石墨烯与磷酸三苯酯对紫贻贝（Mytilus galloprovincialis） 的复合毒理效应研究
孟祥敬
学位类型博士
导师吴惠丰
2020-11-26
培养单位中国科学院烟台海岸带研究所
学位授予单位中国科学院大学
学位授予地点中国科学院烟台海岸带研究所
学位名称理学博士
学位专业海洋生物学
关键词石墨烯磷酸三苯酯复合毒性组学紫贻贝
摘要近年来，随着沿海工业的快速发展和海洋资源的过度开发，我国近岸海域污染日益加重，严重威胁海洋生态安全。不同污染物（如有机物和纳米材料等）通常以相互作用方式对海洋生物产生复合毒性。其中，石墨烯是一种具有优良吸附性能和表面性质的纳米材料，易与其它污染物发生相互作用，进而影响其自身和其它污染物的毒性效应。磷酸三苯酯（TPP）是一种典型的有机磷酸酯类阻燃剂，可显著影响生物体的生殖发育和内分泌调节等过程。TPP分子结构中含有三个苯环，易与石墨烯发生相互作用，但目前关于石墨烯和TPP对海洋生物复合毒性的研究较少。本研究以海洋环境监测指示生物紫贻贝（Mytilus galloprovincialis）为研究对象，整合利用转录组学、代谢组学以及传统生态毒理学等研究方法，探讨了石墨烯和TPP单一及复合暴露对紫贻贝的毒性效应及其作用机制，并初步构建了紫贻贝产生免疫和生殖响应的有害结局路径（AOP）。研究结果如下：1. TPP在紫贻贝体内的富集紫贻贝经石墨烯和TPP暴露7天后，采用气相色谱-质谱串联（GC-MS）检测发现，与对照组相比（0.47 ± 0.16 μg/g脂重），TPP单一暴露组（404.57 ± 258.22 μg/g脂重）和石墨烯 + TPP暴露组（770.77 ± 425.69 μg/g脂重）紫贻贝肝胰腺中TPP富集量显著增加（P < 0.01）。其中，石墨烯 + TPP暴露组中TPP含量约为TPP单一暴露组的1.9倍，表明石墨烯可能通过吸附作用增加了TPP在紫贻贝肝胰腺中的富集量。2. 紫贻贝对石墨烯和TPP暴露响应的组学研究石墨烯和TPP暴露7天后，以紫贻贝肝胰腺为目标器官，在分子水平进行转录组学和代谢组学分析。转录组学分析表明，石墨烯和TPP单一及复合暴露均诱导紫贻贝肝胰腺产生免疫反应、内分泌干扰、生殖发育、物质代谢以及能量代谢等相关的差异表达基因（DEGs）。进一步分析石墨烯对紫贻贝肝胰腺的毒性作用机制发现，石墨烯通过上调促凋亡基因（Caspase3），同时激活细胞转化生长因子（TGF）信号通路、丝裂原活化蛋白激酶（MAPK）信号通路以及Toll样受体信号通路导致细胞死亡，并诱导机体产生免疫应答。在能量代谢方面，石墨烯暴露后紫贻贝通过上调三羧酸（TCA）循环（Idh3b）和糖代谢基因（Treh、Idh3b、Gpi、Ggt1和Gusb）增加机体的能量消耗，通过上调氨基酸代谢基因（Got2和Ggt1）增强机体的渗透压调节能力和胞内谷胱甘肽（GSH）的稳定性，以缓解石墨烯造成的氧化胁迫。此外，石墨烯通过上调Notch基因增强紫贻贝卵泡细胞的增殖分化。TPP暴露后，紫贻贝通过上调Xkr7、Fibcd1、Tp53i3和Otud4基因增强其免疫防御能力。在内分泌干扰和生殖方面，TPP通过上调Pck1和Pck2基因影响紫贻贝激素分泌，并通过下调Cpeb1基因抑制卵母细胞成熟。TPP暴露后紫贻贝肝胰腺产生的物质和能量代谢DEGs与石墨烯暴露结果相似，也主要诱导紫贻贝肝胰腺产生糖代谢和氨基酸代谢DEGs。进一步分析石墨烯 + TPP对紫贻贝肝胰腺的毒性作用机制发现，石墨烯 + TPP暴露通过上调Notch和Birc基因激活Ras和NF-κB信号传导通路，进而诱导机体产生免疫反应。同时，石墨烯 + TPP通过下调Egfr、Birc3、Cdc42和Smad3抑制细胞增殖分化，并影响其细胞骨架和细胞内物质运输。在内分泌干扰和生殖方面，石墨烯 + TPP暴露后表达上调的EP300基因调控紫贻贝激素分泌，Dll1介导的Notch信号通路调控生殖细胞的形态发生和增殖分化。在物质和能量代谢方面，石墨烯 + TPP通过下调TCA循环（Abat）、糖代谢（Gapdh、Sord和Eno1等）基因降低机体的能量消耗；通过下调GSH代谢基因（Gstm3）调节胞内GSH水平，应对石墨烯 + TPP造成的氧化胁迫。代谢组学分析表明，石墨烯和TPP单一及复合暴露诱导紫贻贝肝胰腺产生了能量代谢和渗透调节紊乱等多种生理响应。整合差异变化的代谢物以及物质和能量代谢DEGs发现，石墨烯能够影响紫贻贝肝胰腺糖酵解、苏氨酸代谢、甘氨酸代谢和TCA循环过程。在TPP暴露组中，渗透调节物（龙虾肌碱）和以TCA循环为代表的能量代谢过程发生变化。在石墨烯 + TPP暴露组中，鸟氨酸循环、丝氨酸代谢、牛磺酸代谢、糖酵解和TCA循环过程受到了影响。3. 石墨烯和TPP对紫贻贝的免疫毒性石墨烯和TPP暴露7天后，以紫贻贝血淋巴为研究对象，在亚细胞水平、细胞水平和基因水平分别检测血细胞的超微结构损伤、免疫响应以及免疫功能基因的表达，探究了石墨烯和TPP单一及复合暴露对紫贻贝的免疫毒性。在亚细胞水平通过透射电镜观察发现，石墨烯进入紫贻贝血细胞，导致细胞膜破裂和核膜增厚。同时，石墨烯和TPP单一及复合暴露均可对血细胞产生氧化胁迫、降低血细胞溶酶体膜稳定性（LMS）、增加血细胞凋亡和DNA损伤程度。但与石墨烯暴露相比，石墨烯 + TPP对紫贻贝血细胞产生的氧化胁迫程度显著降低（抗氧化酶活性和抗氧化物含量降低），且通过上调免疫功能基因（NF-κB、Bcl-2和Ras）降低血细胞的凋亡率和DNA损伤程度。推测TPP吸附到石墨烯表面后降低了石墨烯的表面锐度和活性，从而降低了石墨烯对紫贻贝的免疫毒性。在此基础上，以脂质双分子层的物理损伤和抗氧化酶活性的增加为促使免疫响应发生的早期分子事件，以个体出现死亡为最终有害结局，系统整合免疫响应启动的现有数据资源，构建了紫贻贝产生免疫响应的AOP。4. 紫贻贝对石墨烯和TPP暴露的生理和生殖响应研究石墨烯和TPP暴露14天后，采用传统生态毒理学研究方法探究紫贻贝对石墨烯和TPP单一及复合暴露的生理和生殖响应。结果表明，石墨烯和TPP单一及复合暴露均可对紫贻贝产生氧化胁迫，显著升高肝胰腺中抗氧化酶活性和抗氧化物含量，损伤紫贻贝鳃、肝胰腺和性腺组织。与对照组相比，石墨烯显著抑制紫贻贝的呼吸率，并增加其排氨率。同样，TPP暴露也显著增加紫贻贝的排氨率。但石墨烯 + TPP暴露对紫贻贝产生的氧化胁迫、组织损伤及呼吸抑制程度均低于石墨烯单独暴露。此外，与石墨烯单独暴露相比，石墨烯 + TPP暴露组紫贻贝性腺损伤程度降低，且性腺组织中生殖相关基因（Hsd、Vtg和Cyp450）的表达水平显著上调，表明石墨烯 + TPP对紫贻贝的生殖毒性低于石墨烯单独暴露。在此基础上，以脂质双分子层的物理损伤和抗氧化酶活性的增加为促使生殖毒性发生的早期分子事件，以个体繁殖力下降或种群数量降低为最终有害结局，系统整合生殖毒性启动的现有数据资源，构建了紫贻贝产生生殖响应的AOP。关键词：石墨烯，磷酸三苯酯，复合毒性，组学，紫贻贝
