Response of Alveolar Epithelial Cells exposed to Zinc Oxide Nanoparticles: Comparison of Exposure under Air-liquid Interface or Submerged Conditions

Introduction: Inhalation of zinc oxide (ZnO) nanoparticles (NPs) has been associated with pro-inflammatory responses in the lung. However, dosimetric uncertainties limit the relevance of exposures conducted under submerged culture conditions and especially for airborne NPs, cell exposure at the air-liquid-interface (ALI) is more realistic. Methods: A549 cells were exposed to ZnO NPs under both submerged and ALI culture conditions. The cellular response was characterized by measuring mRNA expression (qRT-PCR) of the pro-inflammatory cytokine IL-8 and the oxidative stress marker hemeoxygenase-1 (HO-1). Results: Immediately after the 3h ALI exposure (0h post-incubation) the levels of IL-8 mRNA expression increased 2.4-fold and 3.9-fold relative to control levels for the lower (0.7µg/cm²) and higher (2.2µg/cm²) doses, respectively. A 2h post-incubation resulted in further elevations in IL-8 to 3.9-fold and 10.4-fold over control levels, respectively. In contrast, HO-1 mRNA expression was not induced for any of the two doses even after 2h post-incubation. Using doses of 0.7 and 2.5µg ZnO/cm² under submerged conditions, similar IL-8 and HO-1 mRNA expression values and trends were observed with an increase in response from 0h to 2h post-incubation for both IL-8 (for 0.7µg/cm² no changes compared to control levels, for 2.5µg/cm² at 0h: 1.9-fold and at 2h: 4.3-fold) and HO-1 (for 0.7µg/cm² no changes compared to control levels, for 2.5µg/cm² at 0h: 1.2-fold and at 2h: 3.3-fold) followed by a decrease in the IL-8 and an increase in the HO-1 response with a 5h post-incubation period. Comparing the dose responses for IL-8 and HO-1, we found the IL-8 response under ALI conditions in relation to submerged conditions to be 2–4 times more sensitive to the ZnO exposure, whereas no difference was observed for HO-1. Discussion: The type of exposure (submerged or ALI) showed only a moderate impact on ZnO-induced IL-8 and HO-1 responses. However, in contrast to submerged conditions, the absence of cell medium on the apical side during ALI exposure avoids possible biases due to (extracellular) dissolution of ZnO (or other nanotoxicologically relevant particles). Since the ZnO nanoparticle doses achieved in these experiments exceeds the permissible exposure limit (PEL) according to OSHA by a factor of ˜400, our results are only relevant for highly polluted occupational settings such as encountered during welding activities.