Oxidation of 3-Butene-1, 2-diol by Alcohol Dehydrogenase

Raymond A. Kemper and Adnan A. Elfarra*. Department of Comparative Biosciences and Center for Environmental Toxicology, School of. Veterinary Medicine...
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Chem. Res. Toxicol. 1996, 9, 1127-1134

1127

Oxidation of 3-Butene-1,2-diol by Alcohol Dehydrogenase Raymond A. Kemper and Adnan A. Elfarra* Department of Comparative Biosciences and Center for Environmental Toxicology, School of Veterinary Medicine, University of WisconsinsMadison, Madison, Wisconsin 53706 Received June 4, 1996X

3-Butene-1,2-diol (BDD) is a metabolite of the carcinogenic petrochemical 1,3-butadiene. BDD is produced by cytochrome P450-mediated oxidation of 1,3-butadiene to butadiene monoxide, followed by enzymatic hydrolysis by epoxide hydrolase. The metabolic disposition of BDD is unknown. The current work characterizes BDD oxidation by purified horse liver alcohol dehydrogenase (ADH) and by cytosolic ADH from mouse, rat, and human liver. BDD is oxidized by purified horse liver ADH in a stereoselective manner, with (S)-BDD oxidized at approximately 7 times the rate of (R)-BDD. Attempts to detect and identify metabolites of BDD using purified horse liver ADH demonstrated formation of a single stable metabolite, 1-hydroxy2-butanone (HBO). A second possible metabolite, 1-hydroxy-3-butene-2-one (HBONE), was tentatively identified by GC/MS, but HBONE formation could not be clearly attributed to BDD oxidation, possibly due to its rapid decomposition in the incubation mixture. Formation of HBO by ADH was dependent upon reaction time, protein concentration, substrate concentration, and the presence of NAD. Inclusion of GSH or 4-methylpyrazole in the incubation mixture resulted in inhibition of HBO formation. Based on these results and other lines of evidence, a mechanism is proposed for HBO formation involving generation of several potentially reactive intermediates which could contribute to toxicity of 1,3-butadiene in exposed individuals. Comparison of kinetics of BDD oxidation in rat, mouse, and human liver cytosol did not reveal significant differences in catalytic efficiency (Vmax/Km) between species. These results may contribute to a better understanding of 1,3-butadiene metabolism and toxicity.

Introduction 1,3-Butadiene (BD)1 is a petrochemical used extensively in the manufacture of synthetic rubber and thermoplastic resins. BD has also been found in gasoline, automobile exhaust, and cigarette smoke (1). Long-term inhalational exposure to BD produces tumors in multiple organs in rodents, with mice being significantly more susceptible to the carcinogenic effects of BD than rats (2-4). Furthermore, several epidemiological studies suggest that BD is also a human carcinogen (5-7). The carcinogenic and mutagenic effects of BD have been largely attributed to its primary metabolite, butadiene monoxide (BMO), which is a direct-acting mutagen (8, 9) and a rodent carcinogen (10). BMO is produced by oxidation of BD primarily by microsomal cytochrome P450 enzymes (11-15). BMO can be metabolized further by three separate enzyme systems: cytochromes P450, glutathione S-transferase, and epoxide hydrolase. Oxidation of BMO by cytochrome(s) P450 results in formation of butadiene diepoxide (16, 17), which is a more potent mutagen and carcinogen in rodents than BMO (8, 10). BMO can also be conjugated with GSH in the presence of cytosolic glutathione S-transferase (18-20). Finally, BMO can undergo enzymatic hydrolysis, mediated by microsomal epoxide hydrolase (20, 21). The latter * To whom correspondence should be addressed at: Department of Comparative Biosciences, School of Veterinary Medicine, University of WisconsinsMadison, 2015 Linden Dr. W., Madison, WI 53706. Phone: (608) 262-6518, Fax: (608) 263-3926, e-mail: elfarraa@ svm.vetmed.wisc.edu. X Abstract published in Advance ACS Abstracts, September 1, 1996. 1 Abbreviations: ADH, alcohol dehydrogenase; BD, 1,3-butadiene; BDD, 3-butene-1,2-diol; BMO, butadiene monoxide; HBAL, 2-hydroxy3-butenal; HBO, 1-hydroxy-2-butanone; HBONE, 1-hydroxy-3-butene2-one; HLADH, horse liver alcohol dehydrogenase; 4-MP, 4-methylpyrazole.

