【作者】 何平；

【导师】 王爱国；

【作者基本信息】 华中科技大学 ， 劳动卫生与环境卫生学， 2009， 博士

【摘要】 多溴联苯醚（Polybrominated diphenyl ethers，PBDEs）作为一种重要的防火材料，被广泛应用于电器、电线、家具、纺织品等产品中。PBDEs和多氯联苯（Polychlorinatedbiphenyls，PCBs）同属于卤代烃类化合物，根据苯环上溴原子和氯原子数目和位置的不同，PBDEs和PCBs在理论上均含有10大类209种同系物。虽然目前两者的生产和使用均受到了限制，尤其是PCBs在很多国家被禁止生产和使用，但在地球生物圈的任何地方，如空气、水、土壤、各种沉淀物以及野生动物、鱼类、家禽和人类的机体组织中都有PBDEs和PCBs存在，其中PBDEs检出值呈逐年上升趋势。由于PBDEs和PCBs具有结构的相似性，因此PBDEs和PCBs具有相似的毒性作用。目前大量的体外、体内研究结果表明，PBDEs和PCBs作用的靶器官主要是脂肪组织、甲状腺、生殖发育及中枢神经系统，两者均具有多种毒性，如神经毒性、生殖毒性、免疫毒性、肝毒性和致癌性，作为一种内分泌干扰物，具有类雌激素活性，可引起甲状腺的增生，导致甲状腺素代谢的改变。多项动物实验研究结果表明，在鼠大脑发育的关键期，暴露于低剂量的PBDEs可导致成年鼠脑功能异常，主要表现为自主行为紊乱，学习记忆缺损等，且随年龄的增长越来越严重。因此，近年来PBDEs的发育神经毒性作用越来越受到人们的关注。然而，PBDEs神经毒性的研究工作仍处于起步阶段，目前研究也多集中于神经行为、认知功能、学习与记忆等神经毒效应方面，而对PBDEs如何引起这些神经毒效应的机制研究较少涉及，因此，针对PBDEs的神经毒性作用机制展开研究，将有利于寻找PBDEs神经毒性作用的靶点，为PBDEs的神经毒性作用的预防和控制提供理论依据。Madia等在体外实验研究中发现PBDE-99和Aroclor1254均可诱导星形细胞瘤细胞发生凋亡，从而对细胞产生明显的毒性作用。本研究前期体外实验结果也表明PBDEs同系物PBDE-47可诱导人神经母细胞瘤SH-SY5Y细胞凋亡。大量研究均已证明在多细胞生物的基本生命活动中，细胞凋亡起着十分重要的作用，许多外源化合物的毒性作用与细胞凋亡有关。目前已知的三个主要的凋亡信号通路是：1)线粒体通路；2)死亡受体通路；3)内质网通路。所有这些信号通路都能激活凋亡执行者Caspase3，通过水解各种各样的细胞成分而使细胞凋亡。细胞凋亡在PBDEs所致神经毒性中的作用，目前尚未见研究报道。Hites等综合分析了全球已公布的人群监测结果，认为与人群关系最密切的5种PBDEs同系物是PBDE-47（2，2′，4，4′-tetrabromodiphenyl ether PBDE-47）、99、100、153、154。而Bi等对中国南方某城市21对婴儿脐带和母亲静脉血样的分析结果显示，PBDE-47和PBDE-153是PBDEs最主要的同系物。2，2′，4，4′，5，5′-六氯联苯（2，2’，4，4’，5，5′-hexachlorobiphenyl，PCB153）是人体组织和人乳中含量最多的PCBs同系物之一，因此，PCB153是PCBs中与人类健康关系密切的同系物之一。流行病学调查发现PCB153和PBDE-47可同时存在于环境和人体组织中，但目前尚未见两者联合毒作用及其机制的研究报道。基于上述理由，PBDE-47和／或PCB153是否通过三条经典凋亡信号通路诱导神经细胞凋亡，进而引发一系列神经毒效应呢，这正是本研究立题的目的所在。本研究采用体外和体内实验，利用四甲基偶氮唑盐（3-（4，5-dimethylthiazol-2-yl）2，5-diphenyltetrazolium bromide，MTT）法、分光光度法、Morris水迷宫实验、超微病理检测、流式细胞术（flow cytometry，FCM）、适时荧光定量反转录聚合酶链式反应（realtime fluorescent quantitation reverse transcriptase polymerase chain reaction，real timeRT-PCR）和Western blot等技术和方法，通过检测神经细胞存活率、乳酸脱氢酶（lactatedehydrogenase，LDH）漏出、细胞内Ca²⁺浓度(intracellular free Ca²⁺ concentration，[Ca²⁺]_i)、大鼠的空间学习记忆能力、大鼠海马神经元超微结构及神经细胞X染色体连锁的凋亡抑制蛋白（X-chromosome-linked inhibitor of apoptosis protein，XIAP）、含半胱氨酸的天冬氨酸酶12（cysteine containing aspartate specific protease 12，Caspase12）、含半胱氨酸的天冬氨酸酶3（cysteine containing aspartate specific protease 3，Caspase3）、死亡相关蛋白激酶（death associated protein kinase，DAPK）和细胞色素C（CytochromeC）等凋亡相关基因mRNA和蛋白质的表达，研究PBDE-47和／或PCB153的神经毒性作用及其可能的毒作用机制。第一部分PBDE-47和／或PCB153对SH-SY5Y细胞的毒性作用及其机制研究目的：由于PBDE-47与PCB153分别作为PBDEs和PCBs的同系物可共存于环境和人组织中，因此本研究旨在探查PBDE-47的神经毒效应及其机制，同时研究PBDE-47与PCB153共同存在时对神经细胞的联合毒作用方式。方法：SH-SY5Y细胞被暴露于PBDE-47（0、1、5和10μmol／L）和／或PCB153（0和5μmol／L）24h后，使用MTT法、分光光度法、流式细胞术、real time RT-PCR技术和Western blot技术分别检测了细胞存活率、LDH漏出、[Ca²⁺]_i、细胞凋亡百分率及DAPK、XIAP、Caspase12、Caspase3以及Cytochrome C等凋亡相关基因mRNA和蛋白质表达水平。结果：和对照组比较，一定剂量的PBDE-47可导致SH-SY5Y细胞存活率显著下降（P＜0.05），LDH漏出、[Ca²⁺]_i和凋亡率明显上升（P＜0.05）。进一步研究发现，DAPK和Capspase3 mRNA，Caspase12和Cytochrome C mRNA和蛋白质表达水平均显著性增加（P＜0.05），而Pro-caspase3蛋白质表达水平是明显下降的（P＜0.05）。相关分析表明，[Ca²⁺]_i与细胞凋亡率间存在正相关关系（r=0.86，P＜0.05）。