用^(32)P后标记方法测定典型有毒污染物的DNA加合物

INVESTIGATION OF DNA-CARCINOGENS ADDUCT IN VITRO, IN VIVO, IN HUMAN BY 32P-POSTLABELING

^(32)P后标记方法测定生物样品里的有毒污染物-DNA加合物的过程是用小球菌核酸酶和脾核酸酶将DNA及加合物酶解成为2’-脱氧核苷3’-单磷酸(3’-dNmP+3’-dXmP).以它为底物,在T_4多核苷酸激酶的催化下,与γ^(32)P ATP进行放射磷的转移反应,形成带放射磷的2’-脱氧核苷3’5’-二磷酸酯(3’5’-dNDP+3’5’-dXDP),用ODS-TLC吸附薄层板和PEI-TLC阴离子交换薄层板将标记过的带放射磷的3’5’-二磷酸酯分离分辨,然后用自显影技术制成加合物的指纹图,液闪计数计算出加合物的含量RAL值(相对加合物标记值).方法的灵敏度可达到1加合物/10^(10)正常核酸.本文对典型的有毒物苯乙烯及代谢物环氧苯乙烯(in vitro,in vivo,in human样品),苯及代谢物酚(in vivo样品),醌(in vitro,in vivo样品),单甲脒(in vitro,in vivo样品),2-硝基芴(in vivo)等的DNA加合物进行研究,其结果发现有毒物的DNA加合物和相应的有毒化合物的性质和剂量有相关性.

The 32P-postlabeling method used for detection DNA adduct was that DNA were enzymatically digested to deoxyribonucleoside 3 -monophosphates which were labeled to deoxyribonucleoside 3 5 -bisphosphates by T4 polynucleotide kinase catalyzed and r32P ATP. The labeled nucleotide were resolved on ODS-TLC and PEI-TLC, and detected by autoradiography. The sensitivity of method was 1 adduct per 1010 nucleotides. The 32P-postlabeling method was applied to analysis DNA adduct of styrene oxide, benzene, phenol, benzoquinone, 2-NF, DMA in vitro, in vivo, in human. A good correlation of DNA adduct and exposed concentration of chemical was found.