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含磷阻燃剂的细胞线粒体损伤及 引发的神经毒性机制研究
Alternative TitleThe mechanism study of organophosphate flame retardants on cellular mitochondrial impairment and neurotoxicity involvement of mitochondria
黄超
Subtype博士
Thesis Advisor马梅 ; 王子健
2018-06
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
Keyword有机磷酸酯阻燃剂(opfrs),线粒体损伤,神经毒性,毒性作用机制, 高内涵筛选(hcs) Organophosphate Flame Retardants (Opfrs), Mitochondrial Impairment, NeuroToxicity, Toxic Mechanism, High Content Screening (Hcs)
Abstract

    有机磷酸酯阻燃剂(organophosphate flame retardants, OPFRs)作为溴代阻燃剂的主要替代品,在全球范围内生产和使用量大,且逐年递增。OPFRs已在多种环境介质中被检出,环境浓度相对较高,但当前仍缺乏足够的毒理学数据来对OPFRs的健康与生态风险进行有效评估。近年的研究发现了线粒体是众多环境污染物中一个重要的毒性作用靶点,而线粒体损伤与神经疾病等的发生密切相关,而已有的也研究显示 OPFRs所诱导的毒性效应与线粒体损伤之间具有强关联性,因此本研究关注 OPFRs的线粒体毒性,并对  OPFRs诱导的线粒体与神经毒性细胞作用机制展开研究。
    本研究分别建立了 1)多参数高内涵线粒体毒性筛选方法、2)基于高内涵筛选的细胞周期检测方法与 3)神经发育毒性评价的高内涵筛选方法。具有检测通量高,一致性强、效率高等特点,能够有效的应用于针对 OPFRs线粒体与神经毒性的研究。
    在中华仓鼠卵巢细胞(CHO-k1)中,通过多参数高内涵线粒体毒性筛选方法分析了 11种非卤代  OPFRs的线粒体损伤,发现了经短时间暴露后长链烷烃类与芳烃类 OPFRs都能造成显著的线粒体损伤,且这类亲脂性 OPFRs对线粒体具有一定的选择富集性与损伤专一性。但聚类分析的结果表明这两类化合物具有不同的细胞死亡类型。因此选择了磷酸三己酯(THP)和磷酸甲苯-二甲苯酯(CDP)
这两种 OPFRs同有机磷农药毒死蜱(CPF)展开了细胞死亡作用机制的研究,验证了 THP(烷烃类 OPFRs)和 CPF的细胞死亡途径是经典的由线粒体介导内源性凋亡途径,但 CDP(芳烃类 OPFRs)所诱导的细胞死亡方式却是 caspase非依赖的。
    OPFRs的暴露能够抑制神经突起生长,进一步研究结果表明  OPFRs神经毒性的产生是多机制共同作用的。虽然仅芳烃类 OPFRs对   AChE活性具有弱的抑制能力,但是亲脂性 OPFRs的线粒体损伤效应则显著强于CPF,同时由OPFRs诱导的 ROS被发现是线粒体来源的并能造成氧化应激。在此研究基础上初步阐明了由OPFRs引起的线粒体损伤导致神经毒性的发生的关键途径。一方面OPFRs暴露能通过影响线粒体功能而抑制神经发育过程,另一方面OPFRs的线粒体损伤能激活 CAMKII,进而引起的钙紊乱与氧化应激造成进一步的神经毒性。

Other Abstract

    Organophosphate flame  retardants (OPFRs) are  increasingly in  their production and consumption worldwide as replacements for  brominated flame retardants (BFRs).High concentrations of OPFRs  have been detected in many environmental  samples in recent decades.  but insufficient available  toxicological information on  OPFRs makes
assessing their  health risks challenging.  Recent study has  revealed that mitochondria are   important   targets   of   various   environmental   pollutants,   and   mitochondrial dysfunction may lead to many common diseases, like nerve diseases. On the other hand,OPFRs induced  toxic effects  seemed to be  related with  mitochondrial damage. As  a result,  mitochondria  impairment-related endpoints  for  OPFRs  were  focused  in the present study.  And the toxic  mechanism  of mitochondrial toxicity  and neurotoxicity induced by OPFRs were further investigated.
      Three bioassays based on high content screening were developed, including: 1) A multi-parameter high-content analysis for mitochondrial toxicity. 2)An algorithm based on  Watson  model  for   fitting  cell  cycle  data  from   high  content  screening.  3)  A quantitative  evaluating developmental  neurotoxicity  method  by  using  high content
screening.
    Mitochondrial impairment of 11  selected non-halogen OPFRs were measured by the high content screening assay in Chinese hamster ovary (CHO-k1) cells. Long-chain alkyl-OPFRs  and all  aryl-OPFRs  were found  to  significantly  induce mitochondrial toxicity, and they would selectively accumulate  and impair mitochondria according to the analysis of QSAR model and Tox21 data. A cluster analysis was used to categorize these OPFRs  into  three groups  according to  their results  from the  HCS  assay. Two groups, containing long-chain  alkyl-OPFRs and all  aryl-                   OPFRs, were found to  cause mitochondrial impairment but showed different mechanisms of toxicity. Due to the high correlation  between   cell  death  and   mitochondrial  impairment,  two   OPFRs  with different structures,  trihexyl phosphate (THP)  and cresyl  diphenyl phosphate (CDP),were selected and  compared with chlorpyrifos (CPF) to  elucidate their mechanism of inducing cell  death. THP (an alkyl-OPFR)  was found  to utilize a  similar pathway as  CPF to  induce apoptosis.  However, cell death  induced by  CDP (an aryl-OPFR)  was caspase independent. OPFRs  could inhibit  neurite  outgrowth,  and our  study  further  found  multiple mechanisms  were involved  in  the  OPFRs induced  neurotoxicity.  Only aryl-OPFRs could  inhibit   acetylcholinesterase  (AChE)   at  high   concentrations.  However,   the mitochondria impairment caused  by OPFRs were more potency than  CPF. Meanwile,the  ROS   induced  by  OPFRs  were   observed  in  this   study  to  be   initiated  from mitochondria and cause oxidative stress. Two neurotoxic mechanisms were  concluded in our  research. The exposure  of OFPRs could  influence mitochondrial function  and inhihit neurogenesis.  On the  other hand, OPFRs  induced mitochondrial impairement could active CAMKII, and cause neurotoxicity through calcium overload and oxidative stress.

Pages134
Language中文
Document Type学位论文
Identifierhttp://ir.rcees.ac.cn/handle/311016/41461
Collection环境水质学国家重点实验室
Recommended Citation
GB/T 7714
黄超. 含磷阻燃剂的细胞线粒体损伤及 引发的神经毒性机制研究[D]. 北京. 中国科学院生态环境研究中心,2018.
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