Chem. Res. Toxicol. 1993,6,669-673
669
Chloroperoxidase-Mediated Oxidation of 1,3-Butadiene to 3-Butenal, a Crotonaldehyde Precursor Renee J. Duescher and Adnan A. Elfarra' Department of Comparative Biosciences and Environmental Toxicology Center, University of Wisconsin, Madison, Wisconsin 53706 Received June 1,199P
Previously, we have shown that 1,3-butadiene,a rodent and possibly a human carcinogen, can be oxidized by chloroperoxidaseHzO2 a t pH 7.4 to yield the potent mutagens, butadiene monoxide and crotonaldehyde. Because the crotonaldehydelbutadiene monoxide ratio from reactions with chloroperoxidase was higher than that obtained from reactions with myeloperoxidase or cytochrome P450 enzymes, in the present study, the chloroperoxidase reaction was further investigated in an attempt to define optimal conditions for catalysis and to possibly obtain direct evidence for the formation of the crotonaldehyde precursor 3-butenal in these incubations. The results showed that butadiene monoxide and crotonaldehyde formation was optimal at pH 6.0. As opposed to incubations carried out a t pH 7.4, GC analyses of incubations carried out a t pH 4.5, 5.0, and 6.0 demonstrated the presence of a new peak which had a retention time different from that of butadiene monoxide and crotonaldehyde. The new peak was identified as 3-butenal by comparison of its retention time and mass spectrum with those of reference material. Evidence for 3-butenal being a precursor of crotonaldehyde was obtained by the findings that 3-butenal was not simply a decomposition product of butadiene monoxide or crotonaldehyde under the incubation or assay conditions, and that the 3-butenallcrotonaldehyde ratio decreased when the incubation time was increased between 5 and 30 min or when the incubation temperature was increased between 10 and 45 O C . The combined 3-butenal and crotonaldehyde concentrations remained constant a t the various incubation temperatures. Furthermore, 3-butenal conversion to crotonaldehyde was faster a t pH 7.4, compared to pH 6.0. Thus, the obtained results provide conclusive evidence for 3-butenal being an oxidative metabolite of L3-butadiene and suggest that any 3-butenal that may be formed in vivo after animal or human exposure to 1,3-b;tadiene is likely to be readily Gutomerized to crotonaldehyde.
Introduction Occupational exposure to 1,3-butadiene,a petrochemical used extensively in the industrial production of rubbers and plastics, has been implicated in the development of human cancer (1-3). In vivo and in vitro studies in rats and mice have suggested that 1,3-butadiene metabolism to yield the mutagenic and carcinogenic metabolites butadiene monoxide (BM', Figure 1)and crotonaldehyde (CA) plays an important role in the mechanism(s)of 1,3butadiene-induced toxicity (4-1 3). Studies conducted in our laboratory have also shown that, in addition to cytochrome P450s, enzymes such as myeloperoxidase and chloroperoxidase,a nonmammalianenzyme which appears to have a cytochrome P450-like active site (14-18), are capable of catalyzing 1,3-butadiene oxidation to BM and CA (5-9). The inability to detect methyl vinyl ketone as a lY3-butadienemetabolite provided evidence for a regioselective transfer of oxygen to a terminal carbon rather than to a central carbon of lY3-butadiene(5,6). The intermediates formed may undergo either a ring closure to form BM or a hydrogen shift to form 3-butenal which tautomerizes to yield CA (Figure 1). Evidence for the common intermediate(s) mechanism of 1,3-butadiene oxidation to BM and CA was provided by the findings that BM and CA were not simply decomposition products
* To whom correspondence should be addressed at the Department of Comparative Biosciences, University of Wisconsin School of Veterinary Medicine, 2015 Linden Drive West, Madison, WI 53706. * Abstract published in Advance ACS Abstracts, August 15, 1993. Abbreviations: BM, butadiene monoxide; CA, crotonaldehyde. 0893-228~/93/2706-0669$04.00/0
