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

1我们建议开发一种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、肺上皮细胞间隙连接通讯（GJIC）的影响 3细胞模型。GJIC是维持组织稳态的重要细胞现象。的 4选择GJIC作为终点是开发基于系统的体外模型的重要步骤，因为 5生物现象对于整合细胞内的信号机制与邻近细胞的信号机制至关重要 6，并且是暴露于抗肿瘤药物的组织内异常细胞增殖的重要早期事件。 7有毒物质GJIC的大多数体外评估依赖于荧光染料转移技术， 8通过刮片加载、显微注射或电穿孔技术引入染料，并使用 9显微镜，都倾向于在开发HTS测定，特别是在3D培养系统的问题。 因此，需要开发和验证生物测定系统，以评估GJIC对环境的响应。 11种毒物和候选药物，有助于与体外细胞模型系统相关的HTS。肺和 肝脏是吸入和摄入毒物的主要靶器官，因此我们将使用小鼠肺 13种肺泡II型上皮细胞和大鼠肝卵圆上皮细胞系。 14我们提出的HTS是为每个细胞系开发供体和受体细胞的子集。受体细胞 15将黄色荧光蛋白（YFP）基因转染供体细胞，并将供体细胞与碘转运蛋白（Iodide transporter 16基因。碘化物的加入通过碘化物转运蛋白进入供体细胞启动生物测定， 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闭合或部分闭合的间隙连接通道防止或部分防止来自碘离子的淬灭。 19个受体细胞。荧光读板仪测量荧光，这使得这种生物测定相当 因此，将解决在使GJIC适应HTS毒理学评估方面的关键差距。 目的-1是（a）用碘转运蛋白（IT）/黄色荧光蛋白转染肺和肝细胞系， （B）通过评估多环化合物的影响来验证这些HTS细胞模型。 23种芳烃和多氟烷基物质，已知对亲本细胞系中的GJIC有影响。目标- 24 2是为了验证这种HTS在评估对GJIC具有未知影响的各种化学品中的实用性， 这将需要制定一个质量控制协议，首先是i）初步筛选，ii）命中确认 26和计数器筛选，iii）命中验证和选择性。 27可以评估化合物对GJIC的作用的体外肺和肝模型HTS将提供关键的 28个重要的新工具来筛选对组织产生不利影响的环境毒物和候选药物 29稳态导致细胞的异常增殖和分化。

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.