Mutagenic Potential of Guanine N2 Adducts of Butadiene Mono- and

Center in Molecular Toxicology, Vanderbilt University, Nashville, Tennessee .... Susith Wickramaratne , Uthpala Seneviratne , Melissa Goggin , Mat...
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Chem. Res. Toxicol. 2000, 13, 18-25

Mutagenic Potential of Guanine N2 Adducts of Butadiene Mono- and Diolepoxide J. Russ Carmical,† Mingzhu Zhang,‡ Lubomir Nechev,‡ Constance M. Harris,‡ Thomas M. Harris,‡ and R. Stephen Lloyd*,† Departments of Preventative Medicine and Community Health and Sealy Center for Molecular Science, The University of Texas Medical Branch, Galveston, Texas 77555, and Department of Chemistry and Center in Molecular Toxicology, Vanderbilt University, Nashville, Tennessee 37235 Received July 14, 1999

To explore the role of guanine N2 adducts of stereoisomeric butadiene metabolites in butadiene-induced mutagenesis, 11-mer deoxyoligonucleotides were prepared containing adducts of (R)- and (S)-monoepoxide and (R,R)- and (S,S)-diolepoxide. These adducted oligonucleotides were utilized in both in vivo and in vitro experiments designed to examine the mutagenic potency of each and their replication by Escherichia coli polymerases. Each of the four adducted deoxyoligonucleotides was ligated into a single-stranded M13mp7L2 vector and transfected into E. coli. The resulting plaques were screened for misincorporation at position 2 of the N-ras 12 codon. Although the mutagenic frequencies were low, different relative mutagenicities of the various stereoisomers were discernible. In addition, the biological effects of each adduct on the three major E. coli polymerases were determined via primer extension assays. The adducted 11-mers were ligated into a 60-mer linear DNA molecule to provide a sufficiently long template for primer elongation. All four guanine adducts were determined to be blocking to each of the three polymerases via primer extension assays.

Introduction Over the past two decades, production of 1,3-butadiene (BD)1 has increased sharply in response to market demand for its use in consumer goods, such as styrenebutadiene rubber, styrene-butadiene latex, thermoplastic resins (acrylonitrile-butadiene-styrene), and polychloroprene (neoprene elastomers) (1). This increase in both production and use has promoted investigations for gaining knowledge about its interactions in biological systems, especially since inhalation and animal carcinogenicity studies have shown that 1,3-butadiene is a potent carcinogen in mice and to a lesser extent in rats (2-5). These data have resulted in an increased concern regarding potential adverse human health effects associated with butadiene exposure. Among these is butadienemediated carcinogenesis which is presumed to be initiated through its reactive metabolites: monoepoxide (BDO), diepoxide (BDO2), and diolepoxide (BDE). This hypothesis is consistent with its observed mutagenicity in several biological systems, including bacteria (6-10), yeast (11, 12), insects (13), and in mammalian systems (14-17). Analyses of butadiene-related mutagenesis have revealed mutations at both A‚T and G‚C base pairs (1827). Molecular analysis of activated K-ras genes derived from butadiene-induced tumors on nude mice revealed †

The University of Texas Medical Branch. Vanderbilt University. Abbreviations: BD, butadiene; BDO, butadiene monoepoxide; BDO2, butadiene diepoxide; BDE, butadiene diolepoxide; BMO (R)dGuo, (R)-N2-(1-hydroxy-3-buten-2-yl)deoxyguanosine; BMO (S)-dGuo, (S)-N2-(1-hydroxy-3-buten-2-yl)deoxyguanosine; BDE (R,R)-dGuo, (R,R)N2-(2,3,4-trihydroxy-1-butyl)deoxyguanosine; BDE (S,S)-dGuo, (S,S)N2-(2,3,4-trihydroxy-1-butyl)deoxyguanosine; MALDI-TOF MS, matrixassisted laser desorption/ionization time-of-flight mass spectroscopy. ‡

