Mutagenesis of the supF Gene by Stereoisomers of 1,2,3,4

However, DEB is much more genotoxic and mutagenic than EB and EBD (9−12). Sasiadek et al. (9) reported that the lowest effective concentrations of E...
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Chem. Res. Toxicol. 2007, 20, 790-797

Mutagenesis of the supF Gene by Stereoisomers of 1,2,3,4-Diepoxybutane Min Young Kim,† Natalia Tretyakova,§ and Gerald N. Wogan*,†,‡ Biological Engineering DiVision and Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts AVenue, Cambridge, Massachusetts 02139, and UniVersity of Minnesota Cancer Center and Department of Medicinal Chemistry, UniVersity of Minnesota, Minneapolis, Minnesota 55455 ReceiVed January 2, 2007

1,2,3,4-Diepoxybutane (DEB) is a key metabolite of the important industrial chemical and environmental contaminant, 1,3-butadiene (BD). Although all three optical isomers of DEB, S,S-, R,R-, and meso-DEB, are produced by metabolic processing of BD, S,S-DEB exhibits the most potent genotoxicity and cytotoxicity, followed by R,R- and then meso-DEB. Our previous studies suggested that the observed differences between the biological effects of DEB optical isomers may be structural in their origin. Although S,S- and R,R-DEB produced mainly 1,3-interstrand 1,4-bis-(guan-7-yl)-2,3-butanediol (bis-N7G-BD) crosslinks, meso-diepoxide induced equal numbers of intrastrand and interstrand bis-N7G-BD lesions. In the present study, the mutagenicity of the three DEB stereoisomers in the supF gene was investigated. We found that S,S-DEB was the most potent mutagen. Interestingly, mutation specificity and mutant spectra were strongly dependent on DEB stereochemistry. Although A:T to T:A transversions were the major form of mutation observed following treatment with each of the three stereoisomers (35-40%), S,SDEB induced higher numbers of G:C to A:T transitions, whereas R,R-DEB treatment resulted in a greater frequency of G:C to T:A transversions. Our results are consistent with the stereospecific induction of promutagenic nucleobase adducts other than G-G cross-links by DEB stereoisomers. Introduction 1,2,3,4-Diepoxybutane (DEB1) is a carcinogenic metabolite of 1,3-butadiene (BD), an important industrial chemical and environmental contaminant (1, 2) that is also present in tobacco smoke (3). Metabolic activation of BD to DEB involves epoxidation reactions catalyzed by cytochrome P450 monooxygenases (Figure 1). The first epoxidation forms 3,4-epoxy-1butene (EB), which can be further oxidized to DEB or hydrolyzed to the corresponding diol, followed by oxidation to 3,4-epoxy-1,2-butanediol (EBD) (Figure 1) (4-7). DEB is the most cytotoxic, genotoxic, and carcinogenic metabolite of BD, and is capable of inducing chromosome aberrations, micronucleus formation, sister chromatid exchanges, and mutations in various animal and human cells (6-8). All BD-derived epoxides are direct-acting mutagens in the Ames test (8). However, DEB is much more genotoxic and mutagenic than EB and EBD (9-12). Sasiadek et al. (9) reported that the lowest effective concentrations of EB and DEB that induced sister chromatid exchanges were 25 and 0.5 µM, respectively, whereas sister chromatid exchanges and chromosome aberrations were induced by EB (20-931 µM) or DEB (2.5-320 µM) in human lymphocytes (10, 11). Cochrane et al. found that at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus, * Corresponding author. Tel: 617-253-3188. Fax: 617-258-0499. Email: [email protected]. † Biological Engineering Division, Massachusetts Institute of Technology. ‡ Department of Chemistry, Massachusetts Institute of Technology. § University of Minnesota. 1 Abbreviations: BD, 1,3-butadiene; bis-N7G-BD, 1,4-bis-(guan-7-yl)2,3-butanediol; DEB, 1,2,3,4-diepoxybutane; EB, 3,4-epoxy-1-butene; EBD, 3,4-epoxy-1,2-butanediol; E. coli, Escherichia coli; H2O2, hydrogen peroxide; MF, mutation frequency; N7-THBG, N7-(2′,3′,4′-trihydroxybut-1′yl)-guanine; TE, transformation efficiency; cfu, colony-forming units; HPRT, hypoxanthine-guanine phosphoribosyltransferase; TK, thymidine kinase.

Figure 1. Metabolism of 1,3-butadiene (BD) to metabolites 3,4-epoxy1-butene (EB), 1,2,3,4-diepoxybutane (DEB), and 3,4-epoxy-1,2butanediol (EBD), and the formation of the 1,4-bis-(guan-7-yl)-2,3butanediol (bis-N7G-BD) adduct derived from DEB through DNADNA cross-links.

an induced mutation fraction of 5 × 10-6 was induced by treatment with 3.5 µM DEB, 150 µM EB, and 450 µM EBD, whereas at the thymidine kinase (TK) locus, a similar increase in mutation fraction was induced by treatment with 1.0 µM DEB, 100 µM EB, and 350 µM EBD in TK6 lymphoblastoid cells (12). The types of mutations induced by these epoxides are distinct. Although EB exposure resulted in base substitutions at G:C base pairs (13), DEB and EBD induced deletions and point mutations at both A:T and G:C base pairs (14-17). The observed differences in genotoxicity and mutational spectra produced by the mono- and di-epoxides of butadiene have been attributed to the ability of DEB, but not EB, to form DNA-DNA cross-links and other bifunctional lesions (7). Both epoxides give rise to similar total numbers of DNA monoadducts at the N7 position of guanine and the N1, N3, N7, and N6

