High-Content Screening Assay for Identification of Chemicals Impacting Spontaneous Activity in Zebrafish Embryos

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• 作者：Tara D. Raftery ; Gregory M. Isales ; Krystle L. Yozzo ; David C. Volz
• 刊名：Environmental Science & Technology
• 出版年：2014
• 出版时间：January 7, 2014
• 年：2014
• 卷：48
• 期：1
• 页码：804-810
• 全文大小：344K
• ISSN：1520-5851

文摘

Although cell-based assays exist, rapid and cost-efficient high-content screening (HCS) assays within intact organisms are needed to support prioritization for developmental neurotoxicity testing in rodents. During zebrafish embryogenesis, spontaneous tail contractions occur from late-segmentation (19 h postfertilization, hpf) through early pharyngula (29 hpf) and represent the first sign of locomotion. Using transgenic zebrafish (fli1:egfp) that stably express eGFP beginning at 14 hpf, we have developed and optimized a 384-well-based HCS assay that quantifies spontaneous activity within single zebrafish embryos after exposure to test chemicals in a concentration鈥搑esponse format. Following static exposure of one embryo per well from 5 to 25 hpf, automated image acquisition procedures and custom analysis protocols were used to quantify total body area and spontaneous activity in live embryos. Survival and imaging success rates across control plates ranged from 87.5 to 100% and 93.3鈥?00%, respectively. Using our optimized procedures, we screened 16 chemicals within the US EPA鈥檚 ToxCast Phase-I library, and found that exposure to abamectin and emamectin benzoate鈥攂oth potent avermectins鈥攁bolished spontaneous activity in the absence of gross malformations. Overall, compared to existing locomotion-based zebrafish assays conducted later in development, this method provides a simpler discovery platform for identifying potential developmental neurotoxicants.