UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Effect of peripubertal androgen treatment on hepatic cytochrome P450 expression in adult female rats Anderson, Mellissa Dawn 1997

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

Item Metadata

Download

Media
831-ubc_1997-0399.pdf [ 7.27MB ]
Metadata
JSON: 831-1.0087781.json
JSON-LD: 831-1.0087781-ld.json
RDF/XML (Pretty): 831-1.0087781-rdf.xml
RDF/JSON: 831-1.0087781-rdf.json
Turtle: 831-1.0087781-turtle.txt
N-Triples: 831-1.0087781-rdf-ntriples.txt
Original Record: 831-1.0087781-source.json
Full Text
831-1.0087781-fulltext.txt
Citation
831-1.0087781.ris

Full Text

EFFECT OF PERIPUBERTAL ANDROGEN TREATMENT ON HEPATIC CYTOCHROME P450 EXPRESSION IN ADULT FEMALE RATS by MELLISSA DAWN ANDERSON B.Sc.(Hons.), Mount Allison University, Sackville, New Brunswick, Canada, 1995 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES FACULTY OF PHARMACEUTICAL SCIENCES Divisions of Pharmacology & Toxicology and Pharmaceutical Chemistry We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 1997 © Mellissa Dawn Anderson, 1997 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 ' Pha^^^U The University of British Columbia Vancouver, Canada •ate ^ t , f y y DE-6 (2/88) ABSTRACT The goal of the current study was to investigate the pubertal period as a time when androgen imprinting of hepatic C Y P enzymes can occur. Female Sprague-Dawley rats were ovariectomized, at 25 days of age, and injected subcutaneously with androgens during the pubertal period, on days 35-49. Plasma testosterone levels were measured following the treatment. Rats were killed at 90 days of age and microsomes were prepared. Daily s.e. injections of testosterone enanthate at 5 umol/kg were able to increase adult testosterone 2a- and 16a- hydroxylase activities and CYP2C11 protein levels to 20% of control male levels. This enzyme is normally expressed in adult male rats only. Protein levels of CYP3A, a male-dominant enzyme, were increased 50%, although testosterone 6p-hydroxylase activity was not significantly increased. The increase in CYP3A was not attributed to CYP3A1 or CYP3A2. Neither CYP2A1 nor its marker activity, testosterone 7a-hydroxylase, was affected by pubertal testosterone enanthate injections. These results were compatible with the hypothesis that adult expression of certain C Y P enzymes can be regulated by pubertal exposure to androgens. Injections of unesterified testosterone at 2.5 umol/kg twice daily had the same effect as a single dose of unesterified testosterone at 5 umol/kg/day in increasing expression of CYP2C11 and CYP3A and decreasing expression of CYP2A1. These results indicated that increases in dosing frequency of pubertal androgen to twice daily at 2.5 umol/kg did not affect adult C Y P expression. Unesterified testosterone did not appear to be as effective as testosterone enanthate at elevating expression of CYP2C11 ii and CYP3A. This indicated a possible pharmacokinetic component, which was further supported by the difference in half-lives measured for the two androgens used. Smaller elevations in CYP2C11 and CYP3A expression were observed at 129 and 169 days of age than at 90 days of age. These results indicated that the effect of pubertal testosterone enanthate treatment was not permanent. The results of this study did not support the hypothesis of pubertal imprinting of hepatic C Y P enzymes. It is likely that pharmacokinetic differences between testosterone and testosterone enanthate resulted in the varied response to the androgen formulations. i i i TABLE OF CONTENTS page Title Page i Abstract i i Table of Contents iv List of Tables vii List of Figures vii i List of Abbreviations x Acknowledgements xi 1. INTRODUCTION 1 1.1. C Y P Enzymes 1 1.1.1. Sex Differences in C Y P Expression 2 1.2. Sex Differences in Hormone Secretion in the Rat 5 1.2.1. Sex Steroid Secretion 5 1.2.2. Growth Hormone Secretion 6 1.2.3. Regulation of G H Secretion through the Hypothalamo-Pituitary 7 Axis 1.3. Hormonal Regulation of C Y P Enzymes g 1.3.1. Involvement of Sex Steroids in C Y P Expression g 1.3.2. Definitions of Imprinting and Adult Androgen Responsiveness 9 1.3.3. Involvement of G H in C Y P Expression \ o 1.4. Neonatal Imprinting ig 1.4.1. Evidence for Neonatal Imprinting of C Y P Enzymes 17 1.5. Evidence for Pubertal Imprinting of C Y P Enzymes \ g 1.6. Hypothesis and Objectives 21 1.6.1. Hypothesis 21 1.6.2. Specific Objectives of this Study 21 1.6.3. Rationale for Experimental Design 22 1.7. Sexually Differentiated C Y P Enzymes Investigated 24 1.7.1. CYP2A1 24 1.7.2. CYP2C11 25 1.7.3. CYP3A 25 2. E X P E R I M E N T A L 27 2.1. Chemicals 27 2.2. Animals and Treatment 3 \ 2.3. Preparation of Hepatic Microsomal Fraction 31 2.4. Determination of Protein Content 3 3 2.5. Determination of Total C Y P Content 3 3 2.6. Plasma Testosterone 3 4 2.7. Testosterone Hydroxylase Assay 3 4 2.8. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) 3 7 2.9. Immunoblots 3 g 2.9.1. CYP2C11 3 9 2.9.2. CYP3A 1 0 iv 2.9.3. CYP2A1 40 2.9. Immunoquantitation 40 2.10. Data Analysis 41 2.11.1. Statistical Analysis of Data 41 2.10.2. Pharmacokinetic Analysis of Data 43 3. RESULTS 44 3.1. Assay Validation 44 3.1.1. Testosterone Radioimmunoassay 45 3.1.2. Testosterone Hydroxylase Assay 50 3.1.3. Immunoblots 63 3.1.3.1. CYP2C11 63 3.1.3.2. CYP3A 65 3.1.3.3. CYP2A1 67 3.2. Effect of Pubertal Androgen Treatment on Hepatic C Y P Enzymes 69 3.2.1. Effect of Pubertal Testosterone Enanthate Treatment 3.2.1.1. Elimination of testosterone following pubertal testosterone 70 enanthate treatment 70 3.2.1.2. Effect of pubertal testosterone enanthate treatment on CYP2C11 protein levels and testosterone 2a- and 16a- 71 hydroxylase activities 3.2.1.3. Effect of pubertal testosterone enanthate treatment on CYP3A protein levels and testosterone 6fi -hydroxylase 77 activities 3.2.1.4. Effect of pubertal testosterone enanthate treatment on CYP2A1 protein levels and testosterone 7a -hydroxylase 80 activities. 3.2.2. Effect of Dosing Frequency during Pubertal Androgen Treatment 3.2.2.1. Elimination of a single subcutaneous androgen injection 84 3.2.2.2. Elimination of testosterone following pubertal testosterone 84 treatment 89 3.2.2.3. Effect of dosing frequency during pubertal androgen treatment and adult CYP2C11 protein levels and testosterone 89 2a- and 16a-hydroxylase activities 3.2.2.4. Effect of dosing frequency during pubertal androgen treatment on adult CYP3A protein levels and testosterone 6/3- 91 hydroxylase activities 3.2.2.5. Effect of dosing frequency during pubertal androgen treatment on adult CYP2A1 protein levels and testosterone 94 7 a-hydroxylase activities 3.2.3. Effect of Pubertal Testosterone Enanthate Treatment on C Y P Expression at Various Ages during Adulthood 96 3.2.3.1. CYP2C11 protein levels and testosterone 2a- and 16a-hydroxylase activities at various ages during adulthood 96 following pubertal testosterone enanthate treatment v 3.2.3.2. CYP3A protein levels and testosterone 613 -hydroxylase 100 activities at various ages during adulthoodfollowing pubertal testosterone enanthate treatment 3.2.3.3. CYP2A1 protein levels and testosterone 7 a -hydroxylase 100 activities at various ages during adulthood following pubertal testosterone enanthate treatment 4. DISCUSSION 106 4.1. Effect of Pubertal Androgen Treatment on Hepatic C Y P Enzymes 108 4.1.1. Effect of Pubertal Testosterone Enanthate Treatment on Adult C Y P Expression 108 4.1.2. Effect of Dosing Frequency of Pubertal Androgen Treatment 4.1.3. Duration of Effect of Pubertal Testosterone Enanthate 112 4.2. Potential Limitations of the Study 114 4.2.1. Relevance of the Androgen Doses 115 4.2.2. Definition of Puberty 115 4.2.3. Sensitivity of RIA Assay 117 4.3. Speculation on the Results 117 4.3.1. Pubertal Imprinting or Long-Term Induction? 118 4.3.2. Relevance of C Y P Imprinting to Humans 118 4.4. Future Areas of Research 121 4.5. Summary and Conclusions 123 126 5. REFERENCES 129 6. APPENDIX 140 vi LIST OF TABLES Table page 1.1. Classification of sex-related differences in expression of hepatic C Y P 3 enzymes. 2.1. Grouping of the animals, treatments, and sacrifice times used in this study. 32 3.1. Steroids that do not cross-react with the testosterone RIA antiserum. 46 3.2. Specificity of the antiserum in the testosterone 1 2 5 I -RIA kit used in this 48 study. 3.3. Selectivity of the antiserum in the testosterone 1 2 5 I -RIA kit used in this 49 study. 3.4. Intra-assay variation of the testosterone hydroxylase assay. 58 3.5. Inter-assay variation of the testosterone hydroxylase assay. 59 3.6. Limits of quantitiation (LOQ) and detection (LOD) of the testosterone 61 hydroxylase assay expressed as both the amount of metabolite and the corresponding enzymes activity. 3.7. Inter-assay and intra-assay variation of the CYP2C11 immunoblot assay. 64 vii LIST OF FIGURES Figure page 1.1. Summary of immunochemical quantitations of hepatic microsomal C Y P 3 levels in untreated adult rats. 1.2. G H plasma profiles in control rats and rats given G H injections. 15 1.3. Treatment protocol of pubertal imprinting model. 20 2.1. Schematic indicating the possible CYP-mediated hydroxylated 35 testosterone metabolites. 3.1. Representative HPLC chromatograms obtained from the testosterone 51 hydroxylase assay. 3.2. Standard curves for the testosterone hydroxylase assay. 56 3.3. Formation of 7oc-hydroxytestosterone with respect to time. 62 3.4. Standard curve for immunoblots probed with anti-CYP2Cl 1 IgG. 64 3.5. Standard curve for immunoblots probed with anti-CYP3A IgG. 66 3.6. Standard curve for immunoblots probed with anti- CYP2A1 IgG. 68 3.7. Elimination of testosterone following pubertal testosterone enanthate 72 treatment (hours). 3.8. Elimination of testosterone following pubertal testosterone enanthate 72 treatment (days). 3.9. Effect of pubertal testosterone enanthate treatment on testosterone 2a- 73 and 16a-hydroxylase activities. 3.10. Immunoblot probed with anti-C YP2C11 IgG: Effect of pubertal androgen 75 treatment on CYP2C11 protein levels. 3.11. Effect of pubertal testosterone enanthate treatment on C YP2C11 protein 76 levels. 3.12. Effect of pubertal testosterone enanthate treatment on testosterone 60- 78 hydroxylase activities and CYP3A protein levels. viii 3.13. Irnmunoblot probed with anti-C YP3 A IgG: Effect of pubertal androgen 79 treatment on CYP3A protein levels. 3.14. Irnmunoblot probed with anti-CYP3 A2 IgG: Effect of pubertal androgen 81 treatment on CYP3A2 protein levels. 3.15. Effect of pubertal testosterone enanthate treatment on testosterone 7a- 82 hydroxylase activities and CYP2A1 protein levels. 3.16. Irnmunoblot probed with anti-CYP2Al IgG: Effect of pubertal androgen 83 treatment on CYP2A1 protein levels. 3.17. Elimination of testosterone following a single androgen injection. 86 3.18. Elimination of testosterone following pubertal testosterone treatment. 88 3.19. Effect of testosterone dosing frequency on testosterone 2a- and 16a- 90 hydroxylase activities. 3.20. Effect of testosterone dosing frequency on CYP2C11 protein levels. 92 3.21. Effect of testosterone dosing frequency on testosterone 6p-hydroxylase 93 activities and CYP3A protein levels. 3.22. Effect of testosterone dosing frequency on testosterone 7a-hydroxylase 95 activities and CYP2A1 protein levels. 3.23. Decline in testosterone 2a- and 16a-hydroxylase activities over time 97 following pubertal testosterone enanthate treatment. 3.24. Irnmunoblot probed with anti-CYP2C 11 IgG: Decline in CYP2C11 98 protein levels over time following pubertal testosterone enanthate. 