10.1021/tx700003b CCC: $37.00 © 2007 American Chemical Society Published on Web 04/12/2007

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Figure 2. Chemical structures of DEB stereoisomers: S,S-DEB (A), R,R-DEB (B), and meso-DEB (C).

positions of adenine (18-22), but only DEB possesses two epoxide functional groups capable of bifunctional DNA alkylation (23). DEB induces DNA-DNA cross-links, giving rise to the formation of 1,4-bis-(guan-7-yl)-2,3-butanediol (bis-N7GBD) adducts (Figure 1). N7-N7G cross-links are more hydrolytically stable than the corresponding monoadducts (24), potentially leading to their accumulation in DNA. All three optical isomers of DEB, S,S, R,R, and meso (Figure 2), are produced by the metabolic processing of BD. S,S-DEB is believed to be the active form of treosulfan (Ovastat), used clinically to treat advanced ovarian cancer (25-27). Among the three isomers, S,S-DEB exhibits the most potent genotoxicity and cytotoxicity, suggesting that it produces a greater number of critical DNA lesions (28-30). We have recently demonstrated that S,S-DEB is the most efficient interstrand DNA-DNA crosslinker, whereas meso-DEB produces equal numbers of intrastrand and interstrand bis-N7G-BD cross-links (31). In the present work, the mutagenicity and mutation spectrum of DEB in the supF gene of the pSP189 shuttle vector system were examined, and our results reveal stereospecific differences among the mutagenic properties of DEB isomers.

Figure 3. Mutation frequency (A) and transformation efficiency (B) induced by DEB stereoisomers. Data represent the mean ( SD for three experiments. The spontaneous mutation frequency was subtracted from the observed mutation frequencies after exposure to DEB stereoisomers to yield the treatment-induced mutation frequencies plotted in A.

Materials and Methods Plasmid Amplification. The pSP189 shuttle vector containing an 8-bp signature sequence was a gift from Dr. Michael M. Seidman (NIH, Bethesda, MD). As described previously (32), the plasmid was amplified in Escherichia coli (E. coli) AB2463 cells grown at 37 °C in LB medium containing 50 µg/mL ampicillin (Sigma) for 12-14 h, with shaking at 250 rpm, and isolated using a Maxi DNA isolation and purification kit (Qiagen, Valencia, CA). Treatment of Plasmid with DEB Stereoisomers. Three DEB stereoisomers (S,S, R,R, and meso; Figure 2) were synthesized as described previously (31). Optically active S,S- and R,R-DEB stereoisomers were prepared, starting with dimethyl 2,3-O-isopropylidene-L-tartrate and dimethyl 2,3-O-isopropylidene-D-tartrate, respectively; meso-DEB was prepared from meso-erythritol according to the published procedure (31). Racemic DEB, purchased from Sigma Aldrich Company (Milwaukee, WI), was used as an internal standard for quantification. Experimental groups included treatment with 0, 5, 10, 20, and 40 µM of one DEB stereoisomer (S,S, R,R, or meso) or racemic DEB. Plasmid DNA (20 µg) was treated at 37 °C for 24 h in a total reaction volume of 100 µL containing an appropriate concentration of DEB in 150 mM sodium phosphate at pH 7.4. At the end of treatment, DNA samples were washed twice with cold diethyl ether to remove any unreacted DEB and re-washed twice with cold, sterile water using Amicon Centricon-30 concentrators (Millipore, Billerica, MA). Transformation of MBL50 E. coli and Selection of supF Mutants. MBL50 cells were prepared and used for electroporation as previously described (32). The supF gene suppresses an amber mutation in the lacZ gene of the MBL50 cells, leading to the formation of blue colonies in the presence of X-gal and IPTG but white or light-blue colonies of mutants. MBL50 cells are an araD araC(Am) mutant strain of E. coli and thus become sensitive to L-arabinose when transformed with a supF+ plasmid but remain resistant on transformation with supF- mutants (33). To select

mutant plasmids, aliquots of transformed MBL50 cells were plated onto medium A (34) with 50 µg/mL ampicillin, 20 µg/mL IPTG (Roche), and 10 µg/mL X-gal (Roche), supplemented with 2 g/L L-arabinose (Sigma). The remaining suspension was diluted and plated onto LB agar containing ampicillin for the determination of the total number of transformants; only transformants containing pSP189 form colonies because MBL50 cells do not carry the ampicillin resistance gene (35). Mutation frequency (MF) and transformation efficiency (TE) were defined as the ratio of total mutants to total transformants and the number of colony-forming units (cfu) produced by 1 µg of pSP189 DNA in a transformation reaction, respectively (32). Analysis of Mutated supF Gene. Mutant plasmids were isolated using Wizard Plus Plasmid Miniprep Kit (Promega, Madison WI). DNA sequencing was carried out by the Harvard University DNA Sequencing Facility (Cambridge, MA) using a 20-mer primer with the following sequence: 5′-GGCGACACGG-AAATGTTGAA-3′ (IDT, Coralville, IA). The signature sequence was identified using the Sequencer program (Gene Codes Corporation, version 4.1.4). Poisson distribution analysis was used to assess the randomness of the distribution of mutations, and hot spots were defined as described previously (32).