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that 81% resulted from G to C transversions (28). Additionally, increased levels of ras oncoproteins in human plasma have been correlated with occupational exposure to 1,3-butadiene (29). Animal studies further support an association between butadiene exposure and an increased level of ras oncogene expression (30). Ras genes are involved in cell cycle regulation and are known to be involved in the initiation of many cancers, particularly chemically induced carcinogenesis (30, 31). Given that epidemiological studies have revealed an increased relative risk for leukemia in styrene-butadiene plant workers (32) and that human ras gene mutations are strongly correlated with leukemia (33), ras genes provide an appropriate DNA sequence context for studying butadiene-induced mutagenesis. Although much research has focused on establishing a mutagenic spectrum specific to butadiene and its reactive metabolites, these systems do not have the capacity to identify the primary DNA lesion or the stereochemistry that gives rise to the observed mutations. The interaction of butadiene metabolites with DNA results in a multitude of adducts which differ in regioand stereochemistry. Butadiene is metabolized by cytochrome P450s (primarily 2E1) to (R)- and (S)-monoepoxide (BDO) which is further oxidized to (R,R)-, (S,S)-, and (RS/SR)-diepoxide (BDO2). In addition, diolepoxides (BDE) [(R,R), (S,S), and (RS/SR)] can be formed either by epoxidation of hydrolyzed BDO or by partial hydrolysis of BDO2. The latter appears to be the major route to BDE (34, 35). All of the epoxide species can react at numerous sites in DNA. The formation of specific stereo- and regioisomeric adducts has been detected via 32P-postlabeling in liver DNA of rats exposed to butadiene (36), giving rise to the possibility that a mutagenic spectrum specific

10.1021/tx9901332 CCC: $19.00 © 2000 American Chemical Society Published on Web 12/14/1999

Mutagenic Spectrum of Guanine1,3-butadiene

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preparing oligonucleotides containing N2 adducts of (R)and (S)-BDO and (R,R)- and (S,S)-BDE adducts, which as noted above, can arise by more than one route (Figure 1).2 Previously, we determined that the analogous adducts on the N6 position of adenine were weakly mutagenic, with stereospecific mutation spectra (42).

Materials and Methods

Figure 1. Structures of 1,3-butadiene-adducted guanine stereoisomers.

to butadiene may be stereospecific. Guanine adducts at N7, N1, and N2 have been isolated (37-41) with the N7 adducts being the most abundant and the only guaninederived species thus far detected in vivo. Although the N7 adducts exist in an approximate 100-fold excess over the N2 lesions in vitro, the modification at N7 renders the glycosyl bond unstable, and therefore, rates of spontaneous depurination are high (40). In contrast, the N2 adducts, which are significantly more abundant than N1 lesions, are very stable. The inherent instability of the N7 butadiene adducts makes studies of their mutagenic potential using site-specifically modified DNAs difficult, if not impossible. Therefore, we chose to initiate our in vitro and in vivo studies of guanine adducts by

