Reactive metabolites of 1,3-butadiene, including 1,2-epoxy-3-butene (BDO), 1,2:3,4-diepoxybutane (BDO2), 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 N6 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 → T transversions followed by A → 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 N6,N6-deoxyadenosine intrastrand cross-links derived from BDO2. These lesions were incorporated site-specifically and stereospecifically 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-BDO2 intrastrand cross-link between N6 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 → G transitions with overall mutagenic frequencies of 59 and 90%, respectively. Collectively, these data establish a molecular basis for A → 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 → T transversions that are the dominant point mutation.
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