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reaction, which results in formation of 3-butene-1,2-diol (BDD), appears to be a major pathway for BMO metabolism in humans (20, 21). When BMO or BDD are administered to rodents, only a small amount of BDD (99.5%) were obtained from Acros Organics (Pittsburgh, PA). HLADH, HBO, 2-butanone, methyl vinyl ketone, 4-MP, GSH, and NAD were obtained from Sigma Chemicals (St. Louis, MO). GC-grade solvents were obtained from Fisher Chemicals (Cincinnati, OH). 1-Butanol (HPLC grade) was obtained from Aldrich Chemicals (Milwaukee, WI). Animals, Human Liver Samples, Preparation of Hepatic Cytosol. Male B6C3F1 mice (20-25 g, Jackson Laboratories, Bar Harbor, ME) and Male Sprague-Dawley rats (150250 g, Charles River Laboratories, Wilmington, MA) were housed under standard laboratory conditions and allowed feed and water ad libitum until sacrifice. Three human liver samples were obtained from SRI International (Menlo Park, CA) and maintained at -80 °C prior to subcellular fractionation. Subjects H-19 and H-50 were both 36 year old, Caucasian, nonsmoking males. Subject H-92 was a 15 year old, Caucasian, nonsmoking female. Mice and rats were sacrificed by cervical dislocation and decapitation, respectively. Livers were excised, weighed, and homogenized in 3 volumes of 0.1 M potassium phosphate buffer (pH 7.4). Cytosol was prepared from whole liver homogenate by differential centrifugation as described previously (11) and stored at -80 °C until use. Protein concentrations were determined by the method of Lowry et al. (29). Determination of BDD Dehydrogenase Activity. Kinetics of BDD oxidation were determined spectrophotometrically, by measuring the rate of reduction of NAD to NADH. Reactions were carried out at 37 °C in disposable 1.5 mL semimicrocuvettes (Fisher Scientific, Cincinnati, OH) and contained 0.05 M Tris-HCl (pH 7.4), 0.0125 mg of HLADH or ∼0.2 mg of rat, mouse, or human liver cytosolic protein, 1.35 mM NAD, and 2.5-200 mM BDD ((R)-, (S)-, or racemic mixture), in a final volume of 1.0 mL. The reaction time was 3 min for HLADH experiments, or 10 min for cytosol experiments. These times were chosen based on the duration of the linear phase of the reaction. For cytosolic experiments, only racemic BDD was used as a substrate. Rates of BDD oxidation were corrected for background NADH production measured in the absence of BDD. For inhibition experiments, 4-MP was added to a final concentration of 0.125 (HLADH) or 0.5 µM (mammalian cytosol). Calculation of Michaelis-Menten Parameters for Oxidation of BDD by HLADH. Initial estimates of Km and Vmax were obtained from double reciprocal plots and used as a starting point for nonlinear curve fitting. The velocity vs substrate concentration data were fitted to the MichaelisMenten equation using the nonlinear curve fitting feature of the Sigmaplot software package (Jandel Scientific, San Raphael, CA). This fitting routine employs the Marquardt-Levenberg algorithm to determine the parameters which provide the optimal fit of the data to the equation. Detection and Quantitation of Products of BDD Oxidation by GC. For initial detection of products of BDD oxidation by HLADH, reactions were carried out in 0.05 M Tris-HCl (pH 7.4) at 37 °C. Reaction mixtures contained 0.25 mg of HLADH, 1.35 mM NAD, and 150 mM racemic BDD in a total volume of 1 mL. Control reactions in which enzyme, cofactor, or substrate was omitted were carried out in parallel. The initial reaction

Kemper and Elfarra time was 30 min. For inhibition experiments, 4-MP was added to a final concentration of 50 µM. Reactions were terminated by immersion in an ice water bath, and the contents were transferred to 3 mL capacity solid phase Extrelut extraction columns (EM Science, Gibbstown, NJ), containing high pore volume diatomaceous earth granules. Columns were tamped lightly with a glass stirring rod prior to sample loading to repack material loosened during shipping and handling. After 3 min, samples were eluted with 12 mL (2 × 6 mL) of methylene chloride. Extracts were concentrated to ∼100 µL by centrifugation under vacuum, and a 3 µL aliquot was removed for GC analysis. GC analysis was carried out using a Hewlett Packard 5890 Series II gas chromatograph equipped with a flame ionization detector (Hewlett Packard, Palo Alto, CA). Separation of reaction components was accomplished using a 12 m × 0.32 mm i.d. DB-1 capillary column (J and W Scientific, Folsom, CA). The injection port and detector temperatures were 200 and 250 °C, respectively. The column head pressure was 6 psi. The column was maintained at 50 °C for 5 min and then increased to 200 °C at a rate of 30 °C min-1. The final temperature was maintained for 2 min. Identification of HBO by GC/MS. Combined extracts of HLADH reactions were used for identification of reaction products. Identification was accomplished using an HP 5790 mass selective detector interfaced with an HP 5890 Series II gas chromatograph (Hewlett Packard, Palo Alto, CA). Separation of reaction components was accomplished using a 10 m × 0.25 mm i.d. DB-1 capillary column (J and W Scientific, Folsom, CA). The temperature program used was the same as described above, except that the initial column temperature was 40 °C, the column head pressure was 7.5 psi, and the detector temperature was 280 °C. The mass spectrometer was programmed for scanning mode with a scan range of 10-400 amu. Quantitation of HBO by GC. For quantitation of HBO, 1-butanol (10 µL of a 1:1000 dilution in water) was added to reaction mixtures just prior to extraction as an internal standard. The concentration of HBO in HLADH reaction mixtures was determined by comparison of the peak area ratio (HBO/1-butanol) with standard curves prepared from known concentrations of HBO in reaction buffer and extracted as described above. The detector response for HBO was linear between 1.5 and 100 nmol mL-1, and the limit of detection was ∼1 nmol. Recovery of HBO under these conditions was ∼70%. Recovery of HBO from HLADH reactions was accounted for by preparing standards in buffer and processing them exactly like the enzyme reactions.

