Mutagenesis, Vol. 14, No. 2, 239-242,
March 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
A new modification of the 32P-post-labeling method to recover IQ–DNA adducts as mononucleotides
1 Biochemistry and 2 Carcinogenesis Divisions, National Cancer Center Research Institute, 1-1 Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan and 3 Nestec Ltd, Research Centre, Vers-chez-les-Blanc, 1000-Lausanne 26, Switzerland
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-yl)-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.
4 To whom correspondence should be addressed. Tel: +81 3 3542 2511; Fax: +81 3 3542 2530; Email: mnagao{at}ncc.go.jp
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