Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Influence of tamoxifen on hormonally regulated cytochrome P450 enzyme expression in rats Ickenstein, Ludger Markus 1999

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_1999-0202.pdf [ 8.25MB ]
Metadata
JSON: 831-1.0099351.json
JSON-LD: 831-1.0099351-ld.json
RDF/XML (Pretty): 831-1.0099351-rdf.xml
RDF/JSON: 831-1.0099351-rdf.json
Turtle: 831-1.0099351-turtle.txt
N-Triples: 831-1.0099351-rdf-ntriples.txt
Original Record: 831-1.0099351-source.json
Full Text
831-1.0099351-fulltext.txt
Citation
831-1.0099351.ris

Full Text

I N F L U E N C E O F T A M O X I F E N O N H O R M O N A L L Y R E G U L A T E D C Y T O C H R O M E P 4 5 0 E N Z Y M E E X P R E S S I O N I N R A T S by L U D G E R M A R K U S I C K E N S T E I N Dip lom Biologist, Friedrich-Alexander Universitat Erlangen / Nurnberg, Germany, 1995 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T F O R T H E D E G R E E O F M A S T E R OF S C I E N C E I N T H E F A C U L T Y OF G R A D U A T E S T U D I E S F A C U L T Y OF P H A R M A C E U T I C A L S C I E N C E S Division of Pharmaceutical Chemistry We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A Apr i l 1999 ® Ludger Markus Ickenstein, 1999 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Pharmaceut ical Sc iences The University of British Columbia Vancouver, Canada Date A p r i l 27 . 1999 DE-6 (2/88) Abstract Tamoxifen, a partial estrogen antagonist in humans, is used widely in the treatment of breast cancer and suppresses growth hormone plasma levels in rats up to seven weeks after treatment. The purpose of the present study was to determine i f tamoxifen treatment results in long-term alterations of hormonally regulated cytochrome P450 enzymes. Female rats were injected subcutaneously with tamoxifen at 0.5, 5, 20, 50, 100, or 200 mg/kg for two consecutive days. Male rats were injected subcutaneously with tamoxifen at 200 mg/kg for two consecutive days. Blood samples were taken at regular time intervals. Rats were kil led 36 days after treatment and hepatic microsomes were prepared. Administration of tamoxifen decreased body weight gain in male and female rats. Testosterone 7a-hydroxylase activity and cytochrome P450 2A1 content were decreased in hepatic microsomes from tamoxifen treated female rats by 40% and 35%, respectively. In hepatic microsomes from tamoxifen treated male rats, testosterone 7a-hydroxylase activity was increased by 70% and testosterone 2a- and 16oc-hydroxylase activities and cytochrome P450 2C11 content was decreased by approximately 44% and 30%, respectively. Administration of tamoxifen decreased peak growth hormone plasma levels in male and female rats, but did not affect the average and nadir growth hormone plasma levels. In plasma of rats treated at 50 mg/kg, tamoxifen was detected at one day and at twelve days after treatment but not at 24 or 36 days after treatment. 4-Hydroxytamoxifen was detected at one and twelve days after treatment. In plasma of rats treated with tamoxifen at a dosage of 200 mg/kg, 4-hydroxytamoxifen was detected up to 36 days after treatment. The results of the present study indicate that tamoxifen treatment suppressed body weight gain, blunted peak growth hormone plasma levels, and resulted in long-term alterations of ii specific hepatic cytochrome P450 enzymes in male and female rats. These effects are different from the previously reported direct, short-term effects of tamoxifen on hepatic cytochrome P450 2B and 3 A enzymes. The effects of tamoxifen on cytochrome P450 enzymes are not believed to be mediated solely by the effects of tamoxifen on growth hormone plasma levels. More likely, the effects of tamoxifen on cytochrome P450 enzymes were caused by its estrogenic or antiestrogenic effects. Alternatively, the effects could have been caused by a residual tamoxifen metabolite that remained in rat tissues at concentrations below the limits of quantitation of the H P L C assay. ii i TABLE OF CONTENTS Page Abstract i i - i i i List of Tables v i i -v i i i List o f Figures ix -x i List of Abbreviations x i i Acknowledgements x i i i 1. I n t r o d u c t i o n 1.1. Cytochrome P450 Enzymes 1-6 1.1.1. Nomenclature of C Y P Enzymes 2-3 1.1.2. The C Y P 2 A Subfamily 3-4 1.1.3. The C Y P 2 B Subfamily 4 1.1.4. The C Y P 2 C Subfamily 4-5 1.1.5. The C Y P 3 A Subfamily 5-6 1.2. Growth Hormone 6-7 1.3. Mechanism of Hormonal Regulation of C Y P Enzymes 8-15 1.3.1. Hormonal Regulation of C Y P 2 A 1 12-13 1.3.2. Hormonal Regulation of C Y P 2 B 1 and C Y P 2 B 2 13-14 1.3.3. Hormonal Regulation of CYP2C11 14 1.3.4. Hormonal Regulation of C Y P 3 A 14-15 1.4. Tamoxifen 16-23 1.4.1. Mechanism of Act ion 16-17 1.4.2. Tamoxifen in Breast Cancer Therapy 17-18 1.4.3. Metabolism of Tamoxifen 18-20 1.4.4. Toxicity of Tamoxifen 21 1.4.5. Direct Effects of Tamoxifen on C Y P Enzymes 22 1.4.6. Effects of Tamoxifen on G H Secretion 22-23 1.5. Hypothesis 24 1.6. Objectives 24 2. E x p e r i m e n t a l 2.1. Chemicals 25-27 2.2. Purified C Y P Enzymes 28 2.3. Antibodies 28 iv Section A 2.4. Animals and Treatment 28-29 2.5. Blood Collection 29-30 2.6. Preparation of Hepatic Microsomes 30 2.7. Determination of Total Protein Content in Hepatic Microsomes 30 2.8. Determination of C Y P Protein Content in Hepatic Microsomes 31 2.9. Determination of C Y P Enzyme Activities in Hepatic Microsomes 31-37 2.10. Immunoquantitation of C Y P Enzymes 38-40 2.10.1. Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis 38 2.10.2. Immunoblots 38-39 2.10.3. Immunoquantitation 39 2.11. Detection and Quantitation of G H in Plasma 39-40 2.11.1. Detection and Quantitation of G H in Rat Plasma with an l 2 5 I Radioimmunoassay for Human G H 39-40 2.11.2. Detection and Quantitation of G H in Rat Plasma with an Enzyme-Linked Immunoassay for Rat G H 40 Section B 2.12. Synthesis of Tamoxifen Metabolites 41-43 2.12.1. Synthesis of N-Desmethyltamoxifen 41-42 2.12.2. Synthesis of Tamoxifen-N-oxide 42-43 2.13. H P L C Assay for Tamoxifen and Its Metabolites 43-47 2.14. Statistical Analysis 48 3. Results Section A 3.1. Development of Subcutaneous Cysts at the Injection Site 49-50 3.2. Effect of Tamoxifen on Body Weight and Liver Weight 51-55 3.3. Effect of Tamoxifen on the Relative Hepatic Content of C Y P Enzymes 56 3.4. Testosterone Hydroxylase Assay 57-65 3.4.1. Validation of the Testosterone Hydroxylase Assay 57-60 3.4.2. Effect of Tamoxifen on the Activity of Hepatic C Y P Enzymes 61-65 3.5. Immunoblots 66-73 3.5.1. Validation of the Immunoblots 66-68 3.5.2. Effect of Tamoxifen on Hepatic C Y P Protein Levels 69-73 v 3.6. Detection and Quantitation of G H in Rat Plasma 74-84 3.6.1. Detection and Quantitation of G H in Rat Plasma with an 1 2 5 I Radioimmunoassay for Human G H 74 3.6.2. Validation of the Enzyme-Linked Immunoassay for Rat G H 74-75 3.6.3. Detection and Quantitation of G H in Rat Plasma with an Enzyme-Linked Immunoassay for Rat G H 76-84 Section B 3.7. Synthesis of Tamoxifen Metabolites 85-90 3.7.1. Synthesis of iV-Desmethyltamoxifen 85 3.7.2. Synthesis of Tamoxifen-JV-oxide 85-90 3.8. H P L C Assay for Tamoxifen and Its Metabolites 91 -97 3.8.1. Validation of the H P L C Assay for Tamoxifen and Its Metabolites 91-94 3.8.2. Detection and Quantitation of Tamoxifen and its Metabolites in Rat Plasma 95-97 4. Discussion Section A 4.1. Development of Subcutaneous Cysts at the Injection Site 98-99 4.2. Effect o f Tamoxifen on Body Weight and Liver Weight 99-100 4.3. Effect of Tamoxifen on Hepatic C Y P Enzymes 100-103 4.4. Effect o f Tamoxifen on Plasma G H Levels 103-105 Section B 4.5. Tamoxifen Metabolite Synthesis 105-106 4.6. Detection and Quantitation of Tamoxifen and its Metabolites in Rat Plasma 106-109 4.7. Correlating the Effects of Tamoxifen on Hepatic C Y P Enzymes with Its Effect on G H Plasma Levels 109-111 4.8. Evaluation of a Possible Mechanism for the Effects of Tamoxifen on Hepatic C Y P Enzymes 111-113 5. Conclusions 114 6. Future Experiments 115-117 7. References 118-132 Appendices I -VI 133-138 vi L I S T O F T A B L E S Page Table 1: Sexual Dimorphic Expression of Hepatic C Y P Enzymes in the Rat 8 Table 2: Regulation of Hepatic C Y P Enzyme Expression in the Rat by G H 10 Table 3: Regulation of Hepatic C Y P Enzyme Expression in the Rat by Sex Steroids 11 Table 4: Metabolism of Tamoxifen in Mouse, Rat, and Human 19 Table 5: Development of Subcutaneous Cysts at the Site of Injection of Tamoxifen Treated Rats 50 Table 6: Mean Body Weight of Female Rats Treated with Various Dosages of Tamoxifen 52 Table 7: Mean Body Weight of Male Rats Treated with Tamoxifen 53 Table 8: Mean Liver Weight of Male and Female Rats Treated with Various Dosages of Tamoxifen 55 Table 9: C Y P Concentration, Protein Concentration, and Relative C Y P Content in Hepatic Microsomes from Male and Female Rats Treated with Various Dosages of Tamoxifen 56 Table 10: Inter-Assay Variability of the Testosterone Hydroxylase Assay 58 Table 11: Intra-Assay Variability of the Testosterone Hydroxylase Assay 58 Table 12: Limits of Quantitation of the Testosterone Hydroxylase Assay 58 Table 13: Inter-Assay Variability of the Immunoblots of C Y P 2 A 1 , C Y P 2 B 1 , C Y P 2 C l l , a n d C Y P 3 A l Standards. 67 Table 14: Limits of Quantitation of the Immunoblots 67 Table 15: Specificity of the Goat Anti-Rat G H IgG 75 Table 16: Inter-Assay Variability of the Enzyme-Linked Immunoassay for Rat G H 75 Table 17: Chemical Shift Values in the N M R Proton Spectrum for Tamoxifen and Tamoxifen-JV-oxide 90 Table 18: Intra-Assay Variability of the H P L C Assay for Tamoxifen and Its Metabolites in Rat Plasma 93 Table 19: Inter-Assay Variability of the H P L C Assay for Tamoxifen and Its Metabolites in Rat Plasma 93 Table I: Mean Testosterone Hydroxylase Activities of Hepatic Microsomes from Female Rats Treated with Various Dosages of Tamoxifen 133 Table II: Mean Testosterone Hydroxylase Activities of Hepatic Microsomes from Male Rats treated with Tamoxifen 134 Table III: Relative Content of Specific C Y P Enzymes (nmol/nmol total C Y P ) in Hepatic Microsomes from Male and Female Rats Treated with Various Dosages of Tamoxifen 135 vii Table IV: Relative Content of Specific C Y P Enzymes (pmol/mg protein) in Hepatic Microsomes from Male and Female Rats Treated with Various Dosages of Tamoxifen 136 Table V : Plasma G H Levels of Male and Female Rats Treated with Various Dosages of Tamoxifen 137 Table V I : Concentrations of Tamoxifen in Rat Plasma as Detected by H P L C 138 Table VI I : Concentrations of 4-Hydroxytamoxifen in Rat Plasma as Detected by H P L C 138 vii i L I S T O F F I G U R E S Page Figure 1: Schematic Diagram of the Mechanism of a C Y P Monooxygenase Reaction 2 Figure 2: G H Secretion Pattern in Male and Female Rats 7 Figure 3: Hepatic Microsomal C Y P Expression Levels in Adult Rats 9 Figure 4: Chemical Structure of Tamoxifen 16 Figure 5: Chemical Structure of Estradiol 16 Figure 6: Metabolites of Tamoxifen in Rats and Humans 20 Figure 7: Short-Term Effects of Tamoxifen on C Y P Enzymes in Rats 22 Figure 8: Effects of Tamoxifen on G H Plasma Levels in Rats 23 Figure 9: Treatment Protocol 29 Figure 10: Regiospecific Hydroxylation of Testosterone by C Y P Enzymes 31 Figure 11: Representative Chromatogram of a Mixture of Hydroxytestosterone Standards 34 Figure 12: Representative Chromatogram of a Zero-Time Control Incubation of the Testosterone Hydroxylase Assay 35 Figure 13: Representative Chromatogram of Testosterone Metabolites Formed by Hepatic Microsomes from Vehicle-Treated Female Rats 36 Figure 14: Representative Chromatogram of Testosterone Metabolites Formed by Hepatic Microsomes from Vehicle-Treated Male Rats 37 Figure 15: Representative Chromatogram of a Zero-Time Control Incubation of the H P L C Assay for Tamoxifen and its Metabolites 45 Figure 16: Representative Chromatogram of a Mixture of Tamoxifen Metabolite Standards 46 Figure 17: Representative Chromatogram of Tamoxifen and Its Metabolites Extracted from Plasma of a Tamoxifen-Treated Female Rat 47 Figure 18: Mean Body Weight Gain of Female Rats Treated with Various Dosages o f Tamoxifen 54 Figure 19: Mean Body Weight Gain of Male Rats Treated with Tamoxifen 54 Figure 20: Mean Liver Weight/Body Weight Ratio of Male and Female Rats Treated with Various Dosages of Tamoxifen 55 Figure 21: Representative Calibration Curves for 6a-, 6(5-, 7a-, and 16a-Hydroxytestosterone 59 Figure 22: Representative Calibration Curves for 2(3- and 16(3-Hydroxytestosterone and Androstenedione 60 Figure 23: Mean Testosterone Hydroxylase Activities of Hepatic Microsomes from Female Rats Treated with Various Dosages of Tamoxifen 62 Figure 24: Mean Testosterone Hydroxylase Activities of Hepatic Microsomes from Female Rats treated with Various Dosages of Tamoxifen 63 Figure 25: Mean Testosterone Hydroxylase Activities of Hepatic Microsomes from Male Rats Treated with Tamoxifen 64 Figure 26: Mean Testosterone Hydroxylase Activities of Hepatic Microsomes from Male Rats Treated with Tamoxifen 65 Figure 27: Calibration Curves of Purified C Y P 2 A 1 , C Y P 2 B 1 , C Y P 2 C 1 1 , and C Y P 3 A 1 68 Figure 28: Representative Immunoblots of Hepatic Microsomes from Female Rats Probed with Polyclonal IgGs Against Rat C Y P 2 A 1 , C Y P 2 B 1 , or C Y P 3 A 1 70 Figure 29: Representative Immunoblots of Hepatic Microsomes from Male Rats Probed with Polyclonal IgGs Against Rat C Y P 2 A 1 , C Y P 2 B 1 , C Y P 2 C 1 1 , or C Y P 3A1 71 Figure 30: Relative Content of Specific C Y P Enzymes in Hepatic Microsomes from Female Rats Treated with Various Dosages of Tamoxifen 72 Figure 31: Relative Content of Specific C Y P Enzymes in Hepatic Microsomes from Male Rats Treated with Tamoxifen 73 Figure 32: Representative Calibration Curve of the Enzyme-Linked Immunoassay for Rat G H 75 Figure 33: G H Plasma Levels of Female Control Rats 77 Figure 34: G H Plasma Levels of Female Rats Treated with Tamoxifen at a Dosage of 5 mg/kg. 78 Figure 35: G H Plasma Levels of Female Rats Treated with Tamoxifen at a Dosage of 20 mg/kg 79 Figure 36: G H Plasma Levels of Female Rats Treated with Tamoxifen at a Dosage of 200 mg/kg 80 Figure 37: G H Plasma Levels of Male Control Rats 81 Figure 38: G H Plasma Levels of Male Rats Treated with Tamoxifen at a Dosage of 200 mg/kg 82 Figure 39: Mean, Nadir, and Peak Plasma G H Levels of Female Rats Treated with Various Dosages of Tamoxifen 83 Figure 40: Mean, Nadir, and Peak Plasma G H Levels of Male Rats Treated with Tamoxifen 84 Figure 41: Positive Electrospray Mass Spectrum of the Product of the N-Desmethyltamoxifen Synthesis 86 Figure 42: Positive Electrospray Mass Spectrum of the Product of the Tamoxifen-TV-oxide Synthesis 87 Figure 43: IR Spectrum of Tamoxifen 88 Figure 44: IR Spectrum of the Product of the Tamoxifen-A^-oxide Synthesis 88 Figure 45: N M R Proton Spectrum of Tamoxifen and the Product of the Tamoxifen-JV-oxide Synthesis 89 Figure 46: Representative Calibration Curves for Z 4-Hydroxytamoxifen, E 4-Hydroxytamoxifen, Tamoxifen-jV-oxide, and Tamoxifen 94 Figure 47: Plasma Concentrations of Tamoxifen in Female Rats Treated with Tamoxifen at a Dosage of 50 mg/kg 97 Figure 48: Plasma Concentrations of for Z 4-Hydroxytamoxifen in Female Rats Treated with Tamoxifen at a Dosage of 50, 100, and 200 mg/kg 97 Figure 49: Regulation of Hepatic Microsomal C Y P Enzymes in the Rat 113 x i L I S T O F A B B R E V I A T I O N S A androstenedione A C T H adrenocorticotropic hormone A N O V A analysis of variance B C I P 5-bromo-4-chhloro-3-indolylphosphate disodium salt Bis N, A^-methylene-bis-acrylamide B S A bovine serum albumin b.w. body weight C Y P cytochrome P450 E D T A ethylenediamine tetraacetic acid E L I S A enzyme-linked immunosorbant assay G H growth hormone G H R H growth hormone releasing hormone G C gas chromatography H P L C high performance liquid chromatography i.d. inner diameter IgG immunoglobulin G IR infrared IS internal standard L Q limit o f quantitation M S mass spectroscopy M S A monosodium aspartate M S G monosodium glutamate n number of samples N A D P H nicotinamide adenine dinucleotide phosphate N B T nitro blue tetrazolium chloride N M R nuclear magnetic resonance O D optical density P probability of a type I error p.o. per os (oral) P A G E polyacrylamide gel electrophoresis P B S phosphate buffered saline ppm parts per mil l ion s.c. subcutaneous S E M standard error of the mean SDS sodiumdodecyl sulfate SS somatostatin T testosterone T 3 triiodothyronine T 4 thyroxine T E M E D TV, N, N', N'-tetramethylethylenediamine T M S tetramethylsilane Tris tris(hydroxymethyl)aminomethane T R H thyrotropin releasing hormone T S H thyroid stimulating hormone U V ultraviolet xii A c k n o w l e d g e m e n t s I thank my supervisor, Dr. Stelvio Bandiera, for his support and guidance during my time in his laboratory. I also appreciate the support and guidance of the members of my committee, Drs. Gail Bellward, Kath MacLeod, Wayne Riggs, and Thomas Chang. Thanks to my external university examiner Dr. K. Wasan. I wish to recognize Dr. Andrew Parkinson for kindly supplying us with purified CYP2A1, Dr. Paul Thomas for his sheep anti-CYP2Al polyclonal antibody, and Andreas Melhorn for his help on the development of the HPLC assay for tamoxifen and its metabolites. Thanks to all my colleagues and friends in the faculty for their lively discussions and for making this research experience even more enjoyable. Thanks to my wife Susanne and my family for their moral and emotional support. x i i i Introduction 1. INTRODUCTION 1.1. Cytochrome P450 Enzymes Cytochrome P450 ( C Y P ) enzymes are the major Phase I drug metabolizing enzymes in the body. They function as the terminal enzymes of an electron transport chain and catalyze the biotransformation o f both endogenous compounds such as such as steroids, eicosanoids, fatty acids, and prostaglandins, and exogenous compounds such as pesticides, carcinogens, therapeutic drugs, and industrial chemicals. C Y P enzymes are the found in bacteria, fungi, plants, and animals (Gonzalez 1989). In mammals their greatest concentration is in the liver, but they are also found in lung, heart, kidneys, adrenal glands, ovary, testis, small intestines, brain, and other tissues (Gonzalez 1989 and 1990). The subcellular location of the hepatic C Y P monooxygenase system is the outer membrane of the endoplasmic reticulum. C Y P enzymes are also found in the inner membrane of mitochondria. Those C Y P enzymes are found mainly in mitochondria of the adrenal cortex and are involved in the synthesis and metabolism of steroids (Gonzalez 1990). The composition of C Y P enzymes in the liver is highly variable and dependent on gender, age, disease, stress, and exposure to external inducers. The expression level o f a single C Y P enzyme is controlled by complex, regulatory systems that can lead to elevation or repression of other C Y P enzymes. Alterations in the content of individual C Y P enzymes in mammalian liver can result in changes in the concentration of steroid hormones in the body and can modify the liver's ability to metabolize potentially toxic exogenous chemicals. A carbon monoxide-binding pigment was first reported in the mammalian liver by Klingenberger (1958) and Garfinkle (1958). The pigment was further characterized by Omura and Sato (1962, 1964a, and 1964b), who named it P-450, because it absorbed light maximally at a wavelength of 450 nm. C Y P enzymes consist of a protoporphyrin I X heme moiety (prosthetic group) and a single polypeptide chain (apoprotein) of 45 to 55 k D a (Guengerich and Martin 1980, Guengerich 1990). The heme moiety is part of the catalytic active site o f the enzyme. The iron ion associated with the heme group is coordinated to the center of the protoporphyrin ring. Four ligands of the heme iron are coordinated to the porphyrin ring. The fifth ligand is a thiolate anion from a cysteinyl residue o f the apoprotein. The sixth ligand acts as the binding site for molecular oxygen (Guengerich 1990). The substrate binds to the apoprotein adjacent to the heme moiety and adjacent to one of the oxygen atoms. The electrons for the monooxygenase reaction are transferred from N A D P H via NADPH-dependent C Y P reductase to the C Y P enzyme. The electrons reduce molecular oxygen 1 Introduction bound to the heme iron and generate a reactive oxygen species, which is then transferred to the substrate (Guengerich 1990, Porter and Coon 1991) (Figure 1). C Y P enzymes catalyze a wide range of reactions such as aromatic and aliphatic hydroxylation, epoxidation, peroxidation, N-, 0-, S-dealkylation, deamination, desulfuration, dehalogenation, and reduction (Guengerich 1990, Porter and Coon 1991). CYP-mediated biotransformation of foreign compounds usually increases the polarity and therefore the water-solubility of the substrate. This process usually leads to detoxification and a higher excretion rate of foreign compounds but C Y P enzymes can also form toxic or reactive metabolites that can bind to macromolecules such as proteins and D N A and may initiate mutagenesis and carcinogenesis (Guengerich 1984). ROH RH (ROH)Fe3* (RH)Fe3* j* / \ { R H ( H ) 2 ( R . K F e . O H ) 3 + - * . y (RH)Fe2+ 2 e - , 2 H + H-,0 (RH)Fef(02) 2 H./(RH)Fe 3*(0|) (RH)Fe3*(C T e - 02-H 2 0 2 Figure 1: Schematic diagram of the mechanism of a C Y P monooxygenase reaction. Fe represents the heme iron, R H the substrate, RH(H)2 the reduction product, R O H the monooxygenation product, and X O O H a peroxy-compound that can serve as an alternative oxygen donor (adapted from Porter and Coon 1991). 1.1.1. Nomenclature of C Y P Enzymes Soon after its characterization, it became apparent that a large number of closely related C Y P enzymes exist (Nelson et al. 1993 and 1996). Today, numerous C Y P enzymes and their corresponding genes have been identified. A systematic nomenclature based on the similarity of amino acid sequences is used to classify C Y P enzymes. Each enzyme is characterized by a 2 Introduction number and letter code starting with C Y P for cytochrome P450 (Cyp for mouse enzymes, CYP for the gene). A n enzyme family is denoted by an Arabic number and is defined as having up to 40% gene sequence homology. A n enzymatic subfamily is denoted by a capital Latin letter and defined as having more than 55% sequence homology. Each individual C Y P enzyme is given an Arabic number, e.g. C Y P 1 A 1 . To date, 12 gene families, 22 subfamilies, and approximately 150 C Y P enzymes have been identified in mammals, of those 50 are found in rats and 34 in humans (Nelson et al. 1996). In the human body, enzymes of the C Y P 1, 2, and 3 gene families are the most important C Y P enzymes involved in the metabolism of xenobiotic compounds, while enzymes of the C Y P 4 gene family are primarily responsible for the metabolism of endogenous compounds. Enzymes of the C Y P 2 A , 2B, 2C, and 3 A subfamilies are discussed below. 1.1.2. The CYP2A Subfamily In the rat, the C Y P 2 A gene subfamily consists of three enzymes, C Y P 2 A 1 , C Y P 2 A 2 , and C Y P 2 A 3 . The CYP2AI and CYP2A2 genes share 93% sequence homology and C Y P 2 A 1 and C Y P 2 A 2 proteins share 88% deduced amino acid similarity. C Y P 2 A 1 and C Y P 2 A 2 are found only in the liver (Ryan and Levin 1993). C Y P 2 A 3 is found in the lung but not in the liver (Gonzalez 1989). CYP2A3 shares 71% and 73% sequence homology with CYP2A1 and CYP2A2, respectively (Gonzalez 1990). Purified C Y P 2 A 1 catalyzes the regioselective hydroxylation of testosterone predominantly at the 7a position and to a minor extent at the 6a position (Ryan and Levin 1990). Testosterone 7 a hydroxylation is specific for C Y P 2 A 1 and used as a marker for C Y P 2 A 1 in hepatic microsomes. C Y P 2 A 1 is a female predominant enzyme that is regulated mainly by sex steroids and growth hormone (GH) (Ryan and Levin 1993, Kato and Yamazoe 1993) and inducible by 3-methylcholanthrene (Matsunaga et al. 1988). It represents approximately 3% of total hepatic C Y P in adult male and 6% of total hepatic C Y P in adult female rats (Waxman et al. 1985). C Y P 2 A 2 hydroxylates testosterone predominantly at the 15a but also at the 15(3, 7a, 16a, 6(3 and 2a positions (Gonzalez 1990). C Y P 2 A 2 is a constitutively expressed male predominant enzyme, mainly regulated by testosterone and G H (Waxman et al. 1988). In humans, three enzymes of the C Y P 2 A subfamily have been identified, C Y P 2 A 6 , C Y P 2 A 7 , and C Y P 2 A 1 3 (Nelson et al. 1996). C Y P 2 A 6 appears to be the only C Y P 2 A enzyme expressed in human liver (Guengerich 1995) and is not expressed in fetal liver or in tissues other than the adult liver. Levels o f C Y P 2 A 6 found in the human liver amount to less than 1% o f the 3 Introduction total C Y P content and vary greatly among individuals (Wrighton and Stevens 1992). C Y P 2 A 6 catalyses coumarin 7-hydroxylation and this reaction can be used as a C Y P 2 A 6 specific marker (Guengerich 1995). C Y P 2 A 6 (and C Y P 2 E 1 ) can activate nitrosamines to mutagenic and cytotoxic metabolites (Guengerich 1995). C Y P 2 A 7 and C Y P 2 A 1 3 have been identified recently but are not well characterized, to date (Nelson et al. 1996). 1.1.3. The CYP2B Subfamily In male and female rats, several C Y P 2 B enzymes have been identified (Nelson et al. 1996). The most important enzymes in this subfamily are C Y P 2 B 1 and C Y P 2 B 2 , which share 97% amino acid sequence homology, are immunochemically crossreactive, and highly inducible in the liver by phenobarbital (Gonzalez 1989). In liver of untreated rats, C Y P 2 B 1 and C Y P 2 B 2 are minor constituents of the total C Y P content (1-2%) with constitutive levels of C Y P 2 B 1 five- to ten-fold lower than those of C Y P 2 B 2 (Waxman and Azaroff 1992). Purified C Y P 2 B 1 catalyzes demethylation of benzphetamine, demethylation of hexobarbital, and hydroxylation of testosterone at the 16a and 16(3 positions (Gonzalez 1989, Ryan and Lev in 1990). Hydroxylation of testosterone at the 16(3 position can be used as a marker for C Y P 2 B 1 in hepatic microsomes. C Y P 2 B 2 has a similar substrate profile but catalyzes the reactions at a 2- to 5-fold lower rate than C Y P 2 B 1 (Gonzalez 1989). C Y P 2 B 3 is constitutively expressed at a low level in the liver of male and female rats. It is not inducible by phenobarbital and is 77% similar in its amino acid sequence to those of C Y P 2 B 1 and C Y P 2 B 2 (Gonzalez 1989). Human CYP2B6 shares approximately 76% sequence homology with rat CYP2B1. It is expressed in human liver at levels lower than 2% of total C Y P content (Guengerich 1995). C Y P 2 B 6 can be induced in primary cultures of human hepatocytes with phenobarbital, dexamethasone, or rifampin (Chang et al. 1997b). 1.1.4. The CYP2C Subfamily The C Y P 2 C subfamily is the largest and most diverse subfamily. In rats, several C Y P 2 C enzymes have been identified (Nelson et al. 1996). CYP2C6, CYP2C7, CYP2C11, CYP2C12, and CYP2CI3 share 68% to 75% sequence homology (Gonzalez 1990). C Y P 2 C 7 , C Y P 2 C 1 1 , C Y P 2 C 1 2 , and C Y P 2 C 1 3 are not inducible by xenobiotics; C Y P 2 C 6 is modestly inducible by phenobarbital. C Y P 2 C 6 and C Y P 2 C 7 are expressed in both adult male and female rats; C Y P 2 C 1 1 and C Y P 2 C 1 3 are expressed in males only; C Y P 2 C 1 2 is expressed in females only (Ryan and Lev in 1993, Kato and Yamazoe 1993). Expression of C Y P 2 C 6 , C Y P 2 C 7 , C Y P 2 C 1 1 , 4 Introduction C Y P 2 C 1 2 , and C Y P 2 C 1 3 is developmentally regulated (Bandiera et al. 1986, Ryan and Levin 1990, 1993, Kato and Yamazoe 1993). The hepatic content o f C Y P 2 C 7 rises from less than 1% of total microsomal C Y P in immature rats to 7% and 14% in mature female and male rats (Bandiera et al. 1986, Gonzalez et al. 1986). The expression level of C Y P 2 C 7 is approximately twice as high in the liver of female rats in comparison to male rats (Ryan and Levin 1993). C Y P 2 C 6 exhibits no sex difference in the rat liver (Gonzalez et al. 1986). Expression of C Y P 2 C 7 , C Y P 2 C 1 1 , C Y P 2 C 1 2 , and CYP2C13 is regulated by G H , estradiol, and testosterone (Morgan et al. 1985, MacGeogh et al. 1985, Bandiera et al. 1986, Westin et al. 1990). Purified C Y P 2 C 7 hydroxylates testosterone only at the 16a position, whereas purified C Y P 2 C 1 1 hydroxylates testosterone mainly at the 2a and 16a positions and catalyses the metabolism of a number of drugs and xenobiotics (Ryan and Levin 1990). Hydroxylation of testosterone at the 2 a position can be used as a marker for CYP2C11 in hepatic microsomes. C Y P 2 C 1 2 is an important enzyme for corticosterone metabolism and responsible for the sexually dimorphic metabolism of steroids (Ryan and Levin 1990). The human C Y P 2 C enzymes, C Y P 2 C 8 , C Y P 2 C 9 , C Y P 2 C 1 8 , and C Y P 2 C 1 9 are not expressed in a gender specific manner (Wrighton and Stevens 1992) and are more than 80% identical with each other (Guengerich 1995). C Y P 2 C enzymes metabolize a variety of clinically important drugs including hypoglycemic, anticonvulsant, antimalarial, anticoagulant, antiulcer, anxiolytic, antidepressant, and antiinflammatory drugs (Goldstein and de Morais 1994). C Y P 2 C 1 8 is of minor clinical importance. C Y P 2 C 9 is probably the most abundant C Y P 2 C enzyme in human liver (Guengerich 1995). C Y P 2 C 9 and C Y P 2 C 1 9 are inducible by rifampicin and barbiturates (Goldstein and de Morais 1994). 7-hydroxylation of S-warfarin can be used as a marker for C Y 2 C 9 and 4'-hydroxylation of S-mephenytoin can be used as a marker for C Y P 2 C 1 9 in hepatic microsomes (Goldstein and de Morais 1994). C Y P 2 C 1 9 exhibits a genetic polymorphism in the human population in which 4'-hydroxylation of S-mephenytoin appears at two different levels. Two to five percent of the Caucasian population and twenty percent of the Oriental population are identified as poor metabolizers with respect to 4'-hydroxylation of S-mephenytoin (Henderson and W o l f 1992, Goldstein and de Morais 1994, Guengerich 1995). 1.1.5. The CYP3A Subfamily In the rat, the C Y P 3 A subfamily consists of several enzymes. In the past, only C Y P 3 A 1 and C Y P 3 A 2 were characterized. Results from recent studies indicate that at least three more C Y P 3 A enzymes exist, named C Y P 3 A 9 , C Y P 3 A 1 8 , and C Y P 3 A 2 3 , whose m R N A s have been found in 5 Introduction untreated male and female rats (Mahnke et al. 1997, Nelson et al. 1996). C Y P 3 A 2 3 might be an allelic variant of C Y P 3 A 1 (Mahnke et al. 1997). C Y P 3 A 1 and C Y P 3 A 2 are 89% similar in their amino acid sequences and immunochemically crossreactive (Gonzalez 1990). In untreated male and female rats, C Y P 3 A 1 is undetectable. C Y P 3 A 2 is expressed constitutively in immature and mature male rats and in immature but not mature female rats (Cooper et al. 1992, Wrighton and Stevens 1992). Dexamethasone and triacetyloleandomycin induce both C Y P 3 A 1 and C Y P 3 A 2 ; rifampicin and phenobarbital induce C Y P 3 A 1 to a higher level than C Y P 3 A 2 . 3-Methylcholanthrene induces C Y P 3 A 2 but has no effect on C Y P 3 A 1 (Cooper et al. 1992). Both C Y P 3 A 1 and C Y P 3 A 2 hydroxylate testosterone at the 2(3 and 6p positions (Gonzalez 1989). Hydroxylation of testosterone at the 2P and 6P position can be used as a marker for C Y P 3 A in hepatic microsomes. In humans, the C Y P 3 A subfamily consists of four closely related enzymes, C Y P 3 A 3 and C Y P 3 A 4 , C Y P 3 A 5 , and C Y P 3 A 7 . C Y P 3 A enzymes are the most abundant enzymes in the human liver. Their contribution to the total C Y P content in the liver is 20-60% (Guengerich 1990). C Y P 3 A 3 and C Y P 3 A 4 are the most abundant and most important C Y P 3 A enzymes in adult liver and responsible for the metabolism of many clinically important drugs such as corticosteroids, antifungal agents, macrolide antibiotics, and antineoplastic agents (Spatznegger and Jaeger 1995). C Y P 3 A 3 and C Y P 3 A 4 are 98% similar in their amino acid sequence and are also found in the kidneys and the intestines (Gonzalez 1989). C Y P 3 A enzymes are inducible by steroid antagonists, endogenous and synthetic glucocorticoids, macrolide antibiotics, various antifungal agents, rifampin, dexamethasone, and phenobarbital (Okey et al. 1989). C Y P 3 A 3 and C Y P 3 A 4 are inducible by rifampin, dexamethasone and phenobarbital (Wrighton and Stevens 1992). C Y P 3 A 5 is found only in 10-20% of adult and fetal livers examined (Gonzalez 1989). The substrate specificity of C Y P 3 A 5 is similar to that of C Y P 3 A 3 and C Y P 3 A 4 , but its metabolic capacity is limited (Wrighton and Stevens 1992). C Y P 3 A 7 is expressed in fetal liver only, where it contributes to 30-50% of the total hepatic C Y P content. C Y P 3 A 7 is absent in adult liver. It catalyzes the 16a-hydroxylation of dehydroepiandrosterone 3-sulfate (Wrighton and Stevens 1992). 