Mutagenic potential of adenine N-6 adducts of monoepoxide and diolepoxide derivatives of butadiene

To determine the biological effects of specific DNA adducts resulting From the interaction of 1,3-butadiene metabolites with DNA, deoxyoligonucleotide s have been synthesized with four different adducts at the N-6 position of adenine, centrally located within the human N-ras codon 61. The adducts are those arising from adduction by either the R or S stereoisomer of the mono epoxide (BDO) or the (R,R) or (S,S) isomer of the diolepoxide (BDE). The di olepoxide can arise from partial hydrolysis of the diepoxide (BDO2) or from epoxidation of hydrolyzed monoepoxide. These adducted oligonucleotides wer e used in in vivo and in vitro assays designed both to determine their muta genic potency and to examine specific interactions with Escherichia coli po lymerases. Each adducted oligonucleotide was ligated into a single-stranded vector M13mp7L2 that was subsequently used to transfect E, coli. The resul ting mutagenic spectrum for these modified DNAs was stereoisomer specific. Both monoepoxide lesions were nonmutagenic, but the mutagenic spectra for t he modified DNAs containing BDE adducts were stereoisomer specific. The mut ations generated by adducts of the R, R enantiomer of the diolepoxide were exclusively A --> G, whereas adducts of the S,S enantiomer of the diolepoxi de yielded exclusively A --> C mutations. None of the Four modifications re sulted in significant blocks to in vivo phage replication, as evidenced by no decrease in plaque-forming ability. Consistent with these data, when eac h of three purified E. coli polymerases was used to replicate DNAs containi ng these adducted deoxyoligonucleotides, the individual polymerases appeare d to be virtually unaffected, such that all lesions were readily bypassed. Whereas previous animal model studies identified the mutagenic spectrum rel ated to butadiene exposure, these studies begin to establish the specific l esions responsible for mutagenesis. This is the first report of stereoselec tivity related to butadiene induced mutagenesis. (C) 2000 Wiley-Liss, Inc.