典型溴系阻燃剂对紫贻贝毒理效应的组学研究
吉成龙
学位类型博士
导师吴惠丰 ; 赵建民
2014-05-21
学位授予单位中国科学院研究生院
学位授予地点北京
学位专业环境科学
关键词紫贻贝 2 2′ 4 4′-四溴联苯醚 四溴双酚a 毒理效应 性别特异
其他摘要
    本研究选择两种典型溴系阻燃剂2,2′,4,4′-四溴联苯醚(BDE 47)和四溴双酚A(TBBPA)为暴露污染物,以海洋环境监测生物紫贻贝为研究对象,采用以代谢组学、蛋白质组学为主的组学研究方法,结合传统生态毒理学的研究内容,探讨典型溴系阻燃剂对雌、雄紫贻贝的毒理效应,筛选表征污染物毒理效应的代谢物及蛋白质生物标记物,为海岸带环境生物监测及评价提供一定的理论基础。
1.海洋环境监测生物蛋白质提取方法的优化和比较
    选择裂解液直接提取法、三氯乙酸-丙酮法以及TRIzol法等三种常用的蛋白质提取方法进行优化,提取紫贻贝Mytilus galloprovincialis、牙鲆Paralichthys olivaceus以及沙蚕Nereis diversicolor的蛋白质,对三种方法的提取效率及重现性进行分析比较,结果显示TRIzol法对紫贻贝鳃和牙鲆肝脏组织的蛋白质提取效率最高,重现性最优,而三氯乙酸-丙酮法对沙蚕整体蛋白的提取效果最好。
2BDE 47对紫贻贝毒理效应的组学研究
    基于核磁共振技术的代谢组学研究结果显示,对照组雌、雄紫贻贝的鳃组织代谢物不同,雄个体鳃中精氨酸含量较高,而雌个体鳃中的亚牛磺酸及谷氨酸盐含量高;紫贻贝经BDE 47暴露30天后,紫贻贝鳃组织代谢物发生显著变化,雌紫贻贝的能量代谢(ATP、赖氨酸)及渗透压调节(甜菜碱、亚牛磺酸)相关的代谢物水平显著变化,雄个体只有能量代谢(谷氨酸盐、组氨酸、葡萄糖、苏氨酸以及赖氨酸)相关的代谢物发生变化;利用双向电泳结合质谱检测技术研究紫贻贝蛋白质的变化,BDE 47暴露后雌、雄紫贻贝鳃的蛋白质组均发生改变,对雌、雄紫贻贝的毒理效应均表现为细胞凋亡和细胞骨架的损伤,雌、雄个体还分别表现为蛋白动态平衡和蛋白水解过程的异常。
3TBBPA对紫贻贝毒理效应的组学研究
    紫贻贝经TBBPA暴露30天后,组织病理学研究发现雌、雄个体的鳃丝均出现纤毛变少和局部细胞凋亡,雄个体鳃丝还表现为血细胞扩散,而在雌个体的鳃丝前端出现粘液分泌物;鳃组织中代谢物的变化呈现性别差异,雌紫贻贝胆碱、甜菜碱、龙虾肌碱、天冬氨酸、牛磺酸、甘氨酸以及ATP等代谢物发生显著变化,这些代谢物涉及能量代谢和渗透压调节等生物学过程,而雄紫贻贝只表现出渗透压调节相关的代谢物如二甲基甘氨酸、胆碱、胆碱磷酸以及龙虾肌碱的显著变化;利用基于iTRAQ技术的蛋白质组学研究发现对照组雌、雄紫贻贝鳃组织中有57个蛋白丰度存在差异;TBBPA暴露后雌、雄个体鳃组织中差异表达的蛋白分别有44个和38个,雌、雄个体差异表达的蛋白均涉及细胞骨架、生长发育、物质和能量代谢、信号传导、基因表达、防御机制及细胞凋亡等生物学活动,但发生响应蛋白谱的表达模式不同。
;     Two typical broiminated flame retardants, 2,2′,4,4′-etrabromodiphenyl ether (BDE 47) and Tetrabromobisphenol A (TBBPA), have been widely used and detected in multiple environmental samples. In this study, the omics appoarches including metabolomics and proteomics and traditional ecotoxicology approaches were used to elucidate the toxicological effects of BDE 47 and TBBPA in female and male mussels.
1Evaluation of protein extraction methods for 2DE marine ecotoxicoproteomics
    In this work, we evaluated three protein extraction systems, straightforward lysis buffer, trichloroacetic acid-acetone and TRIzol reagent with some modifications, for the protein extraction from three animal species including mussel Mytilus galloprovincialis, flounder Paralichthys olivaceus, and polychaete Nereis diversicolor used in marine ecotoxicology. Our results indicated that the method using TRIzol reagent resulted in the most matched protein spots resolved in four replicate 2DE gels and highest reproducibilities for the gill of mussel and liver of flounder. However, a modified trichloroacetic acid-acetone solvent system was best for the whole soft tissue of polychaete.
2Omics analyses reveal toxicological effects of BDE 47 on mussels
    Metabolomics study based on NMR technique revealed that there were significant metablic differences between male and female mussel samples from blank groups. Male mussel samples had signifianctly higher levels of arginine, and lower level of hypotaurine and glutamate than those of female mussel samples. After exposed to BDE 47 for 30 days, the mussels presented significant changes in both metabolites and proteins in gills. Metabolic responses indicated that BDE 47 caused disruption in both osmotic regulation and energy metabolism in female individuals, supported by the variation of betaine, hypotaurine, lysine and ATP. While the changes of threonine, lysine, glutamate, glucose and histidine in male mussel samples revealed disturbance in energy metabolism. An integrated 2D electrophoresis and mass spectrometry proteomics approach showed that BDE 47 induced apoptosis and injuries of cytoskeleton in both male and female mussels, disturbance in protein homeostasis in male mussels as well as disturbance in female mussel proteolysis.
3Omics analyses reveal toxicological effects of TBBPA on mussels
    After exposed to TBBPA for 30 days, apoptosis and reduction of cilia were observed on the gill filaments from both female and male mussels, in addition, gill filaments of female and male individuals showed mucus-like secretion and haemocytes infiltration, respectively. The significant changes of choline, betaine, homarine, taurine, glycine and ATP implied the disturbance in energy metabolism as well as the abnormity of osmotic regulation in female mussel gills, by contrast, in male individual samples only be found the variation of metabolites related to osmotic regulation, such as dimethylglycine, choline, choline phosphate and homarine. Proteomic analysis based on iTRAQ showed that there were a total of 57 proteins with significant abundance between male and female mussel samples from blank groups. TBBPA exposure led to 44 and 38 proteins differentially expressed in female and male mussel gills, respectively. These proteins involved in multiple biological processes including production and development, material and energy metabolism, signal transduction, gene expression, defense mechanism and apoptosis, among which the profiles of protein expression were gender-specific.
语种英语
文献类型学位论文
条目标识符http://ir.yic.ac.cn/handle/133337/7086
专题中国科学院烟台海岸带研究所知识产出_学位论文
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吉成龙. 典型溴系阻燃剂对紫贻贝毒理效应的组学研究[D]. 北京. 中国科学院研究生院,2014.
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