Chem. Res. Toxicol. 1997, 10, 779-785
779
Synthesis, Characterization, and in Vitro Quantitation of N-7-Guanine Adducts of Diepoxybutane Natalia Yu. Tretyakova, Ramiah Sangaiah, Ten-Yang Yen, and James A. Swenberg* Laboratory of Molecular Carcinogenesis and Mutagenesis, Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, North Carolina 27599-7400 Received January 15, 1997X
Diepoxybutane (DEB) is an important metabolite of 1,3-butadiene (BD), a high-volume industrial chemical classified as a probable human carcinogen. Rodent inhalation studies show strikingly high sensitivity of mice to carcinogenic effects of butadiene compared to rats, which has been linked to differences in metabolism. Both species convert BD to 3,4-epoxy-1-butene (EB), but mice further oxidize a significantly greater part of EB to DEB. DEB is a potent bifunctional genotoxic agent which is 100-fold more mutagenic than EB and is likely to be involved in BD-induced carcinogenesis. Identification of specific BD-induced DNA adducts is critical to understanding the mechanism of its biological activity. We have previously described reactions of EB with guanine and adenine as nucleobases, nucleosides, and constituents of DNA. In this work, DEB-induced guanine adducts were isolated and structurally characterized by UV spectroscopy, mass spectrometry, and nuclear magnetic resonance. When guanosine was reacted with DEB in glacial acetic acid followed by hydrolysis in hydrochloric acid, three products were isolated: N-7-(2′,3′,4′-trihydroxybut-1′-yl)guanine (DEB-Gua I, major adduct), N-7-(2′,4′-dihydroxy-3′-chlorobut-1′-yl)guanine (DEB-Gua II), and N-7-(2′,3′-dihydroxy-4′acetoxybut-1′-yl)guanine (DEB-Gua III). We suggest initial formation of the N-7-(2′-hydroxy3′,4′-epoxybut-1′-yl)guanine intermediate followed by nucleophilic substitution at the 3′,4′epoxy ring with hydroxide, chloride, or acetate anions to give DEB-Gua I, II, or III, respectively. DEB-Gua I and the epoxy intermediate were also isolated from hydrolysates of DEB-exposed calf thymus DNA (CT DNA). N-7-Guanine adducts are known to undergo spontaneous and enzymatic depurination producing apurinic sites. If not repaired before DNA replication, apurinic sites can give rise to mutations and ultimately cancer. The extent of alkylation at the N-7 of guanine in DEB-exposed DNA (58.7 ( 1.1 adducts/103 normal guanines) was similar to that previously reported for CT DNA exposed to EB at the same molar ratio. Since EB and DEB appear to induce comparable levels of overall DNA alkylation at the conditions applied in this work, other factors, such as formation of DNA cross-links by DEB but not EB or differences in repair of EB and DEB adducts, may be responsible for the differences in mutagenicity.
Introduction Butadiene (BD)1 is an important industrial chemical widely used as a monomer in production of synthetic rubber and plastics. It is also an environmental pollutant found in cigarette smoke and automobile exhaust (1, 2); therefore, low exposures to BD are characteristic for a large segment of the human population. BD is recognized as a probable human carcinogen (3), but the molecular mechanisms underlying its activity are not well understood. Long term inhalation studies with rodents show markedly higher sensitivity of mice to BDinduced neoplasia compared to rats (about 1000-fold; 4-6). Furthermore, the target organs in rats (thyroid, pancreas, testis for males, uterus for females)are different from those in mice (heart, lungs, liver, hematopoietic system). * Corresponding author. Telephone: (919) 966-6139. Fax: (919) 9666123. X Abstract published in Advance ACS Abstracts, June 15, 1997. 1 Abbreviations: Ade, adenine; BD, 1,3-butadiene; CT DNA, calf thymus DNA; 2′-dAdo, 2′-deoxyadenosine; DAD, diode array detector; DEB, diepoxybutane; DEB-Gua I, N-7-(2′,3′,4′-trihydroxybut-1′-yl)guanine; DEB-Gua II, N-7-(2′,4′-dihydroxy-3′-chlorobut-1′-yl)guanine; DEB-Gua III, N-7-(2′,3′-dihydroxy-4′-acetoxybut-1′-yl)guanine; EB, 3,4epoxy-1-butene; ESI-MS, electrospray ionization-mass spectrometry; Gua, guanine; LSI-MS, liquid secondary ion-mass spectrometry.