Results Stereoselective Oxidation of BDD by HLADH. (R)-, (S)-, and racemic BDD were tested for their ability to serve as substrates for purified HLADH. Representative velocity versus substrate concentration plots are shown in Figure 1. Oxidation was rapid with all three substrates, though high concentrations were required to achieve saturation of the enzyme. Oxidation of (S)-BDD was significantly more rapid than oxidation of the (R)enantiomer over the concentration range examined. The rate of oxidation of the racemic mixture was similar to that of (S)-BDD. Kinetic parameters derived from nonlinear regression of these data to the Michaelis-Menten equation are presented in Table 1. While the Km values for all three substrates were comparable, the Vmax for oxidation of the (S)-enantiomer is almost 7-fold higher than that of the (R)-enantiomer. Consequently, the catalytic efficiency (Vmax/Km) is much greater for (S)-BDD compared to (R)-BDD. The catalytic efficiency of the racemic mixture is similar to that of the (S)-enantiomer, suggesting that the kinetics of oxidation of the racemic mixture are dominated by the (S)-enantiomer. GC Analysis of HLADH-Mediated Metabolism of BDD and Identification of HBO by GC/MS. The

Oxidation of 3-Butene-1,2-diol

Chem. Res. Toxicol., Vol. 9, No. 7, 1996 1129

Figure 1. Stereoselective oxidation of BDD by HLADH. Representative velocity versus substrate concentration plots are shown. (A) (R)-BDD, (B) (S)-BDD, (C) racemic BDD. Reactions were carried out for 3 min at 37 °C in 50 mM Tris buffer (pH 7.4) and contained 1.35 mM NAD+, 125 µg of protein, and substrate in a final volume of 1.0 mL. Data points represent observed velocity. Lines indicate computer generated fit of data points to the Michaelis-Menten equation. Table 1. Michaelis-Menten Parameters for Oxidation of BDD by HLADH substrate

Km (mM)

Vmax (nmol min-1 mg-1)

Vmax/Km

(R)-BDD (S)-BDD (R,S)-BDD

28.1 ( 5.9a 33.8 ( 5.6 34.0 ( 1.3

306 ( 27 2124 ( 191 2496 ( 126

11.1 ( 1.5 63.6 ( 5.8 73.5 ( 3.4

a

Mean (SD for 3-5 separate determinations.

expected reaction products of ADH-mediated oxidation of BDD are 2-hydroxy-3-butenal (HBAL) and 1-hydroxy3-butene-2-one (HBONE). Efforts were made to detect these products in extracts of HLADH reactions by GC analysis. A single unique peak was detected in extracts of reaction mixtures (Figure 2). This peak, which had a retention time of 6.4 min under the GC conditions used, was dependent on the presence of enzyme, NAD+, and BDD and was attenuated in the presence of 4-MP (see below), suggesting that it represented an ADH-dependent metabolite of BDD. Organic extracts of HLADH-BDD reactions were pooled, concentrated, and subjected to GC/ MS analysis. The reaction product was identified as 1-hydroxy-2-butanone (HBO), based on comparison of its GC retention time and its electron impact mass spectrum to those of commercial HBO (Figure 3). These data demonstrate that HBO is a product of ADH-mediated metabolism of BDD. Characterization of Enzymatic HBO Formation by HLADH. For a given substrate concentration, the rate of formation of HBO was very low compared to the

Figure 2. Detection of HBO by GC. Reactions were carried out as described under Materials and Methods and contained 0.25 mg of HLADH, 150 mM racemic BDD, and either 1.35 mM NAD+ (top panel) or an equal volume of buffer (bottom panel). GC conditions are as described under Materials and Methods.

