Comparative Mutagenicity of the Two N2-2’-Deoxyguanosine Adducts Formed by 1-Nitropyrene

DNA is the genetic framework of every living creature. Stability and integrity of DNA are necessary for the survival of any organism. However, certain chemical and environmental factors can damage DNA, for example, PAHs and ultraviolet radiation damage DNA, which in turn give rise to mutations. Accumulations of these mutations result in diseases (such as cancer). Chemical mutagens can enter into the cells of living systems from the environment via exposure to pollutants and also through consumption of foods and drinks. Exposure to PAHs, a major class of chemical mutagens formed by combustion of fossil fuels, and also may occur by tobacco smoking. Nitropyrenes, nitrated polycyclic aromatic compounds and their derivatives, are one group of environmental pollutants which can form adducts with DNA base pairs. Because of their high reactivity to DNA, and their ability to cause mutations, nitropyrenes are classified as Class 2B mutagens, according to International Association for Research on Cancer. Mutagenicity of the most nitroaromatic carcinogen, 1-nitropyrene (1-NP), and its major DNA adduct formed at the C8 position of 2ꞌ-deoxyguanosine (dG) are well documented. However, 1-NP also forms two other adducts at the N2 position of dG, and the mutagenicity of these adducts is not well studied. In this thesis I provide an investigation of the biological effects of these two DNA adducts in human embryonic kidney cells (HEK 293T). Oligonucleotides containing dG-N2-6-aminopyrene (dG-N2-6-AP) or dG-N2-8-aminopyrene (dG-N2-8-AP), the two minor dG adducts of 1-NP, were synthesized and incorporated into a single-stranded plasmid, as ss DNA will not be repaired in the cells and helps to understand the action of Lesion. These constructs were transfected in HEK 293T and AB1157 strain of Escherichia coli cells. The mutation frequency and percent viability of these constructs, in comparison with control (non-mutated) plasmid, are reported here. The mutants included various kinds of targeted and off-target or semi-targeted mutations. We found that dG-N2-6-AP was more mutagenic than dG-N2-8-AP in HEK 293T cells, but for both lesions GàA was the most prevalent targeted mutations, which occurred in comparable levels. Significantly higher frequency of semi-targeted mutations was detected in the progeny from the dG-N2-6-AP construct compared to that from dG-N2-8-AP construct. Mutagenicity of dG-N2-6-AP in E. coli was also higher than that of dG-N2-8-AP, with 2.5-fold GàA mutations detected in the progeny from the former. This work shows that all three 1-nitropyrene-dG adducts are mutagenic and that their mutational specificity and frequency depends on the structure of the adduct