Abstract
BackgroundPredominantly, 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.ResultsOff-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.ConclusionIn 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.
Highlights
Studies of nanoparticle (NPs) toxicology in vitro are based upon the exposure of submerged cell cultures to particle suspensions
An aerosol is a complex mixture of gaseous and particulate components which may both trigger adverse health effects. This characteristic is of special importance in the case of combustion derived particles, as high – temperature processes such as flame synthesis release chemically active gases as by products
Flame off gas was found to elicit a significant decrease of total reduced GSH and to induce further the release of the pro – inflammatory cytokine Tumor Necrosis Factor alpha (TNFα). Such findings demonstrate the contribution of the gas phase on aerosol toxicology
Summary
Studies of nanoparticle (NPs) toxicology in vitro are based upon the exposure of submerged cell cultures to particle suspensions. Such an approach does not reflect particle inhalation. Medium properties (i.e. protein content, ionic strength, viscosity, density, pH) interfere with diffusion and sedimentation driven particle motion (particokinetics) and shape agglomeration processes [15,16,17] To avoid such artificial “matrix effects” originating from culture medium, novel direct aerosol exposure systems have been designed which consider aerosols as multifactorial systems, composed of gas and therein dispersed particle components [12,18,19,20,21,22]. In vitro exposure of aerosols at the air liquid interface (ALI) represents a more realistic exposure scenario as it preserves the physical and chemical characteristics of airborne particles [23]
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