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Hydroxymethylvinyl Ketone: A Reactive Michael Acceptor Formed by the Oxidation of 3-Butene-1,2-diol by cDNA-Expressed Human Cytochrome P450s and Mouse, Rat, and Human Liver Microsomes Renee J. Krause, Raymond A. Kemper,† and Adnan A. Elfarra* Department of Comparative Biosciences and Environmental Toxicology Center, University of Wisconsin School of Veterinary Medicine, Madison, Wisconsin 53706 Received July 20, 2001
The metabolic fate of 3-butene-1,2-diol (BDD), a secondary metabolite of the industrial carcinogen, 1,3-butadiene, is unclear. The current study characterizes BDD oxidation to hydroxymethylvinyl ketone (HMVK), a reactive Michael acceptor. Because of its instability in aqueous medium, HMVK was trapped by conjugation with GSH, a reaction that occurred readily at physiological conditions (pH 7.4, 37 °C) to yield 1-hydroxy-2-keto-4-(S-glutathionyl)butane. The results show that BDD was oxidized to HMVK by mouse, rat, and human liver microsomes and by cDNA-expressed human cytochrome P450s. Eadie-Hofstee plots demonstrated biphasic kinetics of BDD oxidation with mouse and rat liver microsomes and one of three individual human liver microsomes; BDD oxidation by the other two human liver microsomal samples was best described by monophasic kinetics. Of the human P450 enzymes examined, only P450 2E1 exhibited activity at 1 mM BDD. P450 3A4 was capable of catalyzing the reaction at a high BDD (10 mM) concentration; P450 1A1, 1A2, 1B1, 2D6-Met, and 2D6-Val produced only trace amounts of HMVK-GSH whereas P450 2A6, 2C8, 2C9, and 4A11 had no detectable activity. Detection of HMVK or the HMVK-GSH conjugate was dependent on reaction time, protein, and BDD concentrations, and the presence of NADPH. Collectively, the results provide clear evidence for BDD bioactivation to yield HMVK. Because mouse, rat, and human liver microsomes exhibited Km values of 50-80 µM, the results also suggest that HMVK could be formed after rodent or human exposure to BDD or its parent compound, BD.
Introduction 1,3-Butadiene (BD),1 a petrochemical used in the manufacture of synthetic rubber and plastics, has been detected in automobile emissions, gasoline, cigarette smoke, and urban air (1). Several epidemiological studies have associated occupational exposure to BD with an increased risk of hematopoietic cancers (2). Upon the basis of these results and other related studies, the U.S. Department of Health and Human Services-National Toxicology Program has recently upgraded the classification of BD to “Known to be a Human Carcinogen” (3). Long-term inhalation exposure of mice and rats to BD has resulted in development of tumors at multiple sites (4,5). BD requires bioactivation in order to exert its mutagenic and carcinogenic effects (Figure 1). BD is initially metabolized to butadiene monoxide (BMO) by P450s and myeloperoxidase (6-8). BMO is a direct-acting mutagen and an animal carcinogen, and it can directly react with cellular macromolecules such as DNA or hemoglobin at * To whom correspondence should be addressed. E-mail: elfarraa@ svm.vetmed.wisc.edu. † Present address: DuPont Haskell Laboratory for Health and Environmental Sciences, Newark, DE 19714. 1 Abbreviations: BD, 1,3-butadiene; BMO, butadiene monoxide; BDD, 3-butene-1,2-diol; HMVK, hydroxymethylvinyl ketone; DEB, diepoxybutane; GC-FID, gas chromatography-flame ionization detection; ESI-MS, electrospray ionization-mass spectrometry.
Figure 1. Proposed scheme for the formation of HMVK from BDD and BD. BD, 1,3-butadiene; BMO, butadiene monoxide, P450, cytochrome P450; BDD, 3-butene-1,2-diol; EH, epoxide hydrolase; HMVK, hydroxymethylvinyl ketone.
