塔玛亚历山大藻(Alexandrium tamarense)对鲈鱼(Lateolabrax japonicus) 的危害机制研究

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	塔玛亚历山大藻(Alexandrium tamarense)对鲈鱼(Lateolabrax japonicus) 的危害机制研究
其他题名	Toxic effects and its mechanism of dinoflagellate Alexandrium tamarense on perch Lateolabrax japonicus
	谭志军
学位类型	博士
	2006-06-09
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	塔玛亚历山大藻麻痹性贝毒毒素鲈鱼溶血毒性组织结构与功能 na+k+-atpase 抗氧化系统酶异生物质代谢酶
摘要	本论文选择在我国分离得到的一株有毒赤潮甲藻-塔玛亚历山大藻（Alexandrium tamarense，ATHK株），研究了其对一种我国沿海常见和典型养殖鱼类鲈鱼（Lateolabrax japonicus）的危害机制。首先研究了塔玛亚历山大藻（ATHK）对鲈鱼鳃结构的影响及其溶血毒性；然后采用腹腔注射的方法，研究了高剂量塔玛亚历山大藻毒素（ATHK毒素：约为1.6×105 细胞，相应PSP为0.886µg STX Equal，相当于每克湿重的鲈鱼PSP注射量为0.0118µg STX Equal）和低剂量塔玛亚历山大藻毒素（ATHK毒素：约为0.16×105 细胞，相应PSP为0.0886µg STX Equal，相当于每克湿重的鲈鱼PSP注射量为0.00118µg STX Equal）在鲈鱼体内代谢过程中对鲈鱼肝脏、肾脏和鳃组织的超微结构、Na+K+-ATPase活性、肝脏功能和肾脏功能的影响、以及对抗氧化系统酶活性和异生物质代谢酶的影响，以期从不同方面了解塔玛亚历山大藻及其所产水溶性毒素（ATHK毒素）对鲈鱼的毒害效应及机制，为有毒赤潮的有效管理提供一定的科学依据。塔玛亚历山大藻（ATHK）对鲈鱼鳃组织影响的实验结果表明，该藻使鲈鱼鳃组织出现水肿现象，细胞间隙变大；粘液细胞颗粒不规则，颜色加深，颗粒发生凝集，有板结状；氯细胞线粒体内部基质凝集。不产PSP的一种亚历山大藻（AT-6）也使鲈鱼鳃出现水肿，且使细胞出现一定的固缩现象。显微镜观察发现鲈鱼鳃丝间存在有这两种亚历山大藻细胞。由此推测塔玛亚历山大藻ATHK和AT-6的表面结构可能具有能导致鲈鱼鳃组织水肿的机械作用。对人血细胞溶血实验结果表明塔玛亚历山大藻（ATHK）具有较强的溶血毒性，大小与藻的生长阶段和细胞密度都有一定的关系：指数期的溶血毒性最大，随细胞数目的增多，活性逐渐加大；藻细胞、细胞碎片、细胞内容物都有一定的溶血毒性，其中细胞碎片的活性最大。通过11种（株）产PSP的亚历山大藻、不产PSP的亚历山大藻以及标准PSP的实验结果表明这种溶血毒性是由藻细胞的其它非PSP物质造成的，且这种溶血毒性在产PSP的亚历山大藻中具有一定的普遍性。塔玛亚历山大藻（ATHK）对鲈鱼组织超微结构实验结果表明：ATHK毒素（约为1.6×105 细胞，相应PSP为0.886µg STX Equal，相当于每克湿重的鲈鱼PSP注射量为0.0118µg STX Equal）能导致鲈鱼组织细胞超微结构发生剧烈的变化，主要表现在：肝细胞细胞膜有肿胀现象，部分膜边缘溶解；细胞质糖原颗粒化；核糖体脱落，仅见滑面内质网；细胞质和线粒体内都出现空泡，且线粒体的嵴状结构也发生变化；核膜溶解比较严重，核质外溢，且异染色质边际化。前肾细胞超微结构的变化主要是淋巴细胞核质出现空泡，核膜有溶解迹象；Ⅰ型粒细胞颗粒膨大，伪足增多且变长；Ⅱ型颗粒细胞颗粒增多，内部出现空腔，细胞膜和核膜溶解，胞质、细胞器和核质外溢。鳃组织中氯细胞的核膜局部溶解，核仁弥散，线粒体膜溶解，微细小管膨大；粘液颗粒膜溶解，内部结构遭受破坏；扁平细胞核膜及线粒体膜几乎全部溶解。因此，我们的结果表明，ATHK毒素能作用于鲈鱼细胞的内膜和外膜系统，使膜发生溶解、脱落等变化；比较注射同样剂量大小ATHK毒素120h和240h时鲈鱼组织超微结构发现，细胞超微结构在一定程度上能够恢复。 ATHK毒素对Na+K+-ATPase活性、肝脏功能以及肾脏功能的影响结果表明，0.16×105―1.6×105细胞范围内的ATHK毒素可以显著影响肝脏和鳃组织中的Na+K+-ATPase，使这两种组织中的Na+K+-ATPase活性出现不同程度的下降；而且还能够显著抑制肝脏中谷丙转氨酶的活性，最大抑制率为95%。但此范围内的ATHK毒素不能显著影响肾脏中的Na+K+-ATPase活性以及尿素氮含量。因此，ATHK毒素对Na+K+-ATPase活性的抑制则会导致细胞能量的缺失，使细胞进一步发生其它变化，而ATHK毒素对肝脏功能完整性的影响则可能会抑制对蛋白质的分解代谢。 ATHK毒素对鲈鱼肝脏、肾脏和鳃组织中的、超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、谷胱肝肽过氧化物酶（GSH-Px）以及谷胱肝肽转硫酶（GST）活性变化影响的结果表明：高剂量（约为1.6×105 细胞，相应PSP为0.886µg STX Equal，相当于每克湿重的鲈鱼PSP注射量为0.0118µg STX Equal）ATHK毒素能显著诱导鲈鱼肝脏和鳃组织中SOD、GSH-Px以及GST酶活性，最大变化范围为正常状态下的3-4倍，对肝脏中CAT酶活性具有一定的抑制作用，对鳃中的CAT抑制效应则不显著；但此剂量的ATHK毒素仅对肾脏鳃中的GSH-Px活性有一定的诱导作用，对SOD、CAT以及GST的活性没有显著影响。低剂量（约为0.16×105 细胞，相应PSP为0.0886µg STX Equal，相当于每克湿重的鲈鱼PSP注射量为0.00118µg STX Equal）ATHK毒素也能诱导鲈鱼肝脏和鳃组织中SOD、GSH-Px以及GST酶活性，其在第一个24h内的诱导效果与临界致死毒素剂量相似；且连续注射低剂量ATHK毒素则对肝脏和鳃中这三种酶活性具有累加的诱导作用，使这三种酶活性的变化范围为正常的5倍；低剂量ATHK毒素对肝脏中CAT酶活性也具有抑制作用，但对鳃中CAT酶活性的抑制作用并不显著。同样，低剂量ATHK毒素除对肾脏中GSH-Px活性具有一定诱导效应外，对SOD、CAT以及GST都没有显著影响。 SOD、GSH-Px以及GST酶活性的显著升高表明ATHK毒素在鲈鱼体内代谢过程中能诱导鲈鱼产生活性氧自由基，且GST活性的升高则说明作为细胞色素P450依赖的异生物质代谢酶，GST在ATHK毒素代谢过程中可能可以加速ATHK毒素的代谢。推测鲈鱼可以通过这三种酶降低ATHK毒素以及次生毒物活性氧自由基对鲈鱼的危害。
其他摘要	The toxic effects of a PSP-producing strain of dinoflagellate Alexandrium tamarense(ATHK) on the perch Lateolabrax japonicus, a popular commercial fish in China, were studied in this thesis. The effects of the toxic algae on the ultrastructure of branchial cells of the perch after exposing to the cultures of A. tamarense(ATHK), and the hemolytic activity of A. tamarense(ATHK) on human erythrocytes, were studied at first. Then the extracted toxins prepared from A. tamarense (ATHK) cells were intraperitoneally injected into the perches in two