A TRPA1-mediated substance P release assay as an in vitro screening tool for airway irritant potential

Environmental exposure to airway irritants can elicit sensory irritation and contribute to various disease outcomes. Although in vitro assays have been developed to measure skin and eye irritation, evaluation of respiratory sensory irritation relies predominately on in vivo testing using the RD₅₀ assay, or Alarie test. Although the RD₅₀ assay aligns well with human responses, various disadvantages, such as low throughput and interspecies differences in receptors responsible for detecting noxious stimuli, highlights a few of the many requirements for alternative, or complementary in vitro approaches. Recently, increased emphasis has been placed on methods that predict human responses at the mechanistic level. In this study, we have developed a human relevant in vitro model for airway irritant detection based around molecular events of airway irritation, including transient receptor potential channel activation and substance P release. Using alveolar- and bronchial-derived cell lines, we demonstrate endogenous TRPA1-driven Substance P release following exposure to prototypical irritants. Calcium response was confirmed by flow cytometry following exposure to irritants, with substance P release demonstrated at both the gene and protein levels by qPCR, immunocytochemistry and ELISA. Furthermore, our model demonstrates some concordance with in vivo RD₅₀ data.