Synthesis of N-ras 12 Modified Oligonucleotides. The 11-mer oligonucleotides containing N2 guanine adducts of butadiene monoepoxide and diolepoxide were prepared by the postoligomerization methodology developed by Harris et al. (43). The 11-mer sequence (5′-GGCAGXTGGT-3′) was synthesized by the typical DNA automated synthesis methodology on a Millipore Expedite 8909 instrument using PAC-protected phosphoramidites and the phosphoramidite of 2-fluoro-O6-[(trimethylsilyl)ethyl]-2′-deoxyinosine to introduce an O6-protected 2-fluorohypoxanthine residue at the 12,2 position (underlined). Following oligonucleotide synthesis, the O6-protected 2-fluorohypoxanthine-containing oligonucleotide was removed from the beads, deprotected, and purified by HPLC (44, 45). The adducts of the monoepoxide were prepared by reaction of (2R or 2S)-2-amino-3-buten-1-ol (10 mg) with fluoropurinecontaining 11-mer (45-50 A260 units) in anhydrous DMSO (150-200 µL) containing diisopropylethylamine (150 µL) for 18 h at 60 °C. The solvents were removed in vacuo. The residue was dissolved in H2O (200 µL) and purified by HPLC using a reversed-phase column (YMC-ODS-AQ, 10 mm × 250 mm, flow rate of 5.0 mL/min) using a gradient of (A) 0.1 M ammonium formate (pH 6.3) and (B) acetonitrile, from 8 to 17% B over the course of 20 min and from 17 to 70% B over the course of 2 min. The O6-protected adducted oligonucleotides had a retention time of 20-21 min. The product peaks were collected, lyophilized, and treated with 0.1 M acetic acid (2 h, room temperature). The desilylated oligonucleotides were rechromatographed in the same system using a gradient of 5 to 10% B over the course of 18 min; the products eluted at 13-14 min. ESI-MS of 122 R: calculated Mr of 3521.6; measured mass based on 703.9 [M 5H]/5z, 586.4 [M - 6H]/6z ) 3524. MALDI-TOF MS of 122 S: m/z calcd for [M - H]- 3521.6, found 3522.6. The adducts of the diolepoxide were prepared by reaction of (2R,3R)- or (2S,3S)-1-aminobutane-2,3,4-triol (5-6 mg) with fluorohypoxanthine-containing 11-mer (50 A260 units) for 18 h in anhydrous DMSO (200 µL) containing diisopropylethylamine (50-150 µL) at 60 °C. The solvents were removed in vacuo. The residue was dissolved in H2O (200 µL) and purified initially by HPLC as described above for the monoepoxide adduct; the products eluted at ∼15 min. The diolepoxide adducts were deprotected and purified as descibed for the monoepoxide adducts. The desilylated oligonucleotides eluted at 9-10 min. MALDI-TOF MS of 122 R,R: m/z calcd for [M - H]- 3555.6, found 3555.9. MALDI-TOF MS of 122 S,S: m/z calcd for [M H]- 3555.6, found 3554.9. In addition to mass spectrometry, the HPLC-purified products were characterized by capillary gel electrophoresis and enzyme digestion. Examination of the purity by PAGE of 32P-end-labeled product was carried out before biological experiments were performed. Construction of ras 122-Guanine1,3-butadiene-Adducted Closed Circular Template. Single-stranded M13mp7L2 bacteriophage, supplied by C. Lawrence (University of Rochester, Rochester, NY), were used to inoculate Escherichia coli and subsequently purified as described by Sambrook et al. (46). 2 The adducts of BDO can form by either an S 2 or S 1 process; N N the former gives adducts with the opposite configuration compared to the parent epoxide [(R) adduct from (S)-epoxide], whereas the latter gives adducts with both retained and inverted configurations. BDE and BDO2 give adducts with the same configuration as the parent compound because the reaction occurs at the terminal position [(R,R)BDE or -BDO2 gives (R,R) adducts].