rate of BDD oxidation measured spectrophotometrically (∼0.014% of Vmax with racemic BDD), suggesting that very little of the original oxidation product is converted to HBO (Figure 4). HBO formation exhibited first order kinetics at substrate concentrations up to 200 mM (Figure 4C). The reaction was linear with respect to reaction time up to at least 2 h and increased in a linear fashion with increasing enzyme concentration between 0.125 and 1.0 mg mL-1 (Figure 4A,B). Formation of HBO by HLADH was subject to both enzymatic and chemical inhibition (Figure 5). Inclusion of 50 µM 4-MP in the reaction mixture decreased HBO formation by approximately 85% relative to control reactions, as would be expected for an ADH-mediated reaction. Likewise, addition of GSH (5 mM) to HLADH reactions also significantly inhibited formation of HBO (∼60%). Addition of GSH after termination of the enzymatic reaction with ZnSO4, followed by an additional 30 min incubation at 37 °C, did not result in decreased formation of HBO (data not shown). Furthermore, incubation of commercial HBO (100 µM) with GSH (5 mM) at 37 °C for 60 min did not decrease HBO concentrations relative to controls (data not shown), suggesting that conjugation of electrophilic precursor(s) of HBO with GSH is responsible for the observed inhibition by GSH. Because HBONE could be a precursor of HBO, experiments were carried out to determine if HLADH was capable of directly reducing the double bond of BDD or methyl vinyl ketone (150 mM), a structural analog of HBONE, in the presence of NADH (1.35 mM). Neither of the products expected from these reactions (1,2butanediol or 2-butanone) was detected by GC. Furthermore, HBO was not detected in incubations containing NADH and BDD, which is contaminated with HBONE (data not shown).

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Figure 3. Identification of HBO by GC/MS. Reactions were carried out and extracted as described under Materials and Methods and contained 0.25 mg of HLADH, 1.35 mM NAD+, and 150 mM racemic BDD. Extracts were pooled for GC/MS analysis. (A) Pooled reaction extracts. (B) Authentic HBO. Fragments m/z 88, 57, and 29 correspond to M+, M+ - CH2OH, and M+ - C2H3O2 respectively.

Oxidation of BDD by Mouse, Rat, and Human Liver Cytosol. Oxidation of BDD by cytosolic ADH from mouse, rat, and human liver was evaluated using racemic BDD as the substrate. Representative velocity versus BDD concentration plots for the three species are shown in Figure 6, and the corresponding apparent kinetic parameters are presented in Table 2. The apparent Km values observed for oxidation of BDD by rat liver cytosol were comparable to those for purified HLADH, while the lower values observed with mouse and human cytosol suggest a somewhat greater affinity for the substrate. The apparent Vmax for rat liver cytosol was found to be about twice that in mice, which resulted in comparable catalytic efficiencies for both species. The variability for the three human liver cytosol samples was remarkably low. The catalytic efficiency in humans is comparable to that in both of the other two species examined. Thus, these data failed to reveal any marked species differences in hepatic ADH-mediated oxidation of BDD in mice, rats, and humans. To confirm the role of ADH in oxidation of BDD, reactions were carried out in the presence or absence of 4-MP, an inhibitor of ADH (28). The effect of 4-MP on oxidation of BDD by HLADH and rat liver cytosol is illustrated in Figure 7. The data in panel A show that 4-MP (0.125 µM) effectively inhibits HLADH-mediated oxidation of BDD. The data in panel B suggest that ADH is the enzyme system involved in BDD oxidation in rat

Kemper and Elfarra

Figure 4. Characterization of HBO formation by HLADH. Reactions were carried out as described under Materials and Methods. (A) Time dependence. Reactions contained 0.25 mg of HLADH, 1.35 mM NAD+, and 150 mM racemic BDD. (B) Dependence on protein concentration. Reactions contained 1.35 mM NAD+ and 150 mM racemic BDD. Reaction time was 30 min. (C) Dependence on substrate concentration. Reactions contained 0.25 mg of HLADH and 1.35 mM NAD+. The reaction time was 30 min.

Figure 5. Inhibition of HBO formation by 4-MP and GSH. Reactions were carried out as described under Materials and Methods and contained 0.25 mg of HLADH, 1.35 mM NAD+, and 150 mM racemic BDD. The reaction time was 30 min. 4-MP (50 µM) or GSH (5 mM) was added as indicated. Control reactions contained an equal volume of buffer.

liver cytosol. Similar inhibition by 4-MP was observed in mouse and human liver (data not shown). The data shown in Figure 7B demonstrate the effect of 0.5 µM

Oxidation of 3-Butene-1,2-diol

Chem. Res. Toxicol., Vol. 9, No. 7, 1996 1131

Figure 6. Oxidation of racemic BDD by mouse, rat, and human liver cytosol. Representative velocity versus substrate concentration plots are shown. Reactions were carried out as described under Materials and Methods and contained 0.20 mg of cytosolic protein, 0.675 mM NAD+, and racemic BDD in a final volume of 1.0 mL. (A) Mouse; (B) rat; (C) human (sample H-19). Values shown have been adjusted for background NADH formation. Data points represent observed velocity data. Lines indicate computer generated fit of observed data to the MichaelisMenten equation. Table 2. Apparent Kinetic Parameters for Oxidation of BDD by Mouse, Rat, and Human Liver Cytosol species mouse rat humanb H-19 H-50 H-92

Km (mM)

Vmax (nmol min-1 mg-1)

Vmax/Km

20.1 ( 32.7 ( 4.7

10.4 ( 1.2 21.4 ( 1.0

0.53 ( 0.11 0.66 ( 0.09

18.3 19.6 22.5

11.6 10.6 9.1

0.63 0.54 0.40

2.2a

a Mean (SD for three separate determinations. b Values for human samples represent average of two separate determinations.

