【作者】 张倩；

【导师】 金一和； 赵慧敏； 刘薇；

【作者基本信息】 大连理工大学 ， 环境科学， 2016， 博士

【摘要】 全氟辛烷磺酸(perfluorooctane sulfonate, PFOS)是一种最典型的全氟磺酸化合物,由于良好的表面活性和稳定性被广泛应用于日常生活和工业生产等多个领域。PFOS能够通过血脑屏障对发育神经系统造成影响,抑制仔鼠学习记忆能力,但其机制尚不清楚。自2009年PFOS被列入斯德哥尔摩公约,已在多国禁止生产使用,但在某些生产领域仍在持续使用,包括直接向环境中排放的泡沫灭火剂和杀虫剂。此外,PFOS替代品生产应用的快速发展,使得大量短碳链及插入氮、氧等杂原子的全氟／多氟磺酸类化合物的环境水平升高,然而相关的毒理学研究极其有限,环境健康风险未知。研究典型全氟／多氟磺酸化合物的神经毒性效应和机制,对于阐明这类污染物与学习能力损伤、神经退行性疾病之间的关系具有重要意义。本论文利用整体动物建立交叉哺育模型进行发育期暴露,研究PFOS对反映学习记忆能力的关键指标长时程增强(LTP)的影响,结合原代海马神经元细胞离体培养,从α-氨基羟甲基恶唑丙酸(AMPA)受体运输调控探讨PFOS暴露引起LTP损伤的潜在机制。进一步对与学习记忆能力密切相关的Tau磷酸化和p淀粉样蛋白(Aβ)生成过程进行检测,阐明发育期PFOS暴露与神经退行性疾病的可能关联。最后,比较急性侧脑室暴露典型全氟／多氟磺酸化合物对在体LTP的影响,初步评价其神经毒性潜能。主要内容包括：(1)建立整体动物交叉哺育模型,利用电生理技术测定PFOS对大鼠海马CA1区在体LTP的影响,阐明发育期PFOS暴露损伤学习记忆能力的机制。结果显示：发育期PFOS暴露会剂量依赖性抑制LTP的诱发和维持,与PFOS影响大鼠自发行为和损伤学习记忆能力的结果一致,为PFOS的发育神经毒性提供了电生理学依据。高频刺激60 min后5 mg/L和15mg/LPFOS暴露组的场突触后电位(field-excitatory postsynaptic potential, fEPSP)幅值与对照组相比降低20-27%。反映基础突触传递能力的输入输出曲线(I/O)刺激强度在0.3-0.5 mA时PFOS处理组fEPSP幅值比对照组显著降低,双脉冲易化效应(PPF)在最大易化点TT15组显著低于对照组。结果提示PFOS发育期暴露会同时影响突触前细胞和突触后细胞的突触传递效率和突触可塑性,是PFOS损伤学习记忆能力的重要机制。(2)结合体内实验和体外实验,阐明PFOS损伤LTP的AMPA调控机制。PFOS发育期体内暴露引起GluRl和GluR2蛋白和基因的表达水平下调,同时引起磷酸化蛋白GluR1-s831和蛋白激酶CaMKⅡ-α表达水平下调。新生仔鼠原代海马神经元细胞暴露于2μM和20 μM PFOS后,AMPA受体亚基GluR1和GluR2在膜上的表达降低,与体内实验结果一致。PFOS引起谷氨酸作用蛋白GRIP1表达显著下调,与GluR2变化趋势一致,RNA剪辑酶ADAR2 mRNA表达比对照组增加2倍。NBQX抑制PFOS对细胞钙稳态和相关基因表达的影响。结果说明PFOS通过改变AMPA受体亚基的表达调节AMPA受体的动态分布,提高AMPA受体对Ca2+的通透性,损伤神经突触可塑性。(3)通过考察PFOS发育期暴露对Tau磷酸化和Aβ聚集水平的影响,阐明PFOS发育期暴露与神经退行性疾病之间的可能关联。结果发现：PFOS发育期暴露引起Tau总蛋白及mRNA表达水平上调,引起Tau蛋白在S199、T231、S396位点的磷酸化水平升高以及蛋白激酶GSK-3β蛋白含量显著上调。PFOS发育期暴露促进淀粉样蛋白前体App mRNA的表达上调,Aβ1-42表达增加,早老素Ps-1表达下调,引起App剪切过程异常和Aβ聚集。另外,PFOS血清浓度较低的出生前PFOS暴露组和出生后暴露组及出生前后均暴露组对Tau磷酸化和Aβ聚集水平的影响程度相当,提示胚胎期PFOS暴露具有较高的发育神经毒性。(4)比较典型全氟/多氟磺酸类化合物急性侧脑室注射对在体LTP的影响,研究PFOS替代物的潜在神经毒性效应和机制。结果表明,PFOS替代物均对LTP产生抑制作用,并随暴露浓度升高抑制作用增强。高频刺激后60 min,对照组fEPSP幅值仍维持在基线的140%以上,而100 μM PFOS、全氟己烷磺酸(PFHxS)和氯代多氟醚基磺酸(Cl-PFAES)的fEPSP幅值降低至基线的97%和98%,显著低于对照组。全氟丁烷磺酸钾盐(PFBS)对LTP的抑制作用较弱,fEPSP幅值维持在基线的122%,与对照组无显著差异。PFHxS和Cl-PFAES对LTP的损伤程度与PFOS相近,提示PFHxS和Cl-PFAES具有与PFOS相当的损伤神经突触可塑性的潜能。Cl-PFAES对基础波fEPSP有显著的抑制作用,提示Cl-PFAES可能具有与全氟磺酸类化合物不同的神经毒性作用机制。因此有必要进一步研究PFOS替代物的发育神经毒性。发育期PFOS暴露抑制LTP的诱导和维持,AMPA受体调控是其重要机制。PFOS通过抑制蛋白激酶CaMKⅡ-α对GluR1-s831、GluR2-s880的磷酸化,改变AMPA受体亚基GluR1、GluR2在细胞膜上的分布,引起AMPA受体内化,增强AMPA受体对钙离子的通透性。同时PFOS发育期暴露引起Tau磷酸化水平上调及Aβ聚集也提示PFOS发育期暴露与神经退行性疾病的可能关联。初步发现PFOS替代物的神经毒性潜能,提示有必要进一步研究其发育神经毒性效应和机制。研究结果为全氟/多氟化合物的人体健康风险评价提供科学依据。更多还原

【Abstract】 Perfluorooctane sulfonate (PFOS), a typical perfluorinated sulfonate compound, has been extensively used in a variety of consumer applications and industrial processes due to its advantageous surfactant capabilities and stability. It has been demonstrated that PFOS could pass through the blood-brain barrier, affect the development of the nervous system, and inhibit the formation of learning and memory abilities in rat, while the mechanisms remain unclear. In 2009, PFOS was listed in Stockholm Convention and was prohibited for production and application in many countries, while it is still permitted to use for limited purposes including firefighting foams and pesticides which disbursed PFOS directly into the environment. Furthermore, the replacements of PFOS by alternatives are undergoing a fast development. The environmental pollution level of