An improved 32P-postlabelling assay for detection and quantitation of styrene 7,8-oxide-DNA adducts.
Using DNA modified with [7-3H]styrene 7,8-oxide (SO) in vitro we have standardized the 32P-postlabelling assay for detecting SO-DNA adducts. Nuclease P1-enriched adducts were 32P-labelled and purified by high-salt (4.0 M ammonium formate, pH 6.1) C18 reverse-phase TLC. After elution from the layer with 2-butoxyethanol:H2O (4:6), adducts were separated by two-dimensional PEI cellulose TLC in non-urea solvents (2.0 M ammonium formate, pH 3.5, and 2.7 M sodium phosphate, pH 5.6). One major, three minor and several trace adducts were detected. The efficiency of the kinase reaction depended on the ATP concentration. Use of standard labelling conditions ([gamma-32P]ATP, < or = 3000 Ci/mmol; < or = 2 microM) resulted in poor (4-7%) adduct recovery. An ATP concentration of 40 microM, however, increased the labelling efficiency by a factor of 5-8 (35-55%) based on 3H-SO labelled DNA). The results indicate that the new separation technique is suitable for the relatively polar SO-DNA adducts and that high labelling efficiency can be achieved.
Cantoreggi
S
S
Institute of Toxicology, Swiss Federal Institute of Technology, Schwerzenbach.
Gupta
R C
RC
Lutz
W K
WK
D000818:Animals; D002273:Carcinogens / Q000032:analysis / Q000633:toxicity; D002855:Chromatography, Thin Layer / Q000379:methods; D004247:DNA / Q000032:analysis* / Q000187:drug effects; D004249:DNA Damage; D004852:Epoxy Compounds / Q000032:analysis* / Q000633:toxicity; D005069:Evaluation Studies as Topic; D066298:In Vitro Techniques; D008297:Male; D009153:Mutagens / Q000032:analysis / Q000633:toxicity; D010761:Phosphorus Radioisotopes
D002273:Carcinogens; D004852:Epoxy Compounds; D009153:Mutagens; D010761:Phosphorus Radioisotopes; D004247:DNA; C013690:styrene oxide