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

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

Can the Ames test provide an insight into nano-object mutagenicity? Investigating the interaction between nano-objects and bacteria

The aim of this study was to assess the interaction of a series of well characterised nano-objects with the Gram negative bacterium Salmonella typhimurium, and how such an interaction may relate to the potential mutagenicity of nano-objects. Transmission electron microscopy showed that nano-objects (Au-PMA-ATTO NPs, CeO₂ NPs, SWCNTs and MWCNTs), as well as CAFs entered S. typhimurium. Only DEPs did not penetrate/enter the bacteria, however, were the only particle stimulus to induce any significant mutagenicity through the Ames test. Comparison with a sophisticated 3D in vitro cell model showed CAFs, DEPs, SWCNTs and MWCNTs to cause a significant increase in mammalian cell proliferation, whilst both the Au-PMA-ATTO NPs and CeO₂ NPs had not significant adverse effects. In conclusion, these results indicate that various of different nano-objects are able to penetrate the double-lipid bilayer of Gram negative bacteria, although the Ames test may not be a good indicator for nano-object mutagenicity

Direct combination of nanoparticle fabrication and exposure to lung cell cultures in a closed setup as a method to simulate accidental nanoparticle exposure of humans

The tremendous application potential of nanosized materials stays in sharp contrast to a growing number of critical reports of their potential toxicity. Applications of in vitro methods to assess nanoparticles are severely limited through difficulties in exposing cells of the respiratory tract directly to airborne engineered nanoparticles. We present a completely new approach to expose lung cells to particles generated in situ by flame spray synthesis. Cerium oxide nanoparticles from a single run were produced and simultaneously exposed to the surface of cultured lung cells inside a glovebox. Separately collected samples were used to measure hydrodynamic particle size distribution, shape, and agglomerate morphology. Cell viability was not impaired by the conditions of the glovebox exposure. The tightness of the lung cell monolayer, the mean total lamellar body volume, and the generation of oxidative DNA damage revealed a dose-dependent cellular response to the airborne engineered nanoparticles. The direct combination of production and exposure allows studying particle toxicity in a simple and reproducible way under environmental conditions

Cerium oxide nanoparticle uptake kinetics from the gas-phase into lung cells in vitro is transport limited

Nowadays, aerosol processes are widely used for the manufacture of nanoparticles (NPs), creating an increased occupational exposure risk of workers, laboratory personnel and scientists to airborne particles. There is evidence that possible adverse effects are linked with the accumulation of NPs in target cells, pointing out the importance of understanding the kinetics of particle internalization. In this context, the uptake kinetics of representative airborne NPs over 30 min and their internalization after 24 h post-exposure were investigated by the use of a recently established exposure system. This system combines the production of aerosolized cerium oxide (CeO(2)) NPs by flame spray synthesis with its simultaneous particle deposition from the gas-phase onto A549 lung cells, cultivated at the air-liquid interface. Particle uptake was quantified by mass spectrometry after several exposure times (0, 5, 10, 20 and 30 min). Over 35% of the deposited mass was found internalized after 10 min exposure, a value that increased to 60% after 30 min exposure. Following an additional 24 h post-incubation, a time span, after which adverse biological effects were observed in previous experiments, over 80% of total CeO(2) could be detected intracellularly. On the ultrastructural level, focal cerium aggregates were present on the apical surface of A549 cells and could also be localized intracellularly in vesicular structures. The uptake behaviour of aerosolized CeO(2) is in line with observations on cerium suspensions, where particle mass transport was identified as the rate-limiting factor for NP internalization

Comparison of manganese oxide nanoparticles and manganese sulfate with regard to oxidative stress, uptake and apoptosis in alveolar epithelial cells

Due to their physicochemical characteristics, metal oxide nanoparticles (NPs) interact differently with cells compared to larger particles or soluble metals. Oxidative stress and cellular metal uptake were quantified in rat type II alveolar epithelial cells in culture exposed to three different NPs: manganese(II,III) oxide nanoparticles (Mn(3)O(4)-NPs), the soluble manganese sulfate (Mn-salt) at corresponding equivalent doses, titanium dioxide (TiO(2)-NPs) and cerium dioxide nanoparticles (CeO(2)-NPs). In the presence of reactive oxygen species an increased apoptosis rate was hypothesized. Oxidative stress was assessed by detection of fluorescently labeled reactive oxygen species and by measuring intracellular oxidized glutathione. Catalytic activity was determined by measuring catalyst-dependent oxidation of thiols (DTT-assay) in a cell free environment. Inductively coupled plasma mass spectrometry was used to quantify cellular metal uptake. Apoptosis rate was determined assessing the activity of caspase-3 and by fluorescence microscopic quantification of apoptotic nuclei. Reactive oxygen species were mainly generated in cells treated with Mn(3)O(4)-NPs. Only Mn(3)O(4)-NPs oxidized intracellular glutathione. Catalytic activity could be exclusively shown for Mn(3)O(4)-NPs. Cellular metal uptake was similar for all particles, whereas Mn-salt could hardly be detected within the cell. Apoptosis was induced by both, Mn(3)O(4)-NPs and Mn-salt. The combination of catalytic activity and capability of passing the cell membrane contributes to the toxicity of Mn(3)O(4)-NPs. Apoptosis of samples treated with Mn-salt is triggered by different, potentially extracellular mechanisms