32P-LABELING TEST FOR NUCLEIC ACID DAMAGE BY CARCINOGENS

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We shall further develop and apply a 32P-labeling test recently developed by us to detect covalent adducts formed by the reaction of nucleic acids (DNA and, to a lesser extent, RNA) with chemicals. The test involves (i) the reaction of DNA with chemical(s) in vitro or in vivo; (ii) DNA purification; (iii) enzymatic digestion of DNA to deoxynucleoside 3'-monophosphates; (iv) conversion of the latter to 32P-labeled deoxynucleoside 3',5'-bisphosphates by incubation with [Gamma-32P]ATP and T4 polynucleotide kinase; (v) fingerprinting of the 32P-labeled digests by reversed-phase high-performance liquid chromatography and anion-exchange thin-layer chromatorgraphy on polyethyleneimine-cellulose, and (vi) autoradiography. Adducts formed by the covalent binding to DNA of chemical are detected as extra spots on such fingerprints. A similar procedure applies to treatment and analysis of RNA. The test will be applied to carcinogenic alkylating agents, mycotoxins, aminoazo dyes, aromatic amines and amides, polycyclic aromatic hydrocarbons, other aromatics, nitrosamines, chlorinated hydrocarbons, hydrazine derivatives, and antineoplastic agents. A number of noncarcinogens will also be assayed. The results will be compared with published data from mutagenesis and carcinogenesis assays to determine the scope of the test as a prescreen for carcinogens.