【作者】 李静；

【导师】 刘春生；

【作者基本信息】 华中农业大学 ， 水生生物学， 2018， 博士

【摘要】 磷酸三(1,3-二氯异丙基)酯(tris(1,3-dichloro-2-propyl)phosphate,TDCIPP)是一种典型的有机磷酸酯类阻燃剂,因其优良的阻燃性能,以及全球范围内对溴代阻燃剂(如多溴联苯醚)的逐渐禁用,使得TDCIPP作为良好的替代品近年来被大量生产并广泛使用。由于TDCIPP是以物理添加的形式被应用在产品中的,因此极易释放进入环境中。环境监测表明,TDCIPP在空气、自然水体、土壤和野生生物的组织中具有很高的检出率,对野生生物和人类的生存和繁衍可能构成潜在的威胁。已有的毒理学研究表明,TDCIPP能够引起脊椎动物多种毒性效应,然而对低等生物的影响及多代毒性效应仍是未知,故本研究选取营自由生活的低等水生原生动物嗜热四膜虫(Tetrahymena thermophila)为实验生物,通过较高浓度TDCIPP(0.01,0.1和1μmol/L)亚慢性暴露(5 d)和环境相关浓度(300和3000 ng/L)多代暴露60 d(约372代)及恢复实验,以表型观测、转录组测序分析、实时定量PCR检测和透射电子显微镜观察为研究手段,对TDCIPP的毒性效应及分子机制进行了评价和探讨。主要结果如下:(1)采用高浓度(1,10,100,1000和10000μmol/L)急性暴露(8 h),研究了TDCIPP对嗜热四膜虫生物量的剂量-依赖性抑制效应,EC50为615.1μmol/L。0.01,0.1和1μmol/L TDCIPP亚慢性暴露,通过引起细胞密度、细胞大小和纤毛数量剂量-依赖性降低,从而导致生物量显著性降低,表明抑制个体生长和繁殖是TDCIPP的主要毒性效应。RNA-Seq检测及KEGG富集分析,发现差异表达基因显著性富集于核糖体通路,TDCIPP诱导核糖体大、小亚基上的12个核糖体蛋白基因呈现剂量-依赖性的下调表达。此外,伴随着核糖体蛋白基因的下调表达,细胞质和粗面内质网上的核糖体数量减少,细胞质中核糖体增大。本实验结果表明,TDCIPP可能通过靶向作用于核糖体影响嗜热四膜虫的生长和繁殖。(2)多代毒性效应评价实验中,使用环境相关剂量TDCIPP对嗜热四膜虫开展连续约372代(60 d)的暴露实验,随后进行约372代的恢复实验。结果发现,TDCIPP暴露引起嗜热四膜虫种群数量、个体大小和纤毛数量降低;同时,TDCIPP引起纤毛的基体结构发生改变,基体深度和直径显著性降低;此外,TDCIPP诱导了与纤毛组装和维持相关的基因(ift家族的3个基因:ift52,ift81和ift172,tcp-1家族的7个基因:tcp-1-3700.m00089,tcp-1-3706.m00106,tcp-1-3715.m00106,tcp-1-3731.m00045,tcp-1-3698.m00091,tcp-1-2.m02183和tcp-1-16.m00478)表达上调。在60 d的恢复实验中,种群数量恢复至正常水平,然而,TDCIPP对嗜热四膜虫个体大小、纤毛数量、基体结构及相关基因的影响中部分效应有恢复迹象,大多效应依旧显著性存在。因此,本研究首次表明环境相关剂量TDCIPP对嗜热四膜虫多代暴露显示出发育和繁殖毒性,且部分效应在实验时间内不可恢复。(3)为进一步阐明TDCIPP引起多代毒性效应的作用机制,开展环境相关剂量(300和3000 ng/L)TDCIPP对嗜热四膜虫连续约372代的暴露及后续约248代的恢复实验。在此次实验中,TDCIPP对嗜热四膜虫表型的抑制效应与之前的多代暴露结果一致,恢复效应也相同。另外,此次实验还发现,TDCIPP引起细胞粗面内质网池扩张,其上附着的核糖体排列不规则,细胞内这种超微结构变化在恢复实验阶段依旧存在。通过RNA-Seq及KEGG富集分析发现,在暴露和恢复时期,下调表达的差异基因均显著性富集于核糖体通路,TDCIPP诱导核糖体大、小亚基上的核糖体蛋白基因普遍呈现剂量-依赖性下调表达,说明TDCIPP抑制了核糖体功能。此外,在恢复时期,上调表达的差异基因显著性富集于核糖体生物合成通路,推测这可能是嗜热四膜虫对TDCIPP抑制其核糖体功能所表现出的积极的恢复响应。更多还原

