Environmental and Molecular Mutagenesis 47:67^70 (2006) Brief Communication 3,4-Epoxy-1-butene, a Reactive Metabolite of 1,3-Butadiene, Induces Somatic Mutations in Xpc-null Mice J.K.Wickliffe,1* L.A. Galbert,1 M.M. Ammenheuser,1 S.M. Herring,1 J. Xie,2 O.E. Masters III,3 E.C. Friedberg,4 R.S. Lloyd,5 and J.B.Ward Jr1 1 Department of Preventive Medicine and Community Health, University of Texas Medical Branch, Galveston, Texas 2 Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas 3 Department of Zoology, University of Oklahoma, Norman, Oklahoma 4 Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas 5 Center for Research on Occupational and Environmental Toxicology, Oregon Health and Science University, Portland, Oregon Xpc-null (Xpc–/–) mice, deﬁcient in the global genome repair subpathway of nucleotide excision repair (NER-GGR), were exposed by intraperitoneal (IP) injection to a 300 mg/kg mutagenic dose of 3,4-epoxy-1-butene (EB), to investigate NER’s potential role in repairing butadiene (BD) epoxide DNA lesions. Mutagenic sensitivity was assessed using the Hprt assay. Xpc–/– mice were signiﬁcantly more sensitive to EB exposure, exhibiting an average 2.8-fold increase in Hprt mutant frequency (MF) relative to those of exposed Xpcþ/þ (wild-type) mice. As a positive control for NERGGR, additional mice were exposed by IP injection to a 150 mg/kg mutagenic dose of benzo[a]- pyrene (B[a]P). The Xpc–/– mice had MFs 2.9-fold higher than those of exposed Xpcþ/þ mice. These results suggest that NER-GGR plays a role in recognizing and repairing some of the DNA adducts formed following in vivo exposure to EB. Additional research is needed to examine the response of Xpc–/– mice, as well as other NER-deﬁcient strains, to inhaled BD. Furthermore, it is likely that alternative DNA repair pathways also are involved in restoring genomic integrity compromised by BD-epoxide DNA damage. Collaborative studies are currently underway to address these critical issues. Environ. Mol. Mutagen. C 2005 Wiley-Liss, Inc. 47:67–70, 2006. V Key words: epoxybutene; benzo[a]pyrene; Hprt; nucleotide excision repair; Xpc; butadiene INTRODUCTION Oleﬁns mediate their genotoxic effects through epoxide metabolites. One model oleﬁn, 1,3-butadiene (BD), is biotransformed in vivo to three reactive epoxides. These include an initial epoxide, epoxybutene (EB), and two secondary epoxides, diepoxybutane (DEB) and epoxybutenediol (EBD). All are documented mutagens, with DEB presumably representing the ultimate mutagen [reviewed by Jackson et al., 2000]. Comparatively little is known about the recognition and repair of the epoxide-DNA adducts that represent the premutagenic lesions. A number of modiﬁed nucleotides and dinucleotides have been detected in vivo and in vitro, including reaction products resulting from rearrangements of unstable primary lesions. These include alkylated bases, predominantly purines, and interstrand crosslinks [Selzer and Elfarra, 1996, 1999; Tretyakova et al., 1998; Koc et al., 1999; Koivisto et al., 1999; Solomon, 1999; C 2005 Wiley-Liss, Inc. V Park and Tretyakova, 2004; Zhang and Elfarra, 2004]. Mutational spectra in cultured cells, as well as mice and humans exposed to BD or BD-epoxides, reveal increases in speciﬁc point mutations (A/T?T/A transversions and A/T?G/C transitions) and deletions ranging from simple frameshifts to entire gene regions [Ma et al., 2000; Recio et al., 2001; Meng et al., 2004]. Studies examining the mutagenicity of synthesized lesions in vitro indicate that Grant sponsor: NIEHS; Grant number: ARCH-NIEHS 1 P30-ES06676, S11 ES-10018, 2R01ES06015-07, T32-ES07254; Grant sponsor: The Sealy Center for Environmental Health and Medicine. *Correspondence to: Jeffrey K. Wickliffe. E-mail: [email protected] Received 26 April 2005; provisionally accepted 5 May 2005; and in ﬁnal form 11 June 2005 DOI 10.1002/em.20169 Published online 10 August 2005 in Wiley InterScience (www.interscience. wiley.com). 