析因分析表明，在诱导SH-SY5Y细胞存活率下降、[Ca²⁺]_i增加、凋亡和LDH漏出，以及诱导XIAP、Caspase3和Cytochrome C mRNA表达，和XIAP、Pro-caspase3、Caspase12和Cytochrome C蛋白质表达方面，PBDE-47与PCB153之间存在交互作用。结论：PBDE-47具有明显的SH-SY5Y细胞毒性作用。三条经典凋亡途径均可能参与了PBDE-47诱导的SH-SY5Y细胞凋亡，且胞内钙平衡失调可能是其主要诱导因素之一。一定剂量的PBDE-47与PCB153共同作用于SH-SY5Y细胞时其毒效应增加。第二部分PBDE-47和／或PCB153对SD大鼠发育神经毒作用及其机制研究目的：由于PBDE-47与PCB153可在环境和生物组织中共存，因此本研究旨在探讨PBDE-47与PCB153单独和联合对SD大鼠的发育神经毒作用及其机制。方法：在SD大鼠出生第10天，通过灌胃一次性暴露于PBDE-47（0、1、5和10 mg／kg）和／或PCB153（0和5 mg／kg），在大鼠出生后2个月时通过Morris水迷宫实验、超微病理检测、化学发光免疫定量分析技术、real time RT-PCR和Western blot技术分别检测大鼠的空间学习记忆能力、海马CA1区神经元超微结构、甲状腺激素水平和XIAP、DAPK、Caspase3、Caspase12及Cytochrome C等凋亡相关基因mRNA和蛋白质表达水平。结果：与对照组相比，PBDE-47可使处理组大鼠总路径（大鼠到达平台所游经的路径）和潜伏期（大鼠到达平台所用的时间）显著性增加，而路径（时间）比值（在60秒内大鼠游经平台所在相限的路径（时间）与总路径（时间）的比值）则明显降低（P＜0.05）。与对照组相比，PBDE-47可使处理组大鼠神经元超微结构发生明显改变。5 mg／kg PBDE-47剂量组T₄浓度明显低于对照组（P＜0.05）。与对照组比较，PBDE-47可使处理组XIAP mRNA和蛋白质表达水平显著下降（P＜0.05），Caspase12和Cytochrome C（雌性大鼠Cytochrome C mRNA表达除外）mRNA和蛋白质表达水平显著上升（P＜0.05）；PBDE-47可使处理组Caspase3 mRNA表达水平明显上升，而Pro-caspase3蛋白质表达水平显著下降（P＜0.05）；PBDE-47可使处理组雌鼠DAPKmRNA表达水平显著下降，而雄鼠DAPK mRNA表达水平上升（P＜0.05）。在降低大鼠学习记忆能力及诱导Caspase3、XIAP、Caspase12和Cytochrome C（仅在雄性大鼠）mRNA表达，Pro-caspase3、XIAP、Caspase12和Cytochrome C蛋白质表达等方面，PBDE-47与PCB153之间存在交互作用。结论：PBDE-47具有明显的发育神经毒性作用。三条经典凋亡途径均可能参与了PBDE-47诱导的发育神经毒性作用。影响甲状腺发育和降低甲状腺激素分泌可能是其具有发育神经毒性作用的原因之一。在发育的关键期，一定剂量的PBDE-47与PCB153共同作用于SD大鼠时其发育神经毒效应增加。更多还原

【Abstract】 Polybrominated diphenyl ethers （PBDEs）, as additive flame retardants, are widespreadused in electrical equipment, wire, furniture, and textile. PBDEs and polychlorinatedbiphenyls （PCBs） belong to the halogenating hydrocarbon compound. According to thedifferent atomic number and the position of bromine and chlorine atom in the benzene ring,both the PBDEs and PCBs theoretically include 10 main categories and 209 kinds ofhomologs. At present the production and use of the two compounds have been restricted,PCBs is forbidden in many countries to produce and to use in particular, but PBDEs andPCBs exist in all the places in the earth biosphere such as air, water, soil, each kind ofsediments, as well as wild animal, fish, domesticated fowl and in humanity’s organism.Furthermore, the concentration of PBDEs being detected has the trend of escalation year byyear.Because the molecular conformation has the similarity, PBDEs is similar to PCBs intoxicity. To date, some in vitro and in vivo studies have reported that the functionary targetorgans of PBDEs and PCBs are mainly the fatty tissue, the thyroid gland, the reproductiongrowth and the central nervous system. PBDEs and PCBs have neurotoxicity,hepatotoxicity, reproduction toxicity, immunity toxicity, and carcinogenicity. And as a kindof endocrine interferent, which possess analogical estrogen activeness, and can cause thehyperplasia of the thyroid gland, leading to the change of thyroid metabolism.The massive animal experimentation studies indicated