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.
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.
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