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'-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.
journal_name
Carcinogenesisjournal_title
Carcinogenesisauthors
Hegi ME,Sagelsdorff P,Lutz WKdoi
10.1093/carcin/10.1.43subject
Has Abstractpub_date
1989-01-01 00:00:00pages
43-7issue
1eissn
0143-3334issn
1460-2180journal_volume
10pub_type
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