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 7β,8α-di-hydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, respectively, as well as DNA preparations from the livers of rats treated with N-hydroxy-2-acetylaminofluorene and benzo[a]pyrene, respectively, were enzymatically digested to deoxyribonudeoside 3'-monophosphates, which were then converted to [5'-³²P]deoxyribonucleoside 3',5' -bisphosphates by T4 polynucleotide kinase-catalyzed [³²P]phosphate transfer from [γ-³²P]ATP. The ³²P-labeled nucleotides were resolved by anion-exchange t.l.c. on polyethylenelmine-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 nudeotides from adduct nudeotides. This purification enabled the detection of a single adduct in 10⁷–10⁸ 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.