Biomechanical effects of environmental and engineered particles on human airway smooth muscle cells

Abstract

The past decade has seen significant increases in combustion-generated ambient particles, which contain a nanosized fraction (less than 100 nm), and even greater increases have occurred in engineered nanoparticles (NPs) propelled by the booming nanotechnology industry. Although inhalation of these particulates has become a public health concern, human health effects and mechanisms of action for NPs are not well understood. Focusing on the human airway smooth muscle cell, here we show that the cellular mechanical function is altered by particulate exposure in a manner that is dependent upon particle material, size and dose. We used Alamar Blue assay to measure cell viability and optical magnetic twisting cytometry to measure cell stiffness and agonist-induced contractility. The eight particle species fell into four categories, based on their respective effect on cell viability and on mechanical function. Cell viability was impaired and cell contractility was decreased by (i) zinc oxide (40-100 nm and less than 44 microm) and copper(II) oxide (less than 50 nm); cell contractility was decreased by (ii) fluorescent polystyrene spheres (40 nm), increased by (iii) welding fumes and unchanged by (iv) diesel exhaust particles, titanium dioxide (25 nm) and copper(II) oxide (less than 5 microm), although in none of these cases was cell viability impaired. Treatment with hydrogen peroxide up to 500 microM did not alter viability or cell mechanics, suggesting that the particle effects are unlikely to be mediated by particle-generated reactive oxygen species. Our results highlight the susceptibility of cellular mechanical function to particulate exposures and suggest that direct exposure of the airway smooth muscle cells to particulates may initiate or aggravate respiratory diseases.

Figure 1.

The measurement of cell stiffness, contractile response and relaxing response using OMTC. (a) The working mechanism of OMTC. The magnetized bead (M) is twisted by the vertical magnetic field (H); the lateral motion of the bead (d) is inversely related to the cell stiffness (G). (b) Normalized stiffness changes in response to histamine and isoproterenol are shown for over 1000 untreated cells (median ± s.e.). The maximum contractile response (at approx. 10 s after adding histamine) and maximum relaxing response (at approx. 250 s after adding histamine) are denoted C and R, respectively.

Figure 2.

The effect of eight particles on the viability of HASM cells after correcting for the optical interference of particles. (a) At the doses tested, TiO₂, DEP, FS and WF caused little change in cell viability. (b) CuO NPs, but not MPs, caused dose-dependent decrease in viability. (a) and (b) have the same x-axis. (c) Both ZnO NPs and MPs impaired cell viability, with the former being more toxic. Also shown here is the soluble ZnCl₂. In this and all subsequent figures, an asterisk indicates statistically significant difference from the negative control (p < 0.01). (a) Lines with open circles, TiO₂; lines with open inverted triangles, DEP; lines with stars, FS; lines with open diamonds, WF. (b) Lines with open squares, CuO^{5 μm}; lines with filled squares, CuO^{50 nm}. (c) Lines with open triangles, ZnO^{44 μm}; lines with filled triangles, ZnO^{40 nm}; lines with crosses, ZnCl₂ (the actual mass dose was scaled by MW_ZnO/MW_ZnCl2 to match the zinc concentration; MW, molecular weight).

Figure 3.

TiO₂ and DEP caused minor changes in (a) cell stiffness, (b) contractile response induced by histamine and (c) relaxing response induced by isoproterenol. All indices have been normalized to those of untreated cells. Lines with open circles, TiO₂; lines with open inverted triangles, DEP.

Figure 4.

ZnO NPs and MPs caused dose-dependent inhibition of (a) cell stiffness, (b) contractile response and (c) relaxing response. Lines with open triangles, ZnO^{44 μm}; lines with filled triangles, ZnO^{40 nm}.

Figure 5.

CuO NPs, but not MPs, caused dose-dependent inhibition of (a) cell stiffness, (b) contractile response and (c) relaxing response. Lines with open squares, CuO^{5 μm}; lines with filled squares, CuO^{50 nm}.

Figure 6.

FS and WF changed cell mechanics in opposite ways. FS dose dependently inhibited (a) stiffness, (b) contraction and (c) relaxation. By contrast, WF had little effect on (a) stiffness or (c) relaxation, but (b) dose dependently enhanced contractile response to histamine. Lines with stars, FS; lines with diamonds, WF.