32P-postlabeling analysis of non-radioactive aromatic carcinogen--DNA adducts

Abstract

A newly developed enzymatic 32P-postlabeling method was applied to the analysis of DNA's containing non-radioactive arylamine, arylamide, and polycyclic aromatic hydrocarbon adducts. DNA reacted in vitro with N-hydroxy-2-amino-fluorene, N-acetoxy-2-acetylaminofluorene, and 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, respectively, as well as DNA preparations from the liver of rats treated with N-hydroxy-2-acetylaminofluorene and benzo[a]pyrene, respectively, were enzymatically digested to deoxyribonucleoside 3'-monophosphates, which were then converted to [5'-32P]deoxyribonucleoside 3',5'-bisphosphates by T4 polynucleotide kinase-catalyzed [32P]phosphate transfer from [gamma-32P]ATP. The 32P-labeled nucleotides were resolved by anion-exchange t.l.c. on polyethyleneimine-cellulose and detected by autoradiography. Aromatic adduct nucleotides were found to be retained at the origin in aqueous electrolyte solutions, but to migrate as distinct spots in solvents containing 7-8.5 M urea. Advantage was taken of this observation to remove 32P-labeled normal DNA nucleotides from adduct nucleotides. This purification enabled the detection of a single adduct in 10(7)-10(8) normal nucleotides. The method appears applicable to the ultrasensitive detection of a large number of carcinogen--DNA adducts of diverse structure without requiring radioactive carcinogens or specific antibodies.