其他摘要In recent years, the rapid development of coastal industry and the over-exploitation of marine resources have resulted in serious pollution in offshore and costal areas, inducing marine ecological risks. Different pollutants (such as organic pollutants and nanomaterials etc.) usually cause joint toxicity to marine organisms in a common or interactive manner. With excellent adsorption properties, graphene nanomaterials can interact with other pollutants after its release into the environment, thereby affecting the environmental behavior of these pollutants. As a typical organophosphate flame retardant (OPFR), triphenyl phosphate (TPP) has multiplex toxicities, including developmental toxicity and endocrine disruption. TPP can easily interact with graphene, as it has three benzene rings in the molecular structure. However, there is a lack of relevant research on the joint toxicities of graphene and TPP to marine organisms. The bivalve species, mussel Mytilus galloprovincialis has been often used as a bio-indicator in marine environmental assessment. Metabolomics, transcriptomics and traditional ecotoxicological methods were used to analyze the toxicity and its mechanisms of graphene and TPP in M. galloprovincialis. In addaition, the adverse output pathway (AOP) frameworks of immune and reproductive response of M. galloprovincialis were preliminarily constructed. Results were as follows:1. Instrumental analysis of TPP in hepatopancreasAfter exposed to graphene and TPP for 7 days, the concentrations of TPP in mussel hepatopancreas were 0.47 ± 0.16, 404.57 ± 258.22 and 770.77 ± 425.69 μg/g fat weight for the control group, TPP-exposed group and graphene + TPP co-exposed group, respectively. The concentrations of TPP in TPP-exposed group and graphene + TPP co-exposed group were significantly higher than that in the control group (P < 0.01). Compared with the TPP-exposed group, the co-exposed group had a higher average TPP concentration (about 1.9 times). These results implied that graphene might adsorb TPP and co-transferred into mussels, thereby resulting in increased accumulation of TPP in mussels.2. Transcriptomic and metabolomic responses of M. galloprovincialis to graphene and TPPAfter exposed to graphene and TPP for 7 days, hepatopancreas was used for transcriptomic and metabolomic analysis at molecular levels. Transcriptomic analysis revealed that the differentially expressed genes (DEGs) in hepatopancreas after graphene and TPP treatments were mainly involved in immune responses, endocrine disruption and reproductive development, substance and energy metabolism. The mechanisms of graphene-induced toxicity in hepatopancreas were analyzed based on the protein-protein interaction (PPI), indicating that graphene induced apoptosis or necrosis by up-regulating the expression of pro-apoptotic gene Caspase3, and activated cell transforming growth factor (TGF) and mitogen-activated protein kinase (MAPK) signaling pathway, and immune responses by activating Toll-like receptor signaling pathway. For energy metabolism, the DEGs involved in tricarboxylic acid (TCA) cycle (Idh3b) and sugar metabolism (Treh, Idh3b, Gpi, Ggt1 and Gusb, etc.) were up-regulated to enhance energy consumption in hepatopancreas after graphene exposure. Meanwhile, amino acids metabolism genes (Got2 and Ggt1) were up-regulated, enhancing osmotic regulation and maintaining intracellular glutathione (GSH) levels to alleviate the oxidative damage induced by graphene in M. galloprovincialis. In addition, graphene enhanced proliferation and