Development of a new method to assess organophosphate insecticides exposure

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^&lt；-1&gt；·mg^&lt；-1&gt；protein and the molecular mass of the catalytic subunit was 76.8 kDa。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 sectices。Unfortunately，the antibody was not obtained。

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.