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镉和砷对褐牙鲆幼鱼线粒体的毒性效应及其作用机制
路珍
Subtype博士
2020-05-21
Degree Grantor中国科学院烟台海岸带研究所
Place of Conferral中国科学院烟台海岸带研究所
Degree Name理学博士
Keyword线粒体毒性 褐牙鲆 组学
Abstract线粒体是细胞的能量代谢中心,机体大部分所需能量均由线粒体产生。除了在能量代谢中发挥重要作用之外,线粒体还参与调控细胞的增殖分化、氧化还原稳态、钙离子稳态、内分泌以及细胞凋亡等。本实验室前期研究表明,镉(Cd)和砷(As)均显著影响海洋生物(牡蛎、蛤仔、贻贝、鱼类等)的能量代谢,并且蛋白质组学分析发现Cd和As(Ⅴ)胁迫下褐牙鲆幼鱼体内20% - 33%的差异表达蛋白(DEPs)与能量代谢有关。众所周知,能量代谢对维持生物生长和生存至关重要,是增殖分化、免疫应激、渗透压调节和生殖发育等生命活动的重要基础。研究表明,线粒体极易受到细胞内外环境影响,在机体应对外界或内部胁迫过程中发挥重要作用,可能是Cd和As生物毒性作用的靶点之一。然而,目前关于Cd和As对海洋生物线粒体毒性效应及其作用机制的研究依然缺乏。因此,本论文选取渤海典型重金属污染物Cd和As(Ⅴ)为暴露物,以渤海重要渔业物种褐牙鲆幼鱼为研究对象,以鳃和肝脏线粒体为研究靶点,整合代谢组学、蛋白质组学及传统生态毒理学研究方法,旨在从亚细胞水平和分子水平上系统地探究环境相关浓度(5 μg/L和50 μg/L)的Cd和As(Ⅴ)对褐牙鲆幼鱼线粒体的毒性效应,并构建线粒体响应通路图,为渤海Cd和As污染的生物监测和风险评估提供理论依据。研究结果如下: 1. Cd对褐牙鲆幼鱼线粒体的毒性效应及其作用机制 Cd暴露14天后,通过线粒体超微形态观察、线粒体膜电位检测及代谢组学和蛋白质组学分析表征Cd对褐牙鲆幼鱼线粒体的毒性效应。在亚细胞水平上,透射电镜观察发现5 μg/L和50 μg/L Cd均诱导鳃线粒体损伤,主要表现为线粒体膜破裂或脱落等;且鳃线粒体密度随暴露浓度增加而显著增加(P < 0.01),而线粒体面积指数显著降低(P < 0.01),即Cd胁迫不仅导致鳃线粒体损伤,还诱导鳃线粒体数量增多且形态变小。为探究Cd暴露对鳃线粒体功能的影响,首先检测了维持线粒体活性及功能的关键因素——线粒体膜电位,发现5 μg/L和50 μg/L Cd以浓度依赖性方式显著降低(P < 0.05)鳃线粒体膜电位。之后,在分子水平上进行了代谢组学和亚细胞蛋白质组学分析。代谢组学结果表明,Cd诱导鳃中能量代谢相关代谢物(ATP、AMP和磷酸肌酸等)发生显著变化。基于iTRAQ的线粒体蛋白质组学分析显示,Cd诱导鳃线粒体产生128个DEPs,在5 μg/L和50 μg/L Cd暴露组分别共有74个和118个DEPs。其中,大部分DEPs呈上调表达趋势,且主要涉及三羧酸(TCA)循环和氧化磷酸化等能量代谢过程。进一步分析Cd对鳃线粒体的毒性作用机制发现,褐牙鲆幼鱼主要通过增强鳃线粒体TCA循环和氧化磷酸化促进ATP的产生以满足Cd胁迫条件下的能量需求,并通过上调线粒体形态与输入相关蛋白(线粒体接触位点和嵴组织系统复合物、线粒体内膜转位酶等)增强鳃线粒体的蛋白输入和结构稳定性缓解Cd毒性造成的线粒体损伤。在应激与凋亡方面,上调的鳃线粒体活性氧簇调节剂1和蛋白NipSnap同系物2造成ROS过量累积并引发Ca2+超载,而电压依赖性阴离子选择通道蛋白、肽基脯氨酰异构酶F、ADP/ATP转位酶2以及Bcl2相关的细胞死亡激动剂等凋亡相关蛋白的差异表达诱导线粒体通透性转换孔的开放和凋亡诱导因子1的释放,最终诱发细胞凋亡。此外,Cd暴露条件下褐牙鲆幼鱼鳃线粒体的响应还涉及胆固醇代谢等。 Cd暴露也造成褐牙鲆幼鱼肝脏线粒体膜结构损伤,并诱导线粒体密度和线粒体表面积增加即线粒体数量增多(P < 0.05),同时伴随出现大量脂滴,表明Cd不仅损伤肝脏线粒体的结构完整性而且会导致其代谢功能障碍。同时,显著降低的线粒体膜电位(P < 0.01)也预示Cd干扰肝脏线粒体的正常功能。分子水平上,与鳃代谢组学结果相似,在肝脏代谢图谱中鉴定到能量代谢相关的差异代谢物。不同地是,无氧呼吸产物乳酸在肝脏中显著增加,且部分能量代谢相关的差异代谢物(如ATP、AMP和磷酸胆碱等)在5 μg/L和50 μg/L Cd暴露组中呈相反的变化趋势,表明Cd暴露条件下褐牙鲆幼鱼鳃和肝脏能量代谢响应模式不同。蛋白质组学结果表明,Cd诱导肝脏线粒体产生28个与能量代谢、应激与凋亡等过程相关的DEPs。其中,5 μg/L Cd暴露组有14个,50 μg/L Cd暴露组有19个。Cd诱导产生的肝脏线粒体DEPs数量比鳃线粒体少,且大多呈下调表达趋势。进一步分析Cd对褐牙鲆幼鱼肝脏线粒体的毒性作用机制发现,Cd诱导肝脏无氧呼吸增强,抑制线粒体蛋白输入,并通过干扰TCA循环和抑制氧化磷酸化电子传递等影响肝脏线粒体的能量产生。此外,下调的蛋白质磷酸酶1K和钙摄取蛋白1参与调控肝脏线粒体通透性转换孔和Ca2+摄取,而促凋亡蛋白(第二线粒体衍生的半胱天冬酶激活剂同系物)和抗凋亡蛋白(ATP依赖的RNA解旋酶和ATP依赖性锌金属蛋白酶等)的差异表达表明Cd干扰了褐牙鲆幼鱼肝脏中线粒体依赖性的细胞凋亡途径。 2. As(Ⅴ)对褐牙鲆幼鱼线粒体的毒性效应及其作用机制 As(Ⅴ)暴露14天后,透射电镜观察发现5 μg/L和50 μg/LAs(Ⅴ)均诱导褐牙鲆幼鱼鳃线粒体损伤,主要表现为嵴松散、断裂或脱落等;且鳃线粒体密度随暴露浓度增加而显著增加(P < 0.01),而鳃线粒体面积指数呈降低趋势,表明As(Ⅴ)诱导鳃线粒体数量增加但形态变小。之后,通过测定线粒体膜电位结合代谢组学和蛋白质组学分析表征As(Ⅴ)对鳃线粒体功能的影响。结果表明,与对照组相比,As(Ⅴ)暴露组鳃线粒体膜电位显著降低(P < 0.05)。此外,在As(Ⅴ)暴露组鳃代谢图谱中鉴定到ATP、AMP、乳酸和葡萄糖等涉及无氧呼吸和能量代谢的差异代谢物。蛋白质组学结果表明,As(Ⅴ)诱导鳃线粒体产生83个与线粒体形态与输入、能量代谢和应激与凋亡等过程相关的DEPs。其中,5 μg/L As(Ⅴ)暴露组有60个,50 μg/L As(Ⅴ)暴露组有68个,且大部分DEPs呈上调表达趋势。