different modes to study the impacts of toxins on the ultrastructure of different cells and enzyme activities of perches. The high dose injection was carried out only one time during the experiment, using extracted solution prepared from 1.6×105 algae cells with 0.05N acetic acid. The PSP toxicity was about 0.886µg STX Equal，and the dose injected was about 0.0118µg STX Equal/g fish (wet weight). The low dose injection was carried out multiply during the experiment, using the solution prepared from 0.16×105 cells，with the PSP toxicity about 0.0886µg STX Equal, and the dose injected was about 0.00118µg STX Equal/g fish (wet weight;) each time. The ultrastructure of liver, kidney and gill cells were observed, and the activities of the enzymes, including Na+K+-ATPase and xenobiotic-metabolizing enzymes and antioxidant enzymes, were tested to study the mechanism of the toxic effects of extracted toxins on L. japonicus. Experimental results showed that the gills of the juvenile perches exposed to the culture medium of A. tamarense(ATHK, 4 000cells/ml) for about 48 hours were significantly damaged. The most representative symptoms included tissue edema, irregular and congregated particles in mucus cells, and disintegrated mitochondria in the chloride cells. However, similar symptoms could also be observed in perches exposed to the culture medium of a non-PSP-producing strain of A. tamarense (AT-6 strain). The observation of algae cells in the gills of tested perches suggested that the mechanical injuries caused by the carapace of the algae, instead of PSP toxins, lead to the symptoms. In the hemolysis experiment using human erythrocytes, strong hemolytic activity of A. tamarense (ATHK) was found, and the hemolytic activity was related to the growth phase and cell concentration of the culture. It was found that the hemolytic activity in the culture medium increased gradually during the exponential phase and finally reached maximum at the stationary phase. Besides intact algal cells, the cell contents and fragments of A. tamarense also showed hemolytic activity, and the hemolytic activity of cell fragment was the highest. Through the comparison of hemolytic activities of twelve Alexndrium strains and STX standard, it was found that the hemolytic activity of A. tamarense was not caused by PSP toxins, but other unknown toxins. Therefore, factors other than PSP toxins will also do harm to organism, either through the mechanical effects or hemolytic activities. The injection of extracted toxins from A. tamarense (ATHK) cells led to apparent damage on the ultrastructure of cells in perches. The fishes injected with high toxicity solution showed swelled or even disintegrated membranes in hepatic cells, and hepatin particles appeared in the cytoplasm. Vacuoles could be observed in the cytoplasm. The rough endoplasmic reticulum became split, only smooth endoplasmic reticulum existed. The ultrastructure changes of mitochondria, like local edema, disarranged cristae, mitochondrial pupiform and vacuolate degeneration could also be observed. Partially disintegrated membranes of nucleus were found and most chromatin was located close to the brim of nucleolus. Some protoplasm was observed flowed out of nucleus. In pronephros cells, disintegrated membrane of lymphocytes and vacuoles in cytoplasm appeared. Enlarged granules in cytoplasm and increased number of pseudopods were observed in type I granulocytes. In type II granulocytes, disintegrated cellular