Development of an adverse outcome pathway for cranio-facial malformations: A contribution from in silico simulations and in vitro data

Modified Xenopus laevis approach (R-FETAX) as an alternative test for the evaluation of foetal valproate spectrum disorder

“…In WEC, VPA exposure (from 750 to 1500 M, comparable to the concentrations used in our present work) resulted in brain and branchial defects, irregular dorsal midline, and changes in transcriptomics [36][37][38]. Recently, our research group reported dose-related teratogenic effects of VPA (from 31.25 to 375 M) in a WEC model with branchial and extra-branchial (neural tube and axial) defects [39]. However, WEC test does not provide any information on behaviour and/or neurological deficits.…”

“…In the present work results obtained using amphibian model (R-FETAX) were compared with those obtained in the mammalian model (WEC) used in the past to evaluate teratogenic activity of different substances including VPA [39]. The effects of R-FETAX and WEC on the brain were similar (dilated ventricles), and the relative sensitivities of the two methods (as evaluated by PROAST) were comparable.…”

“…Validated and not yet validated behavioural tests have been reported in the literature for both zebrafish and Xenopus [67,68,54,69]. Our swimming test was designed to evaluate behavioural abnormalities at NF stage 46, considering both route and speed variations, and showed behavioural deficits (reduced swimming acquisition period (NF stages [37][38][39][40][41][42][43][44][45][46] [57] at any VPA concentration. This finding is consistent with the most recent evidence reporting VPA-related neurodevelopmental deficits in children not showing the typical morphological facies of FVS [11].…”

“…The Neurobehavioral evaluation was performed on embryos exposed to 0-500-750-1500 µM VPA during the phylotypic stages (NF stages [13][14][15][16][17][18][19][20][21][22][23][24][25][26] or spontaneous swimming acquisition period (NF stages [37][38][39][40][41][42][43][44][45][46]. According to Currie et al [57], NF stages 37-46 represent the transition from dormant life to progressive free-swimming locomotion at the onset of active feeding.…”

“…Tested concentrations of VPA (sodium salt, Sigma) were dissolved in Tyrode solution and added (80µl/bottle) to the culture medium of the treated group to obtain final concentrations of 0-250-500-750 µM. VPA concentrations were selected based on Metruccio et al [39] to obtain marked branchial and extra-branchial defects, considering the human therapeutic plasma concentration range (280-700 µM) and the concentration used in other in vitro models [21,30,29,43]. After 48 h of culture, embryos were morphologically examined under a dissecting microscope (Leica) to evaluate any embryonic abnormalities.…”