S0893-228x(97)00004-0 CCC: $14.00
The observed species differences are likely to be due to differences in the in vivo metabolism of BD. BD is epoxidized by the cytochrome P450-dependent monooxygenase CYP2E1 to its major metabolite, 3,4-epoxy-1butene (EB) (7-9), which undergoes further metabolism. EB can be further oxidized to diepoxybutane (DEB), hydrolyzed to 3-butene-1,2-diol via epoxide hydrolase, or conjugated to glutathione (10-12). 3-Butene-1,2-diol can be either eliminated through glutathione conjugation or metabolized to 3,4-epoxy-1,2-butanediol. In rats, EB is primarily hydrolyzed to 3-butene-1,2-diol with little oxidation to DEB. In contrast, mice oxidize a large fraction of EB to DEB, resulting in 100-fold higher blood concentrations of DEB in exposed mice compared to rats (13). DEB is a highly mutagenic bifunctional electrophilic agent capable of forming DNA-DNA and DNAprotein cross-links (14). Since DEB formation is the major metabolic difference between rats and mice, DEB may be of primary importance for the biological effects of BD. Only limited evidence for carcinogenicity of BD in humans is available (15-20). No exposure-related effects in chromosomal aberrations, micronucleus formation, or sister chromatid exchange were observed in peripheral blood lymphocytes of workers exposed to less than 1 ppm BD (20). However, recent studies demonstrated a detect© 1997 American Chemical Society
780 Chem. Res. Toxicol., Vol. 10, No. 7, 1997
able increase in the incidence of lymphohematopoietic cancers in workers exposed to BD (21). In addition, Ward et al. (22) reported an increased hprt mutant frequency in the peripheral lymphocytes of workers at a BD production plant in Texas, which correlated with the urinary levels of 1,2-dihydroxy-4-(N-acetylcystein-S-yl)butane, a glutathione conjugate of 3-butene-1,2-diol. Experimental mutagenicity studies have shown that both EB and DEB are direct mutagens and genotoxic agents in various in vitro and in vivo systems. Mutation frequency data for human lymphoblastoid cell cultures indicate that DEB is about 2 orders of magnitude more potent than EB (23). DNA sequence analysis of hprt exon 3 from splenic T-cells of exposed B6C3F1 mice showed that about one-half of all the mutations induced by BD and its metabolites were frameshift mutations with a +1 “hotspot” in six consecutive guanines (24). In addition, base pair substitutions at both GC and AT base pairs were observed. DEB was also shown to cause deletions, probably due to the formation of interstrand DNA crosslinks. To date it is not known which DNA adducts are responsible for BD mutagenicity and carcinogenicity. The epoxides of BD can be expected to undergo SN2-type substitution at the carbon with the lowest degree of substitution (terminal carbon). Highly nucleophilic sites in DNA, such as N-7 of guanine and N-3 of adenine, are most likely to participate in these reactions. Indeed, EB has been shown to react with calf thymus DNA at the N-7 of guanine and N-3 of adenine giving pairs of regioisomeric adducts (25-28). Jellito et al. (29) reported N-7-(2′,3′,4′-trihydroxybut-1′-yl)guanine as the major guanine adduct of DEB and detected this adduct in livers of BD-exposed mice, but not rats, by coelution of radioactivity with a known standard. Unfortunately, no spectral data (NMR, MS, UV) supporting the identification of the adduct were presented, and the in vivo adduct levels were very close to the detection limit of the method. Leuratti et al. (30) observed several adducts from reactions of deoxyguanosine monophosphate and poly(dG-dC) with DEB using 32P-postlabeling, but the chemical identities of these have not been determined. The present paper reports synthesis and full structural characterization of DEB-induced N-7-guanine adducts. The synthesized adducts were applied as synthetic markers for identification and quantitation of DEB-guanine adducts in exposed calf thymus DNA (CT DNA). In addition, the unstable monoepoxy intermediate N-7-(2′-hydroxy-3′,4′epoxybut-1′-yl) was isolated from DEB-exposed CT DNA and characterized using various spectral methods.
Experimental Section Caution: DEB is carcinogenic and should be handled with extreme caution. Materials. HPLC-grade water and methanol were purchased from Fisher Scientific (Fair Lawn, NJ) and Malinckrodt (Paris, KY), respectively. DEB was obtained from Aldrich (Milwaukee, WI). Calf thymus DNA (CT DNA) and guanosine (Guo) were acquired from Sigma Chemical Co. (St. Louis, MO). Solid phase extraction columns (ODS-AQ, 500 mg, 3 mL) were obtained from YMC (Wilmington, NC). Other chemicals were purchased from Fisher Scientific (Fair Lawn, NJ). Reaction of Guo with DEB. Guo (5-50 mg) was reacted with DEB (3.5 equiv) in 5 mL of glacial acetic acid at 37 °C for 7 h. At the end of the reaction, the solvents were evaporated under reduced pressure, and the reaction mixtures were heated in 3 mL of 1 N HCl at 100 °C for 1 h to release the free bases. The hydrolysates were neutralized with ammonia, diluted with water, and separated by HPLC as described below.
