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.
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