32P-LABELING TEST FOR NUCLEIC ACID DAMAGE BY CARCINOGENS

我们将进一步开发和应用最近开发的32 P标记试验 通过我们来检测核酸反应形成的共价加合物 (DNA以及在较小程度上，RNA）与化学物质。 测试包括（i） DNA与化学物质在体外或体内的反应;（ii）DNA 纯化;（iii）DNA酶消化为脱氧核苷 3 '-单磷酸;（iv）将后者转化为32 P标记的 脱氧核苷3 '，5'-二磷酸与[γ-32 P]ATP孵育， T4多核苷酸激酶;（v）通过32 P标记的激酶的指纹分析， 反相高效液相色谱-阴离子交换 在聚乙烯亚胺-纤维素上的薄层色谱法，和（vi） 放射自显影 化学物质与DNA共价结合形成的加合物 被检测为这些指纹上的额外斑点。 类似的程序 适用于RNA的处理和分析。 该测试将适用于 致癌烷化剂、霉菌毒素、氨基偶氮染料、芳香胺 和酰胺，多环芳烃，其它芳烃， 亚硝胺、氯代烃、肼衍生物，以及 间谍。 一些非致癌物也将被检测。 将结果与已发表的诱变数据进行比较， 致癌性试验，以确定试验范围，作为 致癌物质。

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.