Development of a transient expression assay for detecting environmental oestrogens in zebrafish and medaka embryos.
Development of a transient expression assay for detecting environmental oestrogens in zebrafish and medaka embryos.
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DOI：
10.1186/1472-6750-12-32
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发表时间：
2012-06-24
影响因子：
3.5
通讯作者：
Kudoh T
中科院分区：
文献类型：
--
作者：
Lee O;Tyler CR;Kudoh T
雌激素污染物广泛存在于水生环境中，并已被证明对野生动物（特别是鱼类）和人类产生不利影响，引起国际关注。现有的检测和测试系统在阐明雌激素信号通路和生理影响方面的能力有限。在这里，我们开发了一种瞬时表达试验，以调查雌激素化学品在鱼类早期生命阶段的影响，并确定雌激素效应的靶器官。为了增强对雌激素的反应敏感性，我们在Tol 2转座子介导的Gal 4ff-UAS系统中采用了多个串联的雌激素反应元件（ERec 38）。构建的质粒（pTol 2_ERE-TATA-Gal 4ff）含有三个拷贝的雌激素应答元件（3ERE），其在暴露于雌激素时诱导Gal 4ff的表达，Gal 4ff进而结合Gal 4应答性上游激活序列（UAS）元件，驱动第二报道基因EGFP（增强型绿色荧光蛋白）的表达。我们的构建体雌激素暴露在斑马鱼胚胎的反应进行了检查，使用瞬时表达试验。将这两种质粒分别注射到1-2细胞期斑马鱼胚胎中，分别暴露于天然甾体雌激素17 α-雌二醇（E2）、合成雌激素17α-炔雌醇（EE 2）和相对较弱的环境雌激素壬基酚（NP），荧光显微镜下观察GFP在后续胚胎中的表达。在未暴露的胚胎中未检测到GFP表达，但在暴露于类固醇雌激素处理（EE 2; 10 ng/L，E2; 100 ng/L，暴露72 h后）的肝脏、心脏、体节肌和一些其他组织细胞中检测到GFP的特异性和嵌合表达。对于NP暴露，在10 μg NP/L下72小时后观察到GFP表达（100 μg NP/L对鱼有毒）。我们还表明，我们的构建工程青，另一种模式鱼测试物种，这表明瞬时测定是适用于测试雌激素化学品在鱼一般。本研究结果表明，瞬时表达检测系统可作为环境雌激素的快速综合检测系统，并可用于鱼类胚胎发育过程中雌激素靶位点的检测。
Oestrogenic contaminants are widespread in the aquatic environment and have been shown to induce adverse effects in both wildlife (most notably in fish) and humans, raising international concern. Available detecting and testing systems are limited in their capacity to elucidate oestrogen signalling pathways and physiological impacts. Here we developed a transient expression assay to investigate the effects of oestrogenic chemicals in fish early life stages and to identify target organs for oestrogenic effects. To enhance the response sensitivity to oestrogen, we adopted the use of multiple tandem oestrogen responsive elements (EREc38) in a Tol2 transposon mediated Gal4ff-UAS system. The plasmid constructed (pTol2_ERE-TATA-Gal4ff), contains three copies of oestrogen response elements (3ERE) that on exposure to oestrogen induces expression of Gal4ff which this in turn binds Gal4-responsive Upstream Activated Sequence (UAS) elements, driving the expression of a second reporter gene, EGFP (Enhanced Green Fluorescent Protein). The response of our construct to oestrogen exposure in zebrafish embryos was examined using a transient expression assay. The two plasmids were injected into 1–2 cell staged zebrafish embryos, and the embryos were exposed to various oestrogens including the natural steroid oestrogen 17ß-oestradiol (E2), the synthetic oestrogen 17α- ethinyloestradiol (EE2), and the relatively weak environmental oestrogen nonylphenol (NP), and GFP expression was examined in the subsequent embryos using fluorescent microscopy. There was no GFP expression detected in unexposed embryos, but specific and mosaic expression of GFP was detected in the liver, heart, somite muscle and some other tissue cells for exposures to steroid oestrogen treatments (EE2; 10 ng/L, E2; 100 ng/L, after 72 h exposures). For the NP exposures, GFP expression was observed at 10 μg NP/L after 72 h (100 μg NP/L was toxic to the fish). We also demonstrate that our construct works in medaka, another model fish test species, suggesting the transient assay is applicable for testing oestrogenic chemicals in fish generally. Our results indicate that the transient expression assay system can be used as a rapid integrated testing system for environmental oestrogens and to detect the oestrogenic target sites in developing fish embryos.
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影响因子：
10.4
作者：
Jobling S;Williams R;Johnson A;Taylor A;Gross-Sorokin M;Nolan M;Tyler CR;van Aerle R;Santos E;Brighty G
通讯作者：
Brighty G
影响因子：
3.7
作者：
Adám, A;Bártfai, R;Orbán, L
通讯作者：
Orbán, L
影响因子：
2.7
作者：
Froehlicher, Mirjam;Liedtke, Anja;Eggen, Rik I. L.
通讯作者：
Eggen, Rik I. L.
影响因子：
2.7
作者：
Higashijima, S;Okamoto, H;Eguchi, G
通讯作者：
Eguchi, G
DOI：
10.1073/pnas.022646899
发表时间：
2002-02-05
影响因子：
11.1
作者：
Hartley, KO;Nutt, SL;Amaya, E
通讯作者：
Amaya, E