Functional Effects of Carbon Nanoparticles on Barrier Epithelial Cell Function

Abstract

As mass production of carbon nanoparticles (CNPs) continues to rise, the likelihood of occupational and environmental exposure raises the potential for exposure‐related health hazards. Although many groups have studied the effects of CNPs on biological systems, very few studies have examined the effects of exposure of cells, tissues or organisms to low, physiologically relevant concentrations of CNPs. Three of the most common types of CNPs are single wall nanotubes (SWNT), multi wall nanotubes (MWNT) and fullerenes (C60). We used electrophysiological techniques to test the effects of CNP exposure (40 μg/cm2 – 4 ng/cm2) on barrier function and hormonal responses of well characterized cell lines representing barrier epithelia from the kidney (mpkCCDcl4) and airways (Calu‐3). mpkCCDcl4 is a cell line representing principal cell type that lines the distal nephron in an electrically tight epithelia that aids in salt and water homeostasis and Calu‐3 is one of the few cell lines that produces features of a differentiated, functional human airway epithelium in vivo. These cell lines respond to hormones that regulate salt/water reabsorption (mpkCCDcl4) and chloride secretion (Calu‐3). In mpkCCDcl4 cells, after 48 hour exposure, the transepithelial electrical resistance (TEER) was unaffected by high concentrations (40 – 0.4 μg/cm2) of C60 or SWNT while lower, more relevant levels (< 0.04 μg/cm2) caused a decrease in TEER. MWNT decreased TEER at both high and low concentrations. CNT exposure for 48 hour did not change the transepithelial ion transport in response to anti‐diuretic hormone (ADH). In Calu‐3 cells, after 48 h of exposure to CNPs, fullerenes did not show any effect on TEER whereas the nanotubes significantly decreased TEER over a range of concentrations (4 μg/cm2‐0.004 ng/cm2). The ion transport response to epinephrine was also significantly decreased by the nanotubes but not by fullerenes. To look at the effect of exposure times, airway cells were exposed to same concentrations of CNPs for 24 and 1h. While the 48 h and 24 h exposures exhibited similar effects, there was no effect seen after 1h in terms of TEER or hormonal responses. In both the cell lines the magnitude of the transepithelial resistance change does not indicate a decrease in cellular viability but would be most consistent with more subtle changes (e.g., modifications of the cytoskeleton or changes in the composition of the cellular membrane). These changes in both the cell lines manifested as an inverse relationship with CNP concentration, were further corroborated by an inverse correlation between dose and changes in protein expression as indicated by proteomic analysis. These results indicate a functional impact of CNPs on epithelial cells at concentrations lower than have been previously studied and suggest caution with regard to increasing CNP levels due to increasing environmental pollution.