鎘和砷對不同發(fā)育階段紫貽貝毒理效應的組學研究
發(fā)布時間：2018-08-17 11:09
【摘要】：近年來渤海的重金屬污染問題日益嚴重。本論文根據(jù)渤海重金屬污染現(xiàn)狀,利用代謝組學和蛋白質組學技術,并結合傳統(tǒng)毒理學方法研究了兩種海洋典型重金屬污染物鎘(Cd)和砷(As)對不同發(fā)育階段紫貽貝(Mytilus galloprovincialis)的毒性效應機制,旨在從整體水平探討Cd和As對不同發(fā)育階段紫貽貝脅迫機制的差異,并從中篩選出表征污染物脅迫效應的代謝物及蛋白質作為生物標志物,為海洋環(huán)境監(jiān)測及評價提供理論依據(jù)。研究結果如下:1.不同濃度Cd和As(V)對紫貽貝D型幼蟲毒理效應的組學研究紫貽貝D型幼蟲經(jīng)不同濃度Cd(5μg/L和50μg/L)和As(V)(5μg/L和50μg/L)暴露48小時后,不同濃度的Cd和As(V)暴露對紫貽貝D型幼蟲均未造成致畸作用。Cd和As(V)對D型幼蟲的致毒機制存在顯著差異,其中Cd對D型幼蟲在分子水平存在劑量-效應關系�；诤舜殴舱窦夹g的代謝組學研究結果表明,Cd和As(V)暴露均影響了D型幼蟲能量代謝、脂肪代謝和滲透壓調節(jié)等生物過程�；陔p向電泳結合質譜檢測技術的蛋白質組學研究發(fā)現(xiàn),5μg/L和50μg/L Cd暴露后紫貽貝D型幼蟲組織中與細胞骨架、能量代謝、基因表達和免疫調節(jié)等生物過程有關的蛋白質發(fā)生顯著變化。5μg/L Cd暴露誘導了D型幼蟲組織中與細胞凋亡、氧化應激和蛋白質折疊相關的蛋白質出現(xiàn)顯著變化。50μg/L Cd暴露誘導了D型幼蟲組織中與解毒和蛋白質代謝相關的蛋白質出現(xiàn)顯著變化。5μg/L As(V)暴露干擾了D型幼蟲的蛋白質代謝等生物過程,50μg/L As(V)暴露影響了D型幼蟲的解毒和免疫調節(jié)等生物過程。2.Cd和As(V)對紫貽貝稚貝毒理效應的組學研究紫貽貝稚貝經(jīng)50μg/L Cd和50μg/L As(V)暴露48小時后,重金屬測定結果表明,Cd暴露組紫貽貝稚貝組織中Cd富集量為極顯著水平(p0.01),而As(V)暴露組As富集不顯著。代謝組學研究結果表明,Cd和As(V)均干擾了紫貽貝滲透壓調節(jié)和能量代謝等過程。Cd影響了稚貝的脂肪代謝過程,支鏈氨基酸的顯著下調表明As(V)干擾了稚貝的免疫調節(jié)過程。蛋白質組學結果發(fā)現(xiàn),Cd暴露干擾了稚貝的細胞骨架、免疫調節(jié)和信號轉導等過程,而As(V)暴露影響了稚貝能量代謝、細胞凋亡、基因表達和氧化應激反應等過程。經(jīng)Cd和As(V)暴露后,紫貽貝稚貝組織中不同抗氧化酶(超氧化物歧化酶、過氧化氫酶和谷胱甘肽轉移酶)活性和丙二醛含量均未出現(xiàn)顯著變化。結果表明,Cd和As(V)暴露對紫貽貝稚貝的致毒機制存在顯著差異。3.Cd和As(V)對紫貽貝成貝毒理效應的組學研究紫貽貝成貝暴露于50μg/L Cd和50μg/L As(V)48小時后發(fā)現(xiàn),Cd和As(V)暴露對紫貽貝成貝的致毒機制存在差異。重金屬含量測定結果表明,Cd暴露組成貝組織中Cd富集量為極顯著水平(p0.01),As(V)暴露組成貝組織中As富集量為顯著水平(p0.05)。代謝組學研究發(fā)現(xiàn),Cd暴露和As(V)暴露均可誘導成貝組織中與滲透壓調節(jié)和能量代謝相關的代謝物發(fā)生顯著變化,但相應的脅迫應答機制不同。蛋白質組學研究結果表明,Cd暴露導致紫貽貝成貝組織中細胞骨架的穩(wěn)定性受到干擾,而As(V)暴露影響了成貝組織中基因表達、脂肪代謝和神經(jīng)傳導等生物過程,并誘導了DNA損傷。綜上,紫貽貝三個發(fā)育階段(D型幼蟲期、稚貝期和成貝期)經(jīng)重金屬Cd和As脅迫后均出現(xiàn)不同代謝物和蛋白質的顯著變化,這些代謝物和蛋白質分別對應不同的生物過程。其中,D型幼蟲對Cd和As暴露反應最為敏感,發(fā)生顯著變化的代謝物和蛋白質數(shù)量最多,涉及到的生物過程最為廣泛,并且D型幼蟲在分子水平對Cd脅迫存在劑量-效應關系;其次,稚貝對Cd和As暴露反應敏感度降低,出現(xiàn)顯著變化的代謝物和蛋白質數(shù)量減少,但仍有多個生物學過程受到干擾,并影響了稚貝生長;最后,紫貽貝成貝對Cd和As暴露的耐受度最高,受到影響的生物過程最少。組學研究結果證明,隨著紫貽貝生長期的延續(xù),紫貽貝體內器官逐步成熟,其應對Cd和As脅迫的能力有所增強。
[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.
【學位授予單位】：中國科學院煙臺海岸帶研究所
【學位級別】：博士
【學位授予年份】：2017
【分類號】：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.
【學位授予單位】：中國科學院煙臺海岸帶研究所
【學位級別】：博士
【學位授予年份】：2017
【分類號】：X171.5
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