Study of DNA methylation by tobacco-specific N-nitrosamines.

Abstract

An enzyme-linked immunosorbent assay (BA-ELISA) involving use of biotin-labeled anti-rabbit IgG and avidin-labeled horseradish peroxidase was developed for the measurement of 06-methyl-2'-deoxyguanosine (06-MedGuo). Up to 5 jg of methylated DNA was enzymatically hydrolyzed, and the extent of inhibition of binding of immobilized 06-MedGuo-bovine serum albumin to rabbit anti-O6-MedGuo was measured. Fifty percent inhibition of antigen-antibody binding was achieved with 2.5 pmole of of 06-MedGuo. Separation of 0-MedGuo from unmodified nucleosides by high-performance liquid chromatography (HPLC-BA-ELISA) allowed detection of 700 fmole 06-MedGuo in 1 mg of DNA.Among the tobacco-related carcinogens, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the most potent. In F344 rats it induces nasal cavity, lung and liver tumors. Four hours after a single IV injection of NNK to F344 rats (87 mg/kg body weight), 0'-MedGuo was present in target organs (,umole 0'-MedGuo/mole dGuo) (nasal mucosa, 219; lung, 13.2; and liver, 34.5) but was not detectable in nontarget organs. F344 rats receiving daily IP injections of NNK (40 mg/kg body weight) for 14 days were sacrificed 24 hr after the last injection. The levels of (06-Medguo/dGuo) were 7.9 and 11.4 ,umole/mole in the nasal mucosa and lung, respectively. In the liver no 06-MedGuo was detected, but 1050 ,umole of 7-MeGua/mole Gua was measured by HPLC-fluorimetry. No DNA methylation was observed in the nasal mucosa or liver of F344 rats treated with the nicotine-derived carcinogen N'-nitrosonornicotine. Reduction of the carbonyl of NNK is a major metabolic pathway, giving rise to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butan-1-ol (NNAI). Nasal mucosae were cultured in vitro with NNK or NNAI. After 1 hr, methylation at the 06-dGuo and 7-dGuo sites were observed with NNK but not with NNAI. Methylation by NNAI after 24 hr was associated with the conversion of NNAI to NNK. These results suggest that NNAI is not associated with the activation of NNK to DNA methylating species.

Biochemistry, Biology, and Carcinogenicity of Tobacco-Specific N-Nitrosamines

“…They are present in the respiratory and olfactory parts of the nasal mucosa, with the respiratory mucosa levels being higher. Consistent with expectations, O 6 -mG is not detected in nasal mucosa or liver of rats treated with NNN (178).…”

“…Other adducts are probably also produced, based on studies of other methylating nitrosamines and nitrosoureas, but these have not been identified from NNK (190). The first studies quantified O 6 -mdG in cultured rat nasal mucosa treated with NNK or NNAL; relatively high levels were detected by HPLCimmunoassay, consistent with the high activity in the nasal mucosa for R-hydroxylation of NNK (177,178). DNA methylation by NNK is observed in a number of in vitro studies with different systems capable of its metabolic activation, including rat lung cells and lung, liver, or nasal mucosal microsomes (with added DNA), rat oral tissue, and hamster lung (50,71,78,91).…”

Lysyl Oxidase, A Critical Intra- and Extra-Cellular Target in the Lung for Cigarette Smoke Pathogenesis

“…Aberrant hypermethylation of genomic DNAs and increased levels of DNA methyltransferase (DNA MeTase) activity have been detected in NNK and Cd treated cells [22,23,96]. Furthermore, both NNK and Cd also suppressed histone H3 acetylation at the LO core promoter region (Figure 4C).…”

“…Metabolic activation of NNK by cytochrome P-450 (CYP) is required to exert its carcinogenic activity [21]. Although it is established that NNK damages DNA via formation of methyl adducts [22,23] and mutation of p53, a tumor suppressor gene [24], the intracellular events elicited by CS/NNK and their underlying pathological mechanisms leading to cell injury and cancer development remain unclear.…”

Evidence for Phosphate Adducts in DNA from Mice Treated with 4-(N-Methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

“…The diazohydroxides formed are the methylating methanediazohydroxide or the pyridyl (oxo or hydroxy) butylating 4-(3-pyridyl)-4-(oxo or hydroxy)-1-butanediazohydroxide (6,12). The formation of methanediazohydroxide and its corresponding diazonium ion has been inferred from demonstration of methylations at different sites in DNA; N7- methylguanine, O 6 -methylguanine, and O 4 -methylthymine were observed (13)(14)(15)(16). The first study on pyridyloxobutylating species showed the presence of radioactivity in DNA from rats treated with [2′- 14 C]NNN (metabolized to the pyridyloxobutylating species only) but did not demonstrate covalent binding and adduct structure (13).…”