【Abstract】 Tris(1,3-dichloro-2-propyl)phosphate(TDCIPP)is a typical organophosphate ester flame retardant.Due to its excellent flame-retardant properties,as well as the phase-out of brominated flame retardants(e.g.,polybrominated diphenyl ethers),the production and usage of TDCIPP has increased in recent years.Because the physical addition of TDCIPP is applied to the associated products,it is easily released into the environment.Environmental monitoring has shown that TDCIPP has a high detection rate in air,natural water,soil,and wildlife organizations,threatening the survival and reproduction of wildlife and humans.Toxicological studies have shown that TDCIPP can cause a variety of toxic effects in vertebrates,but the effects on lower organisms and multi-generation toxic effects remains unknown.In the present study,the free-living ciliate protozoa Tetrahymena thermophila,was used as a model.Tetrahymena thermophila were exposed to TDCIPP at high concentrations(0.01,0.1 and 1 μmol/L)for sub-chronic(5 d),and at environmentally-relevant concentrations of TDCIPP(0,300 or 3000 ng/L)for 60 d(e.g.,theoretically 372 generations)treatment followed by a period of recovery,during which T.thermophila were not exposed to TDCIPP,and phenotypic observation,RNA-Seq,RT-q PCR and transmission electron microscopy imaging technologies were used to evaluate the toxic effects of TDCIPP and explore the molecualr mechanisms of its action.The main results were as follows:(1)Acute exposure(8 h)at high concentrations(1,10,100,1000,and 10000 μmol/L)was used to study the dose-dependent inhibition of TCDCPP on T.thermophila biomass,and the speculated EC50 was found to be 615.1 μmol/L.Subchronic exposure to 0.01,0.1 or 1 μmol/L of TDCIPP caused a significantly decreased the relative biomass by reducing number of cells,size of cells and quantity of cilia in a dose-dependent manner,indicating that inhibition of individual growth and proliferation was the major toxicity of TDCIPP.RNA-Seq analysis found that expression of twenty-one ribosome protein genes of ribosome large and small subunits was down-regulated and these genes were enriched in “ribosome” term in KEGG pathway analysis.Furthermore,down-regulation of these genes was accompanied by decreased ribosome quantity in rough endoplasmic reticulum and cytoplasm and enlarged ribosome size.The results of this experiment indicated that TDCIPP might affect the growth and reproduction of T.thermophila by targeting the ribosome.(2)In a multi-generation toxicity effect evaluation experiment,T.thermophila were exposed environmentally-relevant concentrations of TDCIPP(0,300 or 3000 ng/L)for about 372 generations(60-day),followed by about 372 generations of recovery.The results showed that TDCIPP caused a significantly decreased numbers of populations,sizes of individuals,number of cilia,and depth and diameter of basal bodies of cilia,and up-regulated expressions of genes(ift family of 3 genes: ift52,ift81 and ift172,and tcp-1 family of 7 genes: tcp-1-3700.m00089,tcp-1-3706.m00106,tcp-1-3715.m00106,tcp-1-3731.m00045,tcp-1-3698.m00091,tcp-1-2.m02183 and tcp-1-16.m00478)related to assembly and maintenance of cilia.In the 60-day recovery experiment,the population growth returned to the normal levels,however,the effects on individual growth,cilia number,basal bodies of cilia structure and related genes were partially restored,and most of the effects were still significant.Therefore,this study for the first time indicated that multi-generation of environmentally relevant doses of TDCIPP exposure could cause developmental and reproductive toxicity of T.thermophila,and some of the effects were not recoverable within the experimental time.(3)In order to further elucidate the molecular mechanisms of TDCIPP-causing multigenerational effects,exposure of TDCIPP to 300 and 3000 ng/L in T.thermophila for about 372 generations,and subsequent recovery of about 248 generations were carried out.The inhibitory effects of TDCIPP on the phenotype of T.thermophila were consistent with previous multigenerational exposure results.In addition,this study also found that TDCIPP caused cisternae of the rough endoplasmic reticulum were extended and resulted in loosely interconnected curvilinear profiles,and the intracellular ultrastructural changes in the recovery phase still existed.By RNA-Seq and KEGG enrichment analysis,it was found that the down-regulated differently expressed genes(DGEs)were significantly enriched in “ribosome” term.During exposure and recovery,ribosome protein genes of large and small subunits induced by TDCIPP were generally down-regulated in a dose-dependent manner,indicating that TDCIPP inhibited ribosome function.Additionally,the up-regulated DGEs were significantly enriched in “ribosome biogenesis” term,during the recovery period.It was speculated that this might be a positive recovery response of T.thermophila to the TDCIPP inhibited ribosome function.更多还原