of each other under the incubation and assay conditions, and that, in lY3-butadieneincubations with these hemoproteins, the CA/BM ratios remained constant over time (5-9). Evidence for 3-butenal tautomerization to CA was provided by the finding that 3-buten-1-01, an alternative precursor of 3-butenal, was readily oxidized to CA under incubation conditions similar to those used for 1,3butadiene (5). Whereas cytochrome P4508, myeloperoxidase, and chloroperoxidase were capable of oxidizing l,&butadiene to BM and CA, the magnitude of the obtained BMICA ratio was variable depending on the enzyme (5-9). In experiments with mouse or human liver microsomes, or with cDNA-expressed human cytochrome P45Os, the obtained BM/CA ratios at pH 7.4 were nearly 5011, whereas with human myeloperoxidase and Caldariomyces fumago chloroperoxidase, the obtained BM/CA ratios were nearly 2011 and 311, respectively. Since these studies have shown that chloroperoxidase, an enzyme that displays catalytic activity over a wide range of incubation pH and temperature conditions (14,19-211,was an effective catalyst of CA formation, in the present study, the chloroperoxidasemediated oxidation of l,&butadiene to BM and CA was further investigated. Incubations at different pH and temperature conditions were carried out in an attempt to determine optimal conditions for catalysis and to possibly obtain direct evidence for 3-butenal formation as a consequence of a possible suppression of its tautomerization to CA at some of these conditions. The results provide conclusive evidence for 3-butenal formation and 1993 American Chemical Society
670 Chem. Res. Toxicol., Vol. 6, No. 5, 1993
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Figure 1. Proposed scheme for the oxidation of l,&butadiene by cytochrome P450s, myeloperoxidase, and chloroperoxidase: I, l,3-butadiene; II, B M 111, 3-butenal; IV, CA. Because the source of the oxygen in these reactions was not rigorously determined, other oxygen-transfer reactions (14-21) cannot be ruled out.
support our hypothesis that 3-butend is an intermediate in 1,3-butadiene oxidation to CA.
Experimental Procedures Materials. BM, CA, 3-buten-1-01,methyl vinyl ketone, and chromium(V1) oxide were obtained from Aldrich Chemical Co. (Milwaukee, WI). 1,3-Butadiene (purity 99%) was purchased from Air Products (Chicago, IL). Toluene, ethyl ether, and o-xylene were obtained from American Burdick & Jackson (Muskegon,MI). Chloroperoxidase (approximately 1250 units/ mg of protein from Caldariomyces fumago),monochlorodimedon, and 30% Ha02 were purchased from Sigma Chemical Co. (St. Louis, MO). The enzymaticactivity of chloroperoxidase,assessed spectrophotometrically by monitoring the formation of dichlorodimedon from monochlorodimedon and H202 at 278 nm at 25 OC (6,22),was found to be nearly 80% of the activity reported by Sigma; chloroperoxidase units were not corrected. All other chemicals were of the highest grade commercially available. Caution: 1,3-Butadiene,BM, and CA are known mutagens and carcinogens in laboratory animals and must be handled using proper safety measures. Metabolism of 1,3-Butadieneby Chloroperoxidase. Incubations were carried out as previously described (5). Briefly, typical reactions were carried out by incubating 1,3-butadiene with chloroperoxidase [0.25 mL of 0.2-2.0 units/mL stock solution of the enzyme in phosphate buffer (0.1 M KHzPO,, 0.15 M KC1, 1.5 mM EDTA; pH 6.0)] with 0.25 mL of 0.2-2.0 mM H202 in the same buffer in vials capped with Teflon-lined septa at room temperature with constant magneticstirring. For all experiments, the 1,3-butadiene pressure in the vials was monitored and maintained between 48 and 52 cmHg. Experiments designed to determine the effect of pH variation on chloroperoxidasedependent 1,3-butadiene oxidation were carried out using the phosphate buffer described above adjusted to the proper pH with potassium hydroxide, whereas experiments designed to
