Development and Applications of Integrated Bioassays

危险废物场含有各种有毒化学品的复杂混合物。不幸的是，环境和生物样品中特定化学品或化学类别的快速和廉价检测的发展受到缺乏可用的特定生物测定/生物标志物系统的阻碍。因此，本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/生物标志物，这些生物标志物可用于化学检测和筛选。由于生物测定/生物标志物的有效开发和应用极大地促进了对细胞对给定毒物或一类毒物的特异性反应的理解，因此四种拟议方法中的每一种都将利用来自对基本分子机制的分析的信息，所选化学品通过该基本分子机制影响细胞受体、信号转导途径和细胞凋亡。 细胞/酶功能。在目的1中，将开发稳定转染的细胞系，其响应于二恶英样化学物质、类固醇/甲状腺激素或类二恶英样化学物质，诱导或抑制萤火虫荧光素酶或绿色荧光蛋白报道基因的受体依赖性表达。我们还将开发一种新的便携式体外AhR为基础的生物检测二恶英类化学品。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/类金属和化学品后的基因组、蛋白质组和代谢组学效应，以鉴定被这些化学品特异性改变的潜在生物标志物。在目标3中，基于体外和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的表达和活性的外源性物质。目的4提出开发和验证基于ryanodine受体的体外和完整细胞生物测定法， 查明和说明可影响钙信号传导途径的非共面卤化持久性有机污染物的特性。在最终目标中，这些检测系统将被整合和优化，然后用于一系列验证研究，用于检测和相对定量从各种基质中提取的复杂化学品混合物中存在的有毒化学品。总的来说，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效应的基本知识， 将开发的特定生物测定和生物标志物将提供用于检测有毒物质和有毒物质接触的快速机械筛选系统。

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.