Tretyakova et al. Reaction of DEB with CT DNA. CT DNA (15 mg portions) was reacted with 1.24 mmol of DEB in 4 mL of 5 mM Tris-HCL buffer (pH 7.2) at 37 °C for 18 h. The unreacted DEB was extracted with 3 × 5 mL of diethyl ether; 0.1-0.5 mL aliquots of this solution were hydrolyzed at 100 °C for 30 min (neutral thermal hydrolysis) or in 0.1 N HCl at 70 °C for 30 min (acid hydrolysis) followed by filtration on Centricon 3 microfilters (Amicon, Beverly, MA). The resulting filtrates were subjected to HPLC analysis as described below. The reaction was repeated six times in separate vessels to enable statistical analysis of the data. HPLC Analyses. HPLC analyses were performed using Beckman C18 columns, two Waters 510 pumps equipped with a Hewlett Packard 1040A diode-array detector (DAD), and an HPLC detection computer system. The eluents were 70% aqueous methanol (A) and either water (B) or 20 mM potassium phosphate buffer, pH 5.5 (C), and the gradient was linear from 15% A/85% B to 50% each over 30 min (gradient program 1), 15% A/85% C to 50% each in 30 min (gradient program 2), or 5% A/95% C to 50% each in 30 min (gradient program 3). Primary separation of the reaction mixtures was achieved by using a 250 × 10 mm ODS column (Beckman, Fullerton, CA) eluted at 3 mL/min with gradient program 1. Further purification was performed using a 250 × 4.6 mm ODS column (Alltech, Deerfield, IL) eluted with gradient program 2. Quantitative analyses of DEB-Gua adducts in hydrolysates of DEB-exposed calf thymus DNA were performed using 250 × 4.6 mm ODS columns (Alltech, Deerfield, IL) with gradient program 3; 757 UV absorbance detector (Applied Biosystems, Inc., Ramsey, NJ) was set at λ ) 284 nm corresponding to the maximum absorbance of N-7-guanine adducts. Adduct concentrations were determined by measuring HPLC peak areas using calibration curves constructed with standard solutions. The calibration curves were linear over the range from 50 pmol to 10 nmol of the reference adducts. Solid Phase Extraction. The adducts were desalted by applying to ODS-AQ solid phase extraction columns (500 mg, 3 mL; from YMC, Wilmington, NC). The cartridges were primed with 6 mL of methanol and 6 mL of water, and the samples were loaded by gravity. The columns were washed with 5 mL of water, and the samples were recovered with 5 mL methanol. The resulting solutions were evaporated under reduced pressure, and the purified compounds were desiccated over P2O5 under vacuum. Standard solutions were made in water and stored at -20 °C. UV Spectrophotometry. Small amounts of purified, desalted, and desiccated adducts were weighed and dissolved in 1 mL of distilled water, 0.1 N HCl, or 0.1 N NaOH (2-5 × 10-5 M solution), and ultraviolet (UV) spectra were obtained with a Shimadzu (Columbia, MD) 160U UV spectrophotometer. LC/ESI-MS Analyses. The HPLC fractions corresponding to DEB-guanine adducts were collected, desalted, and redissolved in 100 µL of water; 5-10 µL of aqueous solution was used for LC/ESI-MS analysis. LC/ESI-MS analyses were conducted using a Pharmacia liquid chromatographic system with dual pumps #2248 (Pharmacia LKB Biotechnology, Uppsala, Sweden) coupled to a Finnigan 4000 quadrupole mass spectrometer (Finnigan-MAT, San Jose, CA) retrofitted with a pneumatic electrospray source (Analytica of Branford, Inc., Branford, CT). The solvents (water and/or methanol) were passed through a mixing tee. One end of the tee was connected to pressure balance columns (C18, 150 × 4.6 and 250 × 4.6 mm), while the other end was connected to a capillary C18 column (150 × 0.8 mm, Hypersil, 3 µm particle size; LC Packings, San Francisco, CA). This allowed us to deliver 25 µL/min flow through the capillary column while the pumps were operated at 0.45 mL/ min flow rate. A piece of fused-silica gel capillary (30 cm × 50 µm i.d.) directed the eluent of the capillary column to the electrospray needle. Chromatographic separations were accomplished by changing the mobile phase from 97% water/3% methanol to 25% water/ 75% methanol in 18 min, altering the phase to 2% water/98% methanol in 4 min, and washing the column with 100% methanol for 3 min. A voltage of 3.1 kV was applied to the electrospay needle, and 70 psi of the nebulizer gas (N2) was
Guanine Adducts of Diepoxybutane
Chem. Res. Toxicol., Vol. 10, No. 7, 1997 781
Table 1. Retention Times and UV Spectra of the DEB Adducts with Guanine Compared to Reference Compounds tR, mina
a
λmax/min
compound
10 mm column
4.6 mm column
pH ) 7
pH ) 1
pH ) 12
MW
DEB-Gua I DEB-Gua II DEB-Gua III EB-Gua Ib EB-Gua IIb N-7-Et-Guac
6.8 12.0 12.7 14.4 17.2
3.0 6.9 7.3 9.4 10.8
284, 249/261 284, 249/261 284, 249/261 284, 249/260 284, 249/260 282, 248/260
250, 270/230 250, 270/235 250, 270/238 250, 270/240 250, 270/240 250, 273/240
281/257 282/258 281/260 280/260 280/260 279/260
255 273 297 205 205
250 × 4.6 mm ODS column (Alltech), gradient program 2. b From ref 28. c Commercial standard.
Figure 1. HPLC separation of the reaction mixture of DEB + guanosine after acid hydrolysis (250 × 10 mm C18 column, 3 mL/min, gradient program 2). UV detection at 284 nm.