Duescher and Elfarra determine the effect of temperature variation on 1,3-butadiene oxidation were carried out at pH 6.0. At the end of the enzymatic incubation (0-45 min), the reaction was terminated by stopping the flow of 1,3-butadiene, immediately removing the vials from the water bath, and cooling them slightly in a dry ice/acetone bath. Incubation mixtures were then extracted with toluene (0.25 mL), and aliquots of the toluene extracts were analyzed by capillary GC with a flame ionization detector as described previously (5). Quantification of BM and CA was done by comparison of the peak height to standard curves (r > 0.99) obtained with reference materials. Quantification of 3-butenal was done by comparison of the 3-butenal peak height to that of the CA standard since commercial 3-butenal was not available and attempta to obtain a pure sample of 3-butenal by synthesis (23,24)were hampered by its rapid tautomerization to CA. The effect of pH on 3-butenal conversion to CA was studied by incubating 0.25 mL of a 2.0 unita/mL solution of chloroperoxidase with 0.25 mL of 2 mM H202 at 25 OC at pH 6.0 for 10 min to form 3-butenal. The vials were opened, and then 2 N sodium hydroxide was added to adjust the final pH to 7.4. The vials were capped again and allowed to sit at room temperature for 10,20, or 30 min before being analyzed by GC for 3-butenal and CA concentrations, as described above. In this experiment, control vials were processed in the same way except the pH was maintained at 6.0. For identification of 3-butenal by GC and GC-MS, however, reference material was synthesized by 3-buten-1-01oxidation by either one of the following two methods. In the first method (23),Cr09 solution was prepared by mixing 3 mL of HzO with 0.3 mL of HzSOl and adding CrOs (0.1 g, 1mmol). Acetone (7 mL) was then added, and the solution was placed on ice. 3-Butenl-ol (0.13 mL, 1.5 mmol) was stirred into the solution, and the mixture was allowed to stand for 5-30 min. The reaction mixture was extracted with 15mL of toluene, and the toluene extract was washed twice with 1 N H2SO4 to remove the yellow color and once with H2O and finally dried over MgSO4. In the second method (24),3-buten-1-01 (5 rL) was mixed with 5 mL of HzO2 (10 mM) and chloroperoxidase (5 mL, 30 units/mL) in a 25-mL Erlenmeyer flask at room temperature, and the enzymatic reaction was allowed to proceed for 15 min. The reaction was stopped by cooling slightly in a dry ice/acetone bath followed by extraction with 5 mL of toluene. With either method, the toluene extract was analyzed for 3-butenal by GC as previously described (5),without further purification. Characterization of 3-Butenal by GC-MS. For mass spectral characterization of the 3-butenal GC peak from 3-butenl-ol incubations, incubations were carried out as described above. Vials were extracted with 0.5 mL of ethyl ether, and the ether extracts from 30 vials were combined and evaporated under a nitrogen stream to near dryness. o-Xylene (300 pL) was added, and the solution was analyzed by GC-MS. The instrument used was a Kratos MS25 mass spectrometer with a Carlo Erba GC fitted with a DB-1,30-m, 1.5-pm-film-thicknesscapillarycolumn (Supelco, Bellfonte, PA). The ion source was 300 OC, and the split injector was at 140 "C. The initial oven temperature was held at 35 "C for 5 min and then raised at 25 OC/min to a final temperature of 225 "C for 2 min. Theretention time for 3-butenal was 2.4 min. For mass spectral characterization of the 3-butenal formed in 1,3-butadiene incubations, the enzymatic reaction and GC-MS analysis was carried out as described above except that 40 units/mL chloroperoxidase was used and the ether extracts from 70 vials were combined.
Results and Discussion Whereas previous incubations of 1,3-butadiene with chloroperoxidase and hydrogen peroxide at pH 7.4resulted in the detection of only BM and CA (5),incubations carried out a t pH 4.5,5.0, or 6.0 consistently showed the presence of a new additional peak upon GC analysis (Figure 2). The new peak had a retention time similar to that of reference 3-butenal; retention times for 3-butenal, BM, and CA were 2.06, 2.31, and 2.86 min, respectively (Figure 2). The
Chem. Res. Toxicol., Vol. 6, No. 5, 1993 671
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Figure 2. Typical gas chromatograms using flame ionization detection (FID)of 1,3-butadieneoxidation by chloroperoxidase: (A) - chloroperoxidase, (B) - HzOz, (C)+ chloroperoxidase and H202. For additional incubation and assay conditions, see the Experimental Procedures.
