- Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental contaminants that occur in complex mixtures. These environmental mixtures can consist of both parent PAHs and their derivatives. Several parent PAHs are known or suspected mutagens and/or carcinogens, and a handful of PAH derivatives are known to be more potent mutagens and/or carcinogens than their parent compounds. However, little data exists on the developmental toxicity of the majority of parent PAHs, and even less on the developmental toxicity of PAH derivatives. An understanding of the individual developmental toxicity of PAHs is vital to the accurate modelling of PAH mixture effects. Furthermore, for an accurate assessment of mixture effects, an increased understanding of the diversity of PAH toxicity is needed. This dissertation leverages the zebrafish model to address these gaps in knowledge.
To characterize the developmental toxicity of a wide range of PAHs in a single model, 123 PAHs and PAH derivatives were assessed for morphological and neurobehavioral effects for a range of concentrations between 0.1 and 50 μM, using a high-throughput early life stage zebrafish assay. Tested compounds included 33 parent, 22 nitrated, 17 oxygenated, 19 hydroxylated, 14 methylated, 16 heterocyclic, and 2 aminated PAHs. Additionally, each PAH was evaluated for AHR activation, by assessing CYP1A protein expression using whole animal immunohistochemistry. Responses to PAHs varied in a structurally-dependent manner. High-molecular weight PAHs were significantly more developmentally toxic than the low-molecular weight PAHs, and CYP1A expression was detected in 5 distinct tissues, including vasculature, liver, skin, neuromasts and yolk.
To demonstrate a methodology for using the results of this screening data to test PAH mixtures, a representative mixture was constructed, termed “Supermix10”, from the average relative ratios of the ten most abundant PAHs found at the Portland Harbor Superfund site. Freely dissolved concentrations measured using low density polyethylene passive sampling devices were used to determine the relative ratios used in mixture construction. Developmental toxicity of both the individual PAHs and Supermix10 was determined using the developmental zebrafish model. Sub-mixtures were constructed to assess the contribution of certain groups in the observed toxicity of the mixture. Mixture effects fit the concentration addition model suggesting additivity of individual PAH
toxicities for both Supermix10 and the sub-mixture, Supermix3. Additionally, behavioral effects of developmental exposure were assessed in adults developmentally exposed to Supermix10 below observed developmental effect levels, indicated decreased habituation to a startle stimulus and decreased learning behaviors. Tissue specificity of CYP1A1 expression was determined with immunohistochemistry for both the individual PAHs and Supermix10, and AhR dependence was assessed for Supermix10.
A unique caudal fin phenotype seen in only 4 of 123 PAHs screened suggested an unusual mechanism of toxicity for those PAHs. The phenotype was characterized by the growth of a secondary caudal fin perpendicular to the typical fin growth pattern, and hyperpigmentation at the tip of the tail. Additionally, CYP1A immunohistochemistry revealed unusual expression in the skin and neuromasts of the exposed animals. Windowed exposures determined the window of sensitivity for the phenotype was between 12 and 36 hpf. The role of AHR was investigated using morpholino knocknown of the three zebrafish isoforms, and it was determined that the phenotype is AHR2 dependent. To further investigate what may be driving the formation of this phenotype, caudal fin tissue was isolated at 48, 60, 72, and 96 hpf and the transcriptional response to treatment was determined using RNAseq. Results indicate improper induction of wound healing and fin regeneration pathways that likely play a role in the formation of the phenotype.
Overall, the work presented here demonstrates the diverse developmental effects of PAHs, and the utility of the zebrafish model in revealing and studying these effects of PAHs and PAH mixtures. The information obtained in these studies has expanded our
understanding of the roles AHR can play in developmental toxicity, and can inform PAH mixtures studies in the future.
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