1,N2-Propanodeoxyguanosine Adducts of the 1,3-Butadiene

Oct 10, 2003 - Mispairing of a Site Specific Major Groove (2S,3S)-N-(2,3,4-Trihydroxybutyl)-2'-deoxyadenosyl DNA Adduct of Butadiene Diol Epoxide with...
1 downloads 6 Views 119KB Size
Download PDF
1448

Chem. Res. Toxicol. 2003, 16, 1448-1454

1,N2-Propanodeoxyguanosine Adducts of the 1,3-Butadiene Metabolite, Hydroxymethylvinyl Ketone Mark W. Powley,† Karupiah Jayaraj,‡ Avram Gold,‡ Louise M. Ball,‡ and James A. Swenberg*,†,‡ Department of Pathology and Laboratory Medicine, University of North Carolina-Chapel Hill School of Medicine, and Department of Environmental Sciences and Engineering, University of North Carolina-Chapel Hill School of Public Health, Chapel Hill, North Carolina 27599-7400 Received April 25, 2003

1,3-Butadiene (BD) is a rodent and human carcinogen. While several epoxides formed during BD metabolism are mutagenic and may contribute to BD carcinogenicity, another proposed metabolite, hydroxymethylvinyl ketone (HMVK), could also be involved. A significant quantity of HMVK is likely to be formed since it is a proposed intermediate in the metabolism of 3-butene1,2-diol (BD-diol) to 1,2-dihydroxy-4-(N-acetylcysteinyl)butane, the major mercapturic acid metabolite of BD in humans. In addition, BD-diol is a major BD metabolite in liver perfusion experiments in rodents. By analogy with other R,β-unsaturated carbonyls, HMVK is likely to be mutagenic via formation of promutagenic 1,N2-propanodeoxyguanosine adducts. The objective of the current study was to investigate the formation of such adducts in vitro. The reaction between HMVK and dGuo yielded two major products shown to be identical by positive ion electrospray-MS, having protonated molecular ions with m/z consistent with HMVK-derived 1,N2-propanodeoxyguanosine (HMVK-dGuo). Rechromatography of each fraction yielded two fractions with retention times identical to those initially isolated, suggesting equilibration between two diastereomers. Two partially resolved sets of 1H NMR signals were consistent with a 1:1 mixture of diastereomeric C-6-substituted adducts equilibrating slowly on an NMR time-scale. Following deglycosylation, C-6 substitution was verified by two-dimensional correlation NMR spectroscopy, indicating that the initial adducts were formed by Michael addition of dGuo-N1 to the terminal vinyl carbon followed by cyclization to the 1,N2-propano structure. Reactions with calf thymus DNA under physiological conditions yielded two sets of products. The first set had HPLC retention times and mass spectra identical to those of the previously characterized C-6-substituted HMVK-dGuo diastereomers. The second set had a molecular ion and fragmentation pattern identical to the C-6-substituted adducts and on this basis were assigned as the diastereomeric C-8 adducts. In addition to detecting HMVK-dGuo in treated DNA, the adducts were also present in control DNA. Overall, our research demonstrates that HMVK can form promutagenic DNA adducts and it therefore has the potential to play a role in BD-associated mutagenicity.

Introduction BD1

is carcinogenic in rodents (1, 2), and evidence suggests that it may also be a human carcinogen (3, 4). It is labeled as a known human carcinogen by the National Toxicology Program (5), a probable human carcinogen by the International Agency for Research on Cancer (6), and as carcinogenic to humans by inhalation by the U.S. Environmental Protection Agency (7). BD biotransformation involves several pathways resulting in mutagenic metabolites (Scheme 1). Of particu* To whom correspondence should be addressed. Tel: (919)966-6139. Fax: (919)966-6123. E-mail: [email protected]. † University of North Carolina-Chapel Hill School of Medicine. ‡ University of North Carolina-Chapel Hill School of Public Health. 1 Abbreviations: ADH, alcohol dehydrogenase; BD, 1,3-butadiene; BD-diol, 3-butene-1,2-diol; CT DNA, calf thymus DNA; DEB, 1,2:3,4diepoxybutane; EB, 1,2-epoxy-3-butene; EB-diol, 3,4-epoxy-1,2-butanediol; ESI+, positive ion electrospray; EVK, ethylvinyl ketone; GST, glutathione-S-transferase; HBAL, 2-hydroxy-3-butenal; HMVK, hydroxymethylvinyl ketone; HMVK-dGuo, HMVK-derived 1,N2-propanodeoxyguanosine; mEH, microsomal epoxide hydrolase; MI, 1,2-dihydroxy-4-(N-acetylcysteinyl)butane; MII, 1-hydroxy-2-(N-acetylcysteinyl)3-butene; MVK, methylvinyl ketone; P450, cytochrome P450; SRM, selected reaction monitoring.

