Exploring the potential role of tungsten carbide cobalt (WC-Co) nanoparticle internalization in observed toxicity toward lung epithelial cells in vitro

Abstract

Tungsten carbide cobalt (WC-Co) has been recognized as a workplace inhalation hazard in the manufacturing, mining and drilling industries by the National Institute of Occupational Safety and Health. Exposure to WC-Co is known to cause "hard metal lung disease" but the relationship between exposure, toxicity and development of disease remain poorly understood. To better understand this relationship, the present study examined the role of WC-Co particle size and internalization on toxicity using lung epithelial cells. We demonstrated that nano- and micro-WC-Co particles exerted toxicity in a dose- and time-dependent manner and that nano-WC-Co particles caused significantly greater toxicity at lower concentrations and shorter exposure times compared to micro-WC-Co particles. WC-Co particles in the nano-size range (not micron-sized) were internalized by lung epithelial cells, which suggested that internalization may play a key role in the enhanced toxicity of nano-WC-Co particles over micro-WC-Co particles. Further exploration of the internalization process indicated that there may be multiple mechanisms involved in WC-Co internalization such as actin and microtubule based cytoskeletal rearrangements. These findings support our hypothesis that WC-Co particle internalization contributes to cellular toxicity and suggest that therapeutic treatments inhibiting particle internalization may serve as prophylactic approaches for those at risk of WC-Co particle exposure.

Keywords: Hard metal; Internalization; Lung disease; Nanoparticle; Nanotoxicity; Pulmonary exposure.

Figure 1

WC-Co particle characterization via dynamic light scattering (DLS) of A) nano-WC-Co and B) micro-WC-Co particles suspended in cell culture media (average size = 98 nm and 3.4 µm, respectively) and representative TEM images of C) nano-WC-Co (scale bar = 500 nm) and D) micro-WC-Co (scale bar = 2 µm) particles.

Figure 2

Cell viability after A) nano-WC-Co and B) micro-WC-Co particle exposure and C) oxidative stress indicated by DCF fluorescence after exposure to 1000 µg/mL nano- and micro-WC-Co particles. (^*P < 0.05, ^†P < 0.001 compared to control, ^‡P < 0.05 compared to micro-WC-Co)

Figure 3

Summary of flow cytometry staining profiles after 12 hr WC-Co particle exposure: A) total percentage of apoptotic cells (AV⁺/PI⁺ and AV⁺/PI⁻; sum total of upper and lower right quadrants), B) total percentage of viable cells (AV⁻/PI⁻; lower left quadrant) and C) total percentage of necrotic cells (PI⁺/AV⁻; upper left quadrant) (^*P < 0.05 compared to control, ^‡P < 0.05 compared to micro-WC-Co)

Figure 4

Cell viability after exposure to nano- or micro-WC-Co particles in the presence of 10 µg/mL cytoskeletal inhibitors MDC, colchicine or cytochalasin D after A) 6 hr, B) 12 hr and C) 48 hr. [^*P < 0.05, ^†P < 0.001 compared to control (particles only)]

Figure 5

Representative TEM images of A) non-exposed control cells, B) cells exposed to 100 µg/mL nano-WC-Co for 12 hr, C) cells exposed to 100 µg/mL micro-WC-Co for 12 hr and D) cells exposed to 100 µg/mL nano-WC-Co plus 10 µg/mL cytochalasin D for 12 hr. Arrows denote WC-Co particles; scale bars = 0.5 µm.