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M13mp7L2 has been genetically engineered such that it possesses a hairpin loop structure that encompasses an EcoRI site. The circular single-stranded DNA was linearized by incubating 20 µg of DNA with 80 units of EcoRI in 100 mM Tris-HCl (pH 7.5), 50 mM NaCl, and 5 mM MgCl2 for 1 h at 37 °C. The reaction sample (10 µL) was analyzed by electrophoresis through a 1.4% agarose gel and subsequently stained with ethidium bromide to visualize the efficiency of the digest. In accordance with the protocol supplied by Bio-Rad, approximately 20 µg of linearized DNA was passed over a Nensorb column to remove the restriction endonuclease and excess salts. After lyophilization of the appropriate fraction containing the DNA, it was then reconstituted in 10 mM Tris-HCl (pH 7.5) and 0.1 mM EDTA at a concentration of 0.5 µg/µL and stored at -20 °C. The adducted 11-mers were phosphorylated with T4 polynucleotide kinase to provide a 5′-phosphate for ligation into the M13 vector. Next, a 1:1 molar ratio of complementary 51-mer and 11-mer was mixed. The 11-mer hybridizes to the internal 11 nucleotides of the 51-mer, leaving the flanking ends available for hybridization with the linearized M13 DNA molecule. Subsequently, the 11-mer-51-mer duplex was annealed to M13 DNA in the same molar ratio, thus bringing 5′-phosphates and 3′-hydroxyls close to each other. Finally, the ends were ligated by incubating the mix overnight with 2000 units of T4 DNA ligase at 11 °C in a buffer provided by the supplier (New England Biolabs Inc., Beverly, MA). Approximately 10 µL of the ras 122-guanine1,3-butadienemodified M13mp7L2 was analyzed by electrophoresis through a 1.4% agarose gel and visualized via ethidium bromide staining. Ligation efficiencies of the ras 122-guanine1,3-butadiene-modified M13mp7L2 were quantitated using an Appligene Bioimager. In Vivo Replication of ras 122-Guanine1,3-butadieneAdducted DNA. Ras 122-guanine1,3-butadiene-modified M13mp7L2 was used to transfect repair-deficient AB2480 (uvrA- recA-) E. coli cells via electroporation. A 10 mL volume of Luria-Bertani (LB) broth (Difco) was inoculated with a single colony of AB2480 E. coli and grown overnight at 37 °C. A 1:100 dilution of the liquid culture was made in a total volume of 150 mL and allowed to grow to an optical density of ∼0.50 at 600 nm. An aliquot (25 mL) was placed in a prechilled 25 mL corex tube for each electroporation. The remainder of the culture was allowed to continue growing at 37 °C for use as feeder cells. The cells were then harvested by centrifugation at 7000 rpm for 5 min. The supernatant was decanted and the pellet reconstituted in 25 mL of cold autoclaved H2O. The cells were once again centrifuged and harvested. The pellet was then dissolved in 1/2 volume of H2O. This procedure was repeated twice, followed by resuspension in 400 µL of 10% glycerol at 4 °C. The glycerol/cell suspension was then transferred to a cold microfuge tube and centrifuged at 8000 rpm. The supernatant was removed and the pellet reconstituted in 1/10 volume of 10% glycerol. A volume of 5 µL of the ligation reaction was mixed with the cells prior to transferring them to a cold BTX electroporation cuvette. An electrical pulse at 25 µF, 2.5 kV, and 360 Ω was then passed through the cuvette. Immediately after the pulse, the cells were purged with 1 mL of SOC (0.5% yeast extract, 2% tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 20 mM MgSO4, and 2 mM glucose) medium, which had been prewarmed to 37 °C. Aliquots of 100 µL each were plated on prewarmed LB brothagarose plates in the presence of 500 µL of AB2480 E. coli cells and 5 mL of top agar (LB with 0.7% agarose) and subsequently incubated overnight at 37 °C. The M13 particles were transferred from the plaques to nitrocellulose filters in four successive lifts for each plate. These filters were processed by three sequential washes of the following solutions: A, 0.1 N NaOH and 1.5 M NaCl; B, 0.2 M TrisHCl (pH 7.0); and C, 2× SSC (1.5 M NaCl and 0.2 M sodium citrate). The liberated DNA was then covalently linked to the filter via UV light exposure. Four deoxyoligonucleotides (17mers) were synthesized to be directly complementary to the six nucleotides flanking the insert as well as the 11-nucleotide insert with the exception of the central nucleotide, where each of the four possible nucleotides was incorporated. These deoxyo-