10.1021/tx010117g CCC: $20.00 © 2001 American Chemical Society Published on Web 10/23/2001
Oxidation of 3-Butene-1,2-diol
Chem. Res. Toxicol., Vol. 14, No. 12, 2001 1591 Table 1. Data on Human Liver Microsomal Samplesa
donor
sex
age
race
smoker
P450 content (nmol/mg protein)
cause of death
H1 H2 H3
M M M
36 22 59
caucasian caucasian african american
no 1 pack/day no
0.24 0.26 0.30
subarachnoid hemorrhage closed head trauma pituitary adenoma
a
All data listed here were supplied by the vendor (SRI International, Menlo Park, CA).
multiple sites (9, 10). BMO can be further oxidized by P450s to diepoxybutane (DEB; refs 11 and 12), which is a more potent mutagen and carcinogen than BMO. The other known pathways for BMO metabolism involve conjugation with GSH, mediated by glutathione Stransferases (13, 14), and hydrolysis to 3-butene-1,2-diol (BDD; ref 15) mediated by epoxide hydrolases. The latter pathway has toxicological relevance because of the high expression level of microsomal epoxide hydrolase in humans. Rodents exposed to BD, BMO, or BDD excreted only a small amount of BDD (1-5% of the administered dose) or BDD glucoronide or sulfate conjugates in urine (1517). BDD has previously been shown to be metabolized to 1-hydroxy-2-butanone by hepatic alcohol dehydrogenases in vitro (18). Evidence implicating the involvement of both alcohol dehydrogenases and P450s in BDD metabolism in B6C3F1 mice has also been obtained in vivo (17, 18). These data and the finding that treatment of mice with high doses of BDD depleted hepatic GSH levels (17) suggest that BDD oxidation may lead to the formation of multiple reactive metabolites. One such metabolite that has been postulated is hydroxymethylvinyl ketone (HMVK, 1-hydroxy-3-buten-2-one), which may be formed by the oxidation of the secondary hydroxyl group of BDD (18, 19). Formation of this metabolite has also been inferred from studies in which the mercapturate, 1,2-dihydroxy-4-(N-acetyl-L-cysteinyl)butane was identified in the urine of animals and humans exposed to BD (19). In the current study, we demonstrate the formation of the reactive electrophile, HMVK, both directly and indirectly as its corresponding GSH conjugate in BDD incubations with mouse, rat, and human liver microsomes and cDNA-expressed human P450s. Preliminary results of this study have been previously presented (20, 21).
Experimental Procedures Hazardous Materials. The toxicity of BDD has not been investigated. However, the parent compound, BD, is an animal and human carcinogen. Thus, BDD should be handled with care and protective clothing should be worn at all times. Chemicals. Racemic 3-butene-1,2-diol (BDD) was obtained from Acros Chemicals (Pittsburgh, PA). NADPH, NADP, glucose6-phosphate, glucose-6-phosphate dehydrogenase, butyne-1,4diol, mercury(II) oxide, boron trifluoride diethyl etherate, and GSH were purchased from Sigma-Aldrich Research (Milwaukee, WI, and St. Louis, MO). Fluoraldehyde (o-phthalaldehyde reagent) was obtained from Pierce Chemicals (Rockford, IL). GCgrade methylene chloride was purchased from Fisher Scientific (Itasca, IL). All other chemicals and reagents were of the highest quality commercially available. Synthesis of HMVK. HMVK was synthesized by a modified Meyer-Schuster rearrangement method (22). This reaction is characterized by the isomerization of secondary and tertiary R-acetylenic alcohols to R,β-unsaturated carbonyl compounds. Briefly, a catalyst consisting of mercury(II) oxide (1.0 g), BF3diethyl etherate (710 mg), and trichloroacetic acid (245 mg) was prepared in ethyl acetate (5 mL) using vigorous stirring and