Results Mutagenic Potency of DEB Stereoisomers. Exposure to all three stereoisomers of DEB caused dose-dependent increases in supF MF compared to the unexposed control, whereas corresponding decreases in TE were observed (Figure 3B), presumably attributable to DEB-induced interstrand DNA-DNA cross-linking, which can block DNA replication. S,S-DEB was strongly mutagenic (29 × 10-6) and resulted in a 9.8-fold increase of the MF over the spontaneous MF (3 × 10-6) at a dose of 40 µM. Exposure to 40 µM concentrations of R,R- and

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Figure 4. Mutation spectra in spontaneous mutants from unexposed pSP189 replicated in E. coli MBL50 cells. Open square symbols (0) indicate one base pair deletion.

Figure 5. Types of single base substitutions induced by DEB stereoisomers and racemic DEB in the supF gene of pSP189 replicated in E. coli MBL50 cells. (A) S,S-DEB; (B) R,R-DEB; (C) meso-DEB; (D) racemic DEB. Table 1. Types of Spontaneous and DEB Stereoisomer-Induced Mutations in the supF Gene of pSP189 Replicated in E. coli MBL50 Cells

meso-DEB increased the MFs 4- and 5-fold (11 and 15 × 10-6), respectively, compared with that of the control (2.8 and 3 × 10-6). MF also increased in a dose-dependent manner following racemic DEB treatment (Figure 3A). When plasmids were treated with 40 µM racemic DEB, the MF (12.3 × 10-6) was 4.4-fold higher than that of the control (2.8 × 10-6). Although all stereoisomers were mutagenic in the supF gene, the order of potency was S,S > meso > R,R (Figure 3A). Mutagenic potency is defined here as MF (mutants per transformants) per mole of compound. Types of Mutations Induced by DEB Stereoisomers. Twenty-three spontaneous mutants were isolated from untreated control plasmids. The predominant type of spontaneous mutation was single base pair substitutions (92%) (Figure 4). Seventysix percent of the total spontaneous mutations occurred at G:C base pairs. G:C to T:A transversions were predominant (33%), followed by G:C to A:T transitions (24%), and G:C to C:G and A:T to T:A transversions (19%) (Figure 4).

White and light-blue colonies obtained from cells exposed to 40 µM DEB were isolated and further characterized to determine mutation types and locations. Among a total of 405 mutants obtained from DEB-treated plasmids, the major type was single base pair substitutions (62-78%, Figure 5) (Table 1); other mutation types included deletions (19-31%), insertions (1-4%), and multiple sequence changes (3-8%, Figure 7) (Table 1). The types of single base pair substitutions found in induced mutants are summarized in Figure 5. Although A:T to T:A transversion was the major form of mutation (35.5-40%) observed following treatment with each of the three stereoisomers, different proportions of other single base pair substitutions also occurred (Figure 5A-C). G:C to A:T transitions were more abundant following treatment with S,S-DEB (35.5%) as compared with meso-DEB (15.4%) and R,R-DEB (8%) (Figure 5AC). However, R,R-DEB induced G:C to T:A transversions at a higher frequency (44%) than meso-DEB (26.2%) and S,S-DEB

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Figure 6. Distribution of mutations induced in the supF gene by DEB stereoisomers and racemic DEB in the supF gene of pSP189 replicated in E. coli MBL50 cells. (A) S,S-DEB; (B) R,R-DEB; (C) meso-DEB; (D) racemic DEB. Symbol key: (9), hotspot sites that were common to all treatments; (b), hotspots common to both the S,S- and meso-DEB treatments; (O), hotspots common to both the R,R- and meso-DEB treatments; (4), hotspots common to both the S,S- and racemic DEB treatments; (g), hotspots common to the, R,R-DEB, meso-DEB, and racemic DEB treatments; and ([), present only in an individual treatment. Deletions are denoted by open square symbols (0). Multiple sequence changes illustrated in Figure 7 have been omitted.

(13.2%) but fewer G:C to C:G transversions (6.7%) than S,S(14.5%) and meso-DEB (18.5%) (Figure 5B). G:C to C:G transversions were the most abundant from meso-DEB treatment (18.5%) followed by S,S-DEB treatment (14.5%) (Figure 5C). In plasmids treated with racemic DEB, similar frequencies of single base substitutions at A:T (52%) and G:C (48%) base pairs were observed (Figure 5D). A:T to T:A transversions (50.7%), followed by the G:C to A:T (26.9%), were the major mutations induced by racemic DEB (Figure 5D). Mutation Spectra in the supF Gene. Figure 6 shows the distribution of single base substitutions and deletions along the supF gene following treatment with individual DEB stereoisomers or racemic DEB. S,S-DEB induced six mutational hotspots (A119, C133, A137, G150, G159, and G160) (Figure 6A). The six hot spots induced by R,R-DEB were located at positions A119, A120, G122, G123, C133, and C168, and the 10 induced by meso-DEB occurred at A119, A121, G123, G129, C133, G150, G164, C168, C172,

and A177 (Figure 6B and C). The mutational spectrum induced by racemic DEB included six hotspots (A113, A119, C133, A137, A157, and C168) (Figure 6D). Hotspots common to more than one treatment were as follows: A119 and C133 for all treatments; A137 for S,S- and racemic DEB; G150 for S,S- and meso-DEB; G123 for R,Rand meso-DEB; and C168 for racemic, R,R- and meso-DEB. Hotspot positions unique to individual treatments include G159 and G160 for S,S-DEB; A120 and G122 for R,R-DEB; A121, A129, G164, C172, and A177 for meso-DEB; and A113 and A157 for racemic DEB (Figure 6). Figure 7 displays multiple sequence changes found in individual mutants induced by S,S-DEB (A), R,R-DEB (B), meso-DEB (C), and racemic DEB (D). Among the limited number of mutants containing multiple changes, neither the specific sequence changes observed nor their distribution along the gene show evident relationships to the relative mutagenic potencies of the stereoisomers.

