Study On DNA Damage Of Tetrahymena Thermophila Caused By Pharmaceutical Industry Wastewater

With the development of technology for rapid detection of single cell DNA damage, single cell gel electrophoresis gradually became a new technology in recent years. If the cell DNA damaged, the fragment could get into the agarose gel. The damaged fragment migrated towards the anode after gel electrophoresis, and formed fluorescence tailing phenomenon which was like a comet. So it was also called comet assay. If cell DNA was more damaged, the more fragments would appear. The amount of DNA migration under the electric field was also more, and increased the length and fluorescence intensity of the tail. By measuring fluorescence intensity of DNA migration section or migration length, the technique could analysis the degree of injury of DNA cells of each group. The comet assay was simple and convenient. The experiment only needed small sample volume, which can detect a variety of toxic substances and was applicable to any cell type. At present, it was widely used in water toxicity monitoring.As a model organism, Tetrahymena thermophila had many advantages, such as simple cultivation, short life cycle, and high environmental contaminant sensitivity. This experiment used Tetrahymena thermophila, and combined with the experimental improvement, which made the experiment easier. The experimental improvement established a method of detecting industrial wastewater toxicity, which become rapid and accurate. It had the following content. Experiments proved that the improved method could replace the traditional method. Tetrahymena thermophila comet assay, combining with flow cytometry, analyzed DNA damage of Tetrahymena thermophila by the pharmaceutical industry wastewater, and compared adsorption wastewater with chlorinated wastewater about genetic toxicity. Meanwhile, the technique detected water quality of other industrial wastewater.The conclusions of this experiment were the following.(1) Standard comet assay and modified comet assay operated according to the conventional method. When the cells reached logarithmic phase, added different concentrations of test substances (hydrogen peroxide), at the same time set double distilled water with the same volume as the negative control group. After a period of time, centrifugal collectors made a certain concentration of cell suspension. Then did comet assay and observed results. At the same time of exposure, two methods showed that DNA damage about tail length, tail moment and olive tail moment, was basically consistent. Thus, the self-made template can replace the traditional method.(2) Tetrahymena thermophila comet assay, combining with flow cytometry, analyzed DNA damage induced by the pharmaceutical industry wastewater, which can improve the accuracy of test results. The results showed that the experimental improvement can improve the efficiency and stability, and showed a good dose-effect relationship between dilution ratio and Tetrahymena thermophila DNA damage. This result is consistent with the flow cytometry. In the same case of dilution multiple, genetic toxicity of adsorption wastewater was more than chlorinated wastewater.(3) Improvement of experiment detected other industrial wastewater, such as benzoic acid wastewater, benzonitrile wastewater, benzyl benzoate wastewater and chlor-alkali wastewater. Research showed the four kinds of industrial wastewater had different degrees of DNA damage on Tetrahymena thermophila. The toxicity intensity was:benzonitrile wastewater>benzoic acid wastewater> benzyl benzoate wastewater> chlor-alkali wastewater.In this study, improvement of experiment made the pretreatment and the process simpler. Therefore, Tetrahymena thermophila comet assay had the vital significance for improving the environmental monitoring system and monitoring of environmental catastrophe/carcinogen pollution.