32P-Postlabeling test for covalent DNA binding of chemicals in vivo: application to a variety of aromatic carcinogens and methylating agents

Abstract

Carcinogen - DNA adducts were detected and determined by ³²P-postlabeling assay after exposure of mouse or rat tissues in vivo to a total of 28 compounds comprising 7 arylamines and derivatives, 3 azo compounds, 2 nitroaromatics, 12 polycyclic aromatic hydrocarbons, and 4 methylating agents. DNA was isolated from mouse skin, mouse liver, and rat liver after treatment with the individual carcinogens, then digested enzymatically to deoxyribonucleoside 3'-monophosphates, which were converted to 5'-³²P-labeled deoxyribonucleoside 3',5'-bisphosphates by T4 polynucleotide kinase-catalyzed [³²P]phosphate transfer from [γ-³²P]ATP. The nucleotides were resolved by anion-exchange t.l.c. on polyethyleneimine-cellulose and detected by autoradiography. The determination of low levels of DNA binding of the aromatic carcinogens entailed the removal of normal nucleotides prior to the resolution of adduct nucleotides. For this purpose, an alternative procedure employing reversed-phase t.l.c. was devised which offered advantages for the detection of quantitatively minor adducts. The procedures described enabled the detection of 1 aromatic DNA adduct in ∼10⁸ normal nucleotides, while the limit of detection of methylated adducts was 1 adduct in ∼6 × 10⁵ nucleotides. The results show that a great number of carcinogen-DNA adducts of diverse structure are substrates for ³²P-labeling by polynucleotide kinase-catalyzed phosphorylation. Because covalent DNA adduct formation in vivo appears to be an essential property of the majority of chemical carcinogens, ³²P-postlabeling analysis of carcinogen -DNA adducts in mammalian tissues may serve as a test for the screening of chemicals for potential carcinogenicity.