Ethanol and retinoid metabolism/function

描述(申请人提供)：本项目旨在确定乙醇对正常维生素A(视黄醇)营养状态下体内atRA(全反式维甲酸)浓度的影响，并确定乙醇影响atRA稳态的机制和功能后果。我们建立了一种具有Femtomol灵敏度的LC/MS/MS分析方法，用于小生物样品中atRA的特异性定量，并用它来建立几个品系小鼠血清和多种组织中atRA及其异构体的参考值。该方法用于确定正常维生素A营养期间乙醇对稳态atRA浓度的影响。与预期相反，酒精喂养(Lieber-DeCarli液体饮食，36%卡路里，1个月)不会改变C57BL/6成年小鼠肝脏、肾脏、嗅球、纹状体、丘脑和小脑的atRA浓度，但导致海马区、皮质、睾丸和血清的atRA浓度增加2-50倍。初步数据显示，从E13到E18，以同样的方式喂给DAMS的乙醇，增加了E19胚胎海马区和皮质中的atRA，比例与DAM BAC成比例。0.08%的DAM BAC可使RA大鼠的海马区和皮质区分别增加4倍和30倍。我们还发现，海马星形胶质细胞生物合成全反式维甲酸，全反式维甲酸诱导原代培养的小鼠海马神经元树突状生长。ATRA通过RAR1的形式刺激树突状细胞的生长，RAR1定位于树突状RNA颗粒并刺激翻译。我们试图应用这些技术和洞察力来严格确定DAM乙醇摄入对E19胚胎海马区atRA浓度的影响，并了解乙醇影响的生物学后果。需要检验的假设是：DAMS摄入乙醇会增加胚胎海马区内源性atRA的浓度；这些atRA在海马区发育过程中增加，导致乙醇毒性和/或畸形学。其具体目的是确定慢性乙醇对以下方面的影响：1)胚胎中海马区发育早期(e13-E18)内源性atRA；2)发育中海马区维甲酸调节基因的表达(转录和翻译)；3)发育中海马区的神经发生；4)海马区星形胶质细胞合成和分泌atRA的机制；5)海马神经元中atRA的功能。这项工作旨在利用最近对海马区视黄醇功能的新技术和新见解，为类维A代谢和生物学，以及维生素A和乙醇之间的相互作用提供新的见解。这些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对脊椎动物的生命是必不可少的，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。摄入酒精会导致肝脏中维生素A储存的严重耗尽，并被认为可以抑制维生素A转化为激素形式的全反式维甲酸(AtRA)。使用最近开发的技术，我们有了新的数据，乙醇会增加大脑特定区域的atRA，包括海马体。该项目旨在证实DAMS长期摄入乙醇会增加DAMS在海马区发育期间摄入乙醇的胚胎海马区atRA的浓度，并确定乙醇对海马区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.