OPTIMIZATION OF P-32 POSTLABELING ASSAYS FOR THE QUANTITATION OF O-6-METHYL AND N7-METHYLDEOXYGUANOSINE-3'-MONOPHOSPHATE IN HUMAN DNA

The 3' and 5'-monophosphates of O-6-methyldeoxyguanosine and N7-methyldeoxyguanosine were chemically synthesized. Using these standards with deoxyguanosine-3'-monophosphate (dGp) as an internal standard, conditions were optimized to quantify O-6-methyldeoxyguanosine-3'-monophosphate (O-6-MedGp) and N7-methyldeoxyguanosine-3'monophosphate (N7-MedGp) by P-32-postlabelling. Under optimal conditions, the labelling efficiencies of O-6-MedGp and N7-MedGp were respectively similar to 100 and similar to 15%, with detection limits of similar to 1.1 and similar to 6.0 fmol respectively using 10 pmol dGp or 0.8 fmol of O-6-MedGp if 2 pmol of dGp was used. The assay developed for O-6-MedGp was then applied to the quantitation of [H-3]-O-6-MedGp and O-6-MedGp isolated from DNA digests by immunoaffinity separation. The standard curve generated from the use of [H-3]-O-6-MedGp, thus isolated, was identical to that generated previously using the chemically synthesized O-6-MedGp, indicating that no inhibitory factors co-eluted with the O-6-MedGp. After passage through two immunocolumns, recovery of 4 and 40 fmol of O-6-MedGp was similar to 30%. Four human stomach samples were analysed by combining this immunoaffinity purification with P-32-postlabelling: levels ranged from 0.21 to 0.86 mu mol O-6-MedGp/mol dG. Further DNA samples, isolated from the human colon, were fractionated by anion-exchange HPLC and the N7-MedGp and O-6-MedGp containing fractions were purified by reverse-phase HPLC and immunoaffinity chromatography respectively. Adduct-containing fractions were dried and P-32-postlabelled. Whereas O-6-MedGp was detected at levels between 0.3 and 3.4 mu mol O-6-MedGp/mol dG, no N7-MedGp was detected in these samples, probably due to depurination of N7-MedGp to N7-methylguanine or reduced assay sensitivity resulting from contaminating nucleotides and/or unidentified radioactivity eluting close to the N7-methyldeoxyguanosine-5'-monophosphate.