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Highly Sensitive, Specific Detection of O⁶-Methylguanine, O⁴-Methylthymine, and O⁴-Ethylthymine by the Combination of High-Performance Liquid Chromatography Prefractionation, ³²P Postlabeling, and Immunoprecipitation¹

Department of Cancer Cell Research, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108, Japan [H-i. K., C. K., T. K., N-h. H.], and Institute of Cell Biology (Cancer Research), University of Essen Medical School, D-4300 Essen 1, Germany [G. E., M. F. R.]

A highly sensitive and specific method for the detection of O⁶-methylguanine (O⁶-meG), O⁴-methylthymine (O⁴-meT), and O⁴-ethylthymine (O⁴-etT) has been established by combining prefractionation by high-performance liquid chromatography (HPLC), ³²P 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 [-³²P]ATP 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 antibodies. The detection limits were 1 fmol for all the alkyl nucleotides analyzed, so that one adduct in 10⁸ of its normal counterpart nucleotide can be determined using 100 µg (O⁴-meT and O⁴-etT) or 150 µg (O⁶-meG) of DNA, i.e., 3–5 x 10⁷ cells corresponding to 10 ml of peripheral 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 O-alkyl adduct content. O⁶-meG was detected in all three of the leukocyte samples (O⁶-meG:G molar ratio, 1.1 x, 0.8 x, and 1.6 x 10^-8 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^-7 O⁶-meG:guanine molar ratios). Contrary to the leukocyte DNA, O⁴-meT (3.9 x, 4.3 x, and 7.5 x 10^-8 as O⁴-meT:thymine) and O⁴-etT (1.9 x, 4.9 x, and 8.7 x 10^-8 as 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.

¹ This work was supported in part by grants for Cancer Research and for Special Project Research, Cancer-Bioscience, from the Ministry of Education, Science and Culture of Japan and by the Deutsche Forschungsgemeinschaft (SFB 354/1).

² To whom requests for reprints should be addressed, at Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan.

Received 4/14/92. Accepted 7/22/92.

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