DEVELOPMENT OF RISK ASSESSMENT METHODS ON HAZARD IDENTIFICATION USING CATALASE MUTANT GENE AS A PREDICTIVE INDEX

本研究旨在利用过氧化氢酶突变株建立潜在危害的风险评估方法。首先，我们通过将过氧化氢酶突变小鼠(Cs^b，Cs^c)及其相应的野生型(Cs^a)过氧化氢酶基因的编码子转化到缺乏过氧化氢酶的大肠杆菌UM255中，成功地构建了过氧化氢酶突变菌株。不同过氧化氢酶缺陷型大肠杆菌的过氧化氢酶活力有显著差异。CsA组过氧化氢酶活性较高，Cs^c组为Cs^a组的65.1%，Cs^b组为61.7%，UM255组为Cs^a组的8.8%，对H_2O_2的敏感性与过氧化氢酶活性呈负相关。药敏顺序为UM255&gt；Cs？b&gt；Cs？c&gt；Cs？c&gt；Cs？a。为了评价化合物的细胞毒性，我们采用集落形成试验和抑菌圈试验，结果表明过氧化氢酶缺陷型大肠杆菌突变株的存活率是显著的…。对苯二酚、邻苯三酚和L-多巴呈剂量依赖性降低，各过氧化氢酶突变株的细胞存活率也有显著差异，表现出如下顺序：CS^a&Gt；Cs^c&Gt；Cs^b&Gt；UM255。对苯二酚、邻苯三酚和L-多巴的细胞毒性也通过抑制区试验得到证实，其中UM255最敏感，纸盘周围的生长抑制区最大，其次是Cs^b、Cs^c和Cs^a，这些化合物的毒性可被过氧化氢酶和抗坏血酸完全阻断，且呈浓度依赖关系。重金属(汞、铬、镉、砷、镍、铁、铜)的处理也显著降低了这些菌株的存活率，并呈剂量依赖关系，表明过氧化氢在氢醌细胞毒性过程中产生了越来越多的过氧化氢，过氧化氢酶在抑制细胞损伤方面起到了保护作用。这些结果支持了这些新建立的菌株在风险评估过程中用于确定氧化化学物质的危害。较少

This research was aimed at developing a risk assessment method for potential hazards by using catalase mutant strains. At first, we successfully constructed catalase mutant E coli strains by transforming plasmids encoding the catalase cDNA from catalase mutant mice (Cs^b, Cs^c) and its corresponding wild type (Cs^a) into catalase-deficient Escherichia coli UM255. There was a significant difference in catalase activities among each catalase-deficient E. coli mutant. The level of catalase in Csa appeared higher than that in the others, the catalase level in Cs^c was 65.1% of Cs^a, Cs^b was 61.7%, and UM 255 was 8.8% of Cs^a. Susceptibility to H_2O_2 showed a clear dose dependency with negative correlation to catalase activity levels. The susceptibility order was UM255>Cs^b>Cs^c>Cs^a. To assess the cytotoxicity of the chemicals, we employed colony-forming efficiency test and zones of inhibition test, the results demonstrated that survival of catalase-deficient E. coli mutants was signific … More antly reduced by treatment of hydroquinone, pyrogallol and L-Dopa in a dose-dependent manner and the cell viabilities underexposure to these chemicals were also different significantly among each catalase mutant strain, showing the following orders : Cs^a>Cs^c>Cs^b>UM255. The cytotoxicities induced by hydroquinone, pyrogallol and L-Dopa were also confirmed by zone of inhibition test, in which UM255 was the most susceptible, showing the largest zone of growth inhibition surrounding the paper disc, followed by Cs^b, Cs^c and Cs^a. The toxicities of these chemicals were completely blocked by catalase and ascorbic acid in a concentration-dependent relation. Treatment by some heavy metals (Hg, Cr, Cd, As, Ni, Fe, Cu) also significantly reduced the survival of these strains in a dose-dependent manner, suggesting H_2O_2 was increasingly produced in the process of hydroquinone cytotoxicity and catalase played a protective role in inhibiting the cell damage. The results support the usefulness of these newly established strains for hazard identification of oxidative chemicals in a risk assessment process. Less