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脂环族溴代阻燃剂对SH-SY5Y细胞的神经毒性研究
Alternative TitleIn vitro s tudy on the n eurotoxicity of cycloaliphatic brominated flame retardants in SH SY5Y cells
时晓丽
Subtype博士
Thesis Advisor张淑贞 ; 温蓓
2019-12
Degree Grantor中国科学院生态环境研究中心
Place of Conferral北京
Degree Name理学博士
Degree Discipline环境科学
KeywordHbcd 同分异构体 Hbcd Diastereoisomers Tbco Tbech n Eurotoxicity a Poptosis Tbech Tbco 神经毒性 细胞凋亡
Abstract

        六溴环十二烷( HBCD 为代表的 脂环族 溴代阻燃剂 CBFRs )已经在多种环境介质和生物样品中 被 检出。 HBCD 具有持久性有机污染物的特征, 2013 年5 月 召开的 斯德哥尔摩公约缔约方大会第六次会议通过了 将 其 列入持久性有机污染物( POPs 名单(附件 A )中的决议 。 商用 HBCD 主要含有 α 、 β 和 γ HBCD三种异构体 异构体在环境介质中的分布、在生物中的累积与代谢、在食物链的放大及毒性效应等方面均存在显著差异, 只有在异构体水平的研究才能全面准确地评价 HBCD的生态和健康风险 。 HBCD 已 被证实具有显著的神经毒性, 但是目前尚缺乏 从异构体水平 对其 神经毒性 的认识 。与 HBCD 具有相似结构和性能的脂环族溴代阻燃剂 1,2,5,6 四溴环辛烷( TBCO )和 1,2 二溴 4 1,2 二溴乙基)环己烷( TBECH 是 HBCD 的 潜在替代品。作为替代品其对人类和野生动植物健康 的影响应小于现有产品,因此研究和认识替代品的健康和环境效应至关重要。 但是目前尚缺乏对 TBECH 、 TBCO 神经毒性 的 认识。 针对以上问题本论文开展了如下研究:
       系统研究了 α β γ HBCD 对 人神经母细胞瘤细胞 SH SY5Y 的毒性差异。结果显示 HBCD 同分异构体均能降低细胞活力、增加乳酸脱氢酶( LDH )的释放和破坏细胞骨架。通过观察细胞形态特征和凋亡比率发现 三种 HBCD 异构体均诱导 SH SY5Y 细胞发生凋亡。 分析 与细胞凋亡相关基因和蛋白( Bax 、caspas e 3 、 caspase 9 、 cytochrome c Cyt c 、 Bcl 2 、 X linked inhibitor of apoptosisXIAP ))的表达、细胞周期停滞、 DNA 损伤、三磷酸腺苷( ATP )消耗发现HBCD 同分异构体神经毒性大小顺序为 β HBCD > γ HBCD > α HBCD 三种HBCD 异构体诱导的细胞凋亡与 caspase 蛋白表达水平遵循相同的顺序,表明HBCD 同分异构体诱导的神经毒性是一种结构选择性 caspase 依赖性凋亡。为了进一步 阐述 α β γ HBCD 神经毒性机理, 我们分别检测了胞内活性氧( ROS水平和胞内钙离子( Ca 2+2+)水平 发现 三种 HBCD 异构体均能引起 胞内 ROS 和Ca 2+ 水平显著增加 胞内 ROS 增加的大小顺序为 β HBCD > γ HBCD > α HBCD与神经毒性大小顺序一致 而胞内 Ca 2+ 水平增加的大小顺序为 γ HBCD > βHBCD > α HBCD 。因此 ROS 可能是影响 HBCD 同分异构体神经毒性的关键因素。 本研究首次从异构体水平 阐述 了 HBCD 神经毒性效应和可能机理。
       比较了 HBCD 、 TBECH 、 TBCO 对 SH SY5Y 细胞 神经毒性 的 差异。结 果显示, HBCD 、 TBECH 和 TBCO 均 诱导细胞毒性, 表现为 浓度依赖的细胞活力下降 、 细胞膜通透性的增加 、 细胞骨架发育损坏 以及明显的 细胞凋亡 神经毒性的大小顺序为 HBCD > TBCO > TBECH 。 与细胞凋亡相关基因和蛋白( Bax 、caspase 3 、 caspase 9 、 C yt c 、 Bcl 2 、 XIAP )的表达遵循相同的顺序,表明 caspase依赖的凋亡途径是 HBCD 、 TBECH 和 TBCO 神经毒性的机理之一。为进一步 认识 HBCD 、 TBECH 和 TBCO 神经毒性机理, 分别检测了 HBCD 、 TBECH 和 TBCO诱 导的 ROS 产生和胞内 Ca 2+ 水平,发现暴露 HBCD 、 TBECH 、 TBCO 后 胞内ROS 水平显著上升 ,且 抗氧化剂 NAC 存在 时 HBCD 、 TBECH 、 TBCO 诱导的细胞毒性显著降低,表明氧化应激是 HBCD 、 TBECH 、 TBCO 细胞 凋亡 的诱因之一。 暴露 HBCD 和 TBCO 同时导致 胞内 Ca 2+ 水平显著增加, 但是暴露 TBECH后 胞内 Ca 2+ 水平 无显著 变化。 在 钙离子抑制剂 BAPTA AM 存在时, HBCD 和TBCO 诱导的细胞毒性显著降低。 结果 表 明 HBCD 和 TBCO 诱导的 细胞 凋亡还同时 受 Ca 2+ 介导。
       本论文系统地 从异构体水平研 究了 HBCD 对 SH SY5Y 细胞的神经毒性,比较了 HBCD 的潜在替代品 TBCO 、 TBECH 与 HBCD 的神经毒性差异,揭示了CBFRs 的结构特异性细胞毒性,阐述了 CBFRs 神经毒性的机制。为 CBFRs 生产使用的风险性评价提供了重要 的科学 依据。
 

