Measurement of O6-methyldeoxyguanosine in DNA methylated by the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone using a biotin-avidin enzyme-linked immunosorbent assay.
Rabbit antibodies raised against O6-methyl-2'-deoxyguanosine (O6-MedGuo) were used to develop a competitive Biotin-Avidin enzyme-linked immunosorbent assay (BA-ELISA) for the quantitation of this adduct in DNA from animals exposed to tobacco-specific N-nitrosamines. The assay was able to detect as little as 30 mumol O6-MedGuo per mol deoxyguanosine. The specificity of the assay was at least 10 000-fold greater for O6-MedGuo than for unmodified nucleosides or 7-methyl-2'-deoxyguanosine. The sensitivity of the assay was increased to 1 mumol O6-MedGuo per mol deoxyguanosine by the prior separation of O6-MedGuo from unmodified nucleosides by h.p.l.c. This assay was used to study the methylating ability of the tobacco-specific nitrosamine 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in F344 rats. Four hours after exposure to a single i.v. dose of NNK O6-MedGuo was detected in the target tissues nasal mucosa, lung and liver (219, 13.2 and 34.5 mumol/mol deoxyguanosine) but not in the non-target tissues esophagus, spleen, heart or kidney. Chronic exposure by 14 daily i.p. injections of NNK also resulted in detectable amounts of O6-MedGuo in the lung and nasal mucosa (11.4 and 7.9 mumol/mol deoxyguanosine). These results demonstrate that NNK is capable of methylating DNA in vivo. The methodology developed in these studies will allow us to examine in detail the role of DNA methylation in NNK-mediated carcinogenesis.
D000818:Animals; D001360:Avidin; D001710:Biotin; D002417:Cattle; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism*; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay; D008745:Methylation; D008770:Methylnitrosourea / Q000378:metabolism; D009297:Nasal Mucosa / Q000378:metabolism*; D009602:Nitrosamines / Q000378:metabolism / Q000633:toxicity*; D010947:Plants, Toxic; D051381:Rats; D011916:Rats, Inbred F344; D013950:Thymus Gland; D014018:Tissue Distribution; D014026:Nicotiana
D009602:Nitrosamines; D001360:Avidin; D008770:Methylnitrosourea; D001710:Biotin; C016583:4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone; D004247:DNA; C015558:O(6)-methyl-2'-deoxyguanosine; D003849:Deoxyguanosine