除草剂丁草胺对牙鲆及其鳃细胞系的急性毒性研究
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摘要
除草剂丁草胺(butachlor)是目前国内使用最为广泛的三种除草剂之一,由于丁草胺的大量使用,导致其在水生环境中的普遍残留,因此丁草胺对水生生物的毒性效应引起了人们的高度重视。本文采用牙鲆和牙鲆鳃细胞系两个生物检测系统,比较研究了丁草胺对牙鲆的体内和体外急性毒性效应及其致毒机理。
本文首先研究了丁草胺对体外培养牙鲆鳃细胞(FG)的形态、生长、抗氧化酶活性和胞内ATP水平的毒性效应。研究发现:(1)中性红(NR)吸收、MTT检测和细胞蛋白含量分析的结果表明,丁草胺对FG细胞的24h-IC50(半抑制浓度)分别是43.32、43.86和44.91μmol/L。低于10μmol/L的丁草胺对FG细胞没有毒性,刺激FG细胞的生长。丁草胺浓度高于20μmol/L时,对FG细胞的生长开始表现出抑制作用,并具有剂量效应;(2)30μmol/L丁草胺处理24h后,FG细胞的形态才开始出现明显变化,收缩变圆,空泡增多;(3)30μmol/L丁草胺处理可显著影响FG细胞的抗氧化系统中抗氧化酶的活性。FG细胞内的三种抗氧化酶:超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GPX)和过氧化氢酶(CAT)的活性,在丁草胺处理后的早期(2h)急剧升高,随后逐渐下降,6或8h后降到对照水平,12或24h后不再继续下降,稳定在一个较低的水平;(4)0.1-40μmol/L丁草胺处理FG细胞24h后,所有处理组的细胞内ATP含量均明显下降。以上体外实验结果表明:丁草胺对FG细胞具有较高的急性毒性,它可以抑制FG细胞内的抗氧化酶活性,降低细胞内的ATP水平,从而抑制细胞生长,破坏细胞的形态和结构,最终导致细胞死亡。
其次,本文研究了不同浓度丁草胺对牙鲆活体的急性毒性效应。研究发现:(1)丁草胺对牙鲆高毒,96h-LC50(半致死浓度)是6.55 nmol/L,是FG细胞的24h-IC50值的1/7000,说明牙鲆活体比体外培养FG细胞在检测丁草胺的毒性上更敏感;(2)组织病理学检查的结果表明,鳃是丁草胺对牙鲆体内毒性作用的靶器官。2.56 nmol/L丁草胺处理牙鲆96h后,鳃组织出现明显的病理学变化,包括鳃上皮细胞坏死、肿胀和脱落,柱状细胞结构破坏,鳃丝充血,使鳃的结构和功能受到明显破坏;(3)3.84 nmol/L丁草胺处理后24h、48h和96h的牙鲆鳃组织内的ATP含量均显著降低。以上体内实验结果表明:与体外培养FG细胞相比,丁草胺对牙鲆具有更高的急性致死效应,这可能与丁草胺破坏了鳃的结构和功能,导致呼吸障碍,窒息而死有关。
最后,本文还研究了丁草胺对牙鲆和FG细胞的遗传毒性。结果表明:(1)1.28 nmol/L丁草胺处理96h后,就可观察到牙鲆血细胞的微核率明显升高,并且具有明显的剂量效应关系;(2)30μmol/L丁草胺处理FG细胞,8h后开始观察到基因组DNA的片断化,之后随着处理时间的延长,DNA片断化越来越严重,48h后基本就看不到完整的基因组DNA分子。以上结果表明丁草胺对牙鲆和FG细胞均具有较高的遗传毒性。
以上丁草胺对牙鲆的体内和体外急性毒性研究结果告诉我们,对海水鱼类来说,丁草胺是一种高毒农药,在今后的大田施用中,要尽量避开雨天和远离海边,以避免对海水鱼类的毒害。同时,本文研究结果还为今后利用体外培养鱼类细胞的毒性检测结果来预测鱼类致死效应,提供重要的参考资料。
Butachlor is one of the most commonly used herbicides in China. The large use of it in field has resulted in the ubiquitous leftover of it in aquatic environment. Therefore, the potential toxicity of butachlor to aquatic organisms has attracted great attentions of the society. In this study the acute in vivo and in vitro toxicity of butachlor on flounder, Paralichihys olivaceus, and flounder gill (FG) cells, as well as the possible mechanisms were investigated for the risk assessment of butachlor to marine fish species.
