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32P标记测试 由我们检测由核酸反应形成的共价加合物 (DNA和RNA，在较小的程度上)与化学品。这项测试涉及(I) 脱氧核糖核酸与化学物质的反应(S)；(Ii)脱氧核糖核酸 纯化；(Iii)DNA的酶消化生成脱氧核苷 3‘-单磷酸；(Iv)后者向32P标记的转化 脱氧核苷3‘，5’-二磷酸与[Gamma-32P]ATP和 T4多核苷酸激酶；(V)32P标记的酶切指纹图谱 反相高效液相色谱与阴离子交换 聚乙烯亚胺-纤维素的薄层色谱和(VI) 放射自显影。化学物质与DNA共价结合形成的加合物 被检测为此类指纹上的额外斑点。类似的手术 适用于RNA的处理和分析。这项测试将应用于 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、其他芳香族， 亚硝胺、氯代烃、联氨衍生物和 抗肿瘤药物。一些非致癌物质也将被检测。 结果将与已发表的突变和 致癌试验用于确定试验的范围，作为筛查 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.