Optimization of 32P-postlabelling assays for the quantitation of O6-mettyl and N7-methyldeoxyguanosine-3′ -monophosphates in human DNA

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Abstract
The 3′ and 5′-monophosphates of O6-methyldeoxyguanosine and N7-methyldeoxyguanoslne were chemically synthesized. Using these standards with deoxyguanoslne-3′-monophosphate (dGp) as an internal standard, conditions were optimized to quantify O6 -methyldeoxyguanosine-3′-monophosphate (O6 and N7-methyldeoxyguanosine-3′-monophosphate (N7-MedGp) by 32P-postlabelling Under optimal conditions, the labelling efficiencies of O6 -MedGp and N7-MedGp were respectively ∽100 and ∽15%, with detection limits of ∽1.1 and ∽6.0 fmol respectively using 10 pmol dGp or 0.8 fmol of O6-MedGp if 2 pmol of dGp was used. The assay developed for O6-MedGp was then applied to the quantitation of [3H]-O6-MedGp and O6 isolated from DNA digests by immnunoaffinity separation. The standard curve generated from the use of [3H]-O6MedGp, thus isolated, was identical to that generated previously using the chemically synthesized [3H]-O6-MedGp, indicating that no inhibitory factors co-eluted with the [3H]-O6-MedGp After passage through two immuno columns, recovery of 4 and 40 fmol of [3H]-O6-MedGp was ∽30%. Four human stomach samples were analysed by combining this immunoaffinity purification with 32P-post-labelling: levels ranged from 0.21 to 0.86 µmol O6-MedGp/ mol dG. Further DNA samples, isolated from the human colon, were fractionated by anion-exchange HPLC and the N7-MedGp and O6-MedGp containing fractions were purified by reverse-phase HPLC and immunoaffinity chromatography respectively. Adduct-containing fractions were dried and 32 Whereas O6-MedGp was detected at levels between 0.3 and 3.4 µmol O6-MedGp/mol dG, no N7-MedGp was detected in these samples, probably due to depunnation of N7-MedGp to N7-methylguanine or reduced assay sensitivity resulting from contaminating nucleotides and/or unidentified radioactivity elating close to the N7-methyldeoxyguanosine-5′-monophosphate.

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