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

Raemy DO, Grass RN, Stark WJ, Schumacher CM, Clift MJ, Gehr P, Rothen-Rutishauser B - Part Fibre Toxicol (2012)

Bottom Line: Such an approach however, does not reflect particle inhalation.No direct effects could be attributed to ZnO particles.Exposure to suspensions represents a valuable complementary method and allows investigations on particle-associated toxicity by excluding all gas-derived effects.

View Article: PubMed Central - HTML - PubMed

Affiliation: Adolphe Merkle Institute, Bionanomaterials, University of Fribourg, Rte de l'Ancienne Papeterie, P.O. Box 209, CH-1732, Marly, Switzerland.

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.

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Related in: MedlinePlus

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.
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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.

Mentions: The cellular total glutathione (GSH) content (normalized to cellular protein) provides an indirect estimator for oxidative stress. As illustrated in Figure4, total reduced GSH decreased independent of if cell cultures were either exposed to reactor off-gas (gas – control) or particle aerosols. In the incubator – controls, a GSH to protein ratio of 1.33*10-4 (SD 4.30*10-4, n = 8) and 2.08*10-3 (SD 4.78*10-4, n = 8) was measured after 4 and 24 h post – incubation. Gas – controls exhibited ratios of 9.91*10-5 (SD 5.81*10-5) and 2.79*10-4 (SD 3.60*10-5). This gas – dependent reduction was found to be statistically significant (both p = 0.014) for the 4 and 24 h timepoint. ZnO aerosols produced for 22, 45 and 90 sec led to values of 1.42*10-4 (SD 8.84*10-5), 1.81*10-4 (SD 1.05*10-4) and 1.45*10-4 (SD 7.09*10-5) for 4 h and 3.87*10-4 (SD 1.98*10-4), 4.11*10-4 (SD 1.79*10-4) and 3.99*10-4 (SD 7.45*10-5) for 24 h post – incubation. No statistical difference between gas – control and the different zinc doses was observed. A significant (p = 0.000) difference between 4 and 24 h values was found, indicating a certain recovery of cellular antioxidant capacity after prolonged time. With 9.23*10-5 (SD 7.94*10-5, n = 7) and 8.20*10-5 (SD 1.15*10-4, n = 7), a maximal reduction was observed for the TBHP positive controls.


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

Raemy DO, Grass RN, Stark WJ, Schumacher CM, Clift MJ, Gehr P, Rothen-Rutishauser B - Part Fibre Toxicol (2012)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3585858&req=5

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.
Mentions: The cellular total glutathione (GSH) content (normalized to cellular protein) provides an indirect estimator for oxidative stress. As illustrated in Figure4, total reduced GSH decreased independent of if cell cultures were either exposed to reactor off-gas (gas – control) or particle aerosols. In the incubator – controls, a GSH to protein ratio of 1.33*10-4 (SD 4.30*10-4, n = 8) and 2.08*10-3 (SD 4.78*10-4, n = 8) was measured after 4 and 24 h post – incubation. Gas – controls exhibited ratios of 9.91*10-5 (SD 5.81*10-5) and 2.79*10-4 (SD 3.60*10-5). This gas – dependent reduction was found to be statistically significant (both p = 0.014) for the 4 and 24 h timepoint. ZnO aerosols produced for 22, 45 and 90 sec led to values of 1.42*10-4 (SD 8.84*10-5), 1.81*10-4 (SD 1.05*10-4) and 1.45*10-4 (SD 7.09*10-5) for 4 h and 3.87*10-4 (SD 1.98*10-4), 4.11*10-4 (SD 1.79*10-4) and 3.99*10-4 (SD 7.45*10-5) for 24 h post – incubation. No statistical difference between gas – control and the different zinc doses was observed. A significant (p = 0.000) difference between 4 and 24 h values was found, indicating a certain recovery of cellular antioxidant capacity after prolonged time. With 9.23*10-5 (SD 7.94*10-5, n = 7) and 8.20*10-5 (SD 1.15*10-4, n = 7), a maximal reduction was observed for the TBHP positive controls.

Bottom Line: Such an approach however, does not reflect particle inhalation.No direct effects could be attributed to ZnO particles.Exposure to suspensions represents a valuable complementary method and allows investigations on particle-associated toxicity by excluding all gas-derived effects.

View Article: PubMed Central - HTML - PubMed

Affiliation: Adolphe Merkle Institute, Bionanomaterials, University of Fribourg, Rte de l'Ancienne Papeterie, P.O. Box 209, CH-1732, Marly, Switzerland.

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.

Show MeSH
Related in: MedlinePlus