heating at 55 °C in a three-necked flask to produce a bright orange-colored suspension. Butyne-1,4-diol (8.6 g in 35 mL of ethyl acetate warmed to 45 °C) was added to the catalyst and the reaction was refluxed under vacuum at 45 °C for 1 h. The reaction was terminated by the addition of sodium carbonate (160 mg) and filtered to remove sediments. Identity and purity of the compound was confirmed by 1H NMR, 13C NMR, and GC/ MS. The 1H NMR and 13C NMR were obtained in CDCl3 using a 500 MHz Bruker spectrometer (Karlsruhe, Germany). For NMR analysis, chemical shifts are reported in parts per million (ppm) using CHCl3 as the internal standard. The 13C-spectrum contained the following peaks: 66.4 ppm, methylene carbon; 130 ppm, vinyl carbon; 132.2 ppm, vinyl carbon; and 198.6 ppm, carbonyl carbon. The 1H NMR spectrum was as follows: 2.08 ppm, 1H, s, hydroxy proton; 4.46 ppm, 2H, s, methylene protons; 5.95 ppm, 1H, d, vinyl proton; 6.36 ppm, 2H, m, vinyl protons. The purity of the final product was found to be >97% by GC/ MS. The GC/MS spectra were obtained on a HP 6890 Series GC interfaced with a HP 6890 Series mass selective detector fitted with a 30 m × 0.25 mm i.d. HP-5 capillary GC column (Hewlett-Packard, Palo Alto, CA). Enzymatic HMVK was identified in the combined methylene chloride extracts of six incubations concentrated to approximately 100 µL. Synthesis of the HMVK-GSH Conjugate. The HMVKGSH conjugate, 1-hydroxy-2-keto-4-(S-glutathionyl)butane was synthesized by reacting crude HMVK with 100 mg of GSH (approximately 10:1 ratio) in 10 mL of 0.05 M Tris buffer, pH 8.5 at 37 °C for 1 h. At the end of the reaction, the pH was adjusted to 7 and the reaction mixture was then fractionated on a Beckman Ultrasphere ODS 5 µm column (10 × 250 mm) using isocratic elution with 2.25% acetonitrile, pH 2.5, at a flowrate of 3 mL/min. The fractions containing the HMVK-GSH conjugate were pooled and lyophilized to dryness. The oily residue that was obtained in some cases, was run through a Chelex 100 Na+ column (Bio-Rad, Richmond, CA) and then relyophilized to obtain a solid residue. Identity of the compound was confirmed by 1H NMR (see Results) and electrospray ionization mass spectrometry (ESI-MS). The 1H NMR was carried out at a concentration of 2 mg/mL in D2O. Chemical shifts are reported in parts per million using water as internal standard. ESI-MS of the HMVK-GSH conjugate was obtained on a API365 Perkin-Elmer mass spectrometer (Mass Spectrometry/Bioanalytical Facility of the University of Wisconsin Biotechnology Center, Madison, WI). Animals and Human Liver Samples. Male B6C3F1 mice (20-26 g) and male C/D rats (175-225 g) were obtained from Jackson Laboratories, Bar Harbor, ME, and Charles Rivers, Wilmington, MA, respectively. Human liver samples were obtained from SRI International (Menlo Park, CA); a brief description of the donors is listed in Table 1. Liver microsomes were isolated, washed, and stored at -80 °C until use as previously described (23). Protein concentrations for mouse, rat, and human liver microsomes were determined by the method of Lowry et al. (24). Microsomes from human B-lymphoblastoid cell lines expressing cDNAs encoding 12 individual human P450 enzymes as well as microsomes from the parent cell line as controls were obtained from Gentest (Woburn, MA). Of the P450 enzymes tested, only the 1A2, 2D6-Met, and 4A11-containing microsomes did not contain supplemental P450 reductase. However, according to Gentest, these microsomes contained enough endogenous reductase activity to support the P450 catalytic activity. Protein concentrations of the microsomes containing the cDNA-expressed P450s were carried out by the
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bicinchoninic acid method (Pierce, Rockford, IL). Cytochrome P450 content values per milligram of protein of the cDNAexpressed microsomes were provided by the vendor. Enzymatic Reactions for Direct HMVK Detection. Reactions containing 20 mM BDD and microsomal protein in 0.015 M KH2PO4 were preincubated for 3 min at 37 °C before the reaction was initiated by the addition of the NADPH-generating system consisting of 1 mM NADP+, 10 mM glucose 6-phosphate, and 6 units of glucose 6-phosphate dehydrogenase. Total reaction volume was 1 mL. The reaction was terminated by placing the vial on ice. In some experiments, the effect of inclusion of GSH on HMVK detection was examined. Concurrent control reactions without the NADPH-generating system were carried out to account for trace HMVK contamination in some BDD samples. An aliquot (10 µL) of the internal standard, 1-butanol (5 mM prepared in water), was added, and the solution was applied to 3-mL capacity Extrelut solid phase extraction column (EM Science, Gibbstown, NJ). The columns were eluted with 12 mL of methylene chloride, and the solution was concentrated