poly- and perfluorinated sulfonate compounds with shorter carbon chain length or with N/O atoms insertion in the carbon chain increased, with extremely limited toxicological research and unknown environment risk. To study the neurotoxic effects and mechanism of the typical poly- and perfluorinated sulfonate compounds is helpful to clarify the association of developmental abnormalities and neurodegenerative diseases with these pollutants.In this study, cross-fostered animal model was employed for evaluating the effects of developmental PFOS exposure on long-term potentiation (LTP). The primary hippocampal neurons were also used to elucidate the potential mechanism of PFOS-induced LTP impairment associated with a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors trafficking. Furthermore, the changes of Tau and Aβ expression were used to evaluate the potential relationship between developmental PFOS exposure and neurodegenerative diseases. Lastly, the neurotoxicity and the potential mechanism of typical poly- and perfluorinated sulfonate compounds were compared by LTP in vivo via acute intracerebroventricular injection. The main contents include,(1) Effects of PFOS on LTP in the CA1 area of rat in vivo was evaluated using electrophysiological techniques in cross-fostered animal model, to elucidate the mechanisms of learning and memory injury caused by developmental PFOS exposure. The results showed that developmental PFOS exposure induced dose-dependent suppression of LTP, which was consistent with the previous results that developmental PFOS exposure impaired the spontaneous behavior and the learning and memory abilities of rats. The results provided the electrophysiological evidence for the developmental neurotoxicity of PFOS. Field-excitatory postsynaptic potential (fEPSP) in rats from both 5 mg/L and 15 mg/L PFOS exposed groups were lowered 20-27% than those from control group at 60 min after high-frequency stimulation (HFS). Input/output curve (I/O), which reflects the basal synaptic transmission competency, showed that the fEPSP amplitudes at 0.3-0.5 mA in exposed groups were significantly lower than that in control groups. And the paired-pulse facilitation (PPF) in TT15 group at the peak point was significantly lower than that in control group. These results suggested that developmental PFOS exposure affected the synaptic transmission and plasticity both of pre- and post-synaptic cells, which is a critical mechanism of its impairment on learning and memory abilities.(2) Combining experiments in vivo and in vitro, the role of AMPA receptors regulation in PFOS-induced LTP impairment was elucidated. Developmental PFOS exposure decreased the mRNA and protein expression of GluRl and GluR2, along with the decrease of phosphorylated protein GluR1-s831 and protein kinase CaMKII-α expression. The expression of GluR1 and GluR2 were also decreased when the primary hippocampal neurons of newborn rat were exposed with 2 μM and 20 μM PFOS, consistent with the results in vivo. The mRNA expression of glutamate receptor interacting protein (GRIP1) was decreased, consistent with the tendency of GluR2, and the mRNA level of RNA editing enzyme ADAR2 was increased twice than control group. NBQX, the AMPA receptor antagonist, inhibited the changes of PFOS on cellular calcium homeostasis and the related genes expression. The results indicated that PFOS affected the dynamic distribution of AMPA receptor by changing the expression of AMPA receptors subunits, enhanced the permeability of calcium ion and impaired the synaptic plasticity.