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DEVELOPMENT AND ITS PRACTICAL USE OF SCREENING METHODS ON HAZARD IDENTIFICATION USING CATALASE MUTANT GENE AS A PREDICTIVE INDEX

以过氧化氢酶突变基因为预测指标的危害识别筛选方法的开发及应用

基本信息

批准号：
17390173
负责人：
WANG Da-hong
金额：
$ 4.81万
依托单位：
Okayama University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2007
项目状态：
已结题

项目摘要

This research aimed at developing a screening method for hazard identification using catalase mutant E coli strains constructed by encoding the catalase cDNA from catalase mutant mice (Cs^b) and its corresponding wild type (Cs^a) into catalase-deficient K coli UM255. To assess the cytotoxicity of the chemicals, we employed a CAT assay (a zone of inhibition test of bacterial growth and colony-forming efficiency test using catalase mutant E coli strains). Lawsone exposure inhibited the growth of both Cs^a and Cs^b strains in a dose-dependent manner, in which Cs^b was more susceptible than Cs^a, showing the larger zone of growth inhibition surrounding the paper disc. Natural henna leaves did not show toxic effects, whereas two out of four samples of market henna products were shown toxicity effects. Catalase abolished zone of inhibition of henna products, and low concentration of exogenous MnSOD and Cu/ZnSOD eliminated the toxicity. We also exposed dithiothreitol (DTT) to catalase mutant E coli strains. The results show that DTT was also toxic to the catalase mutant K colis in dose dependent manner and significantly different between Cs^a and Cs^b. Catalase, CuZnSOD, and MnSOD also showed protective ability on tester strains. Treatment with waste disposal extracted by methanol and sterilized water showed a negative relation between the exposure dose and survival of these strains. The results support the usefulness of these newly established strains for hazard identification of oxidative chemicals in a risk assessment process.

本研究将过氧化氢酶突变小鼠（Cs^b）及其对应野生型（Cs^a）的过氧化氢酶cDNA编码到过氧化氢酶缺陷型大肠杆菌UM255中，建立过氧化氢酶突变型大肠杆菌菌株的危害鉴定筛选方法。为了评估这些化学物质的细胞毒性，我们采用了CAT试验（细菌生长的区域抑制试验和使用过氧化氢酶突变型大肠杆菌菌株的集落形成效率试验）。Lawsone暴露对Cs^a和Cs^b菌株的生长均呈剂量依赖性，其中Cs^b比Cs^a更敏感，在纸盘周围表现出更大的生长抑制区。天然指甲花叶子没有显示出毒性作用，而市场上四分之二的指甲花产品样品显示出毒性作用。过氧化氢酶消除了指甲花产物的抑制区，低浓度的外源MnSOD和Cu/ZnSOD消除了毒性。我们也将二硫苏糖醇（DTT）暴露于过氧化氢酶突变型大肠杆菌菌株中。结果表明，DTT对过氧化氢酶突变体大肠杆菌也具有剂量依赖性，且Cs^a和Cs^b之间存在显著差异。过氧化氢酶、CuZnSOD和MnSOD对试验菌株也有保护作用。用甲醇提取的废物处理和灭菌水处理，暴露剂量与这些菌株的存活呈负相关。结果支持这些新建立的菌株在风险评估过程中对氧化化学物质危险识别的有用性。