that animals exposed to lowdose PBDEs in the critical period of cerebrum growth, such as grown-up mouse brainfunction to be unusual, behavior disorder, studying and memory decrease will happen, andit will become more serious every day with the increasing of age. Therefore, the recent years the developmental neurotoxic effects of PBDEs get more and more attention frompeople. However, the neurotoxic effects of PBDEs are still at the starting stage. At presentthe studies are also mainly concentrated in nerve behavior, cognition function, learning andmemory and so on. The further research on the neurotoxic effective mechanism of PBDEsis to be advantageous in seeking the target spot of the neurotoxic effect and also providesthe theory basis for the prevention and control of neurotoxic effect of PBDEs.Regarding to the in vitro experimental studies, Madia et al reported that both PBDE-99and Aroclor1254 may induce the astrocytoma cells to apoptosis and possess obvious celltoxicity. Our early in vitro experimental result indicated that PBDE-47, a typicalpredominant congner of PBDEs, may induce the SH-SY5Y cell to apoptosis. Manyresearches confirmed that apoptosis plays an extremely vital roles in multicellularorganism’s basic vital activity. At present apoptosis include three classic pathways:mitochondria pathway, death receptor pathway, and endoplasmic reticulum pathway. Allthese signal transduction pathways can activate apoptosis performer Caspase3, which canhydrolyze various cell component to lead to cell apoptosis. So far, the action of apoptosis inthe neurotoxicity induced by PBDEs is not yet reported.After synthetically analyze all the announced monitoring results from the global crowd,Hites et al reported that the closest 5 kinds of PBDEs homolog to the crowd are PBDE-47（2, 2’, 4, 4’-tetrabromodiphenyl ether, PBDE-47）, 99, 100, 153, 154. But Bi et al foundthat PBDE-47 and PBDE-153 are the most main homologs after analyzed the 21 pairs ofbaby umbilical cord and the mother vein blood samples from an austral city in China. Asthe PCBs homolog, 2, 2’, 4, 4’, 5, 5’-six chlorine biphenyl （2, 2’, 4, 4’, 5, 5’hexachlorobiphenyl, PCB153） is one of most content homolog in the body tissue and thebreast milk. Therefore, PCB153 is one of PCBs homolog which has the closest relationswith human health. The epidemiology investigation discovered that PCB153 and PBDE-47may simultaneously exist in the environment and human body organize, but not yet see thecombined toxic effects and the mechanism reported at present.Basis above reasons, whether PBDE-47 and/or PCB153 induce nerve cell apoptosisthrough three classics signal pathways, then initiates a series of neurotoxic effect issupposed, which is the main content of this study. The research experiment will go on both in vitro and in vivo, 3-（4, 5-dimethylthiazol-2-yl） 2, 5-diphenyltetrazolium bromide （MTT）,spectrophotometric method, Morris maze experiment, ultramicro pathology detect, flowcytometry （FCM）, real time fluorescent quantitation reverse transcriptase polymerase chainreaction （real time RT-PCR）, and Western blot and so on will be used to study theneurotoxic effect and the mechanism of PBDE-47 and/or PCB153 through detecting theneurocyte survival rate, lactate dehydrogenase （LDH） leakage, intracellular free Ca²⁺concentration ([Ca²⁺]_i, the SD rat’s learning and memory capability, ultrastructure ofhippocampal neuron, and the mRNA and protein expression levels ofX-chromosome-linked inhibitor of apoptosis protein （XIAP）, cysteine containing aspartatespecific protease 3 （Caspase3）, cysteine containing aspartate specific protease 12（Caspase12）, death associated protein kinase （DAPK）, and Cytochrome C in SD rat and inSH-SY5Y cells.Part 1 Studies of toxic effect and mechanism of PBDE-47 and/or PCB153in SH-SY5Y cellsObjective: PBDE-47 and PCB153 can coexist in the environment and human tissuesas dominant congeners of PBDEs and PCBs, respectively. The aims of presental experimentare to explore the neurotoxic effect and the mechanism of PBDE-47 and the mode of actionin combination with PCB153 in SH-SY5Y cells.Methods: MTT, spectrophotometric method, flow cytometry, real time fluorescentquantitation RT-PCR, and Western blot were used to detect the cell viability, LDH leakage,[Ca²⁺]_I, apoptosis, and mRNA and protein expression levels of DAPK, XIAP, Caspase3,Caspase12, and Cytochrome C in SH-SY5Y cells treated with PBDE-47 （0, 1, 5 and 10μmol/L） and/or PCB 153 （0 and 5μmol/L） for 24h.Results: Compared to controls, the cell viabilities were clearly decreased （P＜0.05）,and LDH leakage, [Ca²⁺]_i and apoptosis were significantly increased （P＜0.05）.Furthermore, expression levels of DAPK and Caspase3 mRNA, Caspase12, as well asCytochrome C mRNA and proteins were markedly increased （P＜0.05）, whilePro-caspase3 proteins were significantly decreased （P＜0.05）. A positive correlationbetween [Ca²⁺])i and percentage of apoptotic cells （r=0.86, P＜0.05）. An interaction between PBDE-47 and PCB153 were existed in decreasing SH-SY5Y cell survival,increasing the [Ca²⁺]_i, inducing the apoptosis and LDH leakage, as well as inducing theXIAP, Caspase3 and Cytochrome C mRNA expression, and XIAP, Pro-caspase3,Caspase12 and Cytochrome C proteins expression.Conclusions: PBDE-47 has the obvious toxic effect on SH-SY5Y cell. All threeclassic apoptosis pathway possibly participate in the process of SH-SY5Y cell apoptosisinduced by PBDE-47. Moreover, the calcium disequilibrium in the cell may be one of themain induction factors. The toxic effect is supposed to increase when SH-SY5Y cells areexposed to PBDE-47 and PCB 153 together in certain dosage.Part 2 Studies of developmental neurotoxic effect and mechanism ofPBDE-47 and/or PCB153 in ratsObjective: Because PBDE-47 and