RECOMBINANT DETECTION SYSTEMS FOR DIOXIN LIKE CHEMICALS

卤化芳族烃（HAH），例如多氯二苯并- 对二恶英（PCDDs）、联苯（PCBs）和二苯并呋喃（PCDFs）以及相关 化合物代表了一组广泛的环境 污染物，其中许多是有毒的，并在环境中持久存在。 据观察，接触和生物积累多环芳烃会产生 多种物种和组织特异性毒性和生物效应， 例如肿瘤促进、致死性、出生缺陷、肝毒性 免疫毒性、皮肤毒性、内分泌稳态改变， 许多酶的诱导（1，2）。尽管暴露于特定的有害环烷烃， 例如2，3，7，8-四氯二苯并-对-二恶英（TCDD，二恶英），可以产生 这些在低浓度下的各种各样的影响， 细胞色素P45 O 1A 1通过HAH是一种高度保守的反应， 物种，并作为模式系统，分析的机制， HAH的作用。P45 O 1A 1的诱导是由可溶性的 结合HAH的细胞内蛋白（Ah受体（AhR）） 特异性和高亲和力。在结合后，HAH：AhR复合物是 转化为DNA结合形式，它们随后积累在 并通过高亲和力激活基因转录 与特定DNA序列的相互作用（二恶英反应增强子） P45 O 1A 1基因的上游。除了调解诱导 P45 O 1 A1，构效关系研究已证明 一种化学物质与AhR结合的能力 及其产生毒性的能力，暗示AhR介导了 HAHS的毒性。因此，许多，如果不是全部，有毒和生物的， 对HAH的反应目前似乎是由AhR介导的。整体 该提案的目标是使用该AhR的几个机械方面- 开发用于检测HAH的生物测定系统。 考虑到化学物质的能力与化学物质的毒性之间的高度相关性， 结合AhR，其诱导AhR转化和DNA结合的能力 和基因表达，我们将开发，优化和利用两个AhR DNA 结合测定（凝胶阻滞分析和新型膜过滤 测定）作为检测二恶英类化学物质的生物测定。此外，本发明还提供了一种方法， 我们将稳定地转染几种HAH诱导型表达载体， 含有荧光素酶或碱性磷酸酶报告基因，转化为HAH- 应答的人和小鼠细胞系。这些重组细胞的暴露 二恶英样HAH的诱导表达， 报告基因的水平成比例的HAH剂量。DNA结合 和基因表达生物测定系统将被表征，校准和 使用已知HAH标准品、HAH混合物和未知样品进行验证 提取物含有复杂的混合物的羟基芳烃。这些能力 生物测定/生物标志物系统，以准确预测复杂的TCDD-TBQ 各种生物和非生物样品提取物中存在的HAH混合物 将通过与以下浓度直接比较来评价基质： 通过仪器分析确定这些样品中的HAH。这些 研究不仅会产生几种新的敏感的生物测定/生物标志物， 系统的检测和监测，但他们也将是有用的 为毒物鉴定评价提供新的途径， 研究生物活性HAH对人类和动物的影响。

Halogenated aromatic hydrocarbons (HAHs), such as polychlorinated-dibenzo- p-dioxins (PCDDs), biphenyls (PCBs) and dibenzofurans (PCDFs), and related compounds represent a diverse group of widespread environmental contaminants, many of which are toxic and persistent in the environment. Exposure to and bioaccumulation of HAHs have been observed to produce a wide variety of species and tissue-specific toxic and biological effects, such as tumor promotion, lethality, birth defects, hepatotoxicity, immunotoxicity, dermal toxicity, alterations in endocrine homeostasis and induction of numerous enzymes (1,2). Although exposure to specific HAHs, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD,dioxin), can produce these wide variety of effects at low concentrations, the induction of cytochrome P45O1A1 by HAHs is one response that is highly conserved across species and is used as the model system for analysis of the mechanism of action of HAHs. Induction of P45O1A1 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 are converted into their DNA binding form, they subsequently accumulate within the nucleus and activate gene transcription through a high affinity interaction with a specific DNA sequence (dioxin responsive enhancers) upstream of the P45O1A1 gene. In addition to mediating the induction of P45O1A1, structure-activity relationship studies have demonstrated a strong correlation between the ability of a chemical to bind to the AhR and its ability to produce toxicity, implicating the AhR in mediating the toxicity of HAHs. Thus, many, if not all, of the toxic and biological responses to HAHs currently appear to mediated by the AhR. The overall goal of this proposal is to use several mechanistic aspects of this AhR- dependent system to develop bioassay systems for the detection of HAHs. Given the high degree of correlation between the ability of a chemical to bind to the AhR, its ability to induce AhR transformation and DNA binding and gene expression, we will develop, optimize and utilize two AhR DNA binding assays (gel retardation analysis and a novel membrane filtration assay) as bioassays for detection of dioxin-like chemicals. In addition, we will stably transfect several HAH-inducible expression vectors which contains a luciferase or alkaline phosphatase reporter gene, into HAH- responsive human and mouse cell lines. Exposure of these recombinant cell lines to dioxin-like HAHs will result in the induction of expression of the reporter gene to a level proportional to the HAH dose. The DNA binding and gene expression bioassay systems will be characterized, calibrated and validated using known HAH standards, HAH mixtures and unknown sample extracts containing complex mixtures of HAHs. The ability of these bioassay/biomarker systems to accurately predict the TCDD-TBQs of complex HAH mixtures present in sample extracts from various biotic and abiotic matrices will be evaluated by direct comparison to the 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 they will also be useful for toxicant identification evaluation and will provide new avenues for examining the effects of bioactive HAHs in man and animals.