From Concepts to Cultures: Building a Solid Foundation for IVIVE and the Sustainability of In Vitro Approaches to Inhaled Chemical Testing
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The use of in vitro new approach methodologies (NAMs) to reduce the cost, duration, and use of in vivo animal testing to evaluate the potential toxicity of acute and repeated/sub-chronic exposures to inhaled chemicals is a component of the initiative to reduce the use of mammals in chemical testing as expeditiously as possible. Efforts toward the achievement of this goal for inhaled chemicals have relied heavily on the use of differentiated primary human bronchial epithelial cell (pHBEC) in vitro culture models as a representative model of the human respiratory tract in vivo. While pHBEC models provide several benefits over cell line-based alternatives, they also introduce fundamental questions and challenges to their utility for in vitro to in vivo extrapolation (IVIVE). Thus, clearly defining the applicability domain and context for data interpretation are crucial for the use of in vitro systems for inhaled chemical decision making. Here, data will be presented relating to three key questions to be considered for IVIVE. First, what are the impacts of inter-individual variability and repeated exposures on outcomes? Air-liquid interface (ALI) differentiated pHBEC cultures exhibit greater physiological relevance than cell lines; however, their high cost and limited availability often result in the use of a limited number of donors in chemical evaluation and desire to extrapolate the effects of a single exposure outcome to repeated/sub-chronic exposure outcomes. Data will be presented showing the impact of inter-individual variability and repeated exposures on the induction of key pro-inflammatory genes in differentiated pHBEC cultures following exposure to the model oxidant ozone. Second, how are in vitro outcomes and subsequent in vivo extrapolations affected by test agent delivery? Achieving the physiological relevance of the pHBEC model requires prolonged differentiation under air-liquid interface (ALI) conditions; however, the commonly used dosing method of direct application involves the re-submersion of these cultures in the diluted/resuspended test agent. We evaluated the effect of re-submersion in the absence of a test agent and observed the significant alternative expression of >4,000 and >10,000 gene transcripts at 6 and 24 hours, respectively. These changes in gene expression corresponded to the initiation of an epithelial to mesenchymal transition (EMT) that was further evidenced by significant reduction in epithelial barrier integrity and increases in pro-inflammatory cytokine release. Third, do in vitro monoculture systems that are currently used for inhaled chemical assessment represent the role of different cell types and the tissue microenvironment as targets and mediators of the effects resulting from inhaled chemical exposures? Data will be presented highlighting the similar and divergent effects of model oxidant particles on different cellular compartments within both a co-culture model of the bronchial epithelium and a tri-culture model of the alveolar epithelial-microvascular interface. Understanding the impacts of these, as well as other, considerations is critical for ensuring the defensibility and sustainability of in vitro approaches for inhaled chemical testing. Does not necessarily reflect US Environmental Protection Agency policy.