P-32-POSTLABELING TEST FOR COVALENT DNA-BINDING OF CHEMICALS INVIVO - APPLICATION TO A VARIETY OF AROMATIC CARCINOGENS AND METHYLATING AGENTS
P-32-POSTLABELING TEST FOR COVALENT DNA-BINDING OF CHEMICALS INVIVO - APPLICATION TO A VARIETY OF AROMATIC CARCINOGENS AND METHYLATING AGENTS
复制标题
DOI：
10.1093/carcin/5.2.231
复制
发表时间：
1984-01-01
期刊：
影响因子：
4.7
通讯作者：
RANDERATH, K
中科院分区：
文献类型：
--
作者：
REDDY, MV;GUPTA, RC;RANDERATH, K
在小鼠或大鼠体内暴露于28种化合物（包括7种芳胺及其衍生物、3种偶氮化合物、2种硝基芳烃、12种多环芳烃和4种甲基化剂）后，采用32p标记后测定致癌物质- dna加合物。分别从小鼠皮肤、小鼠肝脏和大鼠肝脏中分离出致癌物后的DNA，酶解为脱氧核糖核苷3”-单磷酸，经T4多核苷酸激酶催化[. γ .-32P]ATP转化为5”-32P标记的脱氧核糖核苷3”，5”-二磷酸。用阴离子交换法在聚乙烯亚胺纤维素上分离核苷酸，用放射自显影法检测。要测定芳香族致癌物的低水平DNA结合，需要在测定加合物核苷酸之前先去除正常核苷酸。为此，设计了一种采用反相t.l.c.的替代程序，该程序为定量检测少量加合物提供了优势。所描述的程序能够检测.apprx中的1个芳香DNA加合物。正常核苷酸108个，甲基化加合物的检出限为1个。6个同学。105个核苷酸。结果表明，大量不同结构的致癌物质dna加合物是多核苷酸激酶催化磷酸化32p标记的底物。由于共价DNA加合物在体内的形成似乎是大多数化学致癌物的基本特性，因此对哺乳动物组织中的致癌物-DNA加合物进行32p标记后分析可以作为筛选潜在致癌性化学品的一种测试。
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 [.gamma.-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 .apprx. 108 normal nucleotides, while the limit of detection of methylated adducts was 1 adduct in .apprx. 6 .times. 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 majorty 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.