BIOASSAY/BIOMARKER SYSTEMS TO DETECT HALOGENATED HYDROCARBONS

用于检测卤代烃的生物测定/生物标记系统

• 批准号: 3777260
• 负责人: M S DENISON
• 金额: --
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3777260
• 关键词: alkaline phosphatase; alternatives to animals in research; animal tissue; bioassay; biomarker; carbopolycyclic compound; cytochrome P450; dioxins; environmental contamination; environmental toxicology; gene expression; gene induction /repression; genetic transcription; guinea pigs; halobiphenyl /halotriphenyl compound; human tissue; laboratory rabbit; laboratory rat; luciferin monooxygenase; method development; protein structure function; reporter genes; transfection; transfection /expression vector; trout /salmon; water pollution

Many hazardous waste sites contain complex mixtures of halogenated aromatic hydrocarbons (HAHs) of the class that includes the polychlorinated- and polybrominated-biphenyls and polychlorinated dibenzo-p-dioxins and dibenzofurans. These persistent, toxic compounds can migrate off-site through surface and ground water flow and once in the surrounding environment they can be readily bioaccumulated and biomagnified. Although exposure to specific HAHs can result in a wide variety of species-specific toxic and biological effects at low concentrations, the induction of cytochrome P450IA1 by HAHs is one response that is highly conserved across species. Induction of P450IA1 is mediated by a soluble intracellular protein (the Ah receptor (AhR)) which binds the HAH specifically and with high affinity. After binding, HAH:AhR complexes accumulate within the nucleus and activate gene transcription through an interaction with specific DNA sequences (dioxin responsive enhancers) upstream of the P450IA1 gene. Studies of structure-activity relationship also implicate the AhR in mediating the toxicity of HAHs. Thus, many, if not all, of the toxic and biological responses to HAHs are mediated by the AhR. The overall goal of this proposal is to use several aspects of this mechanism to develop bioassay/biomarker systems for the detection of HAHs. We will stably transfect an HAH-inducible expression vector, which contains an alkaline phosphatase or luciferase reporter gene, into HAH-responsive human, rat and fish cells. Exposure of bioactive HAHs to these cells will induce expression of the reporter gene to a level proportional to the HAH dose. This bioassay system will be characterized, calibrated and validated using known HAH standards, HAH mixtures and unknown sample extracts containing complex mixtures of HAHs. Gel retardation analysis will be used to develop a sensitive assay system for quantitating the relative amount of HAH-bound ahR complex in peripheral lymphocytes of animals exposed to HAHs in vivo. We will also examine the molecular mechanism of action of HAHs in fishes in order to determine if HAHs act in a manner analogous to that described in mammalian systems. In this way we can evaluate the utility of fishes as potential sentinel species for monitoring for HAH contamination and its relationship to potential adverse effects in human populations. The ability of these bioassay/biomarker systems to accurately predict the TCDD-TEQs of complex HAH mixtures present in sample extracts from various biotic and abiotic matrices will be evaluated by direct comparison to the absolute concentration of HAHs in these samples, as determined by instrumental analysis. These studies will not only produce several new sensitive bioassay/biomarker systems for detection and monitoring of HAHs but will provide new avenues for examining the effects of bioactive HAHs in man and animals.

许多危险废物场含有复杂的卤代芳烃混合物， 碳氢化合物（HAH）的类别，包括多氯-和 多溴联苯和多氯二苯并对二恶英， 二苯并呋喃。 这些持久的有毒化合物可以迁移到其他地方 通过地表水和地下水流动， 环境中，它们可以很容易地被生物积累和生物聚集。 虽然 暴露于特定的HAH可以导致各种各样的物种特异性 低浓度的毒性和生物效应， 细胞色素P450 IA 1通过HAH是一种高度保守的反应， 物种 P450 IA 1的诱导是由可溶性细胞内 蛋白质（Ah受体（AhR）），其特异性结合HAH， 高亲和力。 结合后，HAH：AhR复合物在细胞内积累。 细胞核并通过与 特异性DNA序列（二恶英响应增强子）上游的 P450 IA 1基因。 构效关系的研究也表明 AhR在介导HAH毒性中的作用。 因此，许多人，如果不是全部， 对HAH的毒性和生物学反应由AhR介导。 的 本提案的总体目标是利用该机制的几个方面 发展生物测定/生物标记系统，以检测有害环烷烃。 我们将 稳定转染HAH诱导型表达载体，其含有 碱性磷酸酶或荧光素酶报告基因，转化为HAH反应性 人、大鼠和鱼细胞。 将生物活性HAH暴露于这些细胞将 诱导报告基因表达至与HAH成比例的水平 次给药结束 将对该生物测定系统进行表征、校准和验证 使用已知的HAH标准品、HAH混合物和未知样品提取物 含有杂环芳烃的复杂混合物。 将使用凝胶阻滞分析 开发一种灵敏的测定系统，用于定量 多环芳烃接触动物外周血淋巴细胞中多环芳烃结合ahR复合物的研究 in vivo. 我们还将研究HAH在以下方面的分子作用机制： 鱼，以确定HAH的行为是否与 在哺乳动物系统中描述。 这样我们就可以评估 鱼类作为监测有害环烷烃污染的潜在哨点物种 及其与人群中潜在不良影响的关系。 这些生物测定/生物标志物系统准确预测生物标志物的能力， 各种样品提取物中存在的复杂HAH混合物的TCDD-TEQ 生物和非生物基质将通过直接比较 这些样品中HAH的绝对浓度，由 仪器分析 这些研究不仅会产生一些新的 用于检测和监测HAH的灵敏生物测定/生物标记物系统 但将提供新的途径来检查生物活性HAH的影响， 人与动物