic-metabolizing enzymes can have dramatic effects in influencing the level of cancer from chemical carcinogens, at least in experimental animal models (5). However, effects of genetic polymorphism studied within the framework of epidemiology are often controversial. Aflatoxin B1 (AFB1)1 is a very potent carcinogen contributing to human liver cancer in some parts of the world (6, 7). Its mechanism of genoxicity involves epoxidation, primarily by P450 3A4 (8, 9), and reaction of the exo isomer with the N7 atom of Gua in DNA (10, 11). The endo isomer, a minor product, is much more stable and remarkably less genotoxic (11). We have recently determined the reaction rate of the exo isomer with H2O to be 0.6 s-1 (t1/2 ) 1 s) at 25 °C (12). In another study, we have determined that the reactivity of AFB1 exo-8,9epoxide with DNA is surprisingly high with a maximum rate of 42 s-1 (13). In the context of AFB1 exo-8,9-epoxide detoxication, it is in order to consider the role of epoxide hydrolase. A few indirect approaches have been attempted (14). However, in light of the instability of AFB1 exo-8,9-epoxide (12), epoxide hydrolase would have to show great rate
enhancement over the high rate of nonenzymatic hydrolysis in order to sanction a role for the enzyme toward this carcinogen. Recently McGlynn et al. (15) measured the frequency of a mutant epoxide hydrolase allele (Tyr113His) and also AFB1-albumin adducts in individuals in China and Ghana and made comparisons with incidence of hepatocellular carcinoma. They concluded that the mutant allele was significantly over-represented in patients with the cancer. Surprisingly, they also reported a higher level of AFB1-albumin adducts in the individuals with the epoxide hydrolase allelic variation and concluded that epoxide hydrolase had a role in AFB1 8,9-epoxide more significant than that of GSH transferase M1 (15). The objective of this study was to directly assess the effect of epoxide hydrolase on the hydrolysis of AFB1 8,9-epoxide.
* Author to whom correspondence should be addressed. Telephone: (615) 322-2261. Fax: (615) 322-3141. E-mail: guengerich@ toxicology.mc.vanderbilt.edu. † Vanderbilt University School of Medicine. ‡ Osaka Prefectural Institute of Public Health. § Current address: National Research Institute of Chinese Medicine, 155-1, Li-Nong Street, Sec 2, Taipei 11221, Taiwan, Republic of China. X Abstract published in Advance ACS Abstracts, May 15, 1997. 1 Abbreviations: AFB , aflatoxin B ; b , cytochrome b ; STG, steri1 1 5 5 gmatocystin.
S0893-228x(96)00209-3 CCC: $14.00
Experimental Procedures Chemicals. AFB1 was purchased from Sigma Chemical Co. (St. Louis, MO). AFB1 8,9-epoxide was synthesized using dimethyldioxirane (16), and the exo isomer was purified by recrystallization (10). Phenanthrene 9,10-oxide was a gift of Prof. R. N. Armstrong (Vanderbilt University). Enzymes. Rat liver epoxide hydrolase was prepared as described elsewhere (17) (“A” fraction) and used in some assays.