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Figure 7. Distribution of multiple sequence changes in the supF gene mutated by DEB stereoisomers. The diagram shows the locations of base substitution mutations (unshaded columns), single base insertions (shaded columns), and single base-pair deletions (denoted by D) in the supF gene exposed to DEB stereoisomers and racemic DEB. (A) S,S-DEB; (B) R,R-DEB; (C) meso-DEB; (D) racemic DEB.

Discussion Previous studies involving the structural characterization of DEB revealed that the ability of DEB to produce DNA-DNA cross-links was strongly dependent on its stereochemistry (24, 31, 36). If not repaired, these cross-links blocked DNA replication, transcription, and repair, leading to mutagenic and cytotoxic effects. Our study assessed the roles of various DNA damage products of DEB mutagenicity by comparing the mutation types and locations induced by DEB stereoisomers using the supF shuttle vector system. We found that all three stereoisomeric forms of DEB induced increases in MF compared to that of the unexposed control, and there were subtle differences in the mutagenic responses to the stereoisomers or the racemic mixture at lower exposure levels of 5 and 10 µM (Figure 3A). These findings are consistent with those observed in a recent study (37) that evaluated the cytotoxicity and mutagenicity of stereochemical forms of DEB in human TK6 lymphoblastoid cells. All three stereoisomers of 0-6 µM DEB caused increases in mutation frequency at the HPRT and TK loci compared to those of concurrent controls (37). The mutagenic responses to 6 µM S,S-DEB appeared to be greater than that to R,R- and meso-DEB, although the differences were not statistically significant (37). In our experiments, the S,S stereoisomer was the most strongly mutagenic at high concentrations (20 and 40 µM) (Figure 3A), indicating that larger amounts of promutagenic DNA lesions were produced by this isomer than by the other forms. On the basis of current information, intrastrand DNA-DNA cross-links induced by DEB tend to be mutagenic, whereas interstrand cross-links are cytotoxic (31). Our observation that meso-DEB is less mutagenic than the S,S isomer (Figure 3) is not in accord with that generalization because only meso-DEB induces intrastrand lesions, whereas the S,S form induces larger numbers of interstrand lesions (31). The observed differences in mutagenicity thus cannot be explained by the known DNA cross-linking specificities of DEB isomers. One likely explanation for these findings is that DNA lesions other than bis-N7GBD cross-links may play a key role in DEB mutagenicity. For example, recent studies have shown that DEB is capable of forming exocyclic lesions at adenine and guanine nucleobases (36, 38). These novel lesions are potentially promutagenic because of their inability to form standard Watson-Crick base pairs.

Several earlier studies of mutation spectra induced by BD and its metabolites have examined the molecular mechanisms responsible for its mutagenesis (13-17, 34, 39-42). Saranko et al. (40) presented data indicating that BD- and EB-exposed B6C3F1 lacI transgenic mice had an increased frequency of A:T to T:A transversions, but in contrast to the parent compound BD, G:C to A:T transitions occurred with increased frequency in EB-exposed mice. A:T to T:A transversions were also increased in lacI transgenic fibroblasts and at HPRT in human TK6 lymphoblasts exposed to EB in Vitro (14, 41). In TK6 and CHO-K1 cells, DEB induced an increased frequency of A:T to T:A transversions, partial deletions, and deletions, and G:C to A:T transitions and A:T to T:A transversions in the HPRT gene, respectively (15, 16, 39). A high frequency of G:C to A:T transitions was also observed in ViVo in the hprt gene of T-lymphocytes in DEB-exposed mice and rats (17). Our results are consistent with these findings (Table 1 and Figure 5). The predominant mutations induced by the DEB stereoisomers and racemic DEB were single base pair substitutions (62-78%), followed by deletions (19-31%) (Table 1). A:T to T:A transversions and G-C to A-T transitions were the major forms of single base pair substitutions (Figure 5). Our data showed stereospecificity in the ability of DEB isomers to induce mutations in the supF gene (Figure 5). An interesting difference was observed between S,S- and meso-DEB stereoisomers. Although A:T to T:A transversions were the predominant mutation seen for both isomers, S,S-DEB induced a much higher percentage of G:C to A:T transitions than meso-DEB; conversely, meso-DEB produced more G:C to T:A and G:C to C:G transversions. R,R-DEB induced more G:C to T:A, but fewer G:C to C:G transversions, than meso- and S,S-DEB (Figure 5). The results of mutagenicity studies in the hprt gene of BDexposed male B6C3F1 mice and F344 rats performed by Meng et al. (42) provide useful in ViVo data for comparison with our in Vitro data. Mutation spectrum analysis identified mutations mainly involving A:T base pairs in both hprt and supF genes, consistent with findings in earlier studies of BD and its metabolites as mentioned above: A:T to T:A transversions and G:C to A:T transitions were the base pair substitutions found predominantly and consistently. Moreover, deletions were frequently observed in both hprt and supF genes following BD and DEB treatment. Increases in A:T to T:A transversions