to approximately 100 µL. The concentrated sample was analyzed by gas chromatography-flame ionization detection (GC-FID). GC-FID Conditions. Analysis of the methylene chloride extracts of the enzymatic samples (3 µL) was done on a HewlettPackard capillary 5890A gas chromatograph fitted with a DB-1 capillary GC column (J & W Scientific, Folsum, CA). The injection port and detector temperatures were 200 and 250 °C, respectively. Initial temperature was 50 °C for 5 min. The temperature increased at a rate of 30 °C/min to 95 °C. It then increased at a rate of 70 °C/min to 200 °C, where it was held for 3 min for a total run time of 11 min. Retention times of HMVK and the internal standard, 1-butanol, were 4.67 and 2.73 min, respectively. Only ratios of relative detector response for the two compounds were determined since HMVK was unstable when incubated in the phosphate buffer at 37 °C. Detection of the HMVK-GSH Conjugate in BDD Microsomal Incubations. Because HMVK was unstable in aqueous medium (data not shown), an assay was developed to trap enzymatically formed HMVK with GSH to form a stable conjugate. These incubations were carried out by preincubating 0.2 mM GSH, BDD (0.05-10 mM), and the NADPH generating system described above in a buffer of 0.1 M KH2PO4, 0.15 M KCl, and 1.5 mM EDTA, pH 7.4, for 3 min at 37 °C. The reaction was initiated by the addition of microsomal protein. Total reaction volume was 0.5 mL. To terminate the reaction, 35% perchloric acid (25 µL) was added and the reaction was placed on ice. The samples were centrifuged for 10 min at 3000 rpm. The supernatant was transferred to a clean tube, and an aliquot (100 µL) was placed into a microcentrifuge tube before 300 µL of fluoraldehyde (o-phthalaldehyde) reagent was added. After 1 min at room temperature, the reaction was quenched by addition of 50 µL of 10% acetic acid. The sample was then filtered through an Acrodisc LC-13 membrane syringe filter (Pall Gelman, Ann Arbor, MI) and immediately analyzed by HPLC as described below. Concurrent controls without microsomal protein or the NADPH-generating system were used to account for trace HMVK contamination in some BDD samples. Recovery of HMVK (0.2 mM) was assessed by incubating HMVK in the presence of GSH and quantitating the HMVK-GSH conjugate in the presence/absence of protein. Incubations with cDNA-expressed human P450s 1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2D6-Val, 2D6-Met, 2E1, 3A4, and 4A11 (8-73 pmol of P450/incubation) were carried out as described above with 1 or 10 mM BDD, 0.2 mM GSH, and the NADPH generating system for 15 min in a total reaction volume of 0.5 mL. Incubations containing microsomes prepared from the parent cell line were also carried out as controls. The enzymatic activities were normalized for P450 expression level. HPLC Conditions. Analyses were carried out on a Gilson HPLC system consisting of dual pumps and a Shimadzu RF-10AXL fluorescence detector with an excitation wavelength of 340 nm and an emission wavelength of 455 nm. The column used was a Beckman Ultrasphere ODS 5 µm column (4.6 × 250
Krause et al. mm) with a flow rate of 1 mL/min. The mobile phase used on pump A was 25 mM ammonium acetate, pH 5.0, while 100% methanol was used on pump B. The initial mobile phase composition was 25% B where it was maintained for 5 min. The gradient increased to 50% B over 5 min where it was held for 4.5 min. It increased to 60% B over 1.5 min where it was maintained for 3 min. The gradient then decreased to the initial 25% B over 3 min. Total run time was 26.5 min. The retention time of the HMVK-GSH conjugate was 12.5 min. The injection volume was 20 µL and the limit of detection was 0.05 nmol/mL. Calculation of Kinetic Parameters and Statistical Analyses. Eadie-Hofstee plots were used to visually detect deviations from linearity and to make estimates of kinetic constants (Km and Vmax). Statistical analyses were carried out using the SigmaStat software package (SSPS, Chicago, IL). Comparisons of means was done by ANOVA. When significant differences were determined from ANOVA analysis, the Student-NewmansKeul test was used to determine which means were significantly different. The significance level was set at 0.05.
