Study of DNA methylation by tobacco-specific N-nitrosamines.
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 micrograms 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 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 (mumole 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 mumole/mole in the nasal mucosa and lung, respectively. In the liver no O6-MedGuo was detected, but 1050 mumole 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.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D000906:Antibodies; D002273:Carcinogens / Q000378:metabolism*; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism*; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000032:analysis; D003853:Deoxyribonucleosides / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay; D008745:Methylation; D009092:Mucous Membrane / Q000378:metabolism; D009297:Nasal Mucosa / Q000378:metabolism; D009374:Neoplasms, Experimental / Q000473:pathology; D009602:Nitrosamines / Q000378:metabolism* / Q000633:toxicity; D010947:Plants, Toxic; D051381:Rats; D012907:Smoking; D014026:Nicotiana
D000906:Antibodies; D002273:Carcinogens; D003853:Deoxyribonucleosides; D009602:Nitrosamines; C016583:4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone; D004247:DNA; C015558:O(6)-methyl-2'-deoxyguanosine; D003849:Deoxyguanosine