1.2. Growth Hormone G H is a peptide hormone, consisting of 190-199 amino acids, depending on the species. The G H gene is approximately 80% and 92% identical to those of prolactin and somatomammotropin (placental lactogen), respectively (Miller and Eberhardt 1983). G H is secreted in a pulsatile 6 Introduction pattern from the somatotropes of the anterior pituitary. The secretion pattern is influenced by the central nervous system, hypothalamus, anterior pituitary, estrogen, and testosterone (Schlach and Reichlin 1966 and 1968, Jansson and Frohman 1987, Kerrigan and Rogol 1992, Strobl and Thomas 1994). Pulsatile G H secretion is caused by episodic increases and decreases in the secretion of GH-releasing hormone and GH-release inhibiting factor (somatostatin) (Locatelli et al. 1996). The pattern of G H release in rats is sexually dimorphic (Eden 1979, Jansson et al. 1985) and age-dependent (Eden 1979, Sonntag et al. 1980, Gabriel et al. 1992). In 22-day old rats, G H secretion is not sexually dimorphic and plasma G H levels do not exceed 70 ng/ml (Eden 1979). Starting at about 30 days of age, the G H secretion pattern changes in male and female rats. In adult male rats (> 45 days of age), G H is secreted in a pulsatile and intermittent pattern ("male pattern") with a periodicity o f approximately 3.3 hours or 7 pulses per day and trough levels below 1 ng/ml for 60 to 120 minutes (Tannenbaum and Martin 1976, Eden 1979). In adult female rats, the number of peaks per day is greater (1-2 pulses per 2 hours) and the troughs are less pronounced with G H levels never below 10-20 ng/ml (Eden, 1979, Waxman et a. 1991). Secretion o f G H in female rats is described as more continuous than pulsatile ("female pattern") (Figure 2). The most obvious manifestation of this sexual dimorphism is the higher growth rate of male rats versus female rats. The sexually dimorphic G H secretion pattern together with estrogen and testosterone regulates the sexually dimorphic expression of several hepatic C Y P enzymes (reviewed in Gustafsson et al. 1983, Waxman 1988, Shapiro et al. 1995). Females Males i — i — i — i — i — i — i i — i — i — i — i — i — i 9:00 12:00 15:00 9:00 12:00 15:00 Clock time Figure 2: G H secretion pattern in male and female rats (adapted from Eden 1979). 7 Introduction 1.3. Mechanism of Hormonal Regulation of CYP Enzymes In rats, several C Y P enzymes are expressed in a sexually dimorphic manner (Table 1, Figure 3). The expression levels of sex-dependent hepatic C Y P enzymes are controlled by the endocrine system including estrogen, testosterone, and G H (Gustafsson et al. 1983, Waxman 1988, Shapiro et al. 1995). The male predominant C Y P enzyme is C Y P 2 A 2 , which is expressed at a higher level in the liver of male rats, in comparison to female rats (Ryan and Lev in 1993). Male specific C Y P enzymes are C Y P 2 C 1 1 , C Y P 2 C 1 3 , and C Y P 3 A 2 (Ryan and Lev in 1993), which are expressed in adult male rats only. Female predominant C Y P enzymes are C Y P 2 A 1 (Waxman et al. 1989a), C Y P 2 C 7 (Bandiera et al. 1986), and C Y P 2 E 1 (Waxman et al. 1989b). Although hepatic levels of C Y P 1 A 2 in adult female rats have been reported to be slightly higher in comparison to adult male rats (Katamaki et al. 1993), C Y P 1 A 2 is not regarded as a female predominant C Y P enzyme (Ryan and Levin 1993). The female specific C Y P enzyme is C Y P 2 C 1 2 (MacGeoch et al. 1984). The sexually dimorphic expression of C Y P enzymes leads to differences in the ability of male and female rats to metabolize endogenous compounds and xenobiotics. In humans, the sexually dimorphic expression of C Y P enzymes is not apparent, because differences in hepatic levels of C Y P enzymes among individuals are generally greater than differences between genders. The reasons are greater differences of exposure to C Y P inducing compounds and a higher variability in the genetic makeup of humans in comparison to controlled outbred and inbred rat strains. Table 1: Sexual dimorphic expression of hepatic C Y P enzymes in the rat. Male Specific CYP Enzymes Male Predominant CYP Enzymes C Y P 2 C 1 1 C Y P 2 A 2 C Y P 2 C 1 3 C Y P 3 A 2 Female Specific CYP Enzymes Female Predominant CYP Enzymes C Y P 2 C 1 2 C Y P 2 A 1 C Y P 2 C 7 C Y P 2 E 1 Specific enzymes are expressed in one sex only, predominant enzymes are expressed in both sexes at different levels. 8 Introduction 0.4 -"53 Figure 3: Hepatic microsomal C Y P expression levels in adult rats (adapted from Ryan and Levin 1993). Several studies indicate that in rat liver, the male specific G H secretory pattern induces C Y P 2 C 7 (Westin et al. 1990) and CYP2C11 (Shapiro et al. 1989, Waxman et al. 1991, Katamaki et al. 1985, Morgan et al. 1985, McClellan-Green et al. 1989). It suppresses C Y P 2 A 1 (Waxman et al. 1989a), C Y P 2 B 1 , C Y P 2 B 2 (Yamazoe et al. 1987), and C Y P 2 E 1 (Waxman et al. 1989a). The female-specific G H secretory pattern induces C Y P 2 A 1 (Pampori and Shapiro 1996), C Y P 2 C 7 (Westin et al. 1990, Pampori and Shapiro 1996), and C Y P 2 C 1 2 (MacGeoch et al. 1984 and 1985, Pampori and Shapiro 1996). It suppresses, C Y P 2 B 1 , C Y P 2 B 2 (Yamazoe et al. 1987), C Y P 2C11 (Morgan et al. 1985, Pampori and Shapiro 1996), and C Y P 2 E 1 (Waxman et al. 1989b). The role of G H in the regulation of C Y P 2 A 2 , C Y P 2 C 1 3 , and C Y P 3 A 2 is controversial as in male rats hypophysectomy increases hepatic levels of C Y P 2 A 2 (Waxman et al. 1988), C Y P 2 C 1 3 (McClellan-Green et al. 1989) and 3A2 (Waxman et al. 1988) but monosodium aspartate ( M S A ) treatment decreases hepatic levels of C Y P 2 A 2 , C Y P 2 C 1 3 , and C Y P 3 A 2 (Agrawal and Shapiro 1997). To summarize, numerous studies indicate that the sexually dimorphic expression of C Y P 2 C 1 1 , C Y P 2 C 7 , and C Y P 2 C 1 2 is regulated primarily by G H , whereas the sexually 9 Introduction dimorphic expression of C Y P 2 A 2 , C Y P 2 C 1 3 , and C Y P 3 A 2 is regulated by G H and other contributing factors including thyroxine (T4) (Waxman et al. 1989b) and sex steroids. The sexually dimorphic expression of C Y P 2 B 1 , C Y P 2 B 2 , and C Y P 2 E 1 is not regulated by G H , but G H suppresses these enzymes in both sexes (Table 2). The critical event that triggers G H -mediated C Y P enzyme expression is believed to be the trough interval period between G H pulses with a G H concentration close to zero for 2.5 h rather than the mean G H plasma concentration, the number of peaks, or the peak height as shown for C Y P 2 C 1 1 (Shapiro et al. 1989, Waxman et al. 1991). The regulation of sex specific expression of C Y P 2 C 7 , C Y P 2 C 1 1 , C Y P 2 C 1 2 , and C Y P 2 C 1 3 by G H occurs at the level of transcript initiation (Pampori and Shapiro 1996). The mechanism by which the pattern of G H release regulates sex specific C Y P enzyme expression has been proposed to involve the signal transducer/activator of transcription (STAT) 5 signal transduction pathway (Udy et al. 1997). In response to the male specific G H secretion pattern the S T A T 5 b peptide is tyrosine phosphorylated, then serine/threonine phosphorylated. It dimerizes and translocates from the cytosol to the nucleus where it exhibits D N A binding activity and activates the transcription of a number of sexually dimorphic genes (Waxman et al. 1995, Gebert et al. 1997, Ram and Waxman 1997). Table 2: Regulation of hepatic C Y P enzyme expression in the rat by G H . Male Specific GH Secretion Pattern induces suppresses C Y P 2 A 2 c C Y P 3 A C C Y P 2 A l a , b ' c C Y P 2 C 1 3 a C Y P 2 C 7 a C Y P 2 A 2 a ' b C Y P 2 E 1 a , b C Y P 2 C 1 1 a ' b > 0 C Y P 2 B 1 a > b C Y P 3 A a , b C Y P 2 C 1 3 c C Y P 2 B 2 a ' b Female Specific GH Secretion Pattern induces suppresses C Y P 2 A 1 c C Y P 2 A 2 a ' b C Y P 2 C 1 3 b C Y P 2 A 2 c C Y P 2 B 1 a - b C Y P 2 E 1 b C Y P 2 C 7 a ' b C Y P 2 B 2 a ' b C Y P S A 3 - " C Y P 2 C 1 2 b ' c ' d C Y P 2 C 1 1 a a : in hypophysectomized male rats, b: in hypophysectomized female rats, c: in M S A treated rats, d : in intact male rats. 10 Introduction Estrogens and androgens can alter the pituitary secretion of G H (Kerrigan and Rogol 1992, Painson et al. 1992) and regulate hepatic expression of several C Y P enzymes (Mode and Norstedt 1982). These effects of sex steroids on hepatic C Y P enzymes have been shown for C Y P 2 A 1 (Dannan et al. 1986, Waxman et al. 1989a), C Y P 2 A 2 (Waxman et al. 1988), C Y P 2 C 7 (Bandiera and Dworschak 1992), CYP2C11 (Waxman et al. 1985, Morgan et al. 1985, Cadario et al. 1992, Bandiera and Dworschak 1992, Chang and Bellward, 1996, Anderson et al. 1997), C Y P 2 C 1 2 (Waxman et al. 1985, MacGeoch et al. 1985) C Y P 2 C 1 3 (McClellan-Green, et al. 1989), C Y P 2 E 1 (Waxman et al. 1989a), and C Y P 3 A (Waxman et al. 1988, Ribeiro and Lechner 1992, Anderson et al. 1997) (Table 3). Table 3: Regulation of hepatic C Y P enzyme expression in the rat by sex steroids. Estradiol Testosterone induces suppresses induces suppresses C Y P 2 A 1 " C Y P 2 C 1 1 C C Y P 2 A 2 a C Y P 2 A 1 a C Y P 2 C 7 b C Y P 2 E 1 b C Y P 2 C 7 b ' c C Y P 2 C 1 2 b C Y P 2 C 1 2 a C Y P 2 C 1 1 a - b C Y P 2 E 1 a C Y P 2 C 1 3 a ' b C Y P 3 A a a : in neonatally castrated male rats, b : in neonatally ovarectomized female rats, °: in intact male rats. NADPH-dependent C Y P reductase is also hormonally controlled in a manner distinct from that of C Y P enzyme expression indicating that hepatic metabolism is regulated not only by C Y P enzyme expression but also by regulation of enzyme activity. In hepatic microsomes from hypophysectomized adult male and female rats, hepatic microsomal C Y P reductase levels are decreased to approximately 50% and 75%, respectively, of their original levels in comparison to sham-operated rats. Protein levels of C Y P 3 A and C Y P 2 A 2 increased 2-fold but their associated testosterone 6P- and 15a- hydroxylase activities decreased by 35%. In hepatic microsomes from hypophysectomized female rats, protein levels of C Y P 3 A and C Y P 2 A 2 increased more than ten-fold but the testosterone 6(5- hydroxylase activity increased only two-fold. N o change was observed in the testosterone 15a- hydroxylase activity. Enzyme activities increased by administration of C Y P reductase or T 4 (Waxman et al. 1989b). These results indicate that discrepancies between effects of hypophysectomy on rat hepatic microsomal C Y P activities and protein levels might be explained by changes in C Y P reductase levels and that thyroid hormones 11 Introduction might have a regulatory influence of C Y P reductase expression. In conclusion, each sexually dimorphic expressed C Y P enzyme is regulated in a unique manner and although G H plays a major role in the regulation of many C Y P enzymes, the hepatic expression of many C Y P enzymes is influenced by multiple hormones. In the following chapters, the hormonal regulation of C Y P enzymes investigated in the present study is described in more detail. Hypophysectomy and neonatal administration of monosodium glutamate ( M S G ) or monosodium aspartate ( M S A ) have been used to investigate the effects of pituitary hormones on hepatic expression of C Y P enzymes. Hypophysectomy prevents the release of corticotropin, follicle stimulating hormone, luteinizing hormone, thyroid stimulating hormone, G H , and prolactin. The treatment has been used to study the effects of G H suppression on hepatic C Y P enzyme levels in rats although several hormones other than G H are affected. Neonatal administration of M S G or M S A selectively destroys G H releasing hormone secreting neurons in the arcuate nucleus o f the hypothalamus, which results in a profound suppression of G H release (Terry et al. 1981). The effects of M S G and M S A on the hypothalamus are thought to be more GH-specific than hypophysectomy. 1.3.1. H o r m o n a l Regulation of CYP2A1 C Y P 2 A 1 is present in the liver o f prepubertal male and female rats at similar levels. During the post-suckling period, C Y P 2 A 1 levels decline in male rats by approximately 60-70% (Waxman et al. 1985). In adult male rats, hypophysectomy elevates hepatic C Y P 2 A 1 levels to prepubertal levels. C Y P 2 A 1 levels can be elevated further almost to the level o f control female rats by administration of human G H mimicking the female G H secretion pattern (Waxman et al. 1989a). Administration of human G H mimicking the male G H secretion pattern reduces C Y P 2 A 1 levels but not to that of untreated male rats (Waxman et al. 1989a). In adult female rats, hypophysectomy has no effect on hepatic C Y P 2 A 1 levels, but C Y P 2 A 1 expression can be elevated by administration of G H mimicking the female G H secretion pattern (Waxman et al. 1989a). Administration of G H mimicking the male G H secretion pattern reduces C Y P 2 A 1 levels in adult female rats (Waxman et al. 1989a). C Y P 2 A 1 levels are doubled in male rats and elevated by 50% in female rats treated with M S G during the neonatal period (Waxman et al. 1990) contradicting previous results in hypophysectomized female rats (Waxman et al. 1989a). In neonatally or post-pupertally castrated male rats, hepatic C Y P 2 A 1 levels increase approximately 2.5-fold during adulthood, and decrease back to control male levels after 12 Introduction administration of testosterone at physiological concentrations during adulthood (Waxman et al. 1989a). In neonatally ovariectomized female rats, hepatic C Y P 2 A 1 levels are decreased, relative to intact female rats, to levels similar to neonatally castrated male rats (Waxman et al. 1989a). C Y P 2 A 1 expression can be restored to levels of intact female rats by administration of estradiol at physiological concentrations during adulthood (Waxman et al. 1989a). Peripubertal testosterone treatment has no effect on C Y P 2 A 1 levels in prepubertally ovariectomized adult female rats (Anderson et al. 1998). These results indicate that the hepatic level of C Y P 2 A 1 is positively regulated by the female specific G H secretion pattern and estrogen and negatively regulated by the male specific G H secretion pattern and testosterone. 1.3.2. Hormonal Regulation of CYP2B1 and CYP2B2 C Y P 2 B 1 and C Y P 2 B 2 are expressed constitutively in the rat liver at very low levels and can be induced by phenobarbital-type inducers. C Y P 2 B 1 and C Y P 2 B 2 are not expressed in a sexually dimorphic manner, but hypophysectomy increases hepatic levels of both enzymes in male and female rats. In hypophysectomized male rats hepatic C Y P 2 B 1 levels increase 58-fold and C Y P 2 B 2 levels increase 14- fold. In hypophysectomized female rats hepatic C Y P 2 B 1 levels increase 118- and C Y P 2 B 2 levels increase 30-fold (Yamazoe et al. 1987). This effect has been reported in Sprague-Dawley rats (Yamazoe et al. 1987) and Wistar-Furth rats (Larsen et al. 1994) , but not in Fischer 344 rats (Larsen et al. 1994) suggesting a strain-dependent effect. Administration of human G H mimicking the male or the female specific G H secretion pattern to hypophysectomized or MSG-treated rats decreases the hepatic C Y P 2 B 1 and C Y P 2 B 2 content (Yamazoe et al. 1987, Shapiro et al. 1994). The greatest suppression of C Y P 2 B 1 was achieved by continuous G H infusion (Yamazoe et al. 1987). G H inhibits the inductive effects of phenobarbital on C Y P 2 B enzymes, but does not suppress constitutive levels o f C Y P 2 B 1 and C Y P 2 B 2 (Shapiro et al. 1994). The response of phenobarbital induction of C Y P 2 B 1 is also sexually dimorphic and dependent on sex steroids. Prepubertal ovariectomy enhances the inducibility by phenobarbital o f hepatic C Y P 2 B 1 but not C Y P 2 B 2 , whereas postpubertal administration of testosterone enanthate has no effect on the inducibility of C Y P 2 B 1 or C Y P 2 B 2 in prepubertal ovariectomized rats (Chang et al. 1997a). Castration decreases the inducibility of C Y P 2 B 1 and C Y P 2 B 2 in male rats and this effect can be reversed by administration of testosterone exogenously (Larsen et al. 1995) . 13 Introduction These results indicate that C Y P 2 B 1 and C Y P 2 B 2 are both suppressed by G H . Estradiol suppresses the inducibility of phenobarbital on hepatic C Y P 2 B 1 and testosterone enhances the inducibility of phenobarbital on hepatic C Y P 2 B 1 . 1.3.3. Hormonal Regulation of CYP2C11 In male rats, hypophysectomy and M S G treatment reduce hepatic levels of C Y P 2 C 1 1 (Katamaki et al. 1985, Morgan et al. 1985, McClellan-Green et al. 1989, Waxman et al. 1991, Shapiro et al. 1989). G H infusions mimicking the female pattern of G H secretion reduce the activity of C Y P 2 C 1 1 in the liver of hypophysectomized male rats (Morgan et al. 1985). G H injections mimicking the male pattern of G H secretion restore the activity o f C Y P 2 C 1 1 (Waxman et al. 1991) and its m R N A levels (McClellan-Green et al. 1989) to normal male levels in the liver of hypophysectomized male rats. Neonatal gonadectomy reduces hepatic levels of C Y P 2 C 1 1 in adult male rats (Katamaki et al. 1985, Waxman et al. 1985 and 1988, Dannan, et al. 1986, Bandiera and Dworschak 1992). The effect o f neonatal gonadectomy on C Y P 2 C 1 1 can be reversed by administration of testosterone during puberty (Cadario et al. 1992) and adulthood (Bandiera and Dworschak 1992). In prepubertally ovariectomized rats treated with testosterone during puberty, C Y P 2 C 1 1 levels are increased in adult rats similar to those found in normal male rats (Dannan et al. 1986, Chang and Bellward 1996, Anderson et al. 1998). This increase was no longer observed 80 days after treatment indicating that the effect of testosterone on C Y P 2 C 1 1 is an inductive effect rather than an imprinting effect (Anderson et al. 1998). Neonatal estrogen treatment reduced C Y P 2 C 1 1 expression in adult male rats to a similar extent as neonatal castration (Bandiera and Dworschak 1992). These results indicate that the hepatic level o f C Y P 2 C 1 1 is positively regulated by the male specific G H secretion pattern and testosterone and negatively regulated by the female specific G H secretion pattern and estrogen. 1.3.4. Hormonal Regulation of C Y P 3 A C Y P 3 A 1 and C Y P 3 A 2 are 89% similar in their amino acid sequences and immunochemical^ crossreactive. A specific catalytic reaction that could be used to distinguish between activities of both enzymes has not been found. Results from recent studies indicate that at least three more isoforms exist, C Y P 3 A 9 , C Y P 3 A 1 8 , and C Y P 3 A 2 3 . Therefore, studies on the regulation of C Y P 3 A 1 and C Y P 3 A 2 are not specific for either enzyme. In the following, 14 Introduction statements regarding the hormonal regulation of C Y P 3 A enzymes refer to all enzymes in that subfamily, although this might be an incorrect generalization. In hypophysectomized adult rats, hepatic levels of C Y P 3 A are increased two-fold in male rats and 10- to 20-fold in female rats (Waxman et al. 1988 and 1989b). The effect can be partially reversed in male rats by administration of G H mimicking the male G H secretion pattern, and completely reversed in female rats by administration of G H mimicking the female G H secretion pattern (Waxman et al. 1988). Administration of M S A to male rats suppresses C Y P 3 A expression (Agrawal and Shapiro 1997) contradicting previous findings in hypophysectomized rats (Waxman et al. 1988). In hypophysectomized adult male and female rats C Y P 3 A activities are increased by administration o f C Y P reductase or thyroid hormone (Waxman etal. 1989b). In neonatally castrated adult rats, hepatic C Y P 3 A levels are reduced (Waxman et al. 1988, Riberio and Lechner 1991). In neonatally ovariectomized adult rats hepatic C Y P 3 A levels are unchanged (Waxman et al. 1988). In prepubertally ovariectomized rats treated with testosterone during puberty, C Y P 3 A levels are increased in adult rats 41 days after treatment. This increase was no longer observed 80 days after treatment indicating that the effect of testosterone on C Y P 3 A enzymes is an inductive effect rather than an imprinting effect (Anderson et al. 1998). These results indicate that the hepatic level of C Y P 3 A is positively regulated by testosterone and negatively regulated by the male and female specific G H secretion pattern. 15 Introduction 1.4. Tamoxifen Tamoxifen or Z l-(4-(2-dimethylamino-ethoxy)phenyl)-l,2-di-phenyl-l-butene (Nolvadex®, Zeneca Pharmaceuticals, Wilmington, DE) is a nonsteroidal antiestrogen with a triphenylethylene structure that is distinct from the steroid structure o f the naturally occurring estrogens (Figures 4 and 5). Tamoxifen binds to the estrogen receptor with a binding affinity approximately 2% of that of estradiol (Robertson et al. 1982) and triggers the signal transduction cascade only in certain tissues of some species. Depending on the species or target organ, it can display the whole spectrum of biological activities from full agonist, through partial agonist, to full antagonist. For example, tamoxifen is a potent estrogen antagonist in rat uterus but is a full agonist in the mouse uterus. In humans, tamoxifen exhibits both estrogen agonistic and antagonistic properties, although tamoxifen is conventionally described as an antiestrogen. It acts as a pure estrogen antagonist in the human mammary gland but exhibits mixed estrogenic and antiestrogenic properties in the human uterus (Jordan 1984). The target site specificity is suspected to be caused by the distribution of two different estrogen receptors (ERoc and E R P ) in these tissues (Jordan 1984, MacGregor and Jordan 1998). Figure 4: Chemical structure of tamoxifen Figure 5: Chemical structure of estradiol 1.4.1. Mechanism of Tamoxifen's Action The major mechanism of tamoxifen's action is competition with estrogen for binding to the estrogen receptor (Jordan 1990) leading to a reversible blockade of breast cancer cells at the Gj phase of the cell cycle (Osborne et al. 1983). Thus, tamoxifen is able to inhibit the proliferative stimulation of estrogen in the mammary gland. Tumors may regress because of the altered balance between cell proliferation and cell loss. Tamoxifen may also directly induce apoptosis (Ellis et al. 1997). Tamoxifen inhibits cell growth in a hepatoma cell line lacking the estrogen 16 Introduction receptor and tamoxifen therapy is effective in women with estrogen receptor free tumors, though to a lesser degree. These findings led to speculation about an estrogen receptor independent mechanism of action for tamoxifen (Love 1989, Shun-Yuan et al. 1995). Alternatively, the so called estrogen-receptor free tumors might only have a lower concentration of estrogen receptors, as consistent cutoff values for classifying a tumor as being estrogen receptor negative do not exist. In some laboratories, a tumor in which 20 percent of cells have estrogen receptors are still designated as estrogen receptor negative (Osborne 1998). 1.4.2. Tamoxifen in Breast Cancer Therapy Breast cancer is the most common cancer among women and the leading cause of death among 40- to 50-year old women in North America. In the United States, 12% of women are diagnosed with breast cancer and 3.5% o f all women wi l l die o f this disease (Harris et al. 1992a). This amounts to approximately 46,000 women in the United States per year (Marshall 1993). The incidence of breast cancer has been increasing steadily in North America over the last 50 years but surprisingly, the mortality rate has remained relatively constant (Marshall 1993). Because of the growth stimulating effect of estrogen in mammary gland and its potential stimulus for breast cancer development, antiestrogen therapy is effective in decreasing the risk of developing breast cancer as shown in several clinical trials (Carlson 1997, Osborne 1998). The estrogenic and antiestrogenic effects of tamoxifen were first described in 1966 by Harper and Walpole (Harper and Walpole 1966 and 1967). Today, tamoxifen has become the leading drug in the management of breast cancer. It is used as adjuvant therapy after surgical removal o f the primary tumor and radiation therapy. Tamoxifen adjuvant therapy is especially beneficial in postmenopausal women with tumors that are estrogen positive, are confined to the breast, and have not spread to the axillary lymph nodes (Harris et al. 1992b). In a meta-analysis conducted by the Early Breast Cancer Trialists Collaborative Group (1988) tamoxifen treatment was associated with prolongation of disease free survival, decreased metastases rates, and reduction of mortality after a follow-up of about 10 years. The annual reductions in cancer recurrence and death were 26-39% and 14%, respectively, as compared to a placebo. Cl inical trials revealed that patients have a reduced risk of developing a new cancer in the opposite breast while on tamoxifen therapy (Harris et al. 1992b). Tamoxifen does not destroy tumor cells and tumors regrow in animals treated with estradiol. In humans, tumors regrow after tamoxifen therapy is stopped (Love 1989, Osborne et al. 1985, Gottardis et al. 1988). Therefore, it was suggested in the past that tamoxifen therapy, once 17 Introduction initiated, should be given indefinitely (Love 1989, Fisher et al. 1987, Tormey and Jordan 1984). Older clinical data indicated that long-term tamoxifen therapy for five or ten years offered a greater therapeutic advantage over short-term treatment (Early Breast Cancer Trialists Collaborative Group 1988, Fisher et al. 1989, Love 1989). Tamoxifen can also act as a growth-stimulating agent in secondary mammary gland tumors (Jordan 1992, Marx 1993). A possible explanation of this phenomenon is the occurrence of micro-metastases that are "super-sensitive" to the estrogen agonist effect of tamoxifen. Tamoxifen resistant metastases have been found after tamoxifen therapy (Osborne 1998). These results have led to the current recommendation to stop tamoxifen therapy after five years of treatment and to switch to therapy with a pure antiestrogen (Walkeling 1991, Tornetti and Jordan 1996) or with an aromatase inhibitor (Brodie 1994). Clinical investigations also evaluated the use of tamoxifen as a preventive treatment in healthy women with increased risk for breast cancer. Preliminary data from a five year clinical study including 13,388 women suggest that tamoxifen reduces the incidence of invasive cancer by 49% in healthy women at high risk of developing this disease (Fisher et al. 1998). Because many women with breast cancer and healthy women on a tamoxifen preventive therapy will live for several decades after treatment has begun, the extended use of tamoxifen requires careful analysis and consideration of potential unfavorable pharmacological and toxicological consequences. The recommended daily dosage of tamoxifen for breast cancer therapy is 10 or 20 mg twice daily (Jordan 1990). The drug is readily absorbed after oral administration and accumulates to steady state levels within three to five weeks of continual treatment. The serum half-life of tamoxifen in humans is seven to fourteen days and this relatively long half-life is partly caused by binding to various proteins (Lemer and Jordan 1990, Lien et al. 1991 and 1995, Fuchs et al. 1996). 1.4.3. Metabolism of Tamoxifen Tamoxifen is metabolized extensively in mice, rats, rhesus monkeys and humans to N-desmethyltamoxifen, 4-hydroxytamoxifen, 4'-hydroxytamoxifen, tamoxifen-Af-oxide, 4-hydroxy-Af-desmethyl-tamoxifen, metabolite Y (a primary side chain alcohol), 3,4-dihydroxytamoxifen, metabolite E (O-dealkylated tamoxifen), A^Af-didesmethyltamoxifen, a-hydroxytamoxifen, ct-hydroxy-tamoxifen-A7-oxide and other minor metabolites (Foster et al. 1980, Jordan 1984, McCague and Seago 1986, Robinson and Jordan 1988, Robinson et al. 1991, Lien et al. 1991 and 1995, Poon et al. 1993, Fried and Wainer 1994) (Figure 6). The contribution of the four 18 Introduction major tamoxifen metabolites (i.e. A^-desmethyltamoxifen, 4-hydroxytamoxifen, 4'-hydroxytamoxifen, and tamoxifen-A /-oxide) to the overall metabolism differs among species (Table 4). Tamoxifen metabolites are excreted through the bile after Phase II metabolism. Tamoxifen is not excreted as the parent compound (Poon et al. 1993). 4-Hydroxytamoxifen is a highly active metabolite with a approximately 140 times greater affinity for the estrogen receptor than tamoxifen. A^-Desmethyltamoxifen has a similar affinity to the estrogen receptor as tamoxifen and can accumulate in humans to concentrations about 50 % greater than tamoxifen after chronic treatment (Robertson et al. 1982, Fuchs et al. 1996). In humans, tamoxifen is metabolized to Af-desmefhyltamoxifen by C Y P 3 A 4 (Jacolot et al. 1991, Man i et al. 1993a, Wiseman and Lewis 1996), the same enzyme that metabolizes wafarin. Therefore, tamoxifen and warfarin compete for the same enzyme binding site, which leads to elevated blood levels of warfarin i f both drugs are co-administered and may lead to life-threatening bleeding in patients receiving both drugs (Lodwick et al. 1987). The metabolism of tamoxifen can also be inhibited by erythromycin, cyclosporine, nifedipine, and diltiazem (Michalets 1998). Table 4: Metabolism of tamoxifen in mouse, rat, and human. Metabolite Contribution Enzyme (References) (Lim et al. 1994) Mouse Af-desmefhyltamoxifen 16 % ? 4-hydroxytamoxifen 14 % ? tamoxifen-N-oxide 68 % F M O s (Mani etal. 1993b) 4'-hydroxytamoxifen 1 % Rat N-desmethyltamoxifen 36 % C Y P 3 A , C Y P 1 A 2 , C Y P 2 C 1 1 , C Y P 2 C 6 (Mani etal. 1993a) 4-hydroxytamoxifen 39 % C Y P 1 A 2 ? (Mani etal. 1993a) tamoxifen-A /-oxide 15 % F M O s (Mani etal. 1993b) 4'-hydroxytamoxifen 9.5 % ? Human Af-desmethyltamoxifen 74 % C Y P 3 A (Jacolot et al. 1991, Man i et al. 1993 a, Wiseman and Lewis 1996). 4-hydroxytamoxifen 11 % C Y P 2 C 8 , C Y P 2 C 9 , C Y P 2 D 6 (Daniels et al. 1992, Wiseman and Lewis 1996, Dehal and Kupfer 1997) tamoxifen-A'-oxide 11 % F M O s (Mani etal. 1993b) 4-hydroxytamoxifen 4 % ? The question mark indicates that the enzyme has not been clearly identified. 19 Introduction OH metabolite Z Figure 6: Metabolites of tamoxifen in rats and humans (adapted from Jordan 1984). 20 Introduction 1.4.4. Toxicity of Tamoxifen Tamoxifen exhibits an estrogenic effect in the reproductive tract of rodents and humans. Therefore, administration of tamoxifen includes a risk of promoting tumors in ovaries and endometrium o f women on long-term tamoxifen therapy. Clinical studies reveal that among 8,322 woman under long-term tamoxifen therapy, 50 tamoxifen-treated and 9 untreated woman developed an endometrial tumor (reviewed in Carlson 1997). The development of tumors can be initiated by D N A binding of tamoxifen 3,4- and 3'4'- epoxides. Tamoxifen epoxides bind to calf thymus D N A to form D N A adducts (Davis et al. 1995). Epoxidation of tamoxifen is catalyzed by peroxidases, found in high concentration in the endometrium. In addition to the initiation of tumors by tamoxifen, tumor growth can be promoted by the estrogenic effect of tamoxifen in the endometrium. Tamoxifen is a potent carcinogen in rat liver, which has led to concerns about its use in humans. Administration o f tamoxifen at 45.2 mg/kg/day to six-week-old female Sprague-Dawley rats by daily oral gavage for one year led to the development of hepatoadenomas in 50% and hepatocarcinomas in 75% of all rats (Williams et al. 1993). However, epidemiological data do not indicate that tamoxifen treatment leads to an elevated risk of the development of hepatocarcinomas in women on long-term tamoxifen therapy and no other tamoxifen related carcinogenic effects were found (Carlson 1997). Mechanistic studies on the metabolism of tamoxifen showed that a-hydroxytamoxifen is the reactive intermediate responsible for DNA-adducts and for initiating hepatocarcinogenesis in rats (Potter et al. 1994, Jarman et al. 1995, Phillips et al. 1994, Randerath et al. 1994a and 1994b, Osborne et al. 1996). Subsequent 4-hydroxylation of a-hydroxytamoxifen is thought to stabilize this reactive metabolite by electron donation (Potter et al. 1994). a-Hydroxytamoxifen is found at very low concentrations in plasma from mice, rats, and women undergoing long-term tamoxifen therapy (Poon et al. 1995, Phillips et al. 1996). In contrast to rats, 4-hydroxylation of tamoxifen is a minor metabolic pathway in humans (Table 4). The stabilizing effect on a-hydroxytamoxifen is less pronounced in humans compared to rats. Therefore, the development of liver tumors after tamoxifen administration is specific to rats and the hepatocarcnogenic potency of tamoxifen in rats should not be extrapolated to humans. 21 Introduction 1.4.5. D i rec t Ef fects o f T a m o x i f e n on C Y P E n z y m e s Hepatic protein, mRNA, and activity levels of CYP2B1, CYP2B2, CYP3A1, epoxide hydroxylase, and several Phase II enzymes are induced in rats within one to three days after tamoxifen treatment (White et al. 1993, Nuwaysir et al. 1995 and 1996, Hellriegel et al. 1996). Administration of tamoxifen for four days at a dosage of 45 mg/kg induces CYP2B1, CYP2B2, and CYP3A enzymes two to three-fold relative to control rats 24 h after treatment. Twenty days after treatment, CYP2B and CYP3A associated pentoxy-, benzyloxy-, and ethoxyresorufin-O-deethylase activities and testosterone 2p-, 6P-, and 16a-hydroxylase activities returned back to control levels, demonstrating a transient, fully reversible, and dose-dependent direct inductive effect of tamoxifen on CYP2B and CYP3A enzymes (White et al. 1993) (Figure 7). 400 c 0) •4-1 c — 5 2 $ ° <D O > ^ 4-1 300 H 200 A 100 + " Tamoxifen (45 mg/kg) F i g u r e 7: Short-term effects of tamoxifen on C Y P enzymes in rats (adapted from White et al. 1993). 