Phenytoin-induced teratogenesis: a molecular basis for the observed developmental delay during neurulation.
OBJECTIVE
We wished to determine whether chronic phenytoin (PHT) exposure could impair neural development and if any morphological alterations could be linked to changes in gene expression.


METHODS
Pregnant SWV mice were chronically administered PHT 40 mg/kg/day from gestational day (GD) 0:12 (day:h) until they were killed at various timepoints throughout neural tube closure (NTC). At each timepoint, embryos from both treated and control dams were collected and scored for their progression through NTC. The neural tubes were then isolated and subjected to in situ transcription (IST) and antisense RNA amplification procedures. Using these techniques, we examined the expression of 10 genes: N-cadherin (Ncad), collagen type IV (col-IV), bcl-2, c-jun, PAX-3, collular retinol binding protein-2 (CRBP-2), retinoic acid receptor alpha (RAR alpha), transforming growth factor(beta2) (TGF(beta2)), wee-1, and EMX-2.


RESULTS
Chronic PHT exposure not only caused a delay in NTC whereby exposed embryos lagged behind the controls at each collection timepoint, but also significantly altered the expression of specific genes at distinct times during NTC. Early in NTC, PHT induced a significant reduction in the expression of N-cad, col-IV, and c-jun in exposed embryos as compared with controls. In contrast, during the midstages of NTC, the only significant molecular alterations observed in the PHT-exposed embryos was the continued decreased expression of col-IV and an increase in CRBP-2 expression. Finally, in the latter stages of NTC, PHT caused a significant reduction in the expression of bcl-2, RAR alpha, TGF(beta2), EMX-2, and PAX-3.


CONCLUSIONS
These results show that although the effects of PHT are morphologically subtle, causing a delay in the development of the neural tube, this delay is accompanied by alterations in critical genes at crucial times of neural development that may account for the observed neurological deficits often associated with PHT exposure.
Bennett
G D
GD
Department of Veterinary Anatomy and Public Health, College of Veterinary Medicine, Texas A&M University, College Station 77843-4458, USA.
Lau
F
F
Calvin
J A
JA
Finnell
R H
RH
D000014:Abnormalities, Drug-Induced / Q000209:etiology* / Q000235:genetics; D000818:Animals; D015820:Cadherins / Q000096:biosynthesis / Q000235:genetics; D018797:Cell Cycle Proteins; D002490:Central Nervous System / Q000187:drug effects / Q000196:embryology; D003094:Collagen / Q000096:biosynthesis / Q000235:genetics; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D005260:Female; D005784:Gene Amplification; D015870:Gene Expression / Q000187:drug effects; D016758:Genes, jun / Q000187:drug effects / Q000235:genetics; D018398:Homeodomain Proteins / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D008815:Mice, Inbred Strains; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics; D009436:Neural Tube Defects / Q000139:chemically induced* / Q000235:genetics; D009687:Nuclear Proteins; D000072041:PAX3 Transcription Factor; D051761:Paired Box Transcription Factors; D010672:Phenytoin / Q000633:toxicity*; D011247:Pregnancy; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics; D018168:Receptors, Retinoic Acid / Q000096:biosynthesis / Q000235:genetics; D012177:Retinol-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D054840:Retinol-Binding Proteins, Cellular; D014157:Transcription Factors; D000099260:Empty Spiracles Homeobox Proteins
D015820:Cadherins; D018797:Cell Cycle Proteins; D003094:Collagen; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D000072041:PAX3 Transcription Factor; D051761:Paired Box Transcription Factors; D010672:Phenytoin; D011505:Protein-Tyrosine Kinases; D018168:Receptors, Retinoic Acid; D012177:Retinol-Binding Proteins; D054840:Retinol-Binding Proteins, Cellular; D014157:Transcription Factors; D000099260:Empty Spiracles Homeobox Proteins; C518460:Rbp2 protein, mouse; C068686:Pax3 protein, mouse; C499348:Wee1 protein, mouse