Ethanol action in Japanese medaka: Alteration in specific gene methylation

描述(申请人提供)：本研究的主要重点是研究乙醇对日本青竹胚胎发育中乙醛脱氢酶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。在怀孕期间摄入酒精的母亲的新生儿中，乙醇会导致胎儿酒精谱障碍(FASD)。在FASD中，胎儿酒精综合征(FAS)是临床上最常见的类型，表现为生长迟缓、中枢神经系统(CNS)畸形和功能障碍，并具有独特的颅面、心血管和肢体缺陷模式。乙醇扰乱胎儿发育的分子机制尚不清楚。此外，除了女性在怀孕期间戒酒外，对Fas的预防尚不清楚。此前，我们已经证明在胚胎发生期间暴露于乙醇的青竹胚胎在头面部、心血管和骨骼器官中发展出了早熟的Fas特征，这可以与在人类中观察到的Fas特征进行比较。这些发现促使我们使用青竹作为一种独特的模型来搜索与Fas有关的基因。我们的假设是乙醇通过乙醇脱氢酶(ADH)和乙醛脱氢酶(ALDH)代谢，改变细胞内NAD+/NADH比值，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。在这个应用中，我们将研究Aldh1A2基因启动子区域CpG岛上的DNA甲基化模式，我们已经确定Aldh1A2基因是青竹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在防止这些错误方面的效果。分析将在动物的胚胎、幼虫和成体阶段进行。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。这项研究将为鉴定主要负责Fas的酒精代谢酶基因提供基础，并确定天然产品是否可以将胚胎从表观遗传的乙醇毒性中拯救出来。在这项提案中，我们将重点放在乙醇诱导的表观遗传学事件作为胎儿酒精综合征的主要原因，并评估一种天然产品作为预防Fas的药物。

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.