Size and dose dependent effects of silver nanoparticle exposure on intestinal permeability in an in vitro model of the human gut epithelium

Abstract

Background: The antimicrobial activity of silver nanoparticles (AgNP) has led to interest in their use in consumer products such as food contact materials, utensils, and storage containers. Incorporation of these materials into items intended for food processing and storage suggests that consumer use of these products could result in gastrointestinal exposure to AgNP, should the nanoparticles migrate from the product. The health impact of AgNP exposure is unknown, especially effects related to intestinal epithelial permeability and barrier function. This study examined the effects of AgNP exposure of different sizes (10, 20, 75 and 110 nm) and doses (20 and 100 µg/mL) on the permeability of T84 human colonic epithelial cells, which serve as an in vitro model of the human gut epithelium.

Results: Results showed that effects of AgNP on the T84 epithelial cells were size- and dose-dependent, with the 10 nm AgNP causing the most significant changes. Changes in permeability of the epithelial cell monolayer, as measured by transepithelial electrical resistance, after exposure to 10 nm AgNP were most dramatic at the highest dose (100 µg/mL), but also observed at the lower dose (20 µg/mL). AgNP could be visualized inside cells using transmission electron microscopy and silver was detected in basal wells using inductively coupled plasma-mass spectrometry. Exposure to AgNP significantly affected the expression of genes involved in anchoring tight junctions, cellular proliferation and signaling, endocytosis, and cell-cell adhesion, with the 10 nm AgNP having the greatest effect.

Conclusions: The results of this study show that small-size AgNP have significant effects on intestinal permeability in an in vitro model of the human gastrointestinal epithelium. Such effects have the potential to compromise the integrity of the intestinal epithelium and this disruption of barrier function could have health consequences for the gastrointestinal tract.

Keywords: Barrier function; Cell junctions; Intestinal permeability; Silver nanoparticles.

Fig. 1

TEM image of T84 cells grown on transwells showing polarized cells with villi and junctions between individual cells

Fig. 2

Acridine orange/ethidium bromide staining of T84 cell monolayers showing a control cells and cells after treatment with b 10 nm, c 20 nm, d 75 nm, and e 110 nm AgNP (100 µg/mL) for 48 h. T84 cells after treatment with 100 µg/mL of silver acetate (f) for 3 h are also shown

Fig. 3

Results of ATP-based cell viability assay after treatment with AgNP, silver acetate, and controls for 48 h. ‘Control’ cells were treated only with water (negative control), while ‘Tox C’ cells were treated with 0.5 % hydrogen peroxide (positive control). Error bars represent SEM and presence of asterisk denotes significance at p < 0.05

Fig. 4

Relative changes in TER of T84 cells after exposure to AgNP-20 µg/mL (a) AgNP-100 µg/mL (b), and controls (c). Error bars represent standard error of the mean (SEM) of 6 independent experiments

Fig. 5

TEM images of T84 cells with internalized nanoparticles (arrows) after exposure to a 10 nm and b 75 nm AgNP

Fig. 6

Expression of Occludin protein in T84 cells after 24 h treatment with a control, b 10 nm AgNP, c EGTA, and d silver acetate. Arrows point to suspected nanoparticle agglomerates in AgNP-treated cells

Fig. 7

Changes in relative expression of genes related to cell–cell junctions and epithelial barrier function in T84 cells exposed to 10 nm AgNP, 20 nm AgNP, and silver acetate compared to control cells. Error bars represent SEM and presence of asterisk denotes significance at p < 0.05

Fig. 8

Summary of AgNP effects observed in this study. Exposure to 10 nm AgNP was shown to increase permeability of the epithelial barrier, while exposure to larger particles (20, 75, 110 nm) did not demonstrate any significant effects