Liquid Application Dosing Alters Air-Liquid Interface Bronchial Epithelial Culture Physiology and Toxicity Testing Relevant Endpoints

The in vivo functions of the bronchial epithelium can be recapitulated in vitro through the differentiation of primary human bronchial epithelial cells (pHBEC) under air-liquid interface (ALI) culture conditions.  pHBEC ALI models have rapidly gained popularity for in vitro chemical testing and research; however, there are practical challenges facing the delivery of many test agents to ALI cultures.  These limitations have led to the common practice of dosing differentiated pHBEC cultures by the application of the test agent suspended in an aqueous vehicle (e.g., medium, saline, et cetera).  Given the physiological characteristics that develop during pHBEC ALI differentiation, we hypothesized that liquid application alone (i.e., in the absence of a test agent) would have a significant effect on in vitro toxicity endpoints.  To test our hypothesis, we applied an aqueous vehicle (ALI medium) in the absence of a test agent to the apical surface of differentiated pHBEC ALI cultures.  We then examined endpoints that were representative of in vivo relevant physiology including, global gene expression by RNA-sequencing, stress-responsive signaling pathway phosphorylation, pro-inflammatory cytokine and growth factor secretion, cell membrane damage, ciliary beat frequency, and epithelial barrier integrity.  The application of liquid alone resulted in the significant alternative regulation of 4170 and 10269 genes at 6 and 24 hours, respectively, with many of the most dysregulated genes being involved in stress-responsive cell signaling and inflammation.  Western blot analysis indicated a significant increase in the phosphorylation of the stress-responsive signaling pathways ERK1/2, p38, and p65, as well as activation of the HIF1-alpha signaling pathway.  The transcriptional changes were further complemented by significant increases in the secretion of the pro-inflammatory cytokines IL-8, IL-6, IL-1β, and TNFα, as well as vascular endothelial growth factor alpha (VEGFa) and placental growth factor (PGF).  We also observed a progressive decrease in trans-epithelial electrical resistance and increase in small molecule permeability.  Cumulatively, our findings indicate that liquid application alone causes a phenotype in the bronchial epithelium that is consistent with the effects of well-characterized toxicants, several respiratory tract diseases, and early epithelial-to-mesenchymal transition – a common early step in carcinogenesis.  Given the common usage of liquid application dosing of ALI cultures, our observations suggest that this dosing method alone is likely to confound the observation and interpretation of test agent effects.  Liquid application dosing is used with a wide range of different conditions (e.g., different aqueous vehicles, liquid volumes, treatment durations), thus the use of ALI cultures for in vitro hazard identification would benefit from additional characterization of potential effects related to the dosing method alone and any potential to affect study results. Does not reflect EPA policy.

Impact/Purpose

The in vivo functions of the bronchial epithelium can be recapitulated in vitro through the differentiation of primary human bronchial epithelial cells (pHBEC) under air-liquid interface (ALI) culture conditions.  pHBEC ALI models have rapidly gained popularity for in vitro chemical testing and research; however, there are practical challenges facing the delivery of many test agents to ALI cultures.  These limitations have led to the common practice of dosing differentiated pHBEC cultures by the application of the test agent suspended in an aqueous vehicle (e.g., medium, saline, et cetera).  Given the physiological characteristics that develop during pHBEC ALI differentiation, we hypothesized that liquid application alone (i.e., in the absence of a test agent) would have a significant effect on in vitro toxicity endpoints.  To test our hypothesis, we applied an aqueous vehicle (ALI medium) in the absence of a test agent to the apical surface of differentiated pHBEC ALI cultures.  We then examined endpoints that were representative of in vivo relevant physiology including, global gene expression by RNA-sequencing, stress-responsive signaling pathway phosphorylation, pro-inflammatory cytokine and growth factor secretion, cell membrane damage, ciliary beat frequency, and epithelial barrier integrity.  The application of liquid alone resulted in the significant alternative regulation of 4170 and 10269 genes at 6 and 24 hours, respectively, with many of the most dysregulated genes being involved in stress-responsive cell signaling and inflammation.  Western blot analysis indicated a significant increase in the phosphorylation of the stress-responsive signaling pathways ERK1/2, p38, and p65, as well as activation of the HIF1-alpha signaling pathway.  The transcriptional changes were further complemented by significant increases in the secretion of the pro-inflammatory cytokines IL-8, IL-6, IL-1β, and TNFα, as well as vascular endothelial growth factor alpha (VEGFa) and placental growth factor (PGF).  We also observed a progressive decrease in trans-epithelial electrical resistance and increase in small molecule permeability.  Cumulatively, our findings indicate that liquid application alone causes a phenotype in the bronchial epithelium that is consistent with the effects of well-characterized toxicants, several respiratory tract diseases, and early epithelial-to-mesenchymal transition – a common early step in carcinogenesis.  Given the common usage of liquid application dosing of ALI cultures, our observations suggest that this dosing method alone is likely to confound the observation and interpretation of test agent effects.  Liquid application dosing is used with a wide range of different conditions (e.g., different aqueous vehicles, liquid volumes, treatment durations), thus the use of ALI cultures for in vitro hazard identification would benefit from additional characterization of potential effects related to the dosing method alone and any potential to affect study results. Does not reflect EPA policy.

Citation

Mallek, N., E. Martin, L. Dailey, AND S. McCullough. Liquid Application Dosing Alters Air-Liquid Interface Bronchial Epithelial Culture Physiology and Toxicity Testing Relevant Endpoints. Society of Toxicology Annual Meeting, Nashville, TN, March 19 - 23, 2023.