Carmical et al. ligonucleotides were radioactively labeled by incubating 1 µg of DNA with 0.15 mCi of [γ-32P]ATP and 20 units of T4 polynucleotide kinase, accompanied by its supplied buffer, for 1 h at 37 °C. These deoxyoligonucleotides were used as sitespecific probes for position 2 of ras codon 12. The nitrocellulose filters containing four copies of the M13 plaques were hybridized with the radioactively labeled probes under stringent conditions, such that only the correct complement to the variable nucleotide would anneal. Overnight exposure of autoradiographic film to the hybridized filters allowed for visualization of the location of the radioactively labeled probes. Construction of ras 122-Guanine1,3-butadiene-Adducted Linear Template. The adducted 11-mer deoxyoligonucleotides were utilized in the construction of 60-mers with the adducted guanine approximately centrally located. To create these 60mers containing the adducted lesions, two additional oligonucleotides (20- and 29-mers) were synthesized to serve as flanking sequences to the 11-mer. A 45-mer bridge oligonucleotide, complementary to the 11-mer and the appropriate ends of the 20- and 29-mers, was synthesized to facilitate ligation of the individual deoxyoligonucleotides. The adducted 11-mer and 29mer flanking sequences were phosphorylated at the 5′-terminus prior to annealing with the 45-mer scaffold. To visualize the 60-mer ligation product, the 20-mer was phosphorylated with a 1:10 [γ-32P]ATP/ATP mixture. The four deoxyoligonucleotides were added in equal molar concentrations and heated to 70 °C for 5 min. Subsequently, the mixture was incubated in an ice slurry to facilitate proper annealing. The 5′- and 3′-ends were then ligated by incubating them with 2000 units of T4 DNA ligase in the presence of the supplied buffer at 16 °C overnight. The 60-mer ligation product was purified away from the 45mer scaffold and unligated deoxyoligonucleotides via polyacrylamide gel electrophoresis. The DNAs within the ligation reactions were separated by electrophoresis through a 15% denaturing gel and visualized by exposing the gel to autoradiographic film for 5 h. The bands were then excised, and the DNA was eluted from the gel matrix. The DNA was precipitated with 95% ethanol and recovered by centrifugation. Finally, each sample was lyophilized and reconstituted in 100 µL of 10 mM Tris-HCl (pH 8.0) and 0.1 mM EDTA. In Vitro Replication of ras 122-Guanine1,3-butadieneAdducted DNA. Three deoxyoligonucleotides were synthesized so specific sites could be annealed on the templates, thus providing a 3′-hydroxyl at various distances relative to the adduct and allowing for primer extension by the polymerase. The three primers that were synthesized were specifically designed to position the 3′-hydroxyl at the following positions with respect to the adducted guanine: -1 base, -5 base, and +5 base. Each deoxyoligonucleotide was phosphorylated with T4 polynucleotide kinase incorporating a γ-32P label on the 5′end and diluted to a concentration of 50 fmol/µL. Primer was added to a mixture containing 150 fmol of 60-mer template and reaction salts in a total volume of 5 µL. The mixture was heated to 90 °C for 2 min and was subsequently allowed to slowly cool to 25 °C. A reaction for each template-primer combination was carried out in triplicate to provide a partial duplex for each of the three E. coli polymerases. The polymerases that were assayed and their suppliers were as follows: large fragment of polymerase I (Klenow exo-) purchased from New England Biolabs Inc., polymerase II provided by M. F. Goodman and L. Bloom (University of Southern California, Los Angeles, CA), and polymerase III supplied by M. O’Donnell (Rockefeller University, New York, NY). Next, 4 µL of a reaction mix containing the appropriate salts, dNTPs, and the required buffer was added to the template-primer complex. The polymerases were added at 2-fold molar excess in relation to the partial duplex and allowed to proceed at room temperature for 10 min. An equal volume of loading buffer, consisting of formamide, xylene cyanol, and bromophenol blue, was used to terminate the reaction. The reaction products were then analyzed by electrophoresis through a 15% polyacrylamide sequencing gel and visualized by exposing an autoradiographic film overnight.

Mutagenic Spectrum of Guanine1,3-butadiene

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Figure 2. End-labeled 11-mer deoxyoligonucleotides (nonadducted and adducted): lane 1, nonadducted; lane 2, BDO (R)dGuo; lane 3, BDO (S)-dGuo; lane 4, BDE (R,R)-dGuo; and lane 5, BDE (S,S)-dGuo. The purity of the adducted oligonucleotides was determined by end labeling and polyacrylamide gel electrophoresis.

Figure 3. Ligation products of modified M13mp7L2: lane 1, closed circular L2 DNA; lane 2, linear L2 DNA; lane 3, unadducted ligation reaction; lane 4, BDO (R)-dGuo ligation reaction; lane 5, BDO (S)-dGuo ligation reaction; lane 6, BDE (R,R)-dGuo ligation reaction; and lane 7, BDE (S,S)-dGuo ligation reaction.