© 1997 American Chemical Society
Epoxide Hydrolase and Aflatoxin B1 Most of the studies with rat epoxide hydrolase were done with a recombinant enzyme (unmodified sequence) produced in Escherichia coli [BL21(DE3) cells] using a pET20 plasmid vector.2 Epoxide hydrolase was isolated from each of two human liver (designated “HL”) samples, HL96 and HL105, using modifications of previously described techniques (18). All epoxide hydrolase preparations were electrophoretically homogeneous, and the specific activities of the rat liver, recombinant rat liver, and HL96 and HL105 human liver epoxide hydrolase preparations for phenanthrene 9,10-oxide hydrolysis (4, 19) were 0.49, 0.70, 0.46, and 0.36 s-1, respectively. Recombinant P450 3A4 was expressed in E. coli and purified as described elsewhere (20, 21). Rabbit liver NADPH-P450 reductase (22, 23) and cytochrome b5 (b5) (24, 25) were purified as described previously. Kinetics. Stopped-flow measurements utilized an Applied Photophysics SX-17MV apparatus (Leatherhead, U.K.). The reactions were initiated by rapidly mixing AFB1 exo-8,9-epoxide in anhydrous (CH3)2CO with buffered solutions in a volumetric ratio of 1:10 (12). umu Assay. P450-dependent activation of AFB1 to reactive metabolites that cause induction of umu gene expression in the tester strain Salmonella typhimurium TA1535/pSK1002 was determined in reconstituted P450 3A4-containing systems as described previously (26). The standard reconstituted P450 3A4 system was composed of 10 nM purified recombinant P450 3A4, 20 nM b5, 20 nM NADPH-P450 reductase, 10 µg of a phospholipid mixture consisting of L-R-dilauroyl-sn-glycero-3-phosphocholine, L-R-dioleoyl-sn-glycero-3-phosphocholine, and L-Rphosphatidyl-L-serine (1:1:1, w/w/w) mL-1, 10 mM MgCl2, 0.25 mM sodium cholate, and 5 µM AFB1 or STG (27, 28). When indicated, epoxide hydrolase (0.05-0.8 µM) was included in the reaction mixture. Reactions were performed at 37 °C for 2 h and terminated by cooling the mixtures on ice; umu gene expression was monitored by measuring β-galactosidase activities by the method of Miller (29).
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Figure 1. Reaction kinetics of AFB1 exo-8,9-epoxide hydrolysis (37 °C) in the presence of epoxide hydrolase. The lower exponential curve is a fit to the data from hydrolysis of AFB1 exo-8,9-epoxide (14 µM) in the presence of 10 mM potassium phosphate buffer (pH 7.0) (0.97 ( 0.01 s-1). The slightly faster trace (upper) is for the reaction in the presence of 3 µM rat epoxide hydrolase (1.10 ( 0.01 s-1). The difference in rate is 0.13 s-1. Lines are drawn as fits to first-order kinetics by computer software (Applied Photophysics). Table 1. Observed Hydrolysis Rates of AFB1 exo-8,9-Epoxide in the Presence of Rat and Human Epoxide Hydrolase [epoxide], µM 14 14 7 7 7 7 14 14
Results Enzyme Kinetics. The rate of spontaneous AFB1 exo8,9-epoxide hydrolysis (k0) has been determined using a stopped-flow apparatus and monitoring changes in absorbance and fluorescence (12). This method was employed to assess the rate enhancement due to the enzyme epoxide hydrolase or the rate of catalysis via the enzyme. Since the observed rate is the sum of the individual single exponential rates (kobs ) k0 + kenz),3 any increment of rate elevation is the velocity via the enzyme (kenz) (Figure 1). This is quantified by subtracting the spontaneous rate under the same conditions from the observed rate in the presence of enzyme, i.e., kobs - k0 ) kenz. If the experiment is performed sufficiently above the Km of the enzyme, then the calculated kenz approximates the enzyme kcat. The addition of 3 µM rat epoxide hydrolase yielded a modest yet detectable increase in the hydrolysis rate of AFB1 exo-8,9-epoxide (present at 14 µM) (Figure 1). The error in these determinations is ∼1%, and the enhancement or difference in the rates is about 0.13 s-1, which represents the contribution by the enzyme (13%). These experiments were done at 37 °C and are the first time that the rate at physiological temperatures has been determined, i.e., 0.97 s-1. The above approach was used to determine the effects under a variety of conditions (Table 1). With 14 µM AFB1 2 Laughlin, L. T., and Armstrong, R. N. Manuscript in preparation. Purification involved general procedures established elsewhere. 3 This relationship is valid when the on and off rates are sufficiently faster than the first catalytic step (30), if the substrate is in excess over the enzyme (vide infra), or if the substrate concentration is 90% exo, a ratio of ∼1:1 was obtained as judged by analysis of the GSH conjugation products (10, 32). Experiments monitoring absorbance at 386 and 350 nm exhibit the typical increase and decrease, respectively, seen with the exo isomer (12), but the endo isomer did not show a fluorescence increase after the exo isomer reaction was complete. The data are biexponential because of the distinctly different hydrolysis rates of the isomers, and the fit to the data is shown (Figure 3). The first phase is the hydrolysis of the exo isomer with the typical rate of ∼0.67 s-1 (with a large error due to limited data), and the second phase results from the endo isomer. The rate of hydrolysis of the endo isomer is 0.0088 s-1 (at pH 6.8), 80-fold less than that of the exo isomer. AFB 8,9-dihydrodiol (the hydrolysis product) will form a dialdehyde under basic conditions (pKa 8.2) (12), and this compound exhibits an increased absorbance at 395 nm which could confound the conclusions, although the results at 350 nm agree with the results at 386 nm. In this regard, if there were any contribution from dialdehyde formation, then repeating the experiment at lower
Johnson et al.