Figure 2. UV absorption spectra of DEB-Gua I at acidic, neutral, and alkaline pH. applied to stabilize the spay. The voltage difference between the exit of glass capillary and the first skimmer in the differential pumping region was optimized at 80 V to obtain (M + H)+ ions of the adducts and was set to 160 V to promote collisioninduced dissociation of the (M + H)+ ions. The data were acquired and processed by a Technivent Vector 2 data system (Technivent Corp., Maryland Heights, MO). Full scan mass spectra were obtained by scanning from m/z 70 to 500 every 2 s. NMR Spectrometry. The proton NMR spectra were obtained on a Bruker AMX-500 spectrometer using 0.5-1 mg of purified, desalted, and desiccated adducts, and chemical shifts are reported in ppm relative to the proton resonance of TMS.
Results DEB Reaction with Guo. HPLC analysis of acid hydrolysates of the reaction mixture from reaction of Guo with DEB in glacial acetic acid revealed one major and two minor products which were named DEB-Gua I, DEBGua II, and DEB-Gua III in order of their retention times (Figure 1, Table 1). No unreacted guanine was observed. All three adducts shared the same UV spectrum with λmax of 284 nm at neutral and alkaline conditions and λmax of
250 nm at low pH (Figure 2). These changes of the UV spectrum with pH are characteristic for N-7-substituted guanines (31-34); therefore, DEB-Gua I-III were preliminary identified as N-7-alkylguanines. The ESI+ mass spectrum of DEB-Gua I at low voltage contained one major peak at m/z ) 256, corresponding to the molecular ion of guanine having one trihydroxybutyl substituent (Figure 3a). The deprotonated molecules were seen at m/z ) 254 under ESI--MS conditions (results not shown). ESI+ CID spectrum of the (M + H)+ ions revealed one predominant fragmentation process: elimination of the entire trihydroxybutyl substituent to give protonated guanine ions at m/z ) 152 (Figure 3a). This is consistent with the ESI+-MS fragmentation pattern of other described N-7-alkylguanines (27). Similar results were obtained from LSI-MS and LSI-MS/MS (results not shown). Therefore, DEB-Gua I was identified as N-7-(trihydroxybutyl)guanine. Further support for this assignment and more detailed structure of the side chain were obtained from proton NMR spectra (Table 2) which contained signals for both guanine and trihydroxybutyl moieties. Proton assignment was based on comparison of chemical shifts, multiplicities, coupling constants, and integration ratios with the published spectra for N-7-EB-guanine and N-7-EB-guanosine adducts (2527). The presence of the signals for H-1 (10.9 ppm) and H-8 (7.8 ppm) and a two-proton signal for NH2 (6.2 ppm) were consistent with alkylation at the N-7 of guanine. Since some of the signals of the side chain protons overlapped with the broad solvent signal (3.3 ppm) due to the residual water in DMSO-d6, we also obtained the spectrum in D2O/NaOD. Similar to the N-7-EB-Gua adducts (27), the signals for diastereotopic 1′-CH2 methylene protons appeared as two separate doublets of doublets at 3.88 and 4.12 ppm (Table 2). In DMSO-d6, the 2′- and 3′-OH were seen as a two-proton signal at 4.71 ppm, whereas the primary 4′-OH was located upfield from it at 4.5 ppm. 2′- and 3′-methine proton assignment was accomplished by recording two-dimensional COSY 1H NMR spectrum of DEB-Gua I in D O/NaOD (Figure 2 4). The NMR, MS, and UV data were all consistent with N-7-(2′,3′,4′-trihydroxybut-1′-yl)guanine structure for DEBGua I (Chart 1). Chart 1. Structures of DEB-Guanine Adducts
782 Chem. Res. Toxicol., Vol. 10, No. 7, 1997
Tretyakova et al.
a
b
c
d
Figure 3. LC/ESI+ CID mass spectra of DEB-Gua I (a), DEB-Gua II (b), DEB-Gua III (c), and epoxy intermediate (d).
