菲律宾蛤仔无机砷甲基转化及其分子机制的研究
陈丽竹
学位类型硕士
导师吴惠丰研究员 ; 王清副研究员
2016-05-18
学位授予单位中国科学院大学
学位授予地点北京
学位专业海洋生物学
关键词菲律宾蛤仔 富集 转化 亚细胞分布 Gstω 病理变化
摘要砷是自然界广泛分布的有毒类金属,水体中砷主要以毒性很高的无机砷形式存在。本论文针对我国近海砷污染现状,以近岸广泛分布的海洋环境监测生物菲律宾蛤仔(Ruditapes philippinarum)为研究对象,采用砷酸盐(AsⅤ)和亚砷酸盐(AsⅢ)进行毒性暴露,利用分析化学、分子生物学、生物化学等技术手段研究菲律宾蛤仔对无机砷的富集转化规律,探讨谷胱甘肽-S-转移酶Ω(GSTΩ)在菲律宾蛤仔砷代谢过程中的功能机制,并揭示砷暴露下菲律宾蛤仔的组织病理变化,从而为阐明海洋贝类对无机砷的富集转化规律及其分子机制提供一定依据,同时也可为贝类安全养殖和食用提供理论依据。主要研究结果如下:
(1)菲律宾蛤仔对无机砷生物利用率低,对As(Ⅲ)生物利用率高于As(Ⅴ)。菲律宾蛤仔组织中无机砷的转化过程可能既包括As(Ⅴ)还原和甲基转化,还存在As(Ⅲ)氧化。菲律宾蛤仔肝胰腺和鳃组织对无机砷富集转化存在差异,砷更易于在肝胰腺中富集。砷甜菜碱(AsB)和二甲基胂酸盐(DMA)是组织中主要的砷形态,在肝胰腺中无机砷主要转化为AsB,而鳃组织中无机砷主要转化为DMA。各处理组中金属硫蛋白样蛋白(MTLP)是砷在亚细胞水平的主要结合位点,其次是细胞碎片。金属敏感组分(MSF)中总砷变化较为明显,而生物解毒金属组分(BDM)中总砷含量变化较小,且与肝胰腺相比,鳃组织对砷的解毒能力有限。
(2)克隆获得了菲律宾蛤仔体内无机砷甲基化关键酶GSTΩ基因,并对其进行了体外重组表达。GSTΩ重组蛋白具有砷酸盐还原酶活性,且在底物浓度较高,低pH和常温(37℃)条件下活性较高。将GSTΩ转化到砷敏感菌株E. coli AW3110(DE3) (ΔarsRBC)后,能够提高该菌株对无机砷的耐受性,表明其在无机砷的转化和解毒方面发挥了重要作用。
(3)分别从转录水平和蛋白水平检测了GSTΩ在无机砷暴露后的表达变化情况以及砷酸盐还原酶活性的改变。结果发现多个处理组菲律宾蛤仔肝胰腺和鳃组织GSTΩ相对表达量增加,砷酸盐还原酶活性升高,进一步表明其可能在菲律宾蛤仔无机砷甲基转化和解毒过程中发挥了重要作用。采用免疫组化方法对GSTΩ在组织中的表达进行定位,结果表明GSTΩ蛋白主要在鳃丝上皮细胞以及鳃腔中的血细胞中表达,在消化腔内壁上皮细胞中表达。
(4)不同浓度无机砷暴露一定时期后,菲律宾蛤仔体内GSH含量、GR、GST活性在多个处理组出现活性的显著升高,表明砷暴露会引发机体内抗氧化应激反应。随着暴露时间的增加,在菲律宾蛤仔鳃、肝胰腺、外套膜均已产生不同程度的病理损伤,在100 μg/L As(Ⅲ)暴露30天时肝胰腺出现消化管坏死的现象,肌肉纤维也出现断裂扭曲;鳃丝上皮细胞溶解,出现大量空泡;外套膜内表皮和外表皮均出现严重损伤。As(Ⅲ)暴露造成的损伤程度高于As(Ⅴ),证实As(Ⅲ)毒性强于As(Ⅴ)的结论。
其他摘要Arsenic (As) is a ubiquitous toxic metalloid, and mainly exists in the form of highly toxic inorganic arsenic in water. In view of the severe offshore arsenic pollution in our country, the bioaccumulation, biotransformation and detoxification of the clam Ruditapes philippinarum, which are widely distributed in near shore and estuarine areas and extensively used in biomonitoring programs, were investigated after waterborne exposure to arsenite (AsⅢ) or arsenate (AsⅤ). Techniques of analytical chemistry, molecular biology, and biochemistry, etc. were applied to study the bioaccumulation and biotransformation in tissues of R. philippinarum, and investigated the important role of Glutathione-S-transferase Ω (GST Ω) in the process of arsenic metabolism in the clams, and reveals the physiology and histopathological responses to arsenic exposure in the clams. The results were expected to not only provid an important basis for elcucidating the arsenic bioaccumulation and biotransformation mechanism in R. philippinarum, but also provide a theoretical basis for marine shellfish breeding and food safety. The main results were as follows:
(1) Inorganic arsenic showed low bioavailability, and the bioavailability of As(Ⅲ) was slightly higher than As(Ⅴ) to the clams. Arsenic biotransformation in the clams might include As(Ⅲ) oxidation besides As(Ⅴ) reduction and subsequent methylation. The bioaccumulation and biotransformation for inorganic arsenic shows differences in digestive glands and gills of the clams, and arsenic was apt to be accumulated in digestive glands. Besides, the