1.4.6. E f fects o f T a m o x i f e n on G H Secret ion Tamoxifen inhibits G H release in lamb pituitary cell cultures (Malaab et al. 1992). Tamoxifen has also been reported to suppress G H secretion in female rats to baseline levels (1.2 ng/ml) and to reduce G H secretion in male rats up to seven weeks after administration (Tannenbaum et al. 1992) (Figure 8). Therefore, tamoxifen exhibits a long-term effect on G H secretion pattern in both sexes that might influence the expression level of C Y P enzymes that are controlled by GH. In humans, tamoxifen diminishes the G H level in teenaged boys, three to four days after treatment with 10 mg tamoxifen for four days (Metzger et al. 1994) and decreases blood levels of insulin-like growth factor I (reviewed in Pollak et al. 1992). Insulin-like growth factor I is released after G H secretion by the liver and other tissues. 22 Introduction 9 200 1S0 100 50 0 Peanut Oil Peanut Oil 18 hours pott Tamoxifen 2 weeks post Tamoxifen 200 r ISO ioo H so 7 weeks post Tamoxifen 1000 1100 1200 1300 1400 1S0O 1600 Time (hours) C 200 r w 100 h V o so J 200 0 ISO o « mo 1 so 200 150 100 SO 16 noun post Tamoxifen 1 1 week post Tamoxifen 7 weeks post Tamoxifen 1000 1100 1200 1300 1400 1500 1600 Time (hours) Figure 8: Effects of tamoxifen on GH plasma levels in rats (adapted from Tannenbaum et al. 1992). 23 Introduction 1.5. Hypotheses The hypotheses of my research project are based on the findings that tamoxifen treatment has a prolonged suppressive effect on G H secretion in rats (Tannenbaum et al. 1992). G H and sex steroids regulate the expression level of several C Y P enzymes (Gustafsson et al. 1983, Shapiro et al. 1995). Estrogen and testosterone also contribute to the sexual dimorphism of the G H secretion pattern (Jansson et al. 1984). Therefore, treatment with the partial antiestrogenic drug tamoxifen might have a prolonged effect on the expression levels of hormonally regulated hepatic C Y P enzymes (e.g. C Y P 2 C 1 1 , C Y P 2 A 1 ) in the rat, which is different from the direct short term effect o f tamoxifen on C Y P 2 B and C Y P 3 A enzymes (White et al. 1993). The effect on hormonally regulated C Y P enzymes might be mediated by the prolonged effect o f tamoxifen on plasma G H levels or by tamoxifen or a tamoxifen metabolite that is present in the rat plasma for a prolonged time. The hypotheses of this research project are: 1. Administration of tamoxifen results in long-term alterations in hepatic levels of hormonally regulated C Y P enzymes and the metabolic reactions they catalyze. 2. Administration of tamoxifen results in a prolonged suppression of plasma G H levels. 3. Tamoxifen and its metabolites are eliminated from rat plasma 36 days after treatment. The rat is the experimental model in the experiments outlined in this proposal and the following enzymes are examined for alterations in their hepatic activity and content: C Y P 2 A 1 , C Y P 2 B 1 , C Y P 2 B 2 , C Y P 2 C 1 1 , and C Y P 3 A . 1.6. Objectives The immediate objective of this research project is to investigate the influence o f tamoxifen administration on plasma G H levels and expression of C Y P enzymes in rat liver. The long-term objective is to generate information about the mechanism by which hormones change the content of specific C Y P enzymes in liver. The specific objectives are: 1. Define a dose-response for the effects of tamoxifen administration on hepatic C Y P enzyme expression in the rat. 2. Define a dose-response for the effects of tamoxifen administration on plasma G H levels in the rat. 3. Synthesize two of the major tamoxifen metabolites that are commercially unavailable, i.e. N-desmethyltamoxifen and tamoxifen-A^-oxide. 4. Develop an HPLC-assay for separation, detection, and quantitation of tamoxifen and its major metabolites in plasma of tamoxifen-treated rats. 24 Experimental 2. EXPERIMENTAL 2.1. Chemicals Chemicals were obtained from the following sources: Aldrich Chemical Company, (Milwaukee, WIS, USA): Phenyl chloroformate and 2,2,2-trichloroethyl chloroformate. American Scientific & Chemical (Seattle, WA, USA): Ammonium hydroxide. Ammersham Pharmacia Biotech UK Ltd. (Little Chalfont, Buckinghamshire, UK): Enzyme-linked immunoassay for rat GH. Anachemia (Montreal, Quebec, Canada): Hydrochloric acid and hydrogen peroxide. BDHChemicals (Toronto, Ontario, Canada): Magnesium chloride (MgCb), sodium carbonate (NaiCOs), isopropanol, reagent-grade methanol, ethylenediaminetetra-acetate disodium salt (EDTA), phenol reagent, potassium chloride (KC1), chloroform, light petroleum, liquid paraffin (Nujol), zinc powder, and benzene. BIO RAD (Richmond, CA, USA): N,N'- methylene-bis-acrylamide (BIS) and N,N,N',N'-tetramethylenediamine (TEMED). Boehringer Mannheim, Canada Ltd. (Laval, Quebec, Canada): Nicotindiamide adenine dinucleotide phosphate tetrasodium salt (NADPH). Caledon Laboratories Ltd. (Georgetown, Ontario, Canada): Tetrahydrofuran and cyclohexane. Carnation Inc. (Toronto, Ontario, Canada): Skim milk powder. 25 Experimental Fisher Scientific (Fair Lawn, NJ, USA): Potassium dihydrogen phosphate (KH2PO4), dipotassium hydrogen phosphate (K2HPO4), sodium dihydrogen phosphate (NaP^PC^), disodium hydrogen phosphate (Na2HP04), sodium chloride (NaCl), sodium hydroxide (NaOH), triethylamine, sodium dodecyl sulfate (SDS), tris (hydroxymethyl) aminomethane chloride (Tris), 2-mercaptoethanol, glycine (tissue culture grade), sucrose, sodium sulfate, triethylamine, ethyl ether, hexane, methanol (HPLC-grade), and ethanol. Fisher Scientific (Nepean, Ontario, Canada): Dichloromethane, acetic acid. Hershey Canada Inc. (Mississauga, Ontario, Canada): Peanut o i l . Hoechst Marion Roussel (Laval, Quebec, Canada): Clomiphene. ICN Biomedicals, Inc. (Aurora, OH, USA): Acrylamide, bovine serum albumin ( B S A ) , and ammonium persulfate, ICN Pharmaceuticals, Inc. (Costa Mesa, CA, USA): ImmuChem™ double antibody 1 2 5 I radioimmunoassay for human G H . J. T. Baker Inc. (Phillipsburg, NJ, USA): Sodium dithionite (Na 2S20 4) and potassium bicarbonate. Kirkegaard and Perry Laboratories, Inc (Gaithersburg, Maryland, USA): Affinity isolated rabbit anti-sheep IgG alkaline phosphatase conjugated immunoglobulin (IgG heavy and light chain specific). Mandel Scientific Company Ltd. (Edmonton, Alberta, Canada): Nitrocellulose membrane (0.2 urn pore size) and gel blotting paper. 26 Experimental Pierce (Rockford, IL, USA): Nitro blue tetrazolium chloride (NBT) . Praxair Canada Inc. (Mississauga, Ontario, Canada): Carbon monoxide (CO) and nitrogen (N2). Sigma Chemical Co. (St Louis, MO, USA): Tamoxifen, 4-hydroxytamoxifen (racemic mixture: 70% Z, 30% E isomer), platinum oxide, polyoxyethylenesorbitan (Tween 20™), cupric sulfate ( C u S O ^ t k O ) , n-butanol, and sodium potassium tartrate. Steraloids Inc. (Wilton, NH, USA): Testosterone, androstenedione, 2a-, 20-, 6a-, 60-, 7a-, 11a-, 110-, 15a-, 16a-, 160-, and 19-hydroxytestosterone. TAGOInc. (Burlingame, CA, USA): Human IgG absorbed goat F(ab')2 anti-rabbit IgG alkaline phosphatase conjugated immunoglobulin (IgG heavy and light chain specific). W. A. Hammond Drierite Company (Xenia, OH, USA): Anhydrous calcium sulfate. Whatman (Clifton, NJ, USA): Thin layer chromatography (TLC) plates. Xymotech Biosystems (Mt. Royal, Quebec, Canada): 5-Bromo-4-chloro-3-indolylphosphate disodium salt (BCIP). 27 Experimental 2.2. Purified C Y P Enzymes Purified C Y P 2 A 1 , C Y P 2 B 1 , C Y P 2 C 1 1 , or C Y P 3A1 were included as calibration standards in the S D S - P A G E gels. C Y P 2 A 1 was purified previously from Long-Evans rats and was provided to us by Dr. A . Parkinson (Kansas Medical Center, Kansas City, Kansas, U S A ) . C Y P 2 B 1 , C Y P 2 C 1 1 , and C Y P 3 A 1 were purified over a period of several years in Dr. Bandiera's laboratory. C Y P 2 B 1 was purified from pooled livers of phenobarbital-treated, adult female Long Evans rats according to a method of Guengerich et al. (1982) with slight modifications (Chang et al. 1992). C Y P 2C11 was purified from hepatic microsomes from untreated, adult male Long-Evans rats as described by Bandiera and Dworschak (1992). C Y P 3 A 1 was purified from dexamethasone-treated, adult female Long-Evans rats according to a method of Cooper et al. (1993). 2.3. Antibodies Sheep anti-rat C Y P 2 A 1 polyclonal IgG was provided by Dr. P. E . Thomas (Department of Chemical Biology and Pharmacognosy, Rutgers - The State University of N e w Jersey, Piscataway, N J , U S A ) . Polyspecific rabbit anti-rat C Y P 2 B 1 polyclonal IgG, monospecific rabbit anti-rat C Y P 2 C 1 1 polyclonal IgG, and polyspecific rabbit anti-rat C Y P 3 A 1 polyclonal IgG were prepared in Dr. Bandiera's laboratory as reported previously (Bandiera and Dworschak 1992, Panesar et al. 1996, Wong and Bandiera 1996). Section A 2.4. Animals and Treatment Forty-two post-pubertal female and twelve male Long Evans rats were purchased from Charles River (Montreal, Quebec, Canada). Their weight on the day of arrival was 187-225 g for female rats, which corresponds to an approximate age of 50-70 days. The weight of male rats was 313-364 g, which corresponds to an approximate age of 65-80 days. Rats were housed in pairs on corn-cob bedding in polycarbonate cages with free access to food (Laboratory Rodent Diet 5001, P M I Feeds Inc., Richmond, IN, U S A ) and tap-water. The animal quarters were maintained at a constant temperature (20-23° C) and had a photoperiod of 12 h of light and 12 h of darkness (lights on at 6:30). Female rats were, divided into seven treatment groups with six individuals in each group. Male rats were divided into two treatment groups with six individuals in each group. After three days of acclimation, female rats were injected s.c. with 0.5, 5, 20, 50, 28 Experimental 100, or 200 mg/kg b.w. tamoxifen in peanut oil for two consecutive days. Three female animals of the 50 mg/kg treatment group accidentally received an additional dose of 100 mg/kg on the third day after the start of treatment. Those three animals received a total dose of 200 mg/kg of tamoxifen distributed over three days (50 mg/kg at day 1, 50 mg/kg at day 2, and 100 mg/kg at day 3) and are analyzed in a separate treatment group labeled 100a. A s a result, the original 50 mg/kg treatment group was reduced to three rats. One group of male rats was injected s.c. with 200 mg/kg b.w. for two consecutive days. A control group of female and a control group of male rats received the peanut oi l vehicle alone. The injected volume for all treatment groups was 2 ml/kg b.w. Rats were weighed and blood samples were taken at a regular time interval. Rats were kil led by decapitation 36 days after treatment, livers were excised immediately and weighed (Figure 9). Treatment period Sacrifice . , i Days 0 1 t t Weight Blood sampling for tamoxifen assay 10 12 t t Weight Blood F i g u r e 9: Treatment protocol. sampling for tamoxifen assay 20 t Weight 24 30 33 36 t t t t Blood Weight Blood Weight sampling sampling and for tamoxifen for G H blood assay assay sampling for tamoxifen assay In addition, two male adult Wistar rats (15.5 weeks of age) from a previous experiment, in which they received a dose of steptozotocin in their 9 t h week of life to induce diabetes and a dose of a vanadium compound in their 10 t h week of life, were used in a preliminary experiment to determine G H concentrations in rat plasma using an 1 2 5 I radioimmunoassay for human G H . 2.5. B l o o d C o l l e c t i o n Blood samples for the tamoxifen H P L C assay were taken from all rats. Blood was collected every twelve days after treatment until day 36. A volume o f approximately 1 ml o f blood per rat was drawn from the tip of the tail into a heparin-coated glass capillary and stored on ice until plasma separation. Blood samples for the G H assay were taken on day 33 from female rats 29 Experimental treated with tamoxifen at 0, 5, 20, or 200 mg/kg (three rats/treatment group) and from male rats treated with tamoxifen at 0 or 200 mg/kg (three rats/treatment group). Blood was drawn from the tip of the tail every 20 min for 8 h starting at 9:00 a.m. in heparin-coated glass capillaries and stored on ice until plasma separation. A volume of approximately 250 pi of blood was collected each time. A total of 6 ml of blood was taken from each rat without erythrocyte or saline replacement. Plasma was separated by spinning the blood in a relative centrifugal field of 13,000 g at 4° C for 15 min. Plasma samples were stored at -80° C. 2.6. Preparation of Hepatic Microsomes Hepatic microsomes were prepared from individual rats immediately after decapitation. Livers were removed, weighed, minced, and placed in approximately 20 ml of ice-cold 0.05 M Tris containing 1.15% KC1 (pH 7.4). Livers were homogenized using a Potter-Elvehjem glass motar and a motor driven pestle (Talboys Engineering Corp., Emerson, N J , U S A ) . The homogenate was spun in a relative centrifugal field of 9,000 g for 20 min at 5° C using a Beckman J2-21 centrifuge (Beckman Instruments, Palo Alto , C A , U S A ) . The supernatant was filtered through cheese cloth and spun in a relative centrifugal field of 105,000 g for 60 min at 5° C using a Beckman LE-80 ultracentrifuge. The microsome pellet was separated from glycogen and resuspended in ice-cold 10 m M E D T A containing 1.15% KC1 using a homogenizer. The suspension was spun in a relative centrifugal field of 105,000 g for 60 min at 5° C using a Beckman LE-80 ultracentrifuge. The resulting pellet was separated from glycogen and carefully resuspended in 0.25 M sucrose. Aliquots were stored at -80° C . The microsomal sample from one rat of the 100 mg/kg treatment group was accidentally lost. Therefore, the total number of six microsomal samples was reduced to five in this treatment group. 2.7. Determination of total Protein Content in Hepatic Microsomes Protein concentrations in hepatic microsomes were measured using the Lowry protein assay (Lowry et al. 1951). Bovine serum albumin (BSA) was used as the standard. A l l samples were measured in duplicate at an absorbance of 650 nm using a Shimadzu UV-160 UV-v is ib le recording spectrophotometer (Shimadzu Corporation, Kyoto, Japan). 30 Experimental 2.8. Determination of C Y P Protein Content in Hepatic Microsomes Total microsomal C Y P content was determined by the sodium dithionite reduced carbon monoxide difference spectrum according to the method of Omura and Sato (1964a). Hepatic microsomes were diluted in 0.1 M sodium phosphate buffer (pH 7.4) containing 20% glycerol, and 0.1 m M E D T A . Spectral measurements were performed using a S L M - A m i n c o D W - 2 U V -vis spectrophotometer equipped with a S L M - A m i n c o Midan II kinetic processor controller ( S L M Instruments Inc. Urbana, IL, U S A ) . Total hepatic C Y P content was calculated using a molar extinction coefficient of 91 cm 2 /mmol (Omura and Sato 1964b). 2.9. Determination of C Y P Enzyme Activities in Hepatic Microsomes Hydroxylation of testosterone was measured according to a method of Sonderfran et al. (1987) with slight modifications (Wong and Bandiera 1998). The assay is based on the regiospecific hydroxylation of testosterone catalyzed by specific C Y P enzymes (Figure 10). Figure 10: Regiospecific hydroxylation of testosterone by C Y P enzymes. Arrows indicate selected positions for hydroxylation. The reaction mixture consisted of 0.92 ml of 50 m M potassium phosphate buffer, containing 3 m M M g C h (pH 7.4), 50 pi of microsomes (diluted in 0.25 M sucrose to a concentration of 6 nmol/ml) or metabolite standards, and 10 pi o f 100 m M N A D P H at a volume o f 50 ul . The constituents of the reaction mixture were pipetted into test tubes at 0° C and pre-incubated at room temperature for 10 min before adding 20 pi of 12.5 m M testosterone to start the reaction. Samples from individual rats were analyzed in duplicate. Test tubes containing microsomes from female rats were incubated for 10 min and test tubes containing microsomes from male rats were incubated for 5 min in a water bath at 37° C with shaking. The reaction was stopped by adding 6 31 Experimental ml of dichloromethane. Each test tube was then spiked with 50 pi of 50 u M 110-hydroxytestosterone as the internal standard. A l l testosterone metabolites were extracted into the dichloromethane-layer by shaking the reaction vials vigorously for 3 min. The organic and the aqueous layers were separated by spinning the test tubes in a relative centrifugal field of 3,000 g for 2 min using a Beckman G P centrifuge. The aqueous layer was aspirated and discarded. The remaining organic layer was evaporated under a gentle stream of nitrogen. The residue was reconstituted in 200 ml of methanol and filtered into an autosampler vial using a syringe filter (pore size 0.45 um). Testosterone and its metabolites was separated on a 15 cm x 4.6 m m i.d. reverse-phase Cis column with a bead size of 3 um (Supelco, Bellefonte, P A , U S A ) at a constant temperature of 40° C . The mobile phase was delivered at a flow rate of 1.4 ml/min by a Shimadzu L C - 6 0 0 / L C - 9 A binary gradient H P L C system (Shimadzu Scientific Instruments, Columbia, M D , U S A ) . Solvent A consisted of methanol:water:acetonitrile at 35%:64%:1% (v/v/v). Solvent B consisted of methanol:water:acetonitrile at 80%:18%:2% (v/v/v). The elution program was composed as follows: 100% solvent A was delivered from 0 to 10 min. Solvent B was added to solvent A in a linear gradient from 0 to 40% solvent B from 10 to 30 min followed by a linear gradient from 40 to 55% solvent B from 30 to 35 min and a linear gradient from 55 to 100% solvent B from 35 to 36 min. The column was equilibrated with 100% solvent B from 36 to 40 min followed by a linear gradient back to 100% solvent A from 40 to 41 min. From 41 to 45 min, the column was re-equilibrated with 100%) solvent A . Testosterone and its metabolites were monitored at 254 nm with a Shimadzu S P D - 6 A UV-spectrophotometric detector. Peak areas were integrated by a Shimadzu CR501 chromatopac data processor. Testosterone, 2a-, 20, 6a-, 60-, 7a- , 11a, 110-, 15a, 16a-, 160-hydroxytestosterone, and androstenedione (4-androsten-3,17-dione) could be separated by this assay (Figures 11, 13, and 14). 19-Hydroxytestosterone eluted together with 15a-hydroxytestosterone, but both metabolites were not detectable after metabolism of testosterone by rat hepatic microsomes. Metabolites were identified by comparing their retention times to those of authentic standards. Metabolite formation was quantified by dividing the area under the curve ( A U C ) values of the metabolite peak by the A U C values of the internal standard peak. The resulting ratio was divided by the slope of the calibration curves of testosterone and the metabolite standards to determine the amount of metabolite formed. Calibration curves were generated for 60-, 7a-, 16a-, 160-, 20-hydroxytestosterone, and androstenedione at four concentrations by plotting the ratios of the A U C values of the metabolites and the internal standard against the concentration of the 32 Experimental metabolites in the reaction mixture (Figures 21 and 22). Enzymatic C Y P activities were calculated by dividing the amounts of metabolites produced (nmol) by the incubation time (min) and the amount of total C Y P (nmol) used in the assay. For the determination of the amount of 2oc-hydroxytestosterone produced, a calibration curve from a previous study was used because of the lack of a 2a-hydroxytestosterone standard. A zero-time control incubation was included in each assay. For the zero-time control incubation, a randomly selected microsomal sample was added to the reaction mixture and the reaction was stopped with dichloromethane before adding testosterone to account for metabolite impurities within the testosterone standard or non-enzymatic hydroxylation of testosterone in the reaction mixture (Figure 12). 33 Experimental Figure 11: Representative chromatogram of a mixture of hydroxytestosterone standards. The concentration of each standard is 2 nmol/ml. Standards were added to the reaction mixture containing 0.25 M sucrose instead of microsomes and extracted with dichloromethane. (a) 6a-, (b) 6p-, (c) 7a-, (d) 16a-, (e) 16P-, (IS) l i p - , (g) 2p-hydroxytestosterone, (h) androstenedione, (IS) internal standard = l ip -hydroxy-testosterone. 34 Experimental co OS • in C5 IS *-* OJ c o 1_ •4-1 V) o tn gure 12: Representative chromatogram of a zero-time control incubation of the testosterone hydroxylase assay. A randomly selected microsomal sample was added to the reaction mixture and the reaction was stopped with dichloromethane before adding testosterone, (h) androstenedione, (IS) internal standard = lip-hydroxy-testosterone. 35 Experimental I (Si O > _<L> c ure 13: Representative chromatogram of testosterone metabolites formed by hepatic microsomes from vehicle-treated female rats. (a) 6a- , (b) 6P-, (c) 7a-, (d) 16a-, (e) 160-, (IS) 110-, (f) 2a-, (g) 20-hydroxytestosterone, (h) androstenedione, (IS) internal standard = 110-hydroxy-testosterone. 36 Experimental Figure 14: Representative chromatogram of testosterone metabolites formed by hepatic microsomes from vehicle-treated male rats. (a) 6a-, (b) 6(3-, (c) 7a-, (d) 16a-, (e) 16p-, (IS) l i p - , (f) 2a-, (g) 2p-hydroxytestosterone, (h) androstenedione, (IS) internal standard = l lp -hydroxy-testosterone. 37 Experimental 2.10. Immunoquantitation of C Y P Enzymes 2.10.1. Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis Sodium dodecylsulfate polyacrylamide gel electrophoresis ( S D S - P A G E ) was performed using a Hoefer S E 600 vertical slab gel unit (Hoefer Scientific Instruments, San Francisco, C A , U S A ) according to the procedure of Laemmli (1970). The final concentrations of the reagents in the separating gel were 7.5% (w/v) of acrylamide: ./V,./V'-mefhylene-bis-acrylamide (Bis) (22.2%:0.6% w/w), 0.375 M tris(hydroxymethyl)aminomethane (Tris) HC1 (pH 8.8), 0.1% (w/v) of SDS, 0.042% (w/v) of ammonium persulfate, and 0.03% (v/v) of N.N.N'.N'-tetramethylethylenediamine ( T E M E D ) . Stacking gels were 1 cm in height and contained 3% (w/v) of acrylamide:Bis (22.2%:0.6% w/w), 0.125 M Tr i s -HCl (pH 6.8), 0.1% (w/v) o f SDS, 0.08%) (w/v) of ammonium persulfate, and 0.05% (v/v) of T E M E D . The electrode buffer contained 0.1 M Tris base, 0.767 M glycine and 0.4%> (w/v) of SDS. Microsome samples and purified C Y P standards were diluted in sample dilution buffer containing 0.062 M T r i s - H C l (pH 6.8), 1% (w/v) of SDS, 0.001% (w/v) of bromphenol blue, 10% (v/v) o f glycerol, and 5% (v/v) of 2-mercaptoethanol. Diluted samples and standards were heated for 1.5 min in boiling water and were loaded (20 pi) into each well o f the stacking gel. The proteins migrated through the stacking gel at a constant current of 11 m A per gel for approximately 1 h and through the separating gel at a constant current of 22 m A per gel with cooling for approximately 2 h. A series of four or five concentrations of the purified C Y P standard was included on each gel. 2.10.2. Immunoblots Proteins resolved by S D S - P A G E were transferred electrophoretically (0.2 A overnight at 4° C) onto a 13 x 14 cm nitrocellulose membrane according to the procedure of Towbin et al. (1979) using a Hoefer transphor apparatus, model T E 52. Membranes were placed into blocking buffer consisting of 1% (w/v) of bovine serum albumin ( B S A ) , 3% (w/v) of skim milk powder in modified phosphate buffered saline (PBS) (pH 7.4) and stored at 4° C overnight. Modif ied P B S consisted of 0.137 M N a C l , 2.6 m M KC1, 8.1 m M sodium phosphate, 1.5 M potassium phosphate, and 0.2 m M E D T A . After discarding the blocking buffer, membranes were incubated with the primary antibody (sheep anti-rat C Y P 2 A 1 polyclonal IgG at 10 ug/ml, rabbit anti-rat C Y P 2 B 1 polyclonal IgG at 10 ug/ml, rabbit anti-rat C Y P 2 C 1 1 polyclonal IgG at 15 ug/ml, or rabbit anti-rat C Y P 3 A 1 polyclonal IgG) at 50 ug/ml at 37° C for 2 h with shaking. Membranes were washed with washing buffer (0.05% (v/v) polyoxyethylenesorbitan (Tween 20™) in modified PBS) and were incubated with alkaline phosphatase-linked goat F(ab')2 anti rabbit IgG 38 Experimental (1:3000) or alkaline phosphatase-linked rabbit anti-sheep IgG (1:1000) in antibody dilution buffer (1% (w/v) of B S A , 3% (w/v) of skim milk powder, and 0.05% (v/v) of Tween 20™ in modified PBS) at 37° C for 2 h with shaking. The membranes were washed again in washing buffer and rinsed with distilled water. Under dim light conditions, membranes were incubated with substrate solution (0.03% (w/v) nitro blue tetrazolium chloride (NBT) and 0.015% (w/v) 5-bromo-4-chhloro-3-indolylphosphate disodium salt (BCIP) in 0.1 M Tr i s -HCl buffer containing 0.5 m M M g C b , p H 9.5) at room temperature for 0.5-3 min. The reaction was terminated by discarding the substrate solution and rinsing with distilled water. 2.10.3. Immunoquantitation Staining intensities of protein bands on nitrocellulose membranes were determined by computer image analysis with a pdi 420oe densitometer equipped with an A G F A Arcus II scanner using the pdi Quantity One® 3.0 software program. The optical density multiplied by the stained area (OD x mm ) was determined for each band and was then divided by the O D x mm value of the band corresponding to the internal standard. Calibration curves of the purified proteins were generated by plotting the ratio of the O D x mm values of purified C Y P proteins and the internal standard against the amount (pmol) of protein loaded on the gel. The amount of C Y P enzyme present in the microsomal sample was derived from calibration curves (Figure 27) and presented as percentage of the amount of total C Y P present in the microsomal sample. Amounts of C Y P 2 A 2 were quantified based on the C Y P 2 A 1 calibration curves and expressed as a relative O D value because a C Y P 2 A 2 standard was not available to our laboratory and the anti-C Y P 2 A 1 antibody recognizes C Y P 2 A 2 weakly. Amounts of C Y P 2 B 2 in hepatic microsomes were quantified based on the C Y P 2 B 1 calibration curve because a C Y P 2 B 2 standard was not available to our laboratory and the a n t i - C Y P 2 B l antibody recognizes C Y P 2 B 1 and C Y P 2 B 2 equally well . Amounts of C Y P 3 A were expressed as a relative O D value because the C Y P 3 A antibody recognizes probably all C Y P 3 A enzymes, which could not be separated on the SDS gel. 2.11. Detection and Quantification of G H in Plasma 2.11.1. Detection and Quantification of G H in Rat Plasma with an G H 1 2 5 I Radioimmunoassay for Human G H 125 G H determinations in rat plasma were performed by an I radioimmunoassay for human G H following the procedure described by the manufacturer. A polypeptide diluent (0.2 ml) was added to two disposable glass test tubes, which served as blank-controls. The same polypeptide diluent (0.1 ml) was added to two disposable glass test tubes, which served as zero-controls. To 3 9 Experimental the remainder test tubes, human G H (0.5, 1, 2.5, 5, 10, 25, and 50 ng/ml) or plasma samples and 0.1 ml of anti-human G H were added in duplicate. To all test tubes, 0.1 ml of 1 2 5 I-labled human G H was added. The content of all tubes was mixed vigorously and tubes were incubated at 37° C for two h with shaking. Precipitant solution (0.5 ml) was added to all tubes. The contents of the tubes were mixed vigorously and separated in a relative centrifugal field of 1,000 g for 15 min at 7° C . The supernatant was decanted and the r im of the test tubes was blotted on absorbent paper. The radioactivity of the remaining precipitate was counted in a gamma counter. The percent bound (B/Bo) was calculated for each standard and sample by subtracting the average blank counts from the average counts obtained to yield the blank-corrected counts. The result was divided by the corrected zero-standard counts. A calibration curve was generated by plotting the percent bound as a function of the log human G H concentration. 2.11.2. Detection and Quantification of G H in Rat Plasma with an Enzyme-Linked Immunoassay for Rat G H G H determinations in rat plasma were performed by an enzyme-linked immunoassay for rat G H following the procedure described by the manufacturer. The assay kit contained a 92 well microtitre plate coated with donkey anti-goat IgG. A volume of 50 pi o f assay buffer (0.025M phosphate buffer (pH 7.5) containing 0.1% sodium azide), 50 p i of G H standards, or 50 ul o f diluted plasma sample, and 50 p i of goat anti-GH serum were pipetted into the wells and incubated at room temperature for 3 h. Then, 50 pi of biotin-conjugated rat G H was added and incubated at room temperature for 30 min. The contents were discarded and the wells were washed with wash buffer consisting of 0.01M P B S (pH 7.4), 0.2% (v/v) Tween 20™, and 0.01% (v/v) thimerosal. The Amdex™ amplification reagent (a dextran backbone coupled to horseradish peroxidase and streptavidin) was added to a l l wells (100 ul) and incubated at room temperature for 30 min. After washing the wells with wash buffer, 100 p i of T M B substrate (3,3',5,5-tetramethylbenzidine/hydrogen peroxide solution in dimethylforamide) was added to all wells and incubated at room temperature for 30 min. To stop the reaction, 100 pi o f 1 M sulfuric acid was added to all wells and the optical density of each well was determined at 450 nm using a plate reader. The percent bound (B/Bo) was calculated for each standard and sample by multiplying the O D value of the standard or sample by 100 and dividing the product by the O D value for the blank-standard (0 ng/ml GH) . A calibration curve was generated for each plate by plotting the percent bound as a function of the log rat G H concentration. The ng/mg values of the samples were derived from the regression equation of the linear portion of the graph. 40 Experimental Section B 2.12. Synthesis of Tamoxifen Metabolites It was necessary to synthesize two tamoxifen metabolites because they were needed as standards for the H P L C assay for detection and quantitation of tamoxifen and its metabolites in rat plasma and they were not available from commercial sources or from other laboratories. 2.12.1. Synthesis of JV-Desmethyltamoxifen Instead o f using the traditional von Braun-method for dealkylation of tertiary amines with cyanogen bromide (Hageman 1953), chloroformate esters were used to Af-demethylate tamoxifen as this method is thought to produce cleaner products (Montzka et al. 1974, Olofson et al. 1984). The synthesis of A'-desmethyltamoxifen was conducted by adaptation of methods used to synthesize normorphine (Rice 1975). To combine the chloroformate ester with the methyl group of the nitrogen atom of tamoxifen, 250 mg (0.6725 mmol) of tamoxifen, 20 ml of chloroform, 1.25 g (12 mmol) o f potassium bicarbonate, and 0.85 ml (6.017 mmol) of 2,2,2-trichloroethyl chloroformate were combined in a round flask and refluxed (boiled with condensation of vapor) for 169 h (137.5 h without heat) with magnetic stirring. The color of the solution changed from a clear solution at the beginning to a light brown solution after the first day, to a brown solution on the second day, and to a dark brown solution on the third day, indicating decomposition. Samples were taken frequently during the reaction period and separated on a silica TLC-plate to monitor the reaction. The reaction was not complete after 169 h because the tamoxifen spot on the T L C -plate was still visible. The reaction was stopped after 169 h to avoid further decomposition. The reaction mixture was allowed to cool, filtered, and washed successively with 50 m l of water and 50 ml of 0.1 M HC1. The chloroform phase was separated, dried over anhydrous sodium sulfate, and concentrated to a volume of 5 ml with a gentle stream of nitrogen. To separate the reaction product from decomposition products, the resulting dark liquid was separated on TLC-plates with a mobile phase consisting of chloroform:water 3:7 (v/v). The spot that eluted with the solvent front, believed to be A'-desmethyltamoxifen, was scraped from the plates, dissolved in 25 ml of chloroform, and filtered leading to a clear yellow solution. The solvent was evaporated with gentle heat under a stream of nitrogen, leaving behind 0.439 g of a thick oily brownish liquid. To cleave off the chloroformate ester adduct, 2 ml of methanol, 18 ml of 90% acetic acid, and 0.76 g of zinc powder were added and the mixture was refluxed for 4.5 h. Samples were taken after 15 min, 2 h, 3h, and 4.5 h for analysis by T L C . After 15 min, two spots appeared on 41 Experimental the TLC-plate and after 2 h, three spots appeared, one of which had a similar relative mobility as tamoxifen. After 4.5 h, the solution was allowed to cool, filtered, and 30 ml of water was added. In an attempt to crystallize the reaction product, the mixture was extracted with 20 ml chloroform and the solvent was evaporated. The residue was taken up in 1 m l o f petroleum ethendichloromethane 4:1 (v/v) and cooled to -20° C. After 3 h, the solution separated into brown oily drops and a clear solvent. The brown liquid was separated from the clear solvent. When the solvent was diluted with 1 ml of dichloromethane, white oi ly drops formed from the solution and were separated with a pipette. Mass spectroscopy (MS) o f the white o i l fraction was conducted by M r . Roland Burton (Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, B . C . , Canada) using a V G Quattro Triple Quadropole mass spectrometer with an electrospray interface (Fisons Instruments, Altrinchan, Cheshire, England). 