INTRODUCTION:In some cases only a brief exposure to phenytoin (Dilantin) in early embryogenesis results in birth defects. Although the teratogenic effects of phenytoin are multifactorial, we are studying the molecular interactions of phenytoin by clarifying its effect on P19 murine embryonal cells differentiated into neuron like cells using retinoic acid (RA). Neuronal differentiation in the cells can be monitored by the expression of two neurospecific proteins; growth associated protein-43 (GAP-43) and neurofilament-165 (NF-165). GOAL OF THE STUDY: Our long-range goal is to determine the teratogenic mechanisms of phenytoin. AIM OF THE STUDY: The aim of this study is to characterize the effect of phenytoin and bFGFon development and proliferation of P19 neuron like cells. DESIGN: An experimental design was used to determine the effects of phenytoin and bFGF on P19 neuron like cells. MATERIAL AND METHODS: Cells were grown in growth medium, trypsinized, placed in induction media, with RA or RA + phenytoin, for differentiation. Differentiated cells were trypsinized and plated. Cells grown with and without phenytoin, were solubilized, and the cell lysates collected at 2-day intervals for 8 days. For bFGF studies cells were plated in 24 well plates and grown in the growth media for two days. After 2 days the media was changed and media containing bFGF in concentrations from 0.1ng - 100ng/ml was added to the cultures. Cells were grown for an additional two days then assayed for proliferation. Western blots of lysates were probed with GAP- 43 or NF-160 antibodies. Cell proliferation was determined using a standard NADH assay. FINDINGS: GAP-43 proteins are present in 6 and 8-day lysates and NF-160 in 4, 6, and 8-day lysates. Preliminary findings suggest that phenytoin decreased the proliferation rate of the neuron like cells but that bFGF at 1ng/ml restored the proliferative function especially in the cells both differentiated and grown in the presence of phenytoin. RESULTS AND CONCLUSIONS: Phenytoin appears to pose a threat to proliferation and development of the neuron like cells. This in vitro model may serve for the testing of other teratogenic drugs. Knowledge and counseling regarding the effects of these drugs could decrease the embryopathies and congenital anomalies that occur. (This study was supported in part by a grant from the Neuroscience Nurses Foundation)
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