ENVIRONMENTAL TOXICOLOGY USING TRANSGENIC MOUSE MODELS

描述(改编自申请人的摘要)：总体长期 这些研究的目的是建立和使用转基因动物模型。 研究与环境健康有关的问题的系统。具体地说，我们的 研究的重点是金属硫蛋白(MTS)和转录因子 调节MT基因。我们建议确定分子机制 镉和氧化对MT基因表达的调控作用 应激，以及MT在抗氧化中的功能意义 压力。Cd是一种广泛存在的环境污染物和许多外来生物 造成破坏性的氧化应激。MT代表了记录最好的 细胞内重金属(锌、铜、镉)结合蛋白，但它们也可以 清除羟基自由基，清除超氧阴离子。 氧化应激对小鼠MT-I基因转录的诱导 涉及MTF-1与金属反应元件(MRE)结合的激活 所述启动子以及复合USF/抗氧化反应启动子 元素(USF/ARE)。初步证据表明，CD诱导也是 部分由这种元素介导，而锌的诱导不是。小才是 了解MTF-1或USF/ARE的结构和功能。转基因 过表达MT或靶向去除MT-I/MT-II基因的小鼠 函数已创建，并将用于检查这些 保护蛋白质免受氧化应激。这样做的具体目的是 建议是：1)确定氧化的分子机制 应激和金属激活MTF-1；2)探索MTF-1的功能作用 小鼠MT-I启动子诱导过程中的USF/抗氧化反应元件 通过氧化应激和CD；3)检测蛋白质与USF/ARE的相互作用 氧化应激和镉诱导的基因表达；4)分析 过表达MT或MT的转基因小鼠对氧化应激的抵抗力 靶向是MT基因的消融。分子(诱变、EMSA、 足迹法、转染法)和生化(锌滴定分析、 免疫沉淀、结合部位层析)方法将用于 定义MTF-1的结构-功能关系(锌结合，DNA结合， 蛋白质相互作用和反式激活)。的结构--功能 USF/ARE将通过突变、转基因分析和蛋白质来定义 有约束力的分析。转基因小鼠对氧化应激的抵抗力将是 通过组织病理学和血清酶水平进行监测。

DESCRIPTION (Adapted from the APPLICANT'S ABSTRACT): The overall long-term objective of these studies is to develop and use transgenic animal model systems to study environmental health-related issues. Specifically, our studies focus on the metallothioneins (MTs) and transcription factors that regulate MT genes. We propose to determine the molecular mechanisms regulating expression of MT genes in response to cadmium (Cd) and oxidative stresses, and the functional significance of MT in protection from oxidative stress. Cd is a widespread environmental pollutant and many xenobiotics cause damaging oxidative stress. MTs represent the best documented intracellular heavy metal (Zn, Cu, Cd) binding proteins, but they can also scavenge hydroxyl free radicals, and dismutate superoxide anions. Transcriptional induction of the mouse MT-I gene by oxidative stress involves activation of MTF-1 binding to metal responsive elements (MRE) in the promoter, as well as a composite USF/antioxidant response promoter element (USF/ARE). Preliminary evidence indicates that Cd induction is also mediated, in part, by this element whereas Zn induction is not. Little is known about the structure and function of MTF-1 or the USF/ARE. Transgenic mice that over-express MT or that have targeted ablations of MT-I/MT-II gene function have been created and will be used to examine the roles of these proteins in protection from oxidative stress. The specific aims of this proposal are to: 1) Determine the molecular mechanisms by which oxidative stresses and metals activate MTF-1; 2) Explore the functional roles of the USF/antioxidant response element in the mouse MT-I promoter during induction by oxidative stress and Cd; 3) Examine protein interactions with the USF/ARE during oxidative stress and Cd induction of gene expression; and 4) Analyze resistance to oxidative stress in transgenic mice that over-express MT or that have targeted ablations of the MT genes. Molecular (mutagenesis, EMSA, footprinting, transfection) and biochemical (Zn titration analysis, immunoprecipitation, binding site chromatography) approaches will be used to define structure-function relationships for MTF-1 (Zn binding, DNA binding, protein interactions and transactivation). Structure-function of the USF/ARE will be defined by mutagenesis, transfection assays, and protein binding assays. Resistance to oxidative stress in transgenic mice will be monitored by histopathology and serum enzyme levels.