3.25. Decline in CYP2C11 protein levels over time following pubertal 99 testosterone enanthate treatment. 3.26. Decline in testosterone 6p-hydroxylase activities and CYP3A protein 102 levels over time following pubertal testosterone enanthate treatment. 3.27. Irnmunoblot probed with anti-CYP3A IgG: Decline in CYP3A protein 103 levels over time following pubertal testosterone enanthate treatment. 3.28. Decline in testosterone 7a-hydroxylase activities and CYP2A1 protein 104 levels over time following pubertal testosterone enanthate treatment. 3.29. Irnmunoblot probed with anti-CYP2Al IgG: Decline in CYP2A1 protein 105 levels over time following pubertal testosterone enanthate treatment. ix LIST OF ABBREVIATIONS A androstenedione BCIP 5-bromo-4-chloro-3-indolylphosphate Bis N,N'-methylene-bis-acrylamide B S A bovine serum albumin CNS central nervous system c v coefficient of variation C Y P cytochrome P450 DHT dihydrotestosterone E C L enhanced chemiluminescence E D T A ethylenediaminetetraacetic acid G H growth hormone G H R H growth hormone releasing hormone GxF gonadectomized female rat H P L C high performance liquid chromatography IgG immunoglobulin G L O D limit of detection LOQ limit of quantitation M male rat M S G monosodium glutamate n number of samples in a group N A D P H nicotinamide adenine dinucleotide phosphate, reduced form N B T nitroblue tetrazolium chloride OD optical density P probability of a Type I error, a P A G E polyacrylamide gel electrophoresis PBS phosphate buffered saline RIA radioimmunoassay R N A ribonucleic acid s.e. subcutaneous SDS sodium dodecyl sulphate S E M standard error of the mean SS somatostatin T testosterone TE testosterone enanthate T E M E D N,N ,N ' ,N ' -tetramethyethylenediamine TP testosterone proprionate TRIS 2-amino-2-(hydroxymethyl)-1,3-propanediol U V ultraviolet radiation x ACKNOWLEDGEMENTS I would like to express my gratitude to my co-supervisors, Dr. Stelvio Bandiera and Dr. Gail Bellward, for all of their support and guidance over the last two years (was it really that long?). I also appreciate the helpful advice from my examining committee, Dr. Tom Chang, Dr. Keith McErlane, Dr. Ron Reid, and Dr. Wayne Riggs. I would like to acknowledge the Natural Sciences and Engineering Research Council of Canada for their financial support. I would also like to recognize Dr. Andrew Parkinson for providing us with purified CYP2A1, Dr. Paul Thomas for giving us anti-CYP2A1, anti-CYP3Al and anti-CYP3A2, and Dr. Michael Marshall for the statistical advice. Thanks to my fellow lab-mates with whom I shared many hours of conversation, music, and lab equipment. Ann, Jason, Song, and Ludger who else could better understand the trials and tribulations of grad school? I want to send a big thank-you to the family back on the east coast who dealt with the many distraught phone calls and emails with such patience and love. You guys never felt that far away ever i f an entire country lay between us. Also, these acknowledgements would not be complete without mentioning someone else's unconditional love. Chris, your caring and understanding always seemed to be without limits. You knew that I coulddo it even when I wasn't so convinced! xi 1. INTRODUCTION 1 It has been known for some time that the liver is a sexually dimorphic organ. Sexual differences have been reported in liver enzymes involved in the metabolism of xenobiotics for over sixty years (for a review see Shapiro et al. 1995). This sexual dimorphism can be attributed to differential expression of approximately a dozen hepatic C Y P enzymes between sexes. Dimorphic C Y P expression in rats is the extreme case with greater than a three to twenty fold difference between sexes in expression of hepatic drug metabolizing enzymes, making rats the ideal animal model to study the sexual regulation of C Y P expression. Sex steroids and growth hormone are the primary hormones implicated in the regulation of sex-dependent C Y P enzymes. In fact, testosterone plays an essential role in programming the expression of sex-dependent C Y P enzymes in the adult rat. Studies investigating the underlying role of hormonal regulation of rat C Y P may provide insight into similar regulatory pathways in man and other animals. In addition, knowledge of the mechanism of endocrine control in liver gene expression could explain the importance of hormonal status in xenobiotic metabolism. The current study investigates the influence of pubertally administered testosterone on expression of three sex-dependent C Y P enzymes in ovariectomized female rats. 1.1. CYP Enzymes C Y P enzymes are a ubiquitous family of related enzymes with a diverse range of substrates. They are characterized by an iron-heme prosthetic group that is responsible for their strong absorbance band at 450 nm when in the reduced CO-bound state. C Y P enzymes are the primary catalysts for the oxidation of steroids, drugs, and other lipophilic xenobiotics. Each C Y P enzyme is the product of a separate gene and has a different but overlapping substrate specificity. These proteins have a molecular weight around 50 kDa and are usually associated with membrane systems, in particular the endoplasmic reticulum. C Y P enzymes have been conserved throughout the evolutionary chain, which likely indicates that they serve a vital role in the preservation of life. Many believe the endogenous substrates of C Y P enzymes are steroids, such as cholesterol, testosterone, and estrogens (Guengerich 1991). Others maintain that C Y P enzymes serve a protective role against exogenous chemicals because they can metabolize lipophilic xenobiotics to a more water-soluble form that can be excreted more easily (Guengerich 1991). In mammals, many forms of C Y P enzymes exist and are found predominantly in the hepatic, renal, and gonadal tissues. Over 50 different C Y P enzymes have been identified in rats and as many as 34 confirmed enzymes exist in human tissues (Nelson 1996). C Y P enzymes are organized into families and subfamilies based on their genetic sequence similarity and are named by their familial classification. Differences in the protein levels and activities of enzymes between species and among individuals are controlled by sex, genetics, age, environment, and nutritional status. 1.1.1. Sex Differences in C Y P Expression Hepatic C Y P enzymes can be divided into groups based on their differential expression between sexes (Table 1.1.). Sex-specific forms are only expressed in one sex and sex-dominant forms, which are expressed in both sexes, exhibit a more pronounced expression in one sex over the other. Because protein levels of these particular enzymes are higher in either males or females, sex-related differences in C Y P expression are Table 1.1. Classification of sex-related differences in expression of hepatic C Y P enzymes in the rat (adapted from Kato and Yamazoe 1992). C Y P form Relative Difference C Y P form Relative Difference between sexes between sexes Male Specific Female Specific 2C11 >20 2C12 >20 3A2 >10 2A2 >10 2C13 >10 2C22 >10 Male Dominant Female Dominant 2B1 3-10 2A1 2 2B2 2 2C7 2 3A1 5 2E1 1.5 1A2 2 c "o a 0.5 — a E o 2B2 Fig. 1.1. Summary-of-immunochemical quantitations of hepatic microsomal C Y P levels in untreated adult rats. Rats-were from either Long-Evans or Sprague-Dawley strains and various immunochemical techniques were used. The recommended nomenclature designations are given (adapted from Ryan and Levin 1993). 4 described as dimorphic. Sex-related expression may be present in basal, otherwise known as constitutive, levels or in induced levels of particular C Y P enzymes. For example, C YP2B1 is induced to higher protein levels in males than females by phenobarbital (Agrawal and Shapiro 1996). However, it is important to remember that most C Y P enzymes are not sexually differentiated. A l l sex differences in C Y P levels develop at puberty. Yet, the mechanism underlying the differences between sexes that arise during development is different for the various enzymes (for a review see Ryan and Levin 1993). The male-specific forms, CYP2A2, CYP2C11, and CYP2C13, are not present in either sex at birth but are expressed at relatively high levels in adult males constitutively. In contrast, the male-specific form CYP3 A2 is present in both sexes neonatally but is decreased to undetectable levels in adult females while it continues to be expressed in adult males constitutively. The female-specific CYP2C12, like the male specific CYP2C11, is expressed at relatively high levels only after the onset of puberty. Levels of CYP2C7, a female-dominant enzyme, increase with sexual maturation in both sexes but to a greater extent in females. Another female-dominant form, CYP2A1, is expressed at equally high levels in both sexes during immaturity but its expression decreases with maturation in the male only. This suggests that the regulation of sex-dependent C Y P enzymes is not the same for all of the isozymes. During senescence, male CYP-related activities decrease in males but remain unchanged in females (Imaoka et al. 1991). At this time, the female-specific CYP2C12 appears in male liver microsomes (Kamataki et al. 1985a). Sexual dimorphism in the levels and activities of C Y P enzymes has been reported for many species including rat (Kamataki et al. 1982, McLeod et al. 1987), mouse (Hu et 5 al. 1993), chicken (Pampori and Shapiro 1993), sheep (for a review see Shapiro 1995), and humans (for a review see Bonate 1991). Because the rat is the best characterized animal model in this field, all findings reported here will be from the rat unless stated otherwise. Sex-related expression can be extended to additional Phase I enzymes, such as epoxide hydrolase (Pinot et al. 1995), Phase II enzymes such as UDP-glucuronosyl-transferase (Catania et al. 1995) and other enzymes not involved in drug biotransformation such as carbonic anhydrase (Jeffrey et al. 1990). As shown in figure 1.1., several C Y P enzymes are expressed at different levels between sexes and the magnitude of the difference in expression varies between the various enzymes. The variations in C Y P enzyme composition between sexes result in different abilities to biotransform drugs and xenobiotics. Generally, male animals biotransform drugs more rapidly than their female counterparts (Kamataki et al. 1982, Kamataki et al. 1985b, and Pampori et al. 1993). One exception to this trend is found in some mice strains where females tend to have the higher activity levels (McLeod et al. 1987). 1.2. Sex Differences in Hormone Secretion in the Rat 1.2.1. Sex Steroid Secretion The primary sex steroids in males and females are testosterone and estradiol, respectively. Estradiol levels are high in newborn rats and are high again between nine and 23 days of age in both sexes (Dohler and Wuttke, 1975). Mean estradiol plasma levels are similar in adult male and female rats but the female rats have small cyclical changes in plasma estradiol throughout adulthood that intermittently increase estradiol levels above male values (Dohler and Wuttke, 1975). 6 Plasma testosterone is traditionally regarded as the male sex steroid but is also observed in females at a uniformly low level. In the male rat, levels of testosterone increase after birth to one tenth adult levels for the first three to four days of life and then decrease to a basal level (Forest, 1979). Serum androgens, including testosterone and its major metabolite dihydrotestosterone, remain at higher levels in male than female rats during the prepubertal period. The onset of puberty in males is marked by a large increase in plasma testosterone levels at 35 days of age. Testosterone levels continue to increase to control adult levels until 49 days of age. Adult male rats do not have a constant level of circulating testosterone, rather it is released in a trimodal pattern of secretion that is characterized by elevated levels of testosterone at 0200, 1200h, and 1800h (Mock etal. 1978). 