The objective of this thesis was to evaluate hsp70 expression as an indicator of arsenic exposure in zebrafish larvae and to assess the accuracy of the hsp70-eGFP reporter gene construct as a reliable indicator of endogenous hsp70 expression. The relative toxicity of arsenite and arsenate was also examined and gross developmental effects were recorded following an acute 96 hour range finding exposure. Gross effects observed included edema, trunk abnormalities, immobility, and mortality for both arsenite and arsenate, with arsenite more toxic than arsenate. The median lethal concentrations (LC50) for arsenite and arsenate were calculated from the data obtained in the 96 hour exposure. They were determined to be 771.98 μM and 1347 μM respectively. The effective concentrations (EC50) were determined to be 570 μM for arsenite and 1172 μM for arsenate. Results from the 96 hour exposures were also used to determine concentrations used in subsequent exposures. Induction of hsp70 was examined in wild-type larvae following a three hour exposure to arsenic and subsequent in situ hybridization. It was determined that both trivalent and pentavelant arsenic induced expression in the olfactory rosette, gills and skin, GIT, liver, and pericardial muscle. Expression was found to be dose-dependent and tissue-specific for both. Induction of hsp70 was evident in the skin, liver, and gastrointestinal tract of zebrafish larvae exposed to 700 μM arsenite and in the skin, gills, liver, pericardial muscle, and gastrointestinal tract in those exposed to 1000 μM or 2000 μM arsenite. Exposure to 1500 μM arsenate resulted in expression in skin, liver, and gastrointestinal tract, while induction was observed in skin, gills, liver, pericardial muscle and gastrointestinal tract of larvae exposed to 2500 μM or 7500 μM arsenate. Overall expression patterns of hsp70-eGFP in transgenic zebrafish larvae exposed to arsenic were found to closely mimic that of endogenous hsp70 expression patterns in wild-type larvae suggesting that it is an accurate indicator of endogenous hsp70 expression.

The objective of this thesis was to evaluate hsp70 expression as an indicator of arsenic exposure in zebrafish larvae and to assess the accuracy of the hsp70-eGFP reporter gene construct as a reliable indicator of endogenous hsp70 expression. The relative toxicity of arsenite and arsenate was also examined and gross developmental effects were recorded following an acute 96 hour range finding exposure. Gross effects observed included edema, trunk abnormalities, immobility, and mortality for both arsenite and arsenate, with arsenite more toxic than arsenate. The median lethal concentrations (LC50) for arsenite and arsenate were calculated from the data obtained in the 96 hour exposure. They were determined to be 771.98 μM and 1347 μM respectively. The effective concentrations (EC50) were determined to be 570 μM for arsenite and 1172 μM for arsenate. Results from the 96 hour exposures were also used to determine concentrations used in subsequent exposures. Induction of hsp70 was examined in wild-type larvae following a three hour exposure to arsenic and subsequent in situ hybridization. It was determined that both trivalent and pentavelant arsenic induced expression in the olfactory rosette, gills and skin, GIT, liver, and pericardial muscle. Expression was found to be dose-dependent and tissue-specific for both. Induction of hsp70 was evident in the skin, liver, and gastrointestinal tract of zebrafish larvae exposed to 700 μM arsenite and in the skin, gills, liver, pericardial muscle, and gastrointestinal tract in those exposed to 1000 μM or 2000 μM arsenite. Exposure to 1500 μM arsenate resulted in expression in skin, liver, and gastrointestinal tract, while induction was observed in skin, gills, liver, pericardial muscle and gastrointestinal tract of larvae exposed to 2500 μM or 7500 μM arsenate. Overall expression patterns of hsp70-eGFP in transgenic zebrafish larvae exposed to arsenic were found to closely mimic that of endogenous hsp70 expression patterns in wild-type larvae suggesting that it is an accurate indicator of endogenous hsp70 expression.

Various colloidal delivery systems, including polymeric nanoparticles, metal colloids, liposomes, and microemulsions have been reported to enhance the delivery of therapeutic agents following intranasal administration. However, the mechanisms involved in the uptake of these nanomaterials, especially those in the ultrafine size ranges (diameter < 20 nm) through nasal mucosa and their subsequent biodistribution in the body are not well characterized. The objectives of this study address the knowledge gap regarding ultrafine nanoparticle transfer in the nasal mucosa by quantifying nanoparticle uptake and biodistibution patterns in the presence and absence of known inhibitors of endocytic processes. The uptake of ~ 10 nm fluorescent quantum dots (QDs) was investigated by measuring the concentration of QDs following exposure to bovine respiratory and olfactory mucosal explants. An inductively coupled optical emission spectroscopy method was developed to measure the amount of QDs within the tissues. The results demonstrated that carboxylate-modified QDs (COOH-QDs) show ~2.5 fold greater accumulation in the epithelial and submucosal regions of the olfactory tissues compared to the respiratory tissues. Endocytic inhibitory studies showed that in respiratory tissues clathrin-dependent, macropinocytosis and caveolae-dependent endocytosis process were all involved in the uptake of COOH-QDs. Whereas in olfactory tissues, clathrin-dependent endocytosis was the major endocytic pathway involved in uptake of COOH-QDs. Additional energy-independent pathways appeared to also be active in the transfer of COOH-QDs into the olfactory mucosa. Interestingly, PEGylated quantum dots (PEG-QDs) of similar size ~15 nm were not internalized into the bovine nasal tissues. In vivo fluorescence imaging was used to study the biodistribution of quantum dots following nasal instillation in mice. These studies showed that majority of COOH-QDs remain in the nasal tissues for relatively long periods of time (up to 24 h) whereas PEG-QDs showed no such accumulation. Biodistribution studies of gold nanoparticles (~15 nm) in mice using micro-CT showed that gold nanoparticles were transferred to the posterior turbinate region and a fraction of the administered dose distributed to regions in close proximity to the olfactory bulb. Both NIR imaging and micro-CT imaging were useful tools for visualization of in vivo nanoparticle distribution. A diazepam-containing microemulsion (dispersed phase ~40 nm) was formulated to investigate the uptake mechanisms utilized for fluid-phase colloidal dispersions in the nasal mucosa. The resulting diazepam-containing microemulsion showed enhanced transfer of the drug into the bovine nasal respiratory and olfactory tissues. It is unclear if endocytosis of the fluid-phase nanodispersions played a role in drug absorption from the microemulsions in a manner similar to the uptake of solid-phase nanoparticles, however, since there was significant loss of the epithelial cell layer following exposure to the microemulsion formulation which likely altered the barrier properties of the epithelium. These studies have increased the fundamental understanding of ultrafine nanoparticle uptake in the nasal tissues and the resulting nanoparticle biodistribution patterns. While ultrafine nanoparticles may have limited application in the development of efficient drug delivery systems, an understanding of the size-dependent and tissue-dependent processes responsible for the uptake of particulates into mucosal tissues will contribute to the rational development of nanoparticulate drug delivery strategies investigating the nasal and other routes of administration.

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