Challenges and progress in characterizing in vitro dosimetry for air-liquid interface exposures
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Inhalation is one of the three primary modes of chemical exposure, but key challenges in developing reproducible exposure systems with robust analytical dosimetry methods have limited the use of in vitro models for the assessment of inhalable chemicals. In vitro inhalation assays have been improved with air-liquid interface (ALI) exposure systems which allow direct cell-toxicant interactions, but these systems are complex and vary in their geometry, flow rates, and operational parameters. Physicochemical properties of the inhaled substance must be carefully considered when selecting an ALI exposure system, optimizing its operational parameters, and developing methods to characterize exposure conditions. For example, volatile organic compounds (VOCs) and aerosols possess distinct transport and deposition mechanisms which complicate the reliability and reproducibility of deploying ALI exposure systems to screen diverse inhalable compounds without quantitative methods to assess exposures. Detection methods to quantify deposition are often missing in ALI studies, but when deposition is recorded, it is usually with a cell-free collection method (QCM, filter, etc). As an alternative approach, we aerosolized two fluorescent tracers (sodium fluorescein and rhodamine 6G) and developed methods to directly quantify deposition and cellular uptake on human bronchial epithelial cells grown at the ALI across multiple dynamic and static exposure systems, including the EPA ACCES, MedTec CelTox, and the VITROCELL Cloud α12. These fluorescent tracers revealed that particle deposition is highly variable within and across exposure systems, and cell-free collection methods did not reliably estimate cell deposition in many scenarios. Both tracers were also able to highlight differences in internal dose rates across different exposure methods and cell models utilized. Mucus retention decreased cellular uptake in differentiated primary human bronchial epithelial cultures (HBECs) compared to an immortalized cell line, indicating that further work is needed to determine how cell model selection may influence dosimetry. Overall, these results highlight opportunities to better characterize in vitro exposures and improve dosimetry methods for in vitro to in vivo extrapolation. [Abstract does not reflect views or policies of the U.S. EPA.]