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题名: 新型有机卤代化合物的神经毒理学效应与作用机制研究
作者: 刘倩1
学位类别: 博士
答辩日期: 2017-05
授予单位: 中国科学院大学
授予地点: 北京
导师: 江桂斌 ; 周群芳
关键词: 新型有机卤代化合物,四溴双酚 A衍生物,全氟及多氟烷基化合物,神经毒性,血管舒缓素-激肽系统 ; Emerging halogenated organic compounds, Tetrabromobisphenol Aderivatives,Per-and polyfluoroalkyl substances, Neurotoxicity, Plasma kallikrein-kinin system
其他题名: Neurotoxicological Effects and the Underlying Molecular Mechanisms of Emerging Halogenated Organic Compounds
学位专业: 环境科学
中文摘要: 新型有机卤代化合物应用广泛,在许多环境和生物介质中频繁检出,存在多 种人体暴露途径和潜在健康风险,有些化合物的人体负荷量相当显著。尽管一些 有机卤代母体化合物的环境健康效应已经引起人们的关注,但是其衍生物或同系 物的毒理学效应仍不清晰,特别是其神经毒理学数据十分匮乏。考虑到神经毒性 评价是环境污染物健康风险评估的重要方面,本研究基于离体和活体模型探讨了 新型有机卤代化合物四溴双酚 A(tetrabromobisphenol A,TBBPA)衍生物和全 氟及多氟烷基化合物(per- and polyfluoroalkyl substances,PFASs)的神经毒性效 应及其穿透血管屏障的分子机制。 体外与体内实验均显示,TBBPA具有显著的神经毒理学效应,其衍生物具 有与母体化合物 TBBPA非常相似的化学结构,但其神经毒理学效应与作用机制 却少有报道。本研究首先以大鼠肾上腺嗜铬细胞瘤细胞(rat pheochromocytoma cells,PC12)为毒性测试模型,对 TBBPA及其四种主要衍生物的神经毒性效应 进行了筛选分析。结果显示,四溴双酚 A双(2⁃羟乙基醚)( TBBPA bis(2-hydroxyethyl ether),TBBPA-BHEE)具有比母体化合物 TBBPA更强的神经 毒性效应,这与其相对较高的亲水性有关。以 TBBPA-BHEE为衍生物代表的进 一步研究显示,这种化合物可以干扰 PC12细胞多巴胺分泌过程及乙酰胆碱酯酶 的活性,影响细胞呼吸链氧化磷酸化过程,并通过 ROS介导的 caspases活化引 起 PC12细胞毒性。TBBPA-BHEE暴露可引起有丝分裂原激活蛋白激酶 (mitogen-activated protein kinases,MAPKs)的持久性激活,并调控甲状腺激素 相关信号通路,但与 TBBPA不同的是,这些效应并不是介导 TBBPA-BHEE细 胞死亡的主要诱因,因此 TBBPA衍生物具有不同于其母体化合物的神经毒性作 用机制。 在 TBBPA-BHEE离体神经毒性研究的基础上,本论文进一步探讨了该物质 的活体神经毒性效应。实验以新生 SD乳鼠为模型,进行了为期 3周的连续滴鼻 给药暴露。对实验动物的行为学研究发现,TBBPA-BHEE暴露可以明显降低大 鼠在转轴上的平衡运动能力,并且减少其在旷场中的自发探索行为,诱导大鼠出 现明显的神经行为学改变。对实验动物的组织病理学分析显示,暴露大鼠出现了 大脑皮层神经元凋亡、小脑梨状神经元凋亡及小胶质细胞活化的现象,表明 TBBPA-BHEE长期经鼻给药可造成明显的脑组织损伤效应。对实验动物大脑皮 层的基因芯片分析显示,TBBPA-BHEE暴露可造成 911个基因上调,433个基因 下调,这些差异表达基因(differentially expressed genes,DEGs)涉及了实验动 物脑神经细胞的基础生物学过程与相关信号通路,为实验动物神经毒理学表型的 出现提供了分子机制阐释。 血管生物学屏障是调控污染物进入靶器官产生毒性的重要环节,例如,血脑 屏障功能损伤对污染物神经毒性效应具有重要影响。血液中血管舒缓素 -激肽系 统(kallikrein-kinin system,KKS)对血管生物学屏障具有重要的调控作用。考 虑到 PFASs与 KKS系统内源激活剂饱和脂肪酸的结构相似,并且它们在人体血 液中的存在广泛而持久,PFASs对血浆 KKS系统的调控作用值得关注。通过离 体、体外和体内实验,本研究首先证实了 PFASs对 KKS的级联激活效应。针对 20种具有不同结构因子的 PFASs的研究显示,这类化合物激活 KKS的效应依赖 于化合物的碳链长度和氟原子饱和度,并且末端基团为磺酸或羧酸时 KKS激活 效应显著。PFASs激活 KKS的分子机制研究表明,这类化合物可与 KKS的起始 酶原凝血因子 XII(Hageman factor XII,FXII)结合,诱导其构型改变并发生自 身剪切激活,进而导致下游的血浆激肽释放酶原(plasma prekallikrein,PPK)及 高分子量激肽原(high-molecular-weight kininogen,HK)的级联激活。分子对接 分析显示,PFASs与 FXII的结合力越强,其激活 KKS的能力越强。 为进一步探讨 PFASs诱导的 KKS激活对血管屏障的调控作用,本论文采用 激活效应较强的 PFHxDA作为 KKS活化剂代表,同时采用较为流行的 PFOS和 PFOA作为对照,研究了 PFASs活化血浆对人视网膜内皮细胞( human retina endothelial cells,HRECs)旁路通透性的影响。结果显示,PFHxDA活化血浆暴 露可显著增加细胞间通透性,并且这种通透性改变是由细胞间粘合连接蛋白 VE-Cadherein降解内化引起的。KKS系统抑制剂 aprotinin的共孵育显著改善了 由 PFHxDA活化血浆引起的一系列细胞生物学效应,从而有效证明 PFASs诱导 的血管内皮细胞通透性改变是由 KKS系统激活调控的。 本论文揭示了新型有机卤代化合物 TBBPA衍生物和 PFASs的神经毒理学效 应及调控血浆 KKS穿透血管屏障的分子机制,提出了许多新型有机卤代物的衍 生物或同系物具有比其母体化合物如 TBBPA、PFOS或 PFOA更强的毒理学效应 的新观点。本论文研究工作为新型污染物的环境暴露及其健康风险评价提供了新 思路。