lar importance in humans is the metabolic pathway leading to BD-diol and eventually MI, the primary mercapturic acid metabolite found in the urine of BDexposed workers (8). Furthermore, BD-diol is a major metabolite of BD in single pass liver perfusion experiments in rodents (9). Because the formation and elimination of BD-diol are important routes of BD metabolism, reactive intermediates in this pathway may play significant roles in BD carcinogenicity. While epoxide metabolites formed during BD biotransformation, especially DEB, are known mutagens (10, 11), their role in the carcinogenicity of BD is not fully understood. As a consequence, investigation of other potentially mutagenic metabolites is useful in the context of health risks associated with human exposure to BD. HMVK, an R,β-unsaturated carbonyl, is a proposed intermediate in the metabolism of BD-diol to MI (8, 12). Although it has not been detected in vivo, HMVK has been found in microsomal incubations using BD-diol as the substrate (13). HMVK has mutagenic potential based on comparisons with R,β-unsaturated carbonyls that yield

10.1021/tx030021h CCC: $25.00 © 2003 American Chemical Society Published on Web 10/10/2003

1,N2-Propanodeoxyguanosine Adducts Formed by HMVK

Chem. Res. Toxicol., Vol. 16, No. 11, 2003 1449

Scheme 1. BD Metabolism

positive results in mutagenicity assays (14). In addition, R,β-unsaturated carbonyls very similar in structure to HMVK, including crotonaldehyde (15), acrolein (16), MVK (17, 18), and EVK (18), form promutagenic 1,N2propanodeoxyguanosine adducts. The promutagenic nature of this type of DNA adduct has been demonstrated in a number of cases using bacteria (19-22), simian kidney COS-7 cells (23, 24), and mammalian cells (25, 26). Because HMVK is a likely intermediate in an important BD metabolic pathway, it is critical to determine if it is capable of forming promutagenic DNA adducts. The objective of the current study was to investigate in vitro reactions of HMVK with dGuo and CT DNA to determine whether 1,N2-propanodeoxyguanosine adducts are formed.

Experimental Procedures Chemicals. All chemicals and enzymes were obtained from Sigma Aldrich (St. Louis, MO). Caution: Because the toxicity associated with HMVK is unknown, it should be used in an approved laboratory hood while wearing proper personal protective equipment (i.e., gloves and lab coat). Instrumentation. Full scan ESI+-MS and ESI+-MS/MS spectra were obtained by direct injection of samples using a Finnigan LCQDECA ion trap mass spectrometer. The mobile phase was 1:1 methanol:water (v/v) acidified with 1% acetic acid, with a flow rate of 200 µL/min. LC-ESI+-MS/MS analyses were performed using a Finnigan Quantum triple-quadrupole mass spectrometer. High-resolution mass measurements were obtained using a VG70-250 SEQ mass spectrometer with a FAB source by direct insertion probe. NMR data were obtained on a Varian INOVA 500 spectrometer at 500 MHz for 1H and 125 MHz for 13C. 1H and 13C chemical shifts are referenced to the proton and carbon signals, respectively, of TMS. NMR spectra were recorded in DMSO-d6 unless otherwise stated. Coupling constants for the HMVK-Gua adduct were determined by spectral simulation using MestRe-C 2.3 software ([email protected]). UV spectra of the adducts in distilled deionized water were recorded on a Shimadzu UV106U UV/vis recording spectrophotometer. HPLC Analysis. HPLC analysis was performed using two separate systems. System 1 consisted of a Hewlett-Packard 1040A diode array detector, two Agilent Technologies 1100 Series quaternary pumps, and an Agilent 1100 Series vacuum degasser. System 2 was comprised of an Applied Biosystems model 757 Absorbance Detector and two Waters model 510 HPLC pumps. In both systems, sample components were separated by an Alltech Ultrasphere C18 octadecylsilane semiprep column (5 µm; 10 mm × 250 mm) preceded by a guard cartridge. The mobile phase flow composition was isocratic with

