- All animals have developed the critical ability to detect, respond to, and detoxify a large array of environmental chemicals and stressors that can cause adverse health effects. Important examples of landmark contaminants around the world are polycyclic aromatic hydrocarbons (PAHs) and dioxins, both of which can act via the aryl hydrocarbon receptor (AHR), a protein receptor and ligand activated transcription factor. The AHR is conserved across multiple phyla, is required for proper development, and mediates the adverse developmental effects of several PAHs across vertebrates, including humans. While a plethora of research has been conducted investigating PAH toxicity, significant challenges still persist around our lack of understanding of the diversity in toxicity mechanisms, especially due to our narrow focus on select PAHs and few biomarkers. Our knowledge of AHR-regulated mechanisms of PAH toxicity is still in its infancy.
The zebrafish (Danio rerio) is an established model organism in toxicological and biomedical sciences that is well-suited for investigating AHR-regulated biological processes, especially due to the presence of a functional ortholog (AHR2) of the mammalian AHR. To this end, the overall objective of my dissertation is to leverage the zebrafish model to characterize and classify PAHs, and further our understanding of the downstream signaling events upon AHR activation. To achieve this, I first compiled a comprehensive review (Chapter 2) spanning twenty years of AHR research in zebrafish. The review demonstrates the magnitude of research that has been conducted navigating the complexity of AHR signaling as it relates to zebrafish exposure to PAHs and other xenobiotic AHR ligands, such as 2,3,7,8-Tetrachlorodibenzodioxin (TCDD). The chapter identifies significant knowledge gaps such as our lack of understanding of how different PAHs differentially alter the AHR signaling cascade and AHR’s crosstalk with other signaling pathways. To answer these questions, I conducted two studies (Chapters 3 and 4) that leveraged large compendiums of RNA sequencing data collected from developing zebrafish exposed to a diverse array of chemicals. Chapter 3 compares 16 individual PAHs and couples transcriptomic and developmental toxicity data to both characterize and classify PAHs. Using the Context Likelihood of Relatedness algorithm, I identified two major groups of PAHs, one being more developmentally toxic, predominantly activating AHR2, and leading to transcriptional profiles that had both similarities and differences. One important finding was that the expression of cyp1a (an AHR-dependent phase I metabolic enzyme commonly used as a biomarker for AHR activation) was more indicative of transcriptional profiles and not developmental toxicity phenotypes, suggesting the need for additional biomarkers that can better predict toxicity outcomes. Chapter 3 leverages a novel gene co-expression approach in zebrafish to compare and contrast PAHs and TCDD (AHR2 Activators), along with a large array of diverse flame retardant chemicals (FRCs). I found that the AHR2 Activators and FRCs localized to distinct regions of the network, with the FRCs associated with broad neurobehavioral and vascular developmental processes. On the contrary, the AHR2 Activators localized to one region of the network that was primarily associated with chemical metabolism processes. Guided by the network, I identified several new members of the AHR2 signaling pathway that should be investigated in future research. Both Chapters 3 and 4 leveraged co-expression analyses to narrow down on several potentially important candidates for biomarkers of PAH exposure. In Chapter 5, I investigate one such AHR-regulated gene, wfikkn1 (WAP, Follistatin/Kazal, Immunoglobulin, Kunitz And Netrin Domain Containing 1). I found that the expression of wfikkn1 was AHR2-dependent in developing zebrafish exposed to TCDD. Using a combination of CRISPR-Cas9 to generate a mutant zebrafish line, and transcriptomic, proteomic, and high-throughput neurobehavioral assays, I discovered that this AHR2-regulated gene has potentially important roles in muscle developmental processes. Upon exposure to TCDD, wfikkn1 mutant zebrafish had a significantly altered transcriptome and larval neurobehavioral processes compared to wildtype zebrafish, suggestive of its additional role in AHR-regulated neurodevelopment. These data also highlight potential crosstalk between AHR and other signaling pathways such as Estrogen Receptor signaling via wfikkn1, which should be investigated in future studies.
Overall, within this dissertation, I leveraged the advantages of the zebrafish model organism to characterize and classify PAHs by their transcriptomic and developmental toxicity responses. I also investigated the functional role of a novel AHR-dependent gene that might contribute to AHR-regulated toxicity responses. Increasing our knowledge of biological processes and mechanisms associated with AHR activation can help us better understand how PAHs cause toxicity, which will lead to more guided risk assessment measures and opportunities for susceptibility research.