Faculty Bibliography

Phenytoin (PHT), a widely used anticonvulsant, has been shown to inhibit bone resorption in rodent organ cultures. The drug also has complex effects on bone metabolism including chronic clinical symptoms of osteomalacia. However, the precise mechanism of PHT action in bone is still unclear. Neutral collagenases that specifically cleave native collagen have been implicated in the turnover of connective tissue. The effect of PHT was assessed on collagenase and gelatinase activities from UMR 106-01 rat osteoblastic osteosarcoma cells. Semiconfluent cells were treated with PHT (50 and 10 micrograms/ml) in the presence of bovine parathyroid hormone, b-PTH-(1-34), at 10(-7) M for 24, 48, 72 and 96 h. The media were assayed following concentration, APMA activation, and incubation with native or denatured [3H]-methyl collagen substrate (approximately 100,000 dpm) at 27 degrees C for 18 h and 35 degrees C for 2 h, respectively. Enzyme activities were presented as primary counts per minute for each time point and calculated as % activity of PTH at 10(-7) M. Parathyroid hormone (10(-7) M) stimulated collagenase activity (approximately 65-fold) and gelatinase activity (approximately 400-fold). PHT (50 micrograms/ml) reduced the PTH-stimulated collagenase activity by 18-53% and the gelatinase activity by 58-72%. SDS PAGE and fluorography following PHT treatment indicated a PHT-induced partial inhibition of PTH-stimulated degradation to alpha A chains of Type I collagen. Phenytoin may inhibit bone resorption through its action on the transcription, synthesis, and/or secretion of the collagenolytic enzymes, collagenase and gelatinase

Cells were isolated by sequential collagenase digestion from the parietal segments of one day old mice (Swiss albino BNL strain) and characterized for osteoblast parameters by alkaline phosphatase histochemistry and bovine parathyroid hormone (bPTH-(1-34] induced cAMP activity (protein binding assay). Phenytoin (DPH) reduced PTH stimulated cAMP activity nearly 3-fold in the presence and nearly 1.5-fold in the absence of added calcium. In the absence of PTH, DPH exerted no significant effect. Bay-K-8644, a calcium channel activator, appeared to approximate the PTH stimulation of cAMP activity, even in the presence of DPH. This study demonstrates that DPH has a direct effect on PTH stimulated cAMP activity in cultured murine osteoblasts

5,5-diphenylhydantoin (Phenytoin, PHT), a widely used anticonvulsant, is also a vitamin K antagonist and disrupts bone metabolism, leading to osteomalacia. The vitamin K-dependently synthesized protein, osteocalcin, has been implicated as a key regulatory protein in bone resorption. The purpose of the present study was to determine whether PHT had an effect on osteocalcin secretion. Cells were grown to confluence in Ham's F-12 nutrient mixture, and treated with 1,25 (OH)2 vitamin D3 (2.6 microM to 2.6 pM) or PHT (5-100 micrograms/mL) for either 24 or 48 h of pretreatment. The media were then discarded, replaced with fresh media and test reagents, and quantitated for osteocalcin by radioimmunoassay at 0, 4, and 8 h secretion time points. Results were statistically analyzed by the Student's two-tailed t test. Controls showed a nearly linear secretion rate of osteocalcin, reaching 8-9 ng/10(6) cells by 8 h. Vitamin D3 (2.6 nM) maximally stimulated secretion nearly two-fold after 24 or 48 h of pretreatment in comparison to controls. PHT alone (25-100 micrograms/mL) exerted an inhibitory effect, which appeared dose-dependent and was most evident at 4 and 8 h. PHT (50 micrograms/mL) had a significant effect, in the presence of a range of vitamin D3 concentrations (2.6 microM to 2.6 pM), after 48 h of pretreatment. A maximal PHT dose of 100 micrograms/mL had no effect on either the viability or the numbers of cultured cells. These data indicate that PHT affects osteocalcin secretion from osteoblastic rat osteosarcoma (ROS 17/2.8) cells