镉和砷对不同发育阶段紫贻贝毒理效应的组学研究
发布时间：2018-08-17 11:09
【摘要】：近年来渤海的重金属污染问题日益严重。本论文根据渤海重金属污染现状,利用代谢组学和蛋白质组学技术,并结合传统毒理学方法研究了两种海洋典型重金属污染物镉(Cd)和砷(As)对不同发育阶段紫贻贝(Mytilus galloprovincialis)的毒性效应机制,旨在从整体水平探讨Cd和As对不同发育阶段紫贻贝胁迫机制的差异,并从中筛选出表征污染物胁迫效应的代谢物及蛋白质作为生物标志物,为海洋环境监测及评价提供理论依据。研究结果如下:1.不同浓度Cd和As(V)对紫贻贝D型幼虫毒理效应的组学研究紫贻贝D型幼虫经不同浓度Cd(5μg/L和50μg/L)和As(V)(5μg/L和50μg/L)暴露48小时后,不同浓度的Cd和As(V)暴露对紫贻贝D型幼虫均未造成致畸作用。Cd和As(V)对D型幼虫的致毒机制存在显著差异,其中Cd对D型幼虫在分子水平存在剂量-效应关系。基于核磁共振技术的代谢组学研究结果表明,Cd和As(V)暴露均影响了D型幼虫能量代谢、脂肪代谢和渗透压调节等生物过程。基于双向电泳结合质谱检测技术的蛋白质组学研究发现,5μg/L和50μg/L Cd暴露后紫贻贝D型幼虫组织中与细胞骨架、能量代谢、基因表达和免疫调节等生物过程有关的蛋白质发生显著变化。5μg/L Cd暴露诱导了D型幼虫组织中与细胞凋亡、氧化应激和蛋白质折叠相关的蛋白质出现显著变化。50μg/L Cd暴露诱导了D型幼虫组织中与解毒和蛋白质代谢相关的蛋白质出现显著变化。5μg/L As(V)暴露干扰了D型幼虫的蛋白质代谢等生物过程,50μg/L As(V)暴露影响了D型幼虫的解毒和免疫调节等生物过程。2.Cd和As(V)对紫贻贝稚贝毒理效应的组学研究紫贻贝稚贝经50μg/L Cd和50μg/L As(V)暴露48小时后,重金属测定结果表明,Cd暴露组紫贻贝稚贝组织中Cd富集量为极显著水平(p0.01),而As(V)暴露组As富集不显著。代谢组学研究结果表明,Cd和As(V)均干扰了紫贻贝渗透压调节和能量代谢等过程。Cd影响了稚贝的脂肪代谢过程,支链氨基酸的显著下调表明As(V)干扰了稚贝的免疫调节过程。蛋白质组学结果发现,Cd暴露干扰了稚贝的细胞骨架、免疫调节和信号转导等过程,而As(V)暴露影响了稚贝能量代谢、细胞凋亡、基因表达和氧化应激反应等过程。经Cd和As(V)暴露后,紫贻贝稚贝组织中不同抗氧化酶(超氧化物歧化酶、过氧化氢酶和谷胱甘肽转移酶)活性和丙二醛含量均未出现显著变化。结果表明,Cd和As(V)暴露对紫贻贝稚贝的致毒机制存在显著差异。3.Cd和As(V)对紫贻贝成贝毒理效应的组学研究紫贻贝成贝暴露于50μg/L Cd和50μg/L As(V)48小时后发现,Cd和As(V)暴露对紫贻贝成贝的致毒机制存在差异。重金属含量测定结果表明,Cd暴露组成贝组织中Cd富集量为极显著水平(p0.01),As(V)暴露组成贝组织中As富集量为显著水平(p0.05)。代谢组学研究发现,Cd暴露和As(V)暴露均可诱导成贝组织中与渗透压调节和能量代谢相关的代谢物发生显著变化,但相应的胁迫应答机制不同。蛋白质组学研究结果表明,Cd暴露导致紫贻贝成贝组织中细胞骨架的稳定性受到干扰,而As(V)暴露影响了成贝组织中基因表达、脂肪代谢和神经传导等生物过程,并诱导了DNA损伤。综上,紫贻贝三个发育阶段(D型幼虫期、稚贝期和成贝期)经重金属Cd和As胁迫后均出现不同代谢物和蛋白质的显著变化,这些代谢物和蛋白质分别对应不同的生物过程。其中,D型幼虫对Cd和As暴露反应最为敏感,发生显著变化的代谢物和蛋白质数量最多,涉及到的生物过程最为广泛,并且D型幼虫在分子水平对Cd胁迫存在剂量-效应关系;其次,稚贝对Cd和As暴露反应敏感度降低,出现显著变化的代谢物和蛋白质数量减少,但仍有多个生物学过程受到干扰,并影响了稚贝生长;最后,紫贻贝成贝对Cd和As暴露的耐受度最高,受到影响的生物过程最少。组学研究结果证明,随着紫贻贝生长期的延续,紫贻贝体内器官逐步成熟,其应对Cd和As胁迫的能力有所增强。
[Abstract]:The pollution of heavy metals in the Bohai Sea has become more and more serious in recent years. Based on the present situation of heavy metal pollution in the Bohai Sea, the toxic effects of cadmium (Cd) and arsenic (As) on Mytilus galloprovincialis at different developmental stages were studied by using metabolomics and proteomics techniques and combining with traditional toxicological methods. The aim of this study was to explore the differences of Cd and AS stress mechanisms at different developmental stages and to screen metabolites and proteins as biomarkers for monitoring and evaluating marine environment. The results were as follows: 1. The toxicological effects of Cd (5 ug/L and 50 ug/L) and As (V) (5 ug/L and 50 ug/L) on D-type larvae of mussels were studied by histological method. There was no teratogenic effect on D-type larvae of mussels exposed to Cd (5 ug/L and 50 ug/L) at different concentrations for 48 hours. There is a dose-response relationship. Metabonomic studies based on NMR showed that both Cd and As (V) exposure affected the energy metabolism, fat metabolism and osmotic pressure regulation of D-type larvae. Proteomic studies based on two-dimensional electrophoresis and mass spectrometry showed that the D-type larvae of P. edulis exposed to 5 and 