Development of a predictive in silico model for mixtures of azole fungicides acting on the skeletal craniofacial AOP
Résumé
Single azole fungicides, including triadimefon (FON) and flusilazole (FLUSI), can affect cranio-facial morphogenesis during the early development. Data previously obtained in postimplantation rat whole embryo cultured in vitro described specific teratogenic effects at the branchial structures, while the co-exposure resulted in additive effects, accounting for a common mode of action (MoA) for the azole fungicides. In the context of the skeletal craniofacial adverse outcome pathway (AOP), the proposed molecular initiating event for azole teratogenicity is the inhibition of embryonic CYP26 isozymes involved in retinoic acid (RA) catabolism with the consequent local increase in endogenous RA levels. With the aim of investigating this hypothetical mechanism, experimental data were interpreted developing an in silico tool, combining pathway modelling, molecular docking and in vitro experiments, that can simulate the formation of physiological RA levels in the rat embryo hindbrain and predict their perturbation after exposure to single azole fungicides and to their binary mixtures just on the basis of dose-effect data of the individual substances. This model demonstrated to adequately predict the outcome of in vitro exposure of embryos to mixtures, confirming the accuracy of the hypothesized pathogenic pathway: experimental data and model predictions are in promising agreement. This research will provide a better understanding of the toxicity mechanism of single and combined chemicals affecting RA (not just azole fungicides), and a predictive tool for human risk assessment for all mixtures acting on the skeletal craniofacial AOP.