Results When BDD was incubated with mouse, rat, or human liver microsomes in the presence of the NADPH generating system, a peak which coeluted with synthetic HMVK was detected in methylene chloride extracts analyzed by GC-FID. In the absence of NADPH, this peak is greatly reduced and is thought to be due to low level contamination of commercial BDD with HMVK. The peak was also dependent on incubation time and protein concentration (data not shown). Confirmation of the identity of the HMVK formed enzymatically was obtained by comparing the mass spectra of the peak obtained in mouse liver microsomes with that obtained from the HMVK synthesized from butyne-1,4-diol (Figure 2). The mass spectra exhibited the expected molecular ion at m/z 86. Major fragments’ ions were observed at m/z 55 and 31 and m/z 59 and 27, probably corresponding to fragmentation between C1 and C2 and C2 and C3, respectively. The two spectra exhibited identical fragmentation patterns, providing direct evidence for HMVK formation in BDD incubations. Preliminary experiments indicated that HMVK was unstable in KH2PO4 buffer, pH 7.4 at 37 °C, with disappearance t1/2 of approximately 10 min (data not shown). Because HMVK was expected to act as a Michael acceptor, experiments were carried out to determine the effect of inclusion of GSH on HMVK, directly detected by GC (Figure 3). Inclusion of GSH without the NADPHgenerating system resulted in no HMVK detection. Inclusion of the NADPH-generating system but no GSH resulted in a statistically significant increase in HMVK levels over that detected in the incubations without the NADPH-generating system, consistent with oxidation of BDD to HMVK. Addition of GSH in the presence of NADPH reduced the HMVK levels below that due to contamination of the commercial BDD. HMVK was also shown to react readily with other thiol-containing compounds such as sodium ethanethiolate and N-acetyl cysteine (data not shown). These results suggest that HMVK reacts readily with GSH, and that HMVK could be effectively trapped as a GSH conjugate. To allow more accurate quantitation, the reactivity of HMVK with GSH was utilized to develop a fluorescent HPLC method to analyze for the HMVK-GSH conjugate, 1-hydroxy-2-keto-4-(S-glutathionyl)butane which was expected to be more stable than HMVK itself. Reference HMVK-GSH conjugate, 1-hydroxy-2-keto-4-(S-glutathio-
Oxidation of 3-Butene-1,2-diol
Figure 2. Identification of HMVK by GC/MS. Reactions were carried out as described in the Experimental Procedures. (A) Synthetic HMVK (B) Pooled reaction extracts from mouse liver microsomes. Fragments m/z 59/27 correspond to cleavage between C2 and C3 and fragments m/z 55/31 correspond to cleavage between C1 and C2. Reaction extracts not containing NADPH were also examined and did not contain the HMVK peak.
Figure 3. Effect of GSH (1 mM) on BDD (20 mM) oxidation to HMVK. The experiment was carried out as described in the Experimental Procedures. Bars that have different letter designations are significantly different from each other (p < 0.05).
nyl)butane, was synthesized by the reaction of HMVK and GSH, purified by semipreparative HPLC, and characterized by 1H NMR (please see Figure S1 in Supporting Information) and ESI-MS (Figure 4). The 1H NMR spectrum was as follows: 2.12 ppm, 2H, m, glutamate β
Chem. Res. Toxicol., Vol. 14, No. 12, 2001 1593
Figure 4. ESI-MS of the HMVK-GSH, 1-hydroxy-2-keto-4(S-glutathionyl)butane. (A) MS of synthetic HVMK-GSH conjugate. The fragment ions at m/z 394 and 416 correspond to M + 1 and M + Na. (B) MS/MS of the fragment ion at m/z 394. Fragment ions m/z 394, 319, 247, and 87 correspond to M + 1, loss of glycine, loss of glutamic acid, and loss of GSH, respectively.
