Study of DNA methylation by tobacco-specific N-nitrosamines

A. Castonguay, P. G. Foiles, N. Trushin, S. S. Hecht

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27 Scopus citations


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 O6-methyl-2'-deoxyguanosine (O6-MedGuo). Up to 5 μg of methylated DNA was enzymatically hydrolyzed, and the extent of inhibition of binding of immobilized O6-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 O6-MedGuo. Separation of O6-MedGuo from unmodified nucleosides by high-performance liquid chromatography (HPLC-BA-ELISA) allowed detection of 700 fmole O6-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), O6-MedGuo was present in target organs (μmole O6-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 (O6-MedGuo/dGuo) were 7.9 and 11.4 μmole/mole in the nasal mucosa and lung, respectively. In the liver no O6-MedGuo was detected, but 1050 μmole 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 (NNA1). Nasal mucosae were cultured in vitro with NNK or NNA1. After 1 hr, methylation at the O6-dGuo and 7-dGuo sites were observed with NNK but not with NNA1. Methylation by NNA1 after 24 hr was associated with the conversion of NNA1 to NNK. These results suggest that NNA1 is not associated with the activation of NNK to DNA methylating species.

Original languageEnglish (US)
Pages (from-to)197-202
Number of pages6
JournalEnvironmental health perspectives
VolumeVOL. 62
StatePublished - 1985


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