differentiation of follicular cells by up-regulating the expression of Notch gene.TPP induced immune defense and immune regulation in M. galloprovincialis by up-regulating the expression of immune and inflammatory genes (Xkr7, Fibcd1, Tp53i3 and Otud4). As for endocrine disruption and reproductive development, TPP affected the secretion by up-regulating the expression of Pck1 and Pck2, and inhibited the maturation of oocytes by down-regulating the expression of Cpeb1. In addition, the DEGs induced by TPP on material and energy metabolism of M. galloprovincialis hepatopancreas were similar to those induced by graphene, mainly affecting the sugar metabolism and amino acid metabolism genes.Further analysis on the mechanisms of graphene + TPP-induced toxicity in hepatopancreas suggested that graphene + TPP activated Ras and NF-κB signaling pathways by up-regulating the expression of Notch and Birc genes, thereby inducing immune response in M. galloprovincialis. Meanwhile, graphene + TPP down-regulated the expression of Egfr, Birc3, Cdc42 and Smad3, thereby inhibiting the proliferation and differentiation of M. galloprovincialis hepatopancreas cells. As for endocrine disruption and reproductive development, graphene + TPP up-regulated the expression of EP300 to regulate the secretion of M. galloprovincialis, and affected the morphogenesis, proliferation and differentiation of germ cells through the Notch signaling pathway mediated by Dll1. In energy metabolism, the DEGs involved in tricarboxylic acid (TCA) cycle (Abat) and sugar metabolism genes (Gapdh, Sord and Eno1, etc.) were down-regulated to reduce energy consumption in M. galloprovincialis hepatopancreas after graphene + TPP exposure. Meanwhile, metabolomic analysis found that graphene and TPP could affect several physiological processes in M. galloprovincialis hepatopancreas, such as energy metabolism and osmotic regulation. Metabolites and DEGs were used to analyze the perturbance of graphene in hepatopancreas. In the graphene-exposed group, the processes of glycolysis, glycine metabolism, succinate metabolism and tricarboxylic acid cycle were altered. Moreover, the contents of amino acids in M. galloprovincialis hepatopancreas were also involved in endocrine disruption. Metabolomic analysis revealed that metabolites correlated with energy metabolism (succinate), osmotic regulation (dimethylamine, glycine, homarine) and amino acid metabolism (threonine, glutamate, aspartate and alanine, etc.) were significantly changed after TPP treatment, indicating that TPP affected physiological processes in M. galloprovincialis, such as energy metabolism and osmotic regulation. In graphene + TPP-treated group, the alterations of glycolysis, urea cycle, serine metabolism, taurine metabolism and tricarboxylic acid cycle were observed in M. galloprovincialis hepatopancreas.3. Immunotoxicity of graphene and TPP on M. galloprovincialisAfter exposed to graphene and TPP for 7 days, traditional toxicological analysis methods were used to elucidate the immunotoxicity and the mechanisms for M. galloprovincialis induced by graphene, TPP and graphene + TPP at subcellular, cellular and gene levels. At subcellular level, transmission electron microscopy (TEM) observations showed