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含磷阻燃剂和新型表面活性剂的离体毒性及毒性机制研究
Alternative TitleIn vitro study of toxic effects and action mechanisms of phosphorus containing flame retardants and a novel non ionic surfactant
袁圣武
Subtype博士
Thesis Advisor马梅
2019-06
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含磷阻燃剂 ,新型非离子表面活性剂 氧化损伤, Dna 损伤, 细胞周 期 阻滞 phosphorus Containing Flame Retardants, Novel Non Ionic Surfactant, Oxidative Stress, Dna Damage, Cell Cycle Arrest
Abstract

       随着多溴联苯醚等溴代阻燃剂的禁用,作为主要替代品的含磷阻燃剂(phosphorus containing flame retardants, PFRs) 在全球范围内被广泛的生产和使用,且呈现逐年递增趋势。 PFRs 在多种环境样品中被大量检出,环境浓度相对较高成为一种新型的环境污染物。大量的研究表明, PFRs 能够诱导包括神经毒性、内分泌干扰效应、生殖发育毒性甚至致癌性等一系列生物体有害损伤效应。但目前针对 PFRs 的毒性研究仍相对有限,相关的 毒性 机制 也 尚不 清楚 ,缺乏足够的毒理学数据来支撑生态环境健康风险的有效评估。烷基酚聚氧乙烯醚alkylphenol polyethoxylates APEO s 因其巨大的生物毒性受到禁用和限用后,大量的 新型 表面活性剂被陆续 开发 用来替代 APEO s ,香兰素基聚氧乙烯醚 v anillin ethoxylates VAEOs) 是一种新合成的新型非离子表面活性剂,具备良好的表面活性剂特征,但缺乏 足够的 毒理学数据验证其生态安全性。因此,本研究以 PFRs和 VAEOs 为目标化合物,基于高通量、高内涵及分子生物学等一系列离体毒性测试手段, 对两类替代化学品进行 一系列 毒性效应分析。采取结构相似性分类的研究方法 对目标 化合物的氧化损伤、线粒体损伤和 DNA 损伤等毒性效应 进行 分析, 以 结构 相似 性 对化合物 毒性效应进行归类,并在基因转录水平对 p53 介导的细胞周期和凋亡相关的毒性机制进行探索。得到主要结论如下:
       (1) 通过离体毒性分析,芳基取代基 PFRs 能够 诱导显著的线粒体损伤和DNA 损伤。此外,芳基取代基 PFRs 能够诱导细胞周期 G1 期阻滞效应, 磷酸二苯甲酯 (m ethyl diphenyl phosphate MDPP )), 磷酸甲苯 二苯酯 (c resyl diphenyl phosphate , CDP), 2 乙基己基二苯基磷酸酯 2 ethylhexyldiphenyl phosphate ,EHDPP 和 磷酸异葵基二苯酯 (i sodecyl diphenyl phosphate IDPP 引起细胞周期 S期细胞数 分布相比于 D MSO 对照组明显下降 ,最大下降比 例分别为 94.9%, 89.6%,80.7% 和 73.5% 。结构性归类分析发现含有短链烷烃的芳基取代基 PFRs (MDPP,CDP, 双酚 A 双 二苯基磷酸酯 BDP 能够在低浓度下显著诱导细胞核内活性氧物质 ( 和线粒体内超氧化物的产生,并通过氧化应激途径引发 DNA 损伤。此外,芳基取代基 PFRs 均能够激活 p53 相关的信号转导途径,上调 p53/p21 介导的细胞周期相关转录因子,在转录水平上证实芳基取代基 PFRs 能够造成细胞周期阻滞效应和 DNA 损伤。
       (2) 含氯和含氧 取代基 PFRs 能够诱导细胞内 RO S 过度产生,引起氧化损伤 并通过氧化应激 方式 引起线粒体损伤,表现为线粒体质量的显著上升和线粒体膜电位的显著下降。同时, 磷酸三 丁氧基乙基 酯 磷酸三 (1,3 二氯异丙基酯 和 磷酸三 (1,3 二氯 丙基 酯 暴露后, A549 细胞被阻滞在细胞周期 G1 期,导致S 期细胞数 比例 下降 ,而 其中含最多氯原子数 的 磷酸三 (1,3 二氯异丙基 酯 诱导的线粒体损伤和 DNA 损伤最为显著。 在分子水平上, 含氯和含氧 取代基 PFRs 通过上调 细胞周期相关的 p53/p21/gadd45β 和 p53/p21/mdm2 信号 通路 的 关键转录因子 的表达水平来调 控这类化合物诱导 的 DNA 损伤效应。此外, 磷酸三 (1,3 二氯异丙基 酯 和 磷酸三 (1,3 二氯 丙基 酯 通过引起细胞 sub G1 凋亡峰的增加, 在基因水平上 导致凋亡通路 bax 转录因子 的 表达失调,体现出其诱导凋亡的可能性。
       (3) 烷基取代基 PFRs 对 A549 细胞均显示明显的细胞毒性,且与浓度相关。长链烷基取代基 PFRs 的细胞毒性更强,与其较高的 LogKow 值相关。研究显示,长链烷基取代基 PFRs 是通过氧化应激途径 引起 线粒体损伤,并诱导后续的 DNA损伤和细胞周期阻滞效应;而短链的烷基取代基 PFRs 化合物未发现明显的 ROS上升现象,推测其 是 通过线粒体介导的毒性机制造成细胞周期阻滞效应。 磷酸三异丁酯 通过上调 凋亡通路 bax 转录因子 的表达水平 ,同时诱导sub G1 凋亡峰增加 4.54%4.54%,在分子和细胞水平上体现出其诱导细胞凋亡的潜在性。
      (4) 新型非离子型表面活性剂的离体毒性分析显示 VAEOs 和 壬基酚聚氧乙烯醚 (nonylphenol ethoxylates NPEOs) 的 LC50 分别为 104.7 μg/ mL 和 28.7 μg/ mL 。VAEO s 及其代谢产物香兰素对细胞的氧化损伤、线粒体损伤和 Olive 尾距值均明显低于 NPEOs 和 壬基 酚 4 NP 。此外,除 4 NP 显示潜在的直接遗传毒性外,其他化合物均 未检测到阳性 遗传毒性 结果 。因此,我们从离体细胞毒理学的角度认可了 VAEOs 是替代 APEOs 的潜在新型表面活性剂,但需要更多的毒理学手段的验证和支持。

