Our goal is to determine the effect of exogenously added 5,5-diphenylhydantoin, (DPH; Phenytoin, PHT) on cultured human gingival fibroblasts; rat osteoblasts and a rat osteoblastic osteosarcoma cell line and chick osteoclasts. The investigation will largely focus on the ability of PHT to regulate potential mechanisms which may be operable in bone remodeling. PHT has been widely used in the treatment of convulsive disorders and has been shown to influence bone metabolism. Phenytoin has been shown to inhibit the release of lysosomal enzymes in parallel with bone resorption as indicated by reduced release of 45Ca, Ca2+, Pi, and hydroxyproline from mouse calvaria in vitro. Potential mechanisms operable in bone resorption include collagenase and its inhibitor protein, matrix degrading lysosomal enzymes, and the vitamin K-dependently synthesized calcium-binding amino acid, gamma-carboxyglutamic acid (Gla). Cultures of bone cells will be examined in the presence or absence of Phenytoin. The action of PHT on the activity and secretion of collagenase and its inhibitor protein from osteoblasts and fibroblasts will be analyzed by (1) measurement of the release of 14C-labeled peptides from Type 1 rat skin collagen substrate and (2) visualization of collagenase digestion products by 7.5% SDS gel electrophoresis and fluorography of the radiolabeled peptides. PHT action on the synthesis of Gla protein from osteoblasts and the osteosarcoma cell line will be analyzed by radioimmunoassay for bone Gla protein. The effect of PHT on the secretion and activity of chick osteoclast lysosomal enzyme, tartrate-resistant acid phosphatase, a marker enzyme specific for the osteoclast, and beta-glucuronidase will be measured with substrate and chromogenic couplers spectrophotometrically. An electron microscopic cytochemical study will be conducted to examine the localization of lysosomal acid phosphatase reaction product from cultured chick osteoclasts following PHT exposure. The ability of osteoclasts to form a ruffled border on devitalized chick calvarial bone fragments in vitro will also be evaluated. The use of PHT as a probe in cultured cells may advance our understanding of bone remodeling mechanisms and consequently both the treatment of periodontal disease and the development of craniofacial anomalies.