results demonstrated that arsenobetaine (AsB) and dimethylarsinic acid (DMA) was the major arsenic speciation in all treatments, and inorganic arsenic in gills was mainly converted to DMA, but AsB in digestive glands, indicating the different transformation efficiency in different tissues. Metallothionein-like proteins (MTLP) is the main binding site of arsenic at subcellular level, followed by cell debris in all treatments. Total As concentrations in metal sensitive fraction (MSF) changed more obviously, while total arsenic concentrations in biologically detoxified metal fraction (BDM) showed almost no change. And compared with the digestive glands, the gills tissues of the clams demonstrated limitied detoxification capability.
(2) The recombinant GST Ω protein, the rate-limiting enzyme for biotransformation of inorganic arsenic, functioned as arsenate reductase, and expression of GST Ω enhanced the arsenic tolerance in the arsenate-sensitive strains E. coli AW3110(DE3) (ΔarsRBC), indicating its important role in biotransformation and detoxification of inorganic arsenic.
(3) The expression of GST Ω was tested from the transcription level and protein levels respectively after inorganic arsenic exposure. After the inorganic arsenic exposure, the relative mRNA expression and arsenate reductase activity of GST Ω increased in digestive glands and gills of the clams in many treatments, further suggesting that GST Ω may play an important role in the process of inorganic arsenic methyl transformation and detoxification. Meanwhile, GST Ω was investigated by immunohistochemistry in the gills and digestive glands. The results showed that the GST Ω was mainly located in the epithelial cells of the gill filaments and the blood cells in the gill cavity, and mainly located in the epithelial cells of the digestive tubules.
(4) After different concentrations of inorganic arsenic exposure for a period of time, GSH content, GR and GST activity of the clams in many treatments increased significantly, suggesting a resistance to oxidative stress. However, as exposure time increased, the gills, digestive glands and mantles had been histopathologically damaged to different extent. For example, after 100 μg/L As(Ⅲ) exposure for 30 days, the tubules of digestive glands were disfigured and necrotic, and fracture and distortion of muscle fiber was observed; epithelial cells lining at the gill filaments became disintegrated and vacuolated; inner and outer epithelial cells of mantles damaged severely. As(Ⅲ) exposure caused severer damage than As(Ⅴ) exposure, confirming that As(Ⅲ) is more toxic than As(Ⅴ).
文献类型学位论文
条目标识符http://ir.yic.ac.cn/handle/133337/13843
专题中国科学院烟台海岸带研究所知识产出_学位论文
中国科学院烟台海岸带研究所知识产出
作者单位中国科学院烟台海岸带研究所
第一作者单位中国科学院烟台海岸带研究所
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陈丽竹. 菲律宾蛤仔无机砷甲基转化及其分子机制的研究[D]. 北京. 中国科学院大学,2016.
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