Fetal DNA oxidation and repair in neurodegeneration

描述(由申请人提供)：甲基汞(MeHg)是一种环境制剂，主要通过摄入受污染的海鲜引入人类。产前接触甲基汞会导致神经发育障碍，剂量低于引起全身毒性的剂量。这种化学物质和其他环境化学物质的一个潜在毒理学机制可能涉及活性氧物种(ROS)的形成，因为胎儿的抗氧化保护能力较低。这种毒性可能涉及DNA氧化损伤，其中最突出的是8-氧鸟嘌呤(8-oxoG)，它可导致突变或基因转录改变。后者可能与发育病理学有关。8-oxoG的修复通过碱基切除(BER)和转录偶联(TCR)修复途径进行。我们假设，ROS介导的氧化DNA损伤在胎儿大脑中改变了特定的基因表达水平，导致出生后神经发育缺陷。8-oxoG修复活性基因改变的小鼠模型的后代将在宫内暴露于甲基汞后进行神经发育缺陷评估。氧鸟嘌呤糖基酶1(Ogg1)基因敲除(BER缺陷)和Cockayne综合征B(CSB)基因敲除(TCR缺陷)小鼠将被测试为修复缺陷模型，而表达高活性细菌甲酰胺并嘧啶糖基酶(FPG)的转基因小鼠将被转基因并用作BER增强模型。胎儿ROS和8-oxoG，子宫内氧化和相关基因表达变化的特定基因调控靶点，将在相同的胎儿大脑中进行分析，并与子代出生后CMS的病理进行评估。8-OxoG将通过高效液相色谱和电化学检测来定量，靶基因调控元件的氧化将通过配体介导的PCR来表征，基因表达将通过基因芯片分析来表征，神经发育缺陷将通过行为测试来表征。这些研究将为环境诱导的神经发育缺陷的胎儿起源以及DNA修复活动作为危险因素的作用提供机械性见解。

DESCRIPTION (provided by applicant): Methylmercury (MeHg) is an environmental agent introduced to humans primarily via the ingestion of contaminated seafood. Prenatal MeHg exposure induces neurodevelopmental deficits, at doses below those causing systemic toxicity. One potential toxicological mechanism for this and other environmental chemicals likely involves reactive oxygen species (ROS) formation, as the fetus has low antioxidative protection. This toxicity may involve oxidative DNA lesions, the most prominent of which is 8-oxoguanine (8-oxoG), which can result in mutations or altered gene transcription. The latter is potentially relevant to developmental pathologies. Repair of 8-oxoG is carried out by the base excision (BER) and transcription-coupled (TCR) repair pathways. We hypothesize that ROS-mediated oxidative DNA damage in fetal brain modifies specific gene expression levels, contributing to postnatal neurodevelopmental deficits. The progeny of mouse models with genetic alterations in 8-oxoG repair activity will be assessed for neurodevelopmental deficits after in utero exposure to MeHg. Oxoguanine glycosylase 1 (ogg1) knockout (BER-deficient) and Cockayne syndrome B (CSB) knockout (TCR-deficient) mice will be tested as repair-deficient models, while transgenic mice expressing highly active bacterial formamidopyrimidine glycosylase (Fpg) will be genetically engineered and employed as a BER-enhanced model. Fetal ROS and 8-oxoG, specific gene regulatory targets for oxidation and associated gene expression changes in utero will be analyzed in the same fetal brains, and assessed with respect to postnatal CMS pathologies in the progeny. 8-OxoG will be quantified by HPLC with electrochemical detection, oxidation of target gene regulatory elements will be characterized by ligand- mediated PCR, gene expression by microarray analysis, and neurodevelopmental deficits by behavioral tests. These studies will provide mechanistic insight into the fetal origin of environmentally-induced neurodevelopmental defects, and the role of DNA repair activity as a risk factor.