2.12.2. Synthesis of Tamoxifen-A/-oxide Tamoxifen-A /-oxide was synthesized by adaptation of published methods (Foster et al. 1980, McCague and Seago 1986). To oxidize tamoxifen at its nitrogen atom, tamoxifen (250 mg, 0.6725 mmol), 15 ml of methanol, 5 ml of 30 % aqueous hydrogen peroxide, and 100 mg o f platinum(rV)oxide were combined in a round flask and allowed to react at room temperature for 5 h with stirring. To clean the reaction product, the solution was filtered and partitioned overnight between 50 ml of dichloromethane and 50 ml of water. The dichloromethane phase was separated, dried with anhydrous sodium sulfate, filtered, and concentrated to a volume of 2 ml . The clear yellow solution was taken up in 18 ml of light petroleum:dichloromethane 4:1 (v/v). To crystallize the reaction product, the volume of the solution was reduced to 5 m l with a gentle stream of nitrogen and cooled to 4° C. Crystallization started after initiation by scratching the wall o f the beaker with a glass rod. The solution was kept at 4° C overnight and crystallization was complete after 17 h. To clean the product further, crystals were washed with 5 m l of light petroleum:dichloromethane 4:1 (v/v) at -20° C and filtered, resulting in 254.5 mg of product (97.51% tamoxifen-A /-oxide). The crystals were redissolved in 10 m l of benzene:cyclohexane 1:1 (v/v), stored at 4° C for 2 h until re-crystallization occurred, filtered, and dried at 60° C over anhydrous calcium sulfate (Drierite®) for 24 h resulting in 170.7 mg of product (65.4%) of the expected amount). This final product was analyzed by determination of the melting point, infrared (IR) spectroscopy, M S , and nuclear magnetic resonance ( N M R ) spectroscopy. Determination of the melting point was conducted, using a capillary melting point apparatus (A. H . Thomas Company, Philadelphia, P A . , U S A ) . M S analysis was conducted by 42 Experimental M r . Roland Burton (Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, B . C . , Canada) using a V G Quattro Triple Quadropole Mass Spectrometer with an electrospray interface (Fisons Instruments, Altrinchan, Cheshire, England). N M R analysis was conducted by Dr. D . Lang (Faculty of Medicinal Chemistry, University of Washington, Seattle, W A , U S A ) . 2.13. H P L C Assay for Tamoxifen and Its Metabolites The H P L C assay for the detection and quantitation of tamoxifen and its metabolites in plasma was derived from published methods (Foster et al. 1980, Fried and Wainer 1994, L i m et al. 1994, MacCal lum et al. 1996 and 1997, E l -Yaz ig i and Legayada 1997, Manns et al. 1998). A racemic mixture of E and -Z 4-hydroxytamoxifen (70% Z isomer) was commercially available, and tamoxifen-JV-oxide was synthesized. To 0.3 ml of plasma from tamoxifen treated rats, 20 p i of a racemic mixture of cis and trans clomiphene (100 uM) was added as an internal standard. To 0.3 ml of plasma from untreated rats, 20 pi of a racemic mixture of Z and E 4-hydroxytamoxifen, tamoxifen-A^-oxide, and tamoxifen standards at concentrations of 5, 25, 50, and 100 u M and 20 pi of cis and trans clomiphene (100 u M ) was added. Tamoxifen, its metabolites, and the internal standard were extracted from plasma two times using 2 x 3 ml of 2% n-butanol in hexane by shaking the reaction vials vigorously for 3 min. The organic and the aqueous layers were separated by spinning the test tubes in a relative centrifugal field of 2,000 g for 2 min. The aqueous layer was frozen by placing the test tube in dry-ice cold acetone for 30 sec. The organic layer was poured into a clean test tube and evaporated under a gentle stream of nitrogen. The residue was reconstituted in 150 pi of methanol and filtered into an autosampler vial using a syringe filter (pore size 0.45 um). Tamoxifen and its metabolites were separated on a 15 cm x 4.6 mm i.d. reverse-phase Cjg column with a bead size of 3 um (Supelco, Bellefonte, P A , U S A ) at 40° C . Tamoxifen and its metabolites were eluted at flow rate of 0.75 ml/min with an isocratic mobile phase consisting of methanol:water:triethylamine at 80%:19.9%:0.1% (v/v/v) (pH 10.5). The mobile phase was delivered by a Shimadzu L C - 6 0 0 / L C - 9 A pump (Shimadzu Scientific Instruments, Columbia, M D , U S A ) . Tamoxifen and its metabolites were monitored at 243 nm on a Shimadzu S P D - 6 A UV-spectrophotometric detector. Peak areas were integrated by a Shimadzu CR501 chromatopac data processor. Tamoxifen and its metabolites were identified by comparing their retention times to those of 43 Experimental authentic standards (Figure 16). The E and Z isomers of 4-hydroxytamoxifen, tamoxifen-A/-oxide, tamoxifen, and the cis and trans isomer of the internal standard clomiphene could be separated on the column (Figure 16). Plasma concentrations of tamoxifen and its metabolites were calculated by dividing the A U C values of their peak by the sum of the A U C values of the cis and trans clomiphene peak. The resulting ratio was divided by the slope of the calibration curves of tamoxifen and its metabolite standards (Figure 46). The chromatogram of plasma from untreated rats revealed the presence of up to four small peaks, one of which co-eluted with tamoxifen-A /-oxide (Figure 15). In plasma from tamoxifen treated rats, Z 4-hydroxytamoxifen, tamoxifen-A^-oxide, tamoxifen, and cis and trans clomiphene could be detected (Figure 17). 44 Experimental Figure 15: Representative chromatogram of the H P L C assay for tamoxifen and its metabolites of plasma from untreated rats. 45 Experimental Figure 16: Representative chromatogram of a mixture of tamoxifen metabolite standards. To plasma from untreated rats, 20 pi of a racemic mixture of Z and E 4-hydroxytamoxifen, tamoxifen-TV-oxide, and tamoxifen at concentrations 100 u M and 20 p i of cis and trans clomiphene (100 u M ) as an internal standard was added. Tamoxifen, its metabolites and the internal standard were extracted with 2 x 3 m l of 2% n-butanol in hexane. (a) Z 4-hydroxytamoxifen, (b) E 4-hydroxytamoxifen, (c) tamoxifen-TV-oxide, (d) tamoxifen, (e) cis clomiphene, (f) trans clomiphene. 46 Experimental Figure 17: Representative chromatogram of tamoxifen and its metabolites extracted from plasma of a tamoxifen treated female rat. (a) Z 4-hydroxytamoxifen, (c) tamoxifen-iV-oxide, (d) tamoxifen, (e) cis clomiphene, and (f) trans clomiphene. The plasma sample was taken one day after administration of tamoxifen at a dosage of 50 mg/kg. 47 Experimental 2.14. Statistical Analysis Mean values of the control groups an the treatment groups were analyzed by one way analysis o f variance ( A N O V A ) . Differences between pairs of mean values were tested by the Student Newman-Keuls test. Mean differences with a p value < 0.05 were considered significantly different. 48 Results 3 . R E S U L T S Section A 3.1. Development of Subcutaneous Cysts at the Injection Site Rats were treated with tamoxifen by s.c. injection for two consecutive days. The injected volume was 2 ml/kg (approximately 0.4 ml/female rat and 0.7 ml/male rat). On the second day of treatment, all female rats treated with tamoxifen at dosages of 100 and 200 mg/kg and all male rats treated with tamoxifen at 200 mg/kg appeared to be sensitive to the treatment. Rats squealed and tried to avoid the second injection, in contrast to the first injection that was not noticed by the rats. This sensitivity to the second injection was not apparent in rats treated with tamoxifen at lower dosages and probably indicates inflammation of the tissue near the first injection site. Subcutaneous cysts (distinct liquid-filled sacs) developed at the injection site in rats treated with tamoxifen at dosages greater than 5 mg/kg and were noticed five days after treatment. In rats treated with tamoxifen at dosages of 20 and 50 mg/kg, cysts were smaller than 1.5 cm in diameter. Cysts were no longer detectable in most rats by 36 days after treatment. Examination of the cysts at day 36 after treatment revealed that they contained scar tissue but not peanut oi l (Table 5). The size of the cysts in rats treated with tamoxifen at dosages of 100 and 200 mg/kg ranged between 1.5 and 2.5 cm in diameter. The fur and skin covering of the cysts was oily in some cases, indicating some loss of the injected volume. Cysts were no longer detectable at 36 days after treatment in some rats in these treatment groups. In some rats, cysts were decreased in size, while in other rats cysts remained larger than 1.5 cm in diameter. The smaller cysts contained scar tissue and the larger cysts contained peanut o i l and pus (Table 5). The cysts did not appear to cause pain to the rats when touched 5 days after treatment. Rats that developed cysts did not behave differently than rats that did not develop cysts. 49 Results Table 5: Development of subcutaneous cysts at the site of injection of tamoxifen treated rats. Dosage (mg/kg) Sex Number of animals with cysts at day 10 Size of cyst Number of animals with cysts at day 36 Size of cyst Content of cyst at day 36 0 female 0 0 0 male 0 0 0.5 female 0 0 5 female 0 0 20 female 3 < 1.5 cm 1 < 1.5 cm scar tissue 50 female 3 < 1.5 cm 3 < 1.5 cm scar tissue 100a female 3 < 1.5 cm 1 < 1.5 cm scar tissue 100 female 6 (3 oily) < 2.5 cm 4 (1 without fur) 2 < 1.5 cm 2 < 2.5 cm 3 scar tissue 1 o i l 200 female 6 (5 oily) < 2.5 cm 4 (1 without fur) 3 < 1.5 cm 1 <2.5 cm 3 scar tissue 1 o i l 200 male 6 (2 oily) < 2.5 cm 6 (1 oily) 1 < 1.5 cm 5 < 2.5 cm 1 scar tissue 5 o i l The injection volume was 2 ml/kg. Concentrations injected were 0.25, 2.5.10, 25, 50, and 100 mg/ml. Rats in the 100a mg/kg treatment group received a total dose of 200 mg/kg tamoxifen distributed over three days (50 mg/kg at day one, 50 mg/kg at day two, and 100 mg/kg at day three). Each treatment group contained six rats except for the 50 and 100a mg/kg treatment groups, which contained three rats. 5 0 Results 3.2. Effects of Tamoxifen on Body Weight and Liver Weight Each rat was weighed on an electronic balance on both days of treatment (1 s t injection and 2 n d injection), and on days 10, 20, 30, and 36 (Tables 6, 7). Rats were killed on day 36 and livers were removed, blotted dry, and weighed on an electronic balance (Table 8). Body weight gain was calculated by subtracting the initial body weight (on the first day of the treatment) from the later measurements. The liver weight/body weight ratio was calculated by dividing the liver weight by the final body weight (Figure 20). Tamoxifen treated female rats gained significantly less weight than rats in the control group at all times. Suppression of somatic growth was similar between all treatment groups. A t day 36, the body weight gain of tamoxifen treated female rats was on average approximately 40% of the body weight gain of control rats (Figure 18). The mean liver weight of female rats was similar between all treatment groups, except for livers of rats treated with 20 mg/kg. The liver weight/body weight ratio from female rats of all treatment groups was not different from that of the control group except a slight but significant difference for the 20 mg/kg treatment group (Figure 20). Male rats treated with tamoxifen at 200 mg/kg gained significantly less weight than rats in the control group. A t day 36, the body weight gain of tamoxifen treated male rats was on average approximately 5% of the body weight gain of control rats (Figure 19). The mean liver weight of male rats treated with tamoxifen at 200 mg/kg was significantly smaller than the mean liver weight of control male rats (Table 8). The liver weight/body weight ratio from male rats in the treatment group was not different from that of the control group (Figure 20). 51 Results Table 6: Mean body weight of female rats treated with various dosages of tamoxifen. Days after Mean body weight (g) treatment 0 0.5 5 20 50 100a 100 200 mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg 1 s t 200.7 202 208.8 205.2 207.7 211.0 208.8 215.7 injection (2.2) (3.2) (2.6) (5.5) (3.8) (7.4) (3.6) (1.9) 2 n d 207 201 207.3 203.5 208 210.7 207.3 214.5 injection (1.9) (3.6) (3.4) (7) (4.7) (6.7) (4) (3.1) 10 233.2 210.8* 215.5 205.7** 214.7 214.3 206.3** 218.3 (3.2) (5.1) (4.5) (5.4) (8) (8.7) (4.4) (4.2) 20 252.5 223** 228** 215.3** 223.7** 227.3** 218.7** 225.2** (2.7) (6.3) (3.2) (4.5) (4.1) (10.4) (4.6) (4.7) 30 268.3 234.5** 233** 221.7** 233** 237.7** 224.8** 229.8** (4.6) (6.5) (3.7) (4.5) (8) (13.3) (4.2) (2.5) 36 268 238.7** 232** 220.5** 241.7** 241** 230.2** 236.5** (5.2) (8.5) (4.5) (4.3) (7.3) (10.5) (5.9) (5.1) Each treatment group contained six rats except for the 50 and 100a mg/kg treatment groups, which contained three rats. Rats in the 100a mg/kg treatment group received a total dose of 200 mg/kg tamoxifen distributed over three days (50 mg/kg at day one, 50 mg/kg at day two, and 100 mg/kg at day three). Values in parenthesis indicate the standard errors of the mean. * Mean value of the treatment group is statistically different ( * = / ? < 0.05, ** = p < 0.01) from that of the control group. 5 2 Results Table 7: Mean body weight of male rats treated with tamoxifen. Days after Mean body weight (g) treatment 0 200 mg/kg mg/kg 1 s t injection 325.7 331.8 (26.2) (7.4) 2 n d injection 327.5 321.5 (7.3) (6.8) 10 361.7 317.3** (7.9) (8.4) 20 392.5 331.7** (9.6) (7.6) 30 416.3 333.8** (10.4) (10.2) 36 424 336.7** (11.9) (10) Each treatment group contained six rats. Values in parenthesis indicate the standard errors of the mean. ** Mean value of the treatment group is statistically different (p < 0.01) from that of the control group. 53 Results — • — 0 mg/kg • 0.5 mg/kg 5 mg/kg — X — 20 mg/kg —X— 50 mg/kg — • — 100a mg/kg —1— 100 mg/kg — — 200 mg/kg 20 Time (days) 40 Figure 18: Mean body weight gain of female rats treated with various dosages of tamoxifen. Error bars indicate the standard error of the mean. * Mean value of all treatment groups is statistically different (p < 0.05) from that of the control group. 1 2 0 N Time (days) Figure 19: Mean body weight gain of male rats treated with tamoxifen. Error bars indicate the standard error of the mean. * Mean value of the treatment groups is statistically different (p < 0.05) from that of the control group. 54 Results Table 8: Mean liver weight of male and female rats treated with various dosages of tamoxifen. Mean liver weight (g) female male 0 0.5 5 20 50 100a 100 200 0 200 mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg 10.59 9.21 9.32 7.98* 10.07 10.12 9.03 9.66 17.08 13.7* (0.25) (0.52) (0.34) (0.21) (0.44) (0.57) (0.26) (0.37) (0.89) (0.5) Each treatment group contained six rats except for the 50 and 100a mg/kg treatment groups, which contained three rats. Rats in the 100a mg/kg treatment group received a total dose of 200 mg/kg tamoxifen distributed over three days (50 mg/kg at day one, 50 mg/kg at day two, and 100 mg/kg at day three). Values in parenthesis indicate the standard errors of the mean. * Mean value of the treatment group is statistically different (p < 0.05) from that of the control group. Figure 20: Mean liver weight/body weight ratio of male and female rats treated with various dosages of tamoxifen. Error bars indicate the standard error of the mean. * Mean value of the treatment group is statistically different (p < 0.05) from that of the control group. 55 Results 3.3. Effect of Tamoxifen on the Relative Hepatic Content of CYP Enzymes C Y P enzyme concentrations and total protein concentrations were measured in hepatic microsomes from individual tamoxifen-treated and vehicle-treated male and female rats. Both concentrations are dependent on the final concentration of the microsomes in the sucrose-storage solution. Using these concentrations, the relative C Y P content of hepatic microsomes was determined (Table 9). This ratio is independent of the dilution of the microsomes in the storage solution. The C Y P concentration (nmol/ml) of hepatic microsomes from male and female rats in all treatment groups of was not significantly different from that of the control groups except for the 50 mg/kg treatment group. The total protein concentration (mg/ml) of hepatic microsomes of male and female rats in all treatment groups was not significantly different from that of the control groups. The relative C Y P content (nmol C Y P / m g protein) of hepatic microsomes from male and female rats in all treatment groups was not significantly different from that of the control groups, indicating that tamoxifen treatment had no effect on the relative C Y P content of hepatic microsomes from male and female rats. Table 9: C Y P concentration, protein concentration, and relative C Y P content in hepatic microsomes from male and female rats treated with various dosages of tamoxifen. female male 0 0.5 5 20 50 100a 100 200 mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg 0 200 mg/kg mg/kg C Y P concentration 23.36 23.14 20.31 21.86 16.5* 26.11 22.44 21.01 (nmol/ml) (1.58) (1.16) (1.51) (0.82) (1.34) (0.29) (1.66) (0.85) 49.13 40.88 (1.68) (1.65) Protein concentration 25.85 24.67 20.13 23.48 18.24 28.79 27.26 26.28 (mg/ml) (1.77) (1.74) (1.73) (1.21) (0.65) (1.5) (1.49) (1.84) 46.31 43.07 (1.14) (1.2) Relative C Y P content 0.91 0.95 1.02 0.94 0.9 0.91 0.82 0.81 (nmol C Y P / m g protein) (0.03) (0.05) (0.02) (0.02) (0.03) (0.04) (0.03) (0.03) 1.07 0.95 (0.06) (0.05) Each treatment group contained six rats except for the 50 and 100a mg/kg treatment groups, which contained three rats. Rats in the 100a mg/kg treatment group received a total dose o f 200 mg/kg tamoxifen distributed over three days (50 mg/kg at day one, 50 mg/kg at day two, and 100 mg/kg at day three). Values in parenthesis indicate the standard errors of the mean. * Mean value of the treatment group is statistically different (p < 0.05) from that of the control group. 56 Results 3.4. Testosterone Hydroxylase Assay 3.4.1. Validation of the Testosterone Hydroxylase Assay To validate the testosterone hydroxylase assay, the linearity, variability, sensitivity, and specificity of the assay were determined. The recovery of the assay was not tested but was determined in our laboratory previously to be on average 85.4% (Anderson 1997). The recovery of the assay is assumed to be similar because a standard operating procedure for this assay is followed by all people working in Dr. Bandiera's laboratory. Solutions of 60-, 7a-, 16a-, 160-, and 20-hydroxytestosterone and androstenedione were prepared from commercially available authentic standards at four concentrations (25, 50, 100, and 250 uM) and were used to generate calibration curves. The calibration curves were linear over the investigated concentration range (Figures 21 and 22). The concentrations of metabolite formed by microsomes from female and male rats were within the linear range of the calibration curves. The inter-assay variability was determined from ten calibration curves produced on ten different days (Table 10). The coefficients of variation (standard variation/mean value x 100%) were below the accepted limits of 15% in most cases. Exceptions are the coefficients of variation for standards at the lowest concentration (25 uM) and for 60-hydroxytestosterone (Table. 10). The reason for the higher coefficients of variation for 60-hydroxytestosterone as compared to those of all other testosterone metabolites is unknown. The intra-assay variability was determined on one day from a mixture of six hydroxytestosterone standards that were extracted from four incubation mixtures (Table 11). The coefficients of variation were below the accepted limits of 15% in most cases. The limit o f quantitation (LQ) is defined as the smallest amount of analyte that can be quantified with a coefficient of variation less than 20%. The L Q values for all metabolites were converted into amount of metabolite formed divided by the reaction time (10 min for female rats, 5 min for male rats) divided by the total amount of C Y P enzyme (0.3 nmol) to make the L Q s more applicable to the calculated specific C Y P activities. Values are reported in Table 12. The limit o f detection is defined as the smallest amount of analyte that can be detected with a corresponding peak area three times greater than that of baseline peaks. The baseline in the present assay did not contain any peak and therefore, the limits of detection are the same as the limits of quantitation. A n exception is the limit of detection of 20-hydroxytestosterone. Peaks corresponding to this testosterone metabolite could be detected, in two of four incubation mixtures at a concentration of 0.05 nmol/ml. 57 Results Table 10: Inter-assay variability of the testosterone hydroxylase assay. Concentration of standards Coefficients of variation (%) 66 7a 16a 166 26 A 25 u M (0.5 nmol/ml) 54.7 24.4 9.5 24.6 7.14 28.1 50 u M ( l nmol/ml) 27.7 10.3 9.6 15.7 7.4 16.2 100 u M (2 nmol/ml) 28.7 8.9 9.7 13.8 6.1 17.4 250 u M (5nmol/ml) 26.4 10.5 11.8 14.7 9 13.1 Table 11: Intra-assay variability of the testosterone hydroxylase assay. Concentration of standards Coefficients of variation (% 66 7a 16a 166 26 A 2.5 u M (0.05 nmol/ml) 7.9 8.7 5 uM(0 .1 nmol/ml) 7.3 34.1 5.5 7.2 6.1 1 0 u M ( 0 . 2 nmol/ml) 27.7 11.2 1.8 5.1 7.3 25 u M (0.5 nmol/ml) 19.9 34.1 20.4 7.5 7.8 5.1 Table 12: Limits of quantitation of the testosterone hydroxylase assay. Limits of quantitation 66 7a 16a 166 26 A L Q (nmol/ml) 0.5 0.1 0.2 0.1 0.1 0.05 L Q for female rats (nmol metabolite/ 0.17 0.033 0.067 0.033 0.033 0.017 min/nmol C Y P ) L Q for male rats (nmol metabolite/ 0.33 0.067 0.133 0.067 0.067 0.033 min/nmol C Y P ) Limits o f quantitation (LQ) are converted into amount of metabolite formed divided by the reaction time (5 min for female rats, 10 min for male rats) divided by the total amount of C Y P enzyme (0.3 nmol) to make the L Q s more applicable to the calculated specific C Y P activities. The limit o f quantitation for 2a-hydroxytestosterone was .0.1 nmol/ml as determined previously (Anderson 1997). 58 Results 6p-Hydroxytestosterone 0 1 2 3 4 5 Concentration of Standard (nmol/ml) Figure 21: Representative calibration curves for 6p-, 7a-, and 16a-hydroxytestosterone. 59 Results i 16p-Hydroxytestosterone Concentration of Standard (nmol/ml) Figure 22: Representative calibration curves for 2(3- and 16p-hydroxytestosterone and androstenedione. 60 Results 3.4.2. Effect of Tamoxifen on the Activity of CYP Enzymes Female Rats: The rate of formation of 7a-hydoxytestosterone, catalyzed mainly by C Y P 2 A 1 , was decreased significantly by approximately 40% in female rats that received tamoxifen at dosages greater than 5 mg/kg, as compared to the control group that received the peanut oi l vehicle alone. The rates o f formation of 20-, 60-, 16a-, and 160-hydroxytestosterone and androstenedione were unaffected by administration of tamoxifen (Appendix Table I, Figures 23 and 24). The rate of formation of 2a-hydroxytestosterone was below the limits of detection but seemed not to be affected by tamoxifen treatment. Male Rats: The rate of formation of 7a-hydoxytestosterone was increased significantly by approximately 70%> in the treatment group receiving tamoxifen at 200 mg/kg, as compared to the control group. The rate of formation of 16a-hydoxytestosterone, catalyzed mainly by C Y P 2 B 1 and C Y P 2 C 1 1 , was decreased significantly by approximately 43%) as compared to the control group. The rate of formation of 2a-hydoxytestosterone, catalyzed solely by C Y P 2 C 1 1 , was decreased by approximately 44% as compared to the control group. The rates of formation of 20-and 160-hydoxytestosterone and androstenedione were unaffected by administration of tamoxifen (Appendix Table II, Figures 25 and 26). 61 Results o_ >-o "o E _ | "o E £= > ^ -«—' ;> o < 0.6 0.5 0.4 0.3 0.2 0.1 6 (3 -Hydroxylation m O L O L O o O CO E O C M L O o D o s e (mg/kg b.w.) o o o o CM DL >-o "o E cz "£ E "o E c: >^  ;1 o < 2 1.6 1.2 0.8 0.4 0 7a - H y d r o x y l a t i o n i m m D o s e (mg/kg b.w.) r n O L O L O O O C O O c C N L O o O o o C N 0.2 | 0.16 | 0.12 J 0.08 o E -S 0.04 >. ~ 0 o u < a" 16a -Hydroxy lat ion Ii i i cu o LO LO o o c o o cz r-i CM LO o o D o s e (mg /kg b.w.) o o CN Figure 23: Mean testosterone hydroxylase activities of hepatic microsomes from female rats treated with various dosages of tamoxifen. Error bars indicate the standard error of the mean, n = 6 for the control and the 0.5, 5, 20, and 200 mg/kg treatment groups, n = 3 for the 50 and 100a mg/kg treatment groups, n = 5 for the 100 mg/kg treatment group. Rats in the 100a mg/kg treatment group received a total dose of 200 mg/kg tamoxifen distributed over three days (50 mg/kg at day one, 50 mg/kg at day two, and 100 mg/kg at day three). For the zero-time control incubation, a randomly selected microsomal sample was added and the reaction was stopped with dichlorometane before adding testosterone. * Mean value of the treatment group is statistically different (* =p < 0.05, **=/?< 0.01) from that of the control group. 62 Results 2 p - H y d r o x y l a t i o n >-o "o E "5 E o E c >. ;> o < 0.1 0.08 0.06 0.04 0.02 fo r r D o s e (mg/kg b.w.) 4 m O LO LO O O 03 O O £ Q CM LO O O O CN CL >-o "5 E "5 £ o E < 1 6 p - H y d r o x y l a t i o n 0.12 0.1 0.08 0.06 -\ 0.04 >, 0.02 -I. HI ED 4 E O LO LO o C N O LO ro o o o o o o •<— CN D o s e (mg/kg b.w.) A n d r o s t e n e d i o n e - F o r m a t i o n o_ >-o o E c E "5 E ^—. < 0.7 n 0.6 0.5 0.4 0.3 0.2 0.1 0 rti LO LO o o ro o o CN LO o O O ° O T- CN D o s e (mg/kg b.w.) F i g u r e 24: Mean testosterone hydroxylase activities of hepatic microsomes from female rats treated with various dosages of tamoxifen. Error bars indicate the standard error of the mean, n = 6 for the control and the 0.5, 5, 20, and 200 mg/kg treatment groups, n = 3 for the 50 and 100a mg/kg treatment groups, n = 5 for the 100 mg/kg treatment group. Rats in the 100a mg/kg treatment group received a total dose of 200 mg/kg tamoxifen distributed over three days (50 mg/kg at day one, 50 mg/kg at day two, and 100 mg/kg at day three). For the zero-time control incubation, a randomly selected microsomal sample was added and the reaction was stopped with dichlorometane before adding testosterone. 63 Results CL >-o c ~£ E o E -gr-"5 •4= o < 0.6 0.5 0.4 0.3 0.2 0.1 0 7a-Hydroxylation 0-time 0 200 Dose (mg/kg b.w.) 6(3-Hydroxylation CL >-o c "c E o E & o < CL >-o c "£ 'E ~= o E o < 3 2.5 2 1.5 1 0.5 0 3.5 3 2.5 2 1.5 1 0.5 0 .1-1111111111 Wmim •R • H illlllilli Bllli 0-time 0 200 Dose (mg/kg b.w.) 16a-Hydroxylation £ 1 ^1 MMI 0-time 0 200 Dose (mg/kg b.w.) Figure 25: Mean testosterone hydroxylase activities of hepatic microsomes from male rats treated with tamoxifen. Error bars indicate the standard error of the mean, n = 6 for the control and the treatment group. For the zero-time control incubation, a randomly selected microsomal sample was added and the reaction was stopped with dichlorometane before adding testosterone. * Mean value of the treatment group is statistically different (* = p < 0.05, ** =p < 0.01) from that of the control group. 64 Results >-o o E c "c E := o E c: '3 CL >-a E "-4—» O < 0.12 0.1 0.08 0.06 0.04 0.02 0 0.25 0.2 0.15 0.1 0.05 0 16p-Hydroxylation 0-time 0 200 Dose (mg/kg b.w.) 2p-Hydroxylation T 0-time 0 200 Dose (mg/kg b.w.) 6? 2.5 >-o § 2 I 1.5 £ 1 1 & 0.5 3 0 CL >-o 'E o < 1.5 0.5 2a-Hydroxylation • i r 0-time 0 200 Dose (mg/kg b.w.) Androstenedione-Formation 0-time 0 200 Dose (mg/kg b.w.) Figure 26: Mean testosterone hydroxylase activities ± S E M of hepatic microsomes from male rats treated with tamoxifen. Error bars indicate the standard error of the mean, n = 6 for the control and the treatment group. For the zero-time control incubation, a randomly selected microsomal sample was added and the reaction was stopped with dichlorometane before adding testosterone. ** Mean value of the treatment group is statistically different (p < 0.01) from that of the control group. 6 5 Results 3.5. Immunoblots 3.5.1. Validation of the Immunoblots To validate the analysis of the immunoblots, the linearity, variability, sensitivity and specificity of the immunoblots assay were determined. Immunoblots were validated with purified C Y P standards at four or five different concentrations (0.125, 0.25, 0.375, 0.5 pmol/lane for C Y P 2 A 1 ; 0.05, 0.1, 0.25, 0.5 pmol/lane for C Y P 2 B 1 ; 0.25, 0.5, 0.75, 1 pmol/lane for C Y P 2 C 1 1 ; and 0.0625, 0.125, 0.2, 0.25, 0.5 pmol/lane for C Y P 3 A 1 ) . The calibration curves of C Y P 2 A 1 and C Y P 3 A 1 were linear within the investigated concentration range (Figure 27). The calibration curves of C Y P 2 B 1 and C Y P 2 C 1 1 were linear up to a concentration of 0.1 and 0.5 pmol/lane, respectively (Figure 27). The levels of enzymes detected in microsomal samples were within the linear range of the calibration curves. The inter-assay variability for all blots was determined from three calibration curves produced on three different days (Table 13). Coefficients of variation (standard variation/mean value x 100%) were below the accepted limits of 15% in most cases. Exceptions are the coefficients of variation for standards at the lowest concentration for C Y P 2 B 1 , C Y P 2 C 1 1 , and C Y P 3 A and the second lowest concentration for C Y P 3 A 1 . The limits of quantitation (smallest amount of enzyme that can be quantified with a coefficient of variation less than 20%) are reported in Table 14. The levels of al l enzyme detected in microsomal samples were above the limits of quantitation of the immunoblots. The antibodies reacted with the following C Y P proteins but not with other C Y P enzymes. Anti-rat C Y P 2 A 1 IgG reacted predominantly with C Y P 2 A 1 and slightly with C Y P 2 A 2 . Anti-rat C Y P 2 B 1 IgG reacted equally strong with C Y P 2 B 1 and C Y P 2 B 2 . C Y P 2 A 1 , C Y P 2 A 2 , C Y P 2 B 1 , and C Y P 2 B 2 could be separated on the SDS-polyacrylamide gel. Anti-rat C Y P 2 C 1 1 IgG was back-absorbed extensively and reacted only with C Y P 2 C 1 1 . Anti-rat C Y P 3 A 1 IgG was also back-absorbed and reacted predominantly with C Y P 3 A 1 . The antibody recognized also C Y P 3 A 2 and probably other C Y P 3 A enzymes, which could not be separated from C Y P 3 A 1 on the SDS-polyacrylamide gel and were quantified together in one band. 66 Results Table 13: Inter-assay Variability of the immunoblots of C Y P 2 A 1 , C Y P 2 B 1 , C Y P 2 C 1 1 , and C Y P 3 A 1 standards. Concentration of standards (pmoUlane) CYP2A1 Coefficients of variation (%) CYP2B1 CYP2C11 CYP3A1 0.05 19.3 0.0625 29 0.1 IS 0.125 10.2 18.6 0.2 IS 0.25 IS 11.8 17.9 11.5 0.375 11.4 0.5 11.9 11.9 IS 0.75 6.1 1 8.1 IS = this value was used as the internal standard. Table 14: Limits of quantitation of the immunoblots. Limits of quantitation (pmol/lane) CYP2A1 CYP2B1 CYP2C11 CYP3A1 0.125 0.05 0.25 0.125 67 Results < CN co CL — ° I E x E Q x O Q O O CO CN H O-CM " i i s Q O 3 2 .5 2 1.5 1 0.5 0 * C Y P 2 A 1 y = 3.2962X+ 0 .0857 R 2 = 0 .9873 0 0.1 0.2 0.3 0.4 0.5 0.6 Amount C Y P 2 A 1 (pmol/lane) C Y P 2 C 1 1 y = 2 x + 0.0138 R' = 0.9977 0.8 1 1.2 Amount C Y P 2 C 1 1 (pmol/lane) C Y P 2 B 1 CQ CN CO o_ — ° I CM C -E x E Q x O Q o 3 2 .5 2 1.5 1 0.5 y = 1 0 x + 0 .0214 R 2 = 0 .9945 0 0.1 0.2 0 .3 0.4 0.5 0.6 Amount C Y P 2 B 1 (pmol/ lane) C Y P 3 A 1 0 * 0 0.1 0.2 0.3 0.4 0.5 0.6 Amount C Y P 3 A (pmol/ lane) Figure 27: Calibration curves of purified C Y P 2 A 1 , C Y P 2 B 1 , C Y P 2 C 1 1 , and C Y P 3 A 1 . Values on the Y-axis represent the average results of three calibration curves generated on three individual immunoblots on three different days. Trendlines were generated and regression equations were derived from the linear portion of the calibration curves. 