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题名: 微囊藻毒素在水生食物网内的累积、传递、代谢以及对水产品安全性的潜在威胁
作者: 张大文
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 ; 食物网 ; 铜锈环棱螺 ; 日本沼虾 ; 微囊藻毒素-谷胱甘肽结合产物 ; 微囊藻毒素-半胱氨酸结合产物
其他题名: Bioaccumulation, transfer, and metabolization of microcystins in aquatic food web with the potential risks to the safety of aquatic products
摘要: 近年来,在世界各地淡水水体中,由于大量营养物质的输入而导致的水体富营养化日益严重,与之相随的各种有毒蓝藻水华也频繁发生。由这些有毒藻类产生的毒素(特别是微囊藻毒素)对水生态系统和人类健康的危害已成为生命科学界和社会公众广泛关注的热点问题之一。我国是一个湖泊众多且富营养化严重的国家,有毒蓝藻水华在这些湖泊中频繁发生,给水生生物和人类健康带来潜在的威胁。因此,关于我国富营养化湖泊中水产品中微囊藻毒素的累积、分布、代谢和季节变化规律以及水产品安全性问题急需评估。然而,目前关于微囊藻毒素在动物体内的代谢规律的研究较少,还未见微囊藻毒素的各种代谢产物的定量研究。因此,定量研究水生动物体内微囊藻毒素的代谢产物,探讨微囊藻毒素在生物体内的代谢途径也非常必要。本文系统研究了1)微囊藻毒素在我国大型富营养化湖泊-太湖水生食物网中累积、分布、传递和代谢规律,2)分析了我国长江中下游地区9个有蓝藻水华发生的湖泊中的水产品中微囊藻毒素的累积量,并对水产品安全性进行评估;3)通过对水生动物中累积的MC-LR及其两种代谢(MC-LR-GSH和MC-LR-Cys)定量分析,探讨MC-LR在水生动物中的代谢规律及途径。主要的研究结论如下: 1、2004年11月-2005年10月,在太湖贡湖湾设置了3个采样点,每月定期至设置的3个采样点采集足够量的铜锈环棱螺,分析其肝、肠、性腺、足、余体以及幼螺中的三种MCs(MC-RR、-YR、-LR)含量,探讨了MCs在铜锈环棱螺中的组织分布和季节变化规律。本研究采用LC-MS定性定量分析样品中的MCs含量。研究发现,微囊藻毒素已在铜锈环棱螺幼螺中累积,且幼螺中的累积量与其性腺中的毒素含量之间存在明显的正相关关系,这表明,MCs能够通过母体直接传递至下一代。大多数的微囊藻毒素都累积在肠(53.6%)和肝脏(29.9%)中,其他组织中只累积了16.5%的毒素量。如果将肠除外,高达64.3%的毒素分布在肝脏中。肝、肠、性腺中的微囊藻毒素与水柱中的微囊藻生物量和胞外毒素之间有显著的相关性。各个器官中的毒素含量都有明显的季节变化,其中,肝、肠和性腺中的毒素含量的峰值出现在藻类生物量最高的7月,在冬春季节下降至较低的水平,但是肌肉和余体的毒素峰值却出现在蓝藻水华消逝的11月或12月。假设一个60kg的人,每天消费100g螺的足,那么在我们所分析的样品中,有18.2%的样品中的毒素超过了WHO规定的最大日均允许摄入量(TDI)。 2、为了探讨铜锈环棱螺肝脏中累积的微囊藻毒素含量的时空变化规律,我们于2004年11月至2005年10月,在太湖梅梁湾和贡湖湾设置了5个采样点,分析其中铜锈环棱螺肝脏中的MCs含量。研究发现,不同点铜锈环棱螺肝脏中的MCs含量之间有一定的空间差异,1号点肝脏中累积的毒素量高于其他各点。铜锈环棱螺肝脏中的毒素含量与水柱中的胞内毒素含量、微囊藻生物量之间存在明显的正相关关系,这表明铜锈环棱螺中MCs含量的时空变化是由水柱中的微囊藻生物量的时空分布不均匀导致的。PCCA分析指出,除了微囊藻外,温度也能影响铜锈环棱螺对MCs的累积。 3、2005年6月-11月,每月定期在太湖梅梁湾采集不同食性的六种鱼(滤食蓝藻的鲢鱼、杂食性的鲤鱼和鲫鱼、肉食性的鲌鱼、湖鲚和银鱼),研究MCs在不同食性鱼类不同器官中的累积、传递规律。三种微囊藻毒素(MC-RR,-YR,-LR)的加标回收率分别是67.7%,85.3%和88.6%。研究发现,滤食性的鲢鱼肝脏和肠含物中的毒素含量最高,其次是杂食性的鲤鱼和鲫鱼,最低的是肉食性鱼类,但是在肌肉中,杂食性鱼类的MCs含量最高,其次是滤食性鱼类,最低的仍然是肉食性鱼类。本文首次报道了微囊藻毒素在野生鱼类性腺中累积,同时,我们发现,鱼类对MC-YR吸收的主要途径似乎是通过鳃从水中吸收。我们仅发现了鲤鱼肌肉中累积的毒素含量超过了WHO规定的TDI值。 4、为了探讨微囊藻毒素在水生动物体内的代谢途径,我们设计了两个实验。在第一个实验中,我们定量分析了MC-LR及其两种结合产物(MC-LR-GSH和MC-LR-Cys)在太湖三种不同水生动物-铜锈环棱螺、日本沼虾和鲢鱼肝脏中的累积量,并描述了其季节变化规律。我们的研究发现,铜锈环棱螺、日本沼虾和鲢鱼肝脏中的MC-LR均值分别为6.61μg/g DW, 0.24 μg/g DW, 和 0.027μg/g DW,而MC-LR-Cys在以上三种水生动物中的均值分别为0.50 μg/g DW, 0.97 μg/g DW, 和 5.72 μg/g DW,仅在少数几个样品中定性的检测到了MC-LR-GSH的存在。第二个实验是一个野外和室内相结合的实验,室内实验是腹腔注射鳙鱼纯的MC-LR(500 µg/kg bw)后,于1 h,3 h,6 h,12 h分别取3条鳙鱼解剖,检测其肝脏中的MC-LR及其两种结合产物含量;野外实验是分析2008年7-8月在湖南和江苏5个湖泊中的鳙鱼肝脏样品中MC-LR及其两种结合产物。在野外条件下,鳙鱼肝脏中的MC-LR主要以结合产物MC-LR-Cys的形式存在(LR-Cys是LR的13倍)。仅两个湖的鳙鱼样品中MC-LR-GSH含量超过检测限。腹腔注射的鳙鱼肝脏中MC-LR在12 h之内大部分以游离态形式存在,其两种结合产物MC-LR-Cys和MC-LR-GSH都能定量的检测到,但是MC-LR-Cys的量是MC-LR-GSH的6.6倍。以上的实验结果表明1)在水生动物,尤其是鱼类中,MC-LR-Cys是MC-LR结合态的主要存在形式,是MC-LR-Cys而不是MC-LR-GSH在MC-LR的解毒中起着重要的作用。2)MC-LR在水生动物代谢的可能途径是,MC-LR先与含有Cys残基的蛋白或小肽(例如GSH、PP-1和-2A)结合,再降解为MC-LR-Cys,并以该形式排出,或者也有可能是MC-LR直接与体内的游离的Cys结合。3)不同的水生动物对MC-LR的代谢途径之间存在差异。4)不同暴露途径下,鱼类对微囊藻毒素的解毒途径之间存在差异。 5、2008年7-8月,我们对江苏、湖北、湖南、安徽四省9个有蓝藻水华的湖泊(太湖、淀山湖、滆湖、东氿、巢湖、黄盖湖、芭蕉湖、东湖、武山湖)中的鲢鱼肝脏和肌肉样品进行毒素含量以及MC-LR的两种结合产物含量分析。研究表明,虽然不同湖泊水柱中MCs含量不同,但是其肌肉中的毒素含量差别不大。而且可以看出,鲢鱼肌肉中主要是毒性较低的RR为主,这些样品中LR的含量都低于检测限,而且没有检测到LR结合产物的存在;而在肝脏中发现了LR-Cys的大量存在,仅滆湖和巢湖鲢鱼肝脏样品中的MC-LR-GSH的含量超过检测限。这说明,在自然条件下,微囊藻毒素LR主要在肝中代谢并清除,很少能够进入肌肉组织,进入肌肉中的少量的MC-LR以其他途径代谢。假如一个60kg的成人每天摄食300g鲢鱼肌肉,所有湖泊中的鲢鱼肌肉样品并没有超过WHO规定的TDI值,这说明我国有蓝藻暴发的湖泊中的鲢鱼食用是安全的。然而,如果长时间食用或过量食用受藻毒素污染的鱼类,其长期的慢性毒性是不容忽视的。