Figure 3. Time course of AFB1 endo-8,9-epoxide hydrolysis. A 1:1 mixture of endo and exo isomers was prepared by enrichment of the endo isomer by fractional crystallization, and the absorbance change was monitored using the stopped-flow apparatus. The data are clearly double exponential with the first phase exhibiting a rate of 0.67 ( 0.06 s-1 and an amplitude of 0.0083, which represents the hydrolysis of the exo isomer (12). The second phase exhibits a rate of 0.0088 ( 0.0001 s-1 and an amplitude of 0.007, which represents the hydrolysis of the endo isomer.
Figure 4. Effects of epoxide hydrolase on genotoxicity of AFB1 or STG activated by recombinant P450 3A4 in reconstituted monooxygenase systems in the presence of S. typhimurium TA1535/pSK1002. The standard incubation mixture was used for the activation of AFB1 or STG as described in Experimental Procedures. The system was used in the absence (open bars) or included a 0.05 µM concentration of rat epoxide hydrolase (solid bars) or human epoxide hydrolase isolated from sample HL96 (/ / slashed bars) or HL105 (\\ slashed bars). The results are presented as means of duplicate experiments.
pH would slow the rate. However, the rate increases, consistent with acid catalysis of the epoxide hydrolysis. Hence, this is the direct determination of the hydrolysis rate of the endo isomer. In order to determine the effect of epoxide hydrolase on the rate of hydrolysis of the endo epoxide, the experiment was repeated in the presence of enzyme, and the rate was slightly slower. This effect was seen also by adding bovine serum albumin (1.0 mg mL-1) to the reaction mixture. Therefore, the attenuation appears to be a nonspecific effect of the presence of protein. Consequently, AFB1 endo-8,9-epoxide seems not to be a substrate of rat epoxide hydrolase. Genotoxicity Assays. In order to characterize the effect of epoxide hydrolase on DNA modification, an SOS response-based umu genotoxicity test was employed. Binding of activated carcinogens to DNA evokes the SOS response, activation of the umu region of the plasmid, and concomitant production of a measurable chromophore in the system (26). A system was set up to generate a low concentration of AFB1 exo-8,9-epoxide continuously, through the action of a recombinant P450 3A4 system. The initial experiments indicated that rat epoxide hydrolase reduced the detected response though about one-third remained, even with a very high ratio of epoxide hydrolase to P450. Human epoxide hydrolase (sample HL96) reduced the response slightly; another
Epoxide Hydrolase and Aflatoxin B1
Figure 5. Effects of epoxide hydrolase on genotoxicity of cytochrome P450-activated AFB1. The indicated concentration of epoxide hydrolase was included in a system containing 10 nM P450 3A4: heat-inactivated rat epoxide hydrolase (100 °C for 2 min) (O), rat epoxide (b), human liver epoxide hydrolase HL96 (0), and human liver epoxide hydrolase HL105 (9).
preparation (sample HL105) resulted in no detectable change. Similar results were seen with STG, a biosynthetic precursor of AFB1. Further studies were done with varying concentrations of epoxide hydrolase (Figure 5) and indicate an effect that is roughly hyperbolic. However, the human epoxide hydrolase from sample HL96 showed only a slight effect, less than exibited in Figure 4, and that from HL105 again showed no effect. These results are consistent with the pattern seen in the enzyme kinetic experiments, in which rat epoxide hydrolase produced a modest hydrolysis rate enhancement and the two human enzymes produce no detectable rate change. The effect that catalysis via epoxide hydrolase contributes to formation of AFB1-DNA conjugates was tested in other ways. The amount of (N7-guanyl) AFB1 adduct formed with DNA was measured after adding AFB1 exo8,9-epoxide to solutions of DNA (2 mg mL-1) with various amounts of epoxide hydrolase present. The results indicated that there is a slight trend toward less conjugation with DNA at 20-40 µM epoxide hydrolase (results not shown). The precision for these measurements is such that the differences are nearly within the experimental error. Moreover, bovine serum albumin and heatdenatured rat epoxide hydrolase also yielded a slight reduction in the adduct formation.