68 Wickliffe et al. TABLE1. Hprt MFs in Lymphocytes From Xpcþ/þ and Xpc/ Mice Exposed to EB or B[a]P Exposure groupa Genotype Animal identiﬁcation number EB (300 mg/kg) Xpcþ/þ Xpcþ/þ Xpcþ/þ Xpc/ Xpc/ Xpc/ Xpc/ Xpcþ/þ Xpcþ/þ Xpcþ/þ Xpcþ/þ Xpc/ Xpc/ Xpcþ/þ Xpcþ/þ Xpcþ/þ Xpc/ Xpc/ Xpcþ/þ Xpcþ/þ Xpcþ/þ Xpc/ Xpc/ Xpc/ A28M09 A28M10 A28M11 A28M12 A28M13 A28M14 A28M15 A28M01 A28M02 A28M03 A28M04 A28M05 A28M06 A22M05 A22M11 A22M17 A22M12 A22M18 A22M01 A22M19 A22M25 A22M02 A22M14 A22M20 Saline B[a]P (150 mg/kg)c DMSO Cloning efﬁciency Proportion of TG-resistant clonesb Hprt MF (3 106) 0.10 0.15 0.14 0.09 0.11 0.12 0.07 0.14 0.15 0.10 0.12 0.12 0.08 0.15 0.09 0.19 0.08 0.08 0.12 0.21 0.15 0.19 0.04 0.20 11/576 4/420 8/576 9/575 24/576 27/576 14/468 4/576 4/476 3/576 2/576 5/576 7/552 24/192 11/132 55/540 43/180 37/300 7/576 9/564 10/576 13/432 1/529 26/492 4.80 1.55 2.58 4.62 9.32 9.65 1.04 1.27 1.20 1.32 0.71 1.80 4.23 21.67 33.34 14.14 90.46 42.27 2.64 1.92 2.93 3.96 2.15 6.92 a Mice were exposed by IP injection to EB or B[a]P and to saline or DMSO as vehicle controls. Represents the number of wells in 96-well plates containing mutant clones relative to the total number of wells scored. c Mice treated with B[a]P served as positive NER-GGR controls. b the alkylpurines and their subsequent reaction products may produce point mutations, and an intrastrand crosslink may produce deletions [Carmical et al., 2000a,b; Rodriguez et al., 2001; Kanuri et al., 2002]. Because of the broad spectrum of DNA adducts and mutations caused by the BD-epoxides, multiple DNA repair pathways are likely involved in maintaining genomic integrity following exposure. This includes nucleotide excision repair (NER), which may be essential for the repair of crosslinked adducts that are principally formed by DEB [Friedberg et al., 1995]. To examine the role that NER has in this process, Xpc-null (Xpc–/–) mice, deﬁcient in NER global genome repair (NER-GGR), were exposed to EB by injection. These mice were then examined for the induction of somatic mutations, using the Hprt assay. Increased Hprt mutant frequencies (MFs) in Xpc–/– mice, compared with Xpcþ/þ mice, would indicate that NER-GGR is responsible, in part, for maintaining genomic integrity compromised by BD-epoxides. MATERIALS AND METHODS Xpcþ/þ and Xpc–/– C57BL/6 female mice [Cheo et al., 1997] were exposed by intraperitoneal (IP) injection to a 300 mg/kg mutagenic dose of 3,4-epoxy-1-butene (EB; 98% pure, cat. no. 12,757-4, Sigma-Aldrich, St. Louis, MO). Three injections of 100 mg/kg EB were delivered at 48-hr intervals. Xpc–/– littermates were randomly assigned to treatment groups. Age-matched Xpcþ/þ mice were obtained from Charles River Laboratories (CRL, Wilmington, MA) and were treated with saline alone as injection controls. For positive NER controls, additional mice were exposed by IP injection to 150 mg/kg benzo[a]pyrene (B[a]P; cat. no. B1760, SigmaAldrich) dissolved in dimethylsulfoxide (DMSO). Mice serving as vehicle controls were injected with DMSO alone. Three injections of 50 mg/kg B[a]P were delivered at 48-hr intervals. All mice were treated at 5–6 weeks of age and were maintained for 4 weeks after exposure to EB, before performing the Hprt assay [Skopek et al., 1992; Meng et al., 1998; Wickliffe et al., 2003]. Mice treated with B[a]P were housed for 10 weeks before conducting the Hprt assay. All experiments were conducted in accordance with the Animal Care and Use Committee’s standards, at the University of Texas Medical Branch, under protocol 880202402. Mice were provided water and rodent chow (Prolab RMH 2500, LabDiet, Brentwood, MO) ad libitum and maintained on a 12 hr light–dark cycle. Statistical Analysis Hprt MFs were analyzed by univariate ANOVA followed by post hoc mean comparisons (Bonferroni-corrected), using the SPSS program (SPSS, Chicago, IL). An a < 0.05 was used to determine statistical signiﬁcance. RESULTS AND DISCUSSION Cloning efﬁciencies, proportion of 6-thioguanine-resistant clones, and Hprt MFs for each mouse used in this study are presented in Table I. Two mice (M07 and M08) Nucleotide Excision Repair and Epoxybutene 69 Fig. 1. Mean Hprt MFs in mice exposed to 300 mg/kg EB. Bars represent standard errors of the arithmetic mean. *Signiﬁcantly different from all other groups at P < 0.05. Fig. 