Ethanol action in Japanese medaka: Alteration in specific gene methylation

项目描述（由申请人提供）：本项目主要研究乙醇对日本medaka胚胎发生中醛脱氢酶1A2 （Aldh1A2）基因表达的影响，并确定该基因启动子区CpG岛甲基化模式的改变是否可能是乙醇致畸的原因。乙醇诱导胎儿酒精谱系障碍（FASD）的母亲谁在怀孕期间摄入酒精新生婴儿。在FASD中，胎儿酒精综合征（fetal alcohol syndrome， FAS）是临床最容易识别的形式，以生长发育迟缓、中枢神经系统（central nervous system， CNS）畸形和功能障碍为特征，具有明显的颅面、心血管和肢体缺陷模式。乙醇干扰胎儿发育的分子机制尚不清楚。此外，除了妇女在怀孕期间不饮酒外，预防FAS的措施尚不清楚。先前，我们已经证明，在胚胎发生过程中暴露于乙醇的medaka胚胎在颅面、心血管和骨骼器官中出现了与人类FAS特征相比较的早熟FAS特征。这些发现促使我们使用medaka作为寻找FAS基因的独特模型。我们的假设是酒精脱氢酶（ADH）和醛脱氢酶（ALDH）的乙醇代谢改变了细胞NAD + /NADH的比例并诱导氧化应激。这种改变的氧化还原状态能够诱导特定基因的DNA分子的表观遗传修饰，从而调节胚胎的正常发育。在本应用中，我们将研究Aldh1A2基因启动子区域CpG岛的DNA甲基化模式，我们已经确定Aldh1A2基因是medaka胚胎发生中的乙醇靶基因。我们还将确定天然产品葛根（葛根）在预防这些错误方面的作用。分析将在动物的胚胎、幼虫和成年阶段进行。我们将应用组织学、组织化学和分子生物学技术，包括定量RT-PCR和DNA甲基化分析。该研究将为鉴定主要负责FAS的酒精代谢酶基因提供基础，并确定天然产物是否将胚胎从表观遗传乙醇毒性中拯救出来。在这一建议中，我们关注乙醇诱导的表观遗传事件作为胎儿酒精综合征的主要原因，并评估一种天然产物作为FAS的预防剂。

DESCRIPTION (provided by applicant): The primary focus of the project described herein is to study the effects of ethanol on aldehyde dehydrogenase 1A2 (Aldh1A2) gene expression in Japanese medaka embryogenesis and to determine whether alteration in methylation pattern of the CpG island at the promoter region of this gene is a possible cause of ethanol teratogenesis. Ethanol induces fetal alcohol spectrum disorder (FASD) in the newborn babies of mothers who ingested alcohol during pregnancy. Among FASD, fetal alcohol syndrome (FAS) is the most clinically recognizable form identified by growth retardation, central nervous system (CNS) malformation and dysfunction, and has a distinctive pattern of craniofacial,cardiovascular, and limb defects. The molecular mechanism by which ethanol perturbs fetal development is unknown. Moreover, prevention of FAS, other than women abstaining from drinking alcohol during pregnancy, is not known. Previously, we have demonstrated that medaka embryos exposed to ethanol during embryogenesis have developed precocious FAS features in craniofacial, cardiovascular and skeletal organs which can be compared with FAS features observed in human. These findings prompted us to use medaka as a unique model for searching genes responsible for FAS. Our hypothesis is ethanol metabolism by the enzymes alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) alters cellular NAD + /NADH ratio and induces oxidative stress. This altered redox state is able to induce epigenetic modifications in DNA molecules of specific genes and thus modulate the normal development of the embryo. In this application we will study the DNA methylation pattern in the CpG island in the promoter region of Aldh1A2 gene, which we have identified as an ethanol target gene in medaka embryogenesis. We will also determine the effect of a natural product, kudzu (Pueraria lobota), in preventing these errors. Analyses will be made in embryonic, larval and adult stages of the animal. We will apply histological, histochemical and molecular biological techniques including quantitative RT-PCR and DNA methylation analysis for this study. This study will provide a foundation for identifying an alcohol metabolizing enzyme gene that is primarily responsible for FAS, and determine if a natural product will rescue the embryo from epigenetic ethanol toxicity. In this proposal, we have focused on an ethanol-induced epigenetic event as the major cause of Fetal Alcohol Syndrome and evaluate a natural product as the preventive agent for FAS.