Adolphe Merkle Institute, Bionanomaterials, University of Fribourg, Rte de l'Ancienne Papeterie, P.O. Box 209, CH-1732, Marly, Switzerland.
Part Fibre Toxicol. 2012 Aug 17;9:33. doi: 10.1186/1743-8977-9-33.
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.
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.
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.
目前，体外纳米颗粒（NPs）毒理学研究主要基于将细胞培养物浸泡在颗粒悬浮液中进行暴露。然而，这种方法并不能反映颗粒吸入的情况。为了更真实地模拟这种情况，我们努力通过使用气溶胶暴露系统将气溶胶直接输送到气液界面培养的细胞培养物中。本研究旨在直接比较将氧化锌（ZnO）纳米颗粒以气溶胶或悬浮液形式输送到上皮气道屏障的三重细胞共培养模型时的效果。为了确保剂量等效，通过原子吸收光谱法确定了每种暴露情况下的 ZnO 沉积。在 4 或 24 小时孵育后研究的生物学终点包括细胞毒性、总还原型谷胱甘肽、诱导血红素加氧酶 1（HO-1）等抗氧化基因以及促炎细胞因子 TNFα 的释放。
火焰 ZnO 合成的副产物释放的废气导致总还原型 GSH 显著减少，并进一步诱导细胞因子 TNFα 的释放，表明气相对气溶胶毒理学的影响。不能将 ZnO 颗粒的直接作用归因于 ZnO 颗粒。通过进行悬浮暴露以避免“火焰气体”因素，可以显现出颗粒的特定作用。其他参数，如 LDH 和 HO-1，不受气态化合物的影响：气溶胶暴露后，两种时间点的 LDH 水平均升高，HO-1 转录与沉积的 ZnO 剂量呈正相关。在浸没条件下，4 小时和 24 小时的 HO-1 诱导方案不同，24 小时暴露后发现细胞外 LDH 增加。
在本研究中，通过向细胞培养物暴露等效量的 ZnO，比较了气溶胶和悬浮暴露。两种暴露策略在剂量反应模式上存在根本差异。对于时间因素，还可以发现其他差异：在气溶胶情况下，参数在暴露 4 小时后趋于最大值，而在浸没条件下，主要在 24 小时后，效果最为明显。气溶胶暴露提供了有关吸入毒理学中气溶胶的气相和颗粒相协同相互作用的信息。悬浮暴露是一种有价值的补充方法，通过排除所有源自气体的影响，可以研究与颗粒相关的毒性。
