Centre for Health Protection, National Institute for Public Health and the Environment (RIVM), Bilthoven, the Netherlands.
Bayer AG Crop Science Division, Monheim, Germany.
Toxicol Appl Pharmacol. 2021 Dec 15;433:115781. doi: 10.1016/j.taap.2021.115781. Epub 2021 Oct 29.
The cardiac embryonic stem cell test (ESTc) is an in vitro embryotoxicity screen which uses cardiomyocyte formation as the main differentiation route. Studies are ongoing into whether an improved specification of the biological domain can broaden the applicability of the test, e.g. to discriminate between structurally similar chemicals by measuring expression of dedicated gene transcript biomarkers. We explored this with two chemical classes: morpholines (tridemorph; fenpropimorph) and piperidines (fenpropidin; spiroxamine). These compounds cause embryotoxicity in rat such as cleft palate. This malformation can be linked to interference with retinoic acid balance, neural crest (NC) cell migration, or cholesterol biosynthesis. Also neural differentiation within the ESTc was explored in relation to these compounds. Gene transcript expression of related biomarkers were measured at low and high concentrations on differentiation day 4 (DD4) and DD10. All compounds showed stimulating effects on the cholesterol biosynthesis related marker Msmo1 after 24 h exposure and tridemorph showed inhibition of Cyp26a1 which codes for one of the enzymes that metabolises retinoic acid. A longer exposure duration enhanced expression levels for differentiation markers for cardiomyocytes (Nkx2-5; Myh6) and neural cells (Tubb3) on DD10. This readout gave additional mechanistic insight which enabled previously unavailable in vitro discrimination between the compounds, showing the practical utility of specifying the biological domain of the ESTc.
心脏胚胎干细胞测试（ESTc）是一种体外胚胎毒性筛选方法，它使用心肌细胞形成作为主要分化途径。目前正在研究是否可以通过专门的基因转录生物标志物来更精确地确定生物领域，从而扩大该测试的适用性，例如区分具有相似结构的化学物质。我们使用两类化学物质来探索这一点：吗啡啉（三氯生；苯霜灵）和哌啶（芬普雷司；螺螨酯）。这些化合物会导致大鼠出现腭裂等胚胎毒性。这种畸形可归因于对维甲酸平衡、神经嵴（NC）细胞迁移或胆固醇生物合成的干扰。此外，还研究了 ESTc 中的神经分化与这些化合物的关系。在分化第 4 天（DD4）和第 10 天（DD10），用低浓度和高浓度测量相关生物标志物的基因转录表达。所有化合物在 24 小时暴露后均显示出对胆固醇生物合成相关标志物 Msmo1 的刺激作用，而三氯生则抑制 Cyp26a1 的表达，后者编码一种代谢维甲酸的酶。较长的暴露时间增强了心肌细胞（Nkx2-5；Myh6）和神经细胞（Tubb3）分化标志物在 DD10 的表达水平。这种结果提供了额外的机制见解，使我们能够以前所未有的体外方式区分这些化合物，展示了 ESTc 生物领域指定的实际应用价值。
