Abstract 1697: A study on the reactivity of activated polycyclic aromatic hydrocarbons (PAH) with guanines base pared to C-5 substituted cytosines

Abstract

Abstract All CG dinucleotides within exons 5-8 of the human p53 tumor suppressor gene contain endogenous 5-methylcytosine (MeC). Guanine residues within these sites (e.g. codons 157, 158, 245, 248, and 273) are the major mutational hotspots for smoking induced lung cancer, suggesting that MeC mediates the reactivity of neighboring guanine bases towards tobacco carcinogens. Previous studies have shown that the reactivity of a carcinogenic diol epoxide metabolite of the human carcinogen, benzo[a]pyrene (B[a]P), towards the exocyclic amino group of guanine, is increased when guanine is base paired to MeC as compared to unmethylated cytosine. In the present study, the structural basis for the enhanced reactivity of BPDE towards MeC: G base pairs was investigated using a stable isotope labeling approach and a series of MeC structural analogs, including 5-ethyl-dC, 5-propyl-dC, N4-ethyl-dC, 5-chloro-dC, 5-bromo-dC, 5-iodo-dC, 5-propynyl-dC, difluorotoluene, pyrrolo-dC, phenylpyrrolo-dC, and diaminonaphthyl-derived nucleoside. Synthetic DNA duplexes derived from the frequently mutated region of the p53 tumor suppressor gene (5′-CCCGGCACCCGC[15N3, 13C1-G]TCCGCG-3′, from exon 5) were prepared containing [15N3, 13C1]-labeled guanine opposite C, MeC, or nucleobase analogs. Circular dichroism (CD) and UV melting studies have shown that C-5 substituents on cytosine do not disrupt the structure and stability of the DNA duplex. Following treatment with (±)-anti-benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide [(±)-anti-BPDE], (-)-anti-benzo[a]pyrene-s-7,t-8-dihydrodiol-t-9,10-epoxide [(-)-anti-BPDE], or related PAH diol epoxides, 5-methyl chrysene diol epoxide, benzo[c]phenanthrene diol epoxide, benzo[g]chrysene diol epoxide, and benzo[a,l]pyrene diol epoxide, and enzymatic hydrolysis of the adducted DNA to 2′-deoxynucleosides, the amounts of stereoisomeric N2-guanine adducts formed at the labeled site were determined by capillary HPLC-ESI+-MS/MS. We found that the presence of 5-methylcytosine and nucleobases with extended aromatic systems increases the reactivity of the partner guanine towards BPDE and other PAH diolepoxides, while 5-fluoro-dC and 5-iodo-dC lead to a decreased reactivity. Furthermore, the presence of C-5-cytosine analog modifies the stereoisomeric composition of the resulting adducts. Low temperature fluorescence and molecular docking studies reveal that the presence of MeC and unnatural base analogs with extended aromatic systems facilitate the formation of the pre-covalent BPDE-DNA complexes which place BPDE in a favorable orientation for trans attack by the N2 position of guanine. These results provide a mechanistic insight into the origins of increased reactivity of PAHs towards MeCG dinucleotides. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1697.