Mutagenic Spectrum of Butadiene-Derived N1-Deoxyinosine Adducts and N 6 ,N 6 -Deoxyadenosine Intrastrand Cross-Links in Mammalian Cells Manorama Kanuri, † Lubomir V. Nechev, ‡,§ Pamela J. Tamura, ‡ Constance M. Harris, ‡ Thomas M. Harris, ‡ and R. Stephen Lloyd* ,†, | Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1071, and Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37235 Received August 2, 2002 Reactive metabolites of 1,3-butadiene, including 1,2-epoxy-3-butene (BDO), 1,2:3,4-di- epoxybutane (BDO 2 ), and 3,4-epoxy-1,2-butanediol (BDE), form both stable and unstable base adducts in DNA and have been implicated in producing genotoxic effects in rodents and human cells. N1 deoxyadenosine adducts are unstable and can undergo either hydrolytic deamination to yield N1 deoxyinosine adducts or Dimroth rearrangement to yield N 6 adducts. The dominant point mutation observed at AT sites in both in vivo and in vitro mutagenesis studies using BD and its epoxides has been A f T transversions followed by A f G transitions. To understand which of the butadiene adducts are responsible for mutations at AT sites, the present study focuses on the N1 deoxyinosine adduct at C2 of BDO and N 6 ,N 6 -deoxyadenosine intrastrand cross-links derived from BDO 2 . These lesions were incorporated site-specifically and stereospe- cifically into oligodeoxynucleotides which were engineered into mammalian shuttle vectors for replication bypass and mutational analyses in COS-7 cells. Replication of DNAs containing the R,R-BDO 2 intrastrand cross-link between N 6 positions of deoxyadenosine yielded a high frequency (59%) of single base substitutions at the 3′ adducted base, while 19% mutagenesis was detected using the S,S-diastereomer. Comparable studies using the R- and S-diastereomers of the N1 deoxyinosine adduct gave rise to ∼50 and 80% A f G transitions with overall mutagenic frequencies of 59 and 90%, respectively. Collectively, these data establish a molecular basis for A f G transitions that are observed following in vivo and in vitro exposures to BD and its epoxides, but fail to reveal the source of the A f T transversions that are the dominant point mutation. Introduction Butadiene (BD, 1 CH 2 dCHCHdCH 2 , Scheme 1) is a chemically reactive gas widely used in the manufacture of polymers such as styrene-butadiene and acrylonitrile- butadiene-styrene. It ranks among the top 20 high- volume chemicals produced in the United States (1) and is an environmental pollutant, being found in automobile exhaust and tobacco smoke. Butadiene is carcinogenic in mice, but less so in rats, and is classified as a human carcinogen in a recent report from the National Toxicol- ogy Program (2). The toxicology and epidemiology of butadiene have been previously reviewed (3, 4). The genetic effects of butadiene are attributed to its metabolic activation by cytochrome P450s (primarily 2A6 and 2E1) to the R- and S-monoepoxides (BDO), which can undergo diverse reac- tions including further oxidation to give stereoisomers of butadiene diepoxide (BDO 2 ). BDO 2 can undergo hy- drolysis to form the epoxide of butene-3,4-diol (BDE), which can also be formed by hydrolysis of BDO followed by further oxidation. BDO, BDO 2 , and BDE react with deoxynucleosides and DNA to form many different types of adducts (5-13). Examples include adducts at N1, N 2 , and N7 of deoxyguanosine, and N1 and N3 of deoxy- adenosine and N3 adducts of deoxycytidine. The N1 adducts of deoxyadenosine can undergo Dimroth rear- * To whom the correspondence should be addressed: Phone: (409) 772- 2179. Fax: (409) 772-1790. E-mail: rslloyd@utmb.edu. † University of Texas Medical Branch. ‡ Vanderbilt University. § Present address: Transgenomic, Inc., Boulder, CO 80301. | Dr. R. S. Lloyd holds the Mary Gibbs Jones Distinguished Chair in Environmental Toxicology from the Houston Endowment. 1 Abbreviations: BD, butadiene; BDO, butadiene monoepoxide (1,2- epoxy-3-butene); BDO2, butadiene diepoxide (1,2:3,4-diepoxybutane); BDE, 3,4-epoxy-1,2-butanediol; FAB MS, fast atom bombardment mass spectrometry; MALDI-TOF, matrix-assisted laser desorption mass spectrometry. Scheme 1. Reactive Metabolites of Butadiene a BD, butadiene; BDO, butadiene monoepoxide, 1,2-epoxy-3- butene; BDO2, butadiene diepoxide, 1,2,3.4-diepoxybutane; BDE, 3,4-epoxy-1,2-butanediol. 1572 Chem. Res. Toxicol. 2002, 15, 1572-1580 10.1021/tx025591g CCC: $22.00 © 2002 American Chemical Society Published on Web 11/19/2002