Mutation by DEB Stereoisomers

occurred at a similar frequency in the hprt genes of mice and rats, whereas G:C to C:G transversions were significantly increased only in mice exposed to BD. G:C to C:G and A:T to T:A transversions have been known as base pair substitutions biologically relevant to BD-induced cancer (42), and mice by far are more sensitive than rats (5, 6). The authors suggested that the metabolism of BD to DEB may be one basis for BDinduced G:C to C:G transversions, but precise causes of mutations associated with species differences in susceptibility to BD-induced cancer remain unknown. In the present study, an increased frequency of G:C to C:G transversions was found in the supF gene following treatment with S,S- and meso-DEB stereoisomers, the most efficient interstrand (cytotoxic) and intrastrand (mutagenic) DNA-DNA cross-linkers, respectively (Figure 5). Thus, DEB stereochemistry may be reflected in the generation of G:C to C:G transversions; this possibility remains to be tested. Both adenine and guanine adducts have been detected in DNA after in Vitro and in ViVo exposure to BD and its metabolites; adducts at N1, N2, and N7 of deoxyguanosine, and N1, N3, N7, and N6 of deoxyadenosine have been described (24, 35, 36, 38, 43-55). Carmical et al. (47) showed that DEB-induced N2-N2guanine intrastrand cross-links were strong blocking lesions for DNA polymerases, were poorly repaired by DNA excision repair enzymes, and induced increased MF in bacterial phage assay systems. The mutagenicity of intrastrand DEB cross-links between the N6 positions of adjacent adenosines has also been examined (49). This lesion resulted in a high MF of single base pair substitutions at the 3′-adducted base, and replication in mammalian cells was more highly mutagenic than in E. coli (49). Park et al. recently reported that both guanine-guanine and adenine-guanine conjugates were isolated from DEBtreated DNA (24, 31, 36, 38). They demonstrated that DNA alkylation by DEB mainly produces N7-(2′,3′,4′-trihydroxybut1′-yl)-guanine (N7-THBG) monoadducts, which can form bisN7G-BD lesions (24, 36, 38). They also identified four asymmetrical DNA-DNA cross-links involving both adenine and guanine nucleobases: N1A-N7G-BD, N3A-N7G-BD, N7AN7G-BD, and N6A-N7G-BD (31). Guanine alkylation was specific for the N7 position, resulting in bis-N7-guanylbutanediol adducts. Alkylation at the N7 position of guanine can cause depurination, leading to the generation of abasic sites, which allow replicative bypass and are mutagenic (56-58). The replication of DNA containing apurinic sites often results in the incorporation of deoxyadenosine opposite the apurinic site (59), and thus, depurination of an N7-guanine adduct could lead to G:C to T:A transversions (60). At lower frequencies, thymine and guanine have also been shown to be inserted opposite an abasic site, which would lead to G:C to A:T and G:C to C:G mutations, respectively (61). In addition, it has been demonstrated that N7 alkylation of guanine can cause deprotonation at N1 of guanine, giving rise to a zwitterion capable of pairing with thymine, which would result in G:C to A:T transitions (62). Interestingly, Abu-Shakra et al. (63) demonstrated similarities in the mutation spectra between DEB and hydrogen peroxide (H2O2), suggesting a correlation between epoxide-induced DNA damage and that induced by the hydroxyl radical. Thus, the occurrence of G:C to T:A and G:C to C:G transversions, major base substitution mutations induced by both agents, suggests the possibility of a similar underlying mechanism. DEB mainly alkylates adenine at the N1 position and, to a lesser extent, at N3 and N7. The predominant N1-adenine adduct can undergo deamination to form inosine and also Dimroth rearrangement to produce the corresponding N6-adenine adduct

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(49, 50, 55). Kanuri et al. (55) proposed that N3-adenine adducts are reasonable candidates as contributors to A:T to T:A mutations associated with exposure to BD and its metabolites. The replication of apurinic sites resulting from the depurination of N3 adducts could ultimately yield A:T to T:A transversions through the preferential insertion of adenine opposite apurinic sites as outlined above. However, the exact source of the A:T to T:A transversions identified as the most common mutation at A:T base pairs remain unknown. Although DEB isomers showed stereospecific effects, mutations induced by all three occurred in approximately equal proportions at G:C and A:T base pairs (Figure 5). Thus, it would appear that the stereoisomers were equally capable of alkylating both guanine and adenine. If these lesions were in fact responsible for the mutations observed, the mechanisms described above are likely to be involved in their generation. Figures 6 and 7 also illustrate stereospecific differences among DEB isomers with respect to the distribution of mutations in the supF gene. Almost all hotspots were located at A:T and G:C sites, and hotspots at A119 and C133 were common to all exposures. The distribution of mutations over the target gene was nonrandom in each instance, and sites of mutation varied among stereoisomers. Although the general pattern of mutation spectra induced by the stereoisomers was similar in many respects to that induced by racemic DEB, there were differences in the constellation of hotspots among stereoisomers (Figure 6). Millard et al. (51) and Sawyer et al. (60) demonstrated that racemic DEB preferentially induced interstrand DNA lesions at 5′-GNC sequences. They recently reported that the 5′-GNC consensus sequence for racemic DEB was conserved, but efficiencies in inducing cross-links varied in the order S,S-DEB > R,R-DEB > meso-DEB (61). In contrast to their results, we found that 21-25% of all base substitutions and deletions induced by DEB stereoisomers occurred at such sites (Figures 6 and 7). This finding suggests that if these mutations are due to interstrand cross-links between the N7 positions of deoxyguanosine formed by DEB, then the cross-links may be spontaneously released by depurination events or may be repaired. If repaired, errors may be introduced, leading to mutations in addition to those caused by abasic sites. Given the broad spectrum of mutations seen in experimental systems following exposure to BD or its metabolites and the wide variety of DNA adducts formed in Vitro by them, it is reasonable to hypothesize that no single adduct or type of adduct is responsible for the observed genotoxic effects of DEB. The results of a recent study (36, 38) support this hypothesis, in that N1A-N7G-BD and N6A-N7G-BD are more hydrolytically stable and, if formed in ViVo, may accumulate in target tissues. Thus, even though guanine-adenine lesions are 10 times less abundant than the corresponding bis-N7G cross-links in DEBtreated double-stranded DNA in Vitro, the formation of these adducts seems likely to contribute to mutations following DEB exposure. In summary, we have examined the mutational frequency, mutation types, and distribution induced by individual DEB stereoisomers in the supF gene. Our findings are consistent with stereospecific differences in the mutagenicity of diepoxybutanes and suggest that DNA lesions other than guanine-guanine cross-links (bis-N7G-BD) play a key role in DEB mutagenesis. Further studies employing site-specific mutagenesis are needed to help elucidate mechanisms of DEB-induced genotoxicity and rationalize the observed differences among DEB stereoisomers.

