Abstract
This dissertation falls within the context of the paradigm shift in regulatory toxicology testing which promotes using a mechanistic-based approach based on in vitro tests instead of traditional animal testing to predict chemical hazards to human such as developmental toxicity. The novel research expands the understanding of developmental toxicity pathways
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by studying chemically-induced gene expression changes related to the perturbation of the retinoic acid signaling pathway (RA-SP) in a vertebrate embryo model. By using the zebrafish embryo (ZE) model it was possible to take advantage of the conservation of this biological pathway across vertebrate taxa, to predict potential human developmental toxicity. The ZE model is not new, however it has been primarily used and optimized for its morphology readout due to the transparent eggs enabling morphological observations during chemical exposure. However, improvements and harmonization are necessary to utilize this model with a reliable molecular level readout, to reveal relevant changes in gene expression. In chapter 2, the protocol design was refined to identify gene expression (GE) changes in the ZE. This was done by investigating the optimal exposure duration to study such changes due to the perturbation of the RA-SP. An exposure of ZE to the RA-SP agonist all-trans retinoic acid (ATRA) was performed using 6 different exposure durations, ranging from 2-117 hrs. These results identified that 4h exposure was the optimal exposure duration to study chemically-induced GE regulation specifically related to the RA-SP perturbation, thereby optimizing the ZE protocol for GE analysis. In chapters 3 and 4, the optimized ZE-GE protocol was employed to identify GE biomarker candidates for maldevelopment. After exposing ZE to two teratogenic compounds known to perturb the RA-SP (ATRA and Valproic Acid, VPA) and one non-teratogenic control compound (Folic Acid, FA), the chemically-induced perturbation of the RA-SP was explored using a whole genome scale GE analysis approach (RNAseq). The 3 test compounds each showed a specific mRNA expression profile, with 248 genes commonly regulated by both teratogenic compounds (ATRA and VPA) but not by FA. These 248 genes were implicated in several developmental processes. 62 differentially expressed genes (DEGs) were associated with nervous system development and were further examined in Chapter 3. These 62 genes were identified as potential biomarkers of early neurodevelopmental toxicity. In chapter 4, the perturbation of RA-SP on the GE associated with development of mesoderm derived tissues was investigated using bioinformatics methods. The investigation identified gene ontology (GO)-terms related to 47 DEGs. Literature indicates that these genes were normally expressed among 3 mesodermal sections (paraxial, intermediate, and lateral plate section) and 6 mesodermal tissues (somites, striated muscle, bone, kidney, circulatory system, and blood). These 47 DEGs were identified as potential biomarkers of early mesodermal maldevelopment or novel potential biomarkers for specific mesodermal organs. These proposed biomarker candidates advance the knowledge on the retinoic acid-mediated developmental toxicity mechanism. As their responses become even more broadly characterized by exploring different exposure regimes and the profiles of different chemicals, these biomarkers could contribute to predictive tools in animal-free chemical hazard and risk assessment.
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