A highly sensitive and specific method for quantitation of O-alkylated DNA adducts and its application to the analysis of human tissue DNA.

Formation and accumulation of O6-alkylguanine and O4-alkylthymine in human tissues is possibly the most relevant marker for cancer risk. Because humans are chronically exposed to diverse kinds of chemicals and eventual DNA structural modifications are supposed to be a complex mixture of adducts at very low levels, it is essential to use an assay with extremely high sensitivity and specificity. We have established a quantitation method, called PREPI, for O6-methylguanine, O4-methylthymine, and O4-ethylthymine by the combination of prefractionation by HPLC, 32P-postlabeling, and immunoprecipitation. The detection limit was about 1 fmole for all three adducts, enabling us to analyze about 1 x 10(-8) levels as a molar ratio to normal counterpart using 100 micrograms of DNA. In a pilot experiment, we analyzed 11 peripheral blood samples from healthy volunteers. O6-Methylguanine was detected in all the cases with a mean value of 2.0 +/- 1.3 x 10(-8) (range, 0.78-4.6 x 10(-8)). Neither O4-methylthymine nor O4-ethylthymine was above the detection limit of 0.8 x 10(-8) as a ratio to thymine.