4-MP, though at higher inhibitor concentrations (>5 µM) complete inhibition of the cytosolic reaction is observed (data not shown).

Discussion The present work demonstrates that BDD, a metabolite of the industrial chemical BD, is a substrate for ADH from horse, rat, mouse, and human liver. Furthermore, formation of HBO, a stable metabolite of BDD, was demonstrated and characterized. Since enzymatic hydrolysis of BMO to BDD appears to be a significant metabolic pathway, particularly in primates and humans (20, 21), the fate of this compound may be important in overall toxicity and/or carcinogenicity of BD. HLADH rapidly and stereoselectively oxidized BDD. However, the Michaelis constants for these oxidations

Figure 7. Inhibition of BDD oxidation by 4-MP. Representative velocity versus substrate plots are shown. (A) HLADH. Reactions contained 0.125 mg of HLADH, 1.35 mM NAD+, and substrate in a final volume of 1.0 mL. The concentration of 4-MP was 0.125 µM. (B) Rat liver cytosol. Reactions contained 0.5 mg of cytosolic protein, 0.675 mM NAD+, and substrate in a final volume of 1.0 mL. The concentration of 4-MP was 0.5 µM.

were quite high (28-34 mM), suggesting that metabolism of BDD resulting from exposure to BD will be first order in vivo. This is consistent with values observed for oxidation of ethylene glycol and 1,2-propanediol by HLADH (24). A marked stereoselectivity was evident in HLADHmediated oxidation of BDD. While similar Km values were observed for the (R)- and (S)-enantiomers of BDD, the maximal reaction rate (Vmax) of (S)-BDD was almost 7-fold greater than Vmax for (R)-BDD. This in turn results in much higher catalytic efficiency (Vmax/Km) for oxidation of (S)-BDD compared to (R)-BDD. The catalytic efficiency for oxidation of racemic BDD was comparable to that of the (S)-enantiomer. These data are consistent with the observed stereoselectivity of ADH toward secondary alcohols (30, 31), 1,2-diols, and 2-amino alcohols (25). The major products expected from oxidation of BDD were HBAL and HBONE. The latter compound could arise from direct oxidation of the C-2 hydroxyl group, or from rearrangement of HBAL. We were unable to detect HBAL by GC or GC/MS in organic extracts of ADH reaction mixtures. Probable reasons for this include the relatively high water solubility of short chain hydroxycarbonyl compounds, which would complicate organic extraction, and, more likely, the instability of 2-hydroxyaldehydes in aqueous media, where these compounds are quite prone to hydration, polymerization and rearrangement (25, 32, 33). A second compound (retention time 5.6 min, in Figure 2) found in reaction mixture extracts had an MS fragmentation pattern consistent with the

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structure of HBONE (major fragments: 55 [M+ - CH2OH], 31 [M+ - C3H3O], 86 [M+], data not shown) but was subsequently found to be a contaminant in the commercial BDD (∼0.1-0.5%, depending on lot, as determined by GC analysis). While enzymatic incubations suggested that 30-35% of the compound present could not be accounted for by contamination of BDD, its concentration in ADH reaction extracts could not be clearly associated with enzymatic oxidation of BDD in 4-MP inhibition experiments (data not shown). Attempts to remove this compound from BDD by fractional or vacuum distillation, or by column chromatography, were unsuccessful. Preincubation of BDD in buffer alone led to a rapid decrease in the area of this peak, suggesting that HBONE readily decomposes in aqueous solutions. HBO was the only metabolite of BDD conclusively identified in our experiments. This was somewhat surprising, since formation of HBO from BDD does not involve any change in the overall oxidation state of the molecule. Spontaneous rearrangement of BDD to HBO was ruled out as a mechanism, since formation of HBO was strictly dependent on the presence of BDD, NAD, and enzyme and was inhibited by 4-MP. Another possible explanation for HBO formation was that oxidation of BDD merely was acting to supply NADH for an ADHcatalyzed reduction of some contaminant in the reaction mixture, such as HBONE. However, substitution of NADH for NAD as cofactor in the reaction did not result in formation of HBO (data not shown). Furthermore, HLADH was incapable of reducing methy vinyl ketone to 2-butanone, suggesting that direct reduction of the double bond of BDD or HBONE does not occur in the presence of HLADH. The data are consistent with a mechanism of HBO formation in which ADH-mediated oxidation of BDD is followed by rearrangement and subsequent reduction of the initial product. A possible pathway for this mechanism is illustrated in Figure 8. In this scheme, BDD is assumed to be oxidized to HBAL. This assumption is supported by the findings of Matos et al. (25), who demonstrated that HLADH and yeast aldehyde dehydrogenase, coimmobilized on a polyacrylamide gel, oxidized BDD to 2-hydroxy-3-butenoic acid (vinylglycolic acid). Though quantitative data were not reported, these results suggest that HBAL was an intermediate in this reaction. Rearrangement of HBAL could proceed by the shift of the relatively acidic R-proton from C-2 to C-4. A similar shift has been proposed for the rearrangement of 3-butenal to crotonaldehyde (34, 13). The resulting enol could then rapidly tautomerize to form the 1,2dicarbonyl compound 2-ketobutanal (ethylglyoxal). Reduction of the aldehyde moiety of the latter compound by HLADH would generate HBO. Reduction of glyoxals by ADH has been demonstrated for methyl- and phenylglyoxal, both of which are good substrates (Km ) 0.6 mM and 0.3 mM, respectively) for ADH (35). The rate of HBO formation is extremely low compared to the rate of BDD oxidation, as measured by generation of NADH. The most likely cause for this result is the instability of the hypothesized intermediates involved. HBAL, HBONE, and the other postulated intermediates depicted in Figure 8 are expected to be unstable and/or reactive compared to BDD and HBO. The inhibitory effect of GSH on HBO formation observed in our experiments, along with the observation that HBO does not react directly with GSH, is consistent with the involvement of reactive electrophilic intermediates in HBO

