蚯蚓对三种氯代有机磷阻燃剂的毒性响应机制及其差异研究

This project intends to study the responses of the earthworm species Eisenia fetida to the toxicity induced by OFRs-Cl which have different Kow thresholds both at unequally or equally effective dosages to verify the difference of responsive mechanisms. Toxic effects and effect difference concerning reproduction tissues, skin and intestinal accumulation, antioxidant enzymes, DNA in coelom fluid cells etc., as well as the cell level responses to OFRs-Cl toxicity will be distinguished intensively. At molecular level, RNA-seq sequencing technology is used to obtain the information about the differentiation in gene expressions. Differently expressed genes then will be analyzed on the KEGG pathway after GO functional annotation, in order to clarify the involved biological processes and metabolic and signaling pathways. Combined with qRT-PCR, expression amount of specific function genes are used to validate the accuracy of RNA-seq results for uncovering the mechanisms of the toxic effect and effect difference of OFRs-Cl on earthworm. The project is to provide theoretical support for decision-making that whether OFRs-C1 have to be included in China’s priority control list of environmental pollutants and for ecological risk assessment of OFRs-C1 on human health.

氯代有机磷酸酯阻燃剂OFRs-Cl (TCEP、TCPP和TDCPP) 是难降解且有神经、生殖、内分泌等毒性的新兴污染物，已引起国内外环境领域的极大关注，对蚯蚓的毒害研究却较少。本项目拟研究赤子爱胜蚓对结构相似、Kow不同的三种OFRs-Cl 的毒性响应机制及其同等效应剂量下的机制差异。研究探讨蚯蚓在生殖、表皮和肠道富集、抗氧化酶、体腔细胞DNA等组织、细胞层面对OFRs-Cl的毒性响应及其差异；分子层面上运用RNA-seq测序技术，获取差异表达基因，对差异基因进行GO功能注释和KEGG Pathway分析，解释其参与的生物学过程和代谢、信号通路等路径，并结合qRT-PCR的特定功能基因表达量及验证RNA-seq结果的准确性，探究蚯蚓的毒性响应机制及其差异。项目可为是否将OFRs-C1纳入中国环境污染物优先控制名单及OFRs-Cl可为人体健康的生态风险评估提供理论支持。

土壤中氯代有机磷阻燃剂（OPFRs-Cl）检出频繁，及新兴污染物微塑料被强烈呼吁关注，但其对蚯蚓的毒性研究却较少。本项目通过人工土壤法研究了磷酸三(1，3-二氯-2-丙基)酯(TDCPP)、磷酸三(2-氯-丙基)酯(TCPP)和磷酸三(2-氯乙基)酯(TCEP)及其与0.5mg/kg的低聚乙烯（PE）颗粒（300目）联合暴露对蚯蚓的毒性效应。结果表明，蚯蚓的平均体重随TCEP、TCPP和TDCPP暴露时间增加均先增加后降低；蚯蚓SOD、CAT活性在其低浓度诱导高浓度抑制；Ache活性被诱导增加，GST活性随TCEP和TCPP浓度增加逐渐被抑制，随TDCPP浓度增加先被诱导再被抑制。与对照相比，SOD、HSP90基因均上调，且随OPFRs-Cl浓度增加先增后减；HSP70基因随TCEP和TCPP浓度增大逐渐上调。PE胁迫下3种OPFRs-Cl在蚯蚓亚细胞水平的分布均为颗粒物＞悬浮物；PE略增强OPFRs-Cl在蚯蚓环带的富集，对其在蚯蚓头部和尾部的影响不明显；相比于TCPP和TDCPP，蚯蚓更易富集TCEP，在蚯蚓头部、环部、尾部富集浓度可达190、205、90 mg/kg。AChE在3种OPFRs-Cl低浓度、短时间暴露被诱导，高浓度、长时间暴露被抑制；GST、Na+K+-ATP在TCPP、TDCPP暴露、Ca2+-ATP在3种OPFRs-Cl暴露均被抑制；单一OPFRs-Cl及其与PE 复合暴露，SOD、CAT基本均低于CK和PE暴露； MDA在各处理间差异不大；单一和复合污染对蚯蚓体内酶活的影响各不同，无协同效应。复合污染对SOD、GST、HSP70和TCTP基因表达量与单一污染差异较显著。结论：酶活和基因指标对TCEP、TCPP和TDCPP的毒性响应各不相同；相对单一OPFRs-Cl污染，PE的介导对蚯蚓分子水平综合毒性的影响远大于组织水平，原因仍需探索。