Citation:

Murray, J., E. Carlsten, W. Zander, J. Weinstein, Todd Krantz, A. Speen, AND M. Higuchi. Aerosolized fluorescent tracers provide insight into particle deposition and cellular uptake at the air-liquid interface. American Society for Cellular and Computational Toxicology (ASCCT) 11th Annual Meeting, Chapel Hill, NC, October 19 - 21, 2022.

Impact/Purpose:

There is considerable interest in reducing the need for the 90-day animal inhalation study, but traditional toxicity screening programs are incompatible with inhaled test agents (aerosols, volatile compounds, reactive gases). This study aims to identify best in vitro testing methods for aerosols by quantifying deposition and cellular uptake of particles delivered as aerosols or under submerged, direct-dosing conditions. This will improve our dose-response understanding for in vitro inhalation studies and build confidence in New and Alternative Methods (NAMs).

Description:

Aerosols present unique challenges for in vitro toxicity evaluation with New and Alternative Methods (NAMs). They can be difficult to generate and transport in a reproducible manner and require complex air-liquid interface (ALI) exposure systems to mimic realistic inhalation exposures. Deposition and cellular uptake following in vitro ALI exposures are rarely reported which prevents dose-response modeling and comparison across exposure systems. A possible alternative is to re-submerge ALI cell cultures and utilize a direct-dosing method which simplifies exposure methods for high-throughput screening. However, re-submersion of primary human bronchial epithelial cells (pHBEC) grown at the ALI disrupts airway epithelial barrier integrity and changes physicochemical properties of particles which may affect cellular uptake and toxicity. To assess possible dosimetric differences between exposure methods, we compared cellular uptake and basolateral translocation of two fluorescent tracers (fluorescein and rhodamine) that were delivered as liquid aerosols at ALI or by direct-dosing in a human bronchial epithelial cell line (16HBE) and pHBECs. We found that the delivery method altered cellular uptake, basolateral transport, and mucus retention of fluorescent tracers. In 16HBEs, cellular uptake of rhodamine and basolateral transport of fluorescein reached a maximum at ALI and decreased as direct-dosing volumes increased. We also observed volume-dependent changes in mucus retention of rhodamine in pHBECs. Furthermore, these tracers reveal that cell-free matrices may not accurately estimate cell deposition in ALI exposure systems. Together, these data highlight the need for improved analytical assays to support inhalation NAMs and suggest that direct-dosing studies may not be equivalent to ALI exposures. [Abstract does not reflect views or policies of the U.S. EPA.]  

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