protons; 2.48 ppm, 2H, m, glutamate γ protons; 2.74 ppm, 4H, s, HMVK methylene protons; 2.79 ppm, 1 H, dd, cysteine β proton; 2.97 ppm, 1 H, dd, cysteine β proton; 3.85 ppm, 1 H, dd, glutamate R proton; 3.88 ppm, 2 H, s, glycine R protons; 4.17 ppm, 2H, s, HMVK methylene protons; 4.30 ppm, 1H, m, cysteine R proton. The mass spectrum exhibited fragment ions at m/z 394, 319, 247, and 87 that correspond to the M + 1, loss of glycine, loss of glutamic acid, and loss of the GSH moiety, respectively. These results depicted in Figures S1 and 4 are consistent with the HMVK-GSH conjugate structure. When BDD (10 mM) was incubated with NADPH and mouse, rat, or human microsomes in the presence of GSH, a peak was detected in microsomal incubations that coeluted with reference HMVK-GSH derivatized with o-phthalaldehyde. The formation of this peak was dependent on the NADPH generating system, protein concentration, and incubation time (Figure S2). Recovery of HMVK as HMVK-GSH was nearly 100% in the presence of microsomal protein and GSH, indicating insignificant reaction between HMVK and protein sulfhydryl groups when excess GSH is present. Apparent kinetic constants (Vmax and Km) for the mouse, rat, and human liver microsomal reaction were determined using BDD concentrations from 0.05 to 10 mM. In the case of mouse and rat liver microsomes and one of the human liver samples (Figure 5, panels A-C), biphasic kinetics of BDD oxidation were observed. The
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Krause et al.
Discussion
Figure 5. Oxidation of BDD by mouse, rat, and human liver microsomes. (A) Mouse, (B) rat, (C) H3, (D) H1. Representative data are plotted using Eadie-Hofstee plots. Reactions were carried out as described in the Experimental Procedures using BDD concentrations ranging from 0.05 to 10 mM. Table 2. Apparent Kinetic Constants for HMVK-GSH Formation from BDD by Mouse, Rat, and Human Liver Microsomesa low Km
species
Vmax (nmol/mg of protein/ min)
rat mouse human (H1) (H2) (H3)
0.79 ( 0.20 0.92 ( 0.14 ND ND 0.20
high Km
Km (mM)
Vmax (nmol/mg of protein/ min)
Km (mM)
0.08 ( 0.03 0.05 ( 0.01 ND ND 0.07
1.55 ( 0.23 2.99 ( 0.78 1.19 0.91 1.00
0.61 ( 0.29 0.78 ( 0.09 1.16 0.72 1.76
a Kinetic constants were determined using Eadie-Hofstee plots using 0.05-10 mM BDD. All plots gave good correlation coefficients with r values ranging from 0.86 to 0.99. Values for mouse and rat are from three separate determinations. ND, not detected.
other two human samples exhibited monophasic plots (Figure 5D). The Vmax and Km values obtained from the Eadie-Hofstee plots are listed in Table 2. The relative ability of specific cDNA-expressed human P450 enzymes to catalyze BDD oxidation to HMVK was also investigated using the HMVK-GSH conjugate detection method. P450 2E1 was the only tested enzyme that had detectable activity (61.5 pmol of HMVK-GSH/ pmol of P450/15 min) at 1 mM BDD (data not shown). At a higher BDD concentration (10 mM), 2E1 had an activity of 110.5 pmol of HMVK-GSH/pmol of P450/15 min and 3A4 also exhibited substantial activity, which was approximately 50% of the activity observed with 2E1. Experiments with the other P450 enzymes tested exhibited either undetectable (2A6,2C8, 2C9, and 4A11) or trace levels (1A1, 1A2, 1B1, 2D6-Met, and 2D6-Val) of HMVK-GSH at the high BDD (10 mM) concentration.
