DEVELOPMENT AND APPLICATIONS OF INTEGRATED CELL BASED BIOASSAYS

该项目的重点是基于生物学的危险物质检测 通过利用其特定作用机制的方法。首先， 将为几类物质开发灵敏且快速的生物测定法 对环境有重要影响的毒物。在一种方法中，能够 某些试剂通过结合并激活来刺激基因表达 化学特异性 DNA 转录因子将被用于 某些类别化学品的生物测定。为此，细胞将 他用含有荧光素酶的表达载体稳定转染 报告基因受到一种高亲和力相互作用的诱导调节 几种配体依赖性受体（包括雌激素受体， 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 互补方法，竞争性体外结合微孔板测定 将利用纯化的技术来开发这些化学物质的检测 由杆状病毒大量产生的受体 表达系统。最初的努力将集中在异生素上， 与 Ah 和雌激素受体相互作用，并将延伸至 包括其他化学品（例如金属和过氧化物酶体增殖剂） 它还通过化学依赖性受体改变基因表达。第二， 危险物质扰乱生化过程的能力 对于细胞生长和分化很重要，将使用以下方法进行检查 人表皮角质形成细胞作为目标。样品的直接毒性 将测量样品对培养物敏感的能力 代谢激活剂（如多环）的毒性 芳香烃。此外，根据我们最初的发现 砷、选择激酶的磷酸化状态和水平以及 将检查磷酸酶活性。蛋白激酶C同工酶， 对于正常的角质形成细胞功能至关重要，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 兴趣。此外，蛋白质酪氨酸磷酸水平及其对 特定 DNA 转录因子（AP2、类固醇激素受体）和 将酌情监测分化标记物（外皮蛋白）。 这些信息将有助于追踪信号转导途径 哪些有毒物质会干扰。最后，热休克蛋白亚型表达 和亚细胞定位将作为细胞的衡量标准进行检查 压力。化学处理的效果将与实际同工型进行比较 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观察到的效果将使用人类皮肤来实现 在器官培养中。对于上述每个测定，环境的适用性 其他超级基金组件提供的样本将使用 模型药物的纯化和复杂混合物以及提取物 塑料、金属和其他成分的燃烧残留物 在危险废物场。

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.