进一步分析As(Ⅴ)对褐牙鲆幼鱼鳃线粒体的毒性作用机制发现,在线粒体形态与输入方面,As(Ⅴ)暴露诱导线粒体接触位点和嵴组织系统复合物、无机焦磷酸酶2等差异表达并造成鳃线粒体损伤和线粒体蛋白输入增强。在能量代谢方面,褐牙鲆幼鱼通过增强无氧呼吸、TCA循环及氧化磷酸化电子传递等过程增加了鳃线粒体能量产生以满足As(Ⅴ)胁迫条件下的能量需求。在应激与凋亡方面,电压依赖性阴离子选择通道蛋白、ADP/ATP转位酶和磷酸载体蛋白等的差异表达增强了线粒体膜通透性,而Bcl2相关的细胞死亡激动剂、ATP依赖的RNA解旋酶和丝氨酸蛋白酶等凋亡调控蛋白的差异表达表明As(Ⅴ)抑制了鳃组织中线粒体依赖性的细胞凋亡途径。 As(Ⅴ)暴露诱导褐牙鲆幼鱼肝脏线粒体嵴损伤和线粒体密度呈浓度依赖性方式增加(P < 0.01),线粒体面积指数也呈增加趋势,但仅5 μg/L As(Ⅴ)暴露组与对照组具有显著性差异(P < 0.01),即5 μg/L As(Ⅴ)诱导肝脏线粒体数量增加而50 μg/L As(Ⅴ)诱导肝脏线粒体数量增加且形态变小。此外,5 μg/L和50 μg/L As(Ⅴ)暴露组肝脏线粒体膜电位显著降低(P < 0.05)。代谢组学结果表明,仅在50 μg/L As(Ⅴ)暴露组发现能量代谢相关的差异代谢物(ATP、AMP、乳酸和葡萄糖等),表明50 μg/L As(Ⅴ)与5 μg/L As(Ⅴ)相比对肝脏无氧呼吸等能量代谢过程具有更强的干扰作用。蛋白质组学分析表明,As(Ⅴ)诱导肝脏线粒体产生40个与能量代谢和应激与凋亡等过程相关的线粒体DEPs。其中,5 μg/L As(Ⅴ)暴露组有5个,50 μg/L As(Ⅴ)暴露组有35个。进一步分析As(Ⅴ)对肝脏线粒体的毒性作用机制发现,褐牙鲆幼鱼通过促进肝脏无氧呼吸并增强TCA循环和氧化磷酸化缓解As(Ⅴ)胁迫条件下ATP的过度消耗。在应激与凋亡方面,As(Ⅴ)通过上调ADP/ATP转位酶3、补体成分1Q子结合蛋白和早老素相关的菱形样蛋白等增强线粒体膜通透性并干扰了肝脏中线粒体依赖性的细胞凋亡;另一方面,肝脏线粒体通过上调甲硫氨酸-R-亚砜还原酶B2和硫氧还蛋白等维持ROS稳态以降低As(Ⅴ)毒性造成的线粒体损伤。此外,As(Ⅴ)对褐牙鲆幼鱼肝脏线粒体的毒性机制还涉及蛋白质翻译和修饰以及维生素D合成等。 3. Cd和As(Ⅴ)对褐牙鲆幼鱼线粒体的毒性作用机制比较 Cd和As(Ⅴ)对褐牙鲆幼鱼线粒体的毒性机制存在较大差异,但均诱导线粒体数量增加、线粒体损伤以及线粒体膜电位降低。同时,Cd和As(Ⅴ)均诱导褐牙鲆幼鱼增强鳃线粒体TCA循环和氧化磷酸化过程。此外,Cd诱导褐牙鲆幼鱼显著上调鳃线粒体的形态稳定性和蛋白输入,并促进线粒体依赖性的细胞凋亡;而As(Ⅴ)诱导褐牙鲆幼鱼显著下调鳃线粒体形态稳定性但增强线粒体蛋白输入,促进无氧呼吸,并抑制线粒体依赖性的细胞凋亡。Cd和As(Ⅴ)均诱导褐牙鲆幼鱼肝脏无氧代谢增强,并干扰了线粒体依赖性的细胞凋亡途径。Cd抑制肝脏线粒体蛋白输入和氧化磷酸化,并干扰TCA循环;而As(Ⅴ)诱导褐牙鲆幼鱼增强肝脏线粒体TCA循环和ROS稳态,并造成ATP产生量降低。
Other AbstractMitochondrion is the center of energy metabolism in eukaryon, which generates most of the energy for normal physiological activities. Apart from the vital role in energy metabolism, mitochondria also participate in the regulation of cell proliferation and differentiation, redox homeostasis, calcium homeostasis, endocrine, apoptosis, etc. Our previous studies showed that cadmium (Cd) and arsenic (As) could significantly affect the energy metabolism process in marine animals, such as oysters, clams, mussels and fish. Proteomic analysis indicated that 20% - 33% of the differentially expressed proteins (DEPs) in juvenile olive flounder Paralichthys olivaceus induced by Cd and As were related to energy metabolism. As it is known, energy metabolism plays vital roles in growth and survival, which is the primary basis of proliferation and differentiation, immune responses, osmoregulation, reproduction and development and other processes. Mitochondrion is susceptible and may be the main bio-target of Cd and As, playing an essential role in response to the internal or external stress. However, mitochondrial toxicities of Cd and As have not been fully investigated in marine organisms. In this work, metabolomics, proteomics and traditional ecotoxicological methods were integrated to systematically elucidate the mitochondrial toxicity and its mechanisms in gill and liver tissues of juvenile olive flounder P. olivaceus