3-butenal peak was not simply a decomposition product of BM or CA because when reference materials of both compounds (120 pM BM and 34 pM CA) were incubated with chloroperoxidase (30 units) and H202 (10 mM) for 30 min a t the various temperatures and pH conditions, no 3-butenal was detected. Further evidence for the identity of the new peak was obtained by GC-MS (Figure 3); the fragmentation pattern of the 3-buteanl peak obtained from l,&butadiene incubations was similar to that obtained with the reference material obtained by the oxidation of 3-buten-1-01 by either CrOs or chloroperoxidase-HzO2. These spectra also match the fragmentation pattern listed in the literature for 3-butenal(25). Furthermore, 3-butenal, BM, and CA were not detected when either chloroperoxidaseor HzOz was omitted from the incubation mixture (Figure 2). In an effort to determine optimal incubation conditions for overall metabolite formation and to possibly obtain direct evidence for 3-butenal formation, 1,3-butadiene incubations were carried out with chloroperoxidase at different pH and temperature conditions and at different enzyme and hydrogen peroxide concentrations. The total metabolite concentration was found to be highly dependent on the incubation pH with an optimum at pH 6.0 (Figure 4). The finding that the combined 3-butenal and CA concentrations at pH 6.0 constituted nearly 30% of the amount of BM formed, which is consistent with the previous report that CA constituted nearly 30% of the amounts of BM detected at pH 7.4(5),provides additional evidence for 3-butenal being the precursor for CA under
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our assay conditions. The inability to detect 3-butenal at pH 7.4 as compared to pH 4.5,5.0,or 6.0 suggests enhanced tautomerization of 3-butenal to CA at pH 7.4. When incubations were carried out at pH 6.0 a t room temperature in the presence of various chloroperoxidase and hydrogen peroxide concentrations, metabolite formation exhibited first-order dependence on the enzyme concentration. The enzymatic activity was, however, inhibited when the hydrogen peroxide concentration was increased beyond an optimal 0.25-0.50mM when the enzyme concentration was maintained a t 0.5 unit/incubation. This inhibition of enyzmatic activity, which is possibly caused by enzyme inactivation by the excesshydrogen peroxide, is consistent with previous findings with other substrates (14). Further evidence for 3-butenal being the preucurser of CA was obtained by carrying out l,&butadiene incubations at various temperatures and by examining the time courses of 3-butenal and CA formation (Figure 5). Incubations carried out at temperatures ranging from 10to 45 "C show a distinctive change in the 3-butenal/CA ratio. At 10 OC, the 3-butenal concentration was nearly 8 times greater than that of CA, whereas at 45 "C, the CA concentration was higher than that of 3-butenal and the 3-butenal/CA ratio was reduced to nearly 1.0/2.5. The findings that the combined 3-butenal and crotonaldehyde concentrations remained constant a t the various temperatures and the 3-butenal/CA ratio decreased when the incubation time was increased from 5 to 30 min (Figure 5) provide additional evidence for 3-butenal being the precursor for CA.
672 Chem. Res. Toxicol., Vol. 6, No. 5, 1993
Duescher and Elfarra
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Figure 4. (A) Effect of pH variation on 1,3-butadieneoxidation to BM, 3-butenal,and CA by chloroperoxidase. Incubationswere carried out using 0.5 unit/incubation chloroperoxidaseand 1mM H202 at room temperature for 45 min. (B)Effect of varying both H202 and chloroperoxidase units in a 1/2 ratio on l,&butadiene oxidation to BM, 3-butenal, and CA. Incubations were carried out at pH 6.0 at room temperature for 15 min. (C) Effect of the H202 concentration on incubations carried out at room temperature with 0.5 unit of chloroperoxidase for 15 min at pH 6.0: BM, closed circles; 3-butenal,closed squares; CA, open squares. Values represent means f SD of the results obtained from three experiments. When the pH of the incubation mixture was adjusted at the end of the enzymatic reaction from pH 6.0 to pH 7.4 and samples were allowed to stand at these pH values at room temperature for 10-30 min, conversion of 3-butenal to CA in these vials was faster a t pH 7.4 than at pH 6.0. The amounts of 3-butenal remaining in the vials at 10,20, and 30 min at pH 7.4 were nearly 76,60, and 47 % of those remaining at pH 6.0, respectively, and the decrease in 3-butenal concentrations under these conditions was associated with an increase in CA concentrations (data not shown). These results provide further evidence for the rapid tautomerization of 3-butenal to CA a t pH 7.4 at room temperature. The 3-butenal conversion to CA is expected, however, to occur at faster rates at higher temperatures.
