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Development of a new method to assess organophosphate insecticides exposure

开发评估有机磷杀虫剂暴露的新方法

基本信息

批准号：
14580564
负责人：
KERA Yoshio
金额：
$ 1.98万
依托单位：
Nagaoka University of Technology
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

项目摘要

Since several chemicals such as organophosphate and carbamate insecticides and heavy metals inhibit cholinesterases, the tissue levels of acetylcholinesterase(AChE) in various fishes have been used as a potential biomarker for exposure to these chemicals in aquatic environments. In order to make AChE a more reliable and useful biomarker, it seems essential that a new method to assess organophosphate insecticides exposure, which is not affected by individual difference of fish in tissue levels of AChE, be developed. It is also desirable that the AChE is characterized well after purification, since catalytic properties and inhibitor sensitivities of the enzyme to various chemicals vary from species to species in general. However, there has been no characterized AChE of common fresh-water fishes. Thus in the present study, we purified and characterized AChE from body muscle of Koi carp(Cyprinus carpio), one of the fishes used for environmental monitoring in aquatic environment, and tried to develop the new method described above.At the first step, we purified the collagen-tailed asymmetric forms of AChE, the main forms in Koi carp body muscle, 79700-fold. The purified enzyme had a high specific activity of 14900μmol・min^<-1>・mg^<-1> protein and the molecular mass of the catalytic subunit was 76.8kDa. The characterizations of the purified enzyme in catalytic property, substrate specificity and inhibitor specificity demonstrate that the purified AChE from Koi carp muscle is certainly an AChE.At the second stage, we tried to produce specific polyclonal antibody to the catalytic subunit of purified Koi carp AChE, which is used for the development of the new method to assess organophosphate insecticides exposure. Unfortunately, the antibody was not obtained.

由于一些化学品，如有机磷和氨基甲酸酯类杀虫剂和重金属抑制胆碱酯酶，乙酰胆碱酯酶（AChE）在各种鱼类的组织水平已被用作一个潜在的生物标志物暴露于这些化学品在水生环境中。为了使AChE成为一种更可靠、更有用的生物标志物，有必要开发一种新的方法来评估有机磷农药暴露，该方法不受AChE组织水平个体差异的影响。还希望AChE在纯化后被很好地表征，因为酶对各种化学品的催化性质和抑制剂敏感性通常因物种而异。然而，目前还没有常见的淡水鱼类的乙酰胆碱酯酶的特征。因此，本研究以锦鲤（Cyprinus carpio）为研究对象，从锦鲤肌肉中提取AChE，并对其进行了初步的纯化和鉴定，初步建立了一种新的AChE提取方法。第一步，我们对锦鲤肌肉中主要的AChE胶原蛋白尾部不对称型进行了纯化，纯化倍数为79700倍。纯化的酶比活力为14900μmol·min^<-1>·mg^<-1>protein，催化亚基的分子量为76.8kDa。对纯化酶的催化特性、底物特异性和抑制剂特异性进行了表征，证实了锦鲤肌肉AChE是一种AChE。第二阶段，我们尝试制备针对锦鲤AChE催化亚基的特异性多克隆抗体，用于建立新的有机磷农药暴露评估方法。不幸的是，没有获得抗体。