DEVELOPMENT AND APPLICATIONS OF INTEGRATED CELL BASED BIOASSAYS

危险废物场地含有多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，快速、廉价地检测环境和生物样品中的特定化学物质或化学类别的发展一直受到阻碍。因此，该项目的总体目标是开发和验证一系列应用于化学检测和筛选的基于机械的细胞生物分析/生物标记物。由于生物测定/生物标志物的有效开发和应用在很大程度上是通过了解细胞对给定毒物类毒物的具体反应来实现的，因此这四种方法中的每一种都将利用从分析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中获得的信息。在目标1中，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，建立对一种或多种激素(类固醇、甲状腺和维甲酸)有反应的稳定表达的细胞系。一种特定的化学物质(S)或复杂的化学混合物的内分泌干扰活性可以通过检测它们激活/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阵列技术测量二恶英、砷和铬对人角质形成细胞基因表达的扰动，将有助于识别这些化学物质改变其表达的一系列基因产物(即潜在的生物标志物)。在目标3中，将使用细胞和小鼠评估由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作为暴露/暴露于改变sEH活性的危险环境化学品所导致的影响的相关生物标志物的有效性。DNA阵列和代谢组学方法也将被开发和应用于识别暴露于sEH抑制剂的遗传和代谢生物标记物。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)，作为正常和对环境化学物质导致钙调节和信号功能障碍的高敏感性的生物标志物，并作为鉴定影响钙信号的环境毒物的生物检测手段。总体而言，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，而且这些生物检测和生物标记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.