Common Liquid Application Dosing Conditions Alter Toxicity Testing Endpoints in Air-Liquid Interface Primary Bronchial Epithelial Cultures
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The use of in vitro systems to model inhalation exposures has focused on using primary cells as they can recapitulate key features of the airway epithelium in vivo. For inhaled chemicals, achieving this goal has relied on using differentiated primary human bronchial epithelial cell (dpHBEC) cultures as a representative model of the barrier that separates materials inhaled into the respiratory tract from the underlying lung tissue in vivo. Achieving physiological relevance with these models requires that cultures be maintained under air-liquid interface (ALI) conditions. Evaluating potential adverse effects typically involves applying an aqueous solution containing the test substance to the surface of the ALI culture, thus abolishing ALI conditions. While practical, the impact of liquid application on ALI culture phenotype and response to experimental exposures is poorly understood, limiting the interpretation of these studies and integrating the data into decision making. To address this knowledge gap, we sought to determine the effects of common liquid application dosing conditions on in vivo physiologically-relevant endpoints in dpHBEC cultures. Application of 50mL resulted in dpHBEC cultures remaining fully covered at 24-hours, lower volumes (10 and 30mL) lost full coverage after 1-hour. Application of ALI medium or 0.9% saline to dpHBEC-ALI cultures caused significant transcriptional upregulation of pro-inflammatory cytokines: IL-6, IL-8, and IL-1a, and the growth factor PGF. The hypoxia-responsive regulating transcription factor HIF-1a and glucose transporter GLUT3 were upregulated following application of 30 and 50mL of liquid suggesting induction of hypoxia. Consistent with these findings, application of these volumes led to decreases in dpHBEC-ALI culture barrier integrity, measured by trans-epithelial electrical resistance. These findings support the need to further characterize the effects of liquid application on dpHBEC cultures. Doing so will build confidence in the use of these models to recapitulate the human respiratory tract for chemical testing. [Abstract does not reflect views or policies of the U.S. EPA].