8% methanol and 92% distilled deionized water at a flow rate of 4 mL/min. Both phases were filtered through a 0.45 µm nylon filter prior to use. Following separation by HPLC, absorbance at 254 nm was monitored. HMVK. HMVK was synthesized by a modified MeyerSchuster rearrangement according to a published procedure (13, 27). The catalyst mercuric oxide (500 mg, 2.31 mmol), boron trifluoride diethyl etherate (500 mg, 3.52 mmol), and trichloroacetic acid (150 mg, 1.32 mmol) in 5 mL of ethyl acetate were stirred vigorously at 55 °C giving an orange-colored suspension. 2-Butyne-1,4-diol (10 g, 0.12 mol) in 40 mL of ethyl acetate prewarmed to 55 °C was added slowly, and the reaction was refluxed under vacuum at 45 °C for 1 h. Following addition of sodium carbonate (200 mg, 1.89 mmol) and filtration, the ethyl acetate filtrate was extracted with 40 mL of ddH2O. The aqueous solution of HMVK was stored at 4 °C and used without further purification. The HMVK concentration was determined by 1H NMR. A 1 mL aliquot of the ethyl acetate filtrate was extracted with 1 mL of 2H2O, and integrals of the well-resolved terminal vinyl protons of HMVK were compared with the methyl signal of methanol added as internal standard. Typical HMVK concentrations were 150 mg/mL. HMVK-dGuo Diastereomers (1a,b). An aqueous solution (pH ∼ 11) containing HMVK (∼150 mg, 1.74 mmol) and 2′-deoxyguanosine (40 mg, 0.15 mmol) was incubated for 24 h at 37 °C with shaking. Following incubation, the solution was neutralized with 1 M HCl, filtered through a 0.2 µm disposable syringe filter, and separated using HPLC system 1. Fractions with retention times of 13.8 and 17.6 min were collected and dried by centrifugal lyophilization. Each fraction was rechromatographed, giving two peaks with retention times identical to those of the initial chromatography. The fractions were collected, lyophilized, and stored at -20 °C. Proton assignments are based on a 1:1 mixture of diastereomers and two-dimensional NMR correlation data discussed in the following section. UV (ddH2O): λmax ) 258 nm. ESI+-MS: m/z 392 [M + K]+, 376 [M + Na]+, 354 [M + H]+. Daughter ions of [M + H]+ by ESI+MS/MS: m/z 238 [M - dR]+ and 152 [Gua + H]+. FAB-HRMS [M + H ]+ calcd for C14H20N5O6, 354.14136; found, 354.143. 1H NMR (as 1:1 mixture of diastereomers): 7.95, 7.94 (2 × s, 1H total, H-2), 7.89, 7.86 (2 × s, 1H, N5-H), 6.10 (m, 1H, H-1′), 5.70 (s, 1H, 6-OH), 5.26 (s, 1H, 3′-OH), 4.94 (broad s, 2H, 11OH, 5′-OH), 4.43 (m, 1H, H-8b), 4.34 (s, 1H, H-3′), 3.80 (s, 1H, H-4′), 3.59-3.40 (m, 5H, H-8a, H-5′, H-5′′, 11-CH2), 2.53 (m, 1H, H-2′), 2.16 (m, 1H, H-2′′), and 1.79 (m, 2H, H-7a,b) ppm. Racemic HMVK-Gua (2). Diastereomers 1a,b (2 mg) in 1 mL of 0.1 M HCl were heated at 75 °C for 45 min before being neutralized with 0.1 M NaOH, and the mixture was separated by HPLC system 2. The deglycosylated adduct with a retention time of 7.8 min was collected, lyophilized, and stored at -20 °C. UV (ddH2O): λmax ) 251 nm. ESI+-MS: m/z 260 [M + Na]+, 238 [M + H]+. Daughter ions of [M + H]+ by ESI+-MS/MS: m/z 220 (M - H2O)+ and 152 (Gua + H)+. FAB-HRMS [M + H]+

