Louisiana State University LSU Digital Commons LSU Historical Dissertations and Theses Graduate School 1990 A Comparative Study of the Effects of Sesbania Drummondii on the Hepatic Cytochrome P-450 Monooxygenase Systems of Chickens and Rats. Marcy Iva Banton Louisiana State University and Agricultural & Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_disstheses Recommended Citation Banton, Marcy Iva, "A Comparative Study of the Effects of Sesbania Drummondii on the Hepatic Cytochrome P-450 Monooxygenase Systems of Chickens and Rats." (1990). LSU Historical Dissertations and Theses. 4970. https://digitalcommons.lsu.edu/gradschool_disstheses/4970 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Historical Dissertations and Theses by an authorized administrator of LSU Digital Commons. For more information, please contact [email protected]. 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Ann Arbor, Ml 48106-1346 USA 313/761-4700 800/521-0600 Order Number 9112217 A comparative study of the effects of Sesbania drummondii on the hepatic cytochrome P-450 monooxygenase systems of chickens an d ra ts Banton, Marcy Iva, Ph.D. The Louisiana State University and Agricultural and Mechanical Col., 1990 UMI 300 N. Zeeb Rd. Ann Arbor, MI 48106 A COMPARATIVE STUDY OP THE EFFECTS OF SE8BANIA PRUMMOMPII OH THE HEPATIC CYTOCHROME P-450 MONOOXYGENASE SYSTEMS OF CHICKENS AMD RATS A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Interdepartmental Program in Veterinary Medical Sciences (Toxicology Option) by Marcy I. Banton D.V.M., Louisiana State University, 1981 August 1990 AgKHfiWIiBBgMHT6 I would like to acknowledge the contributions of the following individuals. My sincere appreciation is extended to my major advisor, Dr. W. Flory, for his advice, support, and guidance throughout this course of study. I am especially grateful to Dr. C.R. Short, for taking on the position of co-chairman of my committee in the absence of Dr. W. Flory and for his support and counsel in the preparation of this manuscript. I am most appreciative to Dr. G.w. Winston, for his commitment to the planning, execution, and analysis of this study. I also am grateful to the other members of my advisory committee, Dr. S.S. Nicholson, Dr. K.M. Kleinow and Dr. R.M. Grodner for their constructive criticisms and thoughtful analysis of this study. I am thankful for the financial support received from: Louisiana State University in the form of an assistantship for 3 years; Dr. J.J. England for graciously providing monies for supplies; and Dr. W. Springer for furnishing the White Leghorn chickens and their housing. Superior technical help was received from: Dr. P.L.H. Jowett for aldrin epoxidase method development; Dr. P. Bounds for gel electrophoresis and western blotting techniques; Dr. S. Narayan for cytochrome P-450-mediated activities; Dr. M.J.J. Ronis for providing cytochrome P-450 antibodies and western blots; and finally J. Pawlusiow and T. McManus for animal assistance. I am grateful to Dr. M.A. Freeman for statistical analyses of the data and much help with the preparation of this manuscript. I would also like to thank Dr. L.P. Ruhr for serving as my mentor during the first years of my graduate study. Finally, I thank my husband, Michael, and my children, Rebecca, Amelia, and James for their love, understanding, patience, and sacrifice during the time of this study. TABLE -QF-CflMTEWTP PAGE TITLE PAGE i ACKNOWLEDGEMENTS ii TABLE OF CONTENTS iv LIST OF TABLES vii LIST OF FIGURES X ABSTRACT xi CHAPTER 1 - INTRODUCTION 1 CHAPTER 2 - LITERATURE REVIEW 4 A. Sesbania drummondii 4 1. Botany of Sesbania 4 2. Sesbania in agriculture 7 3. Sesbania in medicine 9 4. Sesbania toxicosis 10 B. Hepatic cytochrome P-450 monooxygenase 18 system 1. Structure of the cytochrome P-450 monooxygenase system 19 2. Function of the cytochrome P-450 monooxygenase system 20 3. Regulation of the cytochrome P-450 