induced by two environmental related concentrations (5 μg/L and 50 μg/L) of Cd and As(Ⅴ) for 14 days, aiming to construct the mitochondrial responsive pathways and providing theoretical basis for biological monitoring and risk assessment of heavy metal pollutions (Cd and As) in the Bohai Sea. The main results were as follows: 1. Cd-induced mitochondrial toxicity and its mechanism in juvenile flounder After exposed to Cd for 14 days, the mitochondrial toxicity of Cd was characterized by the mitochondrial morphology observation, mitochondrial membrane potential detection, as well as metabolomic and proteomic analysis in juvenile flounder. At the subcellular levels, transmission electron microscopy (TEM) observations showed that 5 μg/L and 50 μg/L Cd induced mitochondrial damages in juvenile flounder gills, mainly reflected by injured or shed mitochondrial membrane. Compared with the control, dose-dependent increases of mitochondrial density (P < 0.01) and decreases of mitochondrial area index (P < 0.01) were induced after Cd treatments, indicating that Cd could not only cause mitochondrial damages but also induce more and smaller gill mitochondria. Mitochondrial membrane potential, one of the key factors to maintain mitochondrial activity and function, was detected to evaluate the toxic effects of Cd on mitochondrial function. Both Cd treatments (5 μg/L and 50 μg/L) significantly reduced membrane potentials (P < 0.05) of gill mitochondria in a dose-dependent manner. Metabolomic analysis revealed that metabolites related to energy metabolism, such as ATP, AMP and creatine phosphate, were significantly altered in gills after Cd treatments. iTRAQ-based mitochondrial proteomic analysis showed that Cd induced 74 and 118 DEPs in 5 μg/L and 50 μg/L Cd-treated juvenile flounder gills, respectively. Most of them were up-regulated and mainly involved in energy metabolism processes such as tricarboxylic acid (TCA) cycle and oxidative phosphorylation. Further analysis on the mechanisms of Cd-induced mitochondrial toxicity in juvenile flounder gills suggested that the TCA cycle and oxidative phosphorylation was enhanced to promote ATP production in gill mitochondria to meet the energy demand after Cd treatments. The DEPs involved in mitochondrial morphology and import (mitochondrial contact site and cristae organizing system complex, mitochondrial import inner membrane translocase, etc.) were up-regulated to enhance mitochondrial protein import and mitochondrial network stability in juvenile flounder gills after Cd exposure. For stress resistance and apoptosis, ROS accumulation and Ca2+ overload were induced by up-regulated reactive oxygen species modulator 1 and protein NipSnap homolog 2. Several