1.2.2. Growth Hormone Secretion Relatively high levels of growth hormone (GH) are present in the neonatal rat. Serum G H levels then decrease until puberty when they increase again (Wehrenberg 1986). GFI is released in a sex specific pattern from the pituitary after the onset of puberty (Eden 1979). The profile of G H secretion in the adult male rat is characterized by a pulsatile pattern where G H pulses are released every three to three and a half hours and troughs, or nadir, of undetectable levels occur between each pulse (Tannenbaum 1976). Smaller and more frequent pulses are observed in female rats such that the nadir level is higher and sustained. Consequently, a more continuous profile of secretion results. Although there is a difference in the pattern of secretion, the mean G H level is similar for both sexes. 7 1.2.3. Regulation of G H Secretion through the Hypothalamo-Pituitary Axis Gonadectomy of male rats increases G H baseline levels but the effect can be reversed with testosterone replacement therapy (Jansson et al. 1984). If the surgery is performed during the neonatal period, G H pulse heights in the adult male are reduced to female levels (Jansson et al. 1985a, 1985b). Testosterone administered during adulthood, with or without neonatal testosterone treatment, can fully restore pulse heights to normal male levels (Jansson and Frohman, 1987b). In contrast, estrogens elevate basal growth hormone levels and suppress G H pulses (Jansson et al. 1985a), but do so by antagonizing the effects of testosterone rather than having a direct action on pituitary G H secretion. This is shown by a lack of effect of neonatal androgen treatment on intact females (Jansson and Frohman, 1987a). Neonatal treatment with testosterone causes ovariectomized females to have higher G H pulse heights however, the G H secretion pattern is less regular and has a higher baseline than normal males (Jansson and Frohman, 1987a). Complete conversion to a male pattern of release is only possible with combined neonatal and adult testosterone treatment (Jansson and Frohman, 1987b). These observations indicate that sexually dimorphic G H patterns of secretion are imprinted neonatally and respond to an adult exposure of testosterone. The secretion of G H during adulthood is under direct control of the hypothalamus (Jansson et al. 1985a). Growth hormone releasing hormone (GHRH) released from the hypothalamus is responsible for controlling the amplitude of G H pulses while the presence of somatostatin (SS) determines the G H nadir (Wehrenberg et al. 1982, 1986). Testosterone modulates G H R H and SS neurons in the hypothalamus (for a review see Chowen et al. 1996). The number of G H R H neurons in the adult brain and their 8 responsiveness to adult testosterone exposure is increased by neonatal testosterone. The increase in number of G H R H neurons and their ability to synthesize G H R H is marked by stimulated expression of G H R H mRNA after testosterone activation (Zeitler et al. 1990). Somatostatin neurons are also affected by neonatal testosterone but only in synthetic capacity and not number of neurons. Therefore, the effect of testosterone on G H patterns of secretion is determined by testosterone control over G H R H and SS secretion. 1.3. Hormonal Regulation of CYP Enzymes 1.3.1. Involvement of Sex Steroids in C Y P Expression The developmental regulation of sexual dimorphism in C Y P enzymes suggests the involvement of hormones in controlling expression because the onset of sexual differentiation of CYP-dependent activities coincides with a time of increasing sex steroid secretion (Pak et al. 1984). This is confirmed by the observed effects of gonadectomy on levels of sexually differentiated C Y P enzymes. Castration of male rats decreases the metabolism of exogenously administered xenobiotics such as hexobarbital, coumarin, and ethoxyresorufin to levels more similar to female values (for a review see Skett 1987). In fact, castration results in a "feminization" of all sex-dependent activities, including hydroxylation of steroids. This is confirmed by decreases in protein levels of male-specific enzymes CYP2A2, CYP2C11, CYP2C13, and CYP3A2 (Waxman et al. 1985, Dannan et al. 1986) and an increase in female-dominant CYP2A1 expression (Waxman et al. 1989) following castration of adult male rats. However, complete feminization of enzyme activities is only achieved when castration is performed neonatally (Dannan et al. 1986, for a review see Waxman and Chang 1995). Neonatally 9 ovariectomized females exhibit reduced levels of CYP2C12 and CYP2A1 at adulthood as well as another female-specific non-CYP microsomal enzyme, steroid 5a-reductase (Dannanera/. 1986, Waxman etal. 1989). Adult ovariectomy has no effect on these enzymes. A role for sex steroids in the regulation of sex-dependent cytochromes P450 is further supported by restoration of sexually dimorphic enzyme levels in castrated animals with exogenous testosterone or estradiol treatment. Estradiol treatment during adulthood causes expression of CYP2C12 and steroid 5a-reductase to return to normal in neonatally ovariectomized females, while neonatal estradiol treatment is able only to partially restore expression of these enzymes (Dannan et al. 1986). Neonatal testosterone treatment of castrated male and female rats stimulates expression of CYP2C11 and CYP3A2 to levels significantly less than control male animals. However, protein levels statistically similar to normal males are achieved with additional testosterone treatment during adulthood (Waxman et al. 1989). Like GH, it appears the C Y P enzymes are imprinted neonatally and respond to an adult exposure of testosterone. Adult testosterone treatment alone can increase levels of male C Y P enzymes to control male values but the effect is likely dependent upon continued androgen presence because adult castration decreases these levels (Waxman and Chang 1995). 1.3.2. Definitions of Imprinting and Adult Androgen Responsiveness The effects of gonadectomy and hormone replacement therapy resulted in two new terms being adopted to describe the role of androgens in sex-related development of C Y P enzymes. Adult androgen responsiveness describes the ability of testosterone 10 during the adult period to elevate the levels of the male-specific C Y P enzymes. This is a reversible process, as the continued presence of circulating testosterone is required to maintain high enzyme activities. The second term, imprinting, is borrowed from the field of animal (especially avian) behavior where it is used to describe an "extremely rapid attachment [to the mother] during a critical period of early exposure which becomes an important elicitor of later behavior" (Hess 1973). For use in C Y P studies, imprinting refers to an exposure to testosterone early in life that is crucial for full expression of male-specific enzymes upon a later exposure to testosterone. Neonatal imprinting is described as a permanent increase in the basal level of an enzyme after neonatal exposure to testosterone that continues even after levels of testosterone decrease (Dannan et al. 1986). Both of these terms can be applied to exogenous administration of testosterone or physiological periods of testosterone production in the male, i.e. neonatal or postpubertal. 1.3.3. Involvement of G H in C Y P Expression The observation that androgens have no effect on C Y P levels when rats are hypophysectomized suggests that testosterone elicits its effects through the hypothalamic-pituitary axis (Kamataki et al. 1985b, for a review see Kato and Yamazoe 1993). The pituitary gland has been known to regulate expression of sexually differentiated C Y P enzymes for over twenty years (Gustafsson et al. 1976). Considering the large increase in G H secretion at puberty that coincides with the development of sex-dependent expression of C Y P enzymes, G H was then identified as the responsible factor by Mode et al. (1983). Androgens are now believed to control sex-dependent expression 11 of C Y P enzymes by altering the pattern of G H secretion because both phenomena are similarly masculinized by neonatal and adult exposures to testosterone. Hypophysectomy during any period of development abolishes sex differences in xenobiotic metabolism and C Y P levels but does not result in complete suppression of any C Y P enzymes. Benzo[a]pyrene hydroxylase, 7-propoxycoumarin (3-depropylase, and aminopyrine JV-demefhylase activities decrease in hypophysectomized males and increase in hypophysectomized females (Kamataki et al. 1985b). Surgical removal of the pituitary also causes a decrease in the levels of CYP2C11 in males (Waxman et al. 1991) and a decrease in the levels of CYP2C12 and the activity of 5a-reductase in females (Waxman et al. 1990). Interestingly, levels of CYP2A2 and CYP3A2 increase in both sexes upon hypophysectomy such that there is no longer any sex difference (Kato and Yamazoe 1993). Estrogens, on the other hand, appear to have direct actions on C Y P levels. Unlike testosterone, estrogen does alter C Y P levels in hypophysectomized rats. Thus, estrogens must have some direct action on C Y P levels that do not involve the hypothalamic-pituitary axis. However, estrogen cannot reverse the effects of hypophysectomy on CYP2C12. Rather, this hormone enhances the suppression of male-dominant enzyme activities observed after surgery (Kamataki et al. 1985b). Various rat models have been developed and studied extensively to determine whether the amount of G H or the pattern of G H release controls sex-related differences in C Y P expression. Treatment of neonatal rats with high levels (4mg/g) of monosodium glutamate (MSG) or monosodium aspartate (MSA) every other day for the first nine days of life causes a loss of detectable serum G H as an adult (Waxman et al. 1995). Male 12 MSG-treated rats have decreased levels of CYP2C11, CYP2A2, and CYP3A2 that are comparable to levels found in adult female rats (Waxman et al. 1995, Pampori and Shapiro 1994). In addition, CYP2A1 and steroid 5a-reductase activities are induced (Waxman etal. 1990, Pampori etal. 1991). These changes are consistent with those found in hypophysectomized rats with the exception of the suppression of CYP2 A2 and CYP3A2 as these enzymes are elevated in hypophysectomized rats (Waxman et al. 1990) . As with hypophysectomized rats, no induction of CYP2C12 is observed for MSG-treated male rats. This model supports the hypothesis that G H is involved in sex-related differences of hepatic CYP levels. In contrast to hypophysectomized females, MSG-treated females display no change from control female rats in terms of C Y P expression, as CYP2C12 remains present at relatively high levels (Waxman et al. 1990, 1991) . This observation may be a manifestation of the continued presence of low levels of G H , as evidenced by persistent IGF-1 mRNA transcription in response to G H (Waxman etal. 1995). Differences in C Y P expression between MSG-treated rats and hypophysectomized rats may be a result of differences between the two models. High doses of M S G administered during the neonatal period destroy 80-90% of the neurons located in the arcuate nucleus of the hypothalamus (Lemkey-Johnston and Reynolds, 1974). The loss of G H R H produced in the arcuate nucleus blocks the