Fetal DNA oxidation and repair in neurodegeneration

描述（由申请人提供）：甲基汞（MeHg）是一种环境因子，主要通过摄入受污染的海鲜进入人类。在低于引起全身毒性的剂量下，产前接触甲基汞会导致神经发育缺陷。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（ROS）的形成，因为胎儿的抗氧化保护能力较低。这种毒性可能涉及氧化性DNA损伤，其中最突出的是8-氧代鸟嘌呤（8-oxoG），这可能导致突变或改变基因转录。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。我们推测ROS介导的胎儿脑DNA氧化损伤改变了特定的基因表达水平，导致出生后神经发育缺陷。将评估8-oxoG修复活性发生遗传改变的小鼠模型的后代在子宫内暴露于甲基汞后的神经发育缺陷。氧鸟嘌呤糖基化酶1（ogg 1）敲除（BER缺陷）和Cockayne综合征B（CS B）敲除（TCR缺陷）小鼠将作为修复缺陷模型进行测试，而表达高活性细菌甲酰胺嘧啶糖基化酶（Fpg）的转基因小鼠将进行基因工程改造并用作BER增强模型。将在相同胎脑中分析胎儿ROS和8-oxoG、子宫内氧化的特异性基因调控靶标和相关基因表达变化，并评估子代的出生后CMS病理。8-OxoG将通过具有电化学检测的HPLC定量，靶基因调控元件的氧化将通过配体介导的PCR表征，基因表达通过微阵列分析表征，神经发育缺陷通过行为测试表征。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修复活性作为风险因素的作用提供机制性见解。

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.