DEVELOPMENT AND APPLICATIONS OF INTEGRATED CELL BASED BIOASSAYS

该项目的重点是基于生物的危险物质检测。 通过利用其特定的作用机制的方法。第一, 将为几类人开发灵敏和快速的生物检测方法 对环境有重要影响的毒物。在一种方法中， 某些通过结合和激活来刺激基因表达的试剂 化学特异的DNA转录因子将在 某些类别的化学物质的生物测定。为此，细胞将 他稳定地用含有荧光素酶的表达载体转染 报告基因受One的高亲和力相互作用诱导调控 几种配体依赖的受体(包括雌激素的受体， 二恶英和相关的多环芳烃)与转录 增强子元件插入报告基因的上游。在一个 互补法、竞争性体外结合微板法 为了检测这些化学物质，将开发利用提纯的 杆状病毒大量产生的受体 表达系统。最初的努力将集中在外源生物上， 与Ah和雌激素受体相互作用，并将广泛倾向于 包括其他化学品(如金属和过氧化物酶增殖剂) 这也会改变依赖化学物质的受体的基因表达。第二, 有害物质扰乱生化过程的能力 对细胞生长和分化的重要作用将使用 以人表皮角质形成细胞为靶细胞。样品的直接毒性 将被测量以及样本对培养物敏感的能力 代谢激活剂的毒性，如多环 芳香烃。此外，根据我们最初的发现， 砷、选择蛋白的磷酸化状态和水平 将检查磷酸酶活性。蛋白激酶C同工酶， 对角质形成细胞的正常功能至关重要，并且已知(角质形成细胞 转谷氨酰胺酶)或可疑的蛋白质底物将具有特殊的 利息。此外，蛋白质酪氨酸磷酸水平及其对 特异性DNA转录因子(AP2、类固醇激素受体)和 将酌情监测分化标记物(总环蛋白)。 这些信息将有助于追踪信号转导途径 哪种毒物会干扰。最后，热休克蛋白亚型的表达 而亚细胞定位将作为细胞的一种衡量标准进行检查 压力。化学处理的效果将与实际的异构体进行比较 人类皮肤肿瘤和砷角化病的扰动。验证 在培养的角质形成细胞中看到的效果将使用人类皮肤来执行 在器官培养方面。对于上述每一种化验，对环境的适用性 其他Superfund组件提供的样本将使用以下方法进行测试 模型试剂的纯化和复杂混合物以及黄连提取物 塑料、金属和其他成分产生的燃烧残留物 在危险废物地点。

This project focuses on biologically-based detection of hazardous agents by methods exploiting their specific mechanisms of action. First, sensitive and rapid bioassays will be developed for several classes of environmentally important toxicants. In one approach, the ability of certain agents to stimulate gene expression by binding to and activating chemical-specific DNA transcription factors will be exploited in bioassays for certain classes of chemicals. For this purpose, cells will he stably transfected with an expression vector containing a luciferase reporter gene inducibly regulated by the high affinity interaction of one of several ligand-dependent receptors (including those for estrogens, dioxin and related polycyclic aromatic hydrocarbons) with transcriptional enhancer elements inserted upstream of the reporter gene. In a complementary approach, competitive in vitro binding microplate assays for the detection of these chemicals will be developed utilizing purified receptors which have been produced in large quantities by a baculovirus expression system. Initial efforts will be focused on xenobiotics which interact with the Ah and estrogen receptors and will be ex:tended to include other chemicals (such as metals and peroxisome proliferators) that also alter gene expression by chemical-dependent receptors. Second, the ability of hazardous agents to perturb biochemical processes important for cellular growth and differentiation will be examined using human epidermal keratinocytes as targets. The direct toxicity of samples will be measured as well as the ability of samples to sensitize cultures to toxicity from agents activated by metabolism such as polycyclic aromatic hydrocarbons. In addition, based on our original findings with arsenic, the phosphorylation state and levels of select kinase and phosphatase activities will be examined. Protein kinase C isozymes, critical for proper keratinocyte function, and known (keratinocyte transglutaminase) or suspected protein substrates will be of special interest. Moreover, protein tyrosine phosphate levels and effects on specific DNA transcription factors (AP2, steroid hormone receptors) and differentiation markers (involucrin) will be monitored as appropriate. This information will help in tracing signal transduction pathways with which toxicants interfere. Finally, heat shock protein isoform expression and subcellular localization will be examined as a measure of cell stress. Effects of chemical treatment will be compared to actual isoform perturbation in human skin tumors and arsenical keratoses. Validation of effects seen in cultured keratinocytes will be performed using human skin in organ culture. For each assay above, applicability to environmental samples provided by other Superfund components will be tested using purified and complex mixtures of model agents as well as extracts of combustion residues from plastics, metals and other constituents present at hazardous waste sites.