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Arch Toxicol
2022 Oct 01;9610:2815-2824. doi: 10.1007/s00204-022-03327-w.
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Predictive assays for craniofacial malformations: evaluation in Xenopus laevis embryos exposed to triadimefon.
Battistoni M
,
Metruccio F
,
Di Renzo F
,
Bacchetta R
,
Menegola E
.
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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.
Figure 1. R-FETAX experimental approach. The classical FETAX exposure (1) covers the entire test. R-FETAX windowed approach, by contrast, provides limited times of exposure, corresponding to the main developmental phases: 2 = pre-organogenetic period (midblastula-gastrula); 3 = early organogenetic period (from neurula till phylotypic stages; the striped red area corresponds to neurula); 4 = late organogenetic period (from phylotipic stages till tadpole); 5 = tadpole (very late organogenesis and functional differentiation)
Figure 2. Whole mount alcian blue-stained craniofacial cartilages (ventral view), allowing to detect cartilaginous elements in a normal larva (A) and in an abnormal sample (B) characterized by circular funnel-shaped fused cartilages (*). G = gill basket; I = intestine with degl + (A) and degl− (B) phenotypes (respectively, presence/absence of red microplastics in the intestine). In A, dotted lines indicate the M–PQ angle measurement
Figure 3. Appearance of whole mount (A, C, E) and flat mount (B, D, F) stained cartilages in a normal larva (A, B) and in larvae appearing as normal at the whole mount evaluation (C, E) showing moderate (D) or extended (F) fusions among ethmoidal (Eth), Meckel’s (M) and palatoquadrate (PQ) cartilages. In B note the articular space (arrow) between Meckel’s and palatoquadrate cartilages, not visible in the abnormal larvae (D, F). CH = ceratohyal cartilage
Figure 5. Hill model: dose–response curves obtained considering stage as covariate. The colors/symbols in the plot refer to the different exposure groups: black/upward triangle = classical FETAX exposure; green/diamond = gastrula; light blue/cross-square = neurula; red/ cross = early morphogenesis; dark blue/downward triangle = late morphogenesis; pink/cross-plus tadpole. Model evidenced late morphogenesis as the sensible subgroup
Figure 6. Lateral view of the anterior region of a normal larva (upper panel) with degl + phenotype and of an abnormal sample (lower panel) characterized by round head with bent encephalon (arrow), short snout (#) and degl− phenothype. I = intestine; * = normal linear encephalon
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