Evaluating the e.ects of mixtures of endocrine disruptors on development and gene expression in zebrafish larvae (Danio rerio)

English. Persistent organic pollutants (POPs) and endocrine-disrupting chemicals (EDCs) represent significant environmental and health threats due to their stability, bioaccumulation, and complex interactions in biological systems. This thesis synthesizes the findings from three research publications that investigate the developmental, physiological, and behavioral eDects of POP and EDC exposure using zebrafish (Danio rerio) as a model organism. Zebrafish larvae were exposed to a mixture of 29 POPs, at diDerent concentrations based on what was found in the blood of Scandinavian populations, to study the eDects of these chemicals on growth, skeletal development, behavior, mitochondrial function, and gene expression. Across the studies, zebrafish larvae exposed to POP mixtures exhibited significant skeletal and craniofacial deformities. Developmental issues in cartilage and bone formation were identified, including reduced size of structures such as Meckel’s cartilage and disrupted bone mineralization. The disruption of bone formation was linked to interactions with nuclear receptor pathways, such as androgen, vitamin D, and retinoic acid signaling. Exposure to the POP mixtures also led to mitochondrial toxicity, with more pronounced eDects in those mixtures containing per- and polyfluorinated acids (PFAAs). Increased mitochondrial activity, indicative of oxidative stress, was observed. POP exposure caused pronounced behavioral changes, particularly in response to light- dark transitions (startle response). Hyperactivity was observed in zebrafish exposed to mixtures containing PFAAs, and the presence of brominated (Br) and chlorinated (Cl) compounds seemed to modulate these eDects. Cardiac function was also significantly aDected in all treatments. Furthermore, transcriptomic analysis revealed substantial changes in gene expression following exposure to POP mixtures. Genes involved in insulin signaling, brain and eye development, and stress responses were significantly dysregulated. The most notable molecular finding was the inhibition of the condensin I complex, which is crucial for retinal development. This inhibition led to microphthalmia (small eyes) or pear-like shape in exposed zebrafish larvae, providing insights into the mechanisms underlying POP-induced developmental defects. Finally, using the CRISPR/Cas9 technology, we generated specific mutants to explore the role of estrogen receptors (Esr1, Esr2b, and Gper1) in mediating the eDects of EDCs (antagonist eDects) on zebrafish development. Each receptor was found to play distinct roles in regulating skeletal, cardiac, and behavioral outcomes. Zebrafish mutants with inactivated Esr1 exhibited the most severe craniofacial deformations that were seen in 70% of the population, highlighting the role of estrogen receptors in regulating skeletal development. Zebrafish esr2b mutants had increased heart rates, further highlighting the role of estrogen signaling in regulating cardiovascular development and function. Finally, the gper1 mutants displayed reduced activity and skeletal mineralization, emphasizing the complexity of estrogen receptor-mediated developmental processes. The findings from this research highlights the significant risks posed by exposure to POP mixtures, particularly in terms of skeletal and retinal development, behavior, and endocrine disruption. Our results highlight the need for further mechanistic studies to better understand the long-term impacts of POP exposure on both environmental and human health. However, these results provide critical insights into the molecular mechanisms of POP toxicity, with important implications for environmental risk assessment and public health policy. The collective findings emphasize the importance of considering chemical mixtures rather than individual compounds, as their combined eDects seemed to be often more severe and complex.

Protection Against Endocrine Disruptors