(3) The potential relationship between developmental PFOS exposure and neurodegenerative diseases was clarified by detecting the effects of developmental PFOS exposure on Tau phosphorylation and Aβ accumulation. The results showed that protein and mRNA levels of Tau were elevated by developmental PFOS exposure. Tau phosphorylation at S199, T231 and S396 sites and the amount of protein kinase GSK-3β were also increased. Besides, developmental PFOS exposure promoted the expression of amyloid protein precursor (APP), increased the expression level of Aβ1-42, decreased the expression of presenilin (Ps-1), inducing the abnormity of App regulation and Aβ accumulation. And the prenatal PFOS exposure caused the alterations of involved proteins at comparable levels with the postnatal and both prenatal and postnatal exposure, although the serum PFOS levels in the prenatal exposure group were relatively lower. Thus, relatively high risk of PFOS exposure in the embryonic stage was identified.(4) The potential neurotoxicity and mechanism of PFOS alternatives were evaluated by comparing the effects of typical poly- and perfluorinated sulfonate compounds on LTP induction via acute intracerebroventricular injection. The results showed that PFOS alternatives suppressed LTP with dose-dependent manner. The amplitude of fEPSP in rats from control group kept above 140% of baseline at 60 min after HFS, while the fEPSP amplitude of 100 μM PFHxS and Cl-PFAES groups decreased to 97% and 98% of baseline. The amplitude of fEPSP maintained at 122% of baseline in PFBS-treated rats, with no significant difference from control group. PFHxS and Cl-PFAES posed similar potency as PFOS in impairing LTP, comparable neurotoxic potency of PFHxS and Cl-PFAES to impair synaptic plasticity. Meanwhile, Cl-PFAES significantly inhibited the fEPSP amplitude of baseline, indicating that CI-PFAES might act in a different neurotoxic mechanism from perfluorinated sulfonate compounds. Further study on the developmental neurotoxicity of PFOS alternatives is warranted.Developmental PFOS exposure inhibited the induction and maintaining of LTP, and AMPA receptor regulation was an important mechanism. PFOS affected the dynamic distribution of AMPA receptor GluRl and GluR2 in the membrane by changing the phosphorylation GluRl-s831 and GluR2-s880 by protein kinase CaMKⅡ-α, leading to the internalization of AMPA receptor and the increase of the intracellular calcium levels. Meanwhile, developmental PFOS exposure elevated the level of phosphorylated Tau and Aβ aggregation, which hinted the possible link between early PFOS exposure and neurodegenerative diseases. The present study preliminarily revealed the neurotoxicity potency of PFOS alternatives, suggested the necessity to further evaluate their neurotoxic effects and mechanisms. The results provided scientific basis for human health risk assessment of poly- and perfluorinated compounds.更多还原