Nanoparticle effects on rat alveolar epithelial cell monolayer barrier properties

Abstract

Inhaled nanoparticles have been reported to contribute to deleterious effects on human health. In this study, we investigated the effects of ultrafine ambient particulate suspensions (UAPS), polystyrene nanoparticles (PNP; positively and negatively charged; 20, 100, 120 nm), quantum dots (QD; positively and negatively charged; 30 nm) and single-wall carbon nanotubes (SWCNT) on alveolar epithelial cell barrier properties. Transmonolayer resistance (R(t)) and equivalent short-circuit current (I(eq)) of primary rat alveolar epithelial monolayers were measured in the presence and absence of varying concentrations of apical nanoparticles. In some experiments, apical-to-basolateral fluxes of radiolabeled mannitol or inulin were determined with or without apical UAPS exposure and lactate dehydrogenase (LDH) release was analyzed after UAPS or SWCNT exposure. Results revealed that exposure to UAPS decreased R(t) and I(eq) significantly over 24 h, although neither mannitol nor inulin fluxes changed. Positively charged QD decreased R(t) significantly (with subsequent recovery), while negatively charged QD did not. R(t) decreased significantly after SWCNT exposure (with subsequent recovery). On the other hand, PNP exposure had no effects on R(t) or I(eq). No significant increases in LDH release were observed after UAPS or SWCNT exposure. These data indicate that disruption of alveolar epithelial barrier properties due to apical nanoparticle exposure likely involves alteration of cellular transport pathways and is dependent on specific nanoparticle composition, shape and/or surface charge.

Figure 1

Effects of apical exposure to UAPS on Rt of RAECM (n = 4–9 for each concentration). Rt of all monolayers prior to apical UAPS exposure (at t = 0) was 1.41 ± 0.05 KΩ.cm² (n = 45). At the maximum concentration of UAPS (36 μg/mL) studied, Rt declined significantly by 60% after 2 hours of exposure and did not change further over 24 hours. * = significantly different (p < 0.05) from control (monolayers not exposed to UAPS).

Figure 2

Effects of apical exposure to UAPS on Ieq of RAECM (n = 4–9 for each concentration). Ieq of all monolayers prior to apical UAPS exposure of (at t = 0) was 4.72 ± 0.10 μA/cm² (n = 45). At the maximum concentration of UAPS (36 μg/mL) studied, Ieq declined significantly by ~25% after 30 min of exposure and did not change further for up to 24 hours. * = significantly different (p < 0.05) from control (monolayers not exposed to UAPS).

Figure 3

Effects of removal of apical UAPS (9 μg/mL) on Rt of RAECM after 2 hours of exposure. Rt prior to apical exposure to UAPS (t = 0) was 2.33 ± 0.68 KΩ.cm² (n = 3). After 1 and 2 hours of UAPS exposure, Rt decreased significantly. Rt after replacement of UAPS with fresh culture medium recovered toward control values. * = significantly different (p < 0.05) from control (monolayers not exposed to UAPS).

Figure 4

Effects of apical exposure to QD (176 μg/mL) on Rt of RAECM (n = 6 for each concentration). Rt of all monolayers prior to apical exposure to QD (at t = 0) was 2.68 ± 0.14 KΩ.cm² (n = 19). Exposure to positively charged QD decreased Rt significantly after 1 hour, with recovery to control level by 24 hours. * = significantly different (p < 0.05) from control (monolayers not exposed to QD).

Figure 5

Effects of apical exposure to SWCNT on Rt of RAECM (n = 9 for each concentration). Rt of all monolayers used prior to apical exposure to SWCNT (at t = 0) was 3.24 ± 0.07 KΩ.cm² (n = 33). Exposure to SWCNT up to 88 μg/mL decreased Rt significantly after 1 hour. Rt recovered to control level by 4–24 hours. * = significantly different (p < 0.05) from control (monolayers not exposed to SWCNT).

Figure 6

Effects of apical exposure to PNP (176 μg/mL, 20 nm positively and negatively charged and 100 nm negatively charged) on Rt of RAECM (n = 10–13 for each concentration). Rt of all monolayers prior to apical exposure to PNP (at t = 0) was 3.58 ± 0.11 KΩcm² (n = 47). These three types of PNP, as well as positively charged 120 nm PNP (data not shown), did not cause significant changes in Rt or Ieq (data not shown).

Figure 7

Effects of apical exposure to UAPS (36 μg/mL), SWCNT (88 μg/mL), QD (positively and negatively charged, 176 μg/mL), and PNP (20 nm (negatively and positively charged) and 100 nm (negatively charged), 176 μg/mL) on Rt of RAECM after 2, 4 and 24 hours of exposure (n = 5–9 for each condition). Rt of all monolayers prior to apical exposure to nanoparticles (at t = 0) was 2.13 ± 0.27 KΩ.cm² (n = 66). * = significantly different (p < 0.05) from control (unexposed monolayers).