Ethanol and retinoid metabolism/function

描述（由申请人提供）：本项目旨在确定在正常维生素A（视黄醇）营养期间乙醇对体内全反式维甲酸（全反式维甲酸）浓度的影响，并确定乙醇对atRA稳态影响的机制和功能后果。我们建立了一种具有飞特莫灵敏度的LC/MS/MS方法，用于小生物样品中atRA的特异性定量，并利用该方法生成了几种小鼠血清和多种组织中atRA及其异构体的参考值。该试验用于确定乙醇在正常维生素A营养期间对稳态atRA浓度的影响。与预期相反，乙醇喂养（Lieber-DeCarli液体饮食，36%卡路里，1个月）没有改变C57BL/6成年小鼠肝脏、肾脏、嗅球、纹状体、丘脑和小脑中的atRA浓度，但导致海马、皮质、睾丸和血清中的atRA浓度增加2至50倍。初步数据显示，以相同方式饲喂e13 ~ e18的乙醇，e19胚胎海马和皮质的atRA与坝BAC成比例增加。当BAC浓度为0.08%时，海马和皮质区atRA分别增加4倍和30倍。我们还发现，海马星形胶质细胞可以生物合成atRA， atRA可以诱导小鼠海马原代神经元的树突生长。atRA通过一种定位于树突RNA颗粒并刺激翻译的RAR1形式刺激树突生长。我们试图应用这些技术和见解来严格确定乙醇摄入对e19胚胎海马中atRA浓度的影响，并了解乙醇效应的生物学后果。要验证的假设是：水坝摄入乙醇会增加胚胎海马体中内源性atRA的浓度；这些atRA在海马发育期间的增加会导致乙醇毒性和/或致畸。具体目的是确定慢性乙醇对：(1)早期海马发育过程中胚胎内源性atRA的影响（e13-e18）；2)类维甲酸调节的基因表达（转录和翻译）在发育中的海马；3)发育中的海马神经发生；4)海马星形胶质细胞合成和分泌atRA的机制；5) atRA在海马神经元中起作用。本研究旨在利用新技术和近年来对海马体中类维甲酸功能的新见解，为类维甲酸代谢和生物学以及维生素A与乙醇之间的相互作用提供新的见解。研究结果可能会影响治疗酗酒者的方法。公共卫生相关性：维生素A对脊椎动物生命至关重要，因为它是胚胎发育所必需的，并调节中枢神经系统的功能，以及许多其他功能。酒精（乙醇）的摄入会导致肝脏中维生素A储存的严重消耗，并被认为会抑制维生素A进入激素形式的激活，即全反式维甲酸（atRA）。我们使用最近开发的技术获得了新的数据，乙醇增加了大脑特定区域的atRA，包括海马体。本项目旨在证实一项观察结果，即在海马发育过程中摄入乙醇的胚胎，其长期摄入乙醇会增加海马中atRA的浓度，并确定乙醇对海马中atRA功能的影响。

DESCRIPTION (provided by applicant): This project seeks to determine the impact of ethanol on atRA (all-trans-retinoic acid) concentrations in vivo during normal vitamin A (retinol) nutriture, and to identify mechanisms and functional consequences of ethanol effects on atRA homeostasis. We developed a LC/MS/MS assay with femtomol sensitivity for specific quantitation of atRA in small biological samples, and used it to generate reference values for atRA and its isomers in serum and multiple tissues of several strains of mice. The assay was applied to determine the impact of ethanol on steady-state atRA concentrations during normal vitamin A nutriture. Contrary to expectations, ethanol feeding (Lieber-DeCarli liquid diet, 36% of calories, 1 month) did not alter atRA concentrations in liver, kidney, olfactory bulb, striatum, thalamus and cerebellum of C57BL/6 adult mice, but caused 2 to 50-fold increases in atRA concentrations in hippocampus, cortex, testis and serum. Preliminary data showed that ethanol, fed the same way to dams from e13 through e18, increased atRA in the hippocampus and cortex of e19 embryos in proportion to dam BAC. A dam BAC of 0.08% caused 4 and 30- fold increases, respectively, in hippocampus and cortex atRA. We also showed that hippocampus astrocytes biosynthesize atRA, and atRA induces dendritic growth in primary mouse hippocampus neurons. atRA stimulates dendritic growth through a form of RAR1 that localizes to dendritic RNA granules and stimulates translation. We seek to apply these techniques and insight to determine rigorously the consequences of dam ethanol ingestion on atRA concentrations in hippocampus of e19 embryos, and to understand the biological consequences of the ethanol effects. The hypothesis to be tested is: ethanol ingestion by dams increases endogenous concentrations of atRA in the hippocampus of their embryos; these atRA increases during hippocampus development contribute to ethanol toxicity and/or teratology. The specific aims are to determine the effects of chronic ethanol on: 1) endogenous atRA in the embryo during early hippocampus development (e13-e18); 2) retinoid-regulated gene expression (transcription and translation) in the developing hippocampus; 3) neurogenesis in the developing hippocampus; 4) mechanisms of atRA biosynthesis and secretion by hippocampus astrocytes; 5) atRA functions in hippocampus neurons. This work aims to provide new insight into retinoid metabolism and biology, and the interactions between vitamin A and ethanol, using new technology and new insights generated recently about retinoid function in the hippocampus. The results could influence approaches to treating alcoholics. PUBLIC HEALTH RELEVANCE: Vitamin A is essential for vertebrate life, because it is necessary for embryonic development, and regulates function of the central nervous system, among many other functions. Alcohol (ethanol) ingestion causes severe depletion of vitamin A storage in the liver, and has been postulated to inhibit activation of vitamin A into its hormonal form, all-trans-retinoic acid (atRA). We have new data using recently developed techniques that ethanol increases atRA in specific areas of the brain, including the hippocampus. This project seeks to affirm the observation that chronic ethanol ingestion by dams increases the concentrations of atRA in the hippocampus of embryos whose dams have ingested ethanol during hippocampus development, and to determine the effects of ethanol on atRA function in the hippocampus.