Neurotoxic effects in zebrafish embryos by valproic acid and nine of its analogues: the fish-mouse connection?

Abstract

Since teratogenicity testing in mammals is a particular challenge from an animal welfare perspective, there is a great need for the development of alternative test systems. In this context, the zebrafish (Danio rerio) embryo has received increasing attention as a non-protected embryonic vertebrate in vivo model. The predictive power of zebrafish embryos for general vertebrate teratogenicity strongly depends on the correlation between fish and mammals with respect to both overall general toxicity and more specific endpoints indicative of certain modes-of-action. The present study was designed to analyze the correlation between (1) effects of valproic acid and nine of its analogues in zebrafish embryos and (2) their known neurodevelopmental effects in mice. To this end, zebrafish embryos exposed for 120 h in an extended version of the acute fish embryo toxicity test (FET; OECD TG 236) were analyzed with respect to an extended list of sublethal endpoints. Particular care was given to endpoints putatively related to neurodevelopmental toxicity, namely jitter/tremor, deformation of sensory organs (eyes) and craniofacial deformation, which might correlate to neural tube defects caused by valproic acid in mammals. A standard evaluation of lethal (LC according to OECD TG 236) and sublethal toxicity (EC) merely indicated that four out of ten compounds tested in zebrafish correlate with positive results in mouse in vivo studies. A detailed assessment of more specific effects, however, namely, jitter/tremor, small eyes and craniofacial deformation, resulted in a correspondence of 75% with in vivo mouse data. A refinement of endpoint analysis from an integration of all observations into one LCx or ECx data (as foreseen by current ecotoxicology-driven OECD guidelines) to a differential evaluation of endpoints specific of selected modes-of-action thus increases significantly the predictive power of the zebrafish embryo model for mammalian teratogenicity. However, for some of the endpoints observed, e.g., scoliosis, lordosis, pectoral fin deformation and lack of movement, further experiments are required for the identification of underlying modes-of-action and an unambiguous interpretation of their predictive power for mammalian toxicity.

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

“…Non-mammalian vertebrate models have been proposed instead of mammal models to use lower vertebrate. These models showed that i) in the amphibian X. laevis 1000 -20000 M VPA exposure (concentrations exceedingly above the human therapeutic concentrations more than 5 fold) induced severe effects in different body districts (tadpoles with severe abnormalities in different body districts and severe developmental delays) [40,41]; ii) in zebrafish exposed to therapeutic concentrations (from 5 to 1500 M, in line with the concentrations used in our present work) VPA induced disruption of heart looping, haematopoiesis, cranio-facial development, liver and pancreas development, oedema and brain deformities, shortening, folding of the tail, small eyes [41][42][43][44][45]; and iii) in chicken embryos cultured in vitro, VPA (10 l/embryo of a 300mM solution) interfered with the somitogenesis process 6 [46]; chicken embryos exposed to VPA (2-8 mg/kg for a 50 g egg, considered from the Authors close to the range of possible human exposure) in ovo showed increased mortality, growth delay, neural tube, cardiovascular, cranio-facial, limb, and skeletal anomalies [47].…”

“…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.…”

Zebrafish as an Animal Model for Testing Agents with Antidepressant Potential

“…Over the past twenty years, Danio rerio has been widely used in ecotoxicology studies. Observation of fish enables determination of an influence of different kind of pollutants on the environment or to evaluate toxic and/or teratogenic effects of xenobiotics [ 107 , 108 ]. A standard protocol used for toxicity assessment is called the fish embryo acute toxicity (FET) test, which was described by the Organization for Economic Co-operation and Development (OECD) as test guideline No.…”

Potential of the zebrafish (Danio rerio) embryo test to discriminate between chemicals of similar molecular structure—a study with valproic acid and 14 of its analogues

“…In fact, the data available in literature included in vivo developmental data for VPA and some structural analogues in various mammalian models, whereas for zebrafish only VPA data could be localized (Dai et al 2015;Driessen et al 2013;Hill 2012;McGrath and Li 2008). This gap was closed with experimental data retrieved from 120 h fish embryo acute toxicity tests (FETs) based on OECD TG 236 (OECD 2013); results for 10 compounds were already presented by Brotzmann et al (2020), while data for the remaining 5 compounds were generated in the present study.…”