Development and Applications of Integrated Bioassays

综合生物测定法的开发和应用

• 批准号: 6900544
• 负责人: MICHAEL STEVEN DENISON
• 金额: $ 42.92万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6900544
• 关键词: bioassay; biohazard detection; biomarker; calcium channel; cell line; dioxins; environmental toxicology; functional /structural genomics; green fluorescent proteins; human genetic material tag; inflammation; keratinocyte; laboratory mouse; luciferin monooxygenase; metabolomics; proteomics; reporter genes; steroid hormone; steroid hormone analog; technology /technique development; thyroid hormones; toxicant screening; transfection

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 available 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 that have application for chemical detection and screening. Since effective development and application of bioassays/biomarkers is greatly facilitated by an understanding of the specific response of a cell to a given toxicant or class of toxicants, each of the four proposed approaches will exploit information derived from an analysis of the basic molecular mechanisms by which selected chemicals affect cellular receptors, signal transduction pathways and cellular/enzyme functions. In Aim 1, stably transfected cell lines will be developed which respond to dioxin-like chemicals, steroid/thyroid hormones or hormone-like chemicals with the induction or inhibition of receptor-dependent expression of firefly luciferase or green fluorescent protein reporter genes. We will also develop a novel portable in vitro AhR-based bioassay for detection of dioxin-like chemicals. In Aim 2, human keratinocytes will be used to examine genomic, proteomic and metabolomic effects following exposure to metals/metalloids and chemicals that produce oxidative stress to identify potential biomarkers that are specifically altered by these chemicals. In Aim 3 in vitro and cell based bioassay systems will be used to examine the influence of superfund chemicals on regulatory lipids that control inflammation and to identify xenobiotics that alter expression and activity of soluble epoxide hydrolase. Aim 4 proposes to develop and validate ryanodine receptor-based in vitro and intact cell bioassays to identify and characterize non-coplanar halogenated persistent organic pollutants that can affect calcium signaling pathways. In the final Aim, these assay systems will be integrated and optimized and then used in a series of validation studies for the detection and relative quantitation of toxic chemicals present in complex mixtures of chemicals extracted from a variety of matrices. Overall, the proposed studies will not only increase our basic knowledge of the biological and toxicological effects of a variety of Superfund priority chemicals, but the resulting specific bioassays and biomarkers that will be developed will provide rapid mechanistically-based screening systems for the detection of toxicants and toxicant exposure.

危险废物场含有多种有毒化学品的复杂混合物。不幸的是，由于缺乏可用的特定生物测定/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适用于化学检测和筛选的基于机械的细胞和体外生物测定/生物标记。由于了解细胞对给定毒物或一类毒物的特异性反应，极大地促进了生物测定/生物标志物的有效开发和应用，因此所提出的四种方法中的每一种都将利用从基本分子机制分析中获得的信息：哪些选定的化学物质影响细胞受体、信号转导途径和 细胞/酶功能。在目标1中，将开发稳定转染的细胞系，其对二恶英类化学物质、类固醇/甲状腺激素或类激素化学物质作出反应，诱导或抑制萤火虫荧光素酶或绿色荧光蛋白报告基因的受体依赖性表达。我们还将开发一种新型便携式体外基于 AhR 的生物测定法，用于检测二恶英类化学物质。在目标 2 中，人类角质形成细胞将用于检查暴露于产生氧化应激的金属/类金属和化学物质后的基因组、蛋白质组和代谢组效应，以识别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，体外和基于细胞的生物测定系统将用于检查超级基金化学品对控制炎症的调节脂质的影响，并鉴定改变可溶性环氧化物水解酶表达和活性的外源物质。目标 4 提议开发和验证基于兰尼碱受体的体外和完整细胞生物测定法 识别和表征可影响钙信号通路的非共面卤化持久性有机污染物。在最终目标中，这些测定系统将被集成和优化，然后用于一系列验证研究，以检测和相对定量从各种基质中提取的复杂化学混合物中存在的有毒化学物质。总体而言，拟议的研究不仅将增加我们对各种超级基金优先化学品的生物和毒理学影响的基本了解，而且还将增加我们对各种超级基金优先化学品的生物和毒理学影响的基本了解。 将开发的特定生物测定法和生物标志物将为检测毒物和毒物暴露提供基于机械的快速筛查系统。