1450 Chem. Res. Toxicol., Vol. 16, No. 11, 2003 calcd for C9H12N5O3, 238.0940; found, 238.0951. 1H NMR: 12.25 (bs, 1H, N3-H), 7.68 (s, 1H, N5-H), 7.62 (s, 1H, H-2), 5.62 (s, 1H, 6-OH), 4.93 (s, 1H, 11-OH), 4.45 (dt, 1H, J8b,8a ) 13.5, J8b,7a ) 5.3, J8b,7b ) 3.8 Hz, H-8b), 3.54 (td, 1H, J8a,8b ) 13.5, J8a,7a ) 13.0, J8a,7b ) 3.3 Hz, H-8a), 3.50 (dd, 2H, J ) 11 Hz, 11-CH2), and 1.90-1.78 (m, 2H, J7a,7b ) 13.5, J7a,8b ) 3.8, J7a,8a ) 13.0, J7b,8b ) 3.8, J7b,8a ) 3.3 Hz, H-7a,b) ppm. 13C NMR: 149.0 (C-3a), 134.5 (C-2), 114.8 (C-10a), 78.4 (C-6), 66.2 (C-11), 34.1 (C-8), 28.0 (C-7) ppm. Reaction of HMVK with CT DNA. An aqueous solution (pH ∼ 7.4) of HMVK (∼300 mg, 3.5 mmol) was added to 1 mL of hydrated CT DNA (1 mg/mL) and incubated for 2 h at 37 °C with shaking. A large initial excess of HMVK was necessary because the half-life of this compound in KH2PO4 buffer (pH 7.4) at 37 °C is ∼10 min (13). CT DNA was precipitated by adding 10 mL of isopropyl alcohol and shaking for 1 min. The solution was centrifuged at 2000g for 15 min, and the supernatant was decanted. The DNA pellet was washed with 5 mL of ethanol and centrifuged again at 2000g for 5 min. After the ethanol was discarded and the pellet was allowed to air-dry, the DNA was rehydrated in 1 mL of ddH2O and stored at -80 °C. DNA Sample Preparation. A solution containing 500 µL of CT DNA (500 µg/mL) or 330 (1.5 mg/mL) or 1050 µL of 80 mM Tris-HCl buffer (pH 7) with 20 mM MgCl2, and 50 µL of DNase (200 U) was brought to a final volume of 2100 µL with ddH2O and incubated for 10 min at 37 °C. Fifty microliters each of phosphodiesterase I (0.013 U) and alkaline phosphatase (10 U) were then added, and the mixture was incubated for 1 h at 37 °C with shaking. The enzymes were removed with a Centricon-10 filter by centrifugation for 1 h at 5000g at 4 °C. The filtrate was applied to a pre-equilibrated solid phase extraction column. After the sample was loaded, the column was washed with 1 mL of ddH2O followed by 2 mL of 5% methanol. Samples were then eluted with 1 mL of 10% methanol. This adduct-containing fraction was collected, dried by centrifugal lyophilization, resuspended in 100 µL of ddH2O, and stored at -20 °C. LC-ESI+-MS/MS Analysis. Samples were analyzed on an Aquasil C18 column (5 µm; 2.1 mm × 150 mm) preceded by a guard cartridge, using solvents A, 5 mM ammonium formate (pH 5), and B, acetonitrile, using the following gradient: 0 f 15 min: 100% A; 15.01 f 20 min: 100 f 50% A; 20.01 f 25 min: 50% A; 25.01 f 30 min: 100% A. A 400 µL/min flow rate was used throughout. The LC eluant was analyzed either by ESI+-MS/MS of m/z 354 or by SRM of m/z 354 f 238. LC flow was diverted to waste from 0 f 17.25 min and again from 20.5 f 30 min.

Results and Discussion From the reaction of HMVK with dGuo, fractions with a 1:1 absorbance ratio at 254 nm having retention times 13.8 and 17.6 min were isolated using semi-preparative HPLC (Figure 1). Unreacted dGuo was also observed in this chromatogram at 10.9 min. ESI+-MS spectra of the 13.8 and 17.6 min fractions were identical having ions at m/z 354, 376, and 392. These ions are consistent with the protonated adduct and adducts with sodium and potassium ions. The ESI+-MS/MS spectra showed daughter ions corresponding to loss of deoxyribose, as expected for dGuo adducts. A second chromatographic separation produced two peaks with retention times identical to the fractions initially isolated suggesting epimerization between two interconvertible diastereomers. On this basis, the fractions were combined and characterized by 1H NMR. Epimerization may involve two mechanisms: ring opening via retro addition by the carbonyl at the exocyclic amino group of dGuo (Scheme 2) or reversible dehydration of the carbinolamine of 1 involving a Schiff base

Powley et al.

Figure 1. HPLC trace of products from reaction of HMVK with dGuo monitored at 254 nm.

Scheme 2. Proposed Equilibration of Diastereomeric HMVK-dGuo Adduct via Ring Opening

intermediate (Scheme 3). The latter mechanism was proposed by Chung et al. (17) for the epimerization of the 1,N2-propanodeoxyguanosine adduct formed by MVK. Both mechanisms are involved in the epimerization of acrolein-derived 1,N2-propanodeoxyguanosine (28). The 1H NMR spectrum of the combined fractions was consistent with a 1:1 mixture of diastereomeric C-6substituted 1,N2- propano adducts 1a,b equilibrating slowly on an NMR time-scale. The traces in Figure 2 show two partially resolved guanine imidazole proton signals at 7.95 and 7.94 ppm, two partially resolved signals at 7.89 and 7.86 ppm in the region expected for N5-H of a 1,N2-propano adduct (18), and a multiplet of overlapping signals at 6.10 ppm in the range expected for H-1′, all with equal intensities. The assignment of the N5-H protons was supported by the disappearance of signals at 7.89 and 7.86 ppm on exchange with added 2H O. 2 Resonances at 5.70, 5.26, and 4.94 ppm also vanished on addition of 2H2O and can therefore be attributed to hydroxyl protons. Raising the NMR probe temperature to 50 °C caused signals tentatively assigned to the

1,N2-Propanodeoxyguanosine Adducts Formed by HMVK

Chem. Res. Toxicol., Vol. 16, No. 11, 2003 1451

Scheme 3. Proposed Equilibration of Diastereomeric HMVK-dGuo Adduct via Formation of a Schiff Base Intermediate

Figure 2. 1H NMR spectrum (500 MHz, DMSO-d6) of diastereomeric mixture 1a,b. Insets are expansions of signals clearly indicating the presence of a mixture. Proton signals are identified on trace.