altered apoptosis-related proteins, such as voltage-dependent anion-selective channel protein, peptidyl-prolyl isomerase F, ADP/ATP translocase 2, and Bcl2-associated agonist of cell death, facilitated the opening of mitochondrial permeability transition pore and the release of apoptosis-inducing factor 1, eventually caused apoptosis. In addition, mitochondrial responses to Cd in juvenile flounder gills were also related to cholesterol metabolism. Cd induced mitochondrial injuries, increased mitochondrial density and mitochondrial area index (P < 0.05), accompanied by a large number of lipid droplets in juvenile flounder livers, indicating that Cd could not only damage mitochondrial morphology but also lead to metabolic dysfunction in liver mitochondria. Consistently, the decreased mitochondrial membrane potentials (P < 0.01) also indicated that Cd disturbed the normal function of liver mitochondria. At the molecular levels, like the metabolomic results of gill samples, differentially altered metabolites related to energy metabolism were identified in livers. However, some of them such as ATP, AMP and choline phosphate showed opposite alteration trends in 5 μg/L and 50 μg/L Cd-treated groups, suggesting the different responsive patterns of energy metabolism in founder gill and liver tissues after Cd exposure. Proteomic analysis showed that Cd induced 28 DEPs in liver mitochondria. These DEPs were mainly involved in energy metabolism, stress resistance and apoptosis. There were 14 and 19 DEPs in 5 μg/L and 50 μg/L Cd treatments, respectively. Compared with the gill mitochondria, Cd induced less DEPs in liver mitochondria, and most of them were down-regulated. Further analysis on the mechanisms of Cd-induced mitochondrial toxicity in juvenile flounder livers confirmed that Cd enhanced anaerobic metabolism, suppressed mitochondrial import, and disturbed energy production by regulating TCA cycle and inhibiting the electron transport in oxidative phosphorylation. Besides, down-regulated protein phosphatase 1K and calcium uptake protein 1 meant the alteration of mitochondrial permeability transition pore and Ca2+ uptake in livers of Cd-exposed juvenile flounders. The differentially expressed pro-apoptotic protein (second mitochondrial derived activator of caspase homolog) and anti-apoptosis protein (ATP-dependent RNA helicas and ATP-dependent zinc metalloprotease) illustrated that Cd disturbed mitochondrial-dependent apoptosis pathway in juvenile flounder livers. 