The results of the current study clearly demonstrate enzymatic formation of HMVK by mouse, rat, and human liver microsomes and by cDNA-expressed human P450s. HMVK is a highly reactive and potentially toxic metabolite of BDD whose formation had been previously postulated but never directly demonstrated or quantitated. Enzymatic formation of HMVK was initially characterized using GC and GC/MS methods. The results, which provided the first direct evidence for microsomal HMVK formation are consistent with the previous reports of P450-catalyzed oxidation of other allylic alcohols to yield R,β-unsaturated ketones (25). Since HMVK was unstable at physiological conditions and readily reacted with GSH to yield the corresponding HMVK-GSH conjugate, we utilized this latter reaction to trap HMVK in a stable form for quantitative analysis (Figure S2). Among the human cDNA-expressed P450 enzymes examined, P450 2E1 was the only enzyme that yielded detectable HMVK levels at a BDD concentration of 1 mM. At 10 mM BDD, P450 3A4 was also able to catalyze BDD bioactivation to HMVK, but to a lesser extent than 2E1. These data suggest that 2E1 would likely be the primary enzyme catalyzing this reaction following low-level exposure to BDD. However, biphasic kinetics with one of the human liver samples and the results obtained with the cDNA-expressed enzymes at the high BDD concentration suggest the involvement of multiple P450s. The fact that 3A4 is able to catalyze this reaction may be of significance as human liver typically contains a large amount of this enzyme (26). Both mouse and rat liver microsomes as well as one of the three human liver samples evaluated indicated biphasic kinetics. These results are consistent with the above suggested involvement of multiple P450s in BDD oxidation in these tissues. The Km values of 50-80 µM suggest that HMVK could be formed after rodent or human exposure to BDD or its parent molecule, BD. Only one of the three human liver samples exhibited the low Km component. However, more human liver samples need to be examined in order to account for variability in P450 expression that can affect HMVK formation. The Vmax values obtained with the human liver microsomes were lower than the values obtained in rat or mouse liver microsomes. The formation of both HMVK and the HMVK-GSH conjugate, 1-hydroxy-2-keto-4-(S-glutathionyl)butane is consistent with previous studies that identified 1,2dihydroxy-4-(N-acetyl-L-cysteinyl)butane (M1) in the urine of mice, rats, and humans following inhalation exposure to BD (19, 27). HMVK could give rise to 1,2-dihydroxy4-(N-acetyl-L-cysteinyl)butane via conjugation with GSH, a reaction that we have characterized in this study. The glycine and glutamate moieties of GSH would be cleaved by γ-glutamyltransferase and dipeptidase. The remaining cysteine moiety of GSH would be acetylated. Reduction of the keto group to the corresponding hydroxyl group would result in M1. The point in this process at which the keto reduction occurs is currently unknown, as is the mechanism for this reaction. One possibility involves the enzyme formaldehyde dehydrogenase, thought to be a class III alcohol dehydrogenase which catalyzes the GSHdependent oxidation of formaldehyde to formate (28). As many alcohol dehydrogenases are also known to function as carbonyl reductases, it is possible that this enzyme
Oxidation of 3-Butene-1,2-diol
could catalyze the reduction of carbonyl-containing GSH conjugates. Because BDD was suggested to be a major metabolite of BD and BMO in humans (19, 27), and human microsomes and cDNA-expressed P450s were able to catalyze BDD oxidation to HMVK, HMVK is likely to be a metabolite of BDD and BD in humans. This hypothesis is supported by the detection of mercapturate M1 in the urine of workers following occupational exposure to BD (27). BDD oxidation to HMVK may also be of toxicological significance since R,β-unsaturated ketones such as methyl vinyl ketone and crotonaldehyde have been shown to be cytotoxic and carcinogenic (29). HMVK may contribute to BD-induced toxicity/and or carcinogenicity by alkylating nucleophilic sites on proteins or DNA. Alternatively, HMVK may act as a Michael acceptor in its reaction with GSH and could lead to decreased availability of GSH to detoxify the other reactive BD metabolites such as BMO and DEB. Thus, the metabolism and toxicity of HMVK warrant further investigation.
Acknowledgment. This study was supported by the National Institutes of Health Grant ES06841. The authors acknowledge Kredenna Beverly and Christopher McCullough for their technical assistance on the project. Supporting Information Available: Proton NMR spectrum of the HMVK-GSH conjugate and a graph of the timedependent formation of HMVK-GSH conjugate by mouse, rat, and human liver microsomes. This material is available free of charge via the Internet at http://pubs.acs.org.
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