【作者】 王法琦；

【导师】 金一和；

【作者基本信息】 大连理工大学 ， 环境科学， 2012， 博士

【摘要】 全氟辛烷磺酸(perfluorooctane sulfonate, PFOS),是2009年被列入POPs黑名单的持久性有机污染物,其发育神经毒性已被国内外学者广泛研究,但PFOS神经毒性的分子机制尚不全面,本项目的主要目的是从分子水平揭示PFOS引起发育神经毒性的机制、与神经内分泌系统的相互关系以及预测PFOS潜在干扰的神经功能损伤。主要内容包括：(1)利用基因芯片及RT-PCR技术对发育期大鼠进行体内染毒,建立交叉繁殖模型,观察PFOS胚胎期及泌乳期暴露对发育期大鼠脑基因表达谱的影响,找出对PFOS刺激的敏感发育期,从分子水平揭示PFOS的发育神经毒性。结果显示,PFOS出生前及出生后暴露造成大鼠脑组织基因表达的广泛改变。PFOS暴露后仔鼠在出生后第1、7天分别有842和634个基因显著性差异表达(P<0.05),而出生后35天仅有35个基因显著变化(P<0.05)。受PFOS潜在影响的神经功能和生物过程包括,中枢神经系统发育、神经发生、长时程增强或抑制(LTP/LTD)、学习与记忆、神经递质传递和突触可塑性等(P0.05)。结果提示PFOS的胚胎期暴露对幼鼠神经发育的潜在毒性效应强于泌乳期暴露；PFOS对发育过程中神经系统的影响可能通过干扰神经内分泌系统作为第一靶标既而影响整个神经系统的功能改变。(2)采用miRNA芯片技术从分子水平找出PFOS引发脑基因差异表达的可能因素,确定PFOS敏感性微小RNA及其靶标功能蛋白的表达变化,并与靶基因的变化情况联合分析,进一步揭示PFOS引发神经毒性的分子机制。结果显示,在出生1、7天分别有24、17个1niRNA显著性差异表达(P<0.05),其中5种差异表达高达5倍之多的miRNA分别为miR-466b、miR-672、miR-297、miR-674-3p及miR-207,它们均参与调控了神经发育及突触传递等关键神经功能的反应过程。出生1、7天,p75、TrkC与VGLUT2三种蛋白表达显著下调,且没有上调变化的差异表达蛋白。结果提示,PFOS暴露可能影响谷氨酸代谢循环及谷氨酸兴奋突触前末梢的形成,而miR-297与miR-214和miR-1可能参与了其中的调控过程；本研究中没有观察到miRNA对其靶基因的反向调控作用,推测可能由于神经细胞的自我调控反馈回路被激活,使得靶蛋白反作用地抑制了miRNA的正常调控效应；微小RNA分子可能在神经系统发育早期参与了PFOS诱导的神经毒性效应,其中对神经突触传递路径的影响最为显著。(3)利用放射免疫、RT-PCR及Elisa等方法研究PFOS对发育期大鼠THs及THs调控基因和蛋白表达的影响,建立PFOS与BDE-47的(一种TH类似物,其代谢物是甲状腺激素受体的激动剂)联合染毒模型,探讨PFOS发育神经毒性与甲状腺激素转录调控系统的相互作用,并观察PFOS与BDE-47的可能联合毒性效应。结果显示,PFOS与BDE-47单独及联合暴露对血清THs水平及TH应答基因与蛋白表达的作用受脑区、暴露时间、染毒物质等多种因素的影响。①两种污染物在血清及脑组织中的蓄积模式显著不同；②以相同剂量暴露PFOS与BDE-47,但它们对血清TT3与TT4水平的影响显著不同；③血清THs浓度变化及TH调控基因与蛋白的表达变化具有脑区特异性以及暴露时间与发育期依赖性；④PFOS与BDE-47在影响BDNF蛋白表达过程中相互作用,出生1天脑皮质BDNF表现出PFOS与BDE-47的增强作用,出生14天海马组织BDNF表现出它们的抑制作用。结果表明,PFOS与BDE-47可能对发育期脑组织BDNF相关功能的影响产生联合毒性效应,但这种效应的机制可能并不由TH调控路径介导,而可能与其它生物调控途径有关。(4)根据发育期大鼠脑组织差异表达InRNA、miRNA的相关基因组学和路径分析结果,以及神经功能蛋白的差异表达结果,进一步对其进行比较分析,找出PFOS暴露关联最显著的神经生物路径,揭示PFOS可能导致的发育神经疾病或神经功能障碍。结果显示,PFOS极有可能对LTP的产生、发展、维持等进程中的某些环节产生毒性效应,并且PFOS对mRNA表达的影响、对]miRNA调控的干扰作用以及对LTP相关蛋白或基因表达的影响可能是导致突触传递LTP改变的诱因。LTP是脑学习、记忆的细胞基础,大鼠发育早期暴露PFOS可能对突触传递的LTP产生影响,进而干扰神经系统的突触可塑性,威胁脑的学习、记忆功能,提示胚胎期及出生早期母体避免暴露PFOS对保护后代学习记忆能力及脑健康发育至关重要。更多还原

