Detection by 32P-postlabeling of thymidine glycol in γ-irradiated DNA

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Abstract
The 32P-postlabeling method has been adapted for the analysis of thymidine-cis-glycol-3′-phosphate (cis-dTGp, cis-5,6-dihydroxy-5,6-dihydrothymidine-3′-phosphate). Cis-dTGp was isolated and purified from normal nucleotides by phenylboronate affinity chromatography and phosphorylated by T4 polynucleotide kinase in presence of 1 mM BeCl2 at pH 7.5. These modifications of the postlabeling method resulted in a 5′-phosphorylation of dTGp with a labeling efficiency of up to 20% whereas the natural nucleotides were almost completely dephosphorylated at the 3′ position under these conditions. The reaction products, containing radio-labeled thymidine-cis-glycol-3′ ,5′-bis-[5′-32P]phosphate (cis-*pdTGp), were separated by two-dimensional anion-exchange TLC on polyethyleneimine cellulose sheets. Boric acid was added in the second dimension in order to selectively retard cis-glycols. The method was applied to γ-irradiated nucleotides and calf thymus DNA. In the nucleotide mixture, 330–99 000 thymine glycol (TG) moieties were detected per 106 thymines (T) in a dose range of 14–1000 Gy respectively. In DNA, these values ranged from 400 to 2700 TG/106 T. The data are in good agreement with methods using radiochemical and immunological techniques. Non-irradiated DNA showed a background level of 1OTG/106 T. This practical limit of detection was higher than can be achieved with the postlabeling technique, indicating that the present method might be a sensitive alternative for a determination of oxidative DNA damage.