release of G H from the pituitary while the pituitary level of G H remains unchanged. The levels of other pituitary hormones, including L H and FSH, are also unaffected. Hypophysectomy causes a loss of other pituitary hormones in addition to GH, whereas MSG-treatment selectively decreases G H secretion only. Furthermore, hypophysectomy is often performed on adult 13 animals and causes a loss of G H secretion as an adult, whereas MSG-treatment causes a neonatal loss of G H that could be critical for imprinting effects. Lower doses (2mg/g) of M S G merely decrease the pulse amplitude of G H secretion and either are ineffective in abolishing sexually dimorphic expression of C Y P enzymes (Waxman et al. 1990, Pampori et al. 1991) or cause increased expression of CYP2C11 in males (Pampori and Shapiro 1994). This observation indicates that lower amounts of G H are capable of maintaining sex-related differences in C Y P expression. A strain of dwarf rats used by Bullock et al. (1991) have barely detectable serum levels of pituitary G H that are approximately 5% of normal levels. Yet, the sexually dimorphic patterns of G H secretion were maintained in these dwarf rats. Surprisingly, all C Y P levels and catalytic activities were the same as those of normal rats except that CYP3 A2 expression was half of normal levels (Bullock et al. 1991, Wells etal. 1994). The authors of this study concluded that G H was not important for the regulation of sex-related differences in C Y P levels. However, when the results of the dwarf rat, hypophysectomized rat, and MSG-treated rat models are combined, it is apparent that the amount of G H is not crucial for sex specific expression as low levels of G H can maintain sex differences provided that the sexually differentiated pattern of secretion remains. Because the amount of G H present does not regulate the sexually dimorphic expression of C Y P enzymes, G H replacement therapy using various regimes of administering G H was used to reveal which characteristic of the G H profile of secretion controls expression of these enzymes. Figure 1.2. illustrates the G H plasma profiles measured after the various G H replacement therapy regimes. Six intravenous pulses of G H daily were found to be the most effective replacement therapy for MSG-treated 14 males, but only partially restored levels of CYP2C11, CYP2A2, and CYP3A2 (Waxman etal. 1995). MSG-treated females did not respond to any G H replacement therapy. A protocol of subcutaneous injections every twelve hours produces a nonphysiological G H pattern of sustained pulses that is capable of stimulating male specific CYP2C11 expression (Mode et al. 1989, Shimada etal. 1989, Waxman et al. 1991). Intravenous injections are more quickly eliminated and can produce a more physiological pattern of male G H levels. Intravenous injections at two, four, and six times daily stimulate CYP2C11 levels whereas injections at seven times daily is ineffective because there is not enough time between pulses to allow G H levels to fall below the limit of detection. Yet, seven times daily is not frequent enough to stimulate CYP2C12 expression or steroid 5a-reductase activity. These results indicate that a period of at least 2.5 hours with no detectable G H is required to maintain male-specific C Y P protein levels and that pulse frequency is less crucial (Waxman et al. 1991). This is consistent with the negative results of G H replacement therapy of MSG-treated females. As mentioned above, M S G -treated females have sustained low levels of G H so a period of G H absence required to stimulate expression of male C Y P enzymes is not possible in this animal model. Continuous s.e. infusion delivered by an osmotic minipump mimics the female pattern of G H secretion and in hypophysectomized rats is able to induce female-specific CYP2C12 (Mode et al. 1989, Wells et al. 1994) and female-dominant steroid 5ct-reductase levels, as well as suppress the levels of male-specific forms, CYP2A2, CYP3 A2 (Waxman et al. 1991), and CYP2C11 (Shimada et al. 1989, Wells et al. 1994). CYP2C11 heteronuclear R N A and mRNA levels correlate with protein levels in all treatment protocols, suggesting that G H 15 A MALE - C HX + GH sc. 0 HX + GH cont. T " 1 9:00 11.-00 13:00 15:00 17:00 9:00 11:00 13:00 15:00 17:00 ' Clock-time 225 150 75 0 J+ 225 0 -1+ E 2P/DAY F 4P/DAY 1 a 6 P / M Y 1 9:00 11:00 13:00 15:00 17:00 9:00 11:00 13:00 15:00 17:00 Clock-time Figure 1.2. G H plasma profiles of male (A) and female (B) rats. Hypophysectomized male rats treated with a subcutaneous G H injection (C) or injections via osmotic minipump (D) are commonly used to simulate the physiological secretion patterns. A more accurate simulation of the male pattern is possible with i.v. injections of growth hormone at vairous frequencies of two (E), four (F), six (G), and seven (H) pulses daily (adapted from Waxman etal. 1991). 16 regulates this enzyme at a pretranscriptional step (Shimada et al. 1989, Sundeth et al. 1992). 1.4. Neonatal Imprinting Gonadal hormones imprint sexual characteristics that are under control by the central nervous system (CNS) during the neonatai period (for a review see MacLusky and Naftolin 1981). The CNS is innately organized for the homogametic sex, which is female in mammals, without further modulation by sex steroids (ibid. 1981). Any sexual differentiation of the CNS to the male phenotype requires exposure to male gonadal hormones. Two distinct processes may be involved in imprinting, defeminization and masculinization. Defeminization is the suppression of female characteristics and masculinization is the sensitization to hormones that enhance male characteristics during adulthood. In the rat, the CNS displays increased sensitivity to modulation by gonadal hormones 18-27 days after conception, with birth occurring at 20 to 22 days after conception. The "organizational" effects of early gonadal hormone exposure during this sensitive period permanently alter CNS function. However, the "activational" effects of adult hormone exposure are likely reversible because adult castration partially reverses the observed effects. Defeminization of the CNS by androgens during the neonatal period occurs after aromatization of testosterone to 17(3-estradiol in the brain (McEwen et al. 1982, McCarthy 1994). On the other hand, masculinization appears to involve both the androgen and the estrogen receptor. Yet, endogenous estrogens in the neonatal rat do not defeminize the CNS because they are present in the circulation bound to oc-fetoprotein, 17 which prevents the steroid from crossing the blood-brain barrier into the CNS (Aussel et al. 1973). In addition, endogenous neonatal testosterone in female rats does not cause defeminization. This is possibly the result of prior hormonal exposure (Weisz and Ward 1980) or inhibition by the ovaries (Blizard and Denef 1973). 1.4.1. Evidence for Neonatal Imprinting of C Y P Enzymes The sexually differentiated pattern of G H secretion that is neonatally imprinted by testosterone in the rat during the first few days of life (Gustafsson 1983) controls expression of the sexually differentiated C Y P enzymes at puberty. This suggests there might be a common pathway of imprinting for G H and C Y P expression. Just as defeminization of the CNS, which will affect G H patterns of secretion, is mediated by the estrogen receptor and not the androgen receptor, the estrogen receptor appears to be involved in hepatic enzyme imprinting by testosterone (Reyes and Virgo 1988). Neonatal imprinting of CYP2C11 and CYP3A2 in neonatally gonadectomized males and females by testosterone enhances masculinization of these enzymes in the adult period (Dannan et al. 1986). The neonatal period is critical for testosterone imprinting because any disruption that affects testosterone production during this period creates lasting abnormal sex-specific effects in CYP expression. For example, benzo[a]pyrene exposure after birth results in increased CYP2C11 expression in males and decreased CYP1A2 expression in females that is still observed after 110 days (Fujita et al. 1995). Secondly, newborn rats exposed to hyperoxic conditions display the opposite effect and thus are femininized (Kikkawa et al. 1994). 18 Neonatal imprinting by androgens of microsomal enzymes involved in xenobiotic biotransformation is also documented for alcohol dehydrogenase (Crabb et al. 1986), corticosteroid 5oc-reductase (Reyes and Virgo 1988), and acetohexamide reductase (Imamura et al. 1994). As with CYP2C11 and CYP3A2 expression, adult exposure to testosterone is required before full acetohexamide reductase activity is achieved (Imamura et al. 1994). 1.5. Evidence for Pubertal Imprinting of CYP Enzymes Data from a strain of dwarf rats containing a mutant G H gene, are inconsistent with the need for neonatal imprinting to produce a male pattern of C Y P enzymes (Shimada et al. 1995). The altered G H gene in these rats results in abnormal splicing of the protein so that no functional G H is synthesized. This strain of dwarf rats is different than the dwarf rats used by Bullock et al. (1991) that had low levels of functional hormone. In the dwarf rats analyzed by Shimada et al, the developmental profile for female CYP2C11 levels is similar to the normal male even though there was no neonatal exposure to testosterone. Therefore, the ontogeny of CYP2C11 may not be dependent upon neonatal imprinting by testosterone. Also, it has been shown that, pubertal, not neonatal, imprinting is responsible for higher male levels of benzo[a]pyrene hydroxylase (Vaketal. 1984). Currently, investigations are underway to determine the possibilities of imprinting C Y P outside of the neonatal period. Prepubertally gonadectomized females are used as model animals because they lack neonatal testosterone exposure (figure 1.3.). Fourteen days of treatment with testosterone enanthate during the pubertal period "imprints" 19 CYP2C11 and CYP3A protein levels and their related activities to respond to a second androgen challenge during adulthood (Cardario et al. 1992). Expression of CYP2C11 and CYP3A enzymes is equivalent to control male levels following the two treatment periods. Although the secretion of estrogen is believed to oppose the actions of testosterone on C Y P expression (Bandiera and Dworschak, 1992), the effect of combined pubertal and adult testosterone enanthate treatment is observed in sham-operated females as well. The response in sham-operated females is not surprising because neonatal exposure to testosterone causes levels of CYP2C11 and CYP3A2 to increase and levels of 2C12 and 5a-reductase to decrease in intact adult females (Dannan et al. 1986). The same treatment protocol is able to decrease expression of female-dominant CYP2A1 (Cadario et al. 1992) and steroid 5a-reductase (Chang and Bellward 1996) enzymes to control male levels. Expression of male-specific CYP2C11 is still observed 40 days after the pubertal treatment (Cardario et al. 1992). The mRNA levels of CYP2C11 and steroid 5a-reductase reflect the changes in protein levels suggesting a pretranslational control in pubertal androgen imprinting of these two enzymes (Chang and Bellward 1996). The inductive effect is selective for the male-specific enzymes as CYP2A1 -mediated (a female-dominant form) activity and protein levels are reduced to control male values with the same protocol. 20 0 A Birth Tp 25 A 35 Gonadectomy Or Sham Operation Ta <—> i — i — r 49 81 89 90 T Sacrifice Age (days) Figure 1.3. Schematic outline of the treatment protocol used to create the pubertal imprinting model. Tp and Ta refer to the pubertal and adult testosterone enanthate treatment periods of 5 pmol/kg/d s.e, respectively. 