BIOASSAY/BIOMARKER SYSTEMS TO DETECT HALOGENATED HYDROCARBONS

许多危险废物场地含有卤代芳香化合物的复杂混合物 碳氢化合物(HAHS)，包括多氯-和 多溴联苯和多氯二苯并二恶英和 二苯并呋喃。这些持久性的有毒化合物可以迁移到异地。 穿过地表水和地下水，一旦进入周围 在环境中，它们很容易被生物积累和生物放大。虽然 暴露于特定的HAHS可导致多种特定物种的 在低浓度下的毒性和生物效应，诱导 HAHS的细胞色素P450IA1是一种高度保守的反应 物种。P450IA1的诱导是由一种可溶性的胞内分子介导的 与HAH特异结合的蛋白质(ah受体(AhR))，并与 亲和力强。结合后，HAH：AHR复合体在 核和激活基因转录通过相互作用 基因上游的特定DNA序列(二恶英响应增强子) P450IA1基因。构效关系的研究也牵涉到 AHR在调节HAHS毒性中的作用。因此，即使不是全部，也有许多 对HAHS的毒性和生物学反应是由AhR介导的。这个 这项提议的总体目标是使用这一机制的几个方面 建立检测HAHS的生物检测/生物标志物系统。我们会 稳定地转染HAH诱导的表达载体，该表达载体含有 碱性磷酸酶或荧光素酶报告基因，转入HAH反应 人、鼠和鱼的细胞。将生物活性HAHS暴露在这些细胞中将 诱导报告基因的表达达到与HAH成正比的水平 剂量。将对该生物检测系统进行表征、校准和验证 使用已知的HAH标准、HAH混合物和未知的样品提取物 含有HAHS的复杂混合物。将使用凝胶延迟分析 建立一种灵敏的测定系统，以定量测定 HAHS染毒动物外周血淋巴细胞HAH结合的AHR复合体 在活体内。我们还将研究HAHS在体内的分子作用机制。 鱼类以确定HAHS是否以类似的方式起作用 在哺乳动物系统中描述的。通过这种方式，我们可以评估 鱼类作为监测HAH污染的潜在哨点物种 以及它与人类群体潜在不良影响的关系。 这些生物检测/生物标记物系统准确预测 不同品种样品提取物中复杂HAH混合物的TCDD-TEQs 生物和非生物基质将通过与 HAHS在这些样品中的绝对浓度，由 仪器分析。这些研究不仅将产生几个新的 用于HAHS检测和监测的敏感生物检测/生物标志物系统 但将提供新的途径来检验生物活性HAHS在 人和动物。

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.