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汞对单细胞真核生物四膜虫的生物效应研究
Alternative TitleBiological Effects of Mercury on Unicellular Eukaryotic Tetrahymena
刘成斌
Subtype博士
Thesis Advisor江桂斌 ; 史建波
2018-06
Degree Grantor中国科学院生态环境研究中心
Place of Conferral北京
Degree Name理学博士
Degree Discipline分析化学
Keyword汞,四膜虫,生物效应,拮抗作用,甲基化 Mercury, Tetrahymena, Biological Effects, Antagonism, Methylation
Abstract

      汞是一种在自然界中天然存在的重金属元素,具有长距离迁移性、环境持久性、生物累积性和高毒性,被认为是全球性的有毒污染物。汞的毒性与其形态密切相关,甲基汞是公认的毒性最大的汞化合物。在水环境中,二价汞离子和甲基汞是主要的汞形态,而且汞的这两种形态在化学或生物作用下可以发生相互转化。四膜虫是一类在淡水水体中广泛存在的单细胞真核原生动物,它处于食物链的底端,对水环境中有毒物质反应敏感。四膜虫对水环境中汞的吸收和累积是汞进入食物链并参与生物地球化学循环的一个重要环节,因此研究水环境中不同形态汞在四膜虫体内的摄入累积、迁移转化以及对四膜虫的生物效应具有重要环境意义。

      本论文研究了二价汞离子和甲基汞在五种四膜虫体内的吸收动力学,比较了四膜虫对二价汞离子和甲基汞摄入速率的差异,并深入探讨了两种形态汞化合物对四膜虫的毒性效应及潜在毒性机制;发现了低剂量汞化合物对四膜虫的生长促进作用,探讨了不同生长期暴露对生长促进作用的影响。选取一种四膜虫(T. malaccensis)进行三种形态硒和两种形态汞的联合暴露,研究了不同形态和浓度的硒和汞在四膜虫体内的复合效应。此外,对四膜虫体内甲基汞的分析测定方法进行了优化,研究了多种暴露条件下四膜虫对二价汞离子的甲基化能力。

      首先对二价汞离子和甲基汞在五种四膜虫体内的吸收累积、毒性效应及潜在机制进行了研究。吸收动力学和细胞活力实验结果表明,二价汞离子和甲基汞都可以被四膜虫吸收进而对四膜虫的增殖产生抑制作用。相比于二价汞离子,四膜虫对甲基汞的吸收速率更快。进一步的机制研究表明,尽管二价汞离子和甲基汞都可以引起四膜虫细胞线粒体膜电位的改变,但是它们的毒性机制却不尽相同,二价汞离子能够显著提升四膜虫体内的活性氧簇水平从而导致细胞生长受到抑制,而甲基汞主要是通过破坏四膜虫细胞膜的完整性来影响四膜虫的生存。

       考虑到低剂量暴露在实际环境中的重要意义,依赖于流式细胞技术,研究了低浓度二价汞离子和甲基汞暴露对四膜虫生长增殖的影响。结果表明,低浓度的两种汞化合物对三种四膜虫的增殖产生了显著的促进作用(即“毒物兴奋效应”),而对另外两种四膜虫没有影响。同时,选取另外六种常见重金属离子对其中一种四膜虫(T. thermophila SB210)进行低浓度暴露,发现了类似的生长促进作用,并且这种生长促进作用与暴露的起始时间以及暴露的时间长短均有关。

      选取一种四膜虫(T. malaccensis)对不同形态及浓度组合的硒和汞联合暴露的生物效应进行了研究。结果显示,不同形态的硒对四膜虫的生物效应不同,其中亚硒酸钠毒性最大,硒代蛋氨酸次之,硒酸钠对四膜虫几乎没有毒性。汞硒拮抗作用同样存在于四膜虫中,且表现为相互解毒作用,这种相互解毒作用与硒和汞各自的形态及浓度都密切相关。与其他形态相比,甲基汞和硒代蛋氨酸的拮抗作用在汞硒复合效应中更加显著。

      对四膜虫进行了多种暴露条件下二价汞离子的暴露,建立并优化了四膜虫中甲基汞分析的前处理方法,并用多种分析方法(包括气相色谱-原子荧光联用系统,超高效液相色谱-电感耦合等离子体质谱联用系统,同位素标记法和同位素稀释法等)对四膜虫体内是否有甲基汞产生做出准确判断。多次重复实验后,在四膜虫体内都没有检测到甲基汞的产生,表明四膜虫对二价汞离子的甲基化效应并不明显。