Firstly, the cytotoxic effects of butachlor on the cellular morphology, proliferation, activities of antioxidant enzymes and ATP level of FG cells were examined. Results indicated that: (1) the 24h-IC50 values (median inhibition concentration) of butachlor in FG cells were 43.32, 43.86 and 44.91μmol/L for neutral red (NR) uptake, MTT assay and cell protein assay, respectively. No acute cytotoxicity was observed in FG cells exposed to butachlor, which was lower than 10μM. Above 20μmol/L, the cytotoxicity of butachlor to FG cells was obvious and dose-dependent; (2) Obviously morphological alterations like shrinkage and vacuolation began to appear in FG cells exposed to 30μmol/L butachlor for 24h; (3) Butachlor can markedly affect the activities of antioxidant enzymes of Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) in FG cells exposed to 30μmol/L butachlor. And a quick increase of the enzyme activity was observed in the initial 2h exposure period, then drop to the level of control cells after 6 or 8h treatment, and stop at a constant level after 12 or 24 h treatment with a level lower than that of the control; (4) ATP contents in all the treated FG cells, the doses of butachlor ranging from 0.1 to 40μmol/L, were inhibited with a dose-dependent responsive. All the above in vitro results indicated that butachlor is high toxic to FG cells. It can result in the changes of the cellular morphology, inhibition of the cell proliferation and antioxidant enzyme activity, and drop of the ATP level in treated FG cells.
Secondly, acute in vivo effects of butachlor on flounder were investigated. Results indicated that: (1) butachlor caused much higher acute lethality on live flounder and the 96h-LC50 value (median lethal concentration) for live fish was 6.55 nmol/L, about 1/7000 of 24h-IC50 value in FG cells; (2) In live fish, histopathological examination showed that gill was the primary target organ of butachlor. butachlor, badly impaired the respiration of gills, by formation of lesions, breakdown of pillar cells, edema, lifting and detachment of lamellar epithelium, and blood congestion. The disfunction of gills caused suffocation to fish with extremely high acute lethality to flounder by butachlor; (3) ATP content in the gill tissues of flounder exposed to 3.84 nmol/L butachlor was also decreased for all the treatment intervals. All the in vivo results indicated that butachlor posed much higher lethality on flounder than in vitro cultured FG cells. This might be related to the extreme toxic of butachlor to the structure and function of flounder gill.
In the end, genotoxicity of butachlor on flounder and FG cells were studied. Results indicated that: (1) the fragmentation of genomic DNA of FG cells exposed to 30μmol/L butachlor was first observed after 8h exposure, and as increase of exposure time, the DNA damage became more severe; (2) the ratios of micronucleus in the erythrocytes of flounder exposed to 1.28~3.84 nmol/L butachlor for 96h were significantly increased in contrast to control. The above genetoxicity results indicated that butachlor was genotoxic to flounder and FG cells.
Taken together, butachlor is high toxic to marine fish flounder. Therefore, great caution should be taken in the field use of butachlor, such as keep away from raining and sea shore, to avoid any disaster to marine organism. And also, the results obtained can provide us a good guidance for the forecast of in vivo toxicity from the in vitro cytotoxic data.
本文首先研究了丁草胺对体外培养牙鲆鳃细胞(FG)的形态、生长、抗氧化酶活性和胞内ATP水平的毒性效应。研究发现:(1)中性红(NR)吸收、MTT检测和细胞蛋白含量分析的结果表明,丁草胺对FG细胞的24h-IC50(半抑制浓度)分别是43.32、43.86和44.91μmol/L。低于10μmol/L的丁草胺对FG细胞没有毒性,刺激FG细胞的生长。丁草胺浓度高于20μmol/L时,对FG细胞的生长开始表现出抑制作用,并具有剂量效应;(2)30μmol/L丁草胺处理24h后,FG细胞的形态才开始出现明显变化,收缩变圆,空泡增多;(3)30μmol/L丁草胺处理可显著影响FG细胞的抗氧化系统中抗氧化酶的活性。FG细胞内的三种抗氧化酶:超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GPX)和过氧化氢酶(CAT)的活性,在丁草胺处理后的早期(2h)急剧升高,随后逐渐下降,6或8h后降到对照水平,12或24h后不再继续下降,稳定在一个较低的水平;(4)0.1-40μmol/L丁草胺处理FG细胞24h后,所有处理组的细胞内ATP含量均明显下降。以上体外实验结果表明:丁草胺对FG细胞具有较高的急性毒性,它可以抑制FG细胞内的抗氧化酶活性,降低细胞内的ATP水平,从而抑制细胞生长,破坏细胞的形态和结构,最终导致细胞死亡。
其次,本文研究了不同浓度丁草胺对牙鲆活体的急性毒性效应。研究发现:(1)丁草胺对牙鲆高毒,96h-LC50(半致死浓度)是6.55 nmol/L,是FG细胞的24h-IC50值的1/7000,说明牙鲆活体比体外培养FG细胞在检测丁草胺的毒性上更敏感;(2)组织病理学检查的结果表明,鳃是丁草胺对牙鲆体内毒性作用的靶器官。2.56 nmol/L丁草胺处理牙鲆96h后,鳃组织出现明显的病理学变化,包括鳃上皮细胞坏死、肿胀和脱落,柱状细胞结构破坏,鳃丝充血,使鳃的结构和功能受到明显破坏;(3)3.84 nmol/L丁草胺处理后24h、48h和96h的牙鲆鳃组织内的ATP含量均显著降低。以上体内实验结果表明:与体外培养FG细胞相比,丁草胺对牙鲆具有更高的急性致死效应,这可能与丁草胺破坏了鳃的结构和功能,导致呼吸障碍,窒息而死有关。
