Highly sensitive, specific detection of O6-methylguanine, O4-methylthymine, and O4-ethylthymine by the combination of high-performance liquid chromatography prefractionation, 32P postlabeling, and immunoprecipitation.

ABSTRACT

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 reverse-phase HPLC, radiolabeled at the 5' position with [gamma-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 10(8) of its normal counterpart nucleotide can be determined using approximately 100 micrograms (O4-meT and O4-etT) or approximately 150 micrograms (O6-meG) of DNA, i.e., 3-5 x 10(7) cells corresponding to approximately 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-meGG 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-alkylTthymine molar ratio less than 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-meGguanine molar ratios). Contrary to the leukocyte DNA, O4-meT (3.9 x, 4.3 x, and 7.5 x 10(-8) as O4-meTthymine) and O4-etT (1.9 x, 4.9 x, and 8.7 x 10(-8) as O4-etTthymine) were detected in all the liver samples. These results indicate the validity of the PREPI method for molecular epidemiological studies on DNA alkylation products.