Discussion With the advent of assertions for the role of the detoxicating enzyme epoxide hydrolase in metabolism of AFB1 exo-8,9-epoxide based on epidemiology studies (15), we sought to investigate the contribution of the enzyme. In light of the comparatively fast chemical hydrolysis rate of AFB1 exo-8,9-epoxide recently determined (12), it is not surprising that purified rat epoxide hydrolase provides little enhancement to the rate, as judged by direct measurements. Purified human epoxide hydrolase provided no detectable rate enhancement, and the endo epoxide appears not to be a substrate for either enzyme. The two samples of purified human liver epoxide hydrolase were shown to have catalytic activity toward the model substrate phenanthrene 9,10-oxide. Therefore, the His113 allelic variant (with putative 40% reduced activity) (33) is probably not relevant in this determination. The genotoxicity assays showed little effect until the human epoxide hydrolase was in large excess over the activating enzyme (P450 3A4), although rat epoxide
Chem. Res. Toxicol., Vol. 10, No. 6, 1997 675
hydrolase showed a somewhat greater effect. This result is consistent with the lack of hydrolysis rate enhancement offered by the human enzyme. The slight effect resulting from the very high ratio of epoxide hydrolase to cytochrome P450 is difficult to interpret but may constitute a threshold for optimum binding affinity and certainly ensures a great surplus of accessible active sites at all times. As the hydrolysis product of AFB1 exo-8,9-epoxide, AFB 8,9-dihydrodiol, is postulated to be responsible for formation of protein adducts (34) [via formation of the electrophilic dialdehyde (12, 34)], examination of AFB1-albumin adducts may not be an informative approach for studying epoxide hydrolase contributions to metabolism. Consequently, conclusions based on measurement of levels of AFB1-albumin adducts must be considered unvalidated, and there is no real evidence to support the view that the 113 codon allele is really an issue. Further, examination of the original literature regarding the Tyr113His allelic variant indicates that the apparent difference in catalytic activity is only the result of differences in expression levels in the cell system used (33). However, GSH transferase M1-1 evidently provides a real potential for contribution to metabolism of the AFB1 exo-8,9epoxide (35, 36). This biochemical evidence suggests that epidemiology studies may not be definitive at this point. The large difference in hydrolysis rates between the exo and endo isomers (80-fold) is of interest. The difference is even greater than observed in previous studies on methanolysis of the epoxides (11). However, a satisfying chemical explanation for the difference is still not available. In conclusion, it appears that the enzyme epoxide hydrolase probably does not significantly influence human genotoxicity (or hepatocarcinogenicity) resulting from the activated mycotoxin AFB1 exo-8,9-epoxide, since the enzyme is unable to significantly hasten the hydrolysis rate.
References (1) Miller, E. C., and Miller, J. A. (1981) Searches for ultimate chemical carcinogens and their reactions with cellular macromolecules. Cancer 47, 2327-2345. (2) Guengerich, F. P. (1992) Metabolic activation of carcinogens. Pharmacol. Ther. 54, 17-61. (3) Oesch, F. (1973) Mammalian epoxide hydrases: inducible enzymes catalysing the inactivation of carcinogenic and cytotoxic metabolites derived from aromatic and olefinic compounds. Xenobiotica 3, 305-340. (4) Hammock, B. D., Grant, D. F., and Storms, D. H. (1997) Epoxide hydrolases. In Biotransformation, Vol. 3, Comprehensive Toxicology (Guengerich, F. P., Ed.) pp 283-305, Elsevier Science Ltd., Oxford. (5) Nebert, D. W. (1989) The Ah locus: genetic differences in toxicity, cancer, mutation, and birth defects. Crit. Rev. Toxicol. 20, 153174. (6) Busby, W. F., and Wogan, G. N. (1984) Aflatoxins. In Chemical Carcinogens (Searle, C. E., Ed.) pp 945-1136, American Chemical Society, Washington, DC. (7) Qian, G. S., Ross, R. K., Yu, M. C., Yuan, J. M., Gao, Y. T., Henderson, B. E., Wogan, G. N., and Groopman, J. D. (1994) A follow-up study of urinary markers of aflatoxin exposure and liver cancer risk in Shanghai, People’s Republic of China. Cancer Epidemiol. Biomarkers Prev. 3, 3-10. (8) Shimada, T., and Guengerich, F. P. (1989) Evidence for cytochrome P450NF, the nifedipine oxidase, being the principal enzyme involved in the bioactivation of aflatoxins in human liver. Proc. Natl. Acad. Sci. U.S.A. 86, 462-465. (9) Ueng, Y.-F., Shimada, T., Yamazaki, H., and Guengerich, F. P. (1995) Oxidation of aflatoxin B1 by bacterial recombinant human cytochrome P450 enzymes. Chem. Res. Toxicol. 8, 218-225.
