【作者】 宋超；

【导师】 陈家长；

【作者基本信息】 南京农业大学 ， 水产养殖， 2009， 硕士

【摘要】 水体富营养化造成蓝藻水华,铜绿微囊藻作为水华的主要藻类之一,其次级代谢产物微囊藻毒素对水生生物产生严重的毒性影响,该毒素也会通过生物富集作用和生物放大作用,并伴随食物链的过程对人类的健康造成潜在的威胁。因此对微囊藻毒素的毒性研究已迫在眉睫。本论文以尼罗罗非鱼为研究对象,从抗氧化防御系统的角度研究了微囊藻毒素的毒性作用效应。实验以血清ALT和AST酶作为生物指标来检测了微囊藻毒素对罗非鱼肝脏的损伤。结果显示,ALT和AST酶活性在24h达到最高值,并且该酶活性呈现明显的时间-效应关系和剂量-效应关系。说明MC能够侵入肝脏,引起肝脏一定程度的损伤。实验结果也认为运用血清ALT和AST酶作为罗非鱼养殖中监测微囊藻毒素的生物标志物的合理性。实验通过微囊藻毒素对罗非鱼肝脏P450亚酶活性的影响变化探讨了微囊藻毒素在Ⅰ相反应阶段的毒性作用。从实验的整个过程来看,对照组中P450亚酶(P450 1A2,P450 2E1,P450 3A1)随时间变化平稳,变化波动不及对照组。实验结果认为微囊藻毒素在Ⅰ相反应阶段作用并不明显,或者可能并没有进行Ⅰ相反应阶段的解毒作用。实验接下来探索了微囊藻毒素在罗非鱼机体Ⅱ相反应阶段的解毒作用。以内源性抗氧化酶(过氧化氢酶,CAT和过氧化物岐化酶,SOD)和谷胱甘肽系统组成的抗氧化防御系统在这个阶段发挥了重要的作用。实验结果表明,微囊藻毒素在罗非鱼的肝脏和鳃组织引起了活性氧自由基(ROS)的大量产生,分别在24h和36h达到高峰,并随时间变化,逐渐趋于正常。SOD和CAT的变化呈现明显的时间-效应关系与剂量-效应关系。但是在肾脏中,微囊藻毒素并没有引起ROS、SOD和CAT明显的变化。从谷胱甘肽系统相关酶的活性来看,在肝脏中,由于微囊藻毒素与谷胱甘肽的结合,消耗大量谷胱甘肽,引起谷胱甘肽还原酶和谷胱甘肽过氧化物酶在调节还原型谷胱甘肽和氧化型谷胱甘肽时的明显的变化。消耗谷胱甘肽的过程,并引起了肝脏谷胱甘肽从头合成机制的启动。从罗非鱼各组织抗氧化防御系统的能力来看,肝脏作为靶器官在微囊藻毒素的解毒作用中能力最强,肾脏次之,鳃最弱。微囊毒素在经过肝脏的代谢后可能被转运到了肾脏,是经过肾脏的排出还是再吸收作用,有待进一步研究。上述研究结果认为罗非鱼Ⅱ相反应阶段在微囊藻毒素的解毒过程中发挥了重要的作用。一般研究认为,毒素经过Ⅰ相反应阶段的代谢,为Ⅱ相反应创造条件,也可以不经过Ⅱ相反应直接由肾清除,本实验研究发现,微囊藻毒素在罗非鱼体内的代谢和解毒过程可能刚好相反。更多还原

【Abstract】 Water bloom is a serious problem caused by eutrophication. As a causative alage, Microcystis aeruginosa can metabolize microcystin. A toxicant may pose a very stress on fish and other aquatic animals. It also can become a potential risk to human health with a food chain by bioaccumulation and bioamplification. Great deals of attention have focused on microcystin. This thesis discussed oxidative stress responses to microcysrin in tilapia fish (Oreochromis niloticus).The experiment detected liver damage to microcystin in tilapia fish using glutamic-pyruvic transaminase (ALT) and glutamic-oxalacetic transaminase (AST) as bio-indicators. The results showed that the activities of ALT and AST had maximum values in 24 hours, and its values have time-depend effect and dose-depend effect. It indicated that microcysrin could be metabolized into live and caused liver damage, and we can use ALT and AST as biomarkers to monitor the toxicity of microcrstin to tilapia fish.The experiment discussed detoxification of microcystin in phase Ⅰ reaction in terms of variation of cytochrome P450 enzymes (P450 1A2, P450 2E1, P450 3A1).The results showed that P450 enzymes varied subtly without marked diffidence compared to control treatment in different time. Microcystin may not have detoxification in phase Ⅰ reaction.Then, the experiment discussed detoxification of microcystin in phase Ⅱ reaction. Antioxidtion defence system, combined with endogenous antioxidation enzymes (catalase, CAT and superoxidedismutase, SOD) and glutathione and its related enzymes, played a very important role in this reaction. The results showed that microcystin could cause surplus reactive oxygen species (ROS) in liver and gill. It could reach the maximum values in 24 hours and 36 hours respectively, and the activity values of CAT and SOD had time-depend effect and dose-depend effect. In the respect of glutathione system, microcystin could deplete glutathione when combined with it. Glutathione reductase (GR) and glutathione peroxidase (GPx) regulated reduced glutathione (GSH) and oxidized glutathione (GSSG) which is a very important buffer pair in vivo. The depletion of glutathione also could trigger its de novo synthesis. The results also showed that liver had the most ability in antioxidant defence system, kidney better, and gill worse. May microcystin be transported into kidney for excretion and/or reabsorption? It is still a question.Phase Ⅱ reaction played an essential role in detoxification of microcystin. It is believed that toxicant can be metabolitized in phase Ⅰ reaction, providing essential metabolite for phase Ⅱ reaction, or be excreted by kidney. Our results show a contrary detoxification process. Microcystin only processed in phase Ⅱ reaction.更多还原