and nucleolus membranes, which led to the outflow of cytoplasm, karyoplasms and organelles, could be observed. In the branchial cells, partially disintegrated nucleolus membrane and mitochondrion appeared, and tenuous tubes swelled. The disintegrated membranes in other types of cells could also be observed in the gills. It was concluded that toxins extracted from algae cells would lead to the disintegration of cellular and nuclear membranes, and membranes of other organelles such as mitochondria as well. Free radicals produced during the metabolization of toxins, or the PSP toxins which could lead to the blockage of sodium channels, were considered as the major reasons for the destruction of membranes. However, the damage to the membranes could recover after a period of time when injected with low dose of extracted toxins. To study the functional effects caused by the injection of extracted toxins, the Na+K+-ATPase activity in the liver, kidney and gill, Glutamic-pyruvic transaminase(ALT/GPT) activity in the liver, and the urea nitrogen content in the kidney were analyzed. The results showed that the injection of toxins in both modes significantly decreased the enzyme activity of Na+K+-ATPase in liver and gill. The activity of Glutamic-pyruvic transaminase(ALT/GPT) in liver was also significantly inhibited, which decreased about 95% compared to the control. However, there was no apparent effect on Na+K+-ATPase activity and the urea nitrpogen(BUN) content in kidney by the injected toxins. The effects of extracted toxins on the activities of both detoxification and antioxidant enzymes, including superoxide dismutase(SOD), catalase(CAT), Glutathione peroxidase(GSH-Px) and glutathione-S-transferase(GST) in liver, gill and kidney of perch L. japonicus, were also studied. In fishes injected with high dose of toxins, the enhanced activities of SOD, GSH-Px and GST in liver and gill were observed, with activities 3-4 times higher than the control. The activities of CAT in liver was inhibited, and the change of CAT activities in gill was not very significant. While the change of SOD, CAT and GST activites in kidney was not significant, except for the GSH-Px, which enhanced by high dose of ATHK toxins. Perches injected repeatedly with low dose of toxins showed a 5-10 times increase of enzyme activity of SOD, GSH-Px and GST in liver and gill within 120h, compared to the control, which suggested that continuously exposing to low doses of toxins could dramatically enhance SOD, GSH-Px and GST activities. Similarly, the activity of CAT in liver was inhibited by the injection of multiple sub-lethal doses of extracted toxins, and the change of CAT activities in gill was not very significant. While the change of SOD, CAT and GST activites in kidney also was not significant, except for the GSH-Px, which enhanced when injected repeatedly with low dose of toxins. The induction of SOD, GSH-Px and GST activities suggested that mechanisms to eliminate reactive oxygen species(ROS), which might associate with the metabolism of extracted toxins, and to accelerate the metabolism of toxins, would be employed by the perches to decrease the damages caused by the injection of extracted toxins.
页数	127
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/339
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	谭志军. 塔玛亚历山大藻(Alexandrium tamarense)对鲈鱼(Lateolabrax japonicus) 的危害机制研究[D]. 海洋研究所. 中国科学院海洋研究所,2006.

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