Zebrafish ($Danio$ $rerio$) embryos in developmental neurotoxicity screening : addressing the mechanisms of developmental neurotoxicity utilising multiple behaviour and OMIC methods

Haigis, Ann-Cathrin; Schäffer, Andreas (Thesis advisor); Hollert, Henner (Thesis advisor); Legradi, Jessica (Thesis advisor)

Aachen : RWTH Aachen University (2021, 2022)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2021


Environmental pollution through industrial chemicals or pesticides has emerged as a major health issue for humans and the environment. While several of these chemicals were shown to adversely affect nervous system development, the estimated number of potential neurotoxicants is likely to be much higher. Such developmental perturbations cost society billions of dollars and result in IQ loss in children and several other neurological disorders. As there is currently no cure for such disorders, prevention is essential. Yet, the mechanisms leading to developmental neurotoxicity (DNT) are poorly understood, making prevention difficult. Consequently, methods and strategies are required that allow for a fast and reliable screening of potentially neurotoxic or -active substances. With regards to this the zebrafish ($Danio$ $rerio$) embryo has emerged as a model organism. The embryos provide multiple advantageous features, promoting its use in DNT investigations as a non-mammal in vivo test system.In the work at hand, it was hypothesised that zebrafish embryos can be used to generate comprehensive insights into the mechanisms underlying DNT. To reveal these mechanisms, this thesis defined several objectives. First, to assess the applicability of zebrafish embryo behaviour for DNT screens. Therefore, six different insecticides and four endocrine disruptors (EDs) were used. Subsequently, observed behavioural deficits were to be investigated on multiple molecular levels to clarify underlying mechanisms, thus presenting the second goal. Thirdly the applicability of the embryos to lipidome investigations and their relevance for DNT-testing was to be assessed.To accomplish the first goal, it was aimed to increase the information output of one of the most commonly used behaviour assays, the light/ dark transition (LDT) test. Data evaluation was done by integrating multiple parameters at once, thereby retrieving additional information that was indicative for underlying (neuro-) toxic mechanisms. Furthermore, the experimental implementation of the spontaneous movement assay was improved. This resulted in a detailed description of this behaviour and highlighted new endpoints beneficial for DNT screens. As the increasing number of environmental toxicants makes rapid DNT screens necessary, the applicability of zebrafish embryos for such screens was assessed. An optimised battery of different behaviour assays, covering the entire early embryonic development showed that the embryos behaviour was able to indicate DNT. At the same time, this approach dramatically reduced the number of required test organisms. Hence, zebrafish embryo behaviour was recommended for broad DNT screenings in relevance of an Integrated Approach to Testing and Assessment (IATA). The second aim was accomplished by utilising multiple OMIC techniques (transcriptomics, proteomics, metabolomics). Exposure to acetylcholinesterase (AChE) inhibiting substances resulted in similar effects that were, however, not identical. For instance, dichlorvos exerted muscle apparatus and visual system perturbations while exposure to paraoxon-methyl lead to neurotoxic outcomes (e.g., serotonin dysregulation). On a higher level of organisation (locomotion) both substances decreased embryonic activity. Nervous system dysregulations (e.g., increased GABA levels) were further noticed after expo-sure to the EDs TDCPP, MEHP and 4-NP. Employing lipidomic analysis on exposed zebrafish embryos indicated effects on lipids involved in energy storing, cholesterol homeostasis or lipid-based signalling. A dysregulation of the lipidome can be associated with human-like diseases, demonstrating the relevance of including lipidome investigation into environmental exposure considerations for DNT. The thesis presented here demonstrated how environmental toxicants act on multiple levels of organisation in the zebrafish embryo. Due to the high conservation between e.g., the endocrine or the neuronal system of mammals and lower vertebrates, the results obtained from this research likely possess general relevance for both human and environmental DNT risk assessment. However, further research will be necessary to identify causal relationships. This work further established the applicability of zebrafish embryos as a non-mammal in vivo test system for DNT screens, suitable for a DNT-IATA. Overall, this thesis contributed approaches for addressing and understanding DNT as well as emphasised the need for new strategies, which increase the transferability of this knowledge to eco(toxico)logical research questions.