英文摘要: Recently, eutrophication due to the excessive input of nutrients is often accompanied with the occurrence of toxic cyanobacterial blooms in freshwater systems all over the world. Cyanobacterial blooms can produce various toxins that can cause poisonings of animals and human. Among these toxins, microcystins (MCs) are the most common and dangerous group in eutrophic lakes. Potential risks of these toxins (especially microcystins) to aquatic ecosystem and human health are considered to be one of the hot spots of life and environmental sciences and the public. In China, eutrophication of lakes is quite serious. Cyanobacterial surface blooms occur frequently in eutrophic lakes in the warm seasons each year and pose potential risks to both aquatic animals and human health. Hence, great attention should be paid to bioaccumulation, distribution, metabolization and seasonal changes of microcystins in aquatic products and the effects of MCs on safety of aquatic products need to be evaluated. However, so far, studies on the metabolization of microcystins in animals have been limited, and no quantitative study has been conducted to examine the distribution of various metabolites of MCs in animals. Hence, it is very necessary to determinate the metabolites of microcystins quantitatively and discuss the metabolization pathway of microcystins in aquatic animals. The aims of the present study are to 1) examine the bioaccumulation, distribution, transfer and metabolization of microcystins in the food web in a larage eutrophic lake, Taihu Lake, China; 2) to analyze microcystins content in aquatic products from 9 lakes with dense cyanobacterial blooms along the Yangtze River with evaluation on the potential risks of the contaminated aquatic products to human health;3)to determinate the concentration of MC-LR and its two metabolites (MC-LR-GSH and MC-LR-Cys) in aquatic animals with discussion on the metabolization pathway of MC-LR in aquatic animals . The main results and conclusions are as follows: 1. Contents of three common microcystins (microcystin-RR (MC-RR), microcystin-YR (MC-LR), and microcystin-LR (MC-LR)) were examined monthly in hepatopancreas, intestine, gonad, foot, rest, remaining tissue, and offspring of a freshwater snail, Bellamya aeruginosa, from three sites of Gonghu Bay of Lake Taihu during the period between November 2004 and October 2005. Microcystins were present in the offspring and a high positive correlation in microcystin (MCs (RR+YR+LR)) content was found between the offspring and gonad, indicating that microcystins were able to transfer from adult females to their young snail with physiological connection. The majority of the toxins were present in the intestine (53.6%) and hepatopancreas (29.9%), whereas other tissues comprised only 16.5%. If intestines are excluded, up to 64.3% of the toxin burden was allocated in the hepatopancreas. There were great seasonal variations in MCs content in various tissues of the snail. The highest concentrations in the hepatopancreas, intestine, and gonad were all observed in July when Microcystis biomass in the lake water was the highest, and decresed to low level in the spring and winter, while the maxmum MCs contents in muscle and rest tissues were respectively observed in November and December when Microcystis blooms disappeared. Microcystin contents in the intestine, hepatopancreas and gonad were correlated with Microcystis biomass and intracellular and extracellular toxins. 