ESI+-MS analysis of DEB-Gua II provided the major (M + H)+ peaks at m/z ) 274 and 276 with the intensity ratio 3:1 characteristic for the presence of one chlorine atom in the molecule (Figure 3b) (35). The major fragmentation process at ESI+ CID conditions was the loss of the whole alkyl group from the molecular ion giving protonated guanine ions at m/z ) 152 (Figure 3b). These results combined with the UV absorption data suggest that DEB-Gua II is N-7-(dihydroxychlorobutyl)guanine. The position of chlorine atom in the side chain was determined by the chemical shift and the multiplicity of the protons at carbons 3′ and 4′. Since the 4′methylene proton signals were shifted by only 0.2 ppm compared to DEB-Gua I (Table 2) and the 3′-CH signal in DEB-Gua II (3.64 ppm) appeared sharper than in DEB-Gua I (3.25 ppm), DEB-Gua II was assigned the structure of N-7-(2′,4′-dihydroxy-3′-chlorobut-1′-yl)guanine (Chart 1). The molecular weight of DEB-Gua III was established as 297 Da based on mass spectral analysis by ESI+ (Figure 3c). Collision-induced dissociation of the (M + H)+ protonated molecular ions in LC/ESI-MS mode gave rise to protonated guanine ions at m/z ) 152. This suggests the structure of DEB-Gua III as guanine with a butyl side chain containing two hydroxyls and one acetoxy group. As in the case of DEB-Gua II, the geometry of the side chain was established by comparing the proton NMR signals to the corresponding protons in the spectrum of DEB-Gua I (Table 2). In contrast with DEB-Gua II, the signals for the 4′-methylene protons of
DEB-Gua III were significantly shifted downfield (3.99 ppm), suggesting that the electron-withdrawing acetoxy group was located at the 4′-position. Less pronounced deshielding was observed for the 3′-CH group, whose appearance in the spectra was similar to that in DEBGua I. The signal assignments were confirmed by COSY 2D NMR experiments, since cross-peaks were observed for methine protons and the corresponding hydroxy groups: H-1′-H-2′, H-2′-H-3′, and H-3′-H-4′ (results not shown). All spectral evidence was consistent with the N-7-(2′,3′-dihydroxy-4′-acetoxybut-1′-yl)guanine structure for DEB-Gua III (Chart 1). The presence of DEB-Gua I-III in hydrolyzed reaction mixtures (DEB + Guo) can be explained by the initial formation of N-7-(2′-hydroxy-3′,4′-epoxybut-1′-yl)guanine which participates in nucleophilic substitution reactions involving the remaining epoxy group (Scheme 1). DEBGua I is formed through hydrolysis of the 3′,4′-oxirane group of the intermediate by either the SN1 or SN2 mechanism giving the corresponding triol. DEB-Gua III appears to be produced during the incubation of guanosine with DEB in acetic acid. Reactions of DEB with Guo in the presence of acetic acid give rise to acetate anions which attack the remaining 3′,4′-epoxy group by an SN2 mechanism at the least sterically hindered carbon 4′ to give N-7-(2′,3′-dihydroxy-4′-acetoxybut-1′-yl)guanine. DEB-Gua II is likely to form during the deribosylation step, upon heating in strong acid (1 N HCl), which favors SN1-type reactions. The 3′,4′-epoxy ring opening at these conditions is likely to result in N-7-(2′,4′-hydroxybutyl)-
Guanine Adducts of Diepoxybutane
Chem. Res. Toxicol., Vol. 10, No. 7, 1997 783
Table 2. Proton NMR Spectroscopic Data for DEB-Guanine Adducts chemical shift, ppm
coupling constant, Hz
assignment
Scheme 1. Adduct Formation in the Reactions of DEB with Guanosine in Acetic Acid followed by Acid Hydrolysis (1 N HCl)
DEB-Gua I 3.19 (br s, 1H)a 3.25 (m, 2H)a 3.62 (br d, 1H)a 3.88 (dd, 1H)a 4.12 (dd, 1H)a 4.51 (s, 1H)b 4.71 (s, 2H)b 6.22 (s, 2H)b 7.81 (s, 1H)b 10.90 (br s, 1H)b
J ) 13.6, 9.2 J ) 13.6, 4.0
4′-CH2 4′-CH2, 3′-CHOH 2′-CHOH 1′-CH2 1′-CH2 4′-CHOH 2′,3′-CHOH NH2 H-8 H-1
DEB-Gua IIb 3.45-3.55 (m, 2H) 3.64 (m, 1H) 3.91 (m, 1H) 4.12 (dd, 1H) 4.29 (dd, 1H) 5.05 (two br s, 2H) 6.10 (s, 2H) 7.81 (s, 1H) 10.78 (br s, 1H)
J ) 13.6, 9.0 J ) 13.6, 3.8
4′-CH2 3′-CHCl 2′-CHOH 1′-CH2 1′-CH2 4′,2′-CHOH NH2 H-8 H-1
DEB-Gua IIIb 2.00 (s, 3H) 3.55 (br s, 1H) 3.82 (br s, 1H) 3.99 (br d, 2H) 4.11 (dd, 1H) 4.28 (dd, 1H) 5.00 (br s, 1H) 5.12 (br s, 1H) 6.22 (s, 2H) 7.81 (s, 1H) 10.80 (br s, 1H) a
J ) 7.0 J ) 13.6, 9.0 J ) 13.6, 4.1
CH3CO3′-CHOH 2′-CHOH 4′-CH2OAc 1′-CH2 1′-CH2 3′-CHOH 2′-CHOH NH2 H-8 H-1
D2O/NaOD. b DMSO-d6.
Figure 5. HPLC separation of a neutral thermal hydrolysate of DEB-exposed CT DNA (250 × 4.6 mm C18 column, 1.5 mL/ min, gradient program 3). UV detection at 284 nm.
Figure 4. Two-dimensional double-quantum-filtered COSY 1H NMR spectrum of DEB-Gua I dissolved in D2O/NaOD.
guanine 3′-cation which is more stable than the alternative structure, N-7-(2′,3′-dihydroxybutyl)guanine 2′cation. In the presence of chloride anions this results in N-7-(2′,4′-dihydroxy-3′-chlorobut-1′-yl)guanine (DEB-Gua II). Although the monoepoxy intermediate was not found in the Guo + DEB reaction mixture, it was isolated in hydrolysates of DEB-exposed CT DNA (see below). DEBGua III is not expected to form in vivo, but the formation of DEB-Gua I and II is feasible at physiological conditions. The presence of multiple N-7-guanine adducts from guanosine reaction suggests the high reactivity of the initially formed N-7-(2′-hydroxy-3′,4′-epoxybut-1′-yl)guanine which results in generation of a variety of N-7guanine adducts via reactions of the remaining epoxy ring with any nucleophilic species present in solution.