Other Abstract

       Hexabromocyclododecane (HBCD) is a representative of alicyclic brominated flame retardan ts (CBFRs), which have been frequently found in environments and living organisms. HBCD p ossesses all the char a cterics of persistant organic pollutants and was listed in Annex A (POP for elimination) under the Stockholm Convention on Persistent Organic Po llutants in May 2013. T he c ommercially available HBCD (technical mainly consists of α --, β --, and γ HBCD It has been evidenced that d istribution in the environmental media , bioaccumulation, metabolism, food chain
amplification, and toxic effect s of HBCD are diastereoisomer selective. Therefore, it is necessary to investi gate the toxicities of HBCD at the level of individual isomers in order to evaluate its environmental safety and health risks. I t has been reported that HBCD can cause potential neurotoxicity however, there has been no study to address their diastereoisom er specific neurotoxicity so far. 2 dibromo 4 --(1,2 dibromoethyl) cyclohexane (TBECH) and 1,2,5,6 tetrabromocyclooctane (TBCO), with the same structures and properties as HBCD, were regarded as the potential alternatives of HBCD, which could have major imp lications for their significant increases in production, application and environmental accumulation in the near future. It is essential to assess the environmental and health effects of these potential alternatives .However, the understanding of neurotoxic ities of TBECH and TBCO remains lacking. Therefore, this dissertation includes the following parts of studies.
       The neurotoxicit ies of α --, β --, and γ HBCD in SH SY5Y human neuroblastoma cells were investigated . The r esults showed that the HBCD diastereoisomers decreased cell viability, increased lactate dehydrogenase (LDH) release, and impaired cytoskeleton development. Typical morpholo gical features and apoptosis rates showed that all the HBCD diastereoisomers induced SH SY5Y cell apoptosis. The expression levels of several cell apoptosis related genes and proteins, including Bax, caspase 3,caspase 9, cytochrome c (Cyt c), Bcl 2, and X linked inhibitor of apoptosis (XIAP),as well as the cell cycle arrest, DNA damage, and adenosine triphosphate (ATP)consumption, were examined. The results showed that the HBCD diastereoisomer icneurotoxicity was ranked β HBCD > γ HBCD > α HBCD. The cell apoptosis and caspase expression levels of the three HBCD diastereoisomers followed the same order, suggesting that caspase dependent apoptosis may be one of the mechanisms responsible for the st ructure selective HBCD diastereoisomer neurotoxicity. To further explore the mechanisms of cell cytotoxicity, HBCD diastereoisomer induced reactive oxygen species (ROS) and the intracellular calcium Ca 2+2+) levels were examined. The levels of intracellular Ca 2+ and ROS increased significantly. The ROS levels followed the order β HBCD > γ HBCD > α HBCD, which is the same as their toxicities, whereas those of intracellular Ca 2+ were γ HBCD > β HBCD > α HBCD. Thus, ROS may be a key factor regulating the neurotoxici ty of HBCD diastereoisomers. To the best of our knowledge, this is the first study to report on the diastereoisomer specific toxicity of HBCD in human neural cells and on the possible mechanisms responsible for the selective neurotoxicity of HBCD diastereo isomers.
        The neurotoxicity effects of HBCD, TBECH, and TBCO on human SH SY5Y cells were compared. The r esults showed that HBCD, TBECH, and TBCO induced cytotoxicit ies , including dose dependent cell viability decreases, cell membrane permeability increases , cell cytoskeleton development damage, and cell apoptosis induction. The order of toxicity was HBCD > TBCO > TBECH. The cell apoptosis and caspase (Bax, caspase 3, caspase 9, C yt c, Bcl 2, and XIAP) expression levels of HBCD, TBECH, and TBCO followed the same order, suggesting that caspase dependent apoptosis may be one of the mechanisms responsible for the neurotoxicities of HBCD, TBECH, and TBCO. To further explore the mechanisms of cell cytotoxicity, the levels of intracellular ROS and Ca 2+ induced by th e exposures to HBCD, TBECH, and TBCO were examined. The ROS levels w ere significantly elevated for all the exposures, and t he presence of antioxidant N acetyl L cysteine (NAC) significantly reduced the cytotoxicity The intracellular Ca 2+ concentration was significantly increased for the exposure to HBCD or TBCO, but not for TBECH treatment. The presence of Ca 2+ inhibitor BAPTA AM significantly reduced the cytotoxicit ies induced by HBCD and TBCO. The se observations suggested that oxidative stress contribute d to the cytotoxicit ies of HBCD, TBECH, and TBCO, and HBCD and TBCO m ight also induce Ca 2+ mediated apoptosis.
      In summary diastereoisomer specific neurotoxicity of HBCD in human SH SY5Y neuroblastoma cells was investigated Moreover, the neur otoxicity of HBCD and its potential alternatives, TBCO and TBECH, were compared. This study gained some insight s into the structure dependent cytotoxicity for different CBFRs,provid ing valuable basis for the risk assessment of CBFRs.

Pages129
Language中文
Document Type学位论文
Identifierhttp://ir.rcees.ac.cn/handle/311016/42278
Collection环境化学与生态毒理学国家重点实验室
Recommended Citation
GB/T 7714
时晓丽. 脂环族溴代阻燃剂对SH-SY5Y细胞的神经毒性研究[D]. 北京. 中国科学院生态环境研究中心,2019.
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