18.2% of the analyzed foot samples were above the tolerable daily microcystin intake recommended by World Health Organization (WHO) for human consumption. 2. Spatial and temporal variations of MC-RR, -YR, -LR in the hepatopancreas of a freshwater snail (Bellamya aeruginosa) were studied monthly at five sites of two bays of Lake Taihu during November 2004 and October 2005. The MCs concentrations in hepatopancreas were higher at Site 1 than at other sites, which was in agreement with the changes of intracellular MCs concentrations in the water column. There was a significant correlation between MCs concentrations in the hepatopancreas and that in the seston, suggesting that spatial variances of MCs concentrations in hepatopancreas among the five sites were due to spatial changes of toxic Microcystis cells in the water column. PCCA indicates that in addition to Microcystis, other factors (e.g., water temperature) also substantially affected the accumulation of MCs in hepatopancreas of the snail. 3. Accumulation and distribution of microcystins (MCs) were examined monthly in six species of fish with different trophic levels in Meiliang Bay, Lake Taihu from June to November 2005. Average recoveries of spiked fish samples were 67.7% for MC-RR, 85.3% for MC-YR, and 88.6% for MC-LR, respectively. MCs (-RR, -YR, -LR) concentration in liver and gut content was highest in phytoplanktivorous fish, followed by omnivorous fish, and was lowest in carnivorous fish; while MCs concentration in muscle was highest in omnivorous fish, followed by phytoplanktivorous fish, and was lowest in carnivorous fish. This is the first study reporting MCs accumulation in the gonad of field fish. The main uptake of MC-YR in fish seems to be through the gills from the dissolved MCs. Only MCs content in common carp muscle exceeded the tolerable daily intake suggested by WHO. 4. Two experiments were carried out to examine the metabolization pathway of MC-LR in aquatic animals. In the first experiment, seasonal changes of MC-LR and its two metabolites (MC-LR-Cys and MC-LR-GSH conjugates) were studied quantitatively in three aquatic animals –snail (Bellamya aeruginosa), shrimp (Macrobrachium nipponensis) and silver carp (Hypophthalmichthys molitrix) collected from Lake Taihu. The mean MC-LR concentration in hepatopancreas of snail and shrimp and liver of silver carp were 6.61μg/g DW, 0.24 μg/g DW, and 0.027μg/g DW, respectively, while the average MC-LR-Cys were 0.50 μg/g DW, 0.97 μg/g DW, and 5.72 μg/g DW, respectively. MC-LR-GSH was usually not detectable in these samples. The second experiment included both field and laboratory studies. In the laboratory, bighead carp were injected intraperitoneally with pure MC-LR at a dose of 500 µg MC-LR /kg bw, and then MC-LR and its two metabolites (LR-GSH and LR-Cys) concentration in liver were analyzed at 1h, 