A new modification of the 32P-post-labeling method to recover IQDNA 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.