imidazole-H and N5-H protons to increase in half-width and decrease in intensity, as expected for equilibrating structures. In the 1H COSY NMR spectrum (Figure S1), crosspeaks indicating three diastereotopic methylene groups were evident in addition to the cross-peaks expected from the deoxyribose H-5′ and H-5′′ protons. Overlapping signals of one set of methylene protons were centered at 1.79 ppm. Overlapping signals from a second set of methylene protons accounted for two protons within a five proton complex of resonances clustered around 3.5 ppm. Connectivities of the deoxyribose signals established that the H-5′ and H-5′′ protons account for two additional protons in this cluster. The remaining proton had a strong cross-peak with a one proton multiplet at 4.43 ppm. Connectivities between the methylene signals at 1.79 ppm and the coupled one proton methylene signals at 4.43 and 3.5 ppm, as well as the chemical shifts of these resonances, were very close to those reported for the C-7 and C-8 methylene groups of C-6-substituted 1,N2-propano-dGuo adducts formed by acrolein (29), MVK, and EVK (18). In these compounds, the C-7 methylene resonances were closely spaced between 1.7 and 1.9 ppm and the C-8 methylene resonances, separated by ∼1 ppm, appeared at 3.5 and 4.5 ppm. On the basis of an analogous structure for the HMVK-dGuo adduct, the remaining diastereotopic methylene signals in the complex at 3.5 ppm could be assigned to hydroxymethylene protons at C-11. This assignment was supported by a cross-peak between a hydroxyl proton at 4.94 ppm and the 3.5 ppm complex. In the 1H NOESY NMR spectrum (Figure S2), the only interactions evident, in addition to those of the deoxyribose protons, were between the singlets tentatively assigned to N5-H at 7.89 and 7.86 ppm and the five proton cluster at 3.5 ppm. On the basis of models, the H-5′ and H-5′′ deoxyribose protons and the hydroxy-

methylene protons at C-11 could have cross-peaks with N5-H. A NOESY cross-peak between N5-H and the C-11 methylene protons would establish regiochemistry of the propano ring. However, the signals of the H-5′ and H-5′′ protons completely overlapped the C-11 methylene signals and as a consequence, it was not possible to make a specific assignment to the cross-peak. While twodimensional correlation spectra were consistent with C-6 substitution data derived from the dGuo adduct, it was not possible to definitively rule out addition of HMVK with the opposite orientation since the same sets of crosspeaks could occur with C-8 substitution. To resolve this question, the HMVK-dGuo adduct was deglycosylated by mild acid hydrolysis. In contrast to the nucleoside adduct, HPLC analysis of the hydrosylate produced a single major peak with a retention time of 7.8 min. Mass spectrometric analysis was consistent with the aglycone structure 2, and as expected for racemates, only a single set of resonances was evident in the one-

dimensional 1H NMR spectrum (Figure S3) and the 13C assignments determined from 1H, 13C HSQC, and HMBC spectra. A definitive assignment of regiochemistry was not possible from the 1H,13C HMBC spectrum, because no coupling could be detected between N5-H and any propano carbon or between C-10 and any propano proton. However, in the NOESY spectrum (Figure 3), cross-peaks were present between N5-H and the C-11 methylene group as well as the 6- and 11-hydroxy groups. On this basis, the regiochemistry of addition could be definitively ascribed to the regioisomer from initial Michael addition of dGuo-N1 at the terminal vinyl carbon of HMVK yielding the C-6-substituted propano adduct. This is consistent with the reported formation of an MVKderived C-6-substituted 1,N2-propanodeoxyguanosine adduct via Michael addition at dGuo-N1 (17). Additional support for the structural assignment in 1a,b is the observation that C-6 hydroxy-substituted 1,N2-propanodeoxyguanosine adducts uniquely undergo equilibration between diastereomers through epimerization of the chiral center (16-18).

1452 Chem. Res. Toxicol., Vol. 16, No. 11, 2003

Powley et al.

Figure 3. 1H NOESY cross-peaks showing interaction between N5-H and protons on C-6 substituents of HMVK-Gua. Cross-peaks are identified on spectrum.

Figure 4. Comparison of LC-ESI+-MS/MS (SRM of m/z 354 f 238) chromatograms from (a) HMVK-dGuo standard and (b) CT DNA treated with HMVK.

