RECOMBINANT DETECTION SYSTEMS FOR DIOXIN LIKE CHEMICALS

卤代芳香烃(HAHS)，如多氯二苯并-2，4-二苯基-2，4，4-二苯基-2，4，4-二苯基-2，4，4-二氯代-2，4-二苯基-2，4，4，5-二苯基-2，4，4，5，5，5，5，5，5，5，5，5，5，5，5，5，5，5，5，5，5，5，4，5，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4， 对二恶英(PCDDs)、联苯(PCbs)和二苯并呋喃(PCDf)及相关物质 化合物代表了一组不同的广泛存在的环境 污染物，其中许多是有毒的，在环境中持久存在。 已经观察到，接触和生物积累HAHS会产生一种 种类繁多，具有组织特异性的毒性和生物效应， 如肿瘤促进、致命性、出生缺陷、肝毒性、 免疫毒性、皮肤毒性、内分泌稳态改变和 诱导多种酶(1，2)。尽管暴露在特定的HAHS中， 如2，3，7，8-四氯二苯并对二恶英(TCDD，Dioxin) 这些在低浓度下的各种影响，诱导了 HAHS的细胞色素P45O1A1是一种高度保守的反应 物种，并以此为模型系统进行了机理分析。 HAHS的行动。P45O1A1的诱导是由一种可溶性的 结合HAH的细胞内蛋白(AH受体(AhR)) 具体地说，具有很高的亲和力。HAH：AHR络合物结合后， 转化为DNA结合形式后，它们随后在 细胞核通过高亲和力激活基因转录 与特定DNA序列的相互作用(二恶英反应增强剂) 位于P45O1A1基因上游。除了调解诱导 P45O1A1，构效关系研究表明 一种化学物质与AhR结合的能力之间存在很强的相关性 以及其产生毒性的能力，暗示AhR在调节 HAHS的毒性。因此，许多，如果不是全部，有毒的和生物性的 对HAHS的反应目前似乎是由AhR调解的。整体而言 这项提议的目标是使用这个AhR的几个机械方面- 依赖系统开发检测HAHS的生物检测系统。 考虑到一种化学物质的能力与 与AhR结合，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 结合分析(凝胶延迟分析和新型膜过滤 化验)作为检测二恶英类化学物质的生物化验。此外, 我们将稳定地转染几个HAH诱导的表达载体 含有荧光素酶或碱性磷酸酶报告基因，转入HAH- 反应灵敏的人类和小鼠细胞系。暴露这些重组细胞 二恶英类HAHS的品系会诱导表达 报告基因的水平与HAH剂量成正比。DNA结合 和基因表达生物分析系统将被表征、校准和 使用已知的HAH标准、HAH混合物和未知样品进行验证 含有HAHS复杂混合物的提取物。这些人的能力 生物测定/生物标志物系统准确预测络合物的TCDD-TBQs 存在于各种生物和非生物样品提取物中的HHH混合物 矩阵将通过直接比较以下内容进行评估 这些样品中的HAHS，由仪器分析确定。这些 研究不仅将产生几个新的敏感生物检测/生物标志物 用于检测和监测HAHS的系统，但它们也将是有用的 为毒物鉴定评估提供了新的途径 检测生物活性HAHS对人和动物的影响。

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.