有机磷阻燃剂TDCPP和TPP对斑马鱼的生殖发育毒性及其机制研究

Tris (1,3-dichloropropyl) phosphate (TDCPP) and triphenyl phosphate (TPP) are wildly used organophosphate flame retardantds(OPFRs) that might cause adverse effects to environment and human beings. We already reported that TDCPP and TPP showed anti-estrogenic activities, they could influence the synthesis and metabolite of steroid hormones, and thus imapir the reproductive abilities of zebrafish. Recently, we found that TDCPP and TPP could influence thyroid hormone levels in zebrafish, and the expression of genes along hypothalamus-pituitary-thyroid showed negative feedback mechanisms. So far, the study on their endocrine disrupting effects-reproductive and developmental toxicity are limited, and their endocrine disrupting mechnisms are unknown. our project will conduct zebrafish whole life-cycle exposure experiment to investigate their reproductive and developmental toxicity. At mean time, we will investigate their effects on endocrine hormone levels, and expression of genes along hypothalamus-pituitary-gonad (HPG)/thyroid (HPT) and adrenal (HPA) axes. In addition, we will conduct ex vivo brain exposure test and combine the related gene expression changes with previous data, analyze the cross-talk among HPG, HPT and HPA axes. These data would help to find the no observed effect value frome reproductive and developmental endpoints, and identify their endocrine disrupting mechanisms. In all, our project will provide theoretical foundation for their environment risk assessment and toxicological mechanisms, and also provide new idea and method for endocrine dirupting mechanisms studies.

有机磷阻燃剂TDCPP和TPP的使用广泛，对环境和人体的危害不容忽视。我们已发表文章报道: TDCPP和TPP具有抗雌激素受体活性，可影响类固醇激素的合成和代谢，损害斑马鱼生殖能力。近期预实验我们发现: TDCPP和TPP影响斑马鱼甲状腺激素水平，下丘脑-垂体-甲状腺（HPT）轴基因表达显示负调控机制。目前，TDCPP和TPP的内分泌干扰效应-生殖发育毒性研究缺乏，内分泌干扰机制尚不清楚。本项目拟采用斑马鱼整个生命周期暴露实验，①观察斑马鱼生殖发育毒性；②检测内分泌激素水平下和下丘脑-垂体-性腺（HPG）、HPT及肾上腺（HPA）轴上相关基因表达的变化；③结合大脑体外暴露后上游基因表达的变化，分析HPG, HPT和HPA之间的交互作用。明确生殖发育的NOEC值和内分泌干扰机制。本项将为TDCPP和TPP的生态风险评估和毒理机制提供实验依据，也为内分泌干扰机制的研究提供新的思路和方法。

随着溴代阻燃剂PBDEs的禁用，有机磷阻燃剂（OPFRs）的使用量增加，其对环境及人体的危害成为研究的热点。前期试验显示OPFRs具有不同程度的内分泌干扰潜能，但至今OPFRs的内分泌干扰效应-生殖发育毒性研究缺乏，干扰机制不明。本项目采用U.SEPA两阶段筛查法，以斑马鱼为模式动物，利用H295R和MVLN体外细胞试验，筛查了环境中检出率高的六个OPFRs(TDCPP, TCPP, TCEP, TPP, TBEP和TCP)，发现六种OPFRs均干扰内固醇激素的合成 与代谢，且TDCPP, TCP和TPP具有雌激素受体拮抗活性。整体动物斑马鱼短期暴露试验（21天生殖毒性测试和14天成鱼暴露试验）结果显示TDCPP或TPP可干扰下丘脑-垂体-性腺轴上调控内固醇激素合成的基因表达，打破内固醇激素水平影响斑马鱼生殖能力，同时也可通过下丘脑-垂体-甲状腺轴，干扰甲状腺激素水平且具干扰有性别依赖性。对内分泌干扰作用强且使用量高的TDCPP和TPP实施了第二阶段斑马鱼生命周期暴露试验。TPP或TDCPP对斑马鱼受精卵到成鱼的终身暴露试验显示，TPP或TDCPP可通过下丘脑-垂体-性腺（HPG）轴对斑马鱼雌雄鱼产生雌激素效应，这种化合物对HPT及HPI轴及调控的激素水平影响具有性别依赖性，提示受体与非受体途径都参与了相应的内分泌干扰效应。对长期暴露后生殖发育指标的变化，结果显示TPP可以在5µg/L暴露浓度下显著降低雌鱼性腺指数，而TDCPP在500µg/L浓度下才显现出发育毒性。本项目将为OPFRs的风险评估提供实验依据,也为干扰机制研究提供新的思路与方法。此外用H295R和MVLN细胞研究了2007年Heibei石油泄露事故发生后周围受污染的海岸底泥的内分泌干扰效应。结果显示，事故发生5年后，内分泌干扰效应依然存在，而且主要是通过干扰内固醇合成通道和内固醇激素水平，而不是通过雌激素受体活性产生内分泌干扰效应。这种干扰效应与底泥中多环芳烃的含量有关，主要由中级性及高极性的化合物产生，在自然风化过程中这种干扰效应有所降低。此部分结果表明，这个筛查体系即可筛查外源化合物干扰效应也可研究环境样本中的内分泌干扰效应。