676 Chem. Res. Toxicol., Vol. 10, No. 6, 1997 (10) Raney, K. D., Coles, B., Guengerich, F. P., and Harris, T. M. (1992) The endo-8,9-epoxide of aflatoxin B1: a new metabolite. Chem. Res. Toxicol. 5, 333-335. (11) Iyer, R., Coles, B., Raney, K. D., Thier, R., Guengerich, F. P., and Harris, T. M. (1994) DNA adduction by the potent carcinogen aflatoxin B1: mechanistic studies. J. Am. Chem. Soc. 116, 16031609. (12) Johnson, W. W., Harris, T. M., and Guengerich, F. P. (1996) Kinetics and mechanism of hydrolysis of aflatoxin B1 exo-8,9-oxide and rearrangement of the dihydrodiol. J. Am. Chem. Soc. 118, 8213-8220. (13) Johnson, W. W., and Guengerich, F. P. (1997) Reaction of aflatoxin B1 exo-8,9-epoxide with DNA: kinetic analysis of covalent binding and DNA-induced hydrolysis. Proc. Natl. Acad. Sci. U.S.A., in press. (14) Ch’ih, J. J., Lin, T., and Devlin, T. M. (1983) Activation and deactivation of aflatoxin B1 in isolated rat hepatocytes. Biochem. Biophys. Res. Commun. 110, 668-674. (15) McGlynn, K. A., Rosvold, E. A., Lustbader, E. D., Hu, Y., Clapper, M. L., Zhou, T., Wild, C. P., Xia, X.-L., Baffoe-Bonnie, A., OforiAdjei, D., Chen, G.-C., London, W. T., Shen, F.-M., and Buetow, K. H. (1995) Susceptibility to hepatocellular carcinoma is associated with genetic variation in the enzymatic detoxification of aflatoxin B1. Proc. Natl. Acad. Sci. U.S.A. 92, 2384-2387. (16) Baertschi, S. W., Raney, K. D., Stone, M. P., and Harris, T. M. (1988) Preparation of the 8,9-epoxide of the mycotoxin aflatoxin B1: the ultimate carcinogenic species. J. Am. Chem. Soc. 110, 7929-7931. (17) Guengerich, F. P., Wang, P., Mitchell, M. B., and Mason, P. S. (1979) Rat and human liver microsomal epoxide hydratase. Purification and evidence for the existence of multiple forms. J. Biol. Chem. 254, 12248-12254. (18) Guengerich, F. P., Wang, P., Mason, P. S., and Mitchell, M. B. (1979) Rat and human liver microsomal epoxide hydratase. Immunological characterization of various forms of the enzyme. J. Biol. Chem. 254, 12255-12259. (19) Lacourciere, G. M., and Armstrong, R. N. (1993) The catalytic mechanism of microsomal epoxide hydrolase involves an ester intermediate. J. Am. Chem. Soc. 115, 10466-10467. (20) Gillam, E. M. J., Baba, T., Kim, B.-R., Ohmori, S., and Guengerich, F. P. (1993) Expression of modified human cytochrome P450 3A4 in Escherichia coli and purification and reconstitution of the enzyme. Arch. Biochem. Biophys. 305, 123-131. (21) Guengerich, F. P., Martin, M. V., Guo, Z., and Chun, Y.-J. (1996) Purification of recombinant human cytochrome P450 enzymes expressed in bacteria. Methods Enzymol. 272, 35-44. (22) Yasukochi, Y., and Masters, B. S. S. (1976) Some properties of a detergent-solubilized NADPH-cytochrome c (cytochrome P-450) reductase purified by biospecific affinity chromatography. J. Biol. Chem. 251, 5337-5344. (23) Guengerich, F. P. (1994) Analysis and characterization of enzymes. In Principles and Methods of Toxicology, 3rd ed. (Hayes, A. W., Ed.) pp 1259-1313, Raven Press, Ltd., New York. (24) Imaoka, S., and Funae, Y. (1986) Ion-exchange high-performance liquid chromatography of membrane-bound protein cytochrome P-450. J. Chromatogr. 375, 83-90.