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Acknowledgment. We are grateful to Dr. Soobong Park and Kris Murphy (University of Minnesota, Minneapolis, MN) for the synthesis of the DEB stereoisomers and Laura J. Trudel for manuscript preparation. This work was supported by National Cancer Institute Grants No. 5 P01 CA26731 and No. R01 9CA095039, and MIT Center for Environmental Health Sciences NIEHS P30 ES002109.

References (1) Morrow, N. L. (1990) The industrial production and use of 1,3butadiene. EnViron. Health Perspect. 86, 7-8. (2) Pelz, N., Dempster, N. 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) Hecht, S. S. (1999) Tobacco smoke carcinogens and lung cancer. J. Natl. Cancer Inst. 92, 1194-1210. (4) Malvoisin, E. and Roberfroid, M. (1982) Hepatic microsomal metabolism of 1,3-butadiene. Xenobiotica 12, 137-144. (5) Thornton-Manning, J. R., Dahl, A. R., Bechtold, W. E., Griffith, W. C., Jr, and Henderson, R. F. (1995) Disposition of butadiene monoepoxide and butadiene diepoxide in various tissues of rats and mice following a low-level inhalation exposure to 1,3-butadiene. Carcinogenesis 16, 1723-1731. (6) Henderson, R. F., Thornton-Manning, J. R., Bechtold, W. E., and Dahl, A. R. (1996) Metabolism of 1,3-butadiene: species differences. Toxicology 113, 17-22. (7) Jelitto, B., Vangala, R. R., and Laib, R. J. (1989) Species differences in DNA damage by butadiene: role of diepoxybutane. Arch. Toxicol., Suppl. 13, 246-249. (8) Henderson, R. F., Barr, E. B., Belinsky, S. A., Benson, J. M., Hahn, F. F., and Menache, M. G. (2000) 1,3-Butadiene: cancer, mutations, and adducts. Part I: Carcinogenicity of 1,2,3,4-diepoxybutane. Res. Rep. Health Eff. Inst. 92, 1-43. (9) Sasiadek, M., Norppa, H., and Sorsa, M. (1991) 1,3-Butadiene and its epoxides induce sister-chromatid in human lymphocytes in Vitro. Mutat. Res. 261, 117-121. (10) Kligerman, A. D., DeMarini, D. M., Doerr, C. L., Hanley, N. M., Milholland, V. S., and Tennant, A. H. (1999) Comparison of cytogenetic effects of 3,4-epoxy-1-butene and 1,2:3,4-diepoxybutane in mouse, rat and human lymphocytes following in Vitro G0 exposures. Mutat. Res. 439, 13-23. (11) Kligerman, A. D., and Hu, Y. (2006) Some insights into the mode of action of butadiene by examining the genotoxicity of its metabolites. Chem.-Biol. Interact., 166, 132-139. (12) Cochrane, J. E., and Skopek, T. R. (1994) Mutagenicity of butadiene and its epoxide metabolites: I. Mutagenic potential of 1,2-epoxybutane, 1,2,3,4-diepoxybutane and 3,4-epoxy-1,2-butanediol in cultured human lymphoblasts. Carcinogenesis 15, 713-717. (13) Recio, L., Steen, A., Pluta, L. J., Meyer, K. G., and Sarank, C. J. (2001) Mutational spectrum of 1,3-butadiene and metabolites 1,2epoxybutene and 1,2,3,4-diepoxybutane to assess mutagenic mechanisms. Chem.-Biol. Interact. 135, 325-341. (14) Steen, A. M., Meyer, K. G., and Recio, L. (1997) Characterization of hprt mutations following 1,2-epoxy-3-butene exposure of human TK6 cells. Mutagenesis 12, 359-364. (15) Steen, A. M., Meyer, K. G., and Recio, L. (1997) Analysis of hprt mutations occurring in human TK6 lymphoblastoid cells following exposure to 1,2,3,4-diepoxybutane. Mutagenesis 12, 61-67. (16) Cochrane, J. E., and Skopek, T. R. (1994) Mutagenicity of butadiene and its epoxide metabolites: II. Mutational spectra of butadiene, 1,2epoxybutene and diepoxybutane at the hprt locus in splenic T cells from exposed B6C3F1 mice. Carcinogenesis 15, 719-723. (17) Meng, Q., Singh, N., Heflich, R. H., Bauer, M. J., and Walker, V. E. (2000) Comparison of the mutations at Hprt exon 3 of T-lymphocytes from B6C3F1 mice and F344 rats exposed by inhalation to 1,3butadiene or the racemic mixture of 1,2,3,4-diepoxybutane. Mutat. Res. 464, 169-184. (18) Selzer, R. R., and Elfarra, A. A. (1999) In Vitro reactions of butadiene monoxide with single- and double-stranded DNA: characterization and quantitation of several purine and pyrimidine adducts. Carcinogenesis 20, 285-292. (19) 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. (20) Tretyakova, N., 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, 137147.

