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题名: 微囊藻水华对不同食性鱼类毒理效应的野外研究及微囊藻毒素对大鼠心脏致毒性研究
作者: 邱彤
答辩日期: 2009-06-07
导师: 谢平
授予单位: 中国科学院水生生物研究所
授予地点: 水生生物研究所
学位: 博士
关键词: 微囊藻毒素 ; 鱼类 ; 大鼠 ; 组织病理学 ; 超微结构变化 ; 抗氧化系统 ; 线粒体
其他题名: Field Studies on the Toxicological Effects from Microcystis Blooms on Fishes and the Cardiac Toxicity of Microcystins in Rat
摘要: 随着人类社会生产力的大幅提高及人口压力的不断增大,全球范围的淡水水体富营养化现象日趋严重,由此引起的蓝藻水华在世界范围内频繁暴发,其伴随产生的毒素与动物及人类健康的关系受到了越来越多的关注。微囊藻毒素(Microcystins, MC)是一类出现频率高、产毒量大、危害严重的蓝藻毒素。鱼类是水生态系统的次级或顶级消费者,直接暴露于微囊藻毒素中,与此相关的鱼类死亡和鱼类中毒事件时有报道。目前,较多的研究证实了在微囊藻毒素急性染毒条件下对各种不同食性鱼类的致毒效应。然而,关于有毒蓝藻水华长时间或者是反复暴露后导致的动物慢性毒性研究还十分匮乏。本文通过野外调查探讨微囊藻水华对不同食性鱼类肝脏和肾脏的生理和生物化学影响,并分析可能原因。 一直以来,人们都认为微囊藻毒素导致的哺乳动物死亡原因主要是低血量休克及其引起的循环系统障碍。近来研究指出,心脏毒性可能是微囊藻毒素致死的又一潜在因素。然而,目前对于微囊藻毒素心脏致毒性的研究非常少,尚没有致毒机理的研究报道。本文通过研究静脉注射急性染毒情况下,心脏抗氧化系统的响应和线粒体呼吸链的生理改变,探讨了微囊藻毒素心脏致毒性的机制。 主要结果和结论如下: 1.通过太湖梅梁湾的鱼类控藻围栏实验,研究了自然水体中,有毒蓝藻水华引起的不同食性鱼类(肉食性鲌鱼、杂食性鲫鱼以及滤食性鲢鱼和鳙鱼)肝脏的生理和生物化学响应。在水华暴发期,杂食性和肉食性鱼类肝脏细胞超微结构都出现了明显的病理改变(主要是细胞内膜系统肿胀和细胞核形态畸变),而滤食性鱼类表现出较强抗性。通过生物化学分析,检测了鱼类肝脏主要抗氧化酶活力和抗氧化物质(GSH)含量的周年变化,推测,滤食性鱼类较之肉食性和杂食性鱼类具有对MC更强的抗性可能是源于鱼体肝脏内较高的GSH本底储备及抗氧化系统成员更好的协同性。 2.在太湖梅梁湾的鱼类控藻围栏实验中,不同食性鱼类(包括肉食性鲌鱼、杂食性鲫鱼以及滤食性鲢鱼和鳙鱼)在水华暴发期肾脏均出现了病理变化,主要是肾小球上皮细胞足突融合。同时,四种鱼类肾脏的GST和CAT酶活力在水华期都显著升高。而在对这些鱼类肝脏的生理和生物化学研究中,滤食性鱼类较之杂食性和肉食性鱼类对于有毒蓝藻水华具有更强的抗性。通过比较鱼类肝脏和肾脏抗氧化系统的生理状态,推测毒素次级代谢物在肾脏中的累积和肾脏相对薄弱的抗氧化系统导致其较肝脏更易受到毒害影响。由此看来,长期的有毒蓝藻水华对鱼类产生的慢性毒性,首先是引起鱼类肾脏损伤,然后再是肝脏损伤。 3.在太湖梅梁湾的鱼类控藻围栏实验中,通过监测鲢鱼和鳙鱼血清生物化学指标的变化,证实了蓝藻水华对滤食性鱼类健康的危害,其主要是潜在的基因毒理学效应,肝脏代谢功能障碍和肾脏功能障碍。此外,指示胆管阻塞的主要血清学指标的显著升高表明鳙鱼体内极可能出现了胆汁淤积。通过统计分析方法筛选,鲢鱼AST,LDH,DBIL,URIC组合,鳙鱼ALT,ALP,TBIL,DBIL,URIC组合,分别能较好的表征这两种鱼类在水华暴发期和非水华暴发期的机体生理生化改变,可用于野外鱼类健康评估指标。 4.通过静脉注射微囊藻毒素染毒(0.16LD50和1LD50),在死亡个体中,发现大面积的肝脏内出血,并证实同时有心肌梗死现象。1LD50组动物在染毒24h后,心率下降,血压升高,血清肌酸激酶和肌钙蛋白均升高。病理学观察发现,心脏组织变松散,线粒体出现肿胀和破裂。心肌组织抗氧化酶活性及其mRNA表达水平均升高,GSH含量升高。在两个染毒剂量组中,心肌线粒体LPO水平升高,提示线粒体内出现了严重的氧化应激。同时,染毒后心肌细胞线粒体呼吸链酶复合物I和复合物III均受到抑制,提示线粒体呼吸链产生了电子传递障碍。为此,回溯1996年的那次水污染导致的死亡事件,MC的心脏致毒性可能促使在中毒后机体出现的低血压休克症状更为严重,成为导致病人死亡的又一重要因素。
英文摘要: Dense blooms of cyanobacterial (blue–green) algae are one of the consequences of the increasing eutrophication in many waters worldwide. With frequent occurrence of cyanobacterial blooms in recent years, cyanotoxins have become a great threat to aquatic animals, livestock, and human health. Among these toxins, microcystins (MC) are the most common and dangerous hepatotoxins. Fish, one of the main inhabitants in aquatic systems, is usually subjected to MC directly or passively, which consequently causes fish kill or fish poisoning episodes. Till now, most experimental studies documenting the toxicity of MC exposure to fishes have been prone to acute toxicity experiments. Few investigations have been conducted on post-event of wild animal poisonings in which toxic cyanobacteria are suspected, especially when long-term and/or frequent exposure occurs. The main aim of this study was to examine physiological and biochemical responses of fishes with different trophic levels to toxic cyanobacterial blooms in Meiliang Bay, Lake Taihu, with discussion on the possible mechanisms. Deaths from microcystin toxication have widely been attributed to hypovolemic shock due to hepatic interstitial hemorrhage, while some recent studies suggest that cardiogenic complication is also involved. So far, information on cardiotoxic effects of MC has been rare and the underlying mechanism is still puzzling. We conducted intravenous injection of extracted MC in rats,in order to evaluate the roles of oxidative stress and mitochondrial dysfunction in cardiotoxic effects by MC. The main results and conclusions were summarized as follows: 1. Physiological and biochemical responses of four fishes with different trophic levels to toxic cyanobacterial blooms were studied in a large net cage in Meiliang Bay, a hypereutrophic region of Lake Taihu. We sampled four fishes: the phytoplanktivorous Hypophthalmichthys molitrix and Aristichthys nobilis, the omnivorous Carassius auratus, and the carnivorous Culter ilishaeformis. Alterations of the antioxidant (GSH) and the major antioxidant enzymes (CAT, SOD, GPx, GST) in livers were monitored monthly, and the ultrastructures of