68 Results 3.5.2. E f fec t o f T a m o x i f e n on M i c r o s o m a l C Y P Pro te in Leve l s F e m a l e Rats : The anti-rat C Y P 2 A 1 IgG recognized a single band on immunoblots containing liver microsomes from control and tamoxifen treated rats. This band has the same electrophoretic mobility as purified C Y P 2 A 1 . The anti-rat C Y P 2 B 1 IgG recognized several microsomal enzymes, two of which are C Y P 2 B 1 and C Y P 2 B 2 . C Y P 2 B 1 and C Y P 2 B 2 could be separated in a SDS-polyacrylamide gel. The band corresponding to C Y P 2 B 1 has the same electrophoretic mobility as purified C Y P 2 B 1 . The anti-rat C Y P 3 A 1 antibody recognized probably all C Y P 3 A enzymes, which could not be separated in a SDS-polyacrylamide gel. This band has the same electrophoretic mobility as purified C Y P 3 A 1 (Figure 28). Densitometric quantitation of the immunoblots revealed that the hepatic microsomal content of C Y P 2 B 1 , C Y P 2 B 2 , and C Y P 3 A was unaffected by administration of tamoxifen. The content of C Y P 2 C 1 1 was not determined in female rats. The hepatic microsomal content of C Y P 2 A 1 in hepatic microsomes was decreased significantly in female rats that received tamoxifen at doses of 20 mg/kg, 100 mg/kg, and 200 mg/kg by on average approximately 35% as compared to the control group (Appendix Tables III, IV, Figure 30). M a l e Rats : The anti-rat C Y P 2 A 1 IgG recognized predominantly C Y P 2 A 1 and several other microsomal enzymes, one of which is C Y P 2 A 2 . C Y P 2 A 1 and C Y P 2 A 2 could be separated in a SDS-polyacrylamide gel. This band to C Y P 2 A 1 has the same electrophoretic mobility as purified C Y P 2 A 1 . The anti-rat C Y P 2 B 1 IgG recognized several microsomal enzymes, two of which are C Y P 2 B 1 and C Y P 2 B 2 . C Y P 2 B 1 and C Y P 2 B 2 could be separated in a SDS-polyacrylamide gel. The band corresponding to C Y P 2 B 1 has the same electrophoretic mobility as purified C Y P 2 B 1 . The anti-rat C Y P 3 A 1 antibody recognized probably all C Y P 3 A enzymes, which could not be separated in a SDS-polyacrylamide gel. This band has the same electrophoretic mobility as purified C Y P 3 A 1 . The anti-rat C Y P 2 C 1 1 antibody recognized only one microsomal enzyme represented by one band in the C Y P 2 C 1 1 immunoblot. This band has the same electrophoretic mobility as purified C Y P 2 C 1 1 (Figure 29). Densitometric quantitation of the immunoblots revealed that the specific content of C Y P 2 C 1 1 was decreased by approximately 30% as compared to the control group. The hepatic microsomal content of C Y P 2 A 1 , C Y P 2 B 1 , C Y P 2 B 2 , and C Y P 3 A were unaffected by administration of tamoxifen (Appendix Tables III, IV, Figure 31). 69 Results C Y P 2 A 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 C Y P 2 B 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 C Y P 3 A 10 11 12 13 14 15 16 17 18 Figure 28: Representative immunoblots of hepatic microsomes from female rats probed with polyclonal IgGs against rat C Y P 2 A 1 , C Y P 2 B 1 , or C Y P 3 A 1 . C Y P enzymes were separated by S D S - P A G E . A volume of 20 p i was added in each lane. Lanes 1 and 12 contain microsomes from vehicle-treated rats. Lanes 2 to 7 and lanes 13 to 18 contain microsomes from tamoxifen-treated rats. Lanes 8 to 11 contain the respective purified C Y P enzyme in increasing concentrations. For the C Y P 2 A 1 blot: microsomes were diluted to a concentration of 0.2 nmol/ml. The sheep anti-rat C Y P 2 A 1 IgG was added at 10 pg IgG/ml. The rabbit anti-sheep IgG was added at a dilution of 1:1000. For the C Y P 2 B blot: microsomes were diluted to a concentration of 1 nmol/ml. The rabbit anti-rat C Y P 2 B IgG was added at 10 pg IgG /ml . The goat anti-rabbit IgG was added at a dilution of 1:3000. For the C Y P 3 A blot: microsomes were diluted to a concentration of 1 nmol IgG /ml . The rabbit anti-rat C Y P 3 A IgG was added at 50 ug/ml. The goat anti-rabbit IgG was added at a dilution of 1:3000. 7 0 Results C Y P 2 A 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 C Y P 2 B 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 CYP2C11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 C Y P 3 A 8 9 10 11 12 13 14 15 16 17 Figure 29: Representative immunoblots of rat hepatic microsomes from male rats probed with polyclonal IgGs against rat C Y P 2 A 1 , C Y P 2 B 1 , C Y P 2 C 1 1 , or C Y P 3 A . C Y P enzymes were separated by S D S - P A G E . A volume of 20 p i was added in each lane. For the C Y P 2 A l - b l o t : Lanes 1, 3, 5, 11, 13, and 15 contain microsomes from vehicle-treated rats. Lanes 2, 4, 6, 12, 14, and 16 contain microsomes from tamoxifen-treated rats. Lanes 7 to 10 contain purified C Y P 2 A 1 in increasing concentrations. Microsomes were diluted to a concentration of 1 nmol/ml. The sheep anti-rat C Y P 2 A 1 IgG was added at 10 pg IgG/ml. The rabbit anti-sheep IgG was added at a dilution of 1:1000. For the CYP2B-blo t : Lanes 1, 3, 5, 7, 13, and 15 contain microsomes from vehicle-treated rats. Lanes 2, 4, 6, 8, 14, and 16 contain microsomes from tamoxifen-treated rats. Lanes 9 to 12 contain purified C Y P 2 B 1 in increasing concentrations. Microsomes were diluted to a concentration of 1 nmol/ml. The rabbit anti-rat C Y P 2 B IgG was added at 10 pg IgG /ml . The goat anti-rabbit IgG was added at a dilution of 1:3000. For CYP2C11-blot : Lanes 2, 4, 6, 13, 15, and 17 contain microsomes from vehicle-treated rats. 1, 3, 5, 12, 14, 16, and 18 contain microsomes from tamoxifen-treated rats. Lanes 7 to 10 contain purified C Y P 2 C 1 1 in increasing concentrations. Microsomes were diluted to a concentration of 0.2 nmol/ml. The rabbit anti-rat CYP2C11 IgG was added at 15 pg IgG /ml . The goat anti-rabbit IgG was added at a dilution of 1:3000. For CYP3A-b lo t : Lanes 1, 3, 5, 12, 14, and 16 contain microsomes from vehicle-treated rats. Lanes 2, 4, 6, 13, 15, and 17 contain microsomes from tamoxifen-treated rats. Lanes 7 to 11 contain purified C Y P 3 A in increasing concentrations. Microsomes were diluted to a concentration of 0.2 nmol/ml. The rabbit anti-rat C Y P 3 A IgG was added at 50 pg IgG /ml . The goat anti-rabbit IgG was added at a dilution of 1:3000. 71 Results CYP2A1 0.1 CL o 0.08 co o 0.06 | 0.04 o 0.02 E C 0 LO LO O O CO o r-L CN LO O O 0 O T-Dose (mg/kg b.w.) o o CN CYP2B2 0.01 0.008 0.006 0.004 0.002 0 LO LO O O CO O ,_; CN LO O O Dose (mg/kg b.w.) o o CNI CL > o To .—< o -4—» O E c o E c CYP2B1 0.005 0.004 0.003 0.002 0.001 0 em m i M LO LO d O O CO CN LO O o o o o o CN Dose (mg/kg b.w.) Figure 30: Relative content of specific C Y P enzymes in hepatic microsomes from female rats treated with various dosages of tamoxifen. Error bars indicate the standard error of the mean, n = 6 for the control and the 0.5, 5, 20. and 200 mg/kg treatment groups, n = 3 for the 50 and 100a mg/kg treatment groups, n = 5 for the 100 mg/kg treatment group. * Mean value o f the treatment group is statistically different (* = p < 0.05, ** = p < 0.01) from that of the control group. The C Y P 3 A content of hepatic microsomes is expressed as a relative O D value (OD x m m 2 band/OD x m m 2 IS/nmol total C Y P ) because the anti-rat C Y P 3 A 1 IgG cross-reacted with all other C Y P 3 A enzymes, which could not be separated on the SDS-polyacrylamide gel. 72 Results C Y P 2 A 1 0 . 0 1 5 Q . £ 0 . 0 1 2 5 0 .01 0 . 0 0 7 5 0 . 0 0 5 0 . 0 0 2 5 0 ro -*—• o o E c o E £= CL >-o ro o o E c o E CL >-o ro o o E o E 0 . 0 0 6 0 . 0 0 5 0 . 0 0 4 0 . 0 0 3 0 . 0 0 2 0 . 0 0 1 0 0 . 1 4 0 . 1 2 0.1 0 . 0 8 0 . 0 6 0 . 0 4 0 . 0 2 0 •>„~:,S: - J J v . V A ; • • • • W-•nm •111 • t i l • • • • iui^iiiilii .*.!<[.,.::!;y ta* — •lllll 0 2 0 0 D o s e ( m g / k g b .w. ) C Y P 2 B 1 -ra •I - H 0 2 0 0 D o s e ( m g / k g b .w. ) C Y P 2 C 1 1 SHI 0 2 0 0 D o s e ( m g / k g b .w. ) C Y P 2 A 2 16 1 4 1 2 8 1 0 > ro a> CC CL > o ro o -4—' O E £= o E c Q O a> > ro CD or 8 6 H 4 2 0 0 . 0 0 6 0 . 0 0 5 0 . 0 0 4 0 . 0 0 3 0 . 0 0 2 0 . 0 0 1 0 0 2 0 0 D o s e ( m g / k g b .w . ) C Y P 2 B 2 •Bill fe 0 2 0 0 D o s e ( m g / k g b .w . ) C Y P 3 A 0 2 0 0 D o s e ( m g / k g b .w . ) Figure 31: Relative content of specific C Y P enzymes in hepatic microsomes from male rats treated with tamoxifen. Error bars indicate the standard error of the mean, n = 6 for the control and the 0.5, 5, 20. and 200 mg/kg treatment groups, n = 3 for the 50 and 100a mg/kg treatment groups, n = 5 for the 100 mg/kg treatment group. * Mean value of the treatment group is statistically different (p < 0.05) from that of the control group. The C Y P 2 A 2 and the C Y P 3 A content of hepatic microsomes are expressed as relative O D values (OD x m m 2 band/OD x m m 2 IS/nmol total C Y P ) because purified C Y P 2 A 2 was not available for the generation of a calibration curve and because the anti-rat C Y P 3 A 1 IgG cross-reacted with all other C Y P 3 A enzymes, which could not be separated on the SDS-polyacrylamide gel. 73 Results 3.6. Detection and Quantitation of GH in Rat Plasma 3.6.1. Detection and Quantitation of GH in Rat Plasma with an 1 2 5I Radioimmunoassay for Human GH The aim of this preliminary experiment was to examine i f the frequent blood sampling method required to determine the G H secretion pattern is feasible and tolerated by the rats. Additionally, the possibility of determining rat G H plasma levels with an 1 2 5 I radioimmunoassay for human G H was tested. Blood was collected from the tip of the tail every 20 min for 8 h starting in heparin-coated glass capillaries. A volume of approximately 250 p i o f blood was collected each time. The rats appeared to be calm and healthy after the blood sampling procedure and gained approximately 10 g of weight one day after treatment. The blood sampling method was evaluated to be feasible. The concentration of G H in the blood samples ranged between 1.3 and 2.7 ng/ml, representing baseline noise (data not presented). 3.6.2 Validation of the Enzyme-Linked Immunoassay for Rat GH To validate the enzyme-linked immunoassay for rat G H , the linearity, variability, and sensitivity of the assay was determined. The specificity of the goat anti-rat G H IgG is shown in Table 15 as determined by the manufacturer. Dilutions of rat G H at eight concentrations (0, 1.6, 4.1, 10.24, 25.6, 64, 160, and 400 ng/ml) were added to each plate. The linear range of the calibration curves was between 4.1. and 64 ng/ml (Figure 32). Plasma samples of male and female rats were diluted such that the plasma G H concentrations were within the linear range of the calibration curves. The intra-assay variability was 3.3% (n = 80) as determined by the manufacturer. The inter-assay variability was determined from seven calibration curves produced on seven different days (Table 16). The coefficients of variation (standard deviation/mean value x 100%) were below the accepted limits of 15% up to a G H concentration of 64 ng/ml. The limit of quantitation (smallest amount of metabolite that can be quantified with a coefficient of variation less than 20%) was 1.6 ng/ml (Table 16). Concentrations below 1.6 ng/ml were not tested. 74 Results Table 15: Specificity of the goat anti-rat G H IgG. Cross-reactivity (%) Rat GH Rat GH Rat TSH Rat LH Rat PRL Rat FSH RatACTH (NIH-RP2) 100 189 <0.28 < 0.19 < 0.15 < 0.03 < 0.019 Data shown were provided by the manufacturer. Rat G H (NIH-RP2) = rat G H provided by the National Institutes of Health of the U S A , T S H = thyroid-stimulating hormone, P R L = prolactin, F S H = follicle-stimulating hormone, A C T H = adrenocorticotropic hormone. Table 16: Inter-assay variability of the enzyme-linked immunoassay for rat G H . Coefficients of variation (%) 1.6 ng/ml 4.1 ng/ml 10.24 ng/ml 25.6 ng/ml 64 ng/ml 160 ng/ml 400 ng/ml 6.4 8 8.2 12.8 14.2 17.9 19.3 1 0 0 -i 9 0 -8 0 -o CQ 7 0 -6 0 -m 5 0 -4 0 -3 0 -2 0 -1 0 -0 -1 0 1 0 0 Concentration (ng/ml) 1 0 0 0 Figure 32: Representative calibration curve of the enzyme-linked immunoassay for rat G H . 75 Results 3.6.3. Detection and Quantitation of GH in Rat Plasma with an Enzyme-Linked Immunoassay for Rat GH Blood samples were drawn 33 days after treatment every 20 minutes for 8 hours from the tip of the tail of three female rats treated with tamoxifen at dosages of 0, 5, 20, and 200 mg/kg, respectively and from three male rats treated with tamoxifen at dosages of 0 or 200 mg/kg, respectively. Plasma samples from one out of six rats that received tamoxifen at 5 mg/kg were investigated for GH content. Plasma GH values from female rat H were not reported after 15:20 p.m. because the blood flow from the tip of the tail ceased after that time point (Figure 36). Mean plasma GH levels were determined by calculating the average plasma GH concentration over the 8 h time period. Nadir plasma GH levels were determined by calculating the average of the five lowest GH concentrations. Peak plasma GH levels were determined by calculating the average of the two highest GH concentrations in two distinct peaks. If only one identifiable GH peak was present, this GH concentration was taken as the peak plasma GH level. If no peak was present, the two highest GH concentrations were taken as the peak plasma GH level. The GH concentration in female rat B at 9:20 p.m. was not included in the calculation of the plasma GH peak level because this value was an outlier as determined by the Q-test. Plasma GH mean and nadir levels were not affected by administration of tamoxifen at dosages of 5, 20, and 200 mg/kg to female rats and by administration of tamoxifen at tamoxifen at a dosage of 200 mg/kg to male rats. Peak plasma GH levels were decreased significantly by approximately 43% and 60%, respectively, as compared to the control groups after administration of tamoxifen at 20 mg/kg to female rats and at 200 mg/kg to male rats (Appendix Table V, Figures 33-40). 76 Results Female control rat A 350 -, 300 ^ 250 -o o o CM o o o o CM o M" O o o CM O o o O CM O M" O O O CM O o o o CM o M-o o o CM o o o o CM o M" o o ai 6> ai o o O CM CM CM CO CO CO •if •*r IO LO LO cb D cb Clock time Female control rat B 350 -, 300 -0 H 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 o o o o o o o o o o o o o o o o o o o o o o o o o a i O T C » 0 0 0 ^ ^ ^ < N C N C M J Clock time Female control rat C 350 i 300 -_ 250 -0 -I 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 o o o o o o o o o o o o o o o o o o o o o o o o o O C M ^ O C M N - 0 < N ^ O C M ^ O C M ^ O C M N ; O C N ^ O C M Clock time Figure 33: G H plasma levels of female control rats. 77 Results Female rat D 350 n 300 -0 -I 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 o o o o o o o o o o o o o o o o o o o o o o o o o c r i c n c x i o o o ^ ^ ^ c M c ^ Clock time Figure 34: GH plasma levels of female rats treated with tamoxifen at a dosage of 5 mg/kg. 78 Results Female rat E 350 n 300 -_ 250 50 \ 0 -I 1 1 1 1 1 1 1 1 1 . 1 1 1 1 1 1 1 1 1 . 1 1 > 1 o o o o o o o o o o o o o o o o o o o o o o o o o O C N ^ O O v l ^ O C N T T O C N ^ p C M ^ O C N j ^ p c N ^ p C N ^ p a > C T > a > o o o ^ ^ ^ ( N < N C \ i o c o Clock time Female rat F 350 i 300 -^ 250 -•1. 200 -0 "i 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 < i i i i i i i i o o o o o o o o o o o o o o o o o o o o o o o o o O C N ^ O C N ^ O C \ | ^ O C N | ^ O C M ^ O C M ^ O C M ^ O < N ^ t O Clock time Female rat G 350 -, 300 -^ 250 -o o o o o o o o o o o o o o o o o o o o o o o o o O C N ^ O C N ^ t O C s l ^ O f N ^ O ( N y O C M ^ O C N ' * r O < N ^ r O Clock time Figure 35: G H plasma levels of female rats treated with tamoxifen at a dosage of 20 mg/kg. 79 Results Female rat H 350 -I 300 -_ 250 -o o o o o o o o o o o o o o o o o o o o o o o o o O C N ^ O O N j ^ O C N ^ O r ^ ^ O C N ^ O C N J ^ O C N j T f O C N l T t o C l o c k t i m e C l o c k t i m e C l o c k t i m e Figure 36: G H plasma levels of female rats treated with tamoxifen at a dosage of 200 mg/kg. 80 Results 0 "J i 1 1 1 i 1 l l 1 1 1 1 r i i ] 1 1 1 i 1 1 1 1 O O O O O O O O O O O O O O O O O O O O O O O O O O C N l ^ p C ^ ^ O C ^ ^ O C ^ ^ p C S j ^ p c ^ ^ O C N ^ p C N ^ p Clock time Male control rat B 350 -i 0 "i i 1 1 1 1 1 1 1 1 i 1 1 1 1 1 i 1 1 1 1 1 1 1 1 O O O O O O O O O O O O O O O O O O O O O O O O O O O v j T T O C N ^ O C N J ^ O C N j T f O C N ^ p C N I ^ p C N I ^ p C N j T r p Clock time 50 H 0 n 1 1 1 1 1 1 l ! 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 l O O O O O O O O O O O O O O O O O O O O O O O O O O C N J ^ O C S J N ' O C N i ^ O C N ^ O C N ^ O Clock time Figure 37: G H plasma levels of male control rats. 81 Results Male rat D 350 -300 -^ 250 <> o o o CM o o o o CM o o o O CM O o o O CM O O O o CM O o o o CM O O o o CM o o o o CM O O O ai ai di o o o CM CM CM CO CO CO •sr IO in L O CD CD CD Clock time CD 100 o o o o o o o o o o o o o o o o o o o o o o o o o O C N ^ O C M ^ O C M ^ O C N OTOOOOOT-T-CM CN CM CO CO CO LO LO LD CD CD CO C l o c k t ime Figure 38: G H plasma levels of male rats treated with tamoxifen at a dosage of 200 mg/kg. 82 Results c X 1 5 0 1 2 5 1 0 0 7 5 5 0 2 5 0 M e a n p l a s m a G H l e v e l s in f e m a l e r a t s 5 2 0 D o s e ( m g / k g ) • - •1 BHHH 11111111 l l l l l - H | 2 0 0 O) c X CD 1 5 0 1 2 5 1 0 0 7 5 5 0 2 5 0 N a d i r p l a s m a G H l e v e l s in f e m a l e r a t s 3r. 5 2 0 D o s e ( m g / k g ) 2 0 0 3 0 0 " 'ml) 2 5 0 " CO tz 2 0 0 " X 1 5 0 " O 1 0 0 " 5 0 -0 J P e a k p l a s m a G H l e v e l s in f e m a l e r a t s I;.;;.; i S i i i i i l 5 2 0 D o s e ( m g / k g ) — 2 0 0 Figure 39: Mean, nadir, and peak plasma G H levels of female rats treated with various dosages of tamoxifen. Error bars indicate the standard error of the mean. Each bar represents the mean value of G H determinations from three rats except for the bar of the 5 mg/kg dosage group, representing G H determinations from a single rat. Nadir plasma G H levels were determined by calculating the average of the five lowest G H concentrations. Peak plasma G H levels were determined by calculating the average o f the two highest G H concentrations in two distinct peaks. If only one identifiable G H peak was present, this G H concentration was taken as the peak plasma G H level. If no peak was present, the two highest G H concentrations were taken as the peak plasma G H level. * Mean value of the treatment group is statistically different (*=/?< 0.05) from that of the control group. 83 Results 150-f 125 o) 100-I 75-° 50-25" 0 -M e a n p l a s m a G H l e v e l s i n m a l e r a t s D o s e ( m g / k g ) 200 O) c X CD 150-125" 100-75" 50" 25" 0 -N a d i r p l a s m a G H l e v e l s in m a l e r a t s • • • • • 1 lIlliBllii 1111111 11111111111 ': - ViJ • B i D o s e ( m g / k g ) 200 600-— 500-E TO 400 CD 300" 200" 100" 0 -P e a k p l a s m a G H l e v e l s in m a l e r a t s o D o s e ( m g / k g ) 200 Figure 40: Mean, nadir, and peak plasma G H levels of male rats treated with tamoxifen. Error bars indicate the standard error of the mean. Each bar represents the mean value of G H determinations from three rats. Nadir plasma G H levels were determined by calculating the average of the five lowest G H concentrations. Peak plasma G H levels were determined by calculating the average of the two highest G H concentrations in two distinct peaks. If only one identifiable G H peak was present, this G H concentration was taken as the peak plasma G H level. If no peak was present, the two highest G H concentrations were taken as the peak plasma G H level. * Mean value of the treatment group is statistically different (*=/?< 0.05) from that of the control group. 84 Results Section B 3.7. Synthesis of Tamoxifen Metabolites I intended to synthesize two of the major tamoxifen metabolites that are not available commercially (i.e., A'-desmethyltamoxifen and tamoxifen-A^-oxide) for use as standards in an H P L C assay for detection and quantitation of tamoxifen and its metabolites in rat plasma. The synthesis of A^-desmethyltamoxifen was based on a method for the synthesis of N-demethylhydromorphone (Montzka et al. 1974, Olofson et al. 1984, Rice 1975). The synthesis of tamoxifen-A^-oxide was based on methods by Foster et al. (1980) and McCague and Seago (1986). 3.7.1. Synthesis of A'-Desmethyltamoxifen /V-Desmethyltamoxifen was synthesized using chloroformate esters ((Montzka et al. 191 A, Olofson et al. 1984, Rice 1975). Positive electrospray mass spectrum (MS) analysis of the product revealed a base peak with a m/z value of 358.0, which corresponds to the molecular weight of Af-desmethyltamoxifen (F.W.: 357.53) and minor peaks (Figure 41). The yield of the product was < 1% of the initial amount of tamoxifen used as the starting material. Considerable decomposition and contamination probably occurred as indicated by the brown color of the reaction mixture and minor peaks in the mass spectrum of the reaction product of the N-desmethyltamoxifen synthesis (Figure 41). Because of the low yield of impure product, infrared (IR) and nuclear magnetic resonance ( N M R ) analyses were not conducted. 3.7.2. Synthesis of Tamoxifen-A -^oxide Tamoxifen-iV-oxide was synthesized using platinum oxide-catalyzed oxidization of tamoxifen with hydrogen peroxide (Foster et al. 1980, McCague and Seago 1986). M S - (Figure 42) IR- (Figures 43 and 44) and N M R - (Table 17, Figure 45) analysis of the product confirmed its identity with tamoxifen-A /-oxide and showed very little contamination. The yield was 170.7 mg tamoxifen-iV-oxide, which corresponds to 65.4% of the initial amount of tamoxifen used as the starting material. The melting point of the product (84-86° C) was lower than the melting point of tamoxifen-Af-oxide (134-136° C) as reported by Foster et al. (1980). 85 Results 100-%4 O — C H 2 — C H 2 — N — H N-Desmethyltamoxifen F.W.: 357.53 poo.o 110.8 231.0 129.8139.6 187.0 2010 x 2 4 5 1 358.0 359.1 430.1 I360.2 3 8 6 2 289.2 j t ' ^ ^ i ^ i l i ^ . f t . M < v k T i \ ,4q\fl%#fii>fanM,<U<\ Date r 3 Figure 41: Positive electrospray mass spectrum of the product of the A -^desmethyltamoxifen synthesis. 86 Results 100-, 388.1 O t O — C H 2 — C H 2 — N — C H 3 C H . Tamoxifen-N-Oxide F.W.: 387.53 389.1 V I I l| > f |V f l I f f lTf| 11 tl i At I p li I " i " " f ' ^ i ^ " r 390.1 ^ Da/el Figure 42: Positive electrospray mass spectrum of the product of the tamoxifen-/V-oxide synthesis. 87 Results 76.741 5883 2788 397 Wavenumber (cm*1) Figure 43: IR spectrum of tamoxifen. Wavenumber (cm1) Figure 44: IR spectrum of the product of the tamoxifen-A'-oxide synthesis. 88 Results -N-(CH3)2 O — CH? — C H 2 — N — CHo I CHo aromatic -CH2-CH3 -0-CH2-CH2-N-H A B system -Q-CH2-CH2-N-J i l l -CH2-CH3 TMS H20 / H202 -1 i i i I | — n • » i | I I i \ •] -' I 1 ' ' "| 1 1 1 1 I 5 4 1 ' • I ' 0 B o O -N-(CH3)2 t i TMS O — C H 2 — C H 2 — N — C H 3 C H 3 aromatic H AB system _o-CH2-CH2-N-0-CH2-CH2-N-J j J H20 / H202 -CH2-CH3 JL -CH2-CH3 i • i i i i i 7 'I 1 1 1 1 I 1 1 ' ' I 1 ' • i ! i • i i I i i i i ! i 6 5 4 3 ppm • i ' ' • > •<-2 i I i » T T T T T T r j r - r -1 0 Figure 45: NMR proton spectrum of (A) tamoxifen and (B) the product of the tamoxifen-N-oxide synthesis. TMS = tetramethylsilane (standard). 89 Results Table 17: Chemical shift values in the NMR proton spectrum for tamoxifen and tamoxifen-jV-oxide. Chemical shift values (ppm) Tamoxifen Tamoxifen-N-oxide found (Foster et al. 19801 found (Foster et al. 19801 -CH2-CH3 0.92 0.88 0.92 0.91 H 2 0 2 (?) 1.90 -N-(CH,)2 2.28 2.24 3.25 3.25 CH ? -CH 3 2.45 2.44 2.46 2.45 -O-CH2-CH7-N- 2.64 2.60 3.58 3.60 -0-CH ?-CH 2-N- 3.92 3.88 4.44 4.40 aromatic H 6.56 and 6.76 6.50 and 6.76 6.55 and 6.79 6.49 and 6.75 (AB-system) aromatic H 7.1 -7.38 (7.09 - 7.24) 7.10-7.38 7.09 - 7.24 90 Results 3.8. H P L C Assay for Tamoxifen and Its Metabolites 3.8.1. Validation of the H P L C Assay for Tamoxifen and Its Metabolites To validate the H P L C assay for the detection and quantitation of tamoxifen and its metabolites in plasma, the recovery, linearity, variability, sensitivity, and specificity of the assay were determined. The recovery of tamoxifen and its metabolites was determined as percentage of peak area ratios ( A U C / A U C IS) from a standard mixture of tamoxifen and its metabolites added before extraction as compared to peak area ratios of the components of the standard mixture added after extraction. One set of control plasma (0.35 ml) was spiked with 20 pi o f a 5, 25, 50, 100, or 250 u M mixture o f tamoxifen, its metabolites and 20 p i of 100 u M internal standard (cis and trans clomiphene racemate). A second set of control plasma (0.35 ml) was spiked with 20 p i o f methanol instead of the mixture. Plasma was extracted as described in the experimental section. To the second set of plasma that was spiked with methanol, 20 pi of a 5, 25, 50, 100, or 250 u M mixture of tamoxifen and its metabolites and 20 p i of 100 u M internal standard were added. The solvent was evaporated under a gentle stream of nitrogen and the residue was redissolved in 200 p i of methanol. A volume of 50 pi was injected onto the column. The percent recoveries among tamoxifen and its metabolites were not significantly different from each other except for the Z isomer of 4-hydroxytamoxifen. The recovery of this metabolite (82.5 ± 1.6%) was significantly less than the recovery of the E isomer o f 4-hydroxytamoxifen (96.2 ± 1%) and the recovery of tamoxifen (96.6 ± 2.5%). The recovery of tamoxifen-N-oxide was (85.5 ± 5%). The average recovery of tamoxifen and its metabolites was 90.2 ± 2.5%. Plasma (0.3 ml) of untreated rats containing 20 p i o f a racemic mixture o f Z and E 4-hydroxytamoxifen, tamoxifen-N-oxide, and tamoxifen standards at concentrations of 5, 25, 50, and 100 u M and 20 p i of cis and trans clomiphene (100 uM) were analyzed by H P L C and calibration curves were generated. These concentrations correspond to plasma concentrations of 0.3125, 1.5625, 3.125, and 6.25 nmol/ml. Calibration curves were linear over the investigated concentration range (Figure 46). The concentrations of tamoxifen and its metabolites detected in rat plasma were within the linear range of the calibration curves. The specificity of the assay is shown by the H P L C chromatogram. The chromatogram o f the zero-time incubation revealed the presence of up to four small peaks, one o f which had the same retention time as tamoxifen-N-oxide (Figure 15). The remaining three peaks did not interfere with the peaks of tamoxifen, its metabolites, or the internal standard (Figures 16 and 17). 91 Results The intra-assay variability of the assay expressed as the coefficient of variation (standard deviation/mean value x 100%) was determined from five calibration curves generated on one day. Only two concentrations (25 and 250 pM) were tested. The intra-assay variability was below the acceptable limit of 15% (Table 18). The overall intra-assay variability was 6.4%. The inter-assay variability was determined from six calibration curves produced on six different days. Only two concentrations (25 and 250 pM) were tested. The inter-assay variability was below the acceptable limit of 15% (Table 19). The limit of quantitation of the assay (smallest amount of metabolite that can be quantified with a coefficient of variation less than 20%) were at approximately 1.56 nmol/ml (25 pM). Concentrations below 1.56 nmol/ml (25 pM) were not tested, but in one assay in which lower concentrations of tamoxifen and its metabolites were used, tamoxifen could be detected at a concentration of 0.6 nmol/ml (10 pM) and all tamoxifen metabolites could be detected at a concentration of 0.3 nmol/ml (5 pM). 92 Results Table 18: Intra-assay variability of the HPLC assay for tamoxifen and its metabolites in rat plasma. Concentration of standards Z 4-Hydroxy-tamoxifen Coefficients of variation (%) E 4-Hydroxy- Tamoxifen-tamoxifen ./V-oxide Tamoxifen 25 uM 3.3 12.3 7.3 2.9 250 pM 5.8 9 5.7 5 Table 19: Inter-assay variability of the HPLC assay for tamoxifen and its metabolites in rat plasma. Concentration Coefficients of variation (%) of standards Z 4-Hydroxy- E 4-Hydroxy- Tamoxifen- Tamoxifen tamoxifen tamoxifen A/-oxide 25 pM 7.9 14 10.6 13.5 250 pM 7.6 7.8 5.8 4.8 93 Results Tamox i fen - /V -ox ide co o < i o _Q JS CD o 3 < 0 2 4 6 P l a s m a Concentrat ion (nmol/ml) CO o 3 < o JS CD •> O < T a m o x i f e n y = 0 . 7 1 3 3 x - 0 . 0 5 0 2 4 6 P l a s m a Concentrat ion (nmol/ml) Figure 46: Representative calibration curves for Z 4-hydroxytamoxifen, E 4-hydroxytamoxifen, tamoxifen-/V-oxide, and tamoxifen. 94 Results 3.8.2. Detection and Quanti tat ion of Tamoxifen and Its Metabolites in Ra t Plasma Plasma samples collected from female rats treated with tamoxifen at dosages of 50, 100, and 200 mg/kg at 1, 12, 24, and 36 days after treatment have been analyzed. The presence of N-desmethyltamoxifen in plasma was not determined because a highly purified standard was not available, either commercially or as a result of our efforts at chemical synthesis. Previous reports (Fried and Wainer 1994, L i m et al. 1994, MacCal lum et al. 1996, Manns and Brown 1997) indicate that A'-desmethyltamoxifen elutes from the H P L C column, under similar assay conditions, after 4-hydroxytamoxifen and before tamoxifen. N o peak corresponding to N-desmethyltamoxifen was detected in any chromatogram. In some o f the plasma samples from control rats, a small unidentified peak was present that eluted with the same retention time as tamoxifen-A /-oxide. This peak was detected occasionally in chromatograms from plasma samples of rats treated with tamoxifen at dosages of 0, 50 and 100 mg/kg at all time points, but the size, shape, and area of this peak was not different from the corresponding peak in chromatograms of plasma samples of control rats. Interfering peaks were present in chromatograms of plasma samples from rats treated with tamoxifen at a dosage of 200 mg/kg taken at all time points and in chromatograms of plasma samples from rats treated with tamoxifen at a dosage of 100 mg/kg taken at day one and at day twelve. Therefore, chromatograms from these plasma samples were not analyzed for content of tamoxifen and tamoxifen-A^-oxide. Concentrations of tamoxifen and 4-hydroxytamoxifen were derived from corresponding calibration curves (Figure 46). A racemic mixture of approximately 30% E and 70% Z isomer of and 4-hydroxytamoxifen was commercially available for use as a standard. Previous reports indicate that under similar assay conditions the Z isomer elutes before the E isomer (Manns 1989). The Z 4-hydroxytamoxifen calibration curves were adjusted for the percentage of this isomer in the racemic mixture. Several assay conditions were tested to optimize the assay. F low rates ranged from 0.6 to 1 ml/min. The content of methanol in the mobile phase was changed from 76-95%. The content o f water in the mobile phase was changed from 19.9 to 23.9%. The presence of 0.1 % (v/v) triethylamine in the mobile phase leading to a p H of 10.5 was critical for eluting tamoxifen and its metabolites from the column. Tamoxifen, its metabolites, and the internal standard could not be eluted from the column without the presence of triethylamine in the mobile phase. The H P L C detector wavelengths tested were 243 nm, 254 nm, and 210 nm. The extraction solvents tested were dichloromethane, hexane, and 2% n-butanol in hexane. Assay conditions resulting in the 95 Results best separation of tamoxifen, its metabolites, and the internal standard are as follows: a flow rate of 0.75 ml/min; a mobile phase consisting of methanol:watentriethylamine at 80%:19.9%:0.1% (v/v/v) (pH 10.5); a monitoring wavelength of 243 nm; and an extraction solvent consisting of 2% n-butanol in hexane. Using these assay conditions, tamoxifen was detected in plasma of all six rats treated with tamoxifen at a dosage of 50 mg/kg at one day after treatment and in two of the rats at twelve days after treatment. Tamoxifen was no longer detected in plasma of any rat treated with tamoxifen at a dosage of 50 mg/kg at 24 or 36 days after treatment (Appendix Table VI, Figure 47). 4-Hydroxytamoxifen was detected in plasma of most of the rats treated at dosages of 200, 100, and 50 mg/kg at one day after treatment. Twelve days after treatment, this metabolite was still detectable in plasma of some rats treated with tamoxifen at dosages of 200 and 50 mg/kg. Twenty-four days after treatment, 4-hydroxytamoxifen was detected in plasma of three of six rats treated with tamoxifen at a dosage of 200 mg/kg. Thirty-six days after treatment, this metabolite was still detected in plasma of five of six rats treated with tamoxifen at a dosage of 200 mg/kg. 4-Hydroxytamoxifen was not detected in plasma of rats treated with tamoxifen at dosages of 50 or 100 mg/kg at 24 or 36 days after treatment (Appendix Table VII, Figure 48). 96 Results o E c c o cz CD O CZ o o ro E in TO CL-I O 0.8 0.6 0.4 0.2 0 D o s a g e : 50 mg /kg 12 24 T i m e (days) 36 Figure 47: Plasma concentrations of tamoxifen in female rats treated with tamoxifen at a dosage of 50 mg/kg. Error bars indicate the standard error of the mean, n = 6 for day 1, n = 2 for day 12. D o s a g e : 100 mg /kg E 0.10 o E 0.08 c c o 0.06 ro i c 0.04 Q) CJ c o 0.02 o CO E 0 w co C L 1 12 24 36 T i m e (days) D o s a g e : 50 m g / k g o E cz cz o c CD o cz o o CO E m jo CL o E cz c g co i _ c CD o c o o ro E ID ro C L 0.10 0.08 0.06 0.04 0.02 0 1111 L 12 24 T i m e (days) D o s a g e : 2 0 0 m g / k g 0.10 -j 0.08 -0.06 -0.04 -0.02 111 0 mi 12 24 T i m e (days) 36 36 Figure 48: Plasma concentrations of Z 4-hydroxytamoxifen in female rats treated with tamoxifen at dosages of 50, 100, and 200 mg/kg. Error bars indicate the standard error of the mean. For rats treated with tamoxifen at a dosage of 200 mg/kg: n = 4 for day 1, n = 1 for day 12, n = 3 for day 24, n = 5 for day 36. For rats treated with tamoxifen at a dosage of 100 mg/kg: n = 4 for day 1. For rats treated with tamoxifen at a dosage of 50 mg/kg: n = 3 for day 1, n = 1 for day 12. 