monooxygenase system 24 4. Avian hepatic cytochrome P-450 monooxygenase system 28 CHAPTER 3 - EXPERIMENTAL GOALS AND DESIGN 37 CHAPTER 4 - SPECIFIC OBJECTIVES, MATERIALS AND METHODS, RESULTS AND DISCUSSION 42 A. Phase I - Preliminary investigations of the effects of an extract of Sesbania dnimiBondi i on the hepatic cytochrome P-450 monooxygenase systems of chickens and rats.42 Experiment 1 - Initial characterization studies of the effects of an extract of Sesbania drummondii on hepatic cytochrome P-450 monooxygenase systems of chickens and rats. 42 Experiment 2 - Evaluation of the effects of the bicarbonate-azide extraction buffer/vehicle on the hepatic cytochrome P-450 monooxygenase systems of chickens and rats. 70 Experiment 3 - Comparison of the effects of an extract of Sesbania drummondii on the hepatic cytochrome P-450 monooxygenase systems of male versus female rats. 75 Experiment 4 - Evaluation of the effects of an extract of Sesbania drummondii on microsomal membrane stability. 82 B. Phase II - Characterization of Sesbania drummondii effects on the catalytic properties of the hepatic cytochrome P-450 monooxygenase systems. Comparison with effects caused by phenobarbital and B-naphthoflavone. 91 C. Phase III - Effects of Sesbania drummondii on hepatic NADPH-dependent cytochrome P-450 reductase. Comparison with effects caused by phenobarbital and B-naphthoflavone. 147 D. Phase IV - Induction of microsomal enzymes by Sesbania drummondii as determined by SDS-PAGE and Western blots with anti­ cytochrome P-450 antibodies. Comparison with effects caused by phenobarbital and B-naphthoflavone. 188 CHAPTER 5 - SUMMARY AND CONCLUSIONS 208 v BIBLIOGRAPHY 225 APPENDICES 253 VITA 292 V i List of Tables Pag* Table l. Effect of Sesbania drummondi i-treatment on body and liver weights in chickens and r a t s ........... 57 Table 2. Effect of Sesbania drummondi i-treatment on hepatic microsomal monooxygenase cytochromes and reductases in chickens and rats ........................ 58 Table 3. Effect of Sesbania druromond i i-treatment on selected hepatic microsomal monooxygenase activities .. 60 Table 4. The effect of bicarbonate-azide buffer (BBN) vehicle on cytochrome P-450 content and hepatic microsomal monooxygenase activities in chickens and rats...................................................... 73 Table 5. Cytochrome P-450 content of Sesbania drummondii-treated male and female rats ............... 78 Table 6. Glucose-6-phosphatase activity of Sesbania drummondii-treated chickens and rats.................... 88 Table 7. Optimized conditions for the assay of aminopyrine N-demethylase activity in hepatic microsomes from control chickens and rats............. 97 Table 8. Optimized conditions for the assay of aldrin epoxidase activity in hepatic microsomes from control chickens and rats........................................ 99 Table 9. Optimized conditions for the assay of methoxyphenoxazone O-dealkylase activity in hepatic microsomes from control chickens and r a t s ............. 103 Table 10. Optimized conditions for the assay of ethoxyphenoxazone O-dealkylase activity in hepatic microsomes from control chickens and rats............... 104 Table 11. Optimized conditions for the assay of pentoxyphenoxazone O-dealkylase activity in hepatic microsomes from control chickens and rats............... 105 Table 12. Optimized conditions for the assay of benzyloxyphenoxazone O-dealkylase activity in hepatic microsomes from control chickens and rats............... 106 Table 13. The effect of g.esfranja drummondii. phenobarbital, and B-naphthoflavone on chicken and rat hepatic cytochrome P-450 specific content and absorbance maxima....................... 109 vii Table 14. The effect of Sesbania drummondii. phenobarbital, and 6-naphthoflavone on aminopyrine N- demethylase activity in chicken and rat hepatic microsomes................................................