【Abstract】 PFOS is considered as an emerging persistent organic pollutant in2009. Although the developmental neurotoxicity of PFOS exposure has been widely studied, little is known about how PFOS perturbs the developing central nervous system (CNS) at the molecular level. To investigate and compare the potential molecular effects of prenatal and neonatal exposure to PFOS in the developing nervous system, figure out the relationship of PFOS with neuroendocrine system, and predict the potentially interrupted neuro-dysfunction and nervous system impairment by PFOS exposure, we carried out this study containing that:(1) We evaluate the transcriptional effects of prenatal and neonatal exposure to PFOS in developing rat brain by performing gene expression profiling in the cerebral cortex. Six Illumina RatRef-12Expression BeadChips were used to identify gene expression changes on postnatal days (PNDs)1,7, and35. To compare prenatal and lactational exposure contributions to transcriptional effects, a subset of altered genes obtained from the gene-profile study that represented neurobiological functions was analyzed using RT-PCR in a follow-up cross-foster study from PND1to21. It was observed that prenatal and postnatal exposure to PFOS caused potential neurotoxicity as demonstrated by developmentally different global transcriptional changes.842and634genes were significantly affected by PFOS at PND1and PND7, respectively (P<0.05), whereas only13genes were significantly affected at PND35. Significantly affected genes (P<0.05) were involved in central nervous system development, neurogenesis, long-term potentiation/depression, learning and memory, neurotransmission and synaptic plasticity. These results suggest that prenatal exposure was more effective in altering expression of several genes. Also, transcriptional effects of PFOS exposure on neurodevelopment occurred primarily by disrupting the neuroendocrine system.(2) We used eight miRNA arrays to profile the expression of brain miRNAs in neonatal rats on postnatal days (PND)1and7with0mg/kg (Control) and3.2mg/kg of PFOS feed treatment, and subsequently examined six potentially altered synapse-associated proteins to evaluate presumptive PFOS-responsive functions. A complex TH-mediated gene and protein response to BDE-47and/or PFOS exposure that depends on the region, time and chemical characteristics was observed. The results revealed that1) a significant accumulation difference occurred between the two chemicals;2) On a equimolar basis, BDE-47and PFOS affected serum TT3and TT4differently in adults and offspring;3) there were region-specific and exposure-and time-dependent alterations in TH concentrations and tested gene and protein expression levels; and4) interaction for the combined chemicals was only observed for BDNF, which exhibited a synergistic effect on PND1in the cortex and an antagonistic effect on PND14in the hippocampus. Our results suggest that little combined interaction of co-exposures was observed except on BDNF. The underlying mechanisms remain uncertain but seem to involve more actions than just TH-regulated pathway.(3) To investigate whether an interaction existed between PFOS and BDE-47on TH-mediated pathways, adult female Wistar rats were exposed to3.2and32mg/kg of PFOS or BDE-47in their diet, and co-exposed to a combination of each chemical (3.2mg/kg) from gestational day (GD)1to postnatal day (PND)14. Serum and brain tissues from both male and female neonates were collected on PND1,7, and14to examine TH-regulated gene and protein expression. Twenty-four brain miRNAs on PND1and seventeen on PND7were significantly altered with PFOS exposure (P<0.05), of which, miR-466b,-672, and-297that are critical in neurodevelopment and synapse transmission showed high reduction by more than five fold. Protein levels of p75, TrkC, and VGLUT2were significantly decreased and no protein level increased with PFOS exposure during PND1to7. Our findings indicate that PFOS has the potential to effect the formation of glutamate excitatory pre-synaptic terminals, which may involve the functional regulation of miR-297,-214and-1. Negative regulation of miRNA was rarely observed in the present study, suggesting a possible fail-safe mechanism for the interaction of miRNA and its targets. These data identify the role of miRNAs in PFOS-responsive neurobiological processes, especially the synaptic pathway.(4) Based on the GO and Pathway results associated with differentially expressed mRNAs and miRNAs, and results of neuro-functional protein changes, we further explored the common associated neurological pathways by these miRNAs, mRNAs and proteins with attempt to find out the most potential neurological diseases and neuro-dysfunctions involved in PFOS exposure. It was found that PFOS has the potential of affecting the initiation, development and lasting process of LTP to make a toxic action. Moreover, the underlying mechanism of PFOS-induced potential LTP is likely related to the induction of changes in mRNA and miRNA levels, and alteration of LTP-associated gene and protein expressions. As cell LTP is important in brain functions such as learning and memory, the potential toxicity of PFOS exposure on LTP processes may translate into other neuro-disorders such as interruption of synaptic plasticity and threaten to impair the learning and memory ability, suggesting that protection of offspring from PFOS prenatal and postnatal exposure is very critical for the healthy development of brain.更多还原