Stimulatory Effects of Phenytoin on Osteoblastic Differentiation in Cultured MC3T3-E1 Cells.

Abstract

Phenytoin (diphenylhydantoin, DPH) , an anticonvulsant drug for epileptic patients, has several adverse effects that include calvarial thickening and coarsening of the facial features. These effects often occur with chronic DPH therapy.While previous studies have shown DPH to heve an anabolic action on bone cells in vivo and in vitro, the basis of these effects is not fully understood.In this study, the effect of DPH on osteoblastic differentiation in cultured MC 3 T 3-E 1 cells was investigated by measuring mineralized bone nodule (BN) formation, alkaline phosphatase (ALPase) activity, cell growth, and the expression of matrix proteins, such as osteocalcin (OCN) , and osteopontin (OPN) . Continuous treatment of MC 3 T 3-E 1 cells with DPH for 20 days increased the BNs (9.1 to 19.7-fold) in a dose-dependent manner for concentrations of 10-50 , u M DPH. ALPase activity was also stimulated by DPH (1.4-3.2 hold) in a dose-dependent manner for concentrations of 10-100 , u M. Although a concentration 50 , u M DPH increased the DNA content of the cells on days 5, 10, and 15, the final cell saturation density was not affected by any concentration of DPH. When DPH was added at two different culture stages, days 1-10 (growth stage) and days 11-20 (matrix development stage) , BN formation was markedly stimulated in the case of the latter addition. A maximal effect was observed at a concentration of 200 AtNI DPH. The expression of OCN and OPN mRNA was also augmented by continuous treatment at a concentration of 200 du M DPH on days 1-20, and a remarkable effect was also observed when DPH was added on days 11-20. These findings demonstrate that DPH stimulates osteoblastic markers, such as BNs, ALPase activity, and OCN and OPN expression, mainly during the stage of matrix development in MC 3 T 3-E 1 cells.