CT DNA Reaction with DEB. The synthetic markers obtained from the 2′-dGuo reactions were used to identify and quantitate the corresponding DEB-guanine adducts in exposed CT DNA. HPLC analysis of neutral thermal hydrolysates of CT DNA + DEB reaction mixtures (detection at 284 nm) revealed two major peaks at 8.1 and 12.8 min (Figure 5) with area ratio of 0.97-0.99. The former peak had the same HPLC retention time and UV and mass spectra as the N-7-(2′,3′,4′-trihydroxybut1′-yl)guanine (DEB-Gua I) standard and was therefore identified as DEB-Gua I. The peak at 12.8 min was suspected to be the N-7-(2′-hydroxy-3′,4′-epoxybut-1′-yl)guanine intermediate based on its UV spectrum and the reduction in the peak size when the hydrolysis time and/ or temperature was increased. This identification was confirmed by mass spectral analysis of the product collected from multiple HPLC runs. The adduct had a molecular weight of 237 Da, corresponding to guanine with one hydroxyepoxybutyl substituent, and fragmented to protonated guanine under CID conditions (Figure 3d). When heated in the presence of acid, the epoxy intermediate converted into N-7-(2′,3′,4′-trihydroxybut-1′-yl)guanine (DEB-Gua I). Quantitative analysis of DEB-Gua adducts in DEBexposed CT DNA was performed following mild acid hydrolysis to ensure complete conversion of the epoxy intermediate to the corresponding triol (DEB-Gua I). The amounts of DEB-Gua I in acid hydrolysates (58.7 ( 1.1 adducts/103 normal guanines) were comparable to the amounts of N-7-guanine adducts found in CT DNA
784 Chem. Res. Toxicol., Vol. 10, No. 7, 1997
exposed to EB at the same molar ratio (27). This finding is in disagreement with the data of Bolt et al. (36) who reported DEB to be 10 times less reactive toward DNA than EB by measuring the rate of disappearance of the epoxides in solution containing CT DNA. Since the actual adducts were not measured in that study and the epoxides could have been consumed through hydrolysis and other side reactions, the data presented in this work should give a more accurate picture of reactivity of the two epoxy metabolites of butadiene.
Tretyakova et al.
into the reasons for interspecies differences in the carcinogenic response to butadiene and improve risk assessment for humans.
Acknowledgment. The authors thank Patricia Upton for assistance with the manuscript. We would like to acknowledge Asoka Ranasighe for obtaining LSI-MS mass spectra. This work was sponsored in part by a grant from Chemical Manufacturers Association.
References Discussion DNA alkylation by chemicals or their metabolites represents an early critical step in multistage carcinogenesis. Genotoxicity and mutagenicity studies of BD and its metabolites suggest that DEB formation might be critical for the higher susceptibility of mice to BD. In this study, we synthesized and fully structurally characterized N-7-DEB-guanine adducts using various spectral techniques. N-7-(2′,3′,4′-Trihydroxybut-1′-yl)guanine (DEB-Gua I) was the major adduct in reactions of DEB with free guanosine and guanine in CT DNA. We therefore confirm the earlier findings of Jellito (29) who identified the N-7 position of guanine as the major target for DNA alkylation with DEB. N-7-Guanine adducts have a rather low mispairing potential but are released from DNA (t1/2 ) 50-100 h; 37-39) producing apurinic sites which are capable of causing transition and transversion mutations in mammalian cells (40). Further investigation of the CT DNA reaction with DEB enabled us to isolate the unstable intermediate, N-7-(2′-hydroxy-3′,4′-epoxybut-1′-yl)guanine, which was identified based on its UV and mass spectra and its hydrolysis to the corresponding triol, DEB-Gua I. Nucleophilic substitution with chloride and acetate anions at the 3′,4′-epoxy ring of the intermediate is probably responsible for the formation of the two minor products (DEB-Gua II and III) observed in the guanosine reaction. In vivo, the 3′,4′-epoxy ring of DEB-alkylated guanines can be expected to be slowly opened by various nucleophiles present (hydroxide anion, chloride anion, amino acids, etc.). In addition, it can participate in a second reaction with DNA producing intra- and/or interstrand cross-links (41, 42). Interstrand cross-links are known to cause