DEVELOPMENT AND APPLICATIONS OF INTEGRATED CELL BASED BIOASSAYS

危险废物场址含有各种有毒化学物质的复杂混合物。不幸的是，由于缺乏可用的特定生物测定/生物标志物系统，对环境和生物样品中特定化学物质或化学类别进行快速和廉价检测的发展受到了阻碍。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生物测定/生物标记物，这些生物标记物可用于化学检测和筛选。由于生物测定/生物标记物的有效开发和应用极大地促进了对细胞对某一类有毒物质的特定反应的理解，因此四种建议的方法中的每一种都将利用化学物质影响细胞受体、信号转导途径和细胞功能的基本分子机制的分析所得的信息。在Aim 1中，将开发稳定转染的细胞系，通过诱导萤火虫荧光素酶或绿色荧光蛋白受体基因的受体/依赖性表达，对一种或多种激素（类固醇、甲状腺和视黄酸）产生反应。特定化学物质或复杂化学混合物的内分泌干扰活性可以通过检测其激活/抑制诱导报告基因表达的能力来快速确定。在目标2中，使用减法杂交和DNA阵列技术测量人类角质形成细胞中二恶英、砷和铬对基因表达的扰动，将允许鉴定一系列基因产物（即潜在的生物标志物），其表达被这些化学物质改变。在Aim 3中，将使用细胞和小鼠评估可溶性环氧化物水解酶（sEH）产生的氧脂代谢产物作为相关生物标志物的有效性，因为暴露于危险环境化学品会改变sEH活性，从而导致暴露/影响的相关生物标志物。DNA阵列和代谢组学方法也将被开发和用于鉴定暴露于sEH抑制剂的遗传和代谢生物标志物。在Aim 4中，将开发一系列转基因骨骼肌小管细胞系（野生型和突变型），作为对导致Ca2+调节和信号功能障碍的环境化学物质的正常和高度易感性的生物标志物，并作为识别影响钙信号的环境毒物的生物测定。总的来说，这些方法不仅利用了化学物质激活/抑制细胞内受体介导的信号事件和基因表达的能力，而且这些生物测定和生物标志物提供了快速的基于机械的筛选系统来检测有毒物质和有毒物质暴露。

Hazardous waste sites contain complex mixtures of a wide variety of toxic chemicals. Unfortunately, development of a 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 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 of class of toxicants, each of the four proposed approaches will exploit information derived from an analysis of the basic molecular mechanisms by which chemicals affect cellular receptors, signal transduction pathways, and cell functions. In Aim 1, stably transfected cell lines will be developed which respond to one or more hormones (steroid, thyroid and retinoic acid) with the induction of receptor/dependent expression of firefly luciferase or green fluorescent protein receptor genes. The endocrine disrupting activity of a given chemical(s), or complex chemical mixtures could rapidly be determined by examining their ability to activate/inhibit induction of reporter gene expression. In Aim 2, measurement of perturbations in gene expression by dioxin, arsenic and chromium in human keratinocytes, using subtractive hybridization and DNA array techniques will allow identification of a series of gene products (i.e. potential biomarkers of effect) whose expression is altered by these chemicals. In Aim 3, validation of soluble epoxide hydrolase (sEH)-generated oxylipin metabolites as relevant biomarkers as relevant biomarkers of exposure/effect resulting from exposure to hazardous environmental chemicals which alter sEH activity will be evaluated using cells and mice. DNA array and metanomics approaches will also be developed and employed for identifying genetic and metabolic biomarkers of exposure to sEH inhibitors. In Aim 4, a series of transgenic skeletal myotubule cell lines (wild-type and mutant) will be developed as biomarkers of normal and heightened susceptibility to environmental chemicals causing dysfunction in Ca2+-regulation and signaling and as bioassays for the identification of environmental toxicants which influence calcium signaling. Overall, these approaches not only take advantage of the ability of a chemical to activate/inhibit intracellular receptor mediated signaling events and gene expression, but these bioassays and biomarkers provide rapid mechanistically-based screening systems to detect toxicants and toxicant exposure.