3-氨基苯二酰肼发光法在微粒体和重组细胞色素P450酶系中的毒理学应用(英文)

Toxicological application of luminol-dependent chemiluminescence assay in hepatic microsomes and reconstitutedcytochrome P450 enzyme system

本文分别从微粒体和重组细胞色素P450酶系水平,研究了3-氨基苯二酰肼(luminol)化学发光在毒理学中的应用。研究结果表明luminol化学发光法不仅可用于P450酶系活力的测定,也可用来检测反应体系中由外来化合物代谢转化而产生的自由基。自由基清除剂SOD、过氧化氢酶和二甲亚砜对多种重组酶系的化学发光均有不同程度的淬灭作用,其中以SOD作用最强,其抑制率为93～100％。44种外来化合物的重组酶系化学发光的测定结果表明,已知在代谢转化过程中形成自由基的化合物,其化学发光强度明显增强,如四氯化碳、三氯甲烷、四氯乙烯等;经代谢转化后毒作用增强的化合物,其发光强度有的增强,如二硫化碳、苯、甲苯、甲基对硫磷等,有的不变或减弱,如对硫磷、苯胺、马拉硫磷等;毒作用与代谢转化关系不很密切的化合物,其发光强度与对照比较无变化或减弱,如氨基比林等,多环芳烃类和亚硝胺类致癌物的发光强度,除3-甲基胆蒽、二甲基亚硝胺外,均无明显变化。

A mechanism of luminol-dependent chemiluminescence in microsomal cytochrome P450 enzymes has been studied. The results indicated that the chemiluminescence depended not only on NADPH induction but also on intact cytochrome P450 enzymes, and may be used as an assay of catalytic activity of the enzymes. The study of chemiluminescence in detecting free radicals formed by biotransformation of 44 xenobiotics suggested that some of the compounds known as free radical producing agents by bioactivation increased the light emission in the cytochrome P450 enzyme system, such as carbon tetrachloride, chloroform, tetrachloroethylene, etc., others forming toxic metabolites by biotransformation induced chemiluminescence in some cases, such as carbon disulfide, benzene, methyldursban, methylpara thion, etc. or decreased the light emission, such as parathion, aniline and polychlorinated biphenyls etc., and still others whose toxic actions have no relationship with their biotransformation had no influence or an inhibitory effect on the chemiluminescence, such as aminopyrine, TOCP, and acrylamide. Chemiluminescence in the cytochrome P450 enzyme system remained unchanged during the bioactivation of carcinogens such as diethylnitrosamine, dipropyl nitrosamine and benzo(a)pyrene as compared with the control.