DEVELOPMENT AND APPLICATIONS OF INTEGRATED CELL BASED BIOASSAYS

集成细胞生物测定的开发和应用

基本信息

批准号：
6664551
负责人：
ROBERT H RICE
金额：
$ 16.52万
依托单位：
UNIVERSITY OF CALIFORNIA AT DAVIS
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-09-24 至 2003-03-31
项目状态：
已结题

项目摘要

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.

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适用于化学检测和筛选的基于机械的细胞生物测定/生物标记。由于了解细胞对某一类毒物的特定反应，可以极大地促进生物测定/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，哪些化学物质影响细胞受体、信号转导途径和细胞功能。在目标 1 中，将开发稳定转染的细胞系，其对一种或多种激素（类固醇、甲状腺和视黄酸）做出反应，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。给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查它们激活/抑制报告基因表达诱导的能力来快速确定。在目标 2 中，使用消减杂交和 DNA 阵列技术测量人角质形成细胞中二恶英、砷和铬对基因表达的扰动，将能够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，将使用细胞和小鼠对可溶性环氧化物水解酶 (sEH) 生成的氧脂素代谢物作为相关生物标志物进行验证，作为暴露于改变 sEH 活性的危险环境化学品所产生的暴露/影响的相关生物标志物。 DNA 阵列和元组学方法也将被开发和用于识别暴露于 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管细胞系（野生型和突变型），作为对导致 Ca2+ 调节和信号传导功能障碍的环境化学物质正常和高度易感性的生物标志物，并作为识别环境毒物的生物测定法。影响钙信号传导。总体而言，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基于机械的快速筛选系统来检测毒物和毒物暴露。