LC-ESI+-MS/MS analysis of the hydrosylate from HMVK-treated CT DNA showed the presence of previously characterized C-6-substituted diastereomers at 18.76 and 18.84 min, as well as two additional peaks in a 1:1 ratio present at 18.98 and 19.17 min (Figure 4a,b). The second set of peaks was observed using the same MS transition as the C-6-substituted adduct, and LC-ESI+MS/MS analysis of the molecular ion at m/z 354 (data not shown) showed the same fragmentation pattern as

the C-6-substituted adduct standard. We have, therefore, tentatively assigned the structures of these adducts as C-8-substituted diastereomers of HMVK-dGuo. Results of our experiments also demonstrated that HMVK-dGuo may be an endogenous DNA adduct. Although at lower levels than were observed in HMVKtreated CT DNA, we detected the four HMVK-dGuo diastereomers in the hydrosylate of control CT DNA (Figure 5a,b) and in unexposed rat liver DNA (data not shown). The source of the adducts in these samples is not known. However, it is known that a hemoglobin adduct formed by DEB and EB-diol (30) and the urinary metabolite MI (8) is found in unexposed humans. The source(s) of these biomarkers is also unknown. Albertini et al. (31) hypothesized that MI measured in urine from unexposed humans is derived from endogenous BD-diol. If BD-diol is an endogenous compound, it could also be a source for the hemoglobin adduct of EB-diol and the HMVK-dGuo adducts. It is apparent that HMVK-dGuo does form in duplex DNA under physiological conditions. The formation of both the C-6 and the C-8-substituted regioisomers in DNA is in contrast to the exclusive formation of the C-6substituted adduct in reactions with dGuo at pH ∼ 11. The formation of the C-8-substituted adduct is likely due to stereochemical control of the availability of reactive sites in DNA and not the reaction pH. This conclusion is based on studies demonstrating the exclusive formation of C-6-substituted adduct in reactions of MVK with dGuo at neutral pH (17, 18). The formation of both the C-6- and the C-8-substituted 1,N2-propanodeoxyguanosine adduct is of significance in assessing the mutagenic potential of HMVK. The C-6substituted regioisomer of acrolein has been shown to be mutagenic in human cells (25). A potential mechanism for the mutagenicity of this regioisomer has been described by Yang et al. (32). These authors suggest and there is recent evidence to support the hypothesis (24) that the adduct will be in ring-closed form at the replication fork, thereby blocking DNA polymerase. Even if the adduct does ring open, it will form an N1-oxo-

1,N2-Propanodeoxyguanosine Adducts Formed by HMVK

Chem. Res. Toxicol., Vol. 16, No. 11, 2003 1453

In summary, we have characterized a novel DNA adduct formed by the BD metabolite HMVK and demonstrated its formation in duplex DNA. The formation of this promutagenic adduct in vitro suggests that HMVK has the potential to contribute to BD carcinogenicity. Future studies must be performed to determine how much HMVK is formed in vivo and subsequently reacts with DNA or is detoxified by glutathione conjugation. To accomplish this, an LC-ESI+-MS/MS method to quantify the adducts is currently being developed. By determining the fate of HMVK, we will better understand its possible contribution to BD carcinogenicity and ultimately improve risk assessment.

Acknowledgment. We thank Robert Schoonhoven for his skilled assistance with graphical representation of the two-dimensional NMR data and Drs. Hasan Koc and Yutai Li for assistance with MS analyses. The mass spectrometry facility is partly funded by NIH Grants ES11746, P30-CA16086, and P30-ES10126. Additional support was provided by NIH Grants T32-ES07017-28 and F32-ES012357-01. Supporting Information Available: Figure S1, 2D 1H COSY, and Figure S2, 2D 1H NOESY NMR spectra for HMVKdG diastereomers 1a,b. Figure S3, 1H NMR spectrum for HMVK-Gua. This material is available free of charge via the Internet at http://pubs.acs.org.

References Figure 5. Comparison of LC-ESI+-MS/MS (SRM of m/z 354 f 238) chromatograms from (a) HMVK-dGuo standard and (b) untreated CT DNA.

Figure 6. C-8-substituted HMVK-dGuo.

propanyl adduct that will prevent Watson-Crick base pairing and block synthesis (32, 33). As a result of blocking DNA synthesis, error-prone specialized translesion DNA polymerases such as DNA polymerase η may become involved, possibly leading to the higher mutation rate. This has been demonstrated with the C-6-substituted 1,N2-propanodeoxyguanosine adduct formed by acrolein (34). When paired with dC, the C-8-substituted adduct of acrolein opens to an N2-(3-oxopropyl) structure possessing normal Watson-Crick base pairing properties (35). The mutagenicity data for this regioisomer are mixed. Several site specific mutagenesis studies have demonstrated that this adduct is nonmutagenic in bacteria (33, 36) and in human cells (25, 32). In contrast, there are reports of mutagenicity in simian kidney COS-7 cells (23, 24) and in human cells (26). The C-8-substituted acrolein adduct may be mutagenic based on the formation of DNA-DNA (24, 37, 38) and DNA-protein cross-links (24). Furthermore, Minko et al. (26) have proposed that DNA polymerase η may be involved in both error-free and error-prone synthesis across the C-8-substituted 1,N2propanodeoxyguanosine adduct of acrolein.