最后,本文还研究了丁草胺对牙鲆和FG细胞的遗传毒性。结果表明:(1)1.28 nmol/L丁草胺处理96h后,就可观察到牙鲆血细胞的微核率明显升高,并且具有明显的剂量效应关系;(2)30μmol/L丁草胺处理FG细胞,8h后开始观察到基因组DNA的片断化,之后随着处理时间的延长,DNA片断化越来越严重,48h后基本就看不到完整的基因组DNA分子。以上结果表明丁草胺对牙鲆和FG细胞均具有较高的遗传毒性。
以上丁草胺对牙鲆的体内和体外急性毒性研究结果告诉我们,对海水鱼类来说,丁草胺是一种高毒农药,在今后的大田施用中,要尽量避开雨天和远离海边,以避免对海水鱼类的毒害。同时,本文研究结果还为今后利用体外培养鱼类细胞的毒性检测结果来预测鱼类致死效应,提供重要的参考资料。
Butachlor is one of the most commonly used herbicides in China. The large use of it in field has resulted in the ubiquitous leftover of it in aquatic environment. Therefore, the potential toxicity of butachlor to aquatic organisms has attracted great attentions of the society. In this study the acute in vivo and in vitro toxicity of butachlor on flounder, Paralichihys olivaceus, and flounder gill (FG) cells, as well as the possible mechanisms were investigated for the risk assessment of butachlor to marine fish species.
Firstly, the cytotoxic effects of butachlor on the cellular morphology, proliferation, activities of antioxidant enzymes and ATP level of FG cells were examined. Results indicated that: (1) the 24h-IC50 values (median inhibition concentration) of butachlor in FG cells were 43.32, 43.86 and 44.91μmol/L for neutral red (NR) uptake, MTT assay and cell protein assay, respectively. No acute cytotoxicity was observed in FG cells exposed to butachlor, which was lower than 10μM. Above 20μmol/L, the cytotoxicity of butachlor to FG cells was obvious and dose-dependent; (2) Obviously morphological alterations like shrinkage and vacuolation began to appear in FG cells exposed to 30μmol/L butachlor for 24h; (3) Butachlor can markedly affect the activities of antioxidant enzymes of Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) in FG cells exposed to 30μmol/L butachlor. And a quick increase of the enzyme activity was observed in the initial 2h exposure period, then drop to the level of control cells after 6 or 8h treatment, and stop at a constant level after 12 or 24 h treatment with a level lower than that of the control; (4) ATP contents in all the treated FG cells, the doses of butachlor ranging from 0.1 to 40μmol/L, were inhibited with a dose-dependent responsive. All the above in vitro results indicated that butachlor is high toxic to FG cells. It can result in the changes of the cellular morphology, inhibition of the cell proliferation and antioxidant enzyme activity, and drop of the ATP level in treated FG cells.
Secondly, acute in vivo effects of butachlor on flounder were investigated. Results indicated that: (1) butachlor caused much higher acute lethality on live flounder and the 96h-LC50 value (median lethal concentration) for live fish was 6.55 nmol/L, about 1/7000 of 24h-IC50 value in FG cells; (2) In live fish, histopathological examination showed that gill was the primary target organ of butachlor. butachlor, badly impaired the respiration of gills, by formation of lesions, breakdown of pillar cells, edema, lifting and detachment of lamellar epithelium, and blood congestion. The disfunction of gills caused suffocation to fish with extremely high acute lethality to flounder by butachlor; (3) ATP content in the gill tissues of flounder exposed to 3.84 nmol/L butachlor was also decreased for all the treatment intervals. All the in vivo results indicated that butachlor posed much higher lethality on flounder than in vitro cultured FG cells. This might be related to the extreme toxic of butachlor to the structure and function of flounder gill.
In the end, genotoxicity of butachlor on flounder and FG cells were studied. Results indicated that: (1) the fragmentation of genomic DNA of FG cells exposed to 30μmol/L butachlor was first observed after 8h exposure, and as increase of exposure time, the DNA damage became more severe; (2) the ratios of micronucleus in the erythrocytes of flounder exposed to 1.28~3.84 nmol/L butachlor for 96h were significantly increased in contrast to control. The above genetoxicity results indicated that butachlor was genotoxic to flounder and FG cells.
Taken together, butachlor is high toxic to marine fish flounder. Therefore, great caution should be taken in the field use of butachlor, such as keep away from raining and sea shore, to avoid any disaster to marine organism. And also, the results obtained can provide us a good guidance for the forecast of in vivo toxicity from the in vitro cytotoxic data.
引文
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