Highly Sensitive, Specific Detection of C^-Methylguanine, C^-Methylthymine, and C^-Ethylthymine by the Combination of High-Performance Liquid Chromato- graphy Prefractionation, 32P Postlabeling, and Immunoprecipitation1

A highly sensitive and specific method for the detection of 06-methylguanine (O6-meG), O^-methylthymine (O4-meT), and CH-ethylthymine (O4-etT) has been established by combining prefractionation by high-performance liquid chromatography (III'IX '). '•'!' postlabeling, and immunoprecipitation by monoclonal antibodies (PREPI method). DNA was enzymatically hydrolyzed to 2'-deoxynucleoside-3'-monophosphates (3'-dNps). Each alkyl 3'dNp was separated by reversephase HPLC, radiolabeled at the 5' position with |v "I']A 11' and polynucleotide kinase. After removing 3'-phosphate for better recognition by the antibodies, the resulting alkyl nucleotides were further fractionated by HPLC and finally precipitated specifically with respective antibod ies. The detection limits were 1 fmol for all the alkyl nucleotides ana lyzed, so that one adduct in 10" of its normal counterpart nucleotide can be determined using ~100 fig (O4-meT and O4-etT) or ~ 150 *<g(O6meG) of DNA, i.e., 3-5 x IO7cells corresponding to —¿ 10 ml of periph eral blood or a few hundred milligrams of tissue. By the use of the PREPI method, three leukocyte and three liver DNA samples from Japanese living in the Tokyo area were analyzed with respect to (9-alkyl adduct content. O6-meG was detected in all three of the leukocyte sam ples (O'-meG:G molar ratio, 1.1 x, 0.8 x, and 1.6 x 10 "as molar ratios to guanine). Neither O4-meT nor O4-etT was detected (detection limit, O4-alkylT:thymine molar ratio < 0.5 x 10 8). Among the liver samples analyzed, two cases showed positive O6-meG values (4.2 x and 1.1 x 10 ~ O6-meG:guanine molar ratios). Contrary to the leukocyte DNA, O4-meT (3.9 x, 4.3 x, and 7.5 x 10 * as O4-meT:thymine) and O4-etT (1.9 x, 4.9 x, and 8.7 x 10 8as O4-etT:thymine) were detected in all the liver samples. These results indicate the validity of the PREPI method for molecular epidemiological studies on DNA alkylation products.