2. As(Ⅴ)-induced mitochondrial toxicity and its mechanism in juvenile flounder After exposure for 14 days, ultrastructural damages of gill mitochondria were induced by 5 μg/L and 50 μg/L As(Ⅴ) , indicated by loose, fragmented and detached mitochondrial cristae. Besides, mitochondrial density was significantly increased (P < 0.01) with the increase of As(Ⅴ) concentrations for exposures, while mitochondrial area index decreased, indicating that As(Ⅴ) induced more and smaller gill mitochondria. Then the toxic effects of As(Ⅴ) on mitochondrial functions were characterized by mitochondrial membrane potential detection as well as metabolomic and proteomic analysis. Compared with the control, mitochondrial membrane potentials were significantly decreased (P < 0.05) in As(Ⅴ)-treated juvenile flounder gills. Metabolites related to anaerobic respiration and energy metabolism, such as ATP, AMP, lactate and glucose, were altered in gill samples after As(Ⅴ) exposure. Proteomic analysis discovered 83 DEPs in gill mitochondria after As(Ⅴ) exposures, which were mainly related to mitochondrial morphology and import, energy metabolism, and stress and apoptosis. There were 60 and 68 DEPs in 5 μg/L and 50 μg/L As(Ⅴ)-treated groups, respectively, most of which were up-regulated DEPs. Further analysis on the mechanisms of As(Ⅴ)-induced mitochondrial toxicity in juvenile flounder gills illustrated that As(Ⅴ) damaged mitochondria and enhanced mitochondrial protein import by regulating mitochondrial contact site and cristae organizing system complex, inorganic pyrophosphatase 2, etc. The anaerobic metabolism, TCA cycle and oxidative phosphorylation electron transfer were promoted to enhance energy production in flounder gill mitochondria under As(Ⅴ) stress. Moreover, the mitochondrial membrane permeability was increased due to differentially expressed voltage-dependent anion-selective channel protein, ADP/ATP translocase and phosphate carrier protein. Meanwhile, altered apoptosis-associated proteins (Bcl2-associated agonist of cell death, ATP-dependent RNA helicase and serine protease, etc.) might inhibit the mitochondrial-dependent apoptosis in juvenile flounder gills. As(Ⅴ) treatments injured mitochondrial cristae and increased the mitochondrial density (P < 0.01) and in juvenile flounder livers in a dose-dependent manner. The mitochondrial area indexes were also increased, but there was only significant difference between control and 5 μg/L As(Ⅴ)-treated group (P < 0.01), indicating that 5 μg/L As(Ⅴ) induced more mitochondria and 50 μg/L As(Ⅴ) caused more and smaller liver mitochondria. Besides, liver mitochondrial membrane potentials were significantly decreased (P < 0.05) in As(Ⅴ)-treated groups. Metabolomic analysis showed that differentially altered metabolites such as ATP, AMP, lactate and glucose were only identified in 50 μg/L As(Ⅴ) treatment, suggesting that 50 μg/L As(Ⅴ) had stronger effects on liver anaerobic respiration