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University Micrijfilms International 300 N. Zeeb Road Ann Arbor, Ml 48106 8510561 Cunningham, Mary Jane THE INDUCTION AND INHIBITION OF BENZO(A)PYRENE METABOLISM IN HUMAN EPIDERMAL KERATINOCYTES AND DERMAL FIBROBLASTS The Ohio State University Ph.D. 1985 University Microfilms International 300 N. Zeeb Road, Ann Arbor, Ml 48106 Copyright 1985 by Cunningham, Mary Jane All Rights Reserved PLEASE NOTE: In all cases this material has been filmed in the best possible way from the available copy. Problems encountered with this document have been identified here with a check mark V . 1. Glossy photographs or pages ^ 2. Colored illustrations, paper or print 3. Photographs with dark background _____ 4. Illustrations are poor copy ______ 5. Pages with black marks, not original copy ______ 6. Print shows through as there is text on both sides of page ______ 7. Indistinct, broken or small print on several pages 8. Print exceeds margin requirements _____ 9. Tightly bound copy with print lost in spine ______ 10. Computer printout pages with indistinct print _____ 11. Page(s) ____________ lacking when material received, and not available from school or author. 12. Page(s) ____________ seem to be missing in numbering only as text follows. 13. Two pages numbered ____________. Text follows. 14. Curling and wrinkled pages ______ 15. Other_______________________________________________________ ________________ University M icrofilm s International THE INDUCTION AND INHIBITION OF BENZO(A)PYRENE METABOLISM IN HUMAN EPIDERMAL KERATINOCYTES AND DERMAL FIBROBLASTS DISSERTATION Presented 1n Partial F ulfillm ent of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University by Mary Jane Cunningham, B.A. ***** The Ohio State University 1985 Reading Committee: Approved By Dr. George E. Milo Dr. Ronald W. Trewyn Dr. Dorothy E. Schumm Dr. Donald T. Witiak Adviser Department of Physiological Chemistry Copyright by Mary Jane Cunningham 1935 Dedicated To My Family i i ACKNOWLEDGEMENTS I would like to thank Dr. Milo for his guidance and support and Lalitha, Raman, Ponamma, Irene, and Marty for th e ir advice and input. I am grateful to Inge for the use of her slides of the immunofluorescent staining of human keratinocytes and for teaching me the fine art of tissue culture and also to Prashant and Gerold who aided in starting primaries. I am appreciative to Jeff for sharing his mechanical and technical knowledge of the HPLC (especially in times of malfunctions), to Jenny for showing me how to culture human lung c e lls, and to Aline for typing this dissertation. Finally, I am indebted to my family for th e ir constant support and encouragement, especially Mom, Dad, Caryl, B ill, and Blatts, and in memorium, Grandma Mary and Grandpa, Grandma Jane and Aunt Hilda. VITA October 17, 1958 Born - East Cleveland, Ohio 1980 B.A. (Biology, Chemistry), Western Reserve College, Case Western Reserve University, Cleveland, Ohio 1980-1981 Teaching Associate, Department of Physiological Chemistry, The Ohio State University, Columbus, Ohio 1981-1984 Research Associate, Department of Physiological Chemistry, The Ohio State University, Columbus, Ohio PUBLICATIONS Cunningham, M.J., Kurian, P., Tejwani, R., and Milo, G.E., Metabolism of Benzo(a)pyrene in Human Skin Fibroblasts under Transforming and Non-Transforming Conditions. Mol. Pharmacol. Submitted. Cunningham, M.J., and Milo, G.E., The Effect of Cytochrome P-450 Inducers on the Metabolism of Benzo(a)pyrene in Human Epithelial Keratinocytes in Vitro. In preparation. Cunningham, M.O., Kurian, P., and Milo, G.E., Benzo(a)pyrene Metabolite Distribution and Delivery of a Carcinogenic Insult in Human Fibroblasts. Fed. Proc. 43: 2203 (1984). FIELDS OF STUDY Major Field: Biochemistry Studies in Metabolism and Transformation of Human Cells in V itro by Chemical Carcinogens. Professor George E. Milo TABLE OF CONTENTS Page DEDICATION............................................................................................................. i i ACKNOWLEDGMENTS ..............................................................................................i i i VITA............................................................................................................................. iv LIST OF TABLES........................................................................................................ vi LIST OF FIGURES.................................................................................................... v iii LIST OF ABBREVIATIONS........................................................................................ xi CHAPTER: I. BENZO(A)PYRENE METABOLISM IN RANDOMLY-PROLIFERATING, SYNCHRONIZED, AND CONFLUENT HUMAN NEONATAL FORESKIN FIBROBLASTS ......................................................................................... 1 A. Introduction ................................................................................... 1 B. Materials and Methods .................................................................. 16 C. Results and Discussion .............................................................. 23 I I . GROWTH AND MORPHOLOGICAL CHARACTERISTICS OF HUMAN EPITHELIAL KERATINOCYTES ................................................................. 40 A. Introduction ................................................................................... 40 B. Materials and Methods .................................................................. 48 C. Results and Discussion .............................................................. 52 I I I . EFFECTS OF CYTOCHROME P-450 INDUCERS ON BENZO(A)PYRENE METABOLISM IN HUMAN EPITHELIAL KERATINOCYTES ........................... 68 A. Introduction ................................................................................... 68 B. Materials and Methods .................................................................. 73 C. Results and Discussion .............................................................. 78 CONCLUSIONS..................................................................................................................128 BIBLIOGRAPHY..................................................................................................................131 v LIST OF TABLES Table Page 1. Indices of Transformation in the Different Populations of Human Neonatal Foreskin Fibroblasts ..................................................... 14 2. Retention Times of Benzo(a)pyrene Metabolite Standards on a DuPont Zorbax ODS HPLC Column (6.2 mm ID x 25 c m ) ..........................24 3. Organic-Extractable Metabolites of Benzo(a)pyrene in the Extracellular Fractions of the Different Fibroblast Populations 30 4. Organic-Extractable Metabolites of Benzo(a)pyrene in the Cytoplasmic Fractions of the D ifferent Fibroblast Populations . 34 5. Organic-Extractable Metabolites of Benzo(a)pyrene in the Nuclear Fractions of the D ifferent Fibroblast Populations . 37 6. Equimolar Aliquots of Stock Solutions of Pretreatment Chemicals Added to Epidermal Keratinocyte Medium ........................... 75 7. Groups of Pretreated and Nonpretreated Epithelial Cultures. 76 8. Organic-Extractable Benzo(a)pyrene Metabolites in the Extracellular Fractions from Human Epithelial and Fibroblast Populations ............................................................................. 82 9. Organic-Extractable Benzo(a)pyrene Metabolites in the Intracellular Fractions from Human Epithelial and Fibroblast Populations ....................................................................................84