97 Discussion 4. D I S C U S S I O N The present study investigated the effects of tamoxifen on catalytic activities and protein levels of hepatic microsomal C Y P enzymes and on plasma G H levels in Long Evans rats at five weeks after treatment. A n effort was made to assess whether tamoxifen or one of its metabolites was present in plasma over the investigated time period. A previous study reported that administration of tamoxifen at 5 mg (16-23 mg/kg b.w) for two consecutive days suppressed G H levels in male and female rats up to seven weeks after administration (Tannenbaum et al. 1992). Therefore, we hypothesized that tamoxifen treatment has a prolonged effect on hormonally regulated hepatic C Y P enzymes in rats. Another study reported previously that hepatic protein-, m R N A - , and activity-levels of C Y P 2 B 1 , C Y P 2 B 2 , and C Y P 3 A enzymes were increased two- to three-fold in female rats within 24 h after administration of tamoxifen at 45 mg/kg b.w. for 4 days (White et al. 1993). The effects of tamoxifen investigated in the present study were assumed to be different from these direct short-term effects o f tamoxifen on C Y P enzyme expression. Hepatic microsomal activities and protein levels of C Y P 2 A 1 , C Y P 2 B 1 , C Y P 2 B 2 , C Y P 2 C 1 1 , and C Y P 3 A enzymes were examined. These C Y P enzymes include those induced by tamoxifen directly. C Y P enzymes examined in this study also include two sexually dimorphic enzymes, female predominant C Y P 2 A 1 and male specific C Y P 2 C 1 1 . Expression of these enzymes is controlled by G H and sex steroids. Section A 4.1. Development of Subcutaneous Cysts at the Injection Site Subcutaneous cysts developed at the injection site in rats treated with tamoxifen at dosages of 20 mg/kg and greater. Cysts were examined when rats were kil led 36 days after treatment. Cysts smaller than 1.5 cm in diameter contained mostly scar tissue, while cysts larger than 1.5 cm in diameter contained peanut o i l in most cases. Large cysts (> 1.5 cm in diameter) were found only in female rats that received tamoxifen at dosages of 100 and 200 mg/kg and in male rats that received tamoxifen at 200 mg/kg. The cysts did not appear to cause pain to the rats and rats with cysts did not behave differently than rats without cysts. The development of subcutaneous cysts was not related to the injection volume as the volume was based on body weight of the rats and 98 Discussion not on the concentration of tamoxifen administered. The development of subcutaneous cysts is likely a result of inflammatory processes in the skin. Some rats lost the fur around the cysts, while the fur of other rats with cysts was oily at the site of injection indicating loss of some of the injected tamoxifen preparation (Table 5). Therefore, the dose these rats received was probably smaller than the dose received by rats that did not show evidence of oily fur at the site of injection. In rats with cysts, tamoxifen could have been released slowly leading to a prolonged presence of tamoxifen and its metabolites in plasma following treatment at dosages of 100 and 200 mg/kg. Development of subcutaneous cysts might lead to changes in the pharmacokinetics of the drug. However, the effect of tamoxifen treatment on body weight and hepatic C Y P enzymes was not statistically different between groups treated at dosages of 20 mg/kg or greater. Therefore, the development of cysts in rats treated with tamoxifen at 100 and 200 mg/kg does not appear to have influenced body weight gain and hepatic C Y P enzyme expression. Average peak plasma G H levels were higher and more irregular in rats treated with tamoxifen at 200 mg/kg. It can be speculated that this might be a reflection of the development of subcutaneous cysts but not enough data are available for a conclusive decision on this matter. 4.2. Effect of Tamoxifen on Body Weight and Liver Weight The effect of tamoxifen on body weight was investigated, because a decrease in body weight gain could be an indication of an effect of tamoxifen on plasma G H levels. Alternatively, this effect can be interpreted as a result of treatment on food intake. Male and female rats treated with tamoxifen at all dosages investigated gained significantly less weight than rats in the control group. A t day 36 after treatment, the body weight gain of tamoxifen treated female and male rats was 40% and 5%, respectively, of the body weight gain of control rats. In a recent study, administration of tamoxifen (0.5 mg/kg/day, s.c. for 30 days) was reported to have no effect on food intake in ovariectomized methimazole-induced hypothyroid rats but tamoxifen opposed the growth stimulating effect of T 3 administration (10 pg/kg s.c. for 30 days) (Fitts et al. 1998). In an earlier study, tamoxifen treatment (250 pg/day s.c. for 34 days) decreased food intake, body weight gain, and body fat content in ovariectomized rats. This effect was similar to the effect of estradiol (2 pg/day s.c. for 34 days) (Wade and Heller 1993). Susan Holsmer reported in her thesis conducted in Dr. Bandiera's laboratory that, five weeks after treatment, tamoxifen (5 mg/day s.c. for two days) decreased body weight gain of tamoxifen treated female and male rats to 40% and 13%, respectively, relative to control rats (Holsmer 99 Discussion 1995). The magnitude of the suppressive effect on body weight gain in tamoxifen treated rats in all three studies was similar to the magnitude found in the present study. Whether the weight gain suppression by tamoxifen reported in the present study can be interpreted as a behavioral change of the rats with regards to food intake remains uncertain, as food intake was not controlled. Three other mechanisms could have caused body weight gain suppression in tamoxifen treated rats. First, tamoxifen could have had a suppressive effect on GH secretion as reported by Tannenbaum et al. (1992). Previous studies showed that, body weight gain decreases when plasma GH levels are decreased in dwarf rats (Charlton et al. 1988), by hypophysectomy (Waxman et al. 1991), or by neonatal administration of MSG (Pampori et al. 1991). Second, tamoxifen could have opposed the growth stimulating effect of T 3 as reported by Fitts et al. (1998). Third, the weight gain suppression caused by tamoxifen treatment could be interpreted as an estrogenic effect of the drug as reported by Wade and Heller (1993). Regardless of the mechanism, the effect of tamoxifen treatment on body weight gain was probably an indirect long-term effect of tamoxifen that persisted over the five week investigation period. Alternatively, tamoxifen or a tamoxifen metabolite might have caused the effect directly as a result of its presence in plasma or tissue for an extended period of time, especially when tamoxifen was administered at higher dosages. The effect of tamoxifen on liver weight was investigated because repeated treatment with various drugs (e.g. ethanol or phenobarbital) can cause liver enlargement in rats (Waxman and Azaroff 1992, Perez 1972). The relative liver weight of male and female rats in all treatment groups investigated in this study was not different from that of control rats except for a slight decrease in the 20 mg/kg treatment group of female rats. Thus, tamoxifen treatment had no effect on liver weight. 4.3. Effect of Tamoxifen on Microsomal CYP Enzymes Total protein content, total CYP content, CYP-mediated enzyme activities, and CYP protein levels were measured in hepatic microsomes prepared from rats 36 days after receiving tamoxifen s.c. at dosages ranging from 0.5 to 200 mg/kg for two consecutive days. Tamoxifen treatment had no effect on total microsomal CYP content in male and female rats. Administration of tamoxifen to adult female rats at dosages greater than 20 mg/kg resulted in a 40% decrease of testosterone 7a-hydroxylase activity and a 35% decrease of hepatic microsomal CYP2A1 protein content. Testosterone 7ct-hydroxylation is catalyzed by CYP2A1 in the liver of female rats (Ryan and Levin 1993). The same treatment had no effect on testosterone 100 Discussion 20, 60-, 16a-, and 160-hydroxylase activities and on the hepatic microsomal content of C Y P 2 B 1 , C Y P 2 B 2 , and C Y P 3 A enzymes. Administration of tamoxifen to adult male rats at a dosage of 200 mg/kg resulted in a 70% increase of testosterone 7a-hydroxylase activity. The increase of testosterone 7a-hydroxylase activity was not paralleled by an increase in hepatic microsomal C Y P 2 A 1 protein levels in male rats. The reason for this discordance remains unexplained and could be of statistical nature. The sample size may have been too small to pick up a significant difference between the treatment group and the control group. However, a similar discordance in testosterone 7a-hydroxylase activity and C Y P 2A1 protein levels has been reported previously (Waxman et al. 1989a). In that study, testosterone 7a-hydroxylase activity was unaffected in hepatic microsomes of hypophysectomized adult male rats, but C Y P 2 A 1 protein levels were elevated 3-fold. In hepatic microsomes of hypophysectomized adult female rats, testosterone 7a-hydroxylase activity was decreased by 20%, but C Y P 2 A 1 protein levels were unchanged (Waxman et al. 1989a). Tamoxifen treatment also resulted in a long-term 44% decrease in testosterone 2a - and 16a-hydroxylase activities and a 30%) decrease in the hepatic microsomal protein levels o f male specific C Y P 2 C 1 1 . Testosterone 2a- and 16a- hydroxylation is catalyzed by C Y P 2 C 1 1 in the liver of male rats (Ryan and Levin 1993). C Y P 2 B 1 also hydroxylates testosterone at the 16a-position but C Y P 2 B 1 levels were low in tamoxifen treated rats and C Y P 2 B enzymes do not contribute to a significant extent to the overall testosterone 16a-formation in rats not treated with phenobarbital-type inducers. Tamoxifen treatment had no effect on testosterone 20-, 60-, and 160-hydroxylase activities and on the hepatic microsomal content of C Y P 2 A 2 , C Y P 2 B 1 , C Y P 2 B 2 , and C Y P 3 A enzymes. These results are different from those of a previous study by Susan Holsmer conducted in Dr. Bandiera's laboratory investigating the effect of tamoxifen on hepatic C Y P enzymes (Holsmer 1995). In that study, tamoxifen treatment (5 mg/day s.c. for two days) was reported to increase testosterone 60-hydroxylase activity by 63% and to decrease testosterone 7a-hydroxylase activities by 42%) in female rats five weeks after administration. The same treatment decreased the hepatic microsomal C Y P 2 A 1 content by 30% in female rats but had no effect on C Y P 2 C 7 , C Y P 2 E 1 or C Y P 3 A protein levels or on hepatic microsomal p-nitrophenol hydroxylase activity, used as a marker for C Y P 2 E 1 , or on pentoxyresorufin-O-depentylase activity. Tamoxifen treatment had also no effect on testosterone hydroxylase activities in male rats five weeks after administration. The lack of effect in male rats might be attributed to the ten-fold lower dosage of 101 Discussion tamoxifen used in that study in comparison to the present study. More recently, the effects of neonatal tamoxifen treatment on C Y P enzyme expression in adult rats have been investigated (Kawai et al. 1999). Tamoxifen treatment (20 pg s.c. for five days) decreased testosterone 60- and 7a-hydroxylase activities in hepatic microsomes from female rats by 68% and 23%, respectively. C Y P 2 A 1 and C Y P 3 A protein levels were not affected by tamoxifen treatment but C Y P 3 A 9 m R N A levels and 170 estradiol levels were decreased in female rats. The same treatment increased testosterone 7oc-hydroxylase activities 2.6-fold and increased C Y P 2 A 1 protein levels by 58% in hepatic microsomes from male rats. Testosterone 2a-hydroxylase activities and CYP2C11 levels were decreased by 36% and 26%, respectively, after tamoxifen treatment. Tamoxifen treatment had no effect on steroid 5a-reductase activity in male or female rats or on serum testosterone levels in male rats. In summary, the long-term effects of tamoxifen on C Y P 2 A 1 and C Y P 2 C 1 1 activities and protein levels in the liver of male and female rats are relatively consistent between different studies although activities and protein levels do not always correlate. Tamoxifen treatment results in a modest decrease o f hepatic C Y P 2 A 1 activities and protein levels in female rats. In male rats, tamoxifen treatment increases hepatic C Y P 2 A 1 activities and protein levels and decreases C Y P 2 C 1 1 activities and protein levels. The effects of tamoxifen on testosterone 60-hydroxylase activities are discordant between studies. This difference could reflect the contribution of several C Y P enzymes ( C Y P 1 A 1 , C Y P 1 A 2 , C Y P 2 C 1 3 , and C Y P 3 A ) to overall testosterone 60- hydroxylase activity. In conclusion, tamoxifen treatment does not affect total hepatic C Y P 3 A protein levels but decreases C Y P 3 A 9 levels selectively as indicated by decreased hepatic m R N A levels of C Y P 3 A 9 (Kawai et al. 1999). C Y P 2 C 7 , C Y P 2 E 1 , C Y P 2 B 1 , and C Y P 2 B 2 enzymes are not affected by tamoxifen treatment. The effect of tamoxifen treatment on hepatic microsomal C Y P enzymes is believed to be caused by the drug. In the present study, tamoxifen decreased body weight gain in male and female rats. In previous studies, food deprivation for 48 h increased hepatic microsomal levels of C Y P 2 A 1 , C Y P 2 B 1 , C Y P 2 D 6 , C Y P 2 E 1 , and C Y P 3 A and decreased hepatic microsomal levels of C Y P 1 A 2 , C Y P 2 B 2 , C Y P 2 C 1 1 , and C Y P 2 C 1 3 (Imaoka et al. 1990). Hepatic microsomal levels of C Y P 1 A 1 , C Y P 2 A 2 , and C Y P 2 C 6 were not affected in male rats after food deprivation for 48 h (Imaoka et al. 1990). In female rats, food deprivation for 48 h increased hepatic microsomal levels of C Y P 3 A (Cheesman and Reil ly 1998). Thus, it may be argued that the effect of tamoxifen treatment on hepatic microsomal C Y P enzymes could be a result of caloric intake 102 Discussion restriction. However, tamoxifen treatment decreased body weight gain of female rats in the 0.5 and 5 mg/kg treatment groups but had no effect on hepatic microsomal C Y P activities and protein levels in these treatment groups. Therefore, the effect of tamoxifen treatment on hepatic microsomal C Y P enzymes is not believed to be a result of a possible change in the caloric intake of the rats. 4.4. Effect of Tamoxifen on GH Plasma Levels To investigate i f the effects of tamoxifen treatment on body weight and hepatic C Y P enzymes could be correlated to its effect on G H , G H plasma levels were determined in male and female rats treated with tamoxifen and compared to those in control rats. The apparent concentrations of G H in rat plasma samples measured by using an 1 2 5 I radioimmunoassay for human G H ranged between 1.3 and 2.7 ng/ml. These G H levels are unusually low in comparison to literature G H peak values of 200-800 ng/ml in male rats (Tannenbaum et al. 1992, Eden 1979) and can be regarded as baseline noise. Therefore, the 1 2 5 I radioimmunoassay for human G H was not useful for determination of G H in rat plasma but frequent blood sampling from the tail vein was shown to be feasible and tolerated by the animals. The use of an enzyme-linked immunoassay for rat G H was more successful in measuring plasma G H levels in rats. Mean and nadir G H plasma levels were not affected after administration of tamoxifen at dosages of 5, 20, and 200 mg/kg to female rats and at a dosage of 200 mg/kg to male rats. In contrast, peak plasma G H levels were decreased significantly by approximately 43% and 60%, respectively, as compared to the control group after administration of tamoxifen at a dosage of 20 mg/kg to female rats and at dosage of 200 mg/kg to male rats. The effects of tamoxifen on plasma G H levels in adult male rats determined in the present study are comparable with the previously reported effects of gonadectomy in adult male rats (Painson 1992) indicating that tamoxifen might influence testosterone plasma levels in adult male rats. However, testosterone levels were not measured in the present study. G H profiles of male and female control rats determined in this study are comparable to those reported in previous studies, in which a radioimmunoassay for rat G H supplied by the National Institute of Arthritis, Metabolism and Digestive Disease ( N I A M D D , Bethesda, M D , U S A ) was used (Tannenbaum and Mart in 1976, Eden et al. 1978, Eden 1979, Sonntag et al. 1980, Mode et al. 1982, Jansson et al. 1984 and 1985, Tannenbaum and Ling 1984, Jansson and Frohman 1987, MacLeod et al. 1991, Tannenbaum et al. 1992, Painson et al. 1992, Kerrigan and Rogol 1992). In one study (Kerrigan and Rogol 1992), G H concentrations were measured in serum from male 103 Discussion rats and ranged between nadir values of 2 ng/ml and peak values of 70 ng/ml. In the remainder of the studies mentioned above, G H concentrations were measured in plasma and ranged between nadir values of 2 ng/ml and peak values of 900 ng/ml (usually at approximately 300 ng/ml) in male rats. G H concentrations in plasma from female rats ranged between nadir values of 2 ng/ml and peak values of 400 ng/ml (usually at approximately 250 ng/ml). Plasma G H profiles from female rats are not apparently different when sampled during the day or night (Eden and Wikland 1978). Constant light conditions in the animal quarters also had no effect on plasma G H profiles in male rats (Tannenbaum and Martin 1976). G H profiles determined from plasma sampled from the tip of the tail (Eden and Wikland 1978, Eden 1979, Mode et al. 1982) are not apparently different from G H profiles determined from plasma sampled by an intracardiac venous canula and re-injection of resuspended red blood cells (Jansson and Frohman 1987, Painson et al. 1992, Tannenbaum et al. 1992, Tannenbaum and Ling 1984, Tannenbaum and Mart in 1976, Sonntag et al. 1980) or without re-injection of resuspended red blood cells (Jansson et al. 1984). Peak plasma G H values determined from blood sampled by an intracardiac venous canula with re-injection of resuspended red blood cells were generally constant between rats within one study but varied largely between studies. In adult male rats, peak plasma G H values ranged between 200 ng/ml (Tannenbaum et al. 1992) to 800 ng/ml (Tannenbaum and L ing 1984) showing a large variation between studies conducted by the same research group. In adult female rats, peak plasma G H values were generally more constant at 100-200 ng/ml (Tannenbaum et al. 1992, Painson etal. 1992). In conclusion, the blood sampling technique used in the present study did not effect normal plasma G H levels in male and female control rats. However, the high nadir values determined in the present study, especially in plasma G H profiles from male rats, were never observed in any of the studies mentioned above and must be interpreted as a specific artifact of the enzyme-linked immunoassay for rat G H . These high nadir values could be explained by the fact that the anti-rat G H IgG provided in the enzyme-linked immunoassay cross-reacted with an unidentified protein in rat plasma and the assay did not allow for blank correction. Plasma from hypophysectomized rats could serve as a blank and would be useful to correct for the cross-reactivity of the anti-rat G H IgG. The anti-rat G H IgG reacted more strongly (approximately two-times) with rat G H provided by the National Institutes of Health of the U S A than with the rat G H standard provided by the manufacturer of the immunoassay kit (Table 14). Therefore, G H concentrations determined in this study could be artificially high because the anti-rat G H IgG reacted more strongly with G H in rat plasma in comparison to the rat G H standard. However, the 104 Discussion manufacturer provided no correction factor to account for the difference. The effect of tamoxifen treatment on plasma G H levels is believed to be caused by the drug. In the present study, tamoxifen decreased body weight gain in male and female rats. In a previous study, food deprivation for 24, 48, and 72 h progressively decreased plasma G H levels close to nadir levels in adult female rats (Tannenbaum et al. 1979). Thus, it may be argued that the effect of tamoxifen treatment on plasma G H levels could be a result of caloric intake restriction. However, tamoxifen treatment decreased body weight gain of female rats in the 5 mg/kg treatment group but had no effect on plasma G H levels in this treatment group. Therefore, the effect of tamoxifen treatment on plasma G H levels is not believed to be a result of a possible change in the caloric intake of the rats. Section B 4.5. Tamoxifen Metabolite Synthesis The synthesis of A^-desmethyltamoxifen was based on a method for A^demethylation of hydromorphone using chloroformate esters. Positive electrospray M S analysis of the product revealed a base peak with a m/z value corresponding to the molecular weight of N-desmethyltamoxifen and minor peaks (Figure 41). These minor peaks could be caused by contaminants of the final product as indicated by the brown color of the reaction mixture at the end of the coupling process with chloroformate esters indicating disintegration o f the parent compound or the reaction product. Alternatively, these peaks could represent fragments of N-desmethyltamoxifen that were generated during electrospray M S analysis. However, minor peaks did not appear in the M S spectrum of tamoxifen-N-oxide, which is assumed to be equally stabile as Af-desmethyltamoxifen under the chosen M S conditions. The yield of the product was < 1% of the initial amount of tamoxifen. Because of the low yield of probably impure product, N-desmethyltamoxifen could not be used as a standard for the H P L C assay for tamoxifen and its metabolites. The reaction conditions required to A^-demethylate tamoxifen were probably too harsh and caused too much degradation of the substrate to be a useful route of synthesis. The preferable way of synthesizing N-desmethyltamoxifen would probably be a three-step de novo synthesis using two precursors, i.e. 4-[2-(2-tetrahydropyranyloxy)ethoxy]phenyl bromide and 1,2-diphenyl-1 -butanone (Robertson et al. 1982). Tamoxifen-Af-oxide was synthesized using platinum oxide-catalyzed oxidization of 105 Discussion tamoxifen with hydrogen peroxide. M S - , IR-, and NMR-analyses of the product confirmed its identity with tamoxifen-N-oxide and showed very little contamination. The yield was 170.7 mg of tamoxifen-JV-oxide, which corresponds to 65.4% of the original amount of tamoxifen used. The lower melting point of the product (84-86° C) in comparison to literature values (134-136° C) was probably caused by remaining H2O or H2O2 in the crystal structure. This problem was reported previously by Foster et al. (1980). The presence of an O H group in the reaction product of the tamoxifen-/V-oxide synthesis was indicated in the IR spectrum by a broad peak at a wavenumber of approximately 3500-3900 cm"1 (O-H-stretch) (Figure 44). This peak was not found in the IR spectrum of tamoxifen (Figure 43). The presence of H2O or H2O2 in the crystal structure of tamoxifen-/V-oxide was also indicated by the greater size of peak " g " (1.9 ppm) in the N M R proton spectrum (Figure 45). The crystal-water could not be eliminated entirely by heating the tamoxifen-TV-oxide at 60° C over anhydrous calcium sulfate (Drierite®) for 24 h. The identity of tamoxifen-TV-oxide as the reaction product was confirmed by M S analysis and by comparing the chemical shift values of the N M R proton spectrum of tamoxifen-N-oxide (Table 16) to literature values (Foster et al. 1980). Synthesized tamoxifen-TV-oxide was used as a standard to determine the retention time of tamoxifen-A7-oxide in the H P L C assay for tamoxifen and its metabolites. 4.6. Detection and Quantitation of Tamoxifen and its Metabolites in Rat Plasma A n H P L C assay was developed for detection and quantitation of tamoxifen and its metabolites in plasma to asses i f the effects of tamoxifen treatment on C Y P enzymes could be correlated to the presence of tamoxifen or one of its metabolites in plasma over the investigated time period of five weeks. Tamoxifen was detected at concentrations of 0.75 nmol/ml and 0.23 nmol/ml in plasma of rats treated with tamoxifen at a dosage of 50 mg/kg at one and twelve days after treatment, respectively. Tamoxifen was no longer detectable in plasma of any rat treated with tamoxifen at 24 or 36 days after treatment (Table 19). 4-Hydroxytamoxifen was detected at an average concentration of 0.06 nmol/ml in plasma of most of the rats treated with tamoxifen at dosages of 200, 100, and 50 mg/kg at one day after treatment. A t twelve days after treatment, this metabolite was detected in plasma of some rats treated at 200 and 50 mg/kg. A t 24 and 36 days after treatment, 4-hydroxytamoxifen was still detectable in plasma of rats treated with tamoxifen at a dosage of 200 mg/kg. 4-Hydroxytamoxifen was not detectable in plasma of rats treated with tamoxifen at dosages of 50 or 100 mg/kg at 24 or 36 days after treatment. Previously reported 106 Discussion serum levels of tamoxifen and 4-hydroxytamoxifen (Robinson et al. 1991) are comparable with the results of the present study. Serum levels of tamoxifen were at approximately 2.69 nmol/ml one day after treatment and at nadir levels at four days after treatment in mature female rats treated with a single dose of 200 mg/kg p.o. (Robinson et al. 1991). Serum levels of 4-hydroxytamoxifen were approximately 0.2 nmol/ml at one day after treatment and at nadir levels at four days after treatment in mature female rats treated with a single dose of 200 mg/kg p.o. (Robinson et al. 1991). The limits of quantitation of the present H P L C assay were approximately 1.56 nmol/ml for tamoxifen and its metabolites. In one assay in which lower concentrations of tamoxifen and its metabolites were used, tamoxifen could be detected at a concentration of 0.6 nmol/ml and all tamoxifen metabolites could be detected at a concentration of 0.3 nmol/ml. Concentrations of tamoxifen in plasma of female rats treated with tamoxifen at a dosage of 50 mg/kg are above the limits of quantitation one day after treatment. A t all other times after treatment and at al l other dosages, plasma levels of tamoxifen and 4-hydroxytamoxifen are below the limits of quantitation and definite conclusions about their concentrations in plasma cannot be made. Limits o f quantitation of 0.03 nmol/ml, 50 times lower than in the present study, were reported for an H P L C assay using on-line U V photo-activation of tamoxifen and its metabolites with fluorescence detection (El -Yazig i and Legayada 1997). Limits of quantitation of 0.13-0.75 pmol/ml, approximately 1000 times lower than those determined in the present study, were reported for a G C - M S assay using selected ion monitoring of tamoxifen and its metabolites (Murphy et al. 1987) These alternative assays might be preferable for detection and quantitation of tamoxifen and its metabolites in plasma, as interfering peaks are less likely to appear and both assays have lower limits of quantitation. The H P L C assay developed in the present study is far from ideal. One limitation o f the assay is that pure Z 4-hydroxytamoxifen and A^-desmethyltamoxifen were not available and cannot easily be synthesized. Another limitation is that liquid/liquid extraction with 3% butanol in hexane contained contaminants as indicated by up to four small peaks in the H P L C chromatogram, one of which interfered with the peak corresponding to tamoxifen-Af-oxide. Therefore, tamoxifen-A /-oxide could not be quantified by the present H P L C assay, especially in small concentrations. The presence of A^-desmethyltamoxifen in plasma was not determined because a standard was not available, either commercially or from our effort o f chemical synthesis. Previous reports (Fried and Wainer 1994, L i m et al. 1994, MacCal lum et al. 1996, Manns and Brown 1997) indicate that N-desmethyltamoxifen elutes from the H P L C column, 107 Discussion under similar assay conditions, after 4-hydroxytamoxifen and before tamoxifen. N o peak corresponding to Af-desmethyltamoxifen was detected in any chromatogram but definite conclusions about the presence of iV-desmethyltamoxifen in rat plasma cannot be drawn from the results of this study. After metabolism of tamoxifen by rat hepatic microsomes the Z isomers of 4-hydroxytamoxifen and 4'-hydroxytamoxifen can be detected and separated by H P L C ( L i m et al. 1994). The amounts of 4'-hydroxytamoxifen produced were approximately half of the amounts of 4-hydroxytamoxifen (L im et al. 1994). It was therefore assumed in the present study that the peak that eluted with the same retention time as Z 4-hydroxytamoxifen was Z 4-hydroxytamoxifen and not Z 4'-hydroxytamoxifen. Because of the lack of a 4'-hydroxytamoxifen standard, a conclusive decision regarding the identity of that peak could not be made. Given all the limitations of the H P L C assay mentioned above, the concentrations of tamoxifen and its metabolites could not be determined accurately. However, the major purpose of this assay was to determine whether tamoxifen and its metabolites were present in plasma before preparation of microsomes. The H P L C assay served this purpose. The presence of 4-hydroxytamoxifen in plasma of rats 36 days after tamoxifen treatment at a dosage of 200 mg/kg can be interpreted as an indication that tamoxifen stayed in the rat body for a prolonged time. A dosage of 200 mg/kg corresponds to a blood concentration of approximately 12 pmol/ml, assuming a volume of 10 ml . The serum half-life of tamoxifen, A /-desmethyltamoxifen, and 4-hydroxytamoxifen in rats is 10.3 h, 12.1 h, and 17.2 h, respectively (Robinson et al. 1991). The serum half-life of tamoxifen in humans is approximately seven to ten days (Jordan 1990, Fuchs et al. 1996). This relatively long half-life is partly caused by binding to various proteins (Lerner and Jordan 1990). After five to seven half-lives (approximately three days), tamoxifen should be eliminated from rat plasma. In rats, approximately 39% of tamoxifen is metabolized to 4-hydroxytamoxifen ( L i m et al. 1994) and after five to seven half-lives (approximately five days), 4-hydroxytamoxifen should also be eliminated from rat plasma. The presence of 4-hydroxytamoxifen at a concentration of 0.063 nmol/ml in plasma of rats treated with tamoxifen at 200 mg/kg at 36 days after treatment may be explained by the development of cysts at the site of injection in some of the rats of the highest dosage groups (Table 5). Tamoxifen could have been released slowly from these cysts for a prolonged time leading to a prolonged presence of tamoxifen and its metabolites in plasma. However, based on the results obtained, it can be concluded that the presence of tamoxifen or 4-hydroxytamoxifen in plasma was not responsible for the observed effects of tamoxifen on hepatic C Y P enzymes. Although 4-hydroxytamoxifen 108 Discussion was detected in plasma of rats treated with tamoxifen at 200 mg/kg at 36 days after treatment, the effects o f tamoxifen on hepatic C Y P enzymes of rats in these treatment groups were not different from the effects on hepatic C Y P enzymes of rats in all other treatment groups. Furthermore, the effects of tamoxifen on hepatic C Y P enzymes presented in this study were different from the previously reported direct inductive effects of tamoxifen on hepatic C Y P 2 B and C Y P 3 A enzymes (White et al. 1993). Therefore, the effects observed herein were probably not caused by tamoxifen directly. It can be argued that the effects might have been caused by a tamoxifen metabolite present in liver over the investigated period. Concentrations of tamoxifen and tamoxifen metabolites at 24 h after the last dose (1 mg/kg p.o. for three or fourteen days) are reported to be 8- to 70-fold higher in tissues such as lung, liver, kidneys, fat, and brain (100-1000 ng/g tissue) in comparison to plasma concentrations (1-10 ng/ml) (Lien et al. 1991). Similar elimination rates of tamoxifen in serum and several tissues (except fat) and a similar distribution of tamoxifen metabolites in serum and tissues (except fat) suggest an exchange o f tamoxifen and its metabolites between serum and tissues (Lien et al. 1991). Given a serum half-life in rats of 17.2 h for 4-hydroxytamoxifen and plasma and tissue levels of 4-hydroxytamoxifen lower than those of tamoxifen at all times after administration, it seems unlikely that this tamoxifen metabolite could have stayed for more than five days in rat liver and caused the effect on hepatic C Y P enzymes in rats treated with tamoxifen at dosages of 20 and 50 mg/kg and did not develop cysts. However, definite conclusions about plasma concentrations of tamoxifen and 4-hydroxytamoxifen below the limits of quantitation of the H P L C assay (1.56 nmol/ml) cannot be made. Therefore, it cannot be ruled out that 4-hydroxytamoxifen has stayed in rat plasma or in tissues for 36 days after treatment at concentrations below the limits of quantitation o f the H P L C assay and caused the effect on hepatic C Y P enzymes. This possibility becomes even more important as 4-hydroxytamoxifen has an approximately 140 times greater affinity for the estrogen receptor than tamoxifen (Robertson et al. 1982). 