Other Abstract

      Phosphorus containing flame retardants (PFRs) are one of the widely used substitues after the p rohibition of brominated flame retardants (e.g. polybrominated diphenyl ethers, PBDEs). PFRs have been frequently detected in various environmental samples with relative high concentrations, and have been considered as emerging environmental pollutants. Se veral toxicological studies have shown that PFRs are associated with adverse effects, such as, neurotoxicity, endocrine disrupting effects, reproductive effects and carcinogenicity to human health and ecosystem. But still insufficient toxicological informa tion was available to assess their environmental risks with unclear toxic mode of action. Vanillin ethoxylates (VAEOs) are novel non ionic surfactants and are used to replace alkylphenol polyethoxylates (APEOs),which are prohibited due to their severe bio logical toxicity. But absent toxicological evidence was available to suport their possibility, although VAEOs shown favourable characteristics as surfactants. Therefore, in this study, PFRs and VAEOs were selected as exposure compounds, a battary of in vit ro bioassay were conducted to analyze their oxidative stress, mitochondrial impairment, DNA damage and the involved molecular mechanisms. T he major results are as follows:
       (1) The selected aryl PFRs shown significant mitochondrial and DNA damages,also G2 phase cell cycle arrest effects along with S phase cell ratio reducing 94.9%,89.6%, 80.7% and 73.5% of methyl diphenyl phosphate (MDPP), cresyl diphenyl phosphate (CDP) CDP), 2 ethylhexyldiphenyl phosphate (EHDPP), isodecyl diphenyl phosphate (IDPP) were evide nced, respectively. Moreover, bisphenol A bis (diphenyl phosphate) (BDP), MDPP, CDP containing short alkyl chains showed their potential oxidative stress with intracellular reactive oxygen species (ROS) and mitochondrial superoxide overproduction from a re lative low concentration, suggested that BDP,MDPP, CDP caused oxidative stress mediated DNA damage and mitochondrial impairment. Further real time PCR results showed that all selected aryl PFRs trigger p53/p21 mediated cell cycle pathway, evidenced their cell cycle arrest effects at the transcriptional level.
      (2) Chlorine containing and oxygen containing PFRs induced significantly oxidative damage in A549 cells, and shown their mitochondrial impairments by the increasing mitochondrial mass and decreasing m itochondrial membrane potential in oxidative stress pathway. Moreover, A549 cells were arrested in the G1 phase and decreased in the S phase after exposure to t ris (2 butoxyethyl) phosphate (t ris (1,3 dichloro 2 propyl) phosphate TDCPP ) and t ris ( 2 chloroisopropyl)phosphate ( TCPP). Especially TDCPP containing most chlorine atoms, caused the most significantly mitochondrial and DNA damage. From the perspective of gene transcription, chlorine containing and oxygen containing PFRs shown their cell c ycle arrest induced DNA damage by up regulating the key transcription factors of the
p53/p21/gadd45β and p53/p21/mdm2 mediated cell cycle pathways. In addition,TDCPP and TCPP indicated the possibility of cell apoptosis by causing an increase in the sub G1 apoptotic peak and dysregulating the expression of bax transcriptionfactors.
       (3) A lkyl PFRs indicated structurally related cytotoxicity with high toxicity on long chain alkyl PFRs correlated with their higher LogKow values. Long chain alkyl PFRs caused their mitochondrial and DNA damage through oxidative stress pathway,while short chai n alkyl PFRs were speculated to indicate their cell cycle arrest effects through mitochondria mediated pathway. Triisobutyl phosphate increased sub G1 apoptotic peak with 4.54% compared to control and up regulated bax gene expression,shown its potential c ell apoptosis at the molecular and cellular levels.
       (4) Compare to nonylphenol ethoxylates NPEOs and nonylphenol 4 NP )),VAEOs and their metabolite vanillin shown relative lower cytotoxicity (higher LC50 value), oxidative damage, mitochondrial damage and genotoxicity using a battary of in vitro bioassay. Thus, VAEOs have the potential to substitute A PEOs as suitable surfactants from in vitro toxic perspective, but further studies are required to evaluate the potential adverse effects on populations, commu nities, or ecosystems.

Pages134
Language中文
Document Type学位论文
Identifierhttp://ir.rcees.ac.cn/handle/311016/42342
Collection环境水质学国家重点实验室
Recommended Citation
GB/T 7714
袁圣武. 含磷阻燃剂和新型表面活性剂的离体毒性及毒性机制研究[D]. 北京. 中国科学院生态环境研究中心,2019.
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