High-throughput toxicity screening of environmental contaminants and drug candidates using a novel gap junction intercellular communication bioassay in lung and liver cells

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1 We propose to develop an in vitro high throughput bioassay screening (HTS) system to assess the effects 2 of environmental contaminants on gap junctional intercellular communication (GJIC) in liver and lung epithelial 3 cell models. GJIC is a critical cellular phenomenon instrumental in maintaining tissue homeostasis. The 4 selection of GJIC as an endpoint is a significant step in developing a systems-based in vitro model, as this 5 biological phenomenon is crucial for integrating signaling mechanisms within cells with that of neighboring cells 6 in a tissue, and is an important early stage event in abnormal cell proliferation within tissues exposed to 7 toxicants. Most in vitro assessments of GJIC rely on fluorescent dye transfer techniques that require 8 introduction of the dye through scrape loading, microinjection, or electroporation techniques, and detection with 9 microscopes that all tend to be problematic in developing HTS assays, particularly in 3D culture systems. 10 Thus, there is a need to develop and validate a bioassay system to assess GJIC in response to environmental 11 toxicants and drug candidates that is conducive to HTS relevant to in vitro cell model systems. The lung and 12 liver are the major target organs of exposure to inhaled and ingested toxicants so we will use a mouse lung 13 epithelial alveolar type II and rat liver epithelial oval cell lines. 14 Our proposed HTS is to develop a subset of donor and receptor cells for each cell line. The receptor cells 15 will be transfected with yellow fluorescent protein (YFP) gene, and the donor cells with the iodide transporter 16 gene. The addition of iodide initiates the bioassay by entering the donor cells via the iodide transporter, and 17 then transfers through gap junctions to the receptor cells, in which iodide quenches the YFP-fluorescence. 18 Closed or partially closed gap junction channels prevents or partially prevents quenching from iodide in the 19 receptor cells. Fluorescent plate readers measure the fluorescence, which makes this bioassay quite 20 amendable to HTS, thus will address a critical gap in adapting GJIC to HTS toxicological assessments. 21 Aim-1 is to (a) transfect lung and liver cell lines with the iodide transporter (IT)/ yellow fluorescent protein to 22 assess GJIC using HTS, and (b) authenticate these HTS cell models by assessing the effects of polycyclic 23 aromatic hydrocarbons and polyfluoroalkyl substances with known effects on GJIC in the parent cell lines. Aim- 24 2 is to validate the utility of this HTS in assessing a wide array of chemicals with unknown effects on GJIC, 25 which will entail developing a quality control protocol that begins with i) primary screening, ii) hit confirmation 26 and counter screening, iii) hit validation and selectivity. 27 An in vitro lung and liver model HTS that can assess effects of compounds on GJIC, will offer a critically 28 important new tool to screen for environmental toxicants and drug candidates that adversely affect tissue 29 homeostasis resulting in abnormal proliferation and differentiation of cells.