and energy metabolism than 5 μg/L As(Ⅴ). Proteomic analysis identified 40 DEPs in liver mitochondria after As(Ⅴ) exposures, which were basically involved in energy metabolism and stress and apoptosis, including 5 and 35 DEPs in these two As(Ⅴ)-treated groups, respectively. Further analysis on the mechanisms of As(Ⅴ)-induced mitochondrial toxicity in juvenile flounder livers exhibited that As(Ⅴ) enhanced liver anaerobic metabolism and TCA cycle and inhibited ATP production in oxidative phosphorylation, resulting in energy metabolism disturbance. For stress resistance and apoptosis, As(Ⅴ) exposure increased mitochondrial membrane permeability and disturbed mitochondrial-dependent apoptosis in juvenile flounder livers by up-regulation of ADP/ATP translocase 3, complement component 1 Q subcomponent-binding protein and presenilins-associated rhomboid-like protein. On the other hand, liver mitochondria maintained ROS homeostasis by up-regulating methionine-R-sulfoxide reductase B2 and thioredoxin 2 to reduce mitochondrial damages induced by As(Ⅴ) treatments. Moreover, mitochondrial responses to As(Ⅴ) in juvenile flounder livers were also involved in protein translation and modification, and vitamin D synthesis. 3. Comparison of the toxic mechanisms of Cd and As(V) on juvenile flounder mitochondria There existed differences of toxicity mechanisms between Cd and As(V) on flounder mitochondria. Both Cd and As(V) increased mitochondria number, caused mitochondrial damages and decreased mitochondrial membrane potentials. Meanwhile, they promoted TCA cycle and oxidative phosphorylation. In addition, Cd significantly increased the morphological stability and protein import of gill mitochondria and induced mitochondrial-dependent apoptosis. However, As(V) significantly down-regulated morphological stability of gill mitochondria but enhanced mitochondrial protein import, promoted anaerobic respiration, and inhibited mitochondrial-dependent apoptosis. Both Cd and As(V) facilitated liver anaerobic metabolism and disturbed mitochondrial-dependent apoptosis. Cd inhibited mitochondrial protein import and oxidative phosphorylation of liver mitochondria and disturbed TCA cycle, while As(V) enhanced TCA cycle and ROS homeostasis and decreased ATP production.
Subject Area海洋生物学
MOST Discipline Catalogue理学::海洋科学
Language中文
Document Type学位论文
Identifierhttp://ir.yic.ac.cn/handle/133337/24223
Collection中科院烟台海岸带研究所知识产出_学位论文
Corresponding Author路珍
Recommended Citation
GB/T 7714
路珍. 镉和砷对褐牙鲆幼鱼线粒体的毒性效应及其作用机制[D]. 中国科学院烟台海岸带研究所. 中国科学院烟台海岸带研究所,2020.
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镉和砷对褐牙鲆幼鱼线粒体的毒性效应及其作(11247KB)学位论文 限制开放CC BY-NC-SAApplication Full Text
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