Kim et al. (21) Neagu, I., Koiviston, P., Neagu, C., Kaltia, S., Kostiainen, P., and Peltonen, K. (1994) Alkylation of purine bases with 3,4-epoxy-1butene, a reactive metabolite of 1,3-butadiene. IARC Sci. Publ. 419422. (22) Tretyakova, N., Sangaiah, R., Lin, Y., and Swenberg, J. A. (1997) Synthesis, characterization, and in Vitro quantitation of N-7-guanine adducts of diepoxybutane. Chem. Res. Toxicol. 10, 779-785. (23) Lawley, P. D., and Brookes, P. (1967) Interstrand cross-linking of DNA by difunctional alkylating agents. J. Mol. Biol. 25, 143-160. (24) Park, S., and Tretyakova, N. (2004) Structural characterization of the major DNA-DNA cross-link of 1,2,3,4-diepoxybutane. Chem. Res. Toxicol. 17, 129-136. (25) Schmidmaier, R., Oellerich, M., Baumgart, J., Emmerich, B., and Meinhardf, G. (2004) Treosulfan-induced apoptosis in acute myeloid leukemia cells is accompanied by translocation of protein kinase C delta and enhanced by bryostatin-1. Exp. Hematol. (N. Y.) 32, 7686. (26) Hartley, J. A., O Ä Hare, C. C., and Baumgart, J. (1999) DNA alkylation and interstrand cross-linking by treosulfan. Br. J. Cancer 79, 264266. (27) Runowicz, C. D., Fields, A. L., and Goldberg, G. L. (1995) Promising new therapies in the treatment of advanced ovarian cancer. Cancer 76, 2028-2033. (28) Verly, W. G., Brakier, L., and Feit, P. W. (1971) Inactivation of the T7 coliphage by the diepoxybutane stereoisomers. Biochim. Biophys. Acta 228, 400-406. (29) Matagne, R. (1969) Induction of chromosomal aberrations and mutations with isomeric forms of L-threitol-1,4-bismethanesulfonate in plant materials. Mutat. Res. 7, 241-247. (30) Bianchi, A., and Contin, M. (1962) Mutagenic activity of isomeric forms of diepoxybutane in maize. J. Hered. 53, 277-281. (31) Park, S., Anderson, C., Loeber, R., Seetharaman, M., Jones, R., and Tretyakova, N. (2005) Interstrand and intrastrand DNA-DNA crosslinking by 1,2,3,4-diepoxybutane: role of stereochemistry. J. Am. Chem. Soc. 127, 14355-14365. (32) Kim, M. Y., Dong, M., Dedon, P. C., and Wogan, G. N. (2005) Effects of peroxynitrite dose and dose rate on DNA damage and mutation in the supF shuttle vector. Chem. Res. Toxicol. 18, 76-86. (33) Ariza, R. R., Roldan-Arjona, T, Hera, C., and Pueyo, C. (1993) A method for selection of forward mutations in supF gene carried by shuttle-vector plasmids. Carcinogenesis 14, 303-305. (34) Miller, J. H. (1992) A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria, Cold Spring Harbor, New York. (35) Juedes, M. J., and Wogan, G. N. (1996) Peroxynitrite-induced mutation spectra of pSP189 following replication in bacteria and in human cells. Mutat. Res. 349, 51-56. (36) Antsypovich, S., Quirk Dorr, D., Guza, R., Pitts, C., and Tretyakova, N. Structural characterization of exocyclic dA adducts of 1,2,3,4diepoxybutane. Chem. Res. Toxicol., submitted for publication. (37) Meng, Q., Redetzke, D. L., Hackfeld, L. C., Hodge, R. P., Walker, D. M., and Walker, V. E. (2007) Mutagenicity of stereochemical configurations of 1,2-epoxybutane and 1,2:3,4-diepoxybutane in human cells. Chem.-Biol. Interact., 166, 207-218. (38) Zhang, X. Y., and Elfarra, A. A. (2003) Identification and characterization of a series of nucleoside adducts formed by the reaction of 2′deoxyguanosine and 1,2,3,4-diepoxybutane under physiological conditions. Chem. Res. Toxicol. 16, 1606-1615. (39) Lee, D. H., Kim, T. H., Lee, S. Y., Kim, H. J., Rhee, S. K., Yoon, B., Pfeifer, G. P., and Lee, C. S. (2002) Mutations induced by 1,3butadiene metabolites, butadiene diolepoxide, and 1,2,3,4-diepoxybutane at the Hprt locus in CHO-K1 cells. Mol. Cells 14, 411-419. (40) Saranko, C. J., Meyer, K. G., Pluta, L. J., Henderson, R. F., and Recio, L. (2001) Lung-specific mutagenicity and mutational spectrum in B6C3F1 lacI transgenic mice following inhalation exposure to 1,2epoxybutene. Mutat. Res. 472, 37-49. (41) Saranko, C. J., Pluta, L. J., and Recio, L. (1998) The molecular analysis of lacI mutants from transgenic fibroblasts exposed to the butadiene metabolite, 1,2-epoxybutene. Carcinogenesis 11, 1879-1887. (42) Meng, Q., Walker, D. M., Scott, B. R., Seilkop, S. K., Aden, J. K., and Walker, V. E. (2004) Characterization of Hprt mutations in cDNA and genomic DNA of T-cell mutants from control and 1,3-butadieneexposed male B6C3F1 mice and F344 rats. EnViron. Mol. Mutagen. 43, 75-92. (43) de Meester, C. (1988) Genotoxic properties of 1,3-butadiene. Mutat. Res. 195, 273-281. (44) Melnick, R. L., and Kohn, M. C. (1995) Mechanistic data indicate that 1,3-butadiene is a human carcinogen. Carcinogenesis 16, 157163. (45) Seaton, M. J., Follansbee, M. H., and Bond, J. A. (1995) Oxidation of 1,2-epoxy-3-butene to 1,2,3,4-diepoxybutane by cDNA-expressed human cytochromes P450 2E1 and 3A4 and human, mouse and rat liver microsomes. Carcinogenesis 16, 2287-2293.