livers were compared between the bloom and the post-bloom periods. During the cyanobacterial blooms, the phytoplanktivorous fishes displayed only slight ultrastructural changes in liver, while both carnivorous and omnivorous fishes presented serious injuries such as swollen endomembrane system and morphologically altered nuclei in hepatocytes. Biochemically, the phytoplanktivorous fishes possessed higher basal GSH concentrations and better correlations between the major antioxidant enzymes in liver, which might be responsible for their powerful resistance to MC. 2. Physiological and biochemical responses in kidneys of fishes with different trophic levels (phytoplanktivorous, omnivorous and carnivorous) to toxic cyanobacterial blooms were studied in a large net cage in Meiliang Bay. Catalase and glutathione S-transferase were significantly higher during blooms than before and after blooms. All fishes showed ultrastructural alterations in kidneys during blooms, which mainly are inosculation of foot processes in epithelial cell of glomeruli. The results suggested that kidney impairment from chronic exposure of toxic cyanobacterial blooms might be the first step, and then followed by hepatic failure. Compared with livers in terms of physiological status, the weaker antioxidant ability of kidney made it more susceptible to chronic MC exposure, besides the effective accumulation of MC metabolites in kidney. 3. To evaluate the impact of toxic cyanobacterial blooms on the planktivorous fish, 12 serum chemistry variables were investigated in silver carp and bighead carp, in a large net cage in Meiliang Bay, a hypereutrophic region of Lake Taihu. The results confirmed adverse effects of cyanobacterial blooms on the two phytoplanktivorous fishes, mainly characterized with potential toxicogenomic effects and metabolism disorders in liver, and kidney dysfunction. In addition, cholestasis was intensively implied by distinct elevation of all four related biomarkers (ALP, GGT, DBIL, TBIL) in bighead carp. Based on a multivariate discriminant analysis, the combination of LDH and AST activities and DBIL and URIC contents for silver carp, and the combination of ALT and ALP activities and TBIL, DBIL and URIC concentrations for bighead carps strongly indicate toxic effects from cyanobacterial blooms. 4. A laboratory experiment was conducted to examine toxic effects of microcystins on heart muscle of rats intravenously injected with extracted MC at two doses, 0.16LD50 (14µg MC-LReq kg-1 body weight) and 1LD50 (87 µg MC-LReq kg-1 body weight). In the dead rats, both TTC staining and maximum elevations of troponin I levels confirmed myocardial infarction after MC exposure, besides a serious interstitial hemorrhage in liver. In the 1LD50 dose group, the coincident falls in heart rate and blood pressure were related to mitochondria dysfunction in heart, while increases in creatine kinase and troponin I levels indicated cardiac cell injury. The corresponding pathological alterations were mainly characterized as loss of adherence between cardiac myocytes and swollen or ruptured mitochondria at the ultrastructural level. MC administration at a dose of 1LD50 not only enhanced activities and up-regulated mRNA transcription levels of antioxidant enzymes, but also increased GSH content. At both doses, level of lipid peroxides increased obviously, suggesting serious oxidative stress in mitochondria. Simultaneously, complex I and III were significantly inhibited, indicating blocks in electron flow along the mitochondrial respiratory chain in heart. In conclusion, the findings of this study implicate a role for MC induced cardiotoxicity as a potential factor that should be considered when evaluating the mechanisms of death associated with microcystin intoxication in Brazil.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.ihb.ac.cn/handle/342005/12392
Appears in Collections:中科院水生所知识产出(2009年前)_学位论文

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Recommended Citation:
微囊藻水华对不同食性鱼类毒理效应的野外研究及微囊藻毒素对大鼠心脏致毒性研究.邱彤[d].中国科学院水生生物研究所,2009.20-25
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