(1) Owen, P. E., Glaister, J. R., Graunt, I. F., and Pullinger, D. H. (1987) Inhalation toxicity studies with 1,3-butadiene. 3. Two-year toxicity/carcinogenicity study in rats. Am. Ind. Hyg. Assoc. J. 48, 407-413. (2) Melnick, R. L., Huff, J., Chou, B. J., and Miller, R. A. (1990) Carcinogenicity of 1,3-butadiene in C57BL/6 x C3HF1 mice at low exposure concentrations. Cancer Res. 50, 6592-6599. (3) Delzell, E., Sathiakumar, N., Hovinga, M., Macaluso, M., Julian, J., Larson, R., Cole, P., and Muir, D. C. F. (1996) A follow-up study of synthetic rubber workers. Toxicology 113, 182-189. (4) Divine, B. J., and Hartman, C. M. (1996) Mortality update of butadiene production workers. Toxicology 113, 169-181. (5) NTP (2000) Report on Carcinogens, 9th ed., United States Department of Health and Human Services, Public Health Service, National Toxicology Program, Research Triangle Park, NC. (6) IARC (1999) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Vol. 71, pp 109-225, International Agency for Research on Cancer, Lyon, France. (7) USEPA (2002) Health Assessment of 1,3-Butadiene, Publication 600/P-98/001F, United States Environmental Protection Agency, National Center for Environmental Assessment, Washington DC. (8) 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. (9) Filser, J. G., Faller, T. H., Bhowmik, S., Schuster, A., Kessler, W., Putz, C., and Csanady, G. A. (2001) First-pass metabolism of 1,3-butadiene in once-through perfused livers of rats and mice. Chem.-Biol. Interact. 135-136, 249-265. (10) Cochrane, J. E., and Skopek, T. R. (1994) Mutagenicity of butadiene and its epoxide metabolites: I. Mutagenic potential of 1,2-epoxybutene, 1,2,3,4-deepoxybutane and 3,4-epoxy-1,2-butanediol in cultured human lymphoblasts. Carcinogenesis 15, 713717. (11) 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 hrpt locus in splenic T cells from exposed B6C3F1 mice. Carcinogenesis 15, 719-723. (12) 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.

1454 Chem. Res. Toxicol., Vol. 16, No. 11, 2003 (13) Krause, R. J., Kemper, R. A., and Elfarra, A. A. (2001) 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. Chem. Res. Toxicol. 14, 1590-1595. (14) Eder, E., Scheckenbach, S., Deininger, C., and Hoffman, C. (1993) The possible role of R,β-unsaturated carbonyl compounds in mutagenesis and carcinogenesis. Toxicol. Lett. 67, 87-103. (15) Chung, F.-L., and Hecht, S. S. (1983) Formation of cyclic 1,N2adducts by reaction of R-acetoxy-N-nitrosopyrrolidine, 4-(carbethoxynitrosamino)butanal, or crotonaldehyde. Cancer Res. 43, 1230-1235. (16) Chung, F.-L., Young, R., and Hecht, S. S. (1984) Formation of cyclic 1,N2-propanodeoxyguanosine adducts in DNA upon reaction with acrolein or crotonaldehyde. Cancer Res. 44, 990-995. (17) Chung, F.-L., Roy, K. R., and Hecht, S. S. (1988) A study of reactions of R,β-unsaturated carbonyl compounds with deoxyguanosine. J. Org. Chem. 53, 14-17. (18) Eder, E., Hoffman, C., and Deininger, C. (1991) Identification and characterization of deoxyguanosine adducts of methyl vinyl ketone and ethyl vinyl ketone. Genotoxicity of the ketones in the SOS Chromotest. Chem. Res. Toxicol. 4, 50-57. (19) Benamira, M., Singh, U., and Marnett, L. J. (1992) Site-specific frameshift mutagenesis by a propanodeoxyguanosine adduct positioned in the (CpG)4 hot-spot of Salmonella typhirium his D 3052 carried on an M13 vector. J. Biol. Chem. 267, 392-400. (20) Burcham, P. C., and Marnett, L. J. (1994) Site-specific mutagenesis by a propanodeoxyguanosine adduct carried on an M13 genome. J. Biol. Chem. 269, 28844-28850. (21) Moriya, M., Zhang, W., Johnson, F., and Grollman, A. P. (1994) Mutagenic potency of exocyclic DNA adducts: marked differences between escherichia coli and simian kidney cells. Proc. Natl. Acad. Sci. U.S.A. 91, 11899-11903. (22) Fink, S. P., Reddy, G. R., and Marnett, L. J. (1997) Mutagenicicty in escherichia coli of the major DNA adduct derived from the endogenous mutagen malondialdehyde. Proc. Natl. Acad. Sci. U.S.A. 94, 8652-8657. (23) Kanuri, M., Minko, I. G., Nechev, L. V., Harris, T. M., Harris, C. M., and Lloyd, R. S. (2002) Error prone translesion synthesis past γ-hydroxypropano deoxyguanosine, the primary acrolein-derived adduct in mammalian cells. J. Biol. Chem. 277, 18257-18265. (24) Sanchez, A. M., Minko, I. G., Kurtz, A. J., Kanuri, M., Moriya, M., and Lloyd, R. S. (2003) Comparative revaluation of the bioreactivity and mutagenic spectra of acrolein-derived R-HOPdG and γ-HOPdG regioisomeric deoxyguanosine adducts. Chem. Res Toxicol. 16, 1019-1028. (25) Yang, I.-Y., Chan, G., Miller, H., Huang, Y., Torres, M. C., Johnson, J., and Moriya, M. (2002) Mutagenesis by acroleinderived propanodeoxyguanosine adducts in human cells. Biochemistry 41, 13826-13832. (26) Minko, I. G., Washington, M. T., Kanuri, M., Prakash, L., Prakash, S., and Lloyd, R. S. (2003) Translesion synthesis past acrolein-derived DNA adduct, γ-hydroxypropanodeoxyguanosine,