Kemper and Elfarra

Figure 8. Proposed scheme for ADH-mediated formation of HBO from BDD. Species in brackets are hypothetical.

formation. As mentioned previously, R-hydroxyaldehydes are highly susceptible to hydration, polymerization, and rearrangement and are also known react readily with GSH (36) and other nucleophiles (37). Thus, the amount of ethylglyoxal available for reduction by ADH would likely be quite low, resulting in the first order kinetics of HBO formation observed in our experiments. Given the well documented species differences observed for in vivo and in vitro metabolism of BD (3840) and BMO (41-43), it was of interest to examine possible species differences in oxidation of BDD by cytosolic ADH. Racemic BDD was found to be a substrate for ADH in cytosolic preparations from rat, mouse, and human liver. Further evidence for the involvement of ADH in oxidation of BDD by liver cytosol was obtained by inclusion of 4-MP, an inhibitor of ADH. While both the Km and Vmax for oxidation of BDD were significantly lower in mouse liver cytosol than in rat liver cytosol, the catalytic efficiencies for these two species were comparable. Oxidation of BDD by cytosol from three individual human liver samples yielded kinetic parameters which more closely resembled the mouse parameters rather than rat, but again, the catalytic efficiencies were comparable for all three species examined. Thus, these experiments did not reveal any striking species differences associated with hepatic ADH-mediated oxidation of BDD, in agreement with the well documented similarity in structure and catalytic activity among mammalian ADHs (44, 24). The lack of species differences observed in the present study is in contrast with the marked species differences seen in microsomal metabolism of BD (11, 12) and BMO (12, 17) between rat, mouse, and human tissue preparations. The data presented here suggest that differences in hepatic ADH-mediated oxida-

Oxidation of 3-Butene-1,2-diol

tion of BDD probably do not contribute significantly to observed species differences in carcinogenicity and toxicity of BD. The possible contributions from ADH in other tissues remain to be examined. The data from the experiments reported here suggest that enzymatic dehydrogenation of BDD to potentially reactive carbonyl intermediates by hepatic ADH may be a mechanism for metabolic elimination of BDD. The biological significance of this metabolic pathway, which will probably depend on the availability of alternative routes of BDD metabolism and elimination, remains to be established. Given the apparent importance of BDD as a metabolite of BD in humans (20), further investigation of its metabolic fate is needed.

Acknowledgment. This study was supported by Grant ES 06841 from the National Institutes of Health. The authors would like to thank Dr. Harrell E. Hurst of the University of Louisville Therapeutics and Toxicology Laboratory for providing GC/MS facilities for this study.

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(16)

(17)

(18)

(19)

(20)

