Comparison of TLC- and HPLC-32P-postlabelling assay for cisplatin-DNA adducts.
Calf thymus DNA, treated with the antitumour agent cis-diamminedichloroplatinum(II) (cisplatin), was enzymatically digested with deoxyribonuclease I, snake venom phosphodiesterase and prostatic acid phosphatase. As a result the adducts were released with an unmodified nucleotide at their 5'-side. The adducts were postlabelled with [gamma-32P]ATP and separated by thin-layer chromatography (TLC) and HPLC with on-line detection of 32P. One of the standards, platinated d(TGG), could be analysed with a recovery of 31% with both the TLC and the HPLC separation systems, when the amount of adduct was between 0.5 and 100 fmol. In the case of platinated DNA the linear part of the concentration curve was from 100 fmol down to the lowest amount of adducts measured, 3.2 fmol, with the TLC system and between 8 and 40 fmol with the HPLC system. The recoveries of the adducts were 28% and 16% respectively.
Försti
A
A
Center for Nutrition and Toxicology, Karolinska Institute, Huddinge, Sweden.
Staffas
J
J
Hemminki
K
K
D000135:Acid Phosphatase; D000255:Adenosine Triphosphate; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D002945:Cisplatin / Q000494:pharmacology*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000187:drug effects*; D018736:DNA Adducts / Q000737:chemistry / Q000302:isolation & purification*; D003850:Deoxyribonuclease I; D007553:Isotope Labeling / Q000379:methods*; D009841:Oligonucleotides / Q000737:chemistry / Q000302:isolation & purification; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases; D010761:Phosphorus Radioisotopes; D012680:Sensitivity and Specificity
D003432:Cross-Linking Reagents; D018736:DNA Adducts; D009841:Oligonucleotides; D010761:Phosphorus Radioisotopes; D000255:Adenosine Triphosphate; D004247:DNA; D003850:Deoxyribonuclease I; D000135:Acid Phosphatase; D010727:Phosphoric Diester Hydrolases; D043264:Phosphodiesterase I; C470790:phosphodiesterase I, snake venom; D002945:Cisplatin