Metabolism of Chlorothalonil by Glutathione S-Transferase
| Autor: | Chou, Chen-Ping, 周正平 |
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| Rok vydání: | 1998 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 86 Chlorothalonil is an important broad spectrum contact fungicide that has been widely used to control diseases of plants for about 25 years. One toxicological issue that continues to cause regulatory concern by EPA. U.S.A. is that chronic dietary administration of chlorothaloni to rodents induces a series of nonneoplastic histopathological changes in the epithelial cells of the proximal tubule of the kidney and the squamous epithelium of the forestomach. The mechanisms of the histopathlogical changes are initiated by the GSTs'' enzymatic reaction to produce the GSH-conjugated compounds, which are metabolized to cytotoxic thiols by cysteine conjugated β-lyase pathway. Our study is to identify the glutathionyl conjugates of chlorothalonil 1-3 compounds by using LC/MS and to isolate the GSTs from the non-pathogenic Escherichia coli for assaying the GSH-conjugated compounds formed by this enzyme. The results show that the high purity of chlorothalonil-GSH conjugates 1-3 could be obtained by enzymatical methods. And the molecular weights of the glutathionyl conjugates of chlorothalonil 1-3 are agreeable with LC/MS determined, after clean-up by HPLC. E.coli E1 was used as sources of GST. The bacterial cells were grown at 37℃ in 1.5 liters of LB broth, and were harvested and washed twice with buffer. After the cells were broken by ultra sonication, the cell homogenate was centrifuged at 100,000×g for 1h. The supernatant was used as crude enzyme extract.The enzyme was purified stepwisely by ammonium sulfate fractionation and GSH-affinity chromatography. The yield as measured by enzymatic activity was 12.8 %. Forty μl of partially purified GST ( 1.0 μg/ml ) was reacted with 1.0 mM GSH and 39 μM chlorothalonil for 24h. The amount of GSH-conjugates of chlorothalonil produced was higher than the control ( 1.0 mM GSH and 39 μM chlorothalonil only ). |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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