50 ug/L Cd were affected. Proteins related to cytoskeleton, energy metabolism, gene expression and immune regulation in larval tissues of D-type larvae were significantly changed. Exposure to 5 ug/L Cd induced significant changes in proteins related to apoptosis, oxidative stress and protein folding in larval tissues of D-type larvae. Exposure to 50 ug/L Cd induced detoxification in larval tissues of D-type larvae. Proteins associated with protein metabolism were significantly altered. Exposure to 5 ug/L As (V) interfered with protein metabolism and other biological processes of D-type larvae. Exposure to 50 ug/L As (V) affected detoxification and immune regulation of D-type larvae. 2. Histological studies on toxicological effects of Cd and As (V) on mussel larvae after exposure to 50 ug/L Cd and 50 ug/L After 48 hours of exposure to As (V), the results of heavy metal determination showed that Cd enrichment was extremely significant in Cd exposed group (p0.01), but not in As (V) exposed group. The results of proteomics showed that Cd exposure interfered with cytoskeleton, immune regulation and signal transduction, while As (V) exposure affected energy metabolism, cell apoptosis, gene expression and oxidative stress reaction of juvenile shellfish. The results showed that the toxic mechanism of Cd and AS (V) exposure to mussel juveniles was significantly different. 3. Histological study on toxicological effects of Cd and AS (V) on mussel adults. The toxicity mechanism of Cd and As (V) to adult mussels was different after exposure to 50 ug/L Cd and 50 ug/L As (V) for 48 hours. It was found that both Cd exposure and AS (V) exposure could induce significant changes in osmotic pressure regulation and energy metabolism-related metabolites in adult mussel tissues, but the corresponding stress-response mechanisms were different. Proteomic studies showed that Cd exposure caused disturbance of cytoskeleton stability in adult mussel tissues, whereas as As (V) exposure caused disturbance of cytoskeleton stability. In summary, three developmental stages (D-type larval stage, juvenile stage and adult stage) of mussel (Mytilus edulis) exhibited significant changes in metabolites and proteins after Cd and AS stress, which corresponded to different metabolites and proteins respectively. Among them, D-type larvae were the most sensitive to the exposure of Cd and AS, and the number of metabolites and proteins with significant changes was the largest, involving the most extensive biological processes, and D-type larvae had a dose-response relationship to the stress of Cd at the molecular level; secondly, the sensitivity of juvenile shellfish to the exposure of Cd and AS decreased, showing significant changes. The number of metabolites and proteins decreased, but many biological processes were interfered with and affected the growth of young mussels. Finally, the tolerance of adult mussels to Cd and AS was the highest, and the biological processes were the least affected. And the ability of As stress increased.