4.7. Correlating the Effects of Tamoxifen on Hepatic C Y P Enzymes with Its Effect on G H Plasma Levels When correlating the results of tamoxifen treatment on plasma G H levels with its effects on hepatic microsomal C Y P enzymes, the question arises, whether the effect of tamoxifen on plasma G H levels could act as the mediator for the effects of tamoxifen on C Y P enzymes. In rat liver, the male specific G H secretory pattern induces C Y P 2 C 7 (Westin et al. 1990) and C Y P 2 C 1 1 (Shapiro et al. 1989, Waxman et al. 1991, Morgan et al. 1985, McClel lan-Green et al. 109 Discussion 1989) and suppresses C Y P 2 A 1 (Waxman et al. 1989a), C Y P 2 B 1 , C Y P 2 B 2 (Yamazoe et al. 1987), and C Y P 2 E 1 (Waxman et al. 1989a) (Table 2). The female-specific G H secretory pattern induces C Y P 2 A 1 (Pampori and Shapiro 1996), C Y P 2 C 7 (Westin et al. 1990, Pampori and Shapiro 1996), and C Y P 2 C 1 2 (MacGeoch et al. 1984 and 1985, Pampori and Shapiro 1996) and suppresses, C Y P 2 B 1 , C Y P 2 B 2 (Yamazoe et al. 1987), C Y P 2C11 (Morgan et al. 1985, Pampori and Shapiro 1996), and C Y P 2 E 1 (Waxman et al. 1989b) (Table 2). In male and female dwarf rats, G H concentrations are approximately 10% of those in normal male rats and approximately 6% of those in normal female rats, but the sexually dimorphic G H secretion pattern is essentially normal (Charlton et al. 1988). The sex-dependent and age-related changes in expression and activity levels of C Y P 2 A 1 , C Y P 2 A 2 , C Y P 2 C 1 1 , C Y P 2 C 1 2 , C Y P 3 A , and steroid 5a-hydroxylase in hepatic microsomes from male and female dwarf rats are not different from those in normal rats (Bullock et al. 1991). This discrepancy can be explained by the fact that the critical event that triggers enzyme expression is a trough interval period between G H pulses greater than 2.5 h with a G H concentration close to zero rather than the mean G H concentration, number of peaks, or peak height as shown for C Y P 2 C 1 1 (Waxman et al. 1991, Shapiro etal. 1989). The results from the plasma G H determinations observed in the present study do not allow us to determine i f nadir plasma G H levels were affected. The only apparent effect was a change in the peak plasma G H levels in male and female rats. If the observed effects of tamoxifen on hepatic C Y P enzymes were mediated by elevating plasma G H levels or disturbing the regular G H pulse interval, one would expect CYP2C11 levels to be decreased and C Y P 2 A 1 , C Y P 2 B 1 , and C Y P 2 B 2 levels to be increased in hepatic microsomes of male rats. If nadir plasma G H levels and peak plasma G H levels were decreased in male rats but the regular G H pulse interval was unaffected, there would be no effect on expression levels of C Y P enzymes. If the effects of tamoxifen on hepatic C Y P enzymes were mediated by lowering nadir plasma G H levels in female rats, then one would expect C Y P 2 A 1 levels to be decreased and C Y P 2 C 1 1 , C Y P 2 B 1 , and C Y P 2 B 2 levels to be increased. If nadir plasma G H levels were elevated in female rats or the regular G H pulse interval was disturbed there would be no effect on expression levels of C Y P enzymes. The effect of tamoxifen on peak plasma G H levels cannot explain the observed changes of hepatic C Y P enzyme activity and protein levels in male and female rats. If mean G H levels were decreased by tamoxifen treatment, one would expect similar effects on hepatic C Y P enzymes as caused by hypophysectomy. In hypophysectomized male and female rats, hepatic levels of C Y P 2 B 1 and C Y P 2 B 2 are elevated up to 100-fold (Yamazoe et al. 1987). Effects on 110 Discussion CYP2B enzymes were not observed in the present study. Therefore, the results presented do not support the hypothesis that the effects of tamoxifen on hepatic CYP microsomes were mediated by the influence of tamoxifen on plasma GH levels. Plasma GH levels in rats after tamoxifen treatment might be comparable with plasma GH levels in dwarf rats, in which the secretory dynamics of GH are not changed. Therefore, the modest effects of tamoxifen on hepatic CYP enzymes may be GH independent. In hepatic microsomes from male rats however, the effect of tamoxifen on hepatic CYP2C11 and possibly on CYP2A1 levels could have been mediated by higher nadir plasma GH levels, which could not be determined by the enzyme-linked immunoassay for rat GH. 4.8. Evaluation of a Possible Mechanism for the Effects of Tamoxifen on Hepatic CYP Enzymes Estrogens and androgens can alter the pituitary secretion of GH (Kerrigan and Rogol 1992) In a previous study, gonadectomy decreased peak plasma GH levels by two-fold and resulted in a more irregular GH secretion pattern of adult male rats in comparison to sham operated male rats. Gonadectomy did not alter mean and nadir plasma GH levels (Painson 1992). Administration of estradiol (0.1 mg/kg) for four days increased nadir plasma GH levels by four- to twenty-fold and decreased the duration of the GH peak interval period in both sham-operated and gonadectomized adult male rats. Administration of estradiol increased mean plasma GH levels but had no effect on peak plasma GH levels (Painson 1992). These results indicate that sex steroids are a critical factor for the maintenance of the sexually dimorphic GH secretion pattern in rats. Estrogens and androgens can regulate hepatic expression of several CYP enzymes (Mode and Norstedt 1982). Estradiol has no effect on CYP2B and CYP3A enzymes but neonatal estrogen treatment suppresses CYP2C11 in adult male rats (Bandiera and Dworschak 1992). The effect of estradiol on CYP2A1 is controversial. In one study, administration of estradiol s.c. at one and three days after treatment or by an implanted estrogen-packed capsule at five weeks of age or both treatments decreased hepatic CYP2A1 protein levels in neonatally ovariectomized female rats and increased hepatic CYP2A1 protein levels in neonatally castrated male rats (Dannan et al. 1986). In another study, the opposite effect of estradiol on CYP2A1 in female rats was demonstrated. Administration of estradiol s.c. at one and three days after treatment had no effect on CYP2A1 protein levels but CYP2A1 protein levels were increased by administration of estradiol s.c. together with the implantation of an estrogen-packed capsule at five weeks of age in 111 Discussion neonatally ovariectomized female rats (Waxman et al. 1989a). If the effects of tamoxifen on C Y P enzymes were mediated by its estrogenic effect in the liver, one would expect that in male and female rats, C Y P 2 C 1 1 levels to be decreased and C Y P 2 A 1 levels to be increased or decreased. One would expect no effect on C Y P 2 B , or on C Y P 3 A enzymes. The results of tamoxifen treatment on C Y P enzyme activity and protein levels presented in this study are in accordance with the expected estrogenic effects of tamoxifen in male rats. In female rats, activity and protein levels of C Y P 2 A 1 were decreased and C Y P 2 C 1 1 levels were not affected by tamoxifen treatment. The very low expression level of C Y P 2 C 1 1 in female rats might not have been suppressed further by the estrogenic effect of tamoxifen on C Y P 2 C 1 1 or its suppression might not be detectable by the testosterone hydroxylase assay. The finding that testosterone 7a-hydroxylase activities and C Y P 2 A 1 expression levels in female rats were decreased in the present study could be explained by an estrogenic effect (Dannan et al. 1986) or an antiestrogenic effect (Waxman et al. 1989a) of tamoxifen in female rat liver. Tamoxifen displays the whole spectrum of biological activities from full agonist, through partial agonist, to full antagonist depending on the species or target organ (Jordan 1984). Therefore, tamoxifen might exhibit different actions in different sexes in rats regarding its effect on C Y P enzyme induction. Tamoxifen might act as an antiestrogen in female rats but as an estrogen in male rats. Regardless of whether tamoxifen displays an estrogenic or an antiestrogenic effect in the liver of female rats, it can be argued both ways, that the effects of tamoxifen found in this study on testosterone 7a-hydroxylase activities and C Y P 2 A 1 expression levels in hepatic microsomes of female rats were mediated by its estrogenic or antiestrogenic effect. In the present study, the effect of tamoxifen on plasma G H levels were likely mediated by its antiestrogenic effect (Painson 1992) but based on the effects of tamoxifen on C Y P enzymes, the secretory dynamics of G H might not have been disturbed by tamoxifen. Tamoxifen may exhibit a long-term effect on hepatic C Y P enzymes although neither tamoxifen nor its metabolites are present in plasma 36 days after treatment at concentrations greater than the limits of quantitation of the H P L C assay for tamoxifen and its metabolites. The effects of tamoxifen on hepatic C Y P enzymes must therefore be mediated by an additional unknown long-term effect other than the effect of tamoxifen on G H (Figure 49). Alternatively, the effects of tamoxifen on hepatic C Y P enzymes could have been caused by a tamoxifen metabolite that remained in rat tissues for 36 days after treatment at concentrations below the limits of quantitation of the H P L C assay. 112 Discussion / / TSH ^ - 1P I TU'TARV TRH / GHRH (+) / SS{-) HYPOTHALAMUS k Steroid ^ Hormones serum GH Thyroid Gland \ \ s v . Figure 49: Regulation of hepatic microsomal CYP enzymes in the rat. TSH = thyroid stimulating hormone (thyrotropin), TRH = thyrotropin releasing hormone, GH = growth hormone, GHRH = growth hormone releasing hormone, SS = somatostatin, T 3 = triiodothyronine, 7a OH = 7a-hydroxylation, 2a OH = 2a-hydroxylation (adapted from Waxman 1988). 113 Conclusions 5. CONCLUSIONS 1. Tamoxifen treatment resulted in a prolonged suppression of body weight gain in male and female rats but had no effect on liver weight. 2. Tamoxifen treatment resulted in long-term alterations of hormonally regulated C Y P enzymes in rat liver. CYP2A1-mediated activity and protein levels were decreased modestly in female rats and C Y P 2 A 1 -mediated activity was increased modestly in male rats. Also in male rats, C Y P 2 C 1 1 activities and protein levels were decreased. C Y P 2 B and C Y P 3 A activities and protein levels were not affected in male and female rats after tamoxifen treatment. These effects are different from the direct, inductive effects of tamoxifen on C Y P enzymes as reported previously (White et al. 1993). 3. Thirty three days after tamoxifen treatment, peak plasma G H levels were decreased in male rats treated with tamoxifen at 200 mg/kg and in female rats treated with tamoxifen at 20 mg/kg. Mean and nadir G H plasma levels were unaffected by tamoxifen treatment. 4. Tamoxifen was present in rat plasma at one and twelve days after administration of tamoxifen at a dosage of 50 mg/kg. Tamoxifen was not present in rat plasma at a concentration greater than 1.56 nmol/ml at 24 or 36 days after administration. 5. 4-Hydroxytamoxifen was present in rat plasma up to 36 days after administration of tamoxifen at a dosage of 200 mg/kg. 4-Hydroxytamoxifen was present in rat plasma at one and twelve days after administration of tamoxifen at a dosage of 50 mg/kg. 4-Hydroxytamoxifen was not present in rat plasma at a concentration greater than 1.56 nmol/ml at 24 or 36 days after administration of tamoxifen at a dosage of 50 mg/kg. 6. The effect of tamoxifen on C Y P enzymes in the rat liver five weeks after administration is not believed to be mediated solely by the effect of tamoxifen on plasma G H levels but rather by its estrogenic and antiestrogenic effects on hepatic microsomal C Y P enzymes. Alternatively, the effects could have been caused by a residual tamoxifen metabolite that remained in rat tissues at concentrations below the limits of quantitation of the H P L C assay. 114 Future Experiments 6. F U T U R E E X P E R I M E N T S Further Development of the H P L C Assay for Detection and Quantitation of Tamoxifen and Its Metabolites in Rat Plasma A n H P L C assay using on-line U V photo-activation of tamoxifen and its metabolites and fluorescence detection (El -Yazig i and Legayada 1997), or a G C - M S assay using selected ion monitoring (Murphy et al. 1987), might be preferable for detection and quantitation of tamoxifen and its metabolites in plasma. With those assays, interfering peaks are less likely to appear and both assays have limits of quantitation that are 50- and 1000-times less than the H P L C assay used in the present study. Identification of tamoxifen metabolites by their retention times requires comparison with authentic standards. Thus, standards of pure Z 4-hydroxytamoxifen, Z 4'-hydroxytamoxifen, tamoxifen-A7-oxide, and Z A^-desmethyltamoxifen have to be purchased from commercial sources or obtained from other labs. The pure E or Z isomer of clomiphene or the pure E or Z isomer of 3-hydroxytamoxifen (Droloxifene) should be used as the internal standard. Tissue samples have to be prepared from rats treated with tamoxifen at a dosage o f 20 or 50 mg/kg to investigate the tissue distribution and elimination of tamoxifen and its metabolites. Blood and tissues (liver, lung, kidneys, fat, brain, gonads) should be sampled from tamoxifen : treated rats at 1, 6, 12, 24, 48, 72, and 96 h after treatment and pharmacokinetic data should be derived for plasma and tissues, especially liver. Additionally, T 3 levels could be determined in plasma from these rats. Determination of Plasma G H Levels The high nadir plasma G H values determined in the present study by the use of a commercially available enzyme-linked immunoassay for rat G H are probably a specific artifact of that assay. In addition, the anti-rat G H IgG provided in the enzyme-linked immunoassay kit reacted more strongly with rat G H provided by the National Institutes of Health of the U S A than with the rat G H standard provided by the manufacturer of the immunoassay kit. Therefore, a radioimmunoassay for rat G H from the National Institute of Arthritis, Metabolism, and Digestive Disease ( N I A M D D , Bethesda, M D , U S A ) should be used for measuring plasma G H levels. This assay is probably more useful for determining nadir plasma G H levels and plasma G H levels w i l l 115 Future Experiments be more comparable to those reported in previous studies. To investigate the GH secretory pattern, blood samples should be taken from several control rats and tamoxifen-treated rats every 20 min for 5 to 8 h at one or three days before sacrifice. Blood can be collected from the tip of the tail or by using an indwelling right atrial catheter. Red blood cells should be resuspended in saline and re-injected if a right atrial catheter is used. Determination of a Time-Course for the Effects of Tamoxifen on Hepatic CYP Enzyme Expression and on Plasma GH Levels in the Rat For a time-course study, male and female rats should be treated with tamoxifen at 20 mg/kg s.c. once daily for two consecutive days. The food intake needs to be controlled and rats should be weighed regularly. Groups of rats can be killed at one day, three days, one week, three weeks, five weeks, seven weeks, and ten weeks after treatment and hepatic microsomes should be prepared. The effect of tamoxifen on hepatic CYP enzyme activities and protein levels can be investigated as described in the present thesis. Earlier studies in rats showed that CYP2B and CYP3A enzymes were induced within 24 h after administration of tamoxifen (White et al. 1993). GH secretion is not completely suppressed within this time period (Tannenbaum et al. 1992). Consequently, the observed induction of CYP2B and CYP3A enzymes was not hormone-mediated but a direct effect of tamoxifen administration. This short-term effect can be investigated at the earlier time-periods (one day, three days, one week, and three weeks) when tamoxifen is still present in the blood system (to be determined by HPLC analysis). The long-term effect can be investigated at the later time-periods (five weeks, seven weeks, and ten weeks) when tamoxifen is presumably eliminated from blood (to be determined by HPLC analysis) and short-term enzyme induction has returned to control levels. To investigate the GH secretory pattern, blood samples should be taken from several control rats and tamoxifen treated rats every 20 min for 5 to 8 h at one or three days before sacrifice. Blood can be collected from the tip of the tail or by using an indwelling right atrial catheter. Blood samples can be analyzed by a radioimmunoassay for rat GH. To investigate the presence of tamoxifen and its metabolites in plasma and tissues, plasma and tissues will be analyzed by HPLC at the day of sacrifice. 116 Future Experiments Determination of the Effects of jV-Desmethyltamoxifen, Tamoxifen-iV- oxide, and 4-Hydroxytamoxifen Administration on G H Secretion and Hepatic C Y P Enzyme Expression in the Rat The effects of tamoxifen-treatment on hepatic microsomal C Y P enzymes determined in the present study might have been caused by a tamoxifen metabolite present in liver over the investigated time period. Therefore, the effects of the major tamoxifen metabolites should be determined by administering TV-desmefhyltamoxifen, tamoxifen-Af-oxide, and 4-hydroxytamoxifen directly and separately. The dosing regime of each metabolite should be 20 mg/kg s.c. once daily for two consecutive days. Although blood levels of 4-hydroxytamoxifen and tamoxifen-Af-oxide are generally much lower than those of tamoxifen, its optimal dose for induction of C Y P enzymes w i l l serve to maximize the chances of finding an effect of tamoxifen metabolite administration on plasma G H levels and hepatic C Y P enzyme expression in the rat. Rats should be ki l led at the time period found to be optimal by the time-course study, liver microsomes should be prepared, and G H levels can be measured. After the effects of tamoxifen metabolites on G H secretion and C Y P enzyme induction are investigated, a detailed dose and time response study might follow. If an effect is detected and i f one or more metabolites remain in plasma or tissues for an extended period of time, it w i l l suggest that the observed long-term effects of tamoxifen administration were partially or completely caused by tamoxifen metabolites. 117 References 7. R E F E R E N C E S Anderson, M.D. : Effect of Peripubertal Androgen Treatment on Hepatic Cytochrome P450 Expression in Adult Female Rats, M . Sc. Thesis, Faculty of Pharmaceutical Sciences, University of British Columbia, 1997. Anderson, M.D. , Bandiera, S.M., Chang, T . K . H . , and Bellward, G.D.: Effect of Androgen Administration During Puberty on Hepatic C Y P 2 C 1 1 , C Y P 3 A , and C Y P 2 A 1 Expression in Adult Female Rats, Drug Metabolism and Disposition 26 (10): 1031-1038, 1998. Agrawal, A . K . and Shapiro, B . H . : Gender, Age and Dose Effects of Neonatally Administered Aspartate on the Sexually Dimorphic Plasma Growth Hormone Profiles Regulating Expression o f the Rat Sex-Dependent Hepatic C Y P Isoforms, Drug Metabolism and Dispositions 25 (11): 1249-1256, 1997. Bandiera, S., Ryan, D.E. , Levin, W., and Thomas, P.E.: Age- and Sex-Related Expression of Cytochromes P450f and .P450g in Rat Liver, Archives of Biochemistry and Biophysics 248 (2): 658-676, 1986. Bandiera, S. and Dworschak, C : Effects of Testosterone and Estrogen on Hepatic Levels of Cytochromes P450 2C7 and CYP450 2C11 in the Rat, Archives of Biochemistry and Biophysics 296 (1): 286-295, 1992. Brodie, A . M . H . : Aromatase Inhibitors in the Treatment o f Breast Cancer, Journal of Biochemistry and Molecular Biology 49 (4-6): 281-287, 1994. Bullock, P., Gemzik, B. , Johnson, D., Thomas, P., and Parkinson, A . : Evidence from Dwarf Rats that Growth Hormone may not Regulate the Sexual Differentiation of Liver Cytochrome P450 Enzymes and Steroid 5cc-Reductase, Biochemistry 88: 5227-5231, 1991. Cadario, B.J . , Bellward, G.D., Bandiera, S., Chang, T . K . H . , Ko , W.W.W, Lemieux, E . , and Pak, R . C . K . : Imprinting of Hepatic Microsomal Cytochrome P-450 Enzyme Activities and Cytochrome P-4502C11 by Peripubertal Administration of Testosterone in Female Rats, Molecular Pharmacology 41: 981-988, 1992. Carlson, R.W.: Overview from a Medical Oncologist, Seminars in Oncology 24 (1, Suppl. 1): S1-151-S1-157, 1997. Chang, T . K . H . , Levine, M . , Bandiera, S .M, and Bellward, G.D.: Selective Inhibition of Rat Hepatic Microsomal Cytochrome P-450. I. Effect of the in Vivo Administration of Cimetidine, Journal of Pharmacology and Experimental Therapeutics 260 (3): 1441-1449, 1992. Chang, T . K . H . and Bellward, G.D.: Peripubertal Androgen Imprinting of Rat Hepatic Cytochrome P450 2C11 and Steroid 5a-Reductase: Pretranslational Regulation and Impact on Microsomal Drug Activation, Journal of Pharmacology and Experimental Therapeutics 278 (3): 1383-1391, 1996. 118 References Chang, T . K . H . , Chan, M . M . Y . , Holsmer, S.L., Bandiera, S.M., and Bellward, G.D.: Impact o f Tamoxifen on Peripubertal Androgen Imprinting of Rat Hepatic Cytochrome P450 2C11, Cytochrome P450 3A2, and Steroid 5ct-Reductase, Biochemical Pharmacology 51: 357-368, 1996. Chang, T . K . H . , Anderson, M.D. , Bandiera, S.M., and Bellward, G.D.: Impact of Ovariectomy and Androgen on Phenobarbital Induction of Hepatic C Y P 2 B 1 and C Y P 2 B 2 in Sprague-Dawley Rats, Drug Metabolism and Disposition 25 (8): 994-1000, 1997a. Chang, T . K . H . , Y u , L . , Maurel, P., and Waxman, D.J. : Enhanced Cyclophosphamide and Ifosfamide Activation in Primary Human Hepatocyte Cultures: Response to Cytochrome P-450 Inducers and Autoinduction by Oxazaphosphorines, Cancer Research 57: 1946-1954, 1997b. Charlton, H . M . , Clark, R .G. , Robinson, I .C.A.F. Porter Goff, A . E . , Cox, B.S., Bugnon, C , and Bloch, B .A . : Growth Hormone-Deficient Dwarfism in the Rat: A N e w Mutation, Journal of Endocrinology 119: 51-58, 1988. Cheesman, M . J . , and Reilly, P.E.B. : Differential Inducibility of Specific m R N A Corresponding to Five C Y P 3 A Isoforms in Female rat Liver by RU486 and Food Deprivation, Biochemical Pharmacology 56: 473-481, 1998. Cooper, K . O . , Reik, L . M . , Jayyosi, Z. , Bandiera, S., Kelley, M . , Ryan, D.E. , Daniel, R., McCluskey, S.A., Lewin, W., and Thomas, P.E. : Regulation of Two Members of the Steroid-Inducible Cytochrome P450 Subfamily (3A) in Rats, Archives of Biochemistry and Biophysics 301: 345-354, 1993. Crewe, H . K . , Ellis, S.W., Lennard, M.S. , and Tucker, G.T.: Variable Contribution of Cytochromes P450 2D6, 2C9, and 3A4 to the 4-Hydroxylation of Tamoxifen by Human Liver Microsomes, Biochemical Pharmacology 53: 171-178, 1997. Daniels, L . , Blankson, E.A. , Henderson, C.J . , Harris, A . H . , Wolf, C.R., Lennard, M.S . , and Tucker, G.T.: Delineation of Human Cytochromes P450 Involved in the Metabolism of Tamoxifen, British Journal of Clinical Pharmacology 34: 153P-154P, 1992. Dannan, G.A. , Guengerich, F.P., and Waxman, D.J. : Hormonal Regulation of Rat Liver Microsomal Enzymes, Journal of Biological Chemistry 261 (23): 10728-10735, 1986. Davis, A . M . , Mart in, E .A. , Jones, R . M . , L i m , C .K. , Smith, L . L . , and White, I .N.H.: Peroxidase Activation of Tamoxifen and Toremifene Resulting in D N A Damage and Covalent Bound Protein Adducts, Carcinogenesis 16 (3): 539-545, 1995. Dehal, S.S. and Kupfer, D.: C Y P 2 D 6 Catalyzes Tamoxifen 4-Hydroxylation in Human Liver, Cancer Research 57: 3402-3406, 1997. Early Breast Cancer Trialists Collaborative Group: Effects of Adjuvant Tamoxifen and of Cytotoxic Therapy on Mortality in Early Breast Cancer, New England Journal of Medicine 319 (26): 1681-1692, 1988. 119 References Eden, S., Albertson-Wikland, K . , Isaksson, O.: Plasma Levels of Growth Hormone in Female Rats of Different Ages, Acta Endocrinologica 88: 676-691, 1978. Eden, S.: Age- and Sex-Related Differences in Episodic Growth Hormone Secretion in the Rat, Endocrinology 105 (2): 555-560, 1979. EI-Yazigi, A . and Legayada, E. : Direct Liquid Chromatographic Micro-Measurement of Tamoxifen in Plasma of Cancer Patients, Journal of Chromatography B 691: 457-462, 1997. Ellis, P.A., Saccani-Jotti, G. , Clarke, R., Johnston, S.R.D., Anderson, E . , Howell, A . , A 'Hern , R., Salter, J . , Detre, S., Nicholson, R., Robertson, J. , Smith, I.E., and Dowsett, M . : Induction of Apoptosis by Tamoxifen and ICI 182780 in Primary Breast Cancer, International Journal of Cancer 72: 608-613, 1997. Fitts, J . M . , Klein, R . M . , and Powers, C.A. : Comparison of Tamoxifen Effects on the Actions of Triiodothyronine or Growth Hormone in the Ovariectomized-Hypothyroid Rat, Journal of Pharmacology and Experimental Therapeutics 286 (1): 392-402, 1998. Fisher, B. , Brown, A . , Wolmark, N . , Redmond, C , and other NSABP Investigators: Prolonging Tamoxifen Therapy for Primary Breast Cancer, Annals of Internal Medicine 106 (5): 649-654, 1987. Fisher, B. , Costantino, J. , Redmond, C , and other N S A B P Investigators: A Randomized Cl in ica l Trial Evaluating Tamoxifen in the Treatment of Patients with Node-Negative Breast Cancer who Have Estrogen-Receptor-Positive Tumors, New England Journal of Medicine 320 (8): 479-484, 1989. Fisher, B. , Costantino, J. , Wickerham, D.L. , and other N S A B P Investigators: Tamoxifen for Prevention of Breast Cancer: Report of the National Surgical Adjuvant Breast and B o w l Project P - l Study, Journal of the National Cancer Institute 90 (18): 1371-1388,1998. Foster, A . B . , Griggs, L . J . , Jarman, M . , van Maanen, J .M.S., and Schulten, H.-R.: Metabolism of Tamoxifen by Rat Liver Microsomes: Formation of the Af-Oxide, a N e w Metabolite, Biochemical Pharmacology 29: 1977-1979, 1980. Fried, K . M . and Wainer, I.W.: Direct Determination of Tamoxifen and Its Four Major Metabolites in Plasma Using Coupled Column High-Performance Liquid Chromatography, Journal of Chromatography B 655: 261-268, 1994. Fuchs, W. Leary, W.P., van der Meer, M . J . , Gay, S., Witchital, K . , and von Nieciecki, A : Pharmacokinetics and Bioavailability of Tamoxifen in Postmenopausal Healthy Women, Drug Research 46 (4): 418-422, 1996. Gabriel, S .M., Roncancio, S.R., and Ruiz, N.S.: Growth Hormone Pulsatility and the Endocrine Mi l i eu During Sexual Maturation in Male and Female Rats, Neuroendocrinology 56: 619-628, 1992. Garfinkle, D.: Studies on Pig Liver Microsomes, Archives in Biochemistry and Biophysics, 11: 493-509, 1958. 120 References Gebert, C.A., Park, S.-FL, and Waxman, D.J. : Regulation of Signal Transducer and Activator of Transcription (STAT) 5b Activation by the Temporal Pattern of Growth Hormone Stimulation, Molecular Endocrinology 11 (4): 400-414, 1997. Goldstein, J.A. and DeMorais, S.M.F.: Biochemistry and Molecular Biology of the Human CYP2C Subfamily, Pharmacogenetics 4: 285-299, 1994. Gonzalez, F.J . , Kimura, S., Song, B.J . , Pastewka, J . , Gelboin, H.V. , Hardwick, J.P.: Sequence of two Related P-450 mRNAs Trascriptionally Increased During Rat Development, Journal of Biological Chemistry 261: 10667-10672, 1986. Gonzalez, F .J . : The Molecular Biology of Cytochrome P450s, Pharmacological Reviews 40 (4): 243-288, 1989. Gonzalez, F .J . : Molecular Genetics of the P450 Superfamily, Pharmacology and Therapeutics 45: 1-38, 1990. Gottardis, M . M . , Robinson, S.P. and Jordan, V .C . : Estradiol-Stimulated Growth of MCF-7 Tumors Implanted in Athymic Mice: A Model to Study the Tumorstatic Action of Tamoxifen, Journal of Steroid Biochemistry 30 (1-6): 311-314, 1988. Guengerich, F.P. and Mart in, M . V . : Purification of Cytochrome P450, NADPH-Cytochrome Reductase and Epoxide Hydrolase from a Single Preparation of Rat Liver Microsomes, Archives of Biochemistry and Biophysics 205:365-379, 1980 Guengerich, F.P., Dannan, G.A., Wright, S.T., Mart in, M . V . , and Kaminsky, L .S . : Purification and Characterization of Liver Microsomal Cytochromes P-450: Electrophoretic, Spectral, Catalytic, and Immunochemical Properties and Inducibility of Eight Isozymes Isolated from Rats Treated with Phenobarbital or P-Naphtoflavone, Biochemistry 21: 601-9-6030,1982. Guengerich, F.P. and Lieber, D . C : Enzymatic Activation of Chemicals to Toxic Metabolites, Critical Reviews in Toxicology 14 (3): 259-307, 1984. Guengerich, F.P.: Enzymatic Oxidation of Xenobiotic Chemicals, Critical Reviews in Biochemistry and Molecular Biology 25 (2): 97-152, 1990. Guengerich, F.P.: Human Cytochrome P450 Enzymes, in Ortiz de Montellano, P.R. (ed.), Cytochrome P450, Structure, Mechanism, and Biochemistry, 2nd edition, Plenum Press, New York, 1995, pp. 473-536. Gustafsson, J . -A, Mode, A., Norsted, G., and Skett, P.: Sex Steroid Induced Changes in Hepatic Enzymes, Annual Review in Physiology 45: 51-60, 1983. Hageman, H .A.: The von Braun Cyanogen Bromide Reaction, Organic Reactions, John Wiley & Sons, Inc., New York 7: 198-259, 1953. Harper, M . J . K . and Walpole, A .L . : Contrasting Endocrine Activities of Cis and Trans Isomers in a Series of Substituted Triphenylethylenes, Nature 5057: 87, 1966. 121 References Harper, M . J . K . and Walpole, A . L . : A New Derivative of Triphenylethylene: Effect on Implantations and Mode of Action in Rats, Journal of Reproduction and Fertility 13: 101-119,1967. Harris, J.R., Lippman, M . E . , Veronesi, U . and Willet, W.: Breast Cancer, New England Journal of Medicine 327 (5): 319-328, 1992a. Harris, J.R., Lippman, M . E . , Veronesi, U . and Willet, W.: Breast Cancer, New England Journal of Medicine 327 (7): 327-480, 1992b. Hellriegel, E.T., Matwyshyn, G . A . , Fei, P., Dragnev, K . , Nims, R.W., Lubert, R . A . , and Kong, A - N . T . : Regulation of Gene Expression of Various Phase I and Phase II Drug-Metabolizing Enzymes by Tamoxifen in Rat Liver, Biochemical Pharmacology 52: 1561-1568, 1996. Henderson, C . J . and Wolf, C .R. : Molecular Analysis of Cytochrome P450s in the C Y P 2 Gene Family, in Gibson G . G . (ed.), Progress in Drug Metabolism, Taylor and Francis, London, 1992, pp. 73-139. Holsmer, S.L.: Effect o f Tamoxifen on Hepatic Cytochrome P450 Expression in Adult Female Rats, M . Sc. Thesis, Faculty of Pharmaceutical Sciences, University of British Columbia, 1995. Imaoka, S., Terano, Y . , and Funae, Y . : Changes in the Amount of Cytochrome P450s in Rat Hepatic Microsomes with Starvation, Archives of Biochemistry and Biophysics 278 (1): 168-178, 1990. Jacolot, F. Simon, I., Dreano, Y . , Beaune, P. , Riche, C , and Berthou, F.: Identification of the Cytochrome P450 IIIA Family as the Enzymes Involved in the A'-Desmethylation of Tamoxifen in Human Liver Microsomes, Biochemical Pharmacology 41 (12): 1911-1919, 1991. Jansson, J .-O., Ekberg, S., Iskasson, O .G .P . , and Eden, S.: Influence of Gonadal Steroids on Age- and Sex-Related Secretory Patterns of Growth Hormone in the Rat, Endocrinology 114 (4): 1287-1294, 1984. Jansson, I., Mole, J . , and Schenkmann, J.B.: Purification and Characterization of a N e w Form ( R L M 2 ) of Liver Microsomal Cytochrome P-450 from Untreated Rat, Journal of Biological Chemistry 260: 7084-7093, 1985. Jansson, J . -O. , Eden, S., and Iskasson, O . : Sexual Dimorphism in the Control of Growth Hormone Secretion, Endocrine Reviews 6 (2): 128-150, 1985. Jansson, J . -O. and Frohman, L . A . : Inhibitory Effect of the Ovaries on Neonatal Androgen Imprinting of Growth Hormone Secretion in Female Rats, Endocrinology 121 (4): 1417-1423, 1987. 122 References Jarman, M . , Poon, G .K. , Rowlands, M . G . , Grimshaw, R . M . , Horton, M . N . , Potter, G.A. , and McCague, R.: The Deuterium Isotope Effect for the a-Hydroxylation of Tamoxifen by Rat Liver Microsomes Accounts for the Reduced Genotoxicity o f [D5-Ethyl]Tamoxifen, Carcinogenesis 16 (4): 683-688, 1995. Jordan, V . C . : Biochemical Pharmacology of Antiestrogen Action, Pharmacological Reviews 35 (4): 245-276, 1984. Jordan, V . C . : Long-Term Adjuvant Tamoxifen Therapy for Breast Cancer, Breast Cancer Research and Treatment 15: 125-136, 1990. Jordan, V . C . : Overview from the International Conference on Long-Term Tamoxifen Therapy for Breast Cancer, Journal of the National Cancer Institute 84 (4): 231-234, 1992. Kamataki, T., Maeda, K . , Yamazoe, Y . , Matsuda, N . , Ishii, K . , and Kato, R.: A High-Spin Form of Cytochrome P-450 Highly Purified from Polychlorinated Biphenyl-Treated Rats, Molecular Pharmacology 24: 146-155, 1983. Kamataki, T., Shimada, M . , Maeda, K . , and Kato, R.: Pituitary Regulation of Sex-Specific Forms of Cytochrome P-450 in Liver Microsomes, Biochemical and Biophysical Research Communications 130: 1247-1253, 1985. Kato, R. and Yamazoe, Y . : Hormonal Regulation of Cytochrome P450 in Rat Liver, in Born G . V . R . , Cuatrecasas, P., and Herken, H . (eds.), Handbook of Experimental Pharmacology, Springer-Verlag, Berlin, 1993 (105), pp. 447-459. Kawai, M . , Bandiera, S.M., Chang, T . K . H . , Poulet, F . M . , Vancutsem, P . M . , and Bellward, G.D.: Enzyme-Selective and Long-Lasting Modulation of Rat Hepatic Cytochrome P450 Expression by Neonatal Administration of Tamoxifen, submitted to Cancer Research, 1999. Kerrigan, J.R. and Rogol, A .D . : The Impact of Gonadal Steroid Hormone Act ion on Growth Hormone Secretion During Childhood and Adolescence, Endocrine Reviews 13 (2): 281-298, 1992. Klingenberg, M . : Pigments of Rat Liver Microsomes, Archives in Biochemistry and Biophysics 75: 376-386,1958. Larsen, C , Brake, P.B., Parmar, D., and Jefcoate, C.R.: The Induction of Five Rat Hepatic P450 Cytochromes by Phenobarbital and Similarly Act ing Compounds is Regulated by a Sexually Dimorphic, Dietary-Dependent Endocrine Factor that is Highly Strain Specific, Archives of Biochemistry and Biophysics 315: 24-34, 1994. Larsen, C. and Jefcoate, C.R.: Phenobarbital Induction of C Y P 2 B 1 , C Y P 2 B 2 , and C Y P 3 A 1 in Rat Liver: Genetic Differences in a Common Regulatory Mechanism, Archives of Biochemistry and Biophysics 321: 467-476, 1995. Laemmli U .K . : Cleavage of Structural Proteins During the Assembly of the Head of Bacteriophage T4, Nature 227: 680-685, 1970. 