DEVELOPMENT OF RISK ASSESSMENT 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风险评估方法研究

• 批准号: 14570299
• 负责人: WANG Da-hong
• 金额: $ 2.24万
• 依托单位: OKAYAMA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14570299/
• 关键词: Catalase mutant E coli strains; Acatalasemic mice; Hypocatalasemic mice; Catalase mutant genes; Hydrogen peroxide; Cytotoxicity; Antioxidants; Inorganic heavy metal compounds; アカタラセラミクスマウス; ヒポカタラセミクマウス; カタラーゼ阻害剤; ニトロベンゼン; DNA酸化損傷

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

本研究旨在建立过氧化氢酶突变株潜在危害的风险评估方法。首先，我们将编码过氧化氢酶突变小鼠（Cs^b, Cs^c）及其相应野生型（Cs^a）过氧化氢酶cDNA的质粒转化为缺乏过氧化氢酶的大肠杆菌UM255，成功构建了过氧化氢酶突变型大肠杆菌菌株。过氧化氢酶缺陷大肠杆菌突变体中过氧化氢酶活性差异显著。Csa中过氧化氢酶含量高于其他品种，Cs^c中过氧化氢酶含量为Cs^a的65.1%，Cs^b中过氧化氢酶含量为61.7%，um255中过氧化氢酶含量为Cs^a的8.8%。对H_2O_2的敏感性与过氧化氢酶活性呈明显的剂量依赖性和负相关。磁化率顺序为UM255>Cs^b>Cs^c>Cs^a。为了评估这些化学物质的细胞毒性，我们采用了集落形成效率试验和抑制区试验，结果表明，过氧化氢酶缺陷的大肠杆菌突变株的存活率显著降低，对苯二酚、邻苯三酚和左旋多巴以剂量依赖的方式降低，并且在这些化学物质下，每个过氧化氢酶突变株的细胞存活率也有显著差异，表现出以下顺序：Cs ^ > c ^ c > b c ^ > UM255。对苯二酚、邻苯三酚和左旋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，在纸盘周围的生长抑制区最大，其次是Cs^b、Cs^c和Cs^a。过氧化氢酶和抗坏血酸完全阻断了这些化学物质的毒性，并呈浓度依赖性。某些重金属（Hg、Cr、Cd、As、Ni、Fe、Cu）处理也显著降低了这些菌株的存活率，且呈剂量依赖关系，说明氢醌细胞毒性过程中H_2O_2的产生增多，过氧化氢酶在抑制细胞损伤中起保护作用。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。少

项目成果

Reiko Sunami: "Acatalasemia sensitizes renal tubular epithelial cells to apoptosis and exacerbates renal fibrosis following unilateral ureteral obstruction"Am J Physiol-Renal Physiol. In press. (2004)

Reiko Sunami：“去钙血症使肾小管上皮细胞对细胞凋亡敏感，并在单侧输尿管梗阻后加剧肾纤维化”Am J Physiol-Renal Physiol。

Da-hong Wand, Noriyoshi Masuoka, Shohei Kira: "Animal model for oxidative stress research --Catalase mutant mice"Environ Health Prev Med. 8. 37-40 (2003)

Da-hong Wand、Noriyoshi Masuoka、Shohei Kira：“氧化应激研究的动物模型——过氧化氢酶突变小鼠”Environ Health Prev Med。

Hitoshi Sugiyama, Reiko Sunami, Da-Hong Wang, Shohei Kira, Hirofumi Makino: "Role of acatalasemia in the progression of oxidative stress-mediated renal injury."Kidney and Dialysis. 54(6). 749-752 (2003)

Hitoshi Sugiyama、Reiko Sunami、Da-Hong Wang、Shohei Kira、Hirofumi Makino：“无氧化酶血症在氧化应激介导的肾损伤进展中的作用。”肾脏与透析。

Reiko Sunami: "Acatalasemia sensitizes renal tubular epithelial cells to apoptosis and exacerbates renal fibrosis following unilateral ureteral obstruction"Am J Physiol-Renal Physiol. (発表予定).

Reiko Sunami：“肾小管上皮细胞凋亡使肾小管上皮细胞敏感，并在单侧输尿管梗阻后加剧肾纤维化”Am J Physiol-Renal Physiol（即将发表）。

Noriyoshi Masuoka, Hiroyuki Kodama, Tadashi Abe, Da-hon Wang, Taku Nakano: "Characterization of hydrogen peroxide removal reaction by hemoglobin in the presence of reduced pyridine nucleotides"Biochim Biophys Acta 1637. 1637. 46-54 (2003)

Noriyoshi Masuoka、Hiroyuki Kodama、Tadashi Abe、Da-hon Wang、Taku Nakano：“在还原吡啶核苷酸存在下，血红蛋白去除过氧化氢反应的特征”Biochim Biophys Acta 1637. 1637. 46-54 (2003)