P-32 POSTLABELING DETECTION OF THYMINE GLYCOLS - EVALUATION OF ADDUCT RECOVERIES AFTER ENHANCEMENT WITH AFFINITY-CHROMATOGRAPHY, NUCLEASE P1, NUCLEASE S1, AND POLYNUCLEOTIDE KINASE

Thymine glycol (Tg) is a product of DNA damage by oxygen radicals generated by oxidative mutagens and carcinogens and ionizing radiation. The highly sensitive P-32-postlabeling assay was validated and optimized for the measurement of Tg generated in vitro by the reaction of dTp or calf thymus DNA with osmium tetroxide (OsO4). Adduct detection was enhanced by purification of Tg adducts using phenylboronate affinity chromatography or by preferential dephosphorylation of unmodified 3'-nucleotides with nuclease P1, nuclease S1, or polynucleotide kinase; Tg nucleotides were found to be resistant to limited enzymatic 3'-dephosphorylation. Two adducts were seen with OsO4-modified dTp, which may have been cis-Tg adducts, because they were retained on a phenylboronate column, and because OsO4 selectively forms cis-Tg adducts. With OsO4-modified DNA, several adducts were detected, two major derivatives of which coincided chromatographically with those seen in OsO4-modified dTp. The recoveries of major adducts were similar before and after enrichment by different methods, indicating that Tg adducts were resistant to enzymatic dephosphorylation. The efficacy of labeling of the two major Tg adducts by polynucleotide kinase was optimal at 60-mu-M ATP and higher, whereas it was about 3%, 50%, and 80% of the optimal rate at 2, 10, and 30-mu-M, respectively. This was in contrast to our previous finding that only 0.25-mu-M ATP was needed for optimal labeling of benzoquinone-DNA adducts. Our results show that even with enrichment of Tg adducts, their poor substrate characteristics reduced the sensitivity of detection by one to two orders of magnitude compared to aromatic adducts, which are detectable at levels of one adduct in 10(9)-10(10) DNA bases. No Tg adducts were detected in the Zymbal glands of rats treated with benzene, which is potentially an oxygen radical-generating chemical.