Ethanol action in Japanese medaka: Alteration in specific gene methylation

描述（由申请人提供）：本文所述项目的主要焦点是研究乙醇对日本青鳉胚胎发生中醛脱氢酶1A 2（Aldh 1A 2）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是乙醇致畸的可能原因。乙醇可导致妊娠期饮酒母亲的新生儿出现胎儿酒精谱系障碍（FASD）。在FASD中，胎儿酒精综合征（FAS）是临床上最易识别的形式，可通过生长迟缓、中枢神经系统（CNS）畸形和功能障碍来识别，并具有独特的颅面、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戒酒外，还不知道如何预防FAS。以前，我们已经证明，青鳉胚胎暴露于乙醇在胚胎发育过程中已开发早熟FAS功能颅面，心血管和骨骼器官，可以与FAS功能在人类中观察到的。这些发现促使我们使用青鳉作为寻找FAS基因的独特模型。我们的假设是乙醇脱氢酶（ADH）和乙醛脱氢酶（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。在本申请中，我们将研究Aldh 1A 2基因启动子区CpG岛的DNA甲基化模式，我们已确定为青鳉胚胎发生中的乙醇靶基因。我们还将确定一种天然产品葛根（Pueraria lobota）在预防这些错误方面的效果。将在动物的胚胎、幼虫和成虫阶段进行分析。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。这项研究将为确定主要负责FAS的酒精代谢酶基因提供基础，并确定天然产物是否会从表观遗传乙醇毒性中拯救胚胎。在这项提案中，我们集中在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，并评估天然产品作为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.