【学位授予单位】：中国科学院烟台海岸带研究所
【学位级别】：博士
【学位授予年份】：2017
【分类号】：X171.5
[Abstract]:The pollution of heavy metals in the Bohai Sea has become more and more serious in recent years. Based on the present situation of heavy metal pollution in the Bohai Sea, the toxic effects of cadmium (Cd) and arsenic (As) on Mytilus galloprovincialis at different developmental stages were studied by using metabolomics and proteomics techniques and combining with traditional toxicological methods. The aim of this study was to explore the differences of Cd and AS stress mechanisms at different developmental stages and to screen metabolites and proteins as biomarkers for monitoring and evaluating marine environment. The results were as follows: 1. The toxicological effects of Cd (5 ug/L and 50 ug/L) and As (V) (5 ug/L and 50 ug/L) on D-type larvae of mussels were studied by histological method. There was no teratogenic effect on D-type larvae of mussels exposed to Cd (5 ug/L and 50 ug/L) at different concentrations for 48 hours. There is a dose-response relationship. Metabonomic studies based on NMR showed that both Cd and As (V) exposure affected the energy metabolism, fat metabolism and osmotic pressure regulation of D-type larvae. Proteomic studies based on two-dimensional electrophoresis and mass spectrometry showed that the D-type larvae of P. edulis exposed to 5 and 50 ug/L Cd were affected. Proteins related to cytoskeleton, energy metabolism, gene expression and immune regulation in larval tissues of D-type larvae were significantly changed. Exposure to 5 ug/L Cd induced significant changes in proteins related to apoptosis, oxidative stress and protein folding in larval tissues of D-type larvae. Exposure to 50 ug/L Cd induced detoxification in larval tissues of D-type larvae. Proteins associated with protein metabolism were significantly altered. Exposure to 5 ug/L As (V) interfered with protein metabolism and other biological processes of D-type larvae. Exposure to 50 ug/L As (V) affected detoxification and immune regulation of D-type larvae. 2. Histological studies on toxicological effects of Cd and As (V) on mussel larvae after exposure to 50 ug/L Cd and 50 ug/L After 48 hours of exposure to As (V), the results of heavy metal determination showed that Cd enrichment was extremely significant in Cd exposed group (p0.01), but not in As (V) exposed group. The results of proteomics showed that Cd exposure interfered with cytoskeleton, immune regulation and signal transduction, while As (V) exposure affected energy metabolism, cell apoptosis, gene expression and oxidative stress reaction of juvenile shellfish. The results showed that the toxic mechanism of Cd and AS (V) exposure to mussel juveniles was significantly different. 3. Histological study on toxicological effects of Cd and AS (V) on mussel adults. The toxicity mechanism of Cd and As (V) to adult mussels was different after exposure to 50 ug/L Cd and 50 ug/L As (V) for 48 hours. It was found that both Cd exposure and AS (V) exposure could induce significant changes in osmotic pressure regulation and energy metabolism-related metabolites in adult mussel tissues, but the corresponding stress-response mechanisms were different. Proteomic studies showed that Cd exposure caused disturbance of cytoskeleton stability in adult mussel tissues, whereas as As (V) exposure caused disturbance of cytoskeleton stability. In summary, three developmental stages (D-type larval stage, juvenile stage and adult stage) of mussel (Mytilus edulis) exhibited significant changes in metabolites and proteins after Cd and AS stress, which corresponded to different metabolites and proteins respectively. Among them, D-type larvae were the most sensitive to the exposure of Cd and AS, and the number of metabolites and proteins with significant changes was the largest, involving the most extensive biological processes, and D-type larvae had a dose-response relationship to the stress of Cd at the molecular level; secondly, the sensitivity of juvenile shellfish to the exposure of Cd and AS decreased, showing significant changes. The number of metabolites and proteins decreased, but many biological processes were interfered with and affected the growth of young mussels. Finally, the tolerance of adult mussels to Cd and AS was the highest, and the biological processes were the least affected. And the ability of As stress increased.
【学位授予单位】：中国科学院烟台海岸带研究所
【学位级别】：博士
【学位授予年份】：2017
【分类号】：X171.5
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