References (1) Melnick, R. L., and Kohn, M. C. (1995) Mechanistic data indicate that 1,3-butadiene is a human carcinogen. Carcinogenesis 16, 157-163. (2) Huff, J. E., Melnick, R. L., Solleveld, H. A., Haseman, J. K., Powers, M., and Miller, R. A. (1985) Multiple organ carcinogenicity of 1,3-butadiene in B6C3F1 mice after 60 weeks of inhalational exposure. Science 227, 548-549. (3) Melnick, R. L., Huff, J., Chouo, B. J., and Miller, R. (1990) Carcinogenicity of 1,3-butadiene in C57BL/6 x C3H F1 mice at low exposure concentrations. Cancer Res. 50, 6592-6599. (4) Owen, P. E., Glaister, J. R., Gaunt, I. F., and Pullinger, D. H. (1987) Inhalational toxicity studies with 1,3-butadiene. 3. Two year toxicity/carcinogenicity studies in rats. Am. Ind. Hyg. Assoc. J. 48, 407-413. (5) Meinhardt, T. J., Lemen, R. A., Cransall, M. S., and Young, R. J. (1982) Environmental epidemiological investigation of the styrenebutadiene rubber industry. Scand. J. Work Environ. Health 8, 250-259. (6) Divine, B. J., Wendt, J. K., and Hartman, C. M. (1993) Cancer mortality among workers at a butadiene production facility. In Butadiene and Styrene: Assessment of Health Hazards (Sorsa, M., Peltonin, K., Vainio, H., and Hemminki, K., Eds.) pp 345362, IARC Science Publications, Lyons. (7) Matanoski, G. M., Francis, M., Correa-Villasenor, A., Elliot, E., Santos-Burgoa, C., and Schwartz, L. (1993) Cancer epidemiology among styrene-butadiene rubber workers. In Butadiene and Styrene: Assessment of Health Hazards (Sorsa, M., Peltonin, K., Vainio, H., and Hemminski, K., Eds.) pp 345-362, IARC Science Publications, Lyons. (8) Cochrane, J. E., and Skopek, T. R. (1994) Mutagenicity of butadiene and its epoxide metabolites: I. Mutational potential of 1,2-epoxybutene, 1,2,3,4-diepoxybutane, and 3,4-epoxy-1,2butanediol in cultured human lymphoblasts. Carcinogenesis 15, 713-717. (9) Cochrane, J. E., and Skopek, T. R. (1994) Mutagenicity of butadiene and its epoxide metabolites: II. Mutational spectra of butadiene, 1,2-epoxybutene, and diepoxybutane at the HPRT locus in splenic T cells from exposed B6C3F1 mice. Carcinogenesis 15, 129-160. (10) Van Duuren, B. L., Langseth, L., Oris, L., Teebor, G., Nelson, N., and Kuschner, M. (1966) Carcinogenicity of epoxides, lactones, and peroxy compounds. Part IV. Tumor response in epithelial and connective tissue in mice and rats. J. Natl. Cancer Inst. 37, 825838. (11) Duescher, R. J., and Elfarra, A. A. (1994) Human liver microsomes are efficient catalysts of 1,3-butadiene oxidation: Evidence for major roles by cytochromes P450 2A6 and 2E1. Arch. Biochem. Biophys. 311, 342-349. (12) Csana´dy, G. A., Guengerich, F. P., and Bond, J. A. (1992) Comparison of the biotransformation of 1,3-butadiene and its metabolite butadiene monoepoxide, by hepatic and pulmonary tissues from humans, rats, and mice. Carcinogenesis 13, 11431153. (13) Elfarra, A. A., Duescher, R. J., and Pasch, C. M. (1991) Mechanisms of 1,3-butadiene oxidations to butadiene monoxide and

(21)

(22)

(23)

(24)

(25)

(26)

(27)

(28)

(29)

(30)

(31)

(32)

(33)

(34)

(35)

(36)

(37)