Johnson et al. (25) Shimada, T., Misono, K. S., and Guengerich, F. P. (1986) Human liver microsomal cytochrome P-450 mephenytoin 4-hydroxylase, a prototype of genetic polymorphism in oxidative drug metabolism. Purification and characterization of two similar forms involved in the reaction. J. Biol. Chem. 261, 909-921. (26) Shimada, T., Oda, Y., Yamazaki, H., Mimura, M., and Guengerich, F. P. (1994) SOS function tests for studies of chemical carcinogenesis in Salmonella typhimurium TA 1535/pSK1002, NM2009, and NM3009. In Methods in Molecular Genetics, Vol. 5, Gene and Chromosome Analysis (Adolph, K. W., Ed.) pp 342-355, Academic Press, Orlando, FL. (27) Imaoka, S., Imai, Y., Shimada, T., and Funae, Y. (1992) Role of phospholipids in reconstituted cytochrome P450 3A forms and mechanism of their activation of catalytic activity. Biochemistry 31, 6063-6069. (28) Shimada, T., Iwasaki, M., Martin, M. V., and Guengerich, F. P. (1989) Human liver microsomal cytochrome P-450 enzymes involved in the bioactivation of procarcinogens detected by umu gene response in Salmonella typhimurium TA1535/pSK1002. Cancer Res. 49, 3218-3228. (29) Miller, J. H. (1972) Experiments in Molecular Genetics, pp 352355, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. (30) Tzeng, H.-F., Laughlin, L. T., Lin, S., and Armstrong, R. N. (1996) The catalytic mechanism of microsomal epoxide hydrolase involves reversible formation and rate-limiting hydrolysis of the alkyl-enzyme intermediate. J. Am. Chem. Soc. 118, 9436-9437. (31) Fersht, A. (1985) Enzyme Structure and Mechanism, 2nd ed., p 102, W. H. Freeman, W. H. (32) Raney, K. D., Meyer, D. J., Ketterer, B., Harris, T. M., and Guengerich, F. P. (1992) Glutathione conjugation of aflatoxin B1 exo and endo epoxides by rat and human glutathione S-transferases. Chem. Res. Toxicol. 5, 470-478. (33) Hassett, C., Aicher, L., Sidhu, J. S., and Omiecinski, C. J. (1994) Human microsomal epoxide hydrolase: genetic polymorphism and functional expression in vitro of amino acid variants. Hum. Mol. Genet. 3, 421-428. (34) Sabbioni, G., Skipper, P. L., Bu¨chi, G., and Tannenbaum, S. R. (1987) Isolation and characterization of the major serum albumin adduct formed by aflatoxin B1 in vivo in rats. Carcinogenesis 8, 819-824. (35) Langoue¨t, S., Coles, B., Morel, F., Becquemont, L., Beaune, P. H., Guengerich, F. P., Ketterer, B., and Guillouzo, A. (1995) Inhibition of CYP1A2 and CYP3A4 by oltipraz results in reduction of aflatoxin B1 metabolism in human hepatocytes in primary culture. Cancer Res. 55, 5574-5579. (36) Johnson, W. W., Ueng, Y.-F., Mannervik, B., Widersten, M., Hayes, J. D., Sherratt, P. J., Ketterer, B., and Guengerich, F. P. (1997) Conjugation of highly reactive aflatoxin B1 8,9-exo-epoxide catalyzed by rat and human glutathione S-transferases: estimation of kinetic parameters. Biochemistry 36, 3056-3060.
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