RECOMBINANT DETECTION SYSTEMS FOR DIOXIN LIKE CHEMICALS

二恶英类化学品的重组检测系统

• 批准号: 2444232
• 负责人: MICHAEL STEVEN DENISON
• 金额: $ 7.81万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2444232
• 关键词: DNA binding protein; bioassay; biomarker; carbopolycyclic compound; clone cells; cytochrome P450; dioxins; environmental toxicology; enzyme induction /repression; guinea pigs; northern blottings; recombinant DNA; site directed mutagenesis; southern blotting; toxicant screening; transfection

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.

卤化芳基烃（HAHS），例如多氯联苯 - p-dioxin（PCDD），双苯基（PCB）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，其中许多在环境中具有毒性和持久性。 已经观察到HAH的暴露和生物积累会产生A 各种各样的物种和组织特异性的有毒和生物学作用， 例如肿瘤促进，致死性，出生缺陷，肝毒性， 免疫毒性，皮肤毒性，内分泌稳态的改变和 诱导许多酶（1,2）。虽然暴露于特定的hahs，但 例如2,3,7,8-四氯迪本佐-P-二恶英（TCDD，二恶英）可以产生 这些在低浓度下的各种效果，诱导 HAHS的细胞色素P45O1A1是一个高度保守的响应 物种并用作分析机制的模型系统 哈斯的动作。 P45O1A1的诱导是由可溶性介导的 结合HAH的细胞内蛋白（AH受体（AHR）） 具体，具有高亲和力。绑定后，哈哈：ahr络合物是 转换为其DNA结合形式，随后积聚 细胞核并通过高亲和力激活基因转录 与特定的DNA序列的相互作用（二恶英反应性增强子） p45O1a1基因上游。除了介导 P45O1A1，结构活性关系研究表明 化学物质与AHR结合的能力之间的强相关性 以及产生毒性的能力，牵涉到AHR介导 哈斯的毒性。因此，许多（如果不是全部）有毒和生物学 目前对HAH的反应似乎是由AHR介导的。总体 该提议的目标是使用此AHR的几个机械方面 开发用于检测HAH的生物测定系统的依赖系统。 鉴于化学能力之间的高度相关性 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 结合测定（凝胶延迟分析和新型膜过滤 分析）作为检测二恶英样化学物质的生物测定。此外， 我们将稳定转染几个HAH诱导的表达向量，这些向量 含有荧光素酶或碱性磷酸酶报告基因， 响应的人和小鼠细胞系。这些重组细胞的暴露 二恶英样的线将导致诱导的表达 记者基因的水平与HAH剂量成正比。 DNA结合 和基因表达生物测定系统将被表征，校准和 使用已知的HAH标准，HAH混合物和未知样品验证 含有HAH的复杂混合物的提取物。这些能力 生物测定/生物标志物系统，以准确预测复合物的TCDD-TBQ 来自各种生物和非生物的样品提取物中存在的HAH混合物 矩阵将通过直接比较与 通过工具分析确定这些样品中的HAH。这些 研究不仅会产生几个新的敏感生物测定/生物标志物 用于检测和监视HAH的系统，但它们也将很有用 进行有毒的识别评估，并将为 检查生物活性HAHS对人和动物的影响。