Abstract 428 Neurodevelopmental Disabilities Poster Symposium, Tuesday, 5/4

The mechanism of fetal embryopathology resulting from ethanol ingestion during pregnancy is still unknown. Recent evidence suggests that an interaction between ethanol (EtOH) and retinoids may be involved in the mechanism of Fetal Alcohol Syndrome. Retinoic acid receptor (RAR) beta in fetal rat brain and neuroblastoma cell culture is altered by EtOH. Thus, prenatal EtOH effects might be mediated by changes in retinoids in the developing central nervous system (CNS). The expression of glial fibrillary acidic protein (GFAP), an intermediate filament protein expressed almost exclusively in CNS astrocytes, is essential for normal white matter architecture, vascularization and blood brain barrier integrity. Disrupted GFAP expression, therefore, might be an indicator of the deleterious effects of prenatal EtOH exposure. Some preliminary work has indicated an effect of prenatal ethanol on GFAP in primary cultures of fetal rat brain astrocytes. We followed these experiments with the present work, in which the objective was to determine the effects of EtOH and retinoic acid (RA) on GFAP and RAR beta in an established astrocytoma cell line.

The human astrocytoma cell line, U-373 MG was treated with 100 or 150 mM EtOH, 1 pM-10 uM RA, or a combination of both for 48 hours. In other experiments, cells were treated with cycloheximide (CHX) 4 hours prior to addition of EtOH or RA. RNA was extracted for Northern analyses, using GFAP or RAR beta cDNA as probes. EtOH (100-150 mM) increased GFAP mRNA significantly, whereas RA (0.1-10 uM) significantly decreased GFAP mRNA, while 1 pM-0.01 uM had no effect on GFAP mRNA expression. The expression of GFAP was lowered by the combination of EtOH (100-150 mM) and RA (1 uM). The expression of RAR beta mRNA was significantly decreased by EtOH (150 mM) and increased by RA (0.1-10 uM). The increase in expression of RAR beta caused by RA (at 1 uM) still occurred in the presence of EtOH. Thus, RA appears to overcome the effect of EtOH on both GFAP and RAR beta mRNA. In the presence of CHX, EtOH had no effect on either GFAP or RAR beta expression. CHX, however, did not alter the effect of RA on GFAP and RAR beta mRNA. This CHX experiment suggests that the effect of EtOH on GFAP and RAR beta requires protein synthesis, whereas the effect of RA is independent of protein synthesis. In summary, these results demonstrate that GFAP responds to both EtOH and RA. Further work on this interaction in the astrocyte model is required, and could be relevant to the mechanism leading to the effects of prenatal ethanol exposure on fetal brain development.