Mutation by DEB Stereoisomers (46) Blair, I. A., Oe, T., Kambouris, S., and Chaudhary, A. K. (2000) 1,3Butadiene: cancer, mutations, and adducts. Part IV: Molecular dosimetry of 1,3-butadiene. Res. Rep. Health Eff. Inst. 92, 151-190. (47) Carmical, J. R., Kowalczyk, A., Zou, Y., Van Houten, B., Nechev, L. V., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2000) Butadieneinduced intrastrand DNA cross-links: a possible role in deletion mutagenesis. J. Biol. Chem. 275, 19482-19489. (48) Adler, I. D., Cochrane, J., Osterman-Golkar, S., Skopek, T. R., Sorsa, M., and Vogel, E. (1995) 1,3-Butadiene working group report. Mutat. Res. 330, 101-114. (49) Carmical, J. R., Nechev, L. V., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2000) Mutagenic potential of adenine N(6) adducts of monoepoxide and diolepoxide derivatives of butadiene. EnViron. Mol. Mutagen. 35, 48-56. (50) Carmical, J. R., Zhang, M., Nechev, L. V., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2000) Mutagenic potential of guanine N2 adducts of butadiene mono- and diolepoxide. Chem. Res. Toxicol. 13, 18-25. (51) Millard, J. T., and White, M. M. (1993) Diepoxybutane crosslinks DNA at 5′-GNC sequences. Biochemistry 32, 2120-2124. (52) Tretyakova, N. Y., Sangaiah, R., Yen, T. Y., Gold, A., and Swenberg, J. A. (1997) Adenine adducts with diepoxybutane: isolation and analysis in exposed calf thymus DNA. Chem. Res. Toxicol. 10, 1711179. (53) Zhang, X. Y., and Elfarra, A. A. (2004) Characterization of the reaction products of 2′-deoxyguanosine and 1,2,3,4-diepoxybutane after acid hydrolysis: formation of novel guanine and pyrimidine adducts. Chem. Res. Toxicol. 17, 521-528. (54) Zhang, X. Y., and Elfarra, A. A. (2005) Reaction of 1,2,3,4deoxybutane with 2′-deoxyguanosine: initial products and their stabilities and decomposition pattern under physiological conditions. Chem. Res. Toxicol. 18, 1316-1323.

Chem. Res. Toxicol., Vol. 20, No. 5, 2007 797 (55) Kanuri, M., Nechev, L. V., Tamura, P. J., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2002) Mutagenic spectrum of butadiene-derived N1-deoxyinosine adducts and N6,N6-deoxyadenosine intrastrand crosslinks in mammalian cells. Chem. Res. Toxicol. 15, 1572-1580. (56) Kallama, S., and Hemminki, K. (1986) Stabilities of 7-alkylguanosines and deoxyguanosines formed by phosphoramide mustards and nitrogen mustard. Chem.-Biol. Interact. 57, 85-96. (57) Loeb, L. A. (1985) Apurinic sites as mutagenic intermediates. Cell 40, 483-484. (58) Mudipalli, A., Maccubbin, A. E., Nadadur, S. S., Struck, R. F., and Gurtoo, H. L. (1997) Mutations induced by monofunctional and bifunctional phosphoramide mustards in supF tRNA gene. Mutat. Res. 391, 49-57. (59) Loeb, L. A., and Preston, B. D. (1986) Mutagenesis by apurinic/ apyrimidinic sites. Annu. ReV. Genet. 20, 201-230. (60) Sawyer, G. A., Frederick, E. D., and Millard, J. T. (2004) Flanking sequences modulate diepoxide and mustard cross-linking efficiencies at the 5′-GNC site. Chem. Res. Toxicol. 17, 1057-1063. (61) Millard, J. K., Hanly, T. C., Murphy, K., and Tretyakova, N. (2005) The 5′-GNC site for DNA interstrand cross-linking is conserved for diepoxybutane stereoisomers. Chem. Res. Toxicol. 19, 16-19. (62) Sahasrabudhe, S. R., Luo, X., and Humayan, M. Z. (1991) Specificity of base substitutions induced by the acridine mutagen ICR-191: mispairing by guanine N7 adducts as a mutagenic mechanism. Genetics 129, 981-989. (63) Abu-Shakra, A., McQueen, E. T., and Cunningham, M. L. (2000) Rapid analysis of base-pair substitutions induced by mutagenic drug through their oxygen radical or epoxide derivatives. Mutat. Res. 470, 11-18.

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