Liquid Application Dosing Alters Air-Liquid Interface Bronchial Epithelial Culture Physiology and Toxicity Testing Relevant Endpoints
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The in vivo physiology of the bronchial epithelium can be recapitulated in vitro through the differentiation of primary human bronchial epithelial cells (pHBEC) under air-liquid interface (ALI) conditions. pHBEC ALI models are popular for in vitro chemical testing and research; however, there are practical challenges facing test agent delivery to ALI cultures. These limitations have led to the practice of suspending test agents in an aqueous solution and dosing by liquid application to ALI cultures. Given the characteristics that develop during pHBEC ALI differentiation, we hypothesized that liquid application would have a significant effect on in vitro toxicity endpoints. To test our hypothesis, we applied basal growth medium to pHBEC ALI cultures and examined global gene expression, stress-responsive signaling pathway phosphorylation, pro-inflammatory cytokine and growth factor secretion, and epithelial barrier integrity. Liquid application resulted in the significant alternative regulation of 4170 and 10269 genes at 6 and 24 hours, respectively. Immunoblotting indicated a significant change in the phosphorylation of the stress-responsive pathways ERK1/2, p38, and p65. The transcriptional changes were complemented by significant increases in the secretion of the pro-inflammatory cytokines IL-8, IL-6, IL-1β, and TNFα, as well as the growth factors VEGF-A, PDGF-B, and PGF. We also observed a progressive decrease in trans-epithelial electrical resistance and increase in small molecule permeability. Cumulatively, our findings indicate that the use of liquid application approaches for test agent dosing in ALI cultures is likely to be a confounding factor in chemical toxicity testing that requires further characterization. Does not reflect EPA policy.