21 1.6. Hypothesis and Objectives 1.6.1. Hypothesis In this project, I propose to continue to investigate the hypothesis that the pubertal period is a time when androgen imprinting of hepatic C Y P enzymes can occur. Several questions have arisen from the earlier study by B. Cadario (1990,1992) regarding pubertal imprinting. The overall goal of the present study is to further characterize testosterone imprinting during the pubertal period and to validate the observations made previously. 1.6.2. Specific Objectives of This Study The specific objectives of my thesis project are: (1) To confirm, in prepubertally gonadectomized female rats, the increased expression of male-specific enzymes and suppression of female-dominant enzymes during adulthood by androgen treatment administered pubertally. (2) To determine the effects of more frequent testosterone injections, a treatment protocol more accurately simulating physiological testosterone secretion patterns, on the increased expression of male-specific enzymes and suppression of female-dominant enzymes during adulthood (3) To determine the duration of any androgen-mediated effects on C Y P expression during adulthood, to test the hypothesis of imprinting or the possibility that long-term induction is occurring. 22 (4) To determine the elimination profiles of the two androgen formulations, thus studying the possibility of redistribution to adipose tissues and slow release of the hormones from these tissues as the mechanism for the observed enzymatic changes. 1.6.3. Rationale for Experimental Design Previous studies, in which the possibility of pubertal imprinting was investigated (Cadario et al. 1990, 1992), used prepubertally ovariectomized female rats (surgery on day 25) as model animals. This animal model has been used to ensure that estrogen secretion by the ovaries would not suppress the expression of male C Y P (Bandiera and Dworshak, 1992). The sex-dependent CYP levels in these animals at adulthood were found to be imprinted by testosterone enanthate administered over the pubertal period (days 35-49). The present study used the same animal model and pubertal treatment protocol. The fifteen-day treatment period included daily subcutaneous injections of testosterone, testosterone enanthate, or corn oil. A treatment group was included that is the same as the previous experiment (Cadario 1992) in order to confirm the earlier results and provide values to which variations of the model can be compared. In that study, pubertal testosterone enanthate treatment alone was not effective in elevating male-specific and male-dominant C Y P levels to control male values. It is thought that a pulsatile pattern of pubertal testosterone enanthate secretion is necessary to masculinize adult C Y P expression because a subcutaneous testosterone enanthate implant during puberty that continuously releases testosterone enanthate feminizes C Y P expression. 23 Therefore, it is hypothesized that a number of testosterone surges in a twenty-four hour period, with inter-peak periods of undetectable testosterone, may be required to regulate sex-dependent C Y P expression (Chang and Bellward, 1996). The effects of increasing the dose frequency to two daily doses of testosterone were examined (Table 2.1). Twice daily doses of testosterone enanthate were not studied because the hormone's half-life in ovariectomized females is too long and the hormone would accumulate to nonphysiological concentrations. A single daily dose of testosterone at the same concentration as the testosterone enanthate was included to compare with two daily doses of testosterone. These three animal groups and the two control groups were sacrificed at 90 days of age, the same age as the previous study, and hepatic microsomes were prepared from these animals. Two other animal groups were treated with testosterone enanthate as well, except that the sacrifice times are later in the animals' lives (129 and 169 days of age, respectively). These groups were used to determine the duration of the androgen effects in the adult period. Plasma samples were drawn from the rats' tail veins to profile the elimination of testosterone following the pubertal treatment with testosterone enanthate and testosterone. Blood samples were drawn at timed intervals following the final androgen dose and assayed for testosterone. An additional blood sample before sacrifice was assayed to confirm the absence of detectable testosterone prior to C Y P protein level and activity determination. 24 1.7. Sexually Differentiated CYP Enzymes Investigated in the Present Study 1.7.1. CYP2A1 CYP2A1 is a member of the CYP2A subfamily. There are three members of this subfamily in rats, namely, hepatic CYP2A1, hepatic CYP2A2, and pulmonary CYP2A3 (Nelson 1996). CYP2A1 is a female-dominant enzyme that is responsible for 7a-hydroxylation of progesterone and testosterone (Sonderfan 1987, Ryan and Levin 1990). This enzyme is present in neonatal rats at relatively low levels but CYP2A1 expression increases to peak expression at two weeks of age (Imaoka et al. 1991). In male rats, the expression of this enzyme decreases after two weeks of age to one fourth the level in female rats. Constitutive levels of CYP2A1 represent 3% and 6% of total C Y P levels in untreated adult male and female rats, respectively (Thomas et al. 1981). Although the C Y P 2 A is generally refractory to common inducing agents, phenobarbital and 3-methylcholanthrene can induce CYP2A1 levels two-fold above control (Lewis 1996). CYP2A1 expression is under hormonal control. Castration increases the expression of CYP2A1 in adult males to two and half times control male levels (Waxman et al. 1989). Ovariectomy decreases the expression of CYP2A1 in adult females to a level similar to that of castrated males. Hypophysectomy causes a relatively small increase in CYP2A1 expression in adult males to prepubertal levels but has no effect on adult female expression of this enzyme. Neonatal M S G treatment also causes a small increase in CYP2A1 expression in adult males (Waxman et al. 1990). Conversely, M S G -treated female rats have 50% more CYP2A1 than control female rats. 25 1.7.2. CYP2C11 The CYP2C subfamily is composed of noninducible enzymes that are under developmental or sex-specific regulation (Gonzalez 1989, Lewis 1996). There are nine known forms of CYP2C enzymes in the rat (Nelson et al. 1996). CYP2C11 is a male-specific enzyme that is responsible for the hydroxylation of testosterone and progesterone at the 2a- and 16a-positions (Sonderfan 1987, Ryan and Levin 1990) and is the primary enzyme involved in benzo[a]pyrene hydroxylation in untreated rats (Ohgiya et al. 1989). This enzyme exhibits a broad versatility in catalyzing drugs and other lipophilic xenobiotics. In the male rat, CYP2C11 expression is undetectable in prepubertal rats but increases at the onset of puberty. CYP2C11 expression in adult male rats is decreased to one-tenth control levels by neonatal castration (Waxman et al. 1985) or adult hypophysectomy (Shapiro et al. 1989). CYP2C11 is undetectable in female rats at any age and is not increased by either ovariectomy or hypophysectomy. 1.7.3. CYP3A The CYP3 A subfamily is responsible for the metabolism of many clinical agents. Steroids, macrolide antibiotics, phenobarbital, and imidazole antifungals induce enzymes in this subfamily (Lewis 1996). Members of this subfamily hydroxylate testosterone at the 20- 6fi- and 15p-positions (Sonderfan 1987, Nagata 1990). There are five known genes in the adult rat that belong to the CYP3A subfamily, namely, CYP3A1, CYP3A2, CYP3A9, CYP3A18, and CYP3A28 (Nelson et al. 1996). However, it has been suggested that CYP3A28 is an allelic variant of CYP3A1 because the difference 26 between the protein sequences of these two genes is less than 3% (Ribeiro and Lechner 1992) . At least three members of the CYP3 A subfamily are believed to be under hormonal control. CYP3A1 is not expressed in untreated rats but can be induced by glucocorticoids and the synthetic steroid, pregnenolone 16a-carbonitrile (Cooper et al. 1993) , which suggests that CYP3A1 is possibly sensitive to endogenous hormones. CYP3 A2 is expressed in both sexes of immature rats but is only expressed in adult male rats (Ryan and Levin 1990). This enzyme is not induced by glucocorticoids but phenobarbital can cause a three-fold induction above control levels (Lewis 1996). CYP3 A9 expression develops at puberty and is a female-dominant enzyme in adult rats (Mahnke etal. 1997). Four enzymes with testosterone 6(3-hydroxylase activity have been separated and named, 6p-l, 6p-2, 6p-3, and 6p-4 (Nagata etal. 1990). It is believed that 6P-1 and 6p-3 represent CYP3A2 and that 6P-4 represents CYP3A1, respectively (Strofkamp et al. 1995). The deduced amino acid sequence of a female dominant CYP3A gene is similar to the sequence of 6p-2 but it was not identified as CYP3A9 (Strofkamp et al. 1995). In the current study, the expression of three enzymes, CYP2C11, CYP3A, and CYP2A1, was determined in adult female rats following a pubertal androgen treatment period. Protein levels of individual enzymes were measured by immunoblots probed with anti-CYP IgG. Testosterone 2a- and 16a- hydroxylase activities were used as a measure of CYP2C11 activity. Testosterone 6p- and 7a- hydroxylase activities were used as markers for CYP3A and CYP2A1 activities, respectively. 2. EXPERIMENTAL 27 2.1. Chemicals Chemicals used in this study were obtained from the following sources: Amersham International pic (Little Chalfort, Buckinghamshire, England): E C L Western blotting detection reagents. BDH Chemicals, Inc. (Toronto, Ontario, Canada): Corn Oil , disodium hydrogen orthophosphate (dibasic), ethanol (100%), ethylenediaminetetraacetic acid disodium salt (EDTA), Folin & Ciocalteu phenol reagent, magnesium chloride, potassium chloride, potassium dihydrogen orthophosphate (monobasic), sodium carbonate (anhydrous), sodium chloride, sodium dodecyl sulphate, sodium hydroxide, and sucrose. Bio-Rad Laboratories (Richmond, California, USA): Bis, and N,N,N',N'-tetramethyethylenediamine (TEMED). Boehringer Mannheim Canada, Ltd. (Laval, Quebec, Canada): Nicotinamide adenine dinucleotide phosphate tetra-sodium salt reduced form (NADPH). 28 Carnation, Inc. (Toronto, Ontario, Canada): Skim milk powder. Diagnostic Chemicals, Ltd. (Charlottetown, PEI, Canada): 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (BCIP), and nitro blue tetrazolium (NBT). Fisher Scientific Ltd. (Vancouver, British Columbia, Canada): Acetonitrile (HPLC grade), bromophenol blue, glycerol, glycine, 2-mercaptoethanol, methanol (reagent and HPLC grades), and methylene chloride (HPLC grade). ICNBiomedicals, Ltd. (Aurora, Ohio, USA): Acrylamide, and bovine serum albumin (globulin and fatty acid free). ICN Biomedicals, Ltd (Cleveland, Ohio, USA): N,N-dimethyl-formamide. ICN Biomedicals, Ltd. (Costa Mesa, California, USA): ImmunoChem™ coated tube testosterone [ I]-radioimmunoassay (RIA) kit J. T. Baker Chemical Co. (Phillipsubrg, New Jersey, USA): Sodium dithionite. 