3h, 6h, and 12h postinjection. In the field, MC-LR and its two metabolites (LR-GSH and LR-Cys) concentration were detected in liver of bighead carps from five lakes of Hunan and Jiangsu Provinces during July and August 2008. In the field, LR-Cys is the main form of MC-LR in the liver of bighead carp (LR-Cys concentration was as 13-fold as that of MC-LR), and very low level of LR-GSH was detected in the samples of the two lakes. In the laboratory experiment, free MC-LR was the main form of MC-LR in the liver of bighead carp within 12h, and the two MC-LR metabolites were detected quantitatively in the liver samples during the whole study period, but MC-LR-Cys concentration was as 6.6-fold as that of LR-GSH. The above results suggest that 1) in aquatic animals, especially fish, the main excretion form of MC-LR could be MC-LR-Cys, but not MC-LR-GSH, whereas MC-LR-Cys might play an important role in detoxication of MC-LR; 2) that the main detoxication pathway of MC-LR in aquatic animals is suggested as follows: when MC-LR enters into liver/hepatopancreas, it firstly conjugates with polypeptide or protein (including GSH, PP-1 and 2A) containing Cys residues, perhaps also some free cysteine, and subsequently, MC-LR-Cys is degraded from these polypeptide or protein, and finally is excreted from animals by the compounds of MC-LR-Cys or/and MC-LR conjugates with polypeptide; 3) that the MC-LR detoxification pathway varied among species of aquatic animals; and 4) that the MC-LR detoxification pathway differed among different exposure routs. 5. Distributions of microcystins and MC-LR conjugates were examined in liver and muscle of silver carp (Hypophthalmichthys molitrix) from 9 subtropical shallow lakes (Taihu, Chaohu, Huanggaihu, Bajiaohu, Donghu, Dongjiu, Gehu, Dianshanhu, and Wushanhu) with cyanobacterial blooms along the Yangtze River to evaluate the potential risks of microcystins-contaminated fish to human health during July and August. In spite of significant variation of MCs concentrations in seston, MCs (mainly MC-RR) content in muscle of silver carp from these lakes only showed slight variation. High amount of MC-LR and MC-LR-Cys was found in the liver of silver carp from these lakes, while in muscle samples, no MC-LR-Cys was detected and MC-LR contents were below the detectable levels, suggesting that 1) MC-LR was metabolized and transformed primarily in the liver of fish, and little entered into muscle, and that 2) MC-LR in muscle might be excreted via a different pathway from liver. Assuming that a 60 kg person would consume 300g muscle per day, MC contents in muscle materials from all these lakes did not exceed TDI proposed by WHO, indicating that it is still safe to consume muscle of silver carp from these lakes in spite of the presence of cyanobacteria blooms. However, potential risks from long-term exposure to MC-contaminated fish product can not be ignored.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.ihb.ac.cn/handle/342005/12408
Appears in Collections:中科院水生所知识产出(2009年前)_学位论文

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微囊藻毒素在水生食物网内的累积、传递、代谢以及对水产品安全性的潜在威胁.张大文[d].中国科学院水生生物研究所,2009.20-25
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