PCB153 may coexist in the environment media andhuman tissues, this experiment is to explore the developmental neurotoxic effect and themechanism of PBDE-47 alone and/or combined with PCB153 in the SD rats.Methods: Morris water maze, ultramicropathology technique detect,chemoluminescence, real time RT-PCR and Western blot were used to detect the learningand memory capabilities of rats, ultrastructure of neurons in the hippocampal CA1 region,the level of thyroid hormone and the expression levels of mRNA and proteins of XIAP,DAPK, Caspase3, Caspase12 and Cytochrome C in hippocampus in 2-month-old ratsexposed to a single oral dose of PBDE-47 （0, 1, 5 and 10 mg/kg） and/or PCB153 （0 and 5mg/kg） on post natal day （PND） 10.Results: The results suggested that the total distance swam （distance travelled by ratsto reach the escape platform） and the latency period （time travelled by rats to reach anescape platform） were significantly increased in place navigation test and the ratios（distance （time） in platform quadrant/total distance （time） swam） in spatial probe test werenotably decreased in all treated groups in water maze experiment （P＜0.05） whencompared to the control group. Additionally, compared with the control group,ultrastructure of neurons was prominently changed in the treated groups. Theconcentrations of T₄ in serum were obviously depressed in 5 mg/kg PBDE-47 group than that of the control （P＜0.05）. Compares with the control group, the mRNA and the proteinexpression levels of XIAP in PBDE-47 treated groups were remarkably decreased （P＜0.05）, the mRNA and protein expression levels of Caspase12 and Cytochrome C （femalebig mouse Cytochrome C mRNA expression exception） in PBDE-47 treated groups wereremarkably increased （P＜0.05）. PBDE-47 caused Caspase3 mRNA expression levelsobviously increased and caused Pro-caspase3 protein expression levels remarkablydecreased （P＜0.05）. PBDE-47 caused DAPK mRNA expression level of the female ratsremarkably decreased, but caused DAPK mRNA expression level of the male ratsobviously increased （P＜0.05）. Furthermore, an interaction was found between PBDE-47and PCB153 in decreased the capability of learning and memory, as well as inducingCaspase3, XIAP, Caspase12 and Cytochrome C （only in male rats） mRNA expression, andPro-caspase3, XIAP, Caspase12 and Cytochrome C proteins expression in hippocampus.Conclusions: PBDE-47 has the obvious developmental neurotoxic effect. All threeclassic apoptosis pathway possibly participate in the process of the developmentalneurotoxic effect induced by PBDE-47. Both affecting the development of thyroid glandand reducing the thyroxine secretion are possibly one of the reasons of developmental toxiceffects induced by PBDE-47. In the critical period of development, the toxic effect issupposed to increase when SD rat is exposed to PBDE-47 and PCB153 together in certaindosage.更多还原