Knowledge on mode-of-action (MOA) is required to understand toxicological effects of compounds, notably in the context of risk assessment of mixtures. Such information is generally scarce, and often complicated by the existence of multiple MOAs per compound. Here, MOAs related to developmental craniofacial malformations were derived from literature, and assembled in a MOA network. A selection of gene expression markers was based on these MOAs. Next, these markers were verified by qPCR in zebrafish embryos, after exposure to reference compounds. These were: triazoles for inhibition of retinoic acid (RA) metabolism, AM580 and CD3254 for selective activation of respectively RA-receptor (RAR) and retinoid-X-receptor (RXR), dithiocarbamates for inhibition of lysyl oxidase, TCDD for activation of the aryl-hydrocarbon-receptor (AhR), VPA for inhibition of histone deacetylase (HDAC), and PFOS for activation of peroxisome proliferator-activated receptor-alpha (PPARα). Next, marker gene profiles for these reference compounds were used to map the profiles of test compounds to known MOAs. In this way, 2,4-dinitrophenol matched with the TCDD and RAR profiles, boric acid with RAR, endosulfan with PFOS, fenpropimorph with dithiocarbamates, PCB126 with AhR, and RA with triazoles and RAR profiles. Prochloraz showed no match. Activities of these compounds in ToxCast assays, and in silico analysis of binding affinity to the respective targets showed limited concordance with the marker gene expression profiles, but still confirmed the complex MOA profiles of reference and test compounds. Ultimately, this approach could be used to support modeling of mixture effects based on upfront knowledge of (dis)similarity of MOAs.

要了解化合物的毒理学效应，特别是在混合物风险评估的背景下，需要了解作用模式 (MOA)。此类信息通常很少，并且通常由于每种化合物存在多个 MOA 而变得复杂。在这里，与发育性颅面畸形相关的 MOA 来源于文献，并组装在 MOA 网络中。基因表达标记的选择基于这些 MOA。接下来，在暴露于参考化合物后，通过 qPCR 在斑马鱼胚胎中验证这些标记。它们是：用于抑制视黄酸 (RA) 代谢的三唑类，用于分别选择性激活 RA 受体 (RAR) 和类视黄醇-X-受体 (RXR) 的 AM580 和 CD3254，用于抑制赖氨酰氧化酶的二硫代氨基甲酸盐，用于激活芳基烃受体（AhR），VPA 用于抑制组蛋白脱乙酰酶 (HDAC)，PFOS 用于激活过氧化物酶体增殖物激活受体-α (PPARα)。接下来，这些参考化合物的标记基因谱用于将测试化合物的谱图映射到已知的 MOA。这样，2,4-二硝基苯酚与 TCDD 和 RAR 曲线相匹配，硼酸与 RAR 相匹配，硫丹与 PFOS 相匹配，苯丙吗啉与二硫代氨基甲酸盐相匹配，PCB126 与 AhR 相匹配，RA 与三唑和 RAR 曲线相匹配。Prochloraz 显示不匹配。这些化合物在 ToxCast 检测中的活性，以及 硼酸与 RAR、硫丹与 PFOS、苯丙吗啉与二硫代氨基甲酸盐、PCB126 与 AhR 以及 RA 与三唑和 RAR 概况。Prochloraz 显示不匹配。这些化合物在 ToxCast 检测中的活性，以及 硼酸与 RAR、硫丹与 PFOS、苯丙吗啉与二硫代氨基甲酸盐、PCB126 与 AhR 以及 RA 与三唑和 RAR 概况。Prochloraz 显示不匹配。这些化合物在 ToxCast 检测中的活性，以及对各个靶标的结合亲和力的计算机分析显示与标记基因表达谱的一致性有限，但仍证实了参考和测试化合物的复杂 MOA 谱。最终，这种方法可用于支持基于 MOA （不）相似性的前期知识的混合效应建模。