cytotoxicity, whereas the intrastrand cross-links can be mutagenic (43-45). Molecular modeling studies performed in our laboratory suggest that the formation of DEB-induced interstrand cross-links between N-7 positions of neighboring guanines in DNA requires significant bending of the double helix. However, the reactions of the 3′,4′-epoxy group with other nucleophilic sites of DNA such as N6 of adenine appear to be more favorable. Since DEB and EB appear to form similar amounts of the major DNA adducts in vitro at the conditions applied in this work, the 100-fold higher mutagenic potential of DEB compared to EB (23) may be due to factors other than differences in reactivity. DNA-DNA and DNAprotein cross-link formation by DEB could be important for DEB-induced mutagenicity. In addition, differences in repair could play a role. DEB adducts with guanine and their 13C-labeled analogs synthesized in our laboratory are currently used as marker compounds for analysis of specific DEB-induced adducts in target tissues of rats and mice exposed to butadiene. The comparison of the results of quantitative analyses of butadiene-induced adducts in the two species should provide further insight
(1) Brunnemann, K. D., Kagan, M. R., Cox, J. E., and Hoffmann, D. (1990) Analysis of 1,3-butadiene and other selected gas-phase components in cigarette mainstream and sidestream smoke by gas chromatography-mass selective detection. Carcinogenesis 11, 1863-1868. (2) Pelz, N., Dempster, A. M., and Shore, P. R. (1990) Analysis of low molecular weight hydrocarbons including 1,3-butadiene in engine exhaust gases using an aluminum oxide porous-layer open tubular fused-silica column. J. Chromatogr. Sci. 28, 230-235. (3) International Agency for Research on Cancer (IARC). (1992) 1,3Butadiene. IARC Monogr. Eval. Carcinogen. Risk Chem. Hum. 54, 237-285. (4) 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 inhalation exposure. Science 227, 548-549. (5) 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. (6) Owen, P. E., Glaister, J. R., Gaunt, 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. (7) 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. (8) Malvoisin, E., and Roberfroid, M. (1982) Hepatic microsomal metabolism of 1,3-butadiene. Xenobiotica 12, 137-144. (9) Bolt, H. M., Schmiedel, G., Filser, J. G., Rolzhauser, H. P., Lieser, K., Wistuba, D., and Schurig, V. (1983) Biological activation of 1,3-butadiene to vinyl oxirane by rat liver microsomes and exhalation of the reactive metabolite by exposed rats. J. Cancer Res. Clin. Oncol. 106, 112-116. (10) Csanady, 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, 1143. (11) Malvoisin, E., Mercier, M., and Roberfroid, M. (1982) Enzymatic hydration of butadiene monoxide and its importance in the metabolism of butadiene. Adv. Exp. Med. Biol. 38A, 437-444. (12) Sharer, J. E., Duescher, R. J., and Elfarra, A. A. (1991) Formation, stability, and rearrangements of the glutathione conjugates of butadiene monoxide: evidence for the formation of stable sulfurane intermediates. Chem. Res. Toxicol. 4, 430-436. (13) Henderson, R. F., Thornton-Manning, J. R., Bechtold, W. E., and Dahl, A. R. (1996) Metabolism of 1,3-butadiene: species differences. Toxicology 111, 1-6. (14) Lawley, P. D., and Brookes, P. (1967) Interstrand cross-linking of DNA by bifunctional alkylating agents. J. Mol. Biol. 25, 143160. (15) Meinhardt, T. J., Lemen, R. A., Crandall, M. S., and Young, R. J. (1982) Environmental epidemiological investigation of the styrenebutadiene rubber industry. Mortality patterns with discussion of the hematopoietic and lymphatic malignancies. Scand. J. Work Environ. Health 8, 250-259. (16) 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., Peltonen, K., Vainio, H., and Hemminki, K., Eds.) pp 345362, IARC Scientific Publication No. 127, IARC, Lyon, France. (17) Matanoski, G. M., Santos-Burgoa, C., and Schwartz, L. (1990) Mortality of a cohort of workers in the styrene-butadiene polymer manufacturing industry (1943-1982). Environ. Health Perspect. 86, 107-117. (18) Matanoski, G., Francis, M., Correa-Villasen˜or, A., Elliott, 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., Peltonen, K., Vainio, H., and Hemminki, K., Eds.) pp 363-374, IARC Scientific Publication No. 127, IARC, Lyon, France.
