Developmental neurotoxicity testing of chemical mixtures in zebrafish embryos

Developmental neurotoxicity (DNT) is an understudied problem. Every day, people are exposed to complex mixtures of several chemical substances via food intake, inhalation and dermal contact. Nevertheless, risk assessment is performed on single compounds only under the assumption that the individual exposure levels (below no observed adverse effect levels, NOAELs) are predictive of the mixture effect. In the EuroMix project, a method has been developed to evaluate the effects of mixtures of substances, even at or below NOAELs. This method follows the strategy proposed by the European Food Safety Authority (EFSA), and further implements the Adverse Outcome Pathway (AOP) concept as a basis. Currently, assessment of the DNT potential of compounds is performed in costly and time-consuming in vivo rodent studies involving a large number of animals studied over more than one generation. Therefore, from a 3Rs (Replacement, Reduction, Refinement) perspective an alternative approach is needed. The zebrafish (Danio rerio) embryo (ZFE) provides an interesting and potentially useful model to study DNT as neurodevelopment occurs fast with a large resemblance to the higher vertebrate including the human system. Also, from a legal perspective, experimental work with zebrafish embryos within 120 hours post-fertilization, is not considered an animal experiment. Combined with the ease of culture and the high reproduction rate this renders the ZFE a suitable model for high throughput DNT testing in vitro. One of the suitable readouts for DNT testing is neurobehavior since it provides integrated information on the functionality/status of the full nervous system of the embryo. Within 120 hpf the embryo develops from a fertilized egg to a fully functional embryo responsive to environmental stimuli such as light and sound (vibration). The present Ph.D. study investigated the potential human health risk caused by the simultaneous exposure of chemical substances and the need to include the mixtures in the risk assessment. To obtain a real-life picture ofenvironmental pollution by chemical mixtures, an UHPLC-MS/MS-MRM method was developed and validated for screeining pesticide residues on raw and processed tomatoes. Then, the attention was focused on the potential use of the zebrafish model for assessing the chemical mixtures effects in DNT. Recognised that pharmaceuticals display a well-known MOA and are known to cause DNT, their use as model compounds instead of pesticides was preferred. Therefore, the combined effect of three psychoactive pharmaceuticals of concern, Carbamazepine (CBZ), Fluoxetine (FLX), Venlafaxine (VNX) and their main metabolites, Carbamazepine 10,11 -epoxide (CBZ 10,11E), Norfluoxetine (norFLX), and Desvenlafaxine (desVNX), was studied using the zebrafish embryos as a study model. At first, single-compound concentration-effect relationships were assessed as input for dose-response modelling following the benchmark approach leading to a classification of compounds based on potency. Subsequently, a binary mixture was composed based on the relative potency of the individual compounds and tested for their effect on neurological development. To support the assessment of developmental neurotoxicity, the gene expression of three specific DNT markers was investigated.