Title

The influence of local DNA sequence on the mutagenicity and genotoxicity of the major guanine adducts of benzo[a]pyrene and 1-nitropyrene in Escherichia coli

Date of Completion

January 2000

Keywords

Biology, Molecular|Chemistry, Biochemistry

Degree

Ph.D.

Abstract

Benzo[a]pyrene (B[a]P) and 1-Nitropyrene (1-NP) are two environmental pollutants linked to the occurrence of cancer. Through oxidative metabolism, B[a]P can be converted to its reactive metabolite, (+)-anti-BPDE, which forms the major adduct (+)-trans-anti-B[a]P- N2-dG (dGBP). 1-NP, on the other hand, forms the major lesion N-(deoxyguanosin-8-yl)-1-aminopyrene (dGAP) via nitroreduction. A study has shown that the sequence 5-GCG1G2CCAAAG-3 in the supF gene is a major mutagenic hot spot for (+)-anti-BPDE in E. coli with G1 yielding more mutants than G2 (Rodriguez and Loechler, 1993). For the reductively activated 1-NP, a study on its mutational specificity in E. coli has revealed that the sequences 5-GG, 5-GC, and 5-CG are the primary targets for point mutations, which included one base deletions and insertions as the predominant types (Malia and Basu, 1995). In order to investigate the roles of the major adducts in the occurrence of these mutagenic hot spots, the following oligonucleotides were synthesized: 5-GCG 1BPG2CCAAAG-3, 5-GCG1G2 BPCCAAAG-3, 5-CCATC5G6 APC7TACC-3, CCATG5 APG6C7TACC-3, and 5-CCATG5G6APC7TACC-3. These oligonucleotides were then inserted in a ss M13mp7L2 vector, transfected in E. coli, and the resulting plaques screened for possible mutants. Analysis of the progeny showed that G 1BP resulted in significantly higher percentage of base substitutions (G-to-T and G-to-C) than G2BP in SOS-induced cells. The −1 deletions resulting from -C5G6 APC7- were mostly C5 or C7 deletions, while those resulting from -G5APG6C 7- and -G5G6APC7- included mostly G5/6 deletions regardless of whether the cells were induced for SOS. Targeted base substitutions were detected in plaques from C 5G6APC7 (mostly G-to-T and G-to-C) and G5APG6C7 (mostly G-to-C) but not in G5G6APC7 genomes. No significant difference was observed in the genotoxicity of the two dG BP positions. On the contrary, G5G6AP C7 proved to be much less toxic than both C5G 6APC7 and G5APG 6C7 suggesting that the former is more bypassable by the DNA polymerase than the latter two. ^