Results Experimental Rationale. Metabolism of 1,3-butadiene by cytochrome P450s results in a number of reactive metabolites that can interact with DNA at various sites and with different stereochemistries. To understand the relative mutagenic contribution of each modification, it is desirable to independently evaluate each specific adduct. Our previous studies involving DNAs containing stereoisomers of butadiene adducts at the N6 position of adenine suggested a minimal mutagenic contribution (42), raising the possibility that guanine adducts may play an important role in butadiene-induced mutagenesis. The use of a defined vector system with stereoselective chemistries (Figure 1) has facilitated the analysis of guanine adducts independently. In Vivo Replication of ras 122-Guanine1,3-butadieneAdducted DNA. To examine the in vivo replication fate for each of the stereospecific butadiene-induced guanine adducts (Figure 1), modified 11-mers as well as their unmodified counterpart were ligated into the cloning site of a single-stranded M13 vector (see Materials and Methods). Prior to the ras 122 11-mers being cloned into an M13 replication vector, each deoxyoligonucleotide was examined for purity. A 5′ γ-32P label was added to each, via T4 DNA kinase, and the labeled DNAs were analyzed on a denaturing polyacrylamide gel for the presence of any contaminating bands. The mobility of the adducted deoxyoligonucleotides was retarded with respect to that of the unmodified 11-mer (Figure 2). Overexposure of autoradiographic film did not reveal any visible contaminants, thus significantly reducing the probability that the measured mutation spectrum was influenced by the presence of impurities. In addition, mass spectrometry characterization revealed only a single species for each of the adducted deoxyoligonucleotides. Following successful ligation (Figure 3), the modified M13 vectors were replicated in E. coli and evaluated for resulting mutations via differential hybridization (47-

Figure 4. Ras 122 base substitutions as determined by differential hybridization: (top) G f A transition, (middle) G f T transversion, and (bottom) G f C transversion.

49). Examination of phage DNAs, into which unmodified oligonucleotides had been ligated, revealed no mutations of any type. In contrast, the modified DNAs resulted in an array of mutations. Representative mutations for each possible base substitution are depicted in Figure 4. To gain statistical validity, all experimental procedures were carried out as a minimum of three separate and independent trials, from construction of the adducted M13mp7L2 vector through screening for mutations. The raw scores

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Table 1. In Vivo Replication of Ras 122-Guanine1,3-butadiene-Modified M13mp7L2 in AB2480 (uvrA-, recA-) E. coli adduct

GfG

GfA

GfT

GfC

nonadducted BDO (R)-dGuo BDO (S)-dGuo BDE (R,R)-dGuo BDE (S,S)-dGuo

32 000 30 000 11 000 34 000 24 000

0 2 7 3 6

0 0 10 4 5

0 2 5 3 6

Table 2. Plaque-Forming Efficiency DNA

input DNA (µg)

average no. of plaques/plate

efficiency

unadducted BDO (R)-dGuo BDO (S)-dGuo BDE (R,R)-dGuo BDE (S,S)-dGuo

8 × 10-6 8 × 10-4 8 × 10-4 8 × 10-3 8 × 10-3

1691 1086 676 1276 741

2 × 108 1 × 106 8 × 105 1 × 105 9 × 104

Figure 6. Construction of the 1,3-butadiene-adducted 60-mer template.

Figure 5. Confidence intervals of mutagenic spectra. The confidence intervals for the mutagenic spectrum of each adduct were calculated via binomial analysis to a degree of certainty of 0.995.

shown in Table 1 represent a compilation of these data. Although all the mutation frequencies were less than 1%, it appears that both the (S)-monoepoxide and (S,S)diolepoxide stereoisomers are slightly more mutagenic than their (R) stereoisomeric counterparts. Of the mutations that were found, the (S)-monoepoxide resulted in 45% G f T transversions, 32% G f A transitions, and 23% G f C, whereas the (S,S)-diolepoxide resulted in nearly equal numbers of all three base substitutions. The (R,R)-diolepoxide also resulted in nearly equal numbers of each, while the (R)-monoepoxide resulted in relatively lower frequencies of mutations. To ensure the accuracy of these data, each putative mutant was isolated and replated. The resulting plaques were then screened a second time to verify the mutation again by differential hybridization. Furthermore, a subset of these were confirmed via DNA sequencing. The resulting mutation frequencies were determined to be statistically significant by calculating the binomial confidence intervals at the 0.995 level. The upper and lower confidence intervals were then plotted for each stereospecific adduct and for each possible base substitution (Figure 5). In addition to our previous reports on the relative mutagenicity of butadiene-modified adenine (42), other studies have shown that adducts which do not serve as a block to replication also do not result in a significantly increased mutagenic frequency (48, 49). Conversely, adducts that result in replication blockage and require some bypass mechanism create a scenario in which there is an increased probability of misincorporation. However, this does not mandate a high mutagenic frequency, since the correct nucleotide can always be inserted. Replication efficiency, which is indicative of the cells’ ability to replicate past the lesions, was calculated as the number

