Detection by 32P-postlabeling of thymidine glycol in gamma-irradiated DNA.
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'-phosphat e). 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 gamma-irradiated nucleotides and calf thymus DNA. In the nucleotide mixture, 330-99,000 thymine glycol (TG) moieties were detected per 10(6) thymines (T) in a dose range of 14-1000 Gy respectively. In DNA, these values ranged from 400 to 2700 TG/10(6) T. The data are in good agreement with methods using radiochemical and immunological techniques. Non-irradiated DNA showed a background level of 10TG/10(6) 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.
Hegi
M E
ME
Institute of Toxicology, Swiss Federal Institute of Technology, Schwerzenbach.
Sagelsdorff
P
P
Lutz
W K
WK
D002413:Cations, Divalent / Q000494:pharmacology; D002846:Chromatography, Affinity; D002855:Chromatography, Thin Layer; D004247:DNA / Q000528:radiation effects*; D004249:DNA Damage; D005609:Free Radicals; D005720:Gamma Rays; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D009153:Mutagens / Q000494:pharmacology; D010100:Oxygen / Q000633:toxicity; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D013936:Thymidine / Q000031:analogs & derivatives* / Q000032:analysis
D002413:Cations, Divalent; D005609:Free Radicals; D009153:Mutagens; D010761:Phosphorus Radioisotopes; C029390:thymidine glycol; D004247:DNA; D010100:Oxygen; D013936:Thymidine