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

使用肺和肝细胞中新型间隙连接细胞间通讯生物测定法对环境污染物和候选药物进行高通量毒性筛选

• 批准号: 10056987
• 负责人: Brad L. Upham
• 金额: $ 24.07万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-15 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10056987
• 关键词: Abnormal Cell; Address; Adverse effects; Affect; Alveolar Cell Type I; Animal Testing; Apoptosis; Aromatic Hydrocarbons; Aromatic Polycyclic Hydrocarbons; Biliary; Biological Assay; Biological Models; Biological Phenomena; C10; Cell Communication; Cell Differentiation process; Cell Line; Cell Proliferation; Cell model; Cells; Chemicals; Detection; Development; Dose; Ductal Epithelial Cell; Dyes; Electroporation; Environment; Environmental Pollution; Epithelial; Epithelial Cells; Epithelium; Equilibrium; Event; Exposure to; FDA approved; Fluorescence; Fluorescent Dyes; Functional disorder; Gap Junctions; Gene Proteins; Genes; Growth; Health; Hepatic; Hepatocyte; Homeostasis; Human; In Vitro; Ingestion; Inhalation; Interruption; Iodides; Libraries; Liver; Lung; Malignant Neoplasms; Measures; Microinjections; Microscope; Modeling; Molecular; Mouse Cell Line; Mus; Normal Cell; Organ; Parents; Pathology; Pharmaceutical Preparations; Play; Process; Proteins; Protocols documentation; Quality Control; Rattus; Reaction Time; Reader; Receptor Cell; Role; Sampling; Screening procedure; Signal Pathway; Signal Transduction; System; Techniques; Tissues; Toxic Environmental Substances; Toxic effect; Toxicology; Validation; alveolar epithelium; alveolar type II cell; base; cell growth; drug candidate; gap junction channel; high throughput screening; in vitro Model; in vivo; intercellular communication; novel; oval cell; prevent; quantum; response; screening; self-renewal; small molecule libraries; stem; stem cells; three dimensional cell culture; tool; toxicant; uptake

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.

1 我们建议开发体外高通量生物测定筛选（HTS）系统来评估效果 2 环境污染物对肝和肺上皮间隙连接细胞间通讯（GJIC）的影响 3 细胞模型。 GJIC 是一种有助于维持组织稳态的关键细胞现象。这 4 选择 GJIC 作为终点是开发基于系统的体外模型的重要一步，因为这 5 生物现象对于整合细胞内信号机制与邻近细胞的信号机制至关重要 6 在组织中，并且是暴露于的组织内异常细胞增殖的重要早期事件 7毒物。大多数 GJIC 体外评估依赖于荧光染料转移技术，该技术需要 8 通过刮取上样、显微注射或电穿孔技术引入染料，并用 9 种显微镜在开发 HTS 检测时往往会出现问题，特别是在 3D 培养系统中。 10 因此，需要开发和验证生物测定系统来评估 GJIC 对环境的响应 11种有利于HTS的毒物和候选药物与体外细胞模型系统相关。肺和 12 肝脏是接触吸入和摄入有毒物质的主要靶器官，因此我们将使用小鼠肺 13 上皮肺泡II型和大鼠肝上皮卵圆细胞系。 14 我们提出的 HTS 是为每个细胞系开发供体细胞和受体细胞的子集。受体细胞 15号将转染黄色荧光蛋白（YFP）基因，并带有碘化物转运蛋白的供体细胞 16基因。添加碘化物通过碘化物转运蛋白进入供体细胞来启动生物测定，并且 17 然后通过间隙连接转移到受体细胞，其中碘化物淬灭 YFP 荧光。 18 封闭或部分封闭的间隙连接通道可防止或部分防止碘化物淬灭 19个受体细胞。荧光板读数器测量荧光，这使得该生物测定非常有效 20 可对 HTS 进行修改，因此将解决 GJIC 适应 HTS 毒理学评估方面的关键差距。 21 Aim-1 是 (a) 用碘化物转运蛋白 (IT)/黄色荧光蛋白转染肺和肝细胞系，以 22 使用 HTS 评估 GJIC，并且 (b) 通过评估多环的影响来验证这些 HTS 细胞模型 23 种芳香烃和多氟烷基物质对亲代细胞系中的 GJIC 具有已知影响。目的- 24 2 旨在验证该 HTS 在评估对 GJIC 影响未知的多种化学品方面的实用性， 25 这将需要制定质量控制方案，首先是 i) 初步筛选，ii) 命中确认 26 和反筛选，iii) 命中验证和选择性。 27 体外肺和肝模型 HTS 可以评估化合物对 GJIC 的影响，将提供关键的 28 种重要的新工具可用于筛选对组织产生不利影响的环境毒物和候选药物 29 体内平衡导致细胞异常增殖和分化。