that hemocytes could internalize graphene, thereby resulting in oxidative stress. At cellular level, oxidative stress could be induced by graphene and TPP in mussel hemocytes, which would further result in apoptosis, DNA damage and decrease in the lysosomal membrane stability (LMS). Increased oxidative stress and DNA damage in hemocytes were observed in the graphene-exposed group, but significantly reduced after graphene + TPP exposure. The up-regulated genes, including NF-κB, Bcl-2 and Ras, were mainly associated with reduced apoptosis and DNA damage after co-exposure to graphene and TPP. It seemed that the adsorption of TPP on graphene could inhibit the surface activity of graphene, thereby reducing its immunotoxicity. In addition, the physical damage in lipid bilayer and the increase of antioxidant enzyme activity were likely to be the early molecular event in the occurance of immune response, exhibiting individual’s immunosuppression and death as final adverse outcome. According to the systemic integration of the existing data resources, an AOP framework of immune response of M. galloprovincialis was established.4. Reproductive and physiological responses of M. galloprovincialis to graphene and TPPAfter exposed to graphene and TPP for 14 days, the physiological and reproductive effects and mechanisms of graphene and TPP to M. galloprovincialis were characterized. Oxidative stress was induced by graphene and TPP in hepatopancreas, which further caused damages in gill, hepatopancreas and gonad tissues. Compared with the control group, graphene significantly inhibited oxygen consumption rate (OR) of mussels and increased ammonia excretion rate (NR) (P < 0.05). The NR of M. galloprovincialis increased significantly after TPP exposure (P < 0.05). Compared with the graphene-exposed group, the oxidative stress, tissue damage and respiratory inhibition decreased markedly in graphene + TPP-treated group. Simultaneously, the expression levels in terms of reproductive genes (Vtg, Fkbp and Hsd) were markedly up-regulated after graphene + TPP exposure than those in the graphene-exposed group (P < 0.05). Also, the integrated biomarker response index (IBR) value in graphene + TPP treatment was lower than that in graphene and TPP treatments. In conclusion, the combined exposure of graphene and TPP exhibited lower physiological and reproductive toxicities on M. galloprovinciali, compared with the single exposure of graphene or TPP. In addition, the increase in the activity of antioxidant enzymes and the damage of the phospholipid bilayer were seemed to be the early molecular event in the occurrence of reproductive toxicity, exhibiting decline in individual reproductive capacity or population as the final adverse outcome. By integrating the existing data resources, an AOP framework about the occurrence of reproductive response of M. galloprovincialis was established.Key words: Graphene, Triphenyl phosphate, Joint effects, Omics, Mytilus galloprovincialis
页数178
语种中文
文献类型学位论文
条目标识符http://ir.yic.ac.cnhttp://ir.yic.ac.cn/handle/133337/26143
专题中科院烟台海岸带研究所知识产出

推荐引用方式
GB/T 7714孟祥敬. 石墨烯与磷酸三苯酯对紫贻贝（Mytilus galloprovincialis） 的复合毒理效应研究[D]. 中国科学院烟台海岸带研究所. 中国科学院大学,2020.


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