29 Kirkegaard and Perry Laboratories, Inc. (Gaithersburg, Maryland, USA): Affinity purified rabbit anti-sheep IgG phosphatase conjugate. Mandel Scientific Company, Ltd. (Edmonton, Alberta, Canada): Nitrocellulose membrane. Praxair Canada, Inc. (Mississauga, Ontario, Cananda): Carbon monoxide gas, and nitrogen gas. Sigma Chemical Company (St. Louis, Missouri, USA): Cupric sulphate pentahydrate, N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pyronin Y , sodium potassium tartrate pentahydrate, testosterone, testosterone enanthate, trizma base, and Tween 20. Schwarz/Mann Biotech (Cleveland, Ohio, USA): Ammonium persulfate. Steraloids, Inc. (Wilton, New Hampshire, USA): 4-androsten-17p-diol-3-one (testosterone), 4-androsten-3,17p-diol-3-one (androstenedione), 4-androsten-2a,17p-diol-3-one (2a-hydroxytestosterone), 4-androsten-6p,17p-diol-3-one (6p-hydroxytestosterone), 4-androsten-7a,17p-diol-3-one (7a-hydroxytestosterone), 4-androsten-1 ip,17p-diol-3-one (11 p-hydroxytestosterone), 4-30 androsten-16a,17p-diol-3-one (16a-hydroxytestosterone), and 4-androsten-16p,17p-diol-3-one (16p-hydroxytestosterone). TAGO Immunologicals, Inc. (Burlingame, California, USA): Affinity isolated goat F(ab')2 anti-(rabbit IgG) (gamma and light chains specific) human Ig adsorbed alkaline phosphatase conjugate, affinity isolated goat F(ab')2 anti-(mouse IgG) (gamma and light chains specific) human Ig adsorbed alkaline phosphatase conjugate, and affinity isolated goat F(ab')2 anti-(mouse IgG) (gamma and light chains specific) human Ig adsorbed peroxidase conjugate. Dr. S. M. Bandiera (Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, British Columbia, Canada): Purified rat CYP2C11, partially purified rat CYP3A1, monospecific polyclonal rabbit anti-CYP2Cl 1 IgG, and rabbit anti-CYP3A IgG (backabsorbed). Dr. A. Parkinson (University of Kansas Medical Center, Kansas City, Kansas, USA): Purified rat CYP2A1. Dr. P. E. Thomas (Department of Chemical Biology and Pharmacognosy, State University of New Jersey, Rutgers, Piscataway, New Jersey, USA): Sheep anti-CYP2Al polyclonal IgG, mouse anti-CYP3Al monoclonal IgG, and mouse anti-CYP3A2 monoclonal IgG. 31 2.2. Animals and Treatment Eight male and forty-four female Sprague-Dawley rats were purchased from Charles River Canada, Inc. (Montreal, Quebec, Canada). The animals were housed in polycarbonate cages with corncob bedding under conditions of controlled temperature (23°C) and lighting (lights on from 7 am to 7 pm). Food (Lab Diet, Purina Mills , Inc.) and tap water were available ad libitum. The breeder performed ovariectomies on the female rats when the animals were 25 days old. Following approximately three days recovery, the animals were shipped to the University of British Columbia. After five to seven days acclimatization at the university, ovariectomized female rats were injected subcutaneously with testosterone or testosterone enanthate dissolved in corn oil, or equal volumes of corn oil during the pubertal period (35-49 days of age) as outlined in Table 2.1'. Injection volume was 0.1 ml/lOOg animal body weight. Animals were sacrificed by decapitation at 90, 129 or 169 days of age. 2.3. Preparation of Hepatic Microsomal Fraction Hepatic microsomes were prepared from individual rats following decapitation using the method of Thomas et al. (1983). Livers were quickly removed, placed in 20 ml of ice-cold 0.05 M Tris buffer, pH 7.5, containing 1.15% KC1, and minced with scissors. The tissue was homogenized with a Potter-Elvehj em glass mortar and a motor-driven pestle by five low-speed passes with a loose-fitting pestle and five high-speed passes with a tight-fitting pestle. The homogenate was spun at 9,000 x g for 20 minutes at 5°C in a Beckman Model J2-21 centrifuge. The supernatant was filtered through four layers of 32 Table 2.1. Grouping of the animals, treatments, and sacrifice times used in this study. Group Animals n Treatment (days 35-49) Sacrifice A GxF 8 Corn Oil day 90 8am, 8pm B GxF 7 Testosterone Enanthate (5 pmol/kg) day 90 8am C GxF 7 Testosterone (5 umol/kg) day 90 8am D GxF 8 Testosterone (2.5umol/kg) day 90 8am, 8pm E M 8 Untreated day 90 F GxF 7 Testosterone Enanthate (5 umol/kg) day 129 8am G GxF 7 Testosterone Enanthate (5 umol/kg) day 169 8am The abbreviations Gx, F, and M denote gonadectomized, female, and male rats, respectively. 33 cheesecloth and spun at 105,000 x g for 60 minutes at 5°C in a Beckman ultracentrifuge. The resulting microsomal pellets were resuspended in ice-cold 10 m M EDTA, 1.15% KC1, pH 7.4 using the homogenizer with five passes of the loose-fitting pestle, and centrifuged again at 105,000 x g for 60 minutes at 5°C. The pellets were resuspended in 0.25M sucrose by gentle homogenization and were frozen in cryorubes in 0.5 ml aliquots at -75°C. 2.4. Determination of Protein Content Total hepatic microsomal protein was measured by the method of Lowry et al. (1951). Bovine serum albumin was used as the standard. A l l samples were measured in duplicate at an absorbance of 750 nm. 2.5. Determination of Total CYP Content Total hepatic microsomal C Y P was measured spectrophotometrically as described by Omura and Sato (1964a). Hepatic microsome preparations were diluted 1:10 in 0.1 M sodium phosphate buffer, pH 7.4, containing 20% glycerol and 0.1 M EDTA. Each diluted microsomal preparation was placed into two cuvettes, the sample and reference cuvettes. Both cuvettes were reduced with sodium dithionite. The sample cuvette only was saturated with carbon monoxide and the difference spectrum between 400 and 500 nm was measured between the sample and reference cuvettes. The carbon monoxide-reduced difference spectrum was measured on an SLM-Aminco DW-2 spectrophotometer and determined as the difference between the absorption maximum at approximately 450 nm and the baseline at 490 nm. Total hepatic microsomal C Y P 34 concentration was calculated using a molar extinction coefficient of 91 cm2/mmol (Omura and Sato, 1964b). Total C Y P content was expressed as nmoles CYP/mg protein. 2.6. Plasma Testosterone Total unconjugated plasma testosterone concentration was measured using an ImmunoChem™ coated tube testosterone [125I]-radioimmunoassay (RIA) kit. This assay is a competition RIA which measured the amount of radiolabeled testosterone bound to the test tubes following an equilibrium between the radiolabeled testosterone and the unlabelled testosterone present in the plasma sample. The kits are designed to measure testosterone concentrations within the linear range of their standard curve (0.2 ng/ml to 20.0 ng/ml). Blood samples were collected from the tail vein of rats into heparinized Natelson Blood Collecting Tubes (Fisher Scientific) and were centrifuged at 15,000 x g for 10 minutes at 4°C. The plasma supernatant was carefully removed and stored in cryovials at -20°C until analysis. 2.7. Testosterone Hydroxylase Assay The oxidation of testosterone to its monohydroxylated metabolites was monitored using H P L C according to the method of Sonderfan et al. (1987). This CYP-mediated reaction is shown in figure 2.1. The reaction mixture contained 0.92 ml of 50 m M potassium phosphate buffer, pH 7.4, with 3 m M MgCk, 50 pi of microsomes diluted to 6 nmol CYP/ml in 0.25 M sucrose, and 10 ul of 100 m M N A D P H . The mixture was preincubated at room temperature for ten minutes. The reaction was initiated at 37°C with 20 ul of testosterone 35 Figure 2.1. Schematic indicating the possible CYP-mediated hydroxylated testosterone metabolites. (12.5 mM) and was allowed to proceed for ten minutes. Dichloromethane (6.0 ml) was used to stop the reaction. Then, each sample was spiked with 50 pi of 50 p M 11 P-hydroxytestosterone, the internal standard, and mixed vigorously for two minutes. The organic and aqueous phases were separated by centrifugation at 2000 x g for two minutes. The aqueous layer was aspirated and discarded, while the organic layer was evaporated under a gentle stream of nitrogen. The residues were reconstituted in HPLC-grade methanol (200 pi) and filtered through 13 mm (0.45 nm pore size) syringe filters with polytetrafluoroethylene (PTFE) membranes into HPLC autosampler vials with corneal inserts. A 10 pi aliquot of each sample was analyzed using a Shimadzu LC-6A binary gradient HPLC system equipped with an SIL-6B,autosampler, SPD-6A variable U V detector (set at 254 nm) and CTO-6A column heater (Shimadzu Scientific Instruments). 36 Testosterone and its monohydroxylated metabolites were resolved on a Supelco LC-18 (3 um particle size, 15.0 cm x 4.6 mm I.D.) reverse phase column preceded by a Supelguard LC-18 (5 um particle size, 2.0 cm x 4.6 mm I.D.) guard column (Supelco Inc., Bellefonte, PA, USA) at 40°C. The column was eluted at a total flow rate of 1.4 ml/minute with solvent A (methanol:water:acetonitrile, 35:64:1) and solvent B (methanol:water:acetonitrile, 80:18:2) using the following elution program: 100% solvent A from 0 to 10 minutes, followed by linear gradient to 40% solvent B from 10 to 30 minutes, to 55% solvent B from 30 to 35 minutes, and to 100% solvent B from 35 to 36 minutes. The column was equilibrated with 100% solvent B from 35 to 36 minutes. The column was equilibrated with 100% solvent B from 36 to 40 minutes, followed by a linear gradient back to 100% solvent A from 40 to 41 minutes, and re-equilibration of the column with solvent A from 41 to 45 minutes. The presence of testosterone metabolites (2a-, 6P-, 7a-, 16a-, 16p-hydroxytestosterone, and androstenedione) was monitored by absorbance at 254 nm. The identity of the metabolites was confirmed by comparison of retention times to authentic standards. The amount of each metabolite was determined by calculating the ratios of metabolite peak area to the internal standard peak area and interpolating the ratio to a standard curve. Standard curves were generated for each metabolite at four concentrations by plotting the peak area ratios of the authentic standard against the known concentration of the authentic standard. Standard curves were included with each experiment. Standard samples contained the complete reaction mixture except for microsomal protein. A zero time control was included with each assay, which is the 37 same reaction mixture that is stopped with dichloromethane before the addition of testosterone to correct for the presence of endogenous testosterone hydroxylation. Activities are expressed as nmol metabolite formed/minute/nmol total C Y P . Each microsome sample was analyzed in duplicate in each assay on at least two separate occasions. Testosterone hydroxylase activities reported are the mean + standard error of the mean. 2.8. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) SDS-PAGE was performed according to the method of Laemmli (1970) using a Hoefer SE600 vertical slab gel unit. A discontinuous SDS-polyacrylamide gel was prepared with a 3.0% acrylamide stacking gel (1.0 cm long x 0.75 mm thick) and a 7.5% acrylamide separating gel (12.5 cm long x 0.75 mm thick). The stacking gel contained 0.125M Tris-HCl buffer, pH 6.8, 0.1% SDS (w/v), 3.0% acrylamide-Bis (22.2%-0.6%) (w/v), 0.08% ammonium persulphate (w/v), and 0.03% T E M E D (w/v). Final concentrations in the separating gel were: 0.375 M Tris-HCl buffer, pH 8.8, 0.1% SDS (w/v), 7.5% acrylamide-Bis (22.2%-0.6%) (w/v), 0.04% ammonium persulphate (w/v), and 0.03% T E M E D (w/v). Dilutions of microsomes (0.5 -1 .0 nmol/ml) were prepared in buffer containing 0.062 M Tris-HCl buffer, pH 6.8, 1.0% SDS (w/v), 0.001% bromophenol blue (w/v), 10% glycerol (v/v), and 5% p-mercaptoethanol (v/v), and boiled for two minutes. Aliquots (20ul) of boiled microsomes (0.01 - 0.02 nmol) were loaded into each well in the stacking gel. The samples were subjected to electrophoresis with cooling at a current setting of 12 mA per gel until the dye front had moved into the stacking gel and then a 38 setting of 25 mA per gel was used. Electrophoresis buffer contained 0.025 M Trizma base, 0.192 M glycine, and 0.1% SDS (w/v). Following separation, the Hoefer apparatus was disassembled and the separating gel was prepared for transfer to a nitrocellulose membrane. 