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Figure 5. (A) Effect of temperature variation on 3-butenal formation. Incubations were carried out at pH 6.0 for 30 min using0.5 unit/incubationchloroperoxidaseand1 mM HzO2; values represent means f SD of the results obtained from three experiments. (B) Typical time course of 3-butenal and crotonaldehyde formation: 3-butenal, closed squares; CA, open squares. Incubations were carried out at room temperature at pH 6.0 using 0.5 unit/incubation chloroperoxidase and 1 mM H202. The formation of both 3-butenal and CA and the inability to detect the formation of methyl vinyl ketone (retention time 2.25 min) under the various incubation conditions provide further support to the hypothesis that l,&butadiene oxidation occurs predominantly by an oxygen transfer to a terminal carbon rather than to a central carbon of 1,3-butadiene,which would have resulted in the detection of both BM and methyl vinyl ketone. Whereas these results are consistent with the chemical reaction of atomic oxygen in its ground (triplet) state with l,&butadiene (251, the exact mechanism of the oxygentransfer reaction and the source of the oxygen in the chloroperoxidase-catalyzedreactions remain to be established. In summary, the results presented in this paper provide direct evidence for 3-butenal being an oxidative metabolite of l,&butadiene catalyzed by chloroperoxidase. The
1,3-Butadiene Oxidation by Chloroperoxidase
finding that 3-butenal readily tautomerizes to CA at pH 7.4 may explain our inability to detect 3-butenal in incubations with myeloperoxidase, cDNA-expressed human cytochrome P450s, and mouse, rat, and human liver microsomes (5-9). Furthermore, these results suggest that any 3-butenal that may be formed in vivo after animal or human exposure to l,&butadiene is likely to tautomerize readily to CA. Acknowledgment. This research was supported by National Institute of General Medical Sciences Grant GM 40375.
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(1992) Lymphohematopoietic cancer in styrene-butadiene polymerization workers. Am. J. Epidemiol. 136, 843-854. (2) Owen, P. E., Glaister, J. R., Gaunt, I. F., and Pullinger, D. H. (1987) Inhalation toxicity studieswith 1,3-butadiene. 3. Two-year toxicity/ carcinogenicity studies in rata. Am. Ind. Hyg. Assoc. J.,48, 407413. (3) Melnick, R. L., Huff, J., Chou, B. J., and Miller, R. A. (1990) Carcinogeneicityof 1,3-butadiene in C57BL/6 X C3HFl mice at low exposure concentrations. Cancer Res. 50,6592-6599. (4) Bolt, H. M., Schmiedel, G., Filser, J., Rolzhauser, H., Lieser, K., Wistuba, D., and Schurig, V. (1983) Biological activation of 1,3butadiene by rat liver microsomes and expiration of the reactive metabolite by exposed rata. J. Cancer Res. Clin. Oncol. 106,112116. (5) Elfarra, A. A., Duescher, R. J., and Pasch, C. M. (1991)Mechanisms of l,3-butadiene oxidations to butadiene monoxide and crotonaldehyde by mouse liver microsomes and chloroperoxidase. Arch. Biochem. Biophys. 286,244-251. (6) Duescher, R. J., and Elfarra, A. A. (1992)1,3-Butadiene oxidation by human myeloperoxidase: role of chloride ion in catalysis of divergent pathways. J.Biol. Chem. 267, 19859-19865. (7) Sharer, J. E., Duescher, R. J., and Elfarra, A. A. (1992) Species and tissue differences in the microsomal oxidation of 1,3-butadiene and the glutathione conjugation of butadiene monoxide in mice and rata. Poeeiblerolein 1,3-butadieneinducedtoxicity. Drug Metab. Dispos. 20,658-664. (8) Duescher, R. J., and Elfarra, A. A. (1993) 1,3-Butadiene oxidation catalyzed by cDNA-expressedhuman P45Os. Tozicologist, 13,431. (9) Elfarra, A. A., and Duescher, R. J. (1993) Human liver microsomes are more efficient in catalyzing 1,3-butadiene oxidation than mouse or rat liver microsomes. Pharmacologist 35, 202. (10) Sharer, J. E., Duescher, R. J., and Elfarra, A. A. (1991) Formation, stability, and rearrangements of the glutathione conjugates of
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