Defining PAH and Oxygen-substituted PAH-dependent Developmental and Adult Toxicity in Zebrafish

approved: ______________________________________________________________ Robert L. Tanguay Polycyclic aromatic hydrocarbons (PAHs) and oxygen-substituted PAHs (OPAHs) are environmental contaminants present in urban air, dust, soil and water resulting from incomplete combustion of organic materials or fossil fuels; found in crude oil and coal; and formed through photoxidation or biotransformation of microbial. It is widely recognized that PAHs pose risks to human health, especially for the developing fetus and infant, where developmental exposures to PAH have been linked to complex human diseases later in life. To investigate potential developmental toxicity and longterm effects resulting from early in-life stage exposure to PAHs and OPAHs, we utilized the embryonic zebrafish model. In our first study, we conducted a comprehensive toxicity screen of 38 environmentally relevant OPAHs. Zebrafish embryos were exposed throughout development (6 to 120 hours post fertilization, hpf) to a broad concentration range and evaluated for dose response, malformation profiles and CYP1A protein expression. We subsequently clustered the OPAHs based on the concentration that induced 50% adverse effect (EC50) for each endpoint and found distinct groupings based on structure and AHR activation. In addition, further analysis of a selected OPAH from each group revealed oxidative stress to be a main driver of OPAH toxicity. Once we evaluated the developmental toxicity of this large class of OPAHs, we selected three OPAHs: benz(a)anthracene-7,12-dione (7,12B[a]AQ), 1,9-benz-10-anthrone (BEZO), 9,10-phenanthrenequinone (9,10PHEQ), and PAHs: benzo[a]pyrene (B[a]P), and dibenzo[a,l]pyrene (DB[a,l]P) to evaluate adverse long term effects of exposure. Using concentrations that did not result in any visible malformations at 120hpf, we measured in vivo mitochondrial respiration and found at 26hpf, B[a]P, BEZO, and 9,10-PHEQ resulted in a significant decrease in oxygen consumption rates and maximum oxygen capacity. Larval behavior was evaluated at 120hpf, and resulted in a hyperactive phenotype in B[a]P, BEZO, and 9,10-PHEQ, while 7,12-B[a]AQ and DB[a,l]P displayed a hypoactivity in the dark. To further evaluate the effects of developmental exposure, we raised a subset of exposed animals (from 6-120 hpf) to adulthood to evaluate physiological fitness and behavior. Using the AutoResp oxygen probe and swimtunnel (Loligo Systems) we measured differences in oxygen consumption rates, which revealed altered oxygen utilization associated with developmental PAH and OPAH exposures. By using shuttleboxes to test for cognitive learning, we identified that 9,10PHEQ exposed animals were the most adversely affected, and B[a]P, BEZO and DB[a,l]P exposed animals showed a moderate learning deficiency, while 7,12-B[a]AQ exposure resulted in only minor physiological and behavioral changes. Finally, we used mRNA-SEQ analysis at 48hpf to begin to tease apart early transcriptional changes and misregulated biological processes that preceded the onset of these phenotypes and hoped to gain insight into the differential response in adults. B[a]P, BEZO, and 9,10-PHEQ exposed animals showed similar effects in 24hpf oxygen consumption, larval behavior, and adult OCR which we used to correlate to biological processes at 48hpf. Preliminary results illustrated a positive correlation of 5dpf larval behavior and the neurophysiological pathway of visual perception. Further analysis of behavior and mitochondrial function phenotypes will help confirm the biological pathways involved in larval and adult phenotypes and help create a better model for these adverse outcomes. In the work presented here, we used the embryonic and adult zebrafish model to characterize short and long term effects resulting from early exposure to environmentally relevant OPAH and PAH contaminants. ©Copyrighted by Andrea L. Knecht July 31, 2015 All Rights Reserved Defining PAH and Oxygen-substituted PAH-dependent Developmental and Adult Toxicity in Zebrafish by Andrea L. Knecht A DISSERTATION submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Presented July 31, 2015 Commencement June 2016 Doctor of Philosophy dissertation of Andrea L. Knecht presented on July 31, 2015.