2.9. Immunoblots The Western blot, or immunoblot, was performed according to Towbin et al. (1979). Proteins resolved by SDS-PAGE were transferred electrophorectically from the separating gel onto a nitrocellulose membrane (0.45 urn pore size) using a Hoefer TE52 Transphor Unit with Power lid. The transfer was conducted at 0.4 amps for two hours at 4°C in cold transfer buffer (0.025 M Tris base, pH 8.3, 0.192 M glycine, 0.01% SDS (w/v)) and 20% methanol (v/v). After the transfer, the nitrocellulose membrane was incubated in blocking buffer containing 1% B S A and 3% skim milk powder (w/v) in phosphate buffered saline (PBS), pH 6.9, overnight at 4°C. The next day, the blocking buffer was discarded and replaced with 50 ml of primary antibody (anti-CYP IgG) diluted in antibody dilution buffer containing 1% B S A (w/v), 3% skim milk powder (w/v), and 0.05% Tween 20 (v/v) in PBS. The membrane was incubated with the primary antibody solution for two hours at 37°C with shaking. The primary antibody solution was then removed and the membrane was washed with three 10-minute incubations of 0.05% Tween 20 (v/v) in PBS at 37°C with shaking. The secondary antibody, an alkaline phosphatase conjugated F(ab')2 immunoglobulin, was diluted in antibody dilution buffer and incubated with the 39 membrane for two hours at 37°C with shaking. The washing step was repeated at this point to prepare the membrane for reaction with a substrate solution. The presence of bound primary antibody was visualized by incubating the membrane with a substrate for the enzyme conjugated to the secondary antibody. A substrate solution for alkaline phosphatase (0.01% NBT, 0.0005% BCIP in 0.1 M Tris-HC1, 0.5 m M MgCk, pH 9.5) was prepared under subdued light immediately prior to use and then added to the membrane for a timed period. The reaction was stopped by rinsing the membrane with distilled water. 2.9.1. CYP2C11 For immunodetection of CYP2C11, a monospecific rabbit anti-CYP2Cl 1 polyclonal IgG was used. The primary antibody was used at a concentration of 50 pg/ml, the secondary antibody (goat anti-rabbit IgG) was used at a dilution of 1:3000, and the phosphatase reaction was terminated after four minutes. 2.9.2. C Y P 3A For immunodetection of CYP3A, a rabbit anti-CYP3A polyclonal IgG was used. This antibody reacts with predominantly CYP3A1 but also reacts with CYP3A2 and an unidentified female CYP3A form which are unresolved by SDS-PAGE (Holsmer, 1994). The primary antibody was used at a concentration of 50 pg/ml, the secondary antibody (goat anti-rabbit IgG) was used at a dilution of 1:3000, and the phosphatase reaction was terminated after five minutes. For specific immunodetection of CYP3A1 or CYP3A2, monoclonal antibodies to C Y P 3 A l or to CYP3A2 (mouse anti-CYP3Al and anti-40 CYP3A2 monoclonal IgG) were used at a concentration of 1 pg/ml, the secondary antibody (goat anti-mouse IgG) was used at a dilution of 1:3000, and the phosphatase reaction was stopped after four minutes. In addition to visual detection with BCIP and NBT, enhanced chemiluminescence (ECL) detection of immunoblots probed with monoclonal antibodies to CYP3A1 and CYP3A2 was also used. Blots were incubated with the same dilution of the primary antibody as the alkaline phosphatase detection system and a-1:3000 dilution of the second antibody, a goat anti-mouse IgG peroxidase conjugate. The substrate reaction with the E C L substrate kit was terminated after one minute. The membrane was then placed in intimate contact with a piece of film in a cassette tray and the film was exposed to the blot for 20 minutes. Then, the film was developed for two minutes with Kodak developer solution and fixed for two minutes with Kodak fixer solution. 2.9.3. C Y P 2 A 1 For immunodetection of CYP2A1, a sheep anti-CYP2Al polyclonal IgG was used. This antibody cross-reacts with CYP2A2, but these two C Y P forms are resolved by SDS-P A G E . The primary antibody was used at a concentration of 10 pg/ml, the secondary antibody (goat anti-sheep IgG) was used at a dilution of 1:3000, and the phosphatase reaction was terminated after three to five minutes. 2.10. Immunoquantitation Densitometric quantitation of stained protein bands present in each sample was measured with a pdi 420 oe™ scanner connected to an IBM-type personal computer using 41 Quantity One® Ver 3.0 software to analyze the scanned images (pdi Inc., Huntington Station, N Y ) . The scanner scans a series of horizontal rows of pixels across the stained band. The area under the curve, defined as OD x mm, for row average OD versus row position in millimeters is then determined for each band. For quantitation of CYP2C11 and CYP2A1, an internal standard of purified rat CYP2C11 or CYP2A1, respectively, was included at a defined concentration on each blot to allow calculation of relative optical density (OD x mm of sample -f- OD x mm of internal standard). Calibration curves were constructed for each of the isoforms by plotting the relative optical density of the stained bands against the known amounts of purified C Y P isozymes loaded. The amount of protein in each sample was then determined by interpolating the relative optical density of the stained band to the amount of C Y P on the standard curve. Relative quantities of CYP3A protein levels in the microsome samples were reported as relative optical density. Calculating the quantities of individual CYP3 A enzymes was not possible because the anti-CYP3A IgG cross-reacts with at least three unresolved CYP3A enzymes. A purified CYP3A1 sample was used as an internal standard. 2.11. Data Analysis 2.11.1. Statistical Analysis of Data A l l data is presented as the mean ± standard error of the mean for values from individual rats in a group. The mean values of groups were compared using the unpaired one-tailed Mann-Whitney test (GraphPAD InStat, 1990). This nonparametric test was 42 used instead of the conventional Student's t-test because there were unequal variances between the treatment groups. Five comparisons were made between groups: 1) Testosterone enanthate (5pmol/kg/d) treated females at 90 days versus corn oil treated females at 90 days 2) Testosterone (5u,mol/kg/d) treated females at 90 days versus corn oil treated females at 90 days 3) Testosterone (2.5pmol/kg, 2x) treated females at 90 days versus testosterone (5u.mol/kg/d) treated females at 90 days 4) Testosterone enanthate (5pmol/kg/d) treated females at 90 days versus testosterone enanthate (5pmol/kg/d) treated females at 129 days 5) Testosterone enanthate (5umol/kg/d) treated females at 90 days versus testosterone enanthate (5pmol/kg/d) treated females at 169 days. Results were considered to be significant if p<0.1. A significance level of 0.1 was chosen because this was an exploratory study designed to answer several questions at once. When the power of the test is evenly distributed over the five comparisons, significant differences were noted when p<0.02 for each individual comparison. The protein levels and enzyme activities measured in this study were near the limits of quantitation for the assays used. When detectable levels were measured that were below the limits of quantitation, a value of LOQ/2 was used in statistical analysis so that a number could be assigned to the measurement. This was to avoid the use of the number zero that would falsely exaggerate differences between quantified and unqualified measurements. Undetectable levels were entered as zeros in statistical calculations. 43 2.11.2. Pharmacokinetic Analysis of Data Elimination of testosterone was plotted as the logarithm of plasma testosterone against the time following the final dose of androgen. Pharmacokinetic data was analyzed with a one-compartmental model. Elimination constants (k) were derived from the linear slope of the elimination phase of the curve (slope= -k/2.303). Half-lives were calculated from the elimination constants (ti/=0.693/k). 44 3. RESULTS 3.1. Assay Validation Validation of a quantitative analytical assay is necessary to ensure that the data obtained from the assay are reliable and reproducible (Shah et al. 1992). Assays that satisfy validation requirements will allow statistical detection of differences arising from the groups under study and not differences resulting from variability in the assay method. To properly validate an analytical method, it is important to characterize the recovery, linearity, reproducibility (precision), sensitivity, and selectivity (specificity). The general principles of validation are common to all types of analytical methods. Recovery is defined as the percentage of analyte present in the sample following sample processing expressed as a percentage of the amount of analyte measured in a sample not subject to processing (i.e. extraction). Ideally, recovery should not be less than 80%, especially if measuring trace amounts (Karnes et al. 1991). A standard curve containing four to eight concentrations must provide a defined relationship between sample response and sample concentration over the range of expected concentrations. The fit of the data to a mathematical function describing the relationship between the response and analyte concentration must be statistically significant. Intra-assay variation and inter-assay variation are used to measure reproducibility. Generally, a minimum of five determinations per concentration of standard analyte are required to determine the degree of variability. Variability is expressed as a coefficient of variation (CV), which is the standard deviation divided by the mean of the replicate 45 values. Intra-assay variation describes the variability of the assay when the assay is conducted within a single day. Inter-assay variation describes the variability of the assay when the assay is conducted on different days. The coefficients of variation for both of these parameters should not exceed 15% at any concentration of the standard analyte (Shah etal. 1992). The limit of quantitation (LOQ) and limit of detection (LOD) characterize the sensitivity of the assay. The LOQ is the lowest concentration of analyte that can be measured with acceptable accuracy, precision, and variability (Shah et al. 1992). It is necessary that the coefficient of variation at the LOQ be <20%. The LOD is the lowest concentration of analyte that can be detected at a level that is significantly different from the background noise. Any response that is larger than three standard deviations from the blank measurement is generally acceptable (Karnes et al. 1991). Specificity and selectivity are often confused because they describe two similar phenomena (Karnes et al. 1991). Selectivity refers to the ability to distinguish between a number of analytes. The term specific refers to a method that detects only one analyte. 3.1.1. Testosterone Radioimmunoassay The [ I]-testosterone assay kit used to detect plasma levels of testosterone was validated by the manufacturer (ICN Biomedical Inc., Costa Mesa, CA, USA) for recovery, intra-assay variation, inter-assay variation, and antibody specificity. The recovery of testosterone, or the percentage of testosterone that is detected by the assay, is 100.4 ± 1.3%. The intra-assay variation and the inter-assay variation are reported to be 10.9 ± 1.1% and 11.2 ± 2.4%, respectively. The antiserum included in the kit claimed to 46 be specific for testosterone. Cross-reactivity was less than 1% for all of the steroids and testosterone metabolites tested (Table 3.1.) with the exception of 5ct-dihydrotestosterone (7.8%), 11-oxotestosterone (2.0%), and 5a-androsterone-3p,17p-diol (2.2%). Table 3.1. Steroids that do not cross-react with the testosterone RIA antiserum. 