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Ann Arbor, MI 48106 THE DIRECT EFFECTS OF Dl PHENYLH YDANTOIN (DPH) AND DPH ANALOGUES ON f i b r o b l a s t PROLIFERATION. dissertation Presented In Partial Fulfillment of the Requirements for the Degree Doctor of philosophy in the Graduate School of The Ohio State University B y Daniel A. von Deutsch, D.D.S., Ph.D. * t * * * The Ohio State University 1991 Dissertation Committee: Approved by Ralph Stephens ^ - Richard Fertel (dviser Norton Neff Department of Pharmacology. C o - A d v i s ^ / Department of Pharmacology. DEDICATIONS To my wife Deborah for her help and understanding. Also, this is dedicated to the memories of my mentors, my father Albert L. von Deutsch and to my advisor, Dr. Daniel Couri. They will be missed greatly. I also dedicate this work to my son, Albert Wolfgang and to all my cats. ACKNOWLEDGEMENTS I would like to thank Dr. Sarah Tjioe for her help, support and guidance, especially after the death of Dr. Couri. Your help will never be forgotten. My special thanks to Drs. Andrej Rotter, my advisor, and Adrian Frostholm for their help and understanding through the difficult times following the death of Dr. Couri. Thank you for giving me the guidance necessary for completing my project. To Dr. Ralph Stephens for his help and guidance through out this project, I give my thanks. His help and support were critical to my learning and to the project. To Dr. Nick Gerber, I give my special thanks. Without his support this work could never have been done. Thank you. My thanks to Dr. Richard Fertel for his help and .advice through out this study and especially in the preparation of this document. I want to thank my good friends Dr. Mark Brose, Dr. Carl Miller, Dan Mullett, Neil Smith and Greg Miller for their help, encouragement and many fun times. CURRICULUM VITAE EDUCATION 1972-1976 Cleveland State University Cleveland, Ohio B.S. in Biology and Chemistry. 1979-1983 The Ohio State University Columbus, Ohio Doctor of Dental Surgery (D.D.S.) College of Dentistry North East Regional Boards for DDS (1983). 1984-1991 The Ohio State University Columbus, Ohio Graduate Research Fellow / Research Associate Department of Pharmacology, Collage of Medicine. PUBLICATIONS - Journal Abstracts 1) Rieger,M.R; von Deutsch,D.A & Brown,W.T (1982): The Diametrical Tensile Strength of an Undercondensed Amalgam. J.Dental Res. 61 (Special): 84. 2) von Deutsch,D.A; Brose,M.O; Couri,D. and Rieger,M.R (1988): Effects of Implant Materials and Phenytoin Upon Cellular attachment. J.Dental Res. 67 (Special Issue): 384. 3) von Deutsch,D.A.; Rotter,A.; Tjioe,S. and Couri,D. (1990): The Effect of Diphenylhydantoin (DPH) Upon Cultured Fibroblasts. The Pharmacologist: 32,#3: 360, pg 186. FIELDS OF STUDY Major Field: Pharmacology. Toxicology and Instrumental Analysis: Dr. Daniel Couri Drug Metabolism: Drs. D. Feller, S. Black & D. Couri. Radioisotope Methodology: Drs. D. Feller and L. Malspeis. Immunology: Dr. B. Zwilling Cell Biology and Culture: Dr. Ralph Stephens TABLE OF CONTENTS Page DEDICATIONS........................................ ii ACKNOWLEDGEMENTS i i i CURRICULUM VITA.................................... iv LIST OF TABLES..................................... viii LIST OF FIGURES.................................... CHAPTER I, INTRODUCTION ........................... 1 a. Barbiturates and Phenobarbital................ 1 b. Diphenylhydantoin............................ 3 c. Therapeutic uses for DPH...................... 5 d. Clinical Toxicology.......................... 8 e. DPH induction of Gingival Hyperplasia.......... 10 f. DPH Metabolism (P-450 & PGS Pathway............ 13 8. The PGS Pathway.............................. 16 9. DPH-Induced DNA Single Strand Breaks........... 20 10. Statement of the Problem...................... 22 11. Hypothesis................................... 23 CHAPTER II, MATERIALS............................ 25 v CHAPTER III, METHODS 27 1. Media........ 