英文摘要: Emerging halogenated organic compounds are now being highly concerned due to their wide application, environmental occurrence, potential human exposure risks and considerable human body burden. Despite the diverse toxicities reported for the parent compounds or prevalent congeners, toxicological effects of their derivatives or substitutes still remain unclear. In this study, we selected tetrabromobisphenol A (TBBPA) and per- and polyfluoroalkyl substances (PFASs) as the representative halogenated organic compounds, and studied their potential neurotoxiclogical effects and the underlying molecular mechanisms for the potential vascular penatration. The research mainly included four parts. TBBPA can cause diverse adverse effects including neurotoxicity, while the toxicological effects of its derivatives remain unknown. Considering these emerging chemicals have high structural similarity to the parent compound, their potential hazards are now being highly concerned. Using rat pheochromocytoma cells (PC12), the potential neurotoxicity of TBBPA and four derivatives was studied and compared. TBBPA bis(2-hydroxyethyl ether) (TBBPA-BHEE) exhibited relatively higher cellular toxicity to PC12 than the other tested compounds, which was correlated with its hydrophobicity. Functional assays showed that TBBPA-BHEE disturbed dopamine (DA) secretion process and altered acetylcholinesterase (AChE) enzymatic activity in PC12 cells. The molecular mechanism study indicated that TBBPA-BHEE induced dysfunction of the respiratory chain oxidative phosphorylation, and caused cellular toxicity to PC12 cells through ROS-mediated caspase activation to a large extent, which was partially attenuated by the antioxidation of Vitamin E. Moreover, in contrast to TBBPA, TBBPA-BHEE induced toxicity to PC12 was not attributed to sustained activation of mitogen-activated protein kinases (MAPKs) or thyroid hormone (TH) regulated signaling pathway. Considering the potential neurotoxicity of TBBPA derivatives which has been demonstrated in vitro, what could happen in vivo is worthy of being studied. In this study, TBBPA-BHEE, a representative TBBPA derivative, was selected for a 21-day exposure experiment on neonatal Sprague Dawley (SD) rats through intranasal administration. The neurobehavioral, histopathological changes, and differentially expressed genes (DEGs) based on RNA microarray were investigated to evaluate the neurological effects of this chemical. The results indicated that TBBPA-BHEE exposure significantly compromised the motor co-ordination performance and the locomotor activities. The neurobehavioral phenotype could be attributed to the obvious histopatholgical changes in both cerebrum and cerebellum, such as neural cell swelling, microglial activation and proliferation. A total of 911 genes were up-regulated, whereas 433 genes were down-regulated. Gene set enrichment analysis showed multiple signaling pathways, including ubiquitin-mediated proteolysis and Wnt signaling pathway etc. were involved due to TBBPA-BHEE exposure. The gene ontology (GO) enrichment analysis showed the basic cellular function and the neurological processes like synaptic transmission were influenced. The vascular barrier is important for regulating target organ toxicities such as neurotoxicity. The plasma kallikrein-kinin system (KKS) physiologically regulates multiple biological processes, i.e. vascular permeability. PFASs are ubiquitous and persistent in human blood, and they have high structure similarity to fatty acid, an endogenous KKS activator. How PFASs interact with KKS would be of interest in explaining their potential hematological effects. We screened the effects of 20 PFASs and the related long-chain aliphatic compounds on KKS cascade activation using ex vivo, in vivo, and in vitro assays. The binding affinities of the tested compounds with the initial zymogen, Hagmen factor XII (FXII) in the KKS, were evaluated by molecular docking analysis. PFASs were demonstrated to activate the KKS in a structure-dependent mode. More specifically, PFASs with longer carbon chain length, higher fluorine atom saturation degree, and terminal acid group exhibited relatively higher activities in activating the KKS. The binding affinities of PFASs with FXII determined their capabilities for inducing KKS activation. The binding modes, involved with Van der Waals and hydrogen bonds, were different for the tested compounds. How PFASs regulate the possible vascular biological effects through the activation of the KKS is an important issue. As the long-carbon chain compound, perfluorohexadecanoic acid (PFHxDA), showed relatively higher capacity in activating the KKS than the prevalent congeners, i.e. perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA), it was selected for the subsequent study on endothelial paracellular permeability. Using human retina endothelial cells (HRECs) as the experimental model, PFHxDA activated plasma was found to cause significant increase in the paracellular permeability through the degradation of adherens junctions. As evidenced by the antagonistic effect of aprotinin, PFHxDA-involved effects on vascular permeability were mediated by KKS activation. This research have revealed the neurotoxicological effects of halogenated organic compounds and the mechanistic pathway for vascular barrier permeation, which would be of great help in risk assessment for their unintended exposure. When compared to the prevalent parent compounds like TBBPA, PFOS or PFOA, their derivatives and congeners exhibited distinct toxicological effects, which provided new insights on potential hazardous influcences from these emerging chemicals.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/38664
Appears in Collections:环境化学与生态毒理学国家重点实验室_学位论文

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作者单位: 1.中国科学院生态环境研究中心

Recommended Citation:
刘倩. 新型有机卤代化合物的神经毒理学效应与作用机制研究[D]. 北京. 中国科学院大学. 2017.
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