Predictive assays for craniofacial malformations: evaluation in Xenopus laevis embryos exposed to triadimefon

Abstract

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 Xenopuslaevis 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.

Advantages and disadvantages of the use of Xenopus laevis embryos and zebra fish as alternative methods to assess teratogens

“…Special procedures have also been described by Battistoni and colleagues [18*]: skeletal evaluation allows the cartilage examination processing fixed samples with Alcyan Blue; swimming test allows neuro-behavioural evaluation. An extra functional test (deglutition test) has been described and allows the indirect evaluation of craniofacial defects [19].…”