A new modification of the P-32-post-labeling method to recover IQ-DNA adducts as mononucleotides

To obtain accurate estimates of DNA adduct levels yielded by genotoxic comp ounds, it is essential to completely digest adducted nucleotides to mononuc leotides, We previously developed a suitable method, called modified method I, to obtain DNA adducts of heterocyclic amines as P-32-labeled-mononucleo side adduct 5'-phosphate forms, by use of nuclease P1 (NP1) and phosphodies terase I (PDEI) to digest adducted oligonucleotides, In this study, we appl ied method I to 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)DNA adduct anal ysis and found that one of the IQ-DNA adducts, 5-(deoxyguanosin-N-2-yl)-2-a mino-3-methylimidazo [4,5-f]quinoline 3',5'-diphosphate (pdGp-N-2-IQ), was resistant to the 3'-phosphatase activity of NP1, but sensitive to that of T 4 polynucleotide kinase (PNK), DNA obtained from the liver of rats fed IQ w as P-32-labeled by the standard method and the P-32-labeled nucleotides obt ained were incubated with PNK and NP1 to remove 3'-phosphate groups and the n digested with PDEI, Three spots were obtained. One major spot was identif ied as N-(deoxyguanosin-8-yl)-2-amino-3-methylimidazo[4,5-f] 5'-phosphate ( pdG-C8-IQ) and a second abundant adduct as pdG-N-2-IQ. The third spot, of w hich 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 che micals.