Molecular analysis of toxicant mediated teratogenesis

本研究项目的总体目标是评估特定的发育毒物(如三氯乙烯(TCE)和砷(As))是否扰乱了一个或多个导致形态改变的发育途径，从而导致特定的出生缺陷。我们的主要目的是研究TCE改变导致大鼠胚胎心脏畸形的分子途径。我们的假设是，分子水平上的标志物分析将提供环境暴露的敏感指数。当这些标志物是通过对表达变化的经验观察而选择的时候，它们可能表明特定的发育途径受到毒物暴露的干扰。作为标记，这些分子可用于启动子分析和对组织中更近端受影响的分子的持续研究。对正常发育至关重要的分子最终可以通过基因操作作为缺陷的直接来源进行测试。此外，我们将研究发育中胚胎中一种单独的潜在致畸因素(AS与TCE)的异同。初步数据表明，在发育过程中，暴露于三氯乙烯和砷的几个标志物在心脏和肺中都受到干扰。我们实验室已成功用于三氯乙烯的聚合酶链式反应-选择消减杂交技术也将用于分离AS暴露的标记。我们建议实现以下特定目标：1.确定四个特定克隆的基因表达的分布和时间，这些克隆是在致畸剂量TCE暴露后通过筛选获得差异表达的。假说是，在具有发育意义的模式中，缺失表达的分子在产生心脏缺陷的过程中发挥了功能作用。2.评估暴露于母亲饮用水中不同水平的毒物对胚胎显著表达改变的敏感性。我们的目标是确定导致基因表达改变的最低暴露水平，并使用最敏感的标记物进行风险评估。3.克隆AIM 1中确定的标记的启动子区域。鉴定共有基序将有助于鉴定更接近TCE致畸活性的分子。4.测试三氯乙烯暴露与NMDA(N-甲基-D-天冬氨酸)谷氨酸受体和/或NFATc通路之间的潜在关系，作为三氯乙烯致畸的机制。假设TCE介导的心脏缺陷可能是由两种分子所涉及的钙离子流量的变化引起的。5.鉴定和鉴定发育中胚胎砷暴露的标志物。

The overall goal of this research project is to assess whether specific developmental toxicants (e.g. trichloroethylene (TCE) and (As) arsenic) perturb one or more developmental pathways leading to morphologic alterations resulting in specific birth defects. Our main objective is to investigate is to investigate the molecular pathways altered by TCE that cause cardiac malformations in rat embryos. Our hypothesis is that analysis of markers at the molecular level will provide a sensitive index of environmental exposure. When these markers are chosen by empirical observation of altered expression, they are likely to indicate specific developmental pathways that are perturbed by toxicant exposure. As markers, these molecules can by used for promoter analysis and continued investigations into more proximally affected molecules in the tissue. Molecules critical to normal development can eventually be tested by genetic manipulation as a direct source of defects. Furthermore, we shall examine the similarities and differences of a separate potential teratogen (As versus TCE) in the developing embryos. Preliminary data indicate that several markers of exposure to TCE and As are perturbed in both heart and lungs during development. The PCR-Select subtractive hybridization technique that has been successfully used in our laboratory for TCE will also be used to isolate markers for As exposure. We propose to carry out the following specific aims: 1. Identify the distribution and timing of gene expression for four specific clones that were selected by screens for differential expression after exposure to teratogenic doses of TCE. The hypothesis is that miss-expressed molecules during developmentally significant patterns play a functional role in producing in producing heart defects. 2. Evaluate the sensitivity of altered marked expression in embryos exposed to varying levels of toxicants in maternal drinking water. Our objective is to determine minimum exposure levels that result in altered gene expression and use the most sensitive markers for risk assessment. 3. Clone the promoter region of the markers identified in aim 1. Identification of shared motifs would enable identification of molecules more proximal to the teratogenic activity of TCE. 4. Test the potential relationship between TCE exposure and the NMDA (N-Methyl-D-Aspartate) glutamate receptor and/or NFATc pathways as a mechanism of TCE teratogenicity. The hypothesis is that TCE-mediated heart defects may be caused by alterations in Ca++ flux involved with both molecules. 5. Identify and characterize markers of As exposure in developing embryos.