Effects of flame made zinc oxide particles in human lung cells - a comparison of aerosol and suspension exposures

Abstract

Background: Predominantly, studies of nanoparticle (NPs) toxicology in vitro are based upon the exposure of submerged cell cultures to particle suspensions. Such an approach however, does not reflect particle inhalation. As a more realistic simulation of such a scenario, efforts were made towards direct delivery of aerosols to air-liquid-interface cultivated cell cultures by the use of aerosol exposure systems.This study aims to provide a direct comparison of the effects of zinc oxide (ZnO) NPs when delivered as either an aerosol, or in suspension to a triple cell co-culture model of the epithelial airway barrier. To ensure dose-equivalence, ZnO-deposition was determined in each exposure scenario by atomic absorption spectroscopy. Biological endpoints being investigated after 4 or 24h incubation include cytotoxicity, total reduced glutathione, induction of antioxidative genes such as heme-oxygenase 1 (HO-1) as well as the release of the (pro)-inflammatory cytokine TNFα.

Results: Off-gases released as by-product of flame ZnO synthesis caused a significant decrease of total reduced GSH and induced further the release of the cytokine TNFα, demonstrating the influence of the gas phase on aerosol toxicology. No direct effects could be attributed to ZnO particles. By performing suspension exposure to avoid the factor "flame-gases", particle specific effects become apparent. Other parameters such as LDH and HO-1 were not influenced by gaseous compounds: Following aerosol exposure, LDH levels appeared elevated at both timepoints and the HO-1 transcript correlated positively with deposited ZnO-dose. Under submerged conditions, the HO-1 induction scheme deviated for 4 and 24h and increased extracellular LDH was found following 24h exposure.

Conclusion: In the current study, aerosol and suspension-exposure has been compared by exposing cell cultures to equivalent amounts of ZnO. Both exposure strategies differ fundamentally in their dose-response pattern. Additional differences can be found for the factor time: In the aerosol scenario, parameters tend to their maximum already after 4h of exposure, whereas under submerged conditions, effects appear most pronounced mainly after 24h. Aerosol exposure provides information about the synergistic interplay of gaseous and particulate phase of an aerosol in the context of inhalation toxicology. Exposure to suspensions represents a valuable complementary method and allows investigations on particle-associated toxicity by excluding all gas-derived effects.

Figure 1

Aerosol characterization. A. The total aerosol number concentration was determined in parallel by two measuring devices (FMPS and a miniDiSC) for experimental runs with 22, 45 and 90 sec ZnO production (dataset averaged over a 10 sec interval). The cell cultures were exposed to the airborne particles for 30 min (1800 sec). Shortly after this timespan, the glove box chamber was flooded with fresh, pressurized air to clean the atmosphere (air exchange), resulting in an accelerated decay of particle number (disturbed system). B. Contour plot illustrating the temporal variation of particle size distribution (measuring range from 5.6 to 560 nm) in the 22 sec scenario shown in part A. Number concentration ≤ 1E4 pt/ccm is indicated in blue (baseline). Particle size is expressed as electrophoretic mobility diameter. Measurements were performed with a 1 sec resolution.

Figure 2

Aerosol Deposition measurement. ZnO was quantified by atomic absorption spectroscopy, taking advantage of the solubility of zinc in acetic acid. A ZnO mass deposition of 1.3 – 6.1 μg/cm² was measured in the range of 22 – 90 sec reactor runtime, used for subsequent biological experiments. Points indicate individual measurements, bars and whiskers show mean values and standard deviation. Please note the interrupted axis.

Figure 3

Cytotoxicity assessed by measuring the release of intracellular lactate dehydrogenase in aerosol exposed samples. A cell lysate (“Triton”) served as positive control. No difference between incubator – and gas – control could be observed, indicating no influence of the exposure system on basal cytotoxicity. For all ZnO concentrations, elevated LDH levels were measured.

Figure 4

Quantification of total reduced Glutathione content in aerosol exposed samples. A comparable reduction was observed for flame off – gases and particle aerosols. Tert-Butyl hydroperoxide (TBHP) served as positive control.

Figure 5

Real-time PCR on SOD1 and HO-1 in aerosol exposed cultures. White, open bars represent mean values after 4 h post – incubation, black bars indicate 24 h timepoints. Individual data values are expressed as points. No relevant induction was measured for SOD1. After 4 h, SOD 1 values were 1.5 (SD 0.3), 1.7 (SD 0.1), 1.3 (SD 0.1) and 1.5 (SD 0.1) for Gas – control, 22, 45 and 90 sec particle production. The corresponding data for 24 h were 1.3 (SD 0.1), 1.7 (SD 0.6), 1.6 (SD 0.8), 1.4 (SD0.6). In contrast, HO-1 RNA levels were elevated after 4 and 24 h.

Figure 6

TNFα release, measured by a BioPlex assay in aerosol exposed cultures. A significant influence of the gas – atmosphere was found. Lipopolysaccharide (LPS 30 μg/mL) served as positive control.

Figure 7

ZnO deposition onto cell cultures, out of differently concentrated suspensions, measured by atomic absorption spectroscopy. The deposition reaches a plateau over the first 4 h of incubation. 5 – 60 ppm suspensions correspond to an aerosol – exposure for 22 – 90 seconds. Deposition efficiency increases with concentration. Data are presented as mean ± SD.

Figure 8

LDH release measured in the upper and lower well of the cell culture inserts. Absorption values are expressed as optical density (OD), measured at 490 nm, with 630 nm as reference wavelength. LDH is generally elevated after 24 h incubation time.

Figure 9

Total reduced Glutathione content in suspension exposed cultures. No depletion of GSH could be found.

Figure 10

Real-time PCR on SOD1 and HO-1 in suspension exposed cultures. No regulation of SOD1 was found. In contrast, HO-1 is constantly induced (over the entire dose range) after 4 h incubation. Furthermore, a dose dependent increase was observed after 24 h. White, open bars represent mean values after 4 h post – incubation, black bars indicate 24 h timepoints. Individual values are expressed as data points.

Figure 11

TNFα release, quantified by a BioPlex assay. A dose dependent release was found for the apical compartment after 24 h.