DEVELOPMENT AND APPLICATIONS OF INTEGRATED CELL BASED 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，由于缺乏可用的特定生物测定/生物标志物系统，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开发受到阻碍。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。由于有效的开发和应用的生物测定/生物标志物是大大促进了细胞的特定响应的一类毒物的理解，每四个建议的方法将利用来自化学品影响细胞受体，信号转导途径和细胞功能的基本分子机制的分析信息。在目的1中，将开发稳定转染的细胞系，其响应于一种或多种激素（类固醇、甲状腺和视黄酸），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。特定化学品或复杂化学品混合物的内分泌干扰活性可通过检查其激活/抑制报告基因表达诱导的能力来迅速确定。在目标2中，使用消减杂交和DNA阵列技术测量二恶英、砷和铬对人类角质形成细胞中基因表达的干扰，将允许鉴定一系列基因产物（即潜在的效应生物标志物），其表达被这些化学物质改变。在目标3中，将使用细胞和小鼠评估可溶性环氧化物水解酶（sEH）产生的氧脂素代谢物作为相关生物标志物作为暴露于改变sEH活性的有害环境化学品引起的暴露/效应的相关生物标志物的验证。还将开发DNA阵列和元组学方法，用于鉴定暴露于sEH抑制剂的遗传和代谢生物标志物。在目标4中，将开发一系列转基因骨骼肌微管细胞系（野生型和突变型）作为正常和对环境化学物质敏感性提高的生物标志物，这些化学物质导致Ca 2+调节和信号传导功能障碍，并作为生物测定用于鉴定影响钙信号传导的环境毒物。总之，这些方法不仅利用化学物质激活/抑制细胞内受体介导的信号传导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毒物和毒物暴露。

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.