Department of Physics Aldo Pontremoli, Università Degli Studi Di Milano, via Celoria, 16-20133, Milan, Italy.
Department of Biomedical and Clinical Sciences, ICPS, ASST Fatebenefratelli Sacco, Università degli studi di Milano, Via GB Grassi, 74- 20159, Milan, Italy.
Arch Toxicol. 2022 Oct;96(10):2815-2824. doi: 10.1007/s00204-022-03327-w. Epub 2022 Jun 24.
Craniofacial defects are one of the most frequent abnormalities at birth, but their experimental evaluation in animal models requires complex procedures. The aim of the present work is the comparison of different methodologies to identify dose- and stage-related craniofacial malformations in Xenopus laevis assay (R-FETAX, where the full cartilage evaluation, including flat mount technique, is the gold standard for skeletal defect detection). Different methods (external morphological evaluation of fresh samples, deglutition test, whole mount cartilage evaluation and Meckel-palatoquadrate angle measurements) were applied. Triadimefon (FON) was selected as the causative molecule as it is known to induce craniofacial defects in different animal models, including the amphibian X. laevis.FON exposure (0-31.25 μM) was scheduled to cover the whole 6-day test (from gastrula to free swimming tadpole stage) or each crucial developmental phases: gastrula, neurula, early morphogenesis, late morphogenesis, tadpole. Dose-dependent effects (fusions among craniofacial cartilages) were evident for groups exposed during the morphogenetic periods (neurula, early morphogenesis, late morphogenesis); gastrula was insensitive to the tested concentrations, tadpole group showed malformations only at 31.25 μM. The overall NOAEL was set at 3.9 μM. Results were evaluated applying benchmark dose (BMD) approach. The comparison of relative potencies from different methods showed deglutition as the only assay comparable with the gold standard (cartilage full evaluation).In conclusion, we suggest deglutition test as a reliable method for a rapid screening of craniofacial abnormalities in the alternative model X. laevis. This is a rapid, inexpensive and vital test allowing to preserve samples for the application of further morphological or molecular investigations.
颅面畸形是出生时最常见的异常之一，但在动物模型中对其进行实验评估需要复杂的程序。本研究的目的是比较不同方法，以鉴定 Xenopus laevis 检测法（R-FETAX，其中全面软骨评估，包括平置技术，是检测骨骼缺陷的金标准）中与剂量和阶段相关的颅面畸形。应用了不同的方法（新鲜样本的外部形态学评估、吞咽试验、全软骨评估和 Meckel-palatoquadrate 角测量）。三唑酮（FON）被选为致病分子，因为它已知在包括两栖动物 X. laevis 在内的不同动物模型中引起颅面缺陷。FON 暴露（0-31.25μM）计划涵盖整个 6 天的测试（从原肠胚到自由游动的蝌蚪阶段）或每个关键的发育阶段：原肠胚、神经胚、早期形态发生、晚期形态发生、蝌蚪。在形态发生期（神经胚、早期形态发生、晚期形态发生）暴露的组中，出现了剂量依赖性效应（颅面软骨融合）；原肠胚对测试浓度不敏感，仅在 31.25μM 时，蝌蚪组显示出畸形。总体无观察到不良作用水平（NOAEL）设定为 3.9μM。应用基准剂量（BMD）方法评估结果。不同方法的相对效力比较表明，吞咽试验是唯一可与金标准（软骨全面评估）相媲美的试验。总之，我们建议吞咽试验作为一种可靠的方法，用于快速筛选替代模型 X. laevis 中的颅面异常。这是一种快速、廉价且重要的试验，可以保存样本，用于进一步的形态学或分子研究。