123 References Legraverend, C , Mode, A. , Westin, S., Strom, A. , Eguchi, H . , Zaphiropulos, P.G. , and Gustafsson, J.-A.: Transcriptional Regulation of Rat P-450.2C Gene Subfamily Members by the Sexually Dimorphic Pattern of Growth Hormone Secretion, Molecular Endocrinology 6 (2): 259-266, 1992. Lerner, L . J . and Jordan, V . C . : Development of Antiestrogens and Their Use in Breast Cancer: Eight Chain Memorial Award Lecture, Cancer Research 50: 4177-4189, 1990. Lien, E .A. , Solheim, E. , and Ueland, P . M . : Distribution of Tamoxifen and its Metabolites in Rat and Human Tissues During Steady-State Treatment, Cancer Research 51: 4837-4844, 1991. Lien, E .A. , Anker, G. , and Ueland, P . M . : Pharmacokinetics of Tamoxifen in Premenopausal Women with Breast Cancer, Journal of Steroid Biochemistry and Molecular Biology 55 (2): 229-231, 1995. L i m , C .K . , Yuan, Z . X . , Lamb, J .H. , White, N .H . , DeMatteis, F., and Smith, L . L . : A Comparative Study of Tamoxifen Metabolism in Female Rat, Mouse, and Human Liver Microsomes, Carcinogenesis 15 (4): 589-593, 1994. Locatelli, V . , Torsello, A . , Redaelli, M . , Chigo, E . , Massara, F., and Muller, E .E . : Cholinergic Agonist and Antagonist Drugs Modulate the Growth Hormone Response to Growth Hormone Releasing Hormone in the Rat: Evidence for Mediation by Somatostatin, Journal of Endocrinology 111: 271-278, 1996. Lodwick, R., McConkey, B. , Brown, A . M . : Life Threatening Interaction between Tamoxifen and Warfarin, British Medical Journal 295: 1141, 1987. Love, R.R.: Tamoxifen Therapy in Primary Breast Cancer: Biology, Efficacy and Side Effects, Journal of Clinical Oncology 7(6): 803-815, 1989. Lowry, O . H , Risebrough, N.J . , Farr, A . C . , and Randall, R.J . : Protein Measurement with the Fol in Reagent, Journal of Biological Chemistry 193: 265-275, 1951. MacCallum, J . , Cummings, J . , Dixon, J . M . , and Miller, W.R.: Solid-Phase Extraction and High-Performance Liquid Chromatographic Determination of Tamoxifen and Its Major Metabolites in Plasma, Journal of Chromatography B 678 : 317-323, 1996. MacCallum, J . , Cummings, J . , Dixon, J . M . , and Miller, W.R.: Solid-Phase Extraction and High-Performance Liquid Chromatographic Determination of Tamoxifen and Its Major Metabolites in Breast Tumor Tissues, Journal of Chromatography B 698 : 269-275, 1997. MacGeoch, C , Morgan, E.T., and Gustafsson, J.-A.: Hypothalamo-Pituitary Regulation of Cytochrome P450i5p Apoprotein Levels in Rat Liver, Endocrinology 117 (5): 2085-2092, 1985. 124 References MacGeoch, C., Morgan, E.T., Halpert, J . , and Gustafsson, J.-A.: Purification, Characterization and Pituitary Regulation of the Sex-Specific Cytochrome P-450 15p-Hydroxylase from Liver Microsomes of Untreated Female Rats, Journal of Biological Chemistry 259(24): 15433-15439, 1984. MacGregor, J.I. and Jordan, V . C . : Basic Guide to the Mechanisms of Antiestrogen Action, Pharmacological Reviews 50 (2): 151-196, 1998. MacLeod, J .N. , Pampori, N.A. , and Shapiro, B . H . : Sex Differences in the Ultradian Pattern of Plasma Growth Hormone Concentrations in Mice , Journal of Endocrinology 131: 395-399, 1991. Mahnke, A . , Strotkamp, D., Roos, P .H. , Hanstein, W.G. , Chabot, G.G.„ and Nef, P.: Expression and Inducibility of Cytochrome P450 3A9 ( C Y P 3 A 9 ) and other Members of the C Y P 3 A Subfamily in Rat Liver. Archives of Biochemistry and Biophysics 337 (1): 62-68, 1997. Malaab, S., Pollak, M . N . , and Goodyer, C .G . : Direct Effects of Tamoxifen on Growth Hormone Secretion by Pituitary Cells in vitro, European Journal of Cancer 28A (4/5): 788-793, 1992. Mani , C , Gelboin, H.V. , Park, S.S., Pearce, R., Parkinson, A. , and Kupfer, D.: Metabolism of the Antimammary Cancer Antiestrogenic Agent Tamoxifen I., Drug Metabolism and Disposition 21 (4): 645-656, 1993a. Mani , C , Hodgson, E. , and Kupfer, D.: Metabolism of the Antimammary Cancer Antiestrogenic Agent Tamoxifen II., Drug Metabolism and Disposition 21 (4): 657-661, 1993b. Manns, J .E. , Hanks, S. and Brown, J .E.: Optimized Separation of E - and Z - Isomers of Tamoxifen, and Its Principal Metabolites Using Reversed-Phase High Performance Liqu id Chromatography, Journal of Pharmaceutical and Biomedical Analysis 16 : 847-852, 1998. Marshall, E . : Search for a Ki l le r : Focus Shifts from Fat to Hormones, Science 259: 618-621, 1993. Marx, J . : Cellular Changes on the Route to Metastasis, Science 259: 626-629, 1993. Matsunaga, T., Nagata, K . , Holsztynska, E.J . , Lapenson, D.P., Smith, A . , Kato, R., Gelboin, H.V. , Waxman, D.J. , Gonzalez, F.J . : Gene Conversion and Differential Regulation in the Rat P-450 IIA Gene Subfamily, Journal of Biological Chemistry 263: 17995-18002, 1988. Mauvais-Jarvis, P., Kuttenn, F., Malet, C , and Gompel, A . : Normal Breast Cells in Culture, Annals of the New York Academy of Science 595: 117-129, 1991. McCague, R. and Seago, A . : Aspects of Metabolism of Tamoxifen by Rat Liver Microsomes, Biochemical Pharmacology 35: 827-834, 1986. 125 References McClellan-Green, P.D., Linko, P., Yeowell, H.N. , and Goldstein, J .A.: Hormonal Regulation of Male-Specific Rat Hepatic Cytochrome P-450g (P-450IIC13) by Androgens and the Pituitary, Journal of Biological Chemistry 264 (32): 18960-19865, 1989. Metzger, D .L . and Kerrigan, J.R.: Estrogen Receptor Blockade with Tamoxifen Diminishes Growth Hormone Secretion in Boys: Evidence for a Stimulatory Role of Endogenous Estrogen During Male Adolescence, Journal of Clinical Endocrinology and Metabolism 19 (2): 531-518, 1994. Michaelets, E . L . : Update: Clinically Significant Cytochrome P-450 Drug Interactions, Pharmacotherapy 18: 84-112, 1998. Mil ler , M . L . and Eberhard, N . L . : Structure and Evolution of the Growth Hormone Gene Family, Endocrine Reviews 4 (2): 97-130, 1983. Mode, A . and Norstedt, G. : Effect of Gonadal Steroid Hormones on the Hypothalamo-Pituitary-Liver A x i s in the Control of Sex Differences in Hepatic Steroid Metabolism in the Rat, Journal of Endocrinology 95: 181-187, 1982. Montzka, T .A . , Matiskella, J.D. and Partyka, R.A. : 2,2,2-Trichloroethyl Chloroformate: A General Reagent for Demethylation of Tertiary Methylamines, Tetrahedron Letters 14: 1325-1327, 1974. Morgan, E .T. , MacGeoch, C , and Gustafsson, J.-A.: Hormonal and Developmental Regulation of Expression of the Hepatic Microsomal Steroid 16cc-Hydroxylase Cytochrome P450 Apoprotein in the Rat, Journal of Biological Chemistry 260: 11895-11898, 1985. M u r p h y , C , Fotsis, T. , Pantzar, P., Adlercreutz, H . , and Mart in, F.: Analysis of Tamoxifen and Its Metabolites in Human Plasma by Gas Chromatography-Mass Spectroscopy ( G C - M S ) Using Selected Ion Monitoring (SIM), Journal of Steroid Biochemistry 26 (5): 547-555, 1987. Nelson, D., Katamaki, T. , Waxman, D.J., Guengerich, F.P., Estabrook, R.W., Feyereisen, R., Gonzales, F.J . , Coon, M . J . , Gunsalus, I.C., Gotoh, O., Okuda, K . and Nebert, D.W.: The P450 Superfamily: Update on N e w Sequences, Gene Mapping Accession Numbers, Early Trivial Names of Enzymes and Nomenclature, DNA and Cell Biology 12 (1): 1-51, 1993. Nelson, D., Koymans, L . , Katamaki, T. , Stegeman, J.J., Feyereisen, R., Waxman, D.J. , Waterman, M.R. , Gotoh, O., Coon, M . J . , Estabrook, R.W., Gunsalus, I.C., and Nebert, D.W.: P450 Superfamily: Update on New Sequences, Gene Mapping Accession Numbers and Nomenclature, Pharmacogenetics 6: 1-42, 1996. Nuwaysir, E.F., Dragan, Y.P . , Jefcoate, C.R., Jordan, V . G . , and Pilot, H .C . : Effects of Tamoxifen Administration on the Expression of Xenobiotic Metabolizing Enzymes in Rat Liver, Cancer Research 55: 1780-1786, 1995. 126 References Nuwaysir, E.F., Daggett, D.A., Jordan, V . G . , and Pilot, H .C . : Phase II Enzyme Expression in Rat Liver in Response to the Antiestrogen Tamoxifen, Cancer Research 56: 3704-3710, 1996. Okey, A . B : Enzyme Induction in the Cytochrome P450 System, Pharmacology and Therapeutics 45: 241-298, 1990. Olofson, R.A. , Martz, J.T., Senet, J.P., Piteau, M . and Malfroot, T.: A N e w Reagent for the Selective, High Yie ld TV-Dealkylation of Tertiary Amines: Improved Synthesis of Naltrexone and Nalbuphine, Journal of Organic Chemistry 49: 2081-2082, 1984. Omura, T. and Sato, R.: A New Cytochrome in Liver Microsomes, Journal of Biological Chemistry 22,1 (4): PC1375-PC1376, 1962. Omura, T. and Sato, R.: The Carbon Monoxide Binding Pigment o f Liver Microsomes I. Evidence for Its Hemoprotein Nature, Journal of Biological Chemistry 239 (7): 2370-2378, 1964a. Omura, T. and Sato, R.: The Carbon Monoxide Binding Pigment o f Liver Microsomes II. Solubilization and Purification and Properties, Journal of Biological Chemistry 239 (7): 2379-2385, 1964b. Osborne, C . K . , Boldt, D.H. , Clark, G . M . , and Trent, J . M . : Effects of Tamoxifen on Human Breast Cancer Ce l l Cycle Kinetics: Accumulation of Cells in Early G i Phase, Cancer Research 43: 3583-3585, 1983. Osborne, C . K . , Hobbs, K . , and Clark, G . M . : Effect of Estrogens and Antiestrogens on Growth o f Human Breast Cancer Cells in Athymic Nude Mice , Cancer Research 45: 584-590, 1985. Osborne, C . K . : Tamoxifen in the Treatment of Breast Cancer, New England Journal of Medicine 339 (22): 1609-1618,1998. Osborne, M.R . , Hewer, A . , Hardcastle, I.R., Carmichael, P .L. , and Phillips, D .H. : Identification of the Major Tamoxifen-Deoxyguanosine Adduct Formed in the Liver D N A of Rats Treated with Tamoxifen, Cancer Research 56: 66-71, 1996. Pampori, N . A . , Agrawal, A . K . , Waxman, D.J., and Shapiro, B . H . : Differential Effects of Neonatally Administered Glutamate on the Ultradian Pattern of Circulating Growth Hormone Regulating Expression of Sex-Dependent Forms of Cytochrome P450, Biochemical Pharmacology 41 (9): 1299-1309, 1991. Painson, J . - C , Thorner, M.O. , Krieg, R.J . , and Tannenbaum, G.S.: Short-Term Adult Exposure to Estradiol Feminizes the Male Pattern of Spontaneous and Growth Hormone-Releasing Factor - Stimulated Growth Hormone Secretion in the Rat, Endocrinology 130 (1): 511-519, 1992. Panesar, S.K., Bandiera, S.M., Abbott, F.S.: Comparative Effects of Carbamazepine and Carbamazepine-10,11 -Epoxide on Hepatic Cytochromes P450 in the Rat, Drug Metabolism and Disposition 24 (6): 619-627, 1996. 127 References Perez, V . , Schaffner, F., and Popper, H . : Hepatic Drug Reactions, Progress in Liver Disease 4: 597-625, 1972. Phillips, D.H. , Potter, G.A., Horton, M . N . , Hewer, A., Crofton-Slight, C , Jarman, M . , and Venitt, S.: Reduced Genotoxicity of [D5-Ethyl]Tamoxifen Implicates a-Hydroxylation of the Ethyl Group as a Major Pathway of Tamoxifen Activation to a Liver Carcinogen, Carcinogenesis 15 (8): 1487-1492, 1994. Phillips, D.H. , Carmichael, P.L. , Hewer, A., Cole, K . J . , Hardcastle, LR . , Poon, G .K . , Keogh, A., and Strain, A .J . : Activation of Tamoxifen and Its Metabolite a-Hydroxytamoxifen to DNA-Binding Products: Comparison between Human, Rat and Mouse Hepatocytes, Carcinogenesis 17 (1): 89-94, 1996. Pollak, M . N . , Huynh, H.T., and Lefebvre, S.P.: Tamoxifen Reduces Serum Insulin-Like Growth Factor I (IGF-1), Breast Cancer Research and Treatment 22: 91-100, 1992. Poon, G .K. , Chui , Y . C . , McCague, R., Lonning, P.E., Feng, R., Rowlands, M . G . , and Jarman, M . : Analysis of Phase I and Phase II Metabolites of Tamoxifen in Breast Cancer Patients, Drug Metabolism and Disposition 21 (6): 1119-1124, 1993. Poon, G .K. , Walter, B. , Lonning, P.E., Horton, M . N . , and McCague, R.: Identification of Tamoxifen Metabolites in Human HEP G2 Cell Line, Human Liver Homogenate, and Patients on Long-Term Therapy for Breast Cancer, Drug Metabolism and Disposition 23 (3): 377-382, 1995. Porter, T.D and Coon, M . J . : Cytochrome P450, Journal of Biological Chemistry 266 (21): 13469-13472, 1991. Potter, G.A., McCague, R., and Jarman, M . : A Mechanistic Hypothesis for DNA Adduct Formation by Tamoxifen Following Hepatic Oxidative Metabolisms, Carcinogenesis 15 (3): 439-442, 1994. Ram, P.A. and Waxman, D.J. : Interaction of Growth Hormone-Activated STATs with SH2-Containing Phosphotyrosine Phosphatase SHP-1 and Nuclear JAK2 Tyrosine Kinase, Journal of Biological Chemistry 272 (28): 17694-17702, 1997. Randerath, K . , B i , J . , Mabon, N . , Sriram, P., and Moorthy, B. : Strong Intensification of Mouse Hepatic Tamoxifen DNA Adduct Formation by Pretreatment with the Sulfotransferase Inhibitor and Ubiquitous Environmental Pollutant Pentachlorophenol, Carcinogenesis 15 (5): 797-800, 1994a. 32 Randerath, K . , Moorthy, B. , Mabon, N . , and Sriram, P.: Tamoxifen: Evidence by P-Postlabeling and Use of Metabolic Inhibitors for Two Distinct Pathways Leading to Mouse Hepatic DNA Adduct Formation and Identification of 4-Hydroxytamoxifen as a Proximate Metabolite, Carcinogenesis 15 (10): 2087-2094, 1994b. 128 References Ribeiro, V . and Lechner, M . C . : Cloning and Characterization of a Novel C Y P 3 A 1 Al le l i c Variant: Analysis of C Y P 3 A 1 and C Y P 3 A 2 Sex-Hormone-Dependent Expression Reveals that the C Y P 3 A 2 Gene is Regulated by Testosterone, Archives in Biochemistry and Biophysics 293: 147-152, 1992. Rice, K . C . : A n Improved Procedure for the TV-Demethylation of 6,7-Benzomorphans, Morphine and Codeine, Journal of Organic Chemistry 40 (12): 1850-1851, 1975. Robertson, D.W., Katzenellenbogen, J .A . , Long, D.J., Rorke, E . A . , and Katzenellenbogen, B.S.: Tamoxifen Antiestrogens. A Comparison of the Activi ty, Pharmacokinetics and Metabolic Activation of the cis and trans Isomers of Tamoxifen, Journal of Steroid Biochemistry 16: 1-13, 1982. Robinson, SP and Jordan, V . C . : Metabolism of Steroid-Modifying Anticancer Agents, Pharmacology and Therapeutics 36: 41-103, 1988. Robinson, S.P., Langan-Fahey, S.M., Johnson, D.A. , and Jordan, V . C . : Metabolites, Pharmocodynamics and Pharmacokinetics of Tamoxifen in Rats and Mice Compared to the Breast Cancer Patient, Drug Metabolism and Disposition 19 (1): 36-43, 1991. Ryan, D.E. and Levin, W.: Purification and Characterization of Hepatic Micosomal Cytochrome P-450, Pharmacology and Therapeutics 45: 152-239, 1990. Ryan, D.E. and Levin, W. : Age- and Gender-Related Expression of Rat Liver Cytochrome P450, in Born G . V . R . , Cuatrecasas, P., and Herken, H . (eds.), Handbook of Experimental Pharmacology, Springer-Verlag, Berlin, 1993 (105), pp. 461-476. Sasamura, H . Nagata, K . , Yamazoe, Y . , Shimada, M . , Saruta, T., and Kato R.: Effect of Growth Hormone on Rat Hepatic Cytochrome P-450f m R N A : A N e w Mode of Regulation, Molecular and Cellular Endocrinology 68: 53-60, 1990. Schlach, D.S. and Reichlin, S.: Plasma Growth Hormone Concentration in the Rat Determined by Radioimmunoassay: Influence of Sex, Pregnancy, Lactation, Anesthesia, Hypophysectomy and Extrasellar Pituitary Transplants, Endocrinology 79: 275-, 1966. Schlach, D.S. and Reichlin, S.: Stress and Growth Hormone Release, Excerpta Medica, International Congress Series 158: 211-225, 1968. Shapiro, B . H . , MacLeod, J .N. , Pampori, N . A . , Morrissey, J.J., Lapenson, D.P., and Waxman, D.J. : Signaling Elements in the Ultradian Rhythm of Circulating Growth Hormone Regulating Expression of Sex-Dependent Forms of Hepatic Cytochrome P450, Endocrinology 125 (6): 2935-2944, 1989. Shapiro, B . H . , Pampori, N . A . , Ram, P .A. , and Waxman, D.J. : Irreversible Suppression of Growth Hormone-Dependent Cytochrome P-450 2C11 in Adult Rats Neonatally Treated with Monosodium Glutamate, Journal of Pharmacology and Experimental Therapeutics 265 (2): 979-984, 1993. 129 References Shapiro, B.H., Pampori, N.A., Lapenson, D.P., and Waxman, D.J.: Growth Hormone-Dependent and -Independent Sexually Dimorphic Regulation of Phenobarbital-Induced Hepatic Cytochromes P450 2B1 and 2B2, Archives of Biochemistry and Biophysics 312: 234-239, 1994. Shapiro, B.H., Agrawal, A.K., and Pampori, N.A.: Gender Differences in Drug Metabolism Regulated by Growth Hormone, International Journal of Biochemistry and Cell Biology 27 (1): 9-20, 1995. Shun-Yuan, J., Rong-Yaun, S., Ming-Yang, Y., and Jordan, V . C . : Tamoxifen Inhibits Hepatoma Cel l Growth Through an Estrogen Receptor Independent Mechanism, Journal of Hepatology 23: 712-719, 1995. Spatznegger, M, and Jaeger, W: Clinical Importance of Hepatic Cytochrome P450 in Drug Metabolism, Drug Metabolism Reviews 27 (3): 397-417, 1995. Sonderfran, A.J., Arlotto, M.P., Dutton, D.R., McMillen, S.K., and Parkinson, A.: Regulation of Testosterone Hydroxylation by Rat Liver Microsomal Cytochrome P-450, Archives of Biochemistry and Biophysics 255 (1): 27-41, 1987. Sonntag, W.E., Steger, R.W., Forman, L.J., and Meiters, J.: Decreased Pulsatile Release of Growth Hormone in Old Male Rats, Endocrinology 107 (6): 1875-1879, 1980. Strobel, J.S. and Thomas, M.J.: Human Growth Hormone, Pharmacological Reviews 46 (1): 1-34, 1994. Sundseth, S.S. and Waxman, D.J.: Sex-Dependent Expression and Clofibrate Inducibility of Cytochrome P-450 4 A Fatty A c i d ©-Hydroxylases, Journal of Biological Chemistry 267: 3915-3921, 1992. Sundseth, S.S., Alberta, J.A., and Waxman, D.J.: Sex-Specific Growth Hormone-Regulated Transcription of the Cytochrome P450 2C11 and 2C12 Genes, Journal of Biological Chemistry 267 (6): 3907-3914, 1992. Tannenbaum, G.S. and Martin, J.B.: Evidence for an Endogenous Ultradian Rhythm Governing Growth Hormone, Endocrinology 98: 562-570, 1976. Tannenbaum, G.S. and Ling, N.: The Interrelationship of Growth Hormone (GH)-Releasing Factor and Somatostatin in Generation of the Ultradian Rhythm of G H Secretion, Endocrinology 115 (5): 1952-1957, 1984. Tannenbaum, G.S., Rorstad, O., and Brazeau, P.: Effects of Prolonged Food Deprivation on the Ultradian Growth Hormone Rhythm and Immunoreactive Somatostatin Tissue Levels in the Rat, Endocrinology 104 (6): 1733-1738, 1979. Tannenbaum, G.S., Gurd, W., Lapinte, M., and Pollak, M.: Tamoxifen Attenuates Pulsatile Growth Hormone Secretion: Mediation in Part by Somatostatin, Endocrinology 130 (6): 3395-3401, 1992. 130 References Terry, L . C . , Epelbaum, J., and Martin, J.B.: Monosodium Glutamate: Acute and Chronic Effects on Rhythmic Growth Hormone and Prolactin Secretion, and Somatostatin in the Undisturbed Rat, Brain Research 217: 129-142, 1981. Tonetti, D .A . and Jordan, V . C . : Targeted Anti-Estrogens to Treat and Prevent Diseases in Women, Molecular Medicine Today May: 218-223, 1996. Tormey, D .C . and Jordan, V . C . : Long-Term Tamoxifen Adjuvant Therapy in Node-Positive Breast Cancer: A Metabolic and Pilot Clinical Study, Breast Cancer Research and Treatment 4: 297-302, 1984. Towbin, H., Stachelin, T., and Gordon, J.: Electrophoretic Transfer of Proteins from Polyacrylamide Gels to Nitrocellulose Sheets, Procedures and Some Applications, Proceedings of the National Academy of Science of the USA 76: 4350-4354, 1979. Udy, G.B., Towers, R.P., Snell, R.G., Wilkins, R.J., Park, S.-H., Ram, P .A., Waxman, D.J., and Davey, H.W.: Requirement of STAT5b for Sexual Dimorphism of Body Growth Rates and Liver Gene Expression, Proceedings of the National Academy of Science of the USA 94: 7239-7244, 1997. Wade, G . N . and Heller H.W.: Tamoxifen Mimics the Effects of Estradiol on Food Intake, Body Weight, and Body Composition in Rats, American Journal of Physiology 264: R1219-R1232,1993. Wakeling, A.E.: Nove l Pure Antiestrogens, Annals of the New York Academy of Science 595: 348-356, 1991. Waxman, D.J., Dannan, G .A . , and Guengerich, F.P.: Regulation of Rat Hepatic Cytochrome P-450: Age Dependent Expression, Hormonal Imprinting and Xenobiotic Inducibility of Sex-Specific Isoenzymes, Biochemistry 24: 4409-4417, 1985. Waxman, D.J.: Interactions of Hepatic Cytochromes P-450 with Steroid Hormones, Biochemical Pharmacology 37 (1): 71-84, 1988. Waxman, D.J., LeBIanc, G .A . , Morrissey, J.J., Staunton, J., and Lapenson, D.P.: Adult Male-specific and Neonatally Programmed Rat Hepatic P-450 Forms R L M 2 and 2a are not dependent on Pulsatile Plasma Growth Hormone for Expression, Journal of Biological Chemistry 263 (23): 11396-11406, 1988. Waxman, D.J., Morrissey, J.J., and LeBIanc, G .A . : Female Predominant Rat Hepatic P-450 Forms j (IIE1) and 3 (IIA1) are under Hormonal Regulatory Controls Distinct from those of the Sex-Specific P-450 Forms, Endocrinology 124 (6): 2954-2966, 1989a. Waxman, D.J., Morrissey, J.J., and LeBIanc, G .A . : Hypophysectomy Differentially Alters P-450 Protein Levels and Enzyme Activities in Rat Liver: Pituitary Control of Hepatic N A D P H Cytochrome P-450 Reductase, Molecular Pharmacology 35: 519-525, 1989b. 131 References Waxman, D.J. , Morrissey, J.J., MacLeod, J . , and Shapiro, B . H . : Depletion of Serum Growth Hormone in Adult Female Rats by Neonatal Monosodium Glutamate Treatment without Loss of Female Specific Hepatic Enzymes P450 2d (IIC12) and Steroid 5ct-Reductase, Endocrinology 126 (2): 712-720, 1990. Waxman, D.J. , Pampori, N . A . , Ram, P.A., Agrawal, A . K . , and Shapiro, B. : Interpulse Interval in Circulating Growth Hormone Patterns Regulates Sexually Dimorphic Expression of Hepatic Cytochrome P450, Proceedings of the National Academy of Science of the USA 88: 6868-6872, 1991. Waxman, D.J . and Azaroff, L . : Phenobarbital Induction of Cytochrome P-450 Gene Expression, Biochemical Journal 281: 577-592, 1992. Waxman, D.J. , Ram, P.A., Park, S.-H., and Choi, H . K . : Intermittent Plasma Growth Hormone Triggers Tyrosine Phosphorylation and Nuclear Translocation of a Liver-Expressed, Stat 5-related D N A Binding Protein, Journal of Biological Chemistry 270 (22): 13262-13270, 1995. Westin, S., Strom, A. , Gustafsson, J.-A., and Zaphiropoulos, P.: Growth Hormone Secretion of the Cytochrome P-450IIC Subfamily in the Rat: Inductive, Repressive and Transcriptional Effects on P-450f (IIC7) and P -450 P B ] (IIC6) Gene Expression, Molecular Pharmacology 38: 192-197, 1990. White, I .N. , Davies, A. , Smith, L . L . , Dawson, S., and de Matteis, F.: Induction of C Y P 2 B 1 and 3 A 1 , and Associated Monooxigenase Activities by Tamoxifen and Certain Analogues in the Livers of Female Rats and Mice , Biochemical Pharmacology 45 (1): 21-30, 1993. Williams, G . M . , Iatropoulos, M . J . , Djordjevic, M . V . and Kaltenberg, O.P.: The Triphenylethylene Drug Tamoxifen is a Strong Liver Carcinogen in the Rat, Carcinogenesis 14(2): 315-317, 1993. Wiseman, H . and Lewis, F .V. : The Metabolism of Tamoxifen by Human Cytochromes P450 is Rationalized by Molecular Modeling of the Enzyme-Substrate Interactions: Potential Importance to its Proposed Anti-Carcinogenic/Carcinogenic Actions, Carcinogenesis 17 (6): 1357-1360, 1996. Wong, A . , and Bandiera, S .M.: Inductive Effect o f Telazol® on Hepatic Expression of Cytochrome P450 2B in Rats, Biochemical Pharmacology 52: 735-742, 1996. Wrighton S.A. and Stevens, J . C : The Human Hepatic Cytochromes P450 Involved in Drug Metabolism, Critical Reviews in Toxicology 22 (1): 1-21, 1992. Yamazoe, Y . , Shimada, M . , Murayama, N . , and Kato, R.: Suppression of Levels of Phenobarbital-Inducible Rat Liver Cytochrome P450 by Pituitary Hormone, Journal of Biological Chemistry 262 (15): 7423-7428, 1987. 132 Appendix A P P E N D I X I Table I: Mean testosterone hydroxylase activities of hepatic microsomes from rats treated with various doses of tamoxifen. Meta-bolite Mean activity (nmol metabolite formed/min/nmol CYP) 0 mg/kg 0.5 mg/kg 5 mg/kg 20 mg/kg 50 mg/kg 100a mg/kg 100 mg/kg 200 mg/kg 6(3 0.294 0.316 0.331 0.356 0.432 0.323 0.407 0.35 (0.0499) (0.0369) (0.0586) (0.0371) (0.0849) (0.0267) (0.0269) (0.0291) 7 a 1.567 1.364 1.273 0.934** 0.874* 0.93* 0.938** 0.907** (0.1753) (0.0435) (0.1886) (0.1156) (0.0186) (0.0576) (0.0753) (0.0869) 16a 0.121 0.124 0.129 0.14 0.128 0.116 0.145 0.112 (0.0158) (0.0066) (0.0252) (0.0124) (0.0087) (0.0271) (0.0154) (0.0107) 16(3 0.0653 0.0674 0.0652 0.0799 0.0749 0.0847 0.0984 0.0727 (0.0072) (0.0063) (0.0105) (0.0078) (0.0112) (0.0119) (0.0146) (0.0085) 2 a 0.0208 0.0196 0.019 0.0191 0.0192 0.019 0.0203 0.0169 (0.0012) (0.0015) (0.0024) (0.0009) (0.0021) (0.0032) (0.0023) (0.0013) 2(3 0.0513 0.0621 0.0535 0.0422 0.0445 0.0365 0.0433 0.0385 (0.0126) (0.0152) (0.0105) (0.0047) (0.0069) (0.0089) (0.0041) (0.0060) A 0.389 0.372 0.359 0.479 0.538 0.575 0.515 0.429 (0.0556) (0.0450) (0.0682) (0.0573) (0.1038) (0.0915) (0.0909) (0.0713) Values in parenthesis indicate the standard errors of the mean, n = 6 for the control and the 0.5, 5, 20, and 200 mg/kg treatment groups, n = 3 for the 50 and 100a mg/kg treatment groups, n = 5 for the 100 mg/kg treatment group. Rats in the 100a mg/kg treatment group received a total dose of 200 mg/kg tamoxifen distributed over three days (50 mg/kg at day one, 50 mg/kg at day two, and 100 mg/kg at day three). * Mean value of the treatment group is statistically different (* = p < 0.05, ** = p < 0.05) from that of the control group. 7a = 7a-hydroxytestosterone, etc, A = androstenedione. Mean activity values for 2a hydroxytestosterone-formation are below the limits of quantitation of the testosterone hydroxylase assay. 133 Appendix A P P E N D I X I I Table II: Mean testosterone hydroxylase activities of hepatic microsomes from male rats treated with tamoxifen. Meta-bolite Mean activity (nmol metabolite/min/nmol CYP) 0 mg/kg 200 mg/kg 60 2.34 2.36 (0.201) (0.183) 7a 0.252 0.427* (0.0161) (0.0598) 16a 3.05 1.73** (0.208) (0.233) 16p 0.08 0.096 (0.01) (0.008) 2 a 1.8 \ ** (0.115) (0.154) 20 0.203 0.204 (0.011) (0.016) A 1.55 1.45 (0.11) (0.11) Values in parenthesis indicate the standard errors of the mean, n = 6 for the control and the treatment group. * Mean value of the treatment group is statistically different (* = p < 0.05, ** = p < 0.05) from that of the control group. 7a = 7a-hydroxytestosterone, etc, A = androstenedione. 134 Appendix A P P E N D I X I I I Table III: Relative content of specific C Y P Enzymes (nmol/nmol total C Y P ) in hepatic microsomes from male and female rats treated with various doses of tamoxifen. Enzyme Mean microsomal levels of specific CYP (nmol/nmol total CYP) female male 0 0.5 5 20 50 100a 100 200 mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg 0 200 mg/kg mg/kg CYP2A1 0.081 0.082 0.082 0.057* 0.062 0.063 0.051** 0.051** (0.0057) (0.0079) (0.0036) (0.0034) (0.013) (0.0026) (0.0031) (0.0052) 0.012 0.014 (0.0014) (0.0014) CYP2A2 11.6 5.96 (4.22) (2.55) CYP2B1 0.0027 0.0031 0.0032 0.0034 0.0208 0.0044 0.0028 0.0042 (0.0007) 0.0009 0.0009 0.001 0.0017 0.0008 0.0011 0.0012 0.0049 0.0043 0.0007 0.0006 CYP2B2 0.0046 0.0063 0.0058 0.0047 0.0052 0.0051 0.0032 0.0041 (0.0009) (0.0014) (0.0014) (0.001) (0.0043) (0.0023) (0.0013) (0.0007) 0.0049 0.0042 (0.0006) (0.0005) CYP2C11 0.115 0.812* (0.0072) (0.011) CYP3A 68.61 85.44 79.3 59.77 44.52 36.81 57.72 55.5 (8.78) (15.35) (11) (11.27) (4.06) (12.44) (9.76) (6.96) 287.01 282.06 (23.05) (28.44) Values in parenthesis indicate the standard errors of the mean, n = 6 for the control and the 0.5, 5, 20, and 200 mg/kg treatment groups, n = 3 for the 50 and 100a mg/kg treatment groups, n = 5 for the 100 mg/kg treatment group. Rats in the 100a mg/kg treatment group received a total dose of 200 mg/kg tamoxifen distributed over three days (50 mg/kg at day one, 50 mg/kg at day two, and 100 mg/kg at day three). The C Y P 3 A and the C Y P 2 A 2 content of hepatic microsomes is expressed as a relative O D value (OD x m m 2 band/OD x m m 2 IS/nmol total C Y P ) because the anti-rat C Y P 3 A 1 IgG cross-reacted with all other C Y P 3 A enzymes, which could not be separated on the SDS-polyacrylamide gel and because purified C Y P 2 A 2 was not available for generation of a calibration curve. * Mean value of the treatment group is statistically different (* =p < 0.05, ** =p < 0.01) from that of the control group. 135 Appendix APPENDIX IV Table I V : Relative content of specific C Y P enzymes (pmol/mg protein) in hepatic microsomes from male and female rats treated with various doses of tamoxifen. Enzyme Mean microsomal levels of specific CYP (pmol/mg protein) female male 0 0.5 5 20 50 100a 100 200 mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg 0 200 mg/kg mg/kg C Y P 2 A 1 0.629 0.668 0.832 0.5* 0.691 0.438 0.39** 0.403** (0.069) (0.072) (0.087) (0.04) (0.077) (0.035) (0.023) (0.044) 0.238 . 0.299 (0.031) (0.026) C Y P 2 B 1 0.115 0.132 0.16 0.146 0.128 0.15 0.099 0.162 (0.32) (0.041) (0.039) (0.042) (0.106) (0.023) (0.036) (0.052) 0.101 0.092 (0.016) (0.011) C Y P 2 B 2 0.182 0.265 0.29 0.204 0.32 0.174 0.113 0.159 (0.038) (0.062) (0.065) (0.045) (0.27) (0.073) (0.045) (0.027) 0.101 0.09 (0.013) (0.009) C Y P 2 C 1 1 0.494 0.374* (0.028) (0.049) Values in parenthesis indicate the standard errors of the mean, n = 6 for the control and the 0.5, 5, 20. and 200 mg/kg treatment groups, n = 3 for the 50 and 100a mg/kg treatment groups, n = 5 for the 100 mg/kg treatment group. Rats in the 100a mg/kg treatment group received a total dose of 200 mg/kg tamoxifen distributed over three days (50 mg/kg at day one, 50 mg/kg at day two, and 100 mg/kg at day three). * Mean value of the treatment group is statistically different (* = p < 0.05, ** =p < 0.01) from that of the control group. 136 Appendix APPENDIX V Table V: Plasma G H levels of male and female rats treated with various dosages o f tamoxifen. Clock Plasma GH level (ng/ml) time female male 0 5 20 200 mg/kg mg/kg mg/kg mg/kg 0 200 mg/kg mg/kg Rat A B C D E F G H I J A B C D E F 9:00 64.9 113.1 45.3 115.3 137.8 69 71.2 121.5 128.8 79 772.4 39.8 536.5 249.8 157 93.5 9:20 193.9 4£5« 72.8 164 162 98.9 85.9 144.3 165.4 37.5 474.8 56.3 124.9 189.6 93 93.2 9:40 81.2 281.5 86 280.5 121 100.4 48.4 36.8 157.9 83.2 194.9 58.8 104.1 119.4 123.5 80.5 10:00 112.2 199 113.3 160.2 86.8 53.8 32.5 18.9 195.1 85.9 109.1 310 93.8 166.4 132.7 84.2 10:20 105.3 100.6 130.3 74.6 119.4 91.1 76.4 53.7 172.2 101 41.1 147.5 103.7 115 129.4 99.4 10:40 118.4 75.2 67.1 79.9 119 74.5 81.1 38.1 150.1 106.3 91.1 84.4 118.4 155.5 137.5 102.9 11:00 73.4 102.1 97 117 109.9 65.3 72.6 91.3 196.4 86.5 88.2 135.8 110.6 141 155.3 100.8 11:20 64.2 98.6 197.5 84.2 128.7 66.2 64.9 155.7 132.2 94.7 117.9 50.2 102.6 121.2 101.1 81.3 11:40 80.3 131.8 50 106.9 99.5 85.7 67 148.4 209.6 75.7 130.7 82.5 85.5 102.7 102.6 94.5 12:00 61.4 49.7 61.7 35.1 160.3 94.9 73.4 113.2 193.8 106.3 242.3 73 93.5 161.5 114.2 76.5 12:20 64.2 82.7 84.1 140.9 142 63 84.8 33.8 183.1 99 96.5 79.5 108.6 775 143.5 110.5 12:40 48.6 63.1 60.4 140.9 139.1 54.9 46.9 45 178.5 100.3 92.3 97.4 100.4 190.6 129.4 126.7 13:00 37.8 69.3 25.5 102.4 74.5 82.3 44.1 54 168.8 81.6 50.9 185.3 100.4 183.6 131.3 92.7 13:20 31.8 77.8 67.8 96.7 124.9 71.4 74.1 94 159.6 94.8 65.6 150.9 109 120.3 192.4 135 13:40 30.8 62.4 61A 119.2 109.1 60.9 86.5 77.7 164.5 69.7 114.9 81.3 107.5 182.2 122.7 91.8 14:00 47.5 69.7 76.1 100.9 91.7 62.6 67.6 139.5 197.7 97.1 100.3 78.9 112.6 189.2 90.5 79.3 14:20 166.9 90.8 41.3 130.5 111.6 99.4 54.1 139.5 222.4 107 88.2 35 124.4 157.9 74 101.9 14:40 92.9 60.7 44.3 164 128.8 92.5 67 88.8 130.2 118.7 124.9 105.8 101.1 152 94.8 96.6 15:00 40.2 8 8.4 54.4 151.3 75.3 69.5 55.2 105.9 58 109.8 130.7 44.1 83.4 184.9 98.6 154.2 15:20 71.8 79.1 56.8 143.6 102.2 67.6 64.3 98.6 53.4 85.9 104.3 99.6 86.1 148.6 102.6 88.6 15:40 28.8 129.1 81.8 77.3 84.4 55.3 102.3 52.6 125.7 65.6 77.1 105.2 161.5 148.3 94.2 16:00 45.4 104.3 58.4 113.7 85.7 74 104.3 112.9 46 80.6 127.8 105.2 106.6 120.1 77.1 16:20 30 123.7 74.1 84.6 103.5 63 69 173.1 42.4 66.3 93.8 122.7 134.7 110.6 84.9 16:40 37.6 207.4 104.8 114.2 104.3 79 70.5 131.5 91.1 107 99.6 131.3 153.2 128 121.8 17:00 38.8 72.4 81.3 127.5 111.6 96.9 51.9 126.2 88.8 112.7 99.6 118.4 131.7 88.9 79.6 0 70.7 105.5 76 121 113.3 75.7 68.6 89.9 156.6 88.6 146.5 99.8 123.6 153.7 120.9 97.7 Stikethrough value was not included in the calculation of the mean values. Bo ld values represent peak values, bold and italic values represent nadir values. 0 = mean value. 137 Appendix A P P E N D I X V I Table VI: Concentrations of tamoxifen in rat plasma as detected by H P L C . Average plasma concentration (nmol/ml) Day 1 Day 12 Day 24 Day 36 Tamoxifen in plasma of rats 0.746 0.23 treated with tamoxifen at (0.074) (0.026) 50 mg/kg Values in parenthesis indicate the standard errors of the mean, n = 6 for day 1, n = 2 for day 12. Dash indicates that tamoxifen was not detected. Table VII: Concentrations of 4-hydroxytamoxifen in rat plasma as detected by H P L C . Average plasma concentration (nmol/ml) Dayl Day 12 Day 24 Day 36 Z 4-Hydroxytamoxifen in plasma of rats treated with tamoxifen at a dosage of 200 mg/kg 0.046 (0.003) 0.032 0.077 (0.014) 0.063 (0.005) Z 4-Hydroxytamoxifen in plasma of rats treated with tamoxifen at a dosage o f 100 mg/kg 0.057 (0.004) - - -Z 4-Hydroxytamoxifen in plasma of rats treated with tamoxifen at a dosage of 50 mg/kg 0.073 (0.013) 0.016 - -Values in parenthesis indicate the standard errors of the mean. For rats treated with tamoxifen at 200 mg/kg: n = 4 for day 1, n = 1 for day 12, n = 3 for day 24, n = 5 for day 36. For rats treated with tamoxifen at a dosage of 100 mg/kg: n = 4 for day 1. For rats treated with tamoxifen at a dosage of 50 mg/kg: n = 3 for day 1, n = 1 for day 12. Dash indicates that Z 4-hydroxytamoxifen was not detected. 138 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0099351/manifest

Comment

Related Items