In vitro and In vivo toxicity assessment of metal oxide nanomaterials

Metal oxide nanomaterials (NMs) have been used in various applications and they possess unique properties that differ from their bulk materials; however, there are risks associated with inadvertent exposure, especially through inhalation, that need to be assessed and understood. Toxicity assessment of inhaled metal oxide NMs has been widely studied to investigate pulmonary toxicity and inflammation. Copper oxide (CuO) NMs are one of the most toxic metal oxide NMs and are widely used in several fields. Therefore, CuO NMs were chosen as a candidate to further study in terms of biodistribution, translocation to secondary organs, and immunomodulatory effects.The first part of this research was to perform characterizations and in vitro toxicity screening of metal oxide NMs independently comprising ZnO, CuO, CeO2, Fe2O3, WO3, V2O5, TiO2, Al2O3, and MgO. NMs that exhibited the highest toxicity responses in the in vitro screening assays (ZnO, CuO, and V2O5) and the lesser-explored WO3 NMs were tested for acute pulmonary toxicity in vivo. Female and male mice (C57Bl/6J) were exposed to aerosolized metal oxide NMs in a nose-only exposure system, and toxicity outcomes (cytotoxicity, immunotoxicity, inflammation, and lung histopathology) at 4 and 24 hr after the start of exposure were assessed. Among the NMs studied, the highest toxicity was observed for CuO NMs and ZnO NMs in both in vitro and in vivo acute models. Compared to sham-exposed controls, there were significant increases in levels of neutrophils and proinflammatory cytokines, and a loss of macrophage viability, in the bronchoalveolar lavage fluid from mice exposed to ZnO NMs and CuO NMs at both 4 hr and 24 hr but not for mice exposed to V2O5 NMs or WO3 NMs. These effects were observed in both female and male mice. The cell viability assays performed after in vitro exposure to NMs using viable cell counts with propidium iodide staining, and assessment of lung inflammation after acute inhalation exposure in vivo were shown to be sensitive endpoints to predict NM acute toxicity. It has been shown that CuO NMs induce pulmonary toxicity after acute or sub-acute inhalation exposures. However, little is known about the biodistribution and elimination kinetics of inhaled CuO NMs from the respiratory tract. Therefore, the second part of this dissertation aimed to observe the kinetics of pulmonary toxicity during and after CuO NM sub-acute inhalation exposure and investigate copper (Cu) biodistribution and clearance rate from the exposure site as well as homeostasis of selected trace elements in secondary organs of BALB/c mice. Our studies demonstrated that sub-acute inhalation exposure to CuO NMs led to pulmonary toxicity represented by increases in lactate dehydrogenase (LDH), total cell counts, neutrophils, macrophages, inflammatory cytokines, and iron (Fe) levels in bronchoalveolar lavage (BAL) fluid, and lung weight changes. Dosimetry analysis in lung tissues and BAL fluid showed that Cu concentrations increased steadily during exposure and gradually declined after exposure. Cu elimination from the exposure site showed first-order kinetics with a half-life of 6.5 days. Total Cu levels were significantly increased in whole blood and heart, indicating that inhaled Cu could be translocated into the bloodstream and heart and potentially have adverse effects on the kidneys and spleen as there were significant changes in the weights of these organs. Furthermore, concentrations of selenium (Se) in kidneys and Fe in the spleen were decreased, pointing to disruption of trace element homeostasis. The next part of this dissertation was to develop a HDM-induced airway inflammation model and a vaccine (allergen immunotherapy, AIT) protecting against HDM-induced airway inflammation to be used as tools to investigate the immunomodulatory effect of CuO NMs. First, we established a HDM-induced airway inflammatory mouse model that resulted in increased TH2-dominant immunological responses and also boosted the number of TH17 cells. Therefore, it can be used to study allergic airway inflammation and potential treatments. Then, we developed HDM vaccines comprising 50 μg CpG nanoparticles with either 50 or 100 μg Der p1/Der p1 mixture (80:20). Both vaccine doses showed no significant reductions in total cell number, eosinophils, total IgE, and HDM allergen-specific IgE in the HDM-induced airway inflammation model. Nonetheless, a trend of greater IgG2a:IgG1 ratios in the vaccination groups compared to the HDM allergen-exposed group suggested a probable activation of TH1-immune responses. Furthermore, the vaccine 100 μg showed a trend toward fewer TH2-type immune responses compared to the vaccine 50 μg, although the differences were non-significant. Therefore, the vaccine 100 μg was used to investigate the effect of CuO NMs aerosols on immune responses. Lastly, we investigated the immunomodulatory effect of CuO NM aerosols under different health conditions, including naïve, asthmatic, and AIT-treated asthmatic mouse models. CuO NM-exposed naïve mice did not show a significant change in CD4+ T cell subtypes in the lung tissues. However, certain immune cell types such as neutrophils, CD11b+ DCs, and alveolar macrophages (AMs) significantly increased compared to naïve mice, indicating the presence of lung inflammation induced by CuO NM exposure. When asthmatic mice were exposed to CuO NM, increased TH1 and Treg cell levels and decreased TH2 cell levels in the lung tissue were observed compared to the non-exposed asthmatic mice. In AIT-treated asthmatic mice, the effect of CuO NM inhalation exposure had no noticeable effect on any of CD4+ T cell subtypes. The effect of CuO NM exposure on airway hyperresponsiveness was investigated in all 3 models, and there was no significant effect on airway sensitivity. However, there was a trend toward lower resistance and elastance in the asthmatic and AIT-treated asthmatic mice. In conclusion, we have established an appropriate in vitro and in vivo acute inhalation study using simple endpoints that can predict the toxicity of NMs. In addition, we have investigated biodistribution and clearance of CuO NMs; the effect of CuO NMs on extrapulmonary organs; and the immunomodulatory effects of CuO NMs in mouse models exposed to CuO NMs via inhalation.