Project 5: Development and Applications of Integrated in Vitro and Cell-Based

危险废物场含有多种有毒化学品的复杂混合物。不幸的是，由于缺乏特定的生物测定/生物标记系统，对环境和生物样品中特定化学品或化学类别的快速且廉价的检测的发展受到阻碍。因此，该项目的总体目标是开发和验证一系列基于机械的细胞和体外生物测定/生物标记，并应用于化学检测和筛选。自生效以来 了解细胞对给定毒物的具体反应极大地促进了生物测定/生物标志物的开发和应用，所提出的四种方法中的每一种都将利用从分析选定化学物质影响细胞受体、信号转导途径和/或细胞/酶功能的机制中获得的信息。在目标 1 中，将开发稳定转染的细胞系，作为超低水平二恶英类或类固醇激素类化学物质的生物测定，并诱导受体依赖性报告基因表达。将产生化学特异性重组 AhR，以改善基于细胞和体外 AhR 的生物测定系统。在目标 2 中，人类角质形成细胞将用于检查因接触砷而发生的特定细胞内蛋白质组变化，并鉴定被这些化学物质特异性改变的潜在生物标志物。在目标 3 中，将使用高通量体外和基于细胞的生物测定来检查 Superfund 化学品对控制心血管疾病、炎症和 pjn 的调节脂质的产生的影响。在目标 4 中，将为一系列溴化阻燃剂及其代谢物和抗菌剂三氯生建立结构活性关系，因为它们能够改变兰尼碱受体信号传导功能，并对神经元生长和可塑性产生影响。在最终目标中，将使用综合生物测定/生物标记来识别和表征单个化学品和复杂化学品混合物的生化和毒理学效应。

Hazardous waste sites contain complex mixtures of a wide variety of toxic chemicals. Unfortunately, development of rapid and inexpensive detection of specific chemicals or chemical classes in environmental and biological samples has been hampered by the lack of specific bioassay/biomarker systems. Accordingly, the overall goals of this project are to develop and validate a series of mechanistically-based cell and in vitro bioassays/biomarkers with applications to chemical detection and screening. Since effective development and application of bioassays/biomarkers is greatly facilitated by an understanding of the specific responses of cell to a given toxicant(s), each of the four proposed approaches will exploit information derived from analysis of the mechanisms by which selected chemicals affect cellular receptors, signal transduction pathways and/or cellular/enzyme functions. In Aim 1, stably transfected cell lines will be developed as bioassays for ultra-low levels of dioxin-like or steroid hormone-like chemicals with the induction of receptor-dependent reporter gene expression. Chemical-specific recombinant AhRs will be generated to improve both cell and in vitro AhR based bioassay systems. In Aim 2, human keratinocytes will be used to examine specific intracellular proteomic changes that occur in response to exposure to arsenicals and to identify potential biomarkers specifically altered by these chemicals. In Aim 3, high throughput in vitro and cell-based bioassays will be used to examine the influence of Superfund chemicals on the production of regulatory lipids controlling cardiovascular diseases, inflammation and pajn. In Aim 4, structure activity relationships will be established for a series of brominated flame retardants and their metabolites and the antimicrobial agent triclosan for their ability to alter ryanodine receptor signaling functions and the resulting impact on neuron growth and plasticity. In the final Aim, integrated bioassay/biomarkers will be used to identify and characterize the biochemical and toxicological effects of individual chemicals and complex mixtures of chemicals.