Methylmercury treatment (rand-affy-droso-366220)

Methylmercury (MeHg) toxicity in humans manifests deficits in neurological function. Cases of prenatal exposure to mercury have established that the developing nervous system is most highly susceptible to perturbation by MeHg. At a cellular level, MeHg-induced defects result from altered neuronal proliferation, migration and pathfinding. However, the molecular targets of MeHg that give rise to these outcomes are not fully understood. In an overall effort to identify the fundamental molecular targets of MeHg in neural development, we are investigating the effects of MeHg on gene expression and protein function in the Drosophila model. Since the fundamental signaling pathways in development of multicellular animals have been extensively characterized in Drosophila, and demonstrate high degree of conservation in vertebrates, we believe these data will lead us to the most pertinent pathways affected by MeHg and begin to elucidate the mechanism of MeHg neural toxicity relevant to cases of human exposure to this prevalent environmental toxin. Our aim is to identify fundamental signaling pathways in neural development that are targets for MeHg poisoning. Our hypothesis is that MeHg, by direct interaction with cysteine thiol groups, alters the function signaling pathway proteins and subsequently alters transcription of target genes in the respective pathways. Our hypothesis is supported by our recent data demonstrating a direct action of MeHg in activating the Notch receptor pathway and upregulating target gene expression. We anticipate a microarray analysis will elucidate additional fundamental signaling pathways where transcription is affected by this toxin. Fertilized female flies are fed on food containing methylmercury (1-20 micromolar) or solvent control (DMSO) for a period of five days to allow for MeHg penetration in to eggs. Flies are then transfered to a cage for embryo collection for a discrete window of time (e.g. 1hr). Embryos are aged 16-24 hours and collected for total RNA extraction using the Trizol reagent. Total RNA is quantitated by spectrophotometry with a Nanodrop reader. While concentration dependent effects of MeHg are of interest, initial experiments will be conducted on samples exposed to a single concetration known to illicit effect in other bioassays (e.g. 10 micromolar). The goal is to see which genes are up- or down-regulated with MeHg exposure, as compared to control embryos that are not exposed. Emphasis in the analysis stage will be in identifying target genes of known signaling pathways. Keywords: dose response

甲基汞（Methylmercury，MeHg）对人体的毒性表现为神经功能缺损。已有孕期汞暴露病例证实，发育中的神经系统对甲基汞的干扰最为敏感。在细胞层面，甲基汞诱导的神经缺陷源于神经元增殖、迁移及路径选择的异常。然而，介导上述效应的甲基汞分子靶点尚未完全阐明。

为明确神经发育过程中甲基汞的核心分子靶点，本研究以果蝇（Drosophila）为模型，探究甲基汞对基因表达与蛋白质功能的影响。由于多细胞动物发育的核心信号通路已在果蝇模型中得到广泛解析，且此类通路在脊椎动物中具有高度保守性，我们预期本研究数据将帮助我们确定受甲基汞影响的最关键通路，并逐步阐明与人类暴露于这种常见环境毒素相关的甲基汞神经毒性机制。

本研究的目标是识别神经发育过程中作为甲基汞中毒靶点的核心信号通路。我们的假说为：甲基汞可通过与半胱氨酸巯基直接结合，改变信号通路蛋白的功能，进而调控对应通路中靶基因的转录。近期研究数据证实，甲基汞可直接激活Notch受体通路并上调靶基因表达，这为上述假说提供了支持。我们预期通过基因芯片（microarray）分析，可进一步阐明受该毒素影响转录的其他核心信号通路。

实验方案如下：将已受精的雌果蝇饲喂于含有1~20微摩尔浓度甲基汞或溶剂对照（二甲基亚砜，DMSO）的培养基中，持续5天以确保甲基汞渗透进入虫卵。随后将果蝇转移至饲养笼中，在特定时间窗口（例如1小时）内收集胚胎。将胚胎孵育16~24小时后收集，使用Trizol试剂提取总RNA，采用Nanodrop分光光度计进行总RNA定量。尽管我们关注甲基汞的浓度依赖性效应，但初始实验将选用在其他生物检测中已被证实可产生效应的单一浓度（例如10微摩尔）处理样本。本实验的目标是对比暴露组与未暴露对照组胚胎，筛选出受甲基汞调控的上调或下调基因。分析阶段将重点关注已知信号通路的靶基因筛选。
关键词：剂量反应（dose response）