Developmental Neurotoxicity of Environmentally Relevant Pharmaceuticals and Mixtures Thereof in a Zebrafish Embryo Behavioural Test

Abstract

Humans are exposed daily to complex mixtures of chemical substances via food intake, inhalation, and dermal contact. Developmental neurotoxicity is an understudied area and entails one of the most complex areas in toxicology. Animal studies for developmental neurotoxicity (DNT) are hardly performed in the context of regular hazard studies, as they are costly and time consuming and provide only limited information as to human relevance. There is a need for a combination of in vitro and in silico tests for the assessment of chemically induced DNT in humans. The zebrafish (Danio rerio) embryo (ZFE) provides a powerful model to study DNT because it shows fast neurodevelopment with a large resemblance to the higher vertebrate, including the human system. One of the suitable readouts for DNT testing in the zebrafish is neurobehaviour (stimulus-provoked locomotion) since this provides integrated information on the functionality and status of the entire nervous system of the embryo. In the current study, environmentally relevant pharmaceuticals and their mixtures were investigated using the zebrafish light-dark transition test. Zebrafish embryos were exposed to three neuroactive compounds of concern, carbamazepine (CBZ), fluoxetine (FLX), and venlafaxine (VNX), as well as their main metabolites, carbamazepine 10,11-epoxide (CBZ 10,11E), norfluoxetine (norFLX), and desvenlafaxine (desVNX). All the studied compounds, except CBZ 10,11E, dose-dependently inhibited zebrafish locomotor activity, providing a distinct behavioural phenotype. Mixture experiments with these pharmaceuticals identified that dose addition was confirmed for all the studied binary mixtures (CBZ-FLX, CBZ-VNX, and VNX-FLX), thereby supporting the zebrafish embryo as a model for studying the cumulative effect of chemical mixtures in DNT. This study shows that pharmaceuticals and a mixture thereof affect locomotor activity in zebrafish. The test is directly applicable in environmental risk assessment; however, further studies are required to assess the relevance of these findings for developmental neurotoxicity in humans.

Toxicometabolomics and Biotransformation Product Elucidation in Single Zebrafish Embryos Exposed to Carbamazepine from Environmentally-Relevant to Morphologically Altering Doses

“…High LC50s for CBZ (i.e., 1.5 to ≥245 mg/L) determined across several species have led some researchers to conclude that CBZ poses minimal risk to the aquatic environment. , Nevertheless, its ubiquitous occurrence and sublethal effects at low doses have led to renewed concerns that CBZ exposure in fish could lead to adverse effects on the population level. , For example, chronic exposure to 0.5 and 10 μg/L CBZ decreased reproductive output in adult ZF, while similar levels (0.01–100 μg/L) perturbed behavior and reproduction in both ZF embryos and Daphnia magna. , These data point to the need for further information on biochemical perturbations underlying the MoA of CBZ, particularly at environmentally relevant concentrations. To this end, a recent metabolomic investigation of CBZ exposure in Daphnia magna reported a nonmonotonic dose response following exposure to 1.75–14 mg/L CBZ, but these levels are much higher than those typically observed in the environment .…”

Sensory-Motor Perturbations in Larval Zebrafish (Danio rerio) Induced by Exposure to Low Levels of Neuroactive Micropollutants during Development

“…Our results are in this regard consistent with a recent work reporting that fluoxetine at environmentally relevant concentrations induces reduction of visual motor response (VMR) in stress-related swimming activity in 107-h old zebrafish embryos [ 46 ]. Furthermore, a study by Atzei et al also reported that both fluoxetine and venlafaxine disturb embryo activity at 96 hpf during the dark periods as well as the light-dark transition in 5 dpf larvae [ 47 ].…”

“…Interestingly, a recent study by Atzei et al highlighted another important difference between the two studied antidepressants [ 47 ]. The recovery of locomotor functions was observed in larval zebrafish when fluoxetine was removed at 4 dpf but the same exposure scenario for venlafaxine did not lead to any recovery of the behavioral phenotype [ 47 ]. This suggests possible developmental neurotoxicity (DNT) effects of that drug.…”

Exploring Neurobehaviour in Zebrafish Embryos as a Screening Model for Addictiveness of Substances

“…To assess changes in neurobehavior as a result of chemical exposure, a light-dark transition test was performed using a ZebraBox (Viewpoint, Lyon, France). The testing procedure followed a previously documented protocol [ 27 ]. In short, embryos were exposed in a 6-well plate (20 eggs per concentration and solvent control) containing 5 mL of test medium and kept in an incubator at 27.5 ± 0.5 °C up to 120 hpf.…”

“…Recent studies have shown that the ZFE shows a distinct and reproducible pattern of locomotion behavior during light and dark conditions. That is, their activity increases after transitions from light to dark, and decreases after transitions from dark to light [ 19 , 26 , 27 ]. The mechanism behind this behavior is still unknown, however it has been suggested that the increase in activity after a sudden onset of darkness facilitates navigation back to illuminated areas that allow for finding food [ 26 ].…”