EMBRYONIC RETINOID HOMEOSTASIS AND ALCOHOL TERATOGENESIS

胚胎视黄醇稳态和酒精致畸

• 批准号: 2045988
• 负责人: GREGG L DUESTER
• 金额: $ 17.75万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-01 至 1998-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2045988
• 关键词: alcohol dehydrogenase; aldehyde dehydrogenases; cytochrome P450; dosage; embryo /fetus pharmacology; fetal alcohol syndrome; gene expression; genetically modified animals; high performance liquid chromatography; homeobox genes; homeostasis; in situ hybridization; laboratory mouse; northern blottings; retinoids

Fetal alcohol syndrome is defined as a collection of ethanol-induced human neonatal malformations, predominantly of the central nervous system and neural crest, that are the result of excessive maternal ethanol consumption. This syndrome is a leading cause of mental retardation, justifying efforts to understand the teratogenic mechanism in detail. Despite numerous studies designed to analyze ethanol embryotoxicity in humans, rodents, and other vertebrates, no single underlying mechanism for the teratogenic action of ethanol has been widely accepted. A hypothetical mechanism for fetal alcohol syndrome involving an ethanol-induced retinoic acid deficiency has been proposed by this laboratory and will be tested in this proposal. Retinoic acid is the active metabolite of vitamin A and plays a central role in embryonic development as a regulator of gene expression, particularly of-he homeobox gene family which plays a key role in patterning the central nervous system. Since the conversion of retinol (vitamin A alcohol) to retinoic acid by alcohol dehydrogenase is known to be inhibited by ethanol in vitro, it is of great interest to determine if ethanol can effect retinoic acid levels in vivo as well as effect expression of genes known to be regulated by retinoic acid. The similarities between ethanol teratogenesis and retinoid teratogenesis further implicate ethanol as an agent which disturbs retinoic acid levels. Excessive retinoic acid taken during pregnancy is teratogenic to the human fetus, with neonates exhibiting defects similar to those observed for ethanol teratogenesis. Thus, human embryos will undergo normal morphogenesis when converting a small amount of maternally-derived vitamin A (retinol) into retinoic acid in a tissue-specific fashion, but may undergo teratogenesis when responding to events which increase or decrease retinoic acid levels out of the normal range. Goals for this proposal are as follows: (l) Study the effect of various timed doses of prenatal ethanol on retinoid levels in the mouse embryo using HPLC technology to analyze retinoid levels in isolated tissues biochemically, and using a lacZ-retinoic acid indicator cell line to monitor retinoic acid levels in embryo tissues biologically; (2) Analyze by Northern blotting and in situ hybridization the effect of ethanol dose (and timing of dose) on the expression of known retinoic acid target genes in the mouse embryo. The genes studied will include those encoding the homeobox family, the retinoic acid receptor, the cellular retinoic acid binding protein, the cellular retinol binding protein, and various retinoid-metabolizing enzymes including alcohol dehydrogenase, aldehyde dehydrogenase, and cytochrome P450; (3) Examine the in vivo role of alcohol dehydrogenase (ADH) in retinoic acid synthesis by producing ADH mutations in transgenic mice. Three ADH genes have been characterized in the mouse (Adh-1, Adh-2, and Adh-3), and we plan to prepare null mutations in each gene. These knockout mice will be analyzed to determine if loss of a particular ADH results in a change in embryonic development, retinoic acid synthesis, or retinoic acid target gene expression.

胎儿酒精综合征的定义是由酒精引起的人类 新生儿畸形，主要发生在中枢神经系统和 神经峰，这是母体过量酒精的结果 消费。这种综合征是导致精神发育迟滞的主要原因， 证明了详细了解致畸机制的努力是合理的。 尽管许多研究旨在分析乙醇对胚胎的毒性 人类、啮齿动物和其他脊椎动物，没有单一的潜在机制 乙醇的致畸作用已被广泛接受。一种假设 酒精诱导的维甲酸引起胎儿酒精综合征的机制 酸缺乏症已由该实验室提出，并将在 这项提议。维甲酸是维生素A和维生素A的活性代谢物 作为基因的调节者，在胚胎发育中起着核心作用 表达，特别是起关键作用的同源异型盒基因家族 在构筑中枢神经系统的过程中。由于视黄醇的转化 (维生素A酒精)通过酒精脱氢酶转化为维甲酸已知 在体外被乙醇抑制，确定其是否 乙醇对体内维甲酸水平的影响 已知受维甲酸调节的基因的表达。 乙醇致畸与维甲酸致畸的相似性 进一步暗示乙醇是一种干扰维甲酸水平的试剂。 怀孕期间过量服用维甲酸对人类有致畸作用 胎儿，新生儿表现出与观察到的类似的缺陷 乙醇致畸。因此，人类胚胎将经历正常的 转化少量母源维生素时的形态发生 A(视黄醇)以组织特异性的方式转化为维甲酸，但可能 在对增加或减少的事件做出反应时会发生畸形 维甲酸水平超出正常范围。 这项建议的目标如下：(L)研究各种措施的效果 产前限量乙醇对小鼠胚胎维甲酸水平的影响 用高效液相色谱技术分析离体组织中的维甲酸含量 生化，并使用Lacz-维甲酸指示细胞系 从生物学角度监测胚胎组织中的维甲酸水平；(2)分析 Northern印迹和原位杂交检测乙醇剂量的影响 (和剂量时机)对已知的维甲酸靶基因表达的影响 在小鼠胚胎中。所研究的基因将包括那些编码 同源盒家族，维甲酸受体，细胞维甲酸 结合蛋白，细胞视黄醇结合蛋白，以及各种 类维甲酸代谢酶包括酒精脱氢酶、乙醛 脱氢酶和细胞色素P450；(3)检测其在体内的作用 乙醇脱氢酶(ADH)在维甲酸合成中的作用 转基因小鼠的突变。三个ADH基因已被鉴定为 小鼠(Adh-1、Adh-2和Adh-3)，我们计划准备零突变 在每个基因中。这些基因敲除的小鼠将被分析以确定是否丢失了 一种特殊的ADH会导致胚胎发育的变化，维甲酸 酸合成，或维甲酸靶基因表达。