crotonaldehyde by mouse liver microsomes and chloroperoxidase. Arch. Biochem. Biophys. 286, 244-251. Malvoisin, E., and Roberfroid, M. (1982) Hepatic microsomal metabolism of 1,3-butadiene. Xenobiotica 12, 137-144. Malvoisin, E., Lhoest, G., Poncelet, F., Roberfroid, M., and Mercier, M. (1979) Identification and quantitation of 1,2-epoxybutene-3 as the primary metabolite of 1,3-butadiene. J. Chromatogr. 178, 419-425. Elfarra, A. A., and Duescher, R. J. (1995) Abstract. Oxidation of butadiene monoxide to meso- and (()-diepoxybutane by mouse and rat liver microsomes and human cDNA-expressed cytochrome P-450s. Toxicologist 15, 61. Seaton, M. J., Follansbee, M. H., and Bond, J. A. (1995) Oxidation of 1,2-epoxy-3-butene to 1,2:3,4-diepoxybutane by cDNA-expressed human cytochromes P450 2E1 and 3A4 and human, mouse, and rat liver microsomes. Carcinogenesis 16, 2287-2293. Sharer, J. E., Duescher, R. J., and Elfarra, A. A. (1991) Formation, stability, and rearrangement of the glutathione conjugates of butadiene monoxide: Evidence for the formation of stable sulfurane intermediates. Chem. Res. Toxicol. 4, 430-436. Sharer, J. E., and Elfarra, A. A. (1992) S-(Hydroxy-3-buten-1-yl)glutathione and S-(1-Hydroxy-3-buten-2-yl)glutathione are in vivo metabolites of butadiene monoxide: detection and quantitation in bile. Chem. Res. Toxicol. 5, 787-790. Bechtold, W. E., Strunk, M. R., Chang, I.-Y., Ward, J. B., and Henderson, R. F. (1994) Species differences in urinary butadiene metabolites: Comparisons of metabolite ratios between mice, rats, and humans. Toxicol. Appl. Pharmacol. 127, 44-49. Sabourin, P. J., Burka, L. T., Bechtold, W. E., Dahl, A. R., Hoover, M. D., Chang, I.-Y., and Henderson, R. F. (1992) Species differences in urinary butadiene metabolites: Identification of 1,2dihydroxy-4-(N-acetylcysteinyl)butane, a novel metabolite of butadiene. Carcinogenesis 13, 1633-1638. Dalziel, K., and Dickinson, F. M., (1966) The kinetics and mechanism of liver alcohol dehydrogenase with primary and secondary alcohols as substrates. Biochem. J. 100, 34-46. Deetz, J. S., Luehr, C. A., and Vallee, B. L. (1984) Human liver alcohol dehydrogenase isozymes: Reduction of aldehydes and ketones. Biochemistry 23, 6822-6828. Pietruszko, R., Voigtlander, K., and Lester, D. (1978) Alcohol dehydrogenase from human and horse liverssubstrate specificity with diols. Biochem. Pharmacol. 27, 1296-1297. Matos, J. R., Smith, M. B., and Wong, C.-H. (1985) Enantioselectivity of alcohol dehydrogenase-catalyzed oxidation of 1,2-diols and aminoalcohols. Bioorg. Chem. 13, 121-130. Rikans, L. E., and Moore, D. R. (1987) Effect of age and sex on allyl alcohol hepatotoxicity in rats: Role of liver alcohol and aldehyde dehydrogenase activities. J. Pharmacol. Exp. Ther. 243, 20-26. Lenk, W., Lo¨hr, D., and Sonnenbichler, J. (1989) Pharmacokinetics and biotransformation of diethylene glycol and ethylene glycol in the rat. Xenobiotica 19, 961-979. Tolf, B. R., Dahlbom, R., Akeson, A., and Theorell, H. (1985) Synthetic inhibitors of alcohol dehydrogenase. Acta Pharm. Suec. 22, 147-156. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. Stone, C. L., Li, T.-K., and Bosron, W. F. (1989) Stereospecific oxidation of secondary alcohols by human alcohol dehydrogenases. J. Biol. Chem. 264, 11112-11116. Adolph, H. W., Muarer, P., Schneider-Bernlo¨hr, H., Sartorius, C., and Zeppezauer, M. (1991) Substrate specificity and stereoselectivity of horse liver alcohol dehydrogenase Eur. J. Biochem. 201, 615-625. Hassner, A., Reuss, R. H., and Pinnick, H. W. (1975) Hydroxylation of carbonyl compounds via silyl enol ethers. J. Org. Chem. 40, 3427-3429. Blumbergs, P., LaMontagne, M. P., and Stevens, J. I. (1972) The preparation and oxidation of R-hydroxyaldehydes J. Org. Chem. 37, 1248-1251. Duescher, R. J., and Elfarra, A. A. (1993) Chloroperoxidasemediated oxidation of 1,3-butadiene to 3-butenal, a crotonaldehyde precursor. Chem. Res. Toxicol. 6, 669-673. Yang, C. F., and Brush, E. J. (1993) A spectrophotometric assay for R-ketoaldehydes using horse liver alcohol dehydrogenase. Anal. Biochem. 214, 124-127. Vander Jagt, D. L., Daub, E., Krohn, J. A., and Han, L.-P. B. (1975) Effects of pH and thiols on the kinetics of yeast glyoxylase I. An evaluation of the random pathway mechanism. Biochemistry 14, 3669-3675. Lange, L. G., III, Riordan, J. F., and Vallee, B. L. (1974) Functional arginyl residues as NADH binding sites of alcohol dehydrogenases. Biochemistry 13, 4362-4369.

1134 Chem. Res. Toxicol., Vol. 9, No. 7, 1996 (38) Kreiling, R., Laib, R. J., Filser, J. G., and Bolt, H. M. (1986) Species differences in butadiene metabolism between mice and rats evaluated by inhalational pharmacokinetics. Arch. Toxicol. 58, 235-238. (39) Schmidt, U., and Loeser, E. (1985) Species differences in the formation of butadiene monoxide from 1,3-butadiene. Arch. Toxicol. 57, 222-225. (40) Bond, J. A., Dahl, A. R., Henderson, R. F., Dutcher, J. S., Mauderly, J. L., and Birnbaum, L. S. (1986) Species differences in the disposition of inhaled butadiene. Toxicol. Appl. Pharmacol. 84, 617-627. (41) 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 rats. Drug Metab. Dispos. 20, 658-664.

Kemper and Elfarra (42) Elfarra, A. A., Sharer, J. E., and Deuscher, R. J. (1995) Synthesis and characterization of N-acetyl-L-cysteine S-conjugates of butadiene monoxide and their detection and quantitation in urine of rats and mice given butadiene monoxide. Chem. Res. Toxicol. 8, 68-76. (43) Kreuzer, P. E., Kessler, W., Welter, H. F., Bauer, C., and Filser, J. G. (1991) Enzyme specific kinetic of 1,2-epoxybutene-3 in microsomes and cytosol from livers of mouse, rat, and man. Arch. Toxicol. 65, 59-67. (44) Testa, B. (1995) Chapter 2: Dehydrogenation of Alcohols and Aldehydes, Carbonyl Reduction. In The Metabolism of Drugs and Other Xenobiotics: Biochemistry of Redox Reactions (Testa, B., and Caldwell, J., Eds.) pp 41-69, Academic Press, London.

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