Air–Liquid Interface Exposure of Lung Epithelial Cells to Low Doses of Nanoparticles to Assess Pulmonary Adverse Effects

Abstract

Reliable and predictive in vitro assays for hazard assessments of manufactured nanomaterials (MNMs) are still limited. Specifically, exposure systems which more realistically recapitulate the physiological conditions in the lung are needed to predict pulmonary toxicity. To this end, air-liquid interface (ALI) systems have been developed in recent years which might be better suited than conventional submerged exposure assays. However, there is still a need for rigorous side-by-side comparisons of the results obtained with the two different exposure methods considering numerous parameters, such as different MNMs, cell culture models and read outs. In this study, human A549 lung epithelial cells and differentiated THP-1 macrophages were exposed under submerged conditions to two abundant types of MNMs i.e., ceria and titania nanoparticles (NPs). Membrane integrity, metabolic activity as well as pro-inflammatory responses were recorded. For comparison, A549 monocultures were also exposed at the ALI to the same MNMs. In the case of titania NPs, genotoxicity was also investigated. In general, cells were more sensitive at the ALI compared to under classical submerged conditions. Whereas ceria NPs triggered only moderate effects, titania NPs clearly initiated cytotoxicity, pro-inflammatory gene expression and genotoxicity. Interestingly, low doses of NPs deposited at the ALI were sufficient to drive adverse outcomes, as also documented in rodent experiments. Therefore, further development of ALI systems seems promising to refine, reduce or even replace acute pulmonary toxicity studies in animals.

Impact of Nanocomposite Combustion Aerosols on A549 Cells and a 3D Airway Model

“…On the one hand, a comparable toxicity pattern to submerged exposure has been reported applying relatively low doses of 3 µg/cm² not indicating any cytotoxicity [ 45 ] and higher doses of 25 µg/cm² impairing metabolic activity [ 46 , 47 ]. On the other hand, neither cell viability nor genomic stability or gene expression were affected by TiO 2 NP in one study, even at high doses [ 29 ], while low doses induced cyto- and genotoxic effects as well as pro-inflammatory transcriptional changes in another study [ 27 ]. So far, the impact of TiO 2 NP on MucilAir™ tissue has not been published, but other metal oxides such as CeO 2 and CuO were applied using an ALI exposure system [ 48 , 49 , 50 ].…”

“…Additionally, a new tool to reproducibly expose sample grids for transmission electron microscopy (TEM) to obtain dose information with respect to the spatial distribution and the agglomeration state of the deposited particles was developed and applied within this study [ 22 ]. The ALI exposure station has already been used to investigate the toxicological impact of environmental atmospheres and technical emission sources like ship diesel exhaust [ 23 , 24 ], biomass combustion emissions [ 25 ], and suspended nanomaterials as metal oxides and silica [ 15 , 26 , 27 ].…”

A Complete In Vitro Toxicological Assessment of the Biological Effects of Cerium Oxide Nanoparticles: From Acute Toxicity to Multi-Dose Subchronic Cytotoxicity Study

“…As biological responses of lung cells exposed to nanoparticles depend not only on the exposure dose but also on the intrinsic properties of the NPs (e.g., size, shape, chemical composition, surface reactivity, and degree of aggregation), NPs must always be characterized before performing the toxicity assays [ 51 , 52 , 53 ] and, whenever possible the homogeneity of different batches must be ensured [ 54 ].…”

A Complete In Vitro Toxicological Assessment of the Biological Effects of Cerium Oxide Nanoparticles: From Acute Toxicity to Multi-Dose Subchronic Cytotoxicity Study.

“…As biological responses of lung cells exposed to nanoparticles depend not only on the exposure dose but also on the intrinsic properties of the NPs (e.g. size, shape, chemical composition, surface reactivity and degree of aggregation), NPs must always be characterized before performing the toxicity assays [46][47][48] and, whenever is possible, ensure the homogeneity of the different batches [49].…”