27 A. F-12 Medium............................... B. Serum (FBS & Calf)......................... C. Stock & defined Medium..................... D. Growth Factors............................. 1. Fibroblast Growth Factor (FGF)........... 2. Epidermal Growth Factor (EGF)........... 2. cells........................................ 34 A. Normal Human Dermal Fibroblasts (NHDF)...... B. Human Fetal Lung Fibroblasts (WI-38)........ C. BALB/3T3 Clone A31 Fibroblasts.............. 3. Cell Growth Conditions........................ 36 4. Cell Passage................................. 36 5. Cell Counting Methods........................ 37 6. Cryopreservation............................. 38 7. Experimental Design.......................... 38 8. Synthesis of DPH Analogues.................... 41 A. para-Chlorinated Analogue (p-cl-DPH)........ B. para-Brominated Analogue (p-Br-DPH)........ C. Purification & Identification of Product.... 9. Drug Treated Medium & Vehicle.................. 44 10. General Procedure for Lysing Attached Cells 45 11. Total DNA Content............................ 46 12. Total Protein Content........................ 47 13. Statistical Testing.......................... 48 CHAPTER IV, RESULTS.............................. 49 A. DPH Dose Response: Serum Supplemented Medium 49 1. Treatment Schedule for Quiescent Cultures.... 2. Quiescent Serum Supplemented Cells.......... 3. Growth factor Free Studies.................. 4. FGF & EGF Dose Response with DPH............ 5. DPH Dose Response with FGF & EGF............ vi B. Defined Media Studies 75 1. FGF & EGF Free Cell Proliferation with DPH.... 75 a. F-12 Medium............................. b. Defined B-Medium........................ c. B-Medium with 10% FBS................... d. F-14 BC & HCF........................... e. DPH Dose Response (Growth Factor Free).... 2. Structure Activity Relationship in GFF Medium. 79 a. Hydantoin............................... b. Ethotoin............................... c. Mephenytoin............................. d. Phenobarbital........................... e. para substituted halogenated analogues.... 3. Prostaglandin Synthetase Pathway............ 91 1. Hydantoin............................... 2. Dexamethasone........................... 3. Progesterone............................ 4. DPH Dose Response with & without ASA..... 5. ASA Effects on Hydantoin Dose Response.... CHAPTER V, DISCUSSION........................... 102 A. Development of Cell Culture System............. 102 B. Pharmacology of DPH’s Proliferatory Activity 108 1. DPH Dose Response with Serum................ 2. DPH Dose Response with EGF & FGF (serum free). 3. DPH Dose Response with Growth Factor Free Medium and Serum Free C. TOXICOLOGY......................... 113 1. Metabolism...................... a. Cytochrome P-450.............. b. Prostaglandin Synthetase Pathway D. Structure Activity Relationship 120 E. Mechanism of Action 127 CHAPTER VI, CONCLUSIONS 130 REFERENCES 133 APPENDIX A P-450 Metabolism............... 143 APPENDIX B Hemocytometer.................. 150 APPENDIX C Analogues - Instrumental analysis 152 APPENDIX D RU-486 activity with DPH & Dex... 159 vii LIST OF TABLES Table Page 3-1 Comparison of defined media components (salts, vitamins). Comparison of defined media components (amino acids, other). 29 4-1 OPH dose response curve by cell counting. 53 4-2 DPH dose response curve by total DNA content 56 4-3 DPH effect on serum stimulated cell proliferation, (comparison of incubation times) 57 4-4 Effects of serum, growth factors and growth factor free conditions on DPH dose response curve (compar- 74 ison of ED50 & Emax). 4-5 Analogue-induced proliferation by cell counting. 83 4-6 Screen of growth factor free activity for DPH and DPH-analogues 89 4-7 Combined drug & ASA activity compared to ASACtrl. 93 4-8 Combined drug & ASA activity compared to drug Ctrl. 93 4-9 Comparison of growth factor free DPH and Hydantoin 97 dose