2. Mean Hprt MFs in mice exposed to 150 mg/kg B[a]P. Bars represent standard errors of the arithmetic mean. Note the change in scale for MFs, compared with that in Figure 1. *Signiﬁcantly different from DMSO controls at P < 0.05. **Signiﬁcantly different from all other groups at P < 0.05. were excluded from the study. M07 had a cloning efﬁciency of only 0.001, and M08 died immediately following the ﬁrst injection of 100 mg/kg EB. 3,4-Epoxy-1-butene Exposure Mice deﬁcient in NER-GGR were more sensitive than Xpcþ/þ mice to the mutagenic potential of EB, following exposure. The Xpc–/– mice had signiﬁcantly higher Hprt MFs (P < 0.05) than those of Xpcþ/þ mice exposed to EB and control mice (both Xpcþ/þ and Xpc–/–) exposed to saline alone (Fig. 1). The average 2.8-fold increase in Hprt MFs in Xpc–/– mice relative to the MFs of Xpcþ/þ mice is similar to that observed in a recent experiment in which mice deﬁcient in microsomal epoxide hydrolase (Ephx1null) were exposed to 240 mg/kg EB (data unpublished). In that experiment, we observed a signiﬁcant (P < 0.05) 2.9-fold increase in Hprt MFs in Ephx1-null mice, compared with the MFs of exposed Xpcþ/þ mice. This suggests that, at these approximate levels, EB-exposed mice that are incapable of recognizing and repairing the BD-epox- ide DNA lesions that are NER-GGR substrates are as sensitive to the genotoxic effects of BD as Ephx1-null mice that are incapable of detoxifying the BD-epoxides themselves. In this study, exposed Xpcþ/þ mice exhibited Hprt MFs that were 2.7-fold higher than those of unexposed Xpcþ/þ mice. Benzo[a]Pyrene Exposure B[a]P is metabolized to a reactive epoxide that forms bulky DNA adducts that are classic substrates for NER. Therefore, we exposed Xpcþ/þ and Xpc–/– mice, by IP injection, to a mutagenic level of B[a]P. All B[a]Pexposed mice exhibited signiﬁcantly higher Hprt MFs than those of both Xpcþ/þ and Xpc–/– mice injected with vehicle (DMSO) alone (Fig. 2). As hypothesized, exposed Xpc–/– mice were considerably more sensitive to B[a]P, compared with their Xpcþ/þ counterpart mice. Xpc–/– mice exhibited Hprt MFs that were 2.9-fold higher than those of exposed Xpcþ/þ mice and at least 30-fold higher than those of unexposed control mice (Fig. 2). Considering the 70 Wickliffe et al. mutagenic response to the BD-epoxide EB, it was suggested that BD-epoxides are comparatively weak mutagens and/or that alternative DNA repair pathways are efﬁciently removing premutagenic lesions in the Xpc–/– mice. SUMMARY This study indicates that exposure to EB results in the formation of DNA adducts that are recognized and repaired by NER-GGR, but it remains unclear as to which adducts are the NER-GGR substrates. Since mice are capable of generating all of the reactive BD-epoxide intermediates from EB, it is reasonable to assume that DEB, formed in vivo, may be the speciﬁc epoxide responsible for generating these substrates. Additional studies are needed to address this hypothesis. The relevant experiments that are now necessary to better understand the recognition and repair of BD-induced DNA lesions should involve inhalation exposures to BD itself. These future studies, which would allow for the complete in vivo biotransformation of BD, will better address the genotoxicity and enhanced sensitivity associated with deﬁciencies in DNA repair. Characterizing DNA adduct formation in DNA repair-deﬁcient mice and examining mutation induction and mutational spectra in reporter genes, such as Hprt, following exposure to BD will answer important questions regarding formation, recognition, and repair of BD-induced DNA lesions. ACKNOWLEDGMENTS We acknowledge the Advanced Research Cooperation in Environmental Health program (ARCH-NIEHS 1 P30ES06676), the NIEHS Center at UTMB (S11 ES-10018) and an NIH R01 to JBW (2R01ES06015-07) for funding support. JKW was supported by a postdoctoral fellowship from an NIEHS training award to UTMB (T32-ES07254). OEM was supported by the Summer Undergraduate Research Program at UTMB that was funded by the Sealy Center for Environmental Health and Medicine. 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