of plaques produced per microgram of input DNA. Analyses of these data for each of the adduct-containing DNAs revealed a significant reduction in plaque forming ability when compared to that of the unadducted DNA (Table 2). The reduction ranged from ∼200- to 2000-fold, depending on the specific stereoisomer. These data are consistent with the following in vitro replication studies which showed that these are very blocking lesions. Additional studies were carried out using wild-type E. coli both with and without SOS induction. These data showed no significant changes in either the plaque forming ability or the mutagenic spectrum, suggesting that SOS induction does not play a major role in the observed mutagenesis. In Vitro Replication of ras 122-Guanine1,3-butadieneAdducted DNA. The ability of each of the E. coli polymerases to synthesize past the various butadieneguanine adducts was determined via primer extension assays. Each polymerase was incubated with a control and adduct-containing 60-mer template such that each modified strand contained one of the stereospecific monoepoxide or diolepoxide lesions (Figure 6). The purified 60-mer templates were annealed with one of three primers (-1, -5, and +5) in all possible combinations (Figures 7 and 8). The -1 primer represents a “standing start” and the -5 primer a “running start”, and the +5 primer predicts possible downstream affects. Replication synthesis off of the -1 primer (lanes 3 and 4) was extremely poor by all three polymerases with no initiation of synthesis except by the Klenow fragment which terminated one base beyond the adduct. Similarly, replication off of the -5 primer proceeded to one nucleotide prior to the lesion except for Klenow which was able to incorporate opposite the lesion and one nucleotide beyond. The +5 primer was readily extended to full length in all cases.

Discussion Previous studies have revealed that 1,3-butadieneinduced mutations occur at both A‚T and G‚C base pairs

Mutagenic Spectrum of Guanine1,3-butadiene

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Figure 7. Primer extensions of the monoepoxide-adducted templates. Each of the templates [unadducted, BDO (R)-dGuo, and BDO (S)-dGuo] was annealed to one of three primers (-1 primer, -5 primer, and +5 primer). Each possible template-primer combination was extended with each of three polymerases (E. coli Pol I, II, and III), and the products were electrophoresed through a polyacrylamide gel. -1 primer: lane 1, primer alone; lane 2, nonadducted; lane 3, BDO (R)-dGuo; and lane 4, BDO (S)-dGuo. -5 primer: lane 5, primer alone; lane 6, nonadducted; lane 7, BDO (R)-dGuo; and lane 8, BDO (S)-dGuo. +5 primer: lane 9, primer alone; lane 10, nonadducted; lane 11, BDO (R)-dGuo; and lane 12, BDO (S)-dGuo.

(18-21, 23-27, 50). In some of these studies, there was a relatively high background of spontaneous mutations in lacI at CpG sites, and this background rendered it difficult to discern which mutations arose from butadiene exposure. Further studies utilizing lacZ analyzed G‚C base pair mutations at both CpG sites and non-CpG sites and showed an increase in the frequency of G‚C f A‚T transitions (50, 51). In addition to the animal studies, hprt analysis of occupationally exposed individuals showed a high percentage of mutations at G‚C base pairs (52). However, the interpretation of these data was limited by elevated background levels of mutations at G‚C base pairs. These data, in combination with our previous findings (42), indicated that butadiene adducts on guanine deserved further examination. Furthermore, epidemiological studies have shown that styrene-butadiene plant workers have an increased relative risk for leukemia (32). Combined with the fact that mutations at G‚C base pairs in the human ras genes are strongly correlated with leukemia (33), the human N-ras gene was chosen as our sequence context. Through the evaluation of the individual stereospecific adducts, we hoped to establish a hallmark for butadiene-induced mutagenesis that could be used for screening in an exposed population. The mutation spectra of butadiene-induced guanine adducts display an array of base substitutions. On average, the mutagenic frequency of the N2 guanine adducts is 1 order of magnitude greater than that of the N6 adenine

