Endogenous 5-Methylcytosine Protects Neighboring Guanines from N7 and O⁶-Methylation and O⁶-Pyridyloxobutylation by the Tobacco Carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone

Abstract

All CG dinucleotides along exons 5-8 of the p53 tumor suppressor gene contain endogenous 5-methylcytosine (MeC). These same sites (e.g., codons 157, 158, 245, 248, and 273) are mutational hot spots in smoking-induced lung cancer. Several groups used the UvrABC endonuclease incision assay to demonstrate that methylated CG dinucleotides of the p53 gene are the preferred binding sites for the diol epoxides of bay region polycyclic aromatic hydrocarbons (PAH). In contrast, effects of endogenous cytosine methylation on the distribution of DNA lesions induced by tobacco-specific nitrosamines, e.g., 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), have not been elucidated. In the work presented here, a stable isotope labeling HPLC-ESI-MS/MS approach was employed to analyze the reactivity of the N7 and O6 positions of guanines within hemimethylated and fully methylated CG dinucleotides toward NNK-derived methylating and pyridyloxobutylating species. 15N3-labeled guanine bases were placed within synthetic DNA sequences representing endogenously methylated p53 codons 154, 157, and 248, followed by treatment with acetylated precursors to NNK diazohydroxides. HPLC-ESI-MS/MS analysis was used to determine the relative yields of N7- and O6-guanine adducts at the 15N3-labeled position. In all cases, the presence of MeC inhibited the formation of N7-methylguanine, O6-methylguanine, and O6-pyridyloxobutylguanine at a neighboring G, with the greatest decrease observed in fully methylated dinucleotides and at guanines preceded by MeC. Furthermore, the O6-Me-dG/N7-Me-G molar ratios were decreased in the presence of the 5'-neighboring MeC, suggesting that the observed decline in O6-alkylguanine adduct yields is, at least partially, a result of an altered reactivity pattern in methylated CG dinucleotides. These results indicate that, unlike N2-guanine adducts of PAH diol epoxides, NNK-induced N7- and O6-alkylguanine adducts are not preferentially formed at the endogenously methylated CG sites within the p53 tumor suppressor gene.

TP53 and KRAS Mutation Load and Types in Lung Cancers in Relation to Tobacco Smoke: Distinct Patterns in Never, Former, and Current Smokers

“…G:C to A:T may occur through formation of promutagenic O 6 -aklyl adducts induced by tobacco-specific nitrosamines such as NNK, present at significant levels in second-hand tobacco smoke (48). There is experimental evidence that presence of endogenous 5-methylcytosine in TP53 protects neighboring guanine from O 6 -alkylation by NNK (49), explaining the preferential occurrence of these adducts at non-CpG sites. G:C-to-A:T transitions at non-CpG sites in never or former smokers might thus represent a DNA fingerprint for NNK in subjects exposed to secondary smoke.…”

4-Hydroxy-1-(3-pyridyl)-1-butanone-releasing DNA adducts in lung, lower esophagus and cardia of sudden death victims

“…Secondly, the formation of DNA adducts and their role in the development of pulmonary tumors in rodents following exposure to NNK is highly cell-specific (Belinsky et al, 1991). Thirdly, in vivo metabolic activation of NNK in the lung could result in pyridyloxobutylating species which bind to DNA at multiple hotspots of which only some are essential for mutagenesis (Ziegel et al, 2004;Hashimoto et al, 2004;Herzog et al, 2006). Therefore, a high background of total HPB-releasing DNA adducts in samples of peripheral lung from sources other than NNK could mask the presence of biologically relevant adducts at specific sites of DNA in susceptible cell populations.…”

p53 mutation spectra for squamous cell carcinomas at different levels of human bronchial branches

“…10 From these results, the CpGfiCpA mutations observed in our cases were considered related to endogenous carcinogenic mechanisms, and not related to bulky adduct formation by exogenous carcinogens. 23 Comparison of frequencies of different p53 mutation spectra with reference to the tumor location and smoking status generated unexpected results. Thus, the central type, in spite of the highest rate of heavy smokers, had the highest rate of CpGfiCpA transitions.…”

“…Possible reasons are that not only deamination of 5-methylcytosine at methylated CpG sites but also carcinogens, such as nitrosamines, may be associated with or enhance production of CpGfiCpA transitions through other mechanisms different from bulky adduct formation. 23,37 The microenvironment in the tissue which is exposed to high concentrations of tobacco smoke may also influence GfiA mutations by (44) 12 (28) 4 (9) 3 (7) 1 (2) 20 (47) 4 (9) 1 Deletion/Insertion. as yet unidentified mechanisms.…”

“…19,20 Adducts such as those formed from nitrosamines are another likely cause of GfiA transitions. [21][22][23] However, most CpGfiCpA transitions are caused by endogenous mechanisms, if not predominantly observed in nontranscribed strands, because exogenous carcinogens usually produce bulky DNA adducts and consequent mutations show a strand bias. 24 To elucidate whether SCCs arising in different sites of the lung may be caused by different etiological factors and/or carcinogenic mechanisms, we here subclassified a large series of SCC by tumor-growing site, and examined relationships with p53 mutational spectra and smoking history.…”