Guanine Adducts of Diepoxybutane (19) Santos-Burgoa, C., Matanoski, G. M., Zeger, S., and Schwartz, L. (1992) Lymphohematopoietic cancer in styrene-butadiene polymerization workers. Am. J. Epidemiol. 136, 843-854. (20) Ahlberg, R. W., Sorsa, M., Pfa¨ffli, P., Ma¨ki-Paakanen, J., and Viinanen, R. (1992) Genotoxicity and exposure assessment in the manufacture of 1,3-butadiene. WHO Report on Occupational Health in Chemical Industry, pp 199-204, WHO, Copenhagen, Denmark. (21) Delzell, E., Sathiakumar, N., Macaluso, M., Hovinga, M., Larson, R., Barbone, F., Beall, C., and Cole, P. A. (1996) A follow-up study of synthetic rubber workers. Toxicology 113, 182. (22) Ward, J. B., Ammenheuser, M. M., Benchtold, W. E., Whorton, E. B., and Legator, M. S. (1994) Hprt mutant lymphocyte frequencies in workers at a 1,3-butadiene production plant. Environ. Health Perspect. 102 (Suppl. 9), 79-85. (23) Cochrane, J. E., and Skopek, T. R. (1994) Mutagenicity of butadiene and its epoxide metabolites: I Mutagenic potential of butadiene, 1,2-epoxybutene and diepoxybutane in cultured human lymphoblasts. Carcinogenesis 15, 713-717. (24) 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, 719-723. (25) Citti, L., Gervasi, G., Turchi, G., Bellucci, G., and Bianchini, R. (1984) The reaction of 3,4-epoxy-1-butene with deoxyguanosine and DNA in vitro: synthesis and characterization of the main adducts. Carcinogenesis 5, 47-52. (26) Selzer, R. R., and Elfarra, A. A. (1996) Synthesis and biochemical characterization of N1-, N2-, and N7-guanosine adducts of butadiene monoxide. Chem. Res. Toxicol. 9, 126-132. (27) Tretyakova, N. Yu., Lin, Y., Sangaiah, R., Upton, P. B., and Swenberg, J. A. (1997) Identification and quantitation of DNA adducts from calf thymus DNA exposed to 3,4-epoxy-1-butene. Carcinogenesis 18, 137-147. (28) Neagu, I., Koivisto, P., Neagu, C., Kostiainen, R., Stenby, K., and Peltonen, K. (1995) Butadiene monoxide and deoxyguanosine alkylation products at the N7-position. Carcinogenesis 16, 18091813. (29) Jellito, B., Vangala, R. R., and Laib, R. J. (1989) Species differences in DNA damage by butadiene: role of diepoxybutane. Arch. Toxicol. 13 (Suppl.), 246-249. (30) Leuratti, C., Marafante, E., Jones, N. J., and Waters, R. (1993) Detection by HPLC-32P postlabelling of DNA adducts formed after exposure to epoxybutene and diepoxybutane. In Postlabelling Methods for Detection of DNA Adducts (Phillips, D. H. , Castegnaro, M., and Bartsh, H., Eds.) pp 277-283, IARC, Lyon, France. (31) Lawley, P. D., et al. (1975) Isolation and identification of products from alkylation of nucleic acids: Ethyl- and isopropyl-purines. Biochem. J. 145, 73.
Chem. Res. Toxicol., Vol. 10, No. 7, 1997 785 (32) Li, F., Segal, A., and Solomon, J. J. (1992) In vitro reaction of ethylene oxide with DNA and characterization of DNA adducts. Chem.-Biol. Interact. 83, 35-54. (33) Solomon, J. J., Mukai, F., Fedyk, J., and Segal, A. (1988) Reactions of propylene oxide with 2′-deoxynucleosides and in vitro with calf thymus DNA. Chem.-Biol. Interact. 67, 275-294. (34) Singer, B., and Grunberger, D. (1983) Molecular Biology of Mutagens and Carcinogens, Plenum Press, New York. (35) McLafferti, F. W. (1980) Interpretation of Mass Spectra, University Science Books, Mill Valley, CA. (36) Bolt, H. M., Kirkovaski, L., and Capellmann, M. (1995) Differential reactivities of the mono-and di-epoxide of 1,3-butadiene. Presented at the International Symposium “Evaluation of Butadiene and Isoprene Health Effects”, Blaine, WA. (37) Shuker, D. E. G., and Farmer, P. B. (1992) Relevance of urinary DNA adducts as markers of carcinogen exposure. Chem. Res. Toxicol. 5, 450-460. (38) Groopman, J. D., Croy, R. G., and Wogan, G. N. (1981) In vitro reactions of aflatoxin B1-adducted DNA. Proc. Natl. Acad. Sci. U.S.A. 78, 5445-5449. (39) Singer, B. (1979) N-Nitroso alkylating agents: formation and persistence of alkyl derivatives in mammalian nucleic acids as contributing factors in carcinogenesis. J. Natl. Cancer Inst. 62, 1329-1339. (40) Gentil, A., Cabral-Neto, J. B., Mariage-Samson, R., Margot, A., Imbach, J. L., Rayner, B., and Sarasin, A. (1992) Mutagenicity of a unique apurinic/apyrimidinic site in mammalian cells. J. Mol. Biol. 227, 981-984. (41) Vangala, R. R, Laib, R. J., and Bolt, H. M. (1993) Evaluation of DNA damage by alkaline elution technique after inhalation exposure of rats and mice to 1,3-butadiene. Arch. Toxicol. 67, 3438. (42) Lawley, P. D., and Brookes, P. (1965) Molecular mechanism of the cytotoxic action of difunctional alkylating agents and of resistance to this action. Nature 206, 480-482. (43) Yarema, K. J., Lippard, S. J., and Essigmann, J. M. (1995) Mutagenic and genotoxic effects of DNA adducts formed by the drug cis-diamminedichloroplatinum (II). Nucleic Acids Res. 23, 4066-4072. (44) Roberts, J. J., and Thompson, A. J. (1979) The mechanism of action of antitumor platinum compounds. Prog. Nucleic Acid Res. Mol. Biol. 22, 71-129. (45) Skladanovski, A., and Konopa, J. (1994) Interstrand DNA crosslinking induced by anthracyclines in tumour cells. Biochem. Pharmacol. 47, 2269-2278.
TX970004Q