adducts. While there is no distinct mutational signature, it is interesting to note that the (S) stereoisomers appear to be more mutagenic than their (R) stereoisomer counterparts. Although the differences are poorly resolved, in Figure 5 the spectrum associated with the (S,S)diolepoxide adduct concurs with the frequency shown by Recio et al. (50) for butadiene-induced mutations at G‚C base pairs. The following order of relative mutations resulted in both studies: G f T > G f A > G f C. Finally, the in vitro replication data on the ability of polymerases to synthesize past these adducts were in marked contrast to those found with butadiene adducts on adenine N6. Unlike those adducts, all guanine N2 adducts served as blocks to replication by the three E. coli polymerases (Figures 7 and 8). These data were consistent with the decreased plaque forming ability shown in the in vivo replication studies (Table 2). The severity of in vitro replication blockage was not precisely correlated with the in vivo replication studies; however, these differences may be indicative of some replication accessory factors that aid in intracellular replication bypass. The mechanisms responsible for the increased level of bypass in vivo may also be responsible for decreased fidelity associated with the replicative polymerase. Although the N2 guanine adducts were 1 order of magnitude more mutagenic than the N6 adenine adducts, the overall low mutation frequency makes it likely that other adducts may contribute to the mutagenesis associ-

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Figure 8. Primer extensions of the diolepoxide-adducted templates. Each of the templates [unadducted, (R,R)-diolepoxide, and (S,S)-diolepoxide] was annealed to one of three primers (-1 primer, -5 primer, and +5 primer). Each possible template-primer combination was extended with each of three polymerases (E. coli Pol I, II, and III), and the products were electrophoresed through a polyacrylamide gel. -1 primer: lane 1, primer alone; lane 2, nonadducted; lane 3, BDE (R,R)-dGuo; and lane 4, BDE (S,S)-dGuo. -5 primer: lane 5, primer alone; lane 6, nonadducted; lane 7, BDE (R,R)-dGuo; and lane 8, BDE (S,S)-dGuo. +5 primer: lane 9, primer alone; lane 10, nonadducted; lane 11, BDE (R,R)-dGuo; and lane 12, BDE (S,S)-dGuo.

ated with butadiene exposure. Such candidates for mutagenic lesions include those leading to apurinic sites (N7 dGuo and N3 dAdo), N1 dAdo adducts and the related deaminated species, adducts on dCyd and dTHd, regioisomeric adducts of the monoepoxide (attack at C1 instead of C2), and stereoisomers arising from the meso(RS/SR)-diepoxide. In addition, cross-linked species formed by the diepoxide are reasonable candidates for mutagenicity; this possibility is supported by the much greater mutagenicity of the diepoxide (53) and the detection of deletions in some of the mutation studies (18-21, 23, 50).

Acknowledgment. We especially thank our collaborators (Drs. M. F. Goodman and L. Bloom, University of Southern California, and Mike O’Donnell, Rockefeller University) for providing polymerase II and III, respectively. We also acknowledge the work of the staff in the NIEHS Center Molecular Biology Core, The University of Texas Medical Branch, for the production of M13mp7L2 and synthesis of nonadducted templates. We also thank Ms. Rosemary Martinez and Mrs. Lisa Pipper-Stephenson for assistance with the preparation of the manuscript. Finally, we are grateful to Dr. Judah Rosenblatt, The University of Texas Medical Branch, for sharing his expertise in the statistical analysis. This work was supported by National Institutes of Health Grants P30ES06676, ES05355, ES07781, P30-ES00267, S11-ES10018, and T32-ES07253. R.S.L. holds the Mary Gibbs Jones Distinguished Chair in Environmental Toxicology from the Houston Endowment.

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