A new modification of the 32P-post-labeling method to recover IQ-DNA adducts as mononucleotides.
To obtain accurate estimates of DNA adduct levels yielded by genotoxic compounds, it is essential to completely digest adducted nucleotides to mononucleotides. We previously developed a suitable method, called modified method I, to obtain DNA adducts of heterocyclic amines as 32P-labeled-mononucleoside adduct 5'-phosphate forms, by use of nuclease P1 (NP1) and phosphodiesterase I (PDEI) to digest adducted oligonucleotides. In this study, we applied method I to 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-DNA adduct analysis and found that one of the IQ-DNA adducts, 5-(deoxyguanosin-N2-yI)-2-amino-3-methylimidazo[4,5-f]quinoline 3',5'-diphosphate (pdGp-N2-IQ), was resistant to the 3'-phosphatase activity of NP1, but sensitive to that of T4 polynucleotide kinase (PNK). DNA obtained from the liver of rats fed IQ was 32P-labeled by the standard method and the 32P-labeled nucleotides obtained were incubated with PNK and NP1 to remove 3'-phosphate groups and then digested with PDEI. Three spots were obtained. One major spot was identified as N-(deoxyguanosin-8-yl)-2-amino-3-methylimidazo[4,5-f]quinoline 5'-phosphate (pdG-C8-IQ) and a second abundant adduct as pdG-N2-IQ. The third spot, of which the structure is unknown, was minor. The new method is called modified method II. Modified method II could be applicable to a wide variety of chemicals.
Ochiai
M
M
Biochemistry Division, National Cancer Center Research Institute, Tokyo, Japan. mnagao@ncc.go.jp
Nakagama
H
H
Turesky
R J
RJ
Sugimura
T
T
Nagao
M
M
D000818:Animals; D002845:Chromatography; D018736:DNA Adducts / Q000032:analysis*; D008099:Liver / Q000378:metabolism; D008956:Models, Chemical; D009152:Mutagenicity Tests / Q000379:methods*; D011804:Quinolines / Q000032:analysis*; D051381:Rats; D011916:Rats, Inbred F344
D018736:DNA Adducts; C096278:DNA-(2-amino-3-methylimidazo(4,5-f)quinoline) adduct; D011804:Quinolines