32P-Postlabeling/polyacrylamide gel electrophoresis analysis: application to the detection of DNA adducts.
32P-Postlabeling analysis is a powerful technique to detect DNA adducts. Polyethylenimine-cellulose TLC plates are generally used to separate (32)P-labeled adducts using several different buffers. However, separation by TLC is time-consuming and labor-intensive for a large number of DNA samples. To expedite analyses, nondenaturing 30% polyacrylamide gel electrophoresis (PAGE) has been adapted for the (32)P-postlabeling analysis. The major advantages of this technique are as follows: (a) many DNA samples can be loaded concomitantly on the PAGE with standard markers; (b) DNA adducts can be resolved in only a few hours; and (c) exposure to (32)P during handing can be minimized. To show the usefulness of (32)P-postlabeling/PAGE analysis, the formation of a tamoxifen (TAM)-DNA adduct resulting from O-sulfonation of alpha-hydroxytamoxifen was demonstrated. In addition, to quantify TAM adducts, oligodeoxynucleotides containing diastereoisomers of alpha-(N(2)-deoxyguanosinyl)tamoxifen can be used as standards. The detection limit of this assay for 5 microg of DNA was approximately 7 adducts/10(9) nucleotides. The (32)P-postlabeling/PAGE analysis can also be used to detect DNA adducts derived from benzo[a]pyrene diol epoxide, 2-acetylaminofluorene, and 4-hydroxyequilenin.
Terashima
Isamu
I
Laboratory of Chemical Biology, Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794-8651, USA.
Suzuki
Naomi
N
Shibutani
Shinya
S
D018736:DNA Adducts / Q000032:analysis* / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D007553:Isotope Labeling; D010761:Phosphorus Radioisotopes; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000737:chemistry; D013629:Tamoxifen / Q000737:chemistry
D018736:DNA Adducts; D010761:Phosphorus Radioisotopes; D013629:Tamoxifen; D011116:Polynucleotide 5'-Hydroxyl-Kinase