Inflammatory and Oxidative Stress Responses of an Alveolar Epithelial Cell Line to Airborne Zinc Oxide Nanoparticles at the Air-Liquid Interface: A Comparison with Conventional, Submerged Cell-Culture Conditions

Abstract

The biological effects of inhalable nanoparticles have been widely studied in vitro with pulmonary cells cultured under submerged and air-liquid interface (ALI) conditions. Submerged exposures are experimentally simpler, but ALI exposures are physiologically more realistic and hence potentially biologically more meaningful. In this study, we investigated the cellular response of human alveolar epithelial-like cells (A549) to airborne agglomerates of zinc oxide (ZnO) nanoparticles at the ALI, compared it to the response under submerged culture conditions, and provided a quantitative comparison with the literature data on different types of particles and cells. For ZnO nanoparticle doses of 0.7 and 2.5 μg ZnO/cm2 (or 0.09 and 0.33 cm2 ZnO/cm2), cell viability was not mitigated and no significant effects on the transcript levels of oxidative stress markers (HMOX1, SOD-2 and GCS) were observed. However, the transcript levels of proinflammatory markers (IL-8, IL-6, and GM-CSF) were induced to higher levels under ALI conditions. This is consistent with the literature data and it suggests that in vitro toxicity screening of nanoparticles with ALI cell culture systems may produce less false negative results than screening with submerged cell cultures. However, the database is currently too scarce to draw a definite conclusion on this issue.

Biological Reactivity of Zinc Oxide Nanoparticles With Mammalian Test Systems: an Overview

“…A549 cells subcultured under ALI when exposed to ZnO NPs demonstrated enhanced inflammatory cytokine expression (IL-8, IL-6 and GM-CSF) without effect on cytotoxicity as compared with the same cells cultured in submerged conditions. Although the same effect was not observed for oxidative stress-related genes (HO-1 and SOD-2) upregulation, this study reported that ALI subculture systems may provide less falsenegative immunotoxicity results as compared with submerged systems [20]. On the other hand, Xie and coworkers reported an increase in oxidative stress in ALI-subcultured A549 cells and not submersed A549 cell cultures when exposed to aerosolized ZnO NPs.…”

Bridging the Gap Between Science and Clinical Efficacy: Physiology, Imaging, and Modeling of Aerosols in the Lung

“…An overview of the available single-and multi-cell models of the lung is given elsewhere. (94,100,101) Recently, these cell culture models have matured into biomimetic models of the lung by incorporating previously neglected but crucial physiological aspects of the lung such as: 1) cultivation at the air-liquid interface with air on the apical side and liquid on the basal side of the epithelial barrier; (102,103) 2) growing the cells on biocompatible 3D (or 2D) matrices mimicking the elasticity of the pulmonary extracellular matrix; 3) exerting cyclic stretch on the cell layer simulating the mechanical strain profile experienced by the alveolar tissue during breathing activity; (104) and 4) combinations thereof. (105) FIG.…”

Non-animal models of epithelial barriers (skin, intestine and lung) in research, industrial applications and regulatory toxicology

“…Aerosol-to-cell delivery was shown to be highly efficient, reproducible and rapid using aerosolized fluorescein as a surrogate drug. This type of approach may pave the way for screening of inhalable drugs under more physiologically relevant and hence potentially more predictive conditions than the currently used submerged cell culture systems (Lenz et al, 2013). Primary human alveolar epithelial cell (hAEpC) models can also be used for toxicity and transport studies for newly developed compounds and delivery systems (Daum et al, 2012).…”