Hoyberghs Jente, Bars Chloé, Pype Casper, Foubert Kenn, Ayuso Hernando Miriam, Van Ginneken Chris, Ball Jonathan, Van Cruchten Steven
University of Antwerp, Wilrijk, Belgium.
Anju Software, Berchem, Belgium.
MethodsX. 2020 Oct 7;7:101087. doi: 10.1016/j.mex.2020.101087. eCollection 2020.
Several pharmaceutical and chemical companies are using the zebrafish embryo as an alternative to animal testing for early detection of developmental toxicants. Unfortunately, the protocol of this zebrafish embryo assay varies between labs, resulting in discordant data for identical compounds. The assay also has some limitations, such as low biotransformation capacity and fewer morphological endpoints in comparison with the in vivo mammalian developmental toxicity studies. Consequently, there is a need to standardize and further optimize the assay for developmental toxicity testing. We developed a Zebrafish Embryo Developmental Toxicity Assay (ZEDTA) that can be extended with a metabolic activation system and/or skeletal staining to increase its sensitivity. As such, the ZEDTA can be used as a modular system depending on the compound of interest.•Our protocol is customized with a metabolic activation system for test compounds, using human liver microsomes. This system ensures exposure of zebrafish embryos to metabolites that are relevant for human risk and safety assessment. As human liver microsomes are toxic for the zebrafish embryo, we developed a preincubation system with an ultracentrifugation and subsequent dilution step.•Additionally, we developed a skeletal staining protocol that can be added to the ZEDTA modular system. Our live Alizarin Red staining method detects several bone structures in 5-day old zebrafish larvae in a consistent manner.
几家制药和化工公司正在使用斑马鱼胚胎作为动物试验的替代方法，用于早期检测发育毒性物质。不幸的是，这种斑马鱼胚胎试验的方案在不同实验室之间存在差异，导致相同化合物的数据不一致。与体内哺乳动物发育毒性研究相比，该试验也有一些局限性，如生物转化能力低和形态学终点较少。因此，需要对发育毒性测试的试验进行标准化和进一步优化。我们开发了一种斑马鱼胚胎发育毒性试验（ZEDTA），可以通过代谢激活系统和/或骨骼染色进行扩展，以提高其灵敏度。因此，根据感兴趣的化合物，ZEDTA可以用作模块化系统。•我们的方案针对测试化合物定制了代谢激活系统，使用人肝微粒体。该系统确保斑马鱼胚胎暴露于与人类风险和安全评估相关的代谢物中。由于人肝微粒体对斑马鱼胚胎有毒，我们开发了一种预孵育系统，包括超速离心和随后的稀释步骤。•此外，我们开发了一种骨骼染色方案，可以添加到ZEDTA模块化系统中。我们的活茜素红染色方法能够以一致的方式检测5天大斑马鱼幼虫中的几种骨骼结构。