Powley et al.

(27) (28)

(29) (30)

(31)

(32) (33)

(34)

(35)

(36)

(37)

(38)

by yeast and human DNA polymerase η. J. Biol. Chem. 278, 784790. Swaminathan, S., and Narayanan, K. V. (1971) The Rupe and Meyer-Schuster Rearrangements. Chem. Rev. 71, 429-438. Nechev, L. V., Kozekov, I. D., Brock, A. K., Rizzo, C. J., and Harris, T. H. (2002) DNA adducts of acrolein: site-specific synthesis of an oligdeoxynucleotide containing 6-hydroxy-5,6,7,8-tetrahydropyrimido[1,2-a]purin-10(3H)-one, an acrolein adduct of guanine. Chem. Res. Toxicol. 15, 607-613. Huang, Y., and Johnson, F. (2002) Regioisomeric synthesis and characteristics of the R-hydroxy-1,N2-propanodeoxyguanosine. Chem. Res. Toxicol. 15, 236-239. Swenberg, J. A., Christova-Gueorguieva, N. I., Upton, P. B., Ranasinghe, A., Scheller, N., Wu., K.-Y., Yen, T.-Y., and Hayes, R. (2000) Part V: Hemoglobin adducts as biomarkers of 1,3butadiene exposure and metabolism. In 1,3-Butadiene: Cancer, Mutations, and Adducts (Health Effects Institute Research Report Number 92) pp 191-210, Health Effects Institute, Boston. Albertini, R. J., Sram, R. J., Vacek, P. M., Lynch, J., Nicklas, J. A., vam Sittert, N. J., Boogaard, P. J., Henderson, R. F., Swenberg, J. A., Tates, A. D., Ward, J. B., and Wright, M., et al. (2003) Biomarkers in Czech Workers Exposed to 1,3-Butadiene: a Transitional Epidemiologic Study, Research Report 116, Health Effects Institute, Boston, MA. Yang, I.-Y., Johnson, F., Grollman, A. P., and Moriya, M. (2002) Genotoxic mechanism for the major acrolein-derived deoxyguanosine adduct in human cells. Chem. Res. Toxicol. 15, 160-164. VanderVeen, L. A., Hashim, M. F., Nechev, L. V., Harris, T. M., Harris, C. M., and Marnett, L. J. (2001) Evaluation of the mutagenic potential of the principal DNA adduct of acrolein. J. Biol. Chem. 276, 9066-9070. Yang, I.-Y., Miller, H., Wang, Z., Frank, E. G., Ohmori, H., Hanaoka, F., and Moriya, M. (2003) Mammalian translesion DNA synthesis across an acrolein-derived deoxyguanosine adduct: participation of DNA polymerase η in error-prone synthesis in human cells. J. Biol. Chem. 278, 13989-13994. de los Santos, C., Zaliznyak, T., and Johnson, F. (2001) NMR characterization of a DNA duplex containing the major acroleinderived deoxyguanosine adduct γ-OH-1,-N2-propano-2′-deoxyguanosine. J. Biol. Chem. 276, 9077-9082. Yang, I.-Y., Hossain, M., Miller, H., Khullar, S., Johnson, F., Grollman, A., and Moriya, M. (2001) Responses to the major acrolein-derived deoxyguanosine adduct in escherichia coli. J. Biol. Chem. 276, 9071-9076. Kozekov, I. D., Nechev, L. V., Sanchez, A., Harris, C. M., Lloyd, R. S., and Harris, T. M. (2001) Interchain cross-linking of DNA mediated by the principal adduct of acrolein. Chem. Res. Toxicol. 14, 1482-1485. Kozekov, I. D., Nechev, L. V., Moseley, M. S., Harris, C. M., Rizzo, C. J., Stone, M. P., and Harris, T. M. (2003) J. Am. Chem. Soc. 125, 50-61.

TX030021H