Xu Hongyan, Li Caixia, Li Yan, Ng Grace Hwee Boon, Liu Chunsheng, Zhang Xiaoyan, Gong Zhiyuan
Department of Biological Sciences, National University of Singapore, S3-Level 6, 14 Science Drive 4, Singapore, 117543, Singapore.
Pearl River Fishery Research Institute, Chinese Academic of Fishery Sciences, Guangzhou, 510380, China.
Mar Biotechnol (NY). 2015 Dec;17(6):831-40. doi: 10.1007/s10126-015-9669-1. Epub 2015 Sep 26.
Both dioxins/dioxin-like compounds and polycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants and cause multiple adverse health effects on human and wildlife. Cyp1a is the most commonly used biomarker induced by these pollutants through activation of the aryl hydrocarbon receptor (AhR) pathway. Here we generated Tg(cyp1a:gfp) transgenic zebrafish for establishing a convenient in vivo assay for analysing these xenobiotic compounds. The Tg(cyp1a:gfp) larvae at 4 day post-fertilization were tested with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and GFP induction was observed mainly in the kidney, liver and gut. Similar GFP expression was also induced strongly by two dioxin-like chemicals, co-planar polychlorinated biphenyl (PCB126) and polychlorinated dibenzo-p-furan (PeCDF) and relatively weakly by two PAHs, 3-methylcholanthrene (3-MC) and benzo[a]pyrene (BAP). The lowest observed effective concentration (LOEC) of TCDD was estimated to be ∼1 pM and the EC50 (effective concentration to induce GFP in 50 % of Tg(cyp1a:gfp) larvae) was ∼10 pM. PCB126 and PeCDF had ∼10× lower potencies in GFP induction than TCDD, while the potencies for 3-MC and BAP were at least 1000× lower. The sensitivity of Tg(cyp1a:gfp) larvae to respond TCDD was also favourable compared to that of ethoxyresorufin-O-deethylase (EROD) assay in both zebrafish larvae and adult livers. As GFP-based assay in transgenic zebrafish can be easily accommodated in multi-well dishes, the Tg(cyp1a:gfp) zebrafish should provide not only a valuable biomonitoring tool for aquatic contaminants but also a potential high-throughput chemical screening platform for identification of new AhR agonists.
二噁英/二噁英类化合物和多环芳烃（PAHs）均为持久性有机污染物，会对人类和野生动物造成多种不良健康影响。Cyp1a是这些污染物通过激活芳烃受体（AhR）途径诱导产生的最常用生物标志物。在此，我们构建了Tg(cyp1a:gfp)转基因斑马鱼，用于建立一种便捷的体内分析方法，以检测这些外源性化合物。对受精后4天的Tg(cyp1a:gfp)幼虫用2,3,7,8-四氯二苯并对二噁英（TCDD）进行检测，观察到绿色荧光蛋白（GFP）的诱导主要出现在肾脏、肝脏和肠道。两种二噁英类化学物质，共平面多氯联苯（PCB126）和多氯二苯并对呋喃（PeCDF）也能强烈诱导类似的GFP表达，而两种多环芳烃，3-甲基胆蒽（3-MC）和苯并[a]芘（BAP）诱导作用相对较弱。TCDD的最低观察有效浓度（LOEC）估计约为1 pM，半数有效浓度（EC50，即诱导50%的Tg(cyp1a:gfp)幼虫产生GFP的有效浓度）约为10 pM。PCB126和PeCDF诱导GFP的效力比TCDD低约10倍，而3-MC和BAP的效力至少低1000倍。与斑马鱼幼虫和成年肝脏中的乙氧基异吩唑酮-O-脱乙基酶（EROD）检测相比，Tg(cyp1a:gfp)幼虫对TCDD反应的敏感性也更优。由于转基因斑马鱼中基于GFP的检测可以轻松地在多孔板中进行，Tg(cyp1a:gfp)斑马鱼不仅应为水生污染物提供一种有价值的生物监测工具，还应为鉴定新AhR激动剂提供一个潜在的高通量化学筛选平台。