Other Abstract

      Mercury (Hg) is regarded as a global toxic pollutant due to its long-range transport, environmental persistence, bioaccumulation, and high toxicity. The toxicity of mercury is closely related to its species, and methylmercury (MeHg) is recognized as the most toxic mercury compound. In aquatic environments, mercuric ions (Hg2+) and MeHg are the major species of mercury and they can be transformed into one another by chemical or biological processes. Tetrahymena is a type of unicellular eukaryotic protozoa, which is widespread in fresh water bodies. Located at the bottom of the food chain, Tetrahymena is sensitive to toxic substances in aquatic systems. In the case of mercury, the uptake and accumulation by Tetrahymena can be an important point for mercury to enter the food chain and participate in the biogeochemical cycle. Therefore, studies about the accumulation, transportation and transformation as well as the biological effects of different mercury species in Tetrahymena are environmentally significant.

      In this dissertation, the uptake kinetics of Hg2+ and MeHg in Tetrahymena were first studied. The different ingestion rates of Hg2+ and MeHg in Tetrahymena were compared, and the toxic characteristics and potential mechanisms of the two mercury species were further investigated. The growth-promoting effect (known as “hormesis”) of low-dose mercury on Tetrahymena was discovered. Furthermore, the effects of the starting time of exposure on the growth promotion were investigated. Afterwards, one species of Tetrahymena (T. malaccensis) was selected to be exposed to three species of selenium and two species of mercury, and the interactions of selenium and mercury with different species and concentrations in T. malaccensis were studied. In addition, analytical methods for MeHg in Tetrahymena cells were optimized. Based on the analytical methods, the possible transformation of inorganic mercury in Tetrahymena was studied after exposure to a variety of exposure conditions.

      First, the accumulation, toxicity and potential mechanisms of Hg2+ and MeHg in five Tetrahymena species were studied. The results of uptake kinetics and cell viability experiments showed that both Hg2+ and MeHg could be absorbed by Tetrahymena and, thus, induce the inhibition on the proliferation of Tetrahymena. Tetrahymena has a faster ingestion rate for MeHg than Hg2+. Further mechanistic studies have shown that potential toxic mechanisms of Hg2+ and MeHg are significantly different, although both of them can cause changes in the mitochondrial membrane potential of Tetrahymena cells. In detail, Hg2+ can significantly increase the reactive oxygen species in Tetrahymena and then leads to the inhibition of cell growth, while MeHg mainly affects the survival of Tetrahymena by destroying the integrity of Tetrahymena cell membranes.

      Considering the environmental importance of low-dose exposure, the effects of low-dose Hg2+ and MeHg exposure on the growth and proliferation of Tetrahymena were initially explored relying on flow cytometry. The results showed that the low-dose exposure of both two mercury compounds induced significantly growth-promoting effect (i.e. hormesis) on the proliferation of three Tetrahymena species but showed no effect on the other two Tetrahymena species. Six other common heavy metals were further selected and found to display the similar growth-promoting effects on T. thermophila SB210 after exposure to low concentrations. The hormesis was found to be related to both the onset and the duration of exposure.

      One species of Tetrahymena (T. malaccensis) was used to examine the joint effects of selenium and mercury after exposure to different species and concentration combinations. Individual exposure of selenium compounds showed that the biological effects of these selenium species on T. malaccensis were different, with sodium selenite being the most toxic species, followed by selenomethionine, and sodium selenate having almost no toxicity to T. malaccensis. The joint-exposure results showed that the antagonistic effects of mercury and selenium also existed in T. malaccensis and could be defined as mutual detoxification. This mutual detoxification was closely related to the respective species and concentration of selenium and mercury. Compared to other species, MeHg and selenomethionine are more likely to participate in the antagonism of mercury and selenium in T. malaccensis.

      Tetrahymena was exposed to inorganic Hg2+ under various exposure conditions. Pretreatment method and multiple analytical methods for the analysis of MeHg in Tetrahymena cells were established and optimized to make an accurate judgment on the presence of MeHg in Tetrahymena after exposure to Hg2+, including gas chromatography coupled with atomic fluorescence, ultra-high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry, isotope labeling method and isotopes dilution method. After repeated experiments, no MeHg production was detected in Tetrahymena, indicating that the methylating effect of Tetrahymena on Hg2+ was not obvious.

Pages116
Language中文
Document Type学位论文
Identifierhttp://ir.rcees.ac.cn/handle/311016/41486
Collection环境化学与生态毒理学国家重点实验室
Recommended Citation
GB/T 7714
刘成斌. 汞对单细胞真核生物四膜虫的生物效应研究[D]. 北京. 中国科学院生态环境研究中心,2018.
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