ENVIRONMENTAL TOXICOLOGY USING TRANSGENIC MOUSE MODELS

描述（改编自申请人摘要）：总体长期 这些研究的目的是开发和使用转基因动物模型， 研究环境卫生相关问题的系统。 具体来说，我们 研究集中在金属硫蛋白（MT）和转录因子， 调节MT基因。 我们建议确定分子机制 调节MT基因表达响应镉（Cd）和氧化 应激，以及MT在保护氧化应激中的功能意义 应力 镉是一种广泛存在的环境污染物，也是多种外源性物质 会导致有害的氧化应激。 MT代表了记录最完整的 细胞内重金属（Zn，Cu，Cd）结合蛋白，但它们也可以 清除羟基自由基和歧化超氧阴离子。 氧化应激诱导小鼠MT-I基因转录 涉及激活MTF-1与金属响应元件（MRE）的结合， 所述促进剂以及复合USF/抗氧化剂应答促进剂 元素（USF/ARE）。 初步证据表明，镉诱导也是 介导的，部分地由这个元素，而锌诱导不是。 之甚少 了解MTF-1或USF/ARE的结构和功能。 转基因 过表达MT或靶向切除MT-I/MT-II基因小鼠 函数，并将用于检查这些 蛋白质保护免受氧化应激。 具体目标是 建议是：1）确定氧化的分子机制， 应力和金属激活MTF-1; 2）探索 诱导过程中小鼠MT-I启动子中的USF/抗氧化反应元件 通过氧化应激和Cd; 3）检查蛋白质与USF/ARE的相互作用 在氧化胁迫和Cd诱导的基因表达过程中;以及4）分析 抗氧化应激的转基因小鼠，过表达MT或 有针对性地切除MT基因。 分子（诱变，EMSA， 足迹法，转染）和生物化学（Zn滴定分析， 免疫沉淀、结合位点层析）方法将用于 定义MTF-1的结构-功能关系（Zn结合，DNA结合， 蛋白质相互作用和反式激活）。 结构-功能 USF/ARE将通过诱变、转染测定和蛋白质表达来定义。 结合测定。 转基因小鼠的抗氧化应激能力将在 通过组织病理学和血清酶水平监测。

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.