Graduate Thesis Or Dissertation

 

Utilization of the Zebrafish Model for Investigating Nitrated Polycyclic Aromatic Hydrocarbon Developmental Toxicity Public Deposited

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  • Polycyclic aromatic hydrocarbons (PAHs) are among the most widely known and studied environmental contaminants, originating from a range of natural and anthropogenic sources. PAHs are known to occur in the environment as complex mixtures, containing both unsubstituted PAHs, as well as a range of PAH derivatives. Among the less-studied of these derivative PAH classes are nitrated PAHs (NPAHs). NPAHs are known to form from atmospheric reactions with PAHs and can be found in the environment in a variety of matrices. Many NPAHs are known to be mutagenic, in some cases more so than the corresponding unsubstituted PAH. Less is known about the toxicity of NPAHs in whole-animal systems and for non-cancer endpoints, in particular with regard to the developmental toxicity and metabolism across a wide number of NPAH compounds, in a consistent model system. One of the major challenges in studying PAHs, and related compounds, is the high hydrophobicity and low water solubility of these compounds, which can result in losses due to partitioning of the analytes out of the aqueous phase and on to the walls of the container or exposure vessel. Numerous in vitro and in vivo models utilize plastic plates as exposure vessels, including the use of polystyrene 96-well plates for developmental toxicity testing in the developing zebrafish (Danio rerio) model. We directly measured the losses which occur due to sorption to the polystyrene plates during zebrafish testing for a set of PAHs and NPAHs. Sorptive losses in some instances were greater than fifty percent, in particular for the lower of the two exposure concentrations tested. These sorptive losses decrease the concentration of chemical available to the zebrafish embryos, and therefore impact the interpretation of dose-response toxicity data. In an attempt to create a predictive model for sorptive losses, the measured sorption was modeled against the log K[subscript ow], molecular weight, and subcooled liquid solubilities of the corresponding compounds. The correlations between subcooled liquid solubility and PAH sorption was statistically significant (p<0.05) for both concentrations tested, as well as molecular weight at the higher concentrations tested. However, none of the correlations were statistically significant for NPAH sorption, indicating a need for increased research in this area. We utilized the developing zebrafish model to investigate the developmental toxicity, and potential contributing mechanisms of action, of a suite of 27 NPAHs, as well as 10 heterocyclic PAHs (HPAHs) and 2 amino-PAHs (potential metabolites of NPAHs). Results from the toxicity screen indicate that NPAHs and HPAHs have a wide range of bioactivities in the developing zebrafish, from non-toxic at the concentrations tested to acutely toxic at sub-micromolar exposure concentrations. Activation of the aryl hydrocarbon receptor (AHR) pathway was investigated using a transgenic reporter zebrafish line and morpholino oligonucleotide knockdown to isolate specific isoforms of the AHR. The compounds investigated induced cyp1a expression in five distinct tissues (liver, vasculature, skin, yolk, and neuromast), which we determined to be due to different isoforms of the AHR. A subset of NPAHs was also selected for further mechanistic analysis via qPCR, and genes related to xenobiotic metabolism, cardiac stress, and oxidative stress were investigated. Each NPAH resulted in a unique profile of differentially regulated genes, indicating several potential contributing mechanisms of action. Combined, the results indicate that NPAHs and HPAHs are diverse in their bioactivities towards developing zebrafish. To further investigate metabolism as a contributing factor in the developmental toxicity of NPAHs, we explored the use of a transgenic nitroreductase-expressing zebrafish line for developmental toxicity testing of NPAHs, as well as the applicability of this transgenic line for high-throughput toxicity screening for hepatotoxicity. Humans, and other vertebrate model systems, have endogenous nitroreductase activity, which is responsible for the reduction of nitro functional groups to amino functional groups, while zebrafish do not. Published protocols utilized this transgenic line for tissue-specific cell ablation for a specific exposure scenario. We expanded upon the published protocols for the utilization of this transgenic line for tissue-specific cell ablation, as well as explored potential uses beyond cell ablation. Nitroreduction of a NPAH to the corresponding amino-PAH would have resulted in a shift in the toxicity profile. Unfortunately, no such changes were observed, despite validation of the nitroreductase functionality of the transgenic line, suggesting that the nitroreductase does not have a suitably high affinity for NPAHs to allow for this use. We also exposed the transgenic embryos to compounds known to undergo hepatic metabolism (benzo[a]pyrene and retene) or known hepatotoxins (flutamide, acetaminophen), with and without hepatic ablation, to demonstrate the utility of this transgenic line in isolating the role of the liver in observed toxicity. Overall, the uses for this transgenic line expand beyond the intended and published protocol, but would be further expanded with the development of a similar line with utilized a higher-affinity nitroreductase enzyme. Together, these studies show the overall utility and challenges of using the zebrafish model for the investigation of NPAHs and similar hydrophobic or nitro-containing compounds.
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  • description.provenance : Submitted by Anna Chlebowski (chlebowa) on 2017-05-02T20:59:42Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: bb87e2fb4674c76d0d2e9ed07fbb9c86 (MD5) ChlebowskiAnnaC2017.pdf: 3279424 bytes, checksum: 105ff32a4c15527fdced6cb27aafa18e (MD5)
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