Date of Graduation

2014

Document Type

Dissertation

Degree Type

PhD

College

School of Pharmacy

Department

Pharmaceutical Sciences

Committee Chair

Bingyun Li

Committee Co-Chair

Peter Gannett

Committee Member

Rae Matsumoto

Committee Member

Tim Nurkiewicz

Committee Member

Yon Rojanasakul

Abstract

Hard metals, such as tungsten carbide cobalt (WC-Co), are frequently used for a number of industrial applications such as surface coatings for heavy machinery and tools. In particular, WC-Co coatings are prevalent in mining and drilling applications where extensive, repetitive use of these tools causes wear over time. In enclosed environments, WC-Co wear particles become airborne and present an occupational inhalation hazard. It is known that inhalation of WC-Co "dusts", composed of nano- and micro-sized WC-Co particles, contributes to the development of hard metal lung disease and increased risk for lung cancer; however, the relationship between acute WC-Co toxicity and disease progression remains poorly understood. To address this gap in knowledge, we systematically evaluated nano-WC-Co particle toxicity using a combination of in vitro and in vivo models. In Aim 1, we determined the toxicity of nano-WC-Co particles in BEAS-2B lung epithelial cells over concentrations ranging 0.1 to 1000 mug/mL and exposure periods from 0.5 to 48 hr. Our MTT-based cell viability assay indicated that nano-WC-Co exhibits greater toxicity than micro-WC-Co at concentrations ≥ 10 mug/mL. We also found that nano-WC-Co exposure induces oxidative stress at the highest particle concentration tested (1000 mug/mL) using a fluorescence-based (DCF/DHE) assay and that WC-Co particle exposure induced cellular apoptosis, marked by increased annexin-V staining in our flow cytometry apoptosis assay. The potential for nano-WC-Co particle internalization was also investigated using transmission electron microscopy (TEM) and confirmed that nano-WC-Co particles are capable of being internalized by BEAS-2B cells. In Aim 2, we determined the inflammatory response toward nano-WC-Co particles in a co-culture model of BEAS-2B cells and macrophages, to more closely represent the dynamic tissue environment of the lung. The results of our viability assay indicated that macrophages attenuated the toxicity of nano-WC-Co in the co-culture model compared to BEAS-2B alone, which indicated a protective effect of the macrophages. We found that nano-WC-Co exposure caused macrophage polarization toward the M1 pro-inflammatory phenotype and determined that nano-WC-Co exposure also stimulates the secretion of cytokines such as IL-12 and IL-1beta in macrophages, consistent with a pro-inflammatory response. In Aim 3, we investigated the potential systemic (extra-pulmonary) effects of nano-WC-Co exposure in an intra-tracheal instillation (IT) rat model and compared the outcomes with a known pulmonary irritant, cerium dioxide (CeO2). After 24 hr exposure, nano-WC-Co exposure did not induce pulmonary or systemic inflammation at a dose of 50, 250 or 500 mug compared to control or CeO2; this outcome highlights the need for future in vivo studies which examine the inflammatory effects of chronic or repeated nano-WC-Co exposure. Taken together, the results of our studies improve the current understanding of hard metal WC-Co toxicity and may point toward potential therapeutic or diagnostic strategies for the future.

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