11 P-hydroxytestosterone 6p-hydroxytestosterone 11 P-hydroxyandrostenedione Dihydroxytestosterone Androstenedione Estrone 5a-androstane-3,17-dione 17p-estradiol 5 P-androstane-3,17-dione Estriol 5 P-dihydrotestosterone Progesterone 5ce-androsterone-3a, 17p-diol Corticosterone 5-androsterone-3p,l 7p-diol Desoxycorticosterone Dehydroepiandrosterone Danazol However, the manufacturer of the kit did not test for cross-reactivity of the antiserum with testosterone esters. The specificity and selectivity of the assay kit for testosterone and testosterone enanthate was important for interpreting the data in this study. Therefore, solutions of testosterone, testosterone enanthate, testosterone proprionate, and dihydrotestosterone were prepared in 100% ethanol and serially diluted in distilled water until the final concentrations of these solutions were 0.3 ng/ml, 3 ng/ml, 47 and 15 ng/ml (less than 1% ethanol in the final solution). These concentrations represent the low, middle, and upper ranges of the.linear portion of the standard curve. The solutions were tested using the assay kit and the amount of testosterone detected as a percentage of the concentration of androgen present in the solutions is shown in table 3.2. The antiserum was found to be specific for nonesterified forms of testosterone. The kit detected only 1.3% of the highest concentration of testosterone enanthate tested and there was no observable cross-reaction at the lower concentrations. This experiment measured less cross-reactivity with dihydrotestosterone than reported by the manufacturer. At a concentration of competing testosterone compound equal to that of radiolabeled testosterone, no cross-reaction was observed. A 5.9% cross-reaction was detected at 15 ng/ml dihydrotestosterone. The selectivity of the antiserum for testosterone over testosterone enanthate in a mixture was also tested at concentrations relevant to this project. When a mixture of testosterone and testosterone enanthate were tested by the kit, the response was the same as that observed with a solution of pure testosterone at the same concentration (Table 3.3.) indicating no detection of the enanthate. The kit is able to accurately quantify testosterone concentrations between 0.2 ng/ml and 20.0 ng/ml, which is the linear portion of the standard curve. In an attempt to characterize the standard curve below 0.2 ng/ml, the testosterone standards provided by the manufacturer were diluted with distilled water to concentrations below 0.2 ng/ml. These diluted standards were assayed with the kit. Unfortunately, no testosterone was detected in any of the diluted standards. 48 Table 3.2. Specificity of the antiserum in the testosterone I-RIA kit used in this study. Concentration of Testosterone Detected Cross Reaction as a % of Androgen Concentation detected 0.130 3.70 15.86 0.195 1.3 0.886 5.9 Androgen Formulation Concentration Prepared (ng/ml) T TP TE DHT 0.3 3 15 0.3 3 15 0.3 3 15 0.3 3 15 Testosterone (T), testosterone proprionate (TP), testosterone enanthate (TE), and dihydrotestosterone (DHT) were prepared in ethanol and diluted in distilled water and measured by the kit. T and DHT were included as positive controls to ensure proper dilution of the androgen solutions. The kit claims DHT cross reacts 7.8% with the antibody at 3ng/ml. 49 Table 3.3. Selectivity of the antiserum in the testosterone I-RIA used in this study. Androgen Number of Trials % B / B 0 0.3 ng/ml TE 2 100 3 ng/ml TE 2 100 0.3 ng/ml T 2 74.2 ± 4.6 3 ng/ml T 2 34.6 + 0.8 0.3ng/ml T & 0.3ng/ml TE 4 76.2 ± 0.6 3 ng/ml T & 3 ng/ml TE 4 38.8±0.5 Testosterone (T) and testosterone enanthate (TE) were prepared in distilled water and measured by the kit as instructed. % B/Bn is the counts of bound testosterone in each sample expressed as the percentage of counts of bound testosterone in a standard containing no testosterone. 100% B/Bn means that there was no detectable testosterone in the sample. 50 3.1.2. Testosterone Hydroxylase Assay Four solutions containing a mixture of known concentrations of testosterone metabolites (2a-, 60-, 7a-, 16a-, 16P-hydroxytestosterone, and androstenedione) were prepared in methanol and used to construct standard curves to validate the assay. The four concentrations, 25, 50, 100, and 250 uM, are equivalent to 0.5,1.0,2.0, 5.0 nmol/20 ul of authentic standard mixture included in the final reaction mixture. The mixtures were extracted and analyzed by HPLC in the same manner as the microsome reaction mixture. Representative chromatograms are shown in figure 3.1. Baseline separation between all six of the monohydrdoxylated testosterone metabolites and the internal standard is achieved with the elution profile used in this study. A small amount of androstenedione is present in the zero time blank due to contamination present in the testosterone substrate, but is corrected for after sample analysis. A small amount of 7a-hydroxytestosterone is also present in the zero time control, which is likely a result of endogenous metabolism. There are no other interfering peaks in the blank sample. The sharp rise in baseline after 36 minutes is caused by a change in refractive index of the H P L C eluting solvent when the solvent composition is changed. Testosterone enanthate elutes after testosterone and therefore does not interfere with the assay. Figure 3.2. shows the standard curves generated for the six monohydroxylated testosterone metabolites. Only the peak areas of 60- and 160-hydroxytestosterone curves were linear with respect to concentration over the range of concentrations tested. The peak area of the four other metabolites were linear to a maximum level of 100 u M . 51 OJ 11-6 Figure 3.I.A. A representative HPLC chromatogram of 2a-, 60-, 7a-, 16a-, 160-hydroxytestosterone and androstenedione (A) standards separated by HPLC. A mixture of 100 p M of each standard was prepared as described in the Experimental section. The internal standard, 11 0-hydroxytestosterone, was added'(50 pi of 50 u.M stock solution) after the dichloromethane was added. Metabolites were resolved on a reverse phase C i8 column and detected by U V absorption at 254 nm. 52 m to C O T 113 Figure 3.LB. A representative HPLC chromatogram of a zero-time control separated by HPLC. Unmetabolized testosterone (T) eluted as indicated at 35.7 minutes. A reaction mixture containing a random hepatic microsome sample was prepared as described in the Experimental section. Dichloromethane was added to the mixture before the addition of testosterone to prevent the initiation of the reaction. An internal standard, 110-hydroxytestosterqnerwas-included (50 pi of 50 u M stock solution). Testosterone and its metabolites were resolved on a reverse phase Cig column and detected by U V absorption at 254 nm. -, •>•:>•  ±. ' "' •»•-T Figure 3.I.C. A representative HPLC chromatogram of testosterone metabolites produced in hepatic microsomes from gonadectomized female rats. Testosterone was added to the reaction mixture and allowed to incubate for ten minutes at 37°C as .-described in the Experimental section. An internal standard, 11 (3-hydroxytestosterone, was added following the incubation (50 pi of 50 u M stock solution). The metabolites were resolved on a reverse phase Cig column and detected by U V absorption at 254 nm, 54 Figure 3.I.D. A representative H P L C chromatogram of testosterone metabolites produced in hepatic microsomes from testosterone enanthate treated female rats. Testosterone was added to the reaction mixture and allowed to incubate for ten minutes at 37°C as described in the Experimental section. A n internal standard, 11(3-hydroxytestosterone, was added following the incubation (50 pi of 50 u M stock solution). The metabolites were resolved on a reverse phase Ci.8 column and detected by U V absorption at 254 nm. 55 I N . C O T Figure 3.1.E A representative HPLC chromatogram of testosterone metabolites produced in hepatic microsomes from control male rats. Testosterone was added to the reaction mixture and allowed to incubate for ten minutes at 37°C as described in the Experimental section. An internal standard;-! 1 P-hydroxytestosterone, was added following the incubation (50 pi of 50 u M stock solution). The metabolites were resolved on a reverse phase C i8 column and detected-by U V absorption at 254 nm. 56 (A) 1.6 -i CO 0 -I , , , , : , 0 100 200 300 metabolite concentration (uM) (B) metabolite concentration (uM) Figure 3.2. Standard curves for the testosterone hydroxylase assay. The amount of the metabolite is measured as the ratio of the area of the metabolite peak to the area of the internal standard peak. (A) The three testosterone metabolites included in this graph are 60-, 7a-hydroxytestosterone and androstenedione (A), as indicated in the legend. (B) The three testosterone metabolites included in this graph are 160-, 2a-, and 16a-hydroxytestosterone, as indicated in the legend. Each curve is the mean of six standard curves. 57 Metabolite levels formed by microsomes analyzed in this project were within the linear ranges of the standard curves except for the androstenedione values obtained with microsomes prepared from adult male rats. These values were calculated by extrapolating from the curve between 100 and 250 uM. The percent recovery of the metabolites was determined as the percentage of peak area ratios from the standard mixtures as compared to peak area ratios from standard mixtures prepared by adding the internal standard after the extraction procedure. The percent recoveries were not found to vary significantly among the metabolites over the four concentrations tested. The recovery of monohydroxylated testosterone metabolites ranged from 79.1% to 93.5%. The mean recovery of the testosterone metabolites was 85.4 ± 0.9%. The presence of microsomal protein did not alter recovery because the peak areas of the internal standard did not differ significantly between samples with microsomal protein and standard samples without microsomal protein (data not shown). Samples containing the four standard mixtures were prepared six times on the same day and analyzed by HPLC to determine the intra-assay variation. The peak area of the response to each metabolite was expressed relative to the internal standard's peak area and the variability of the six trials was expressed as a coefficient of variation (standard deviation/mean x 100%). The intra-assay variability of detection for the six testosterone metabolites was below the acceptable limit of 15% (Table 3.4.). The coefficients of variation ranged from 9.4% to 15.2% for the six metabolites over the four concentrations tested. There was no significant difference in variability between the six metabolites or the four concentrations. A n overall average intra-assay variability of 11.9% was calculated. 58 Table 3.4. Intra-assay variation of the testosterone hydroxylase assay. Testosterone Metabolites 60 7a 16a 160 2a A 25 u M 12.5 13.2 11.6 10.6 12.6 10.5 50 u M 10.6 11.7 10.5 9.5 9.4 9.7 100 u M 13.7 14.6 13.1 15.2 12.8 14.0 250 u M 11.5 10.7 11.1 11.8 11.8 12.2 Standard mixtures of six testosterone metabolites (6P-, 7a-, 16P-, 2a-, 16a-hydroxytestosterone and androstenedione [A]) were prepared in methanol at the amounts indicated. These mixtures (in six replicates) were extracted into dichloromethane and analyzed by HPLC in a single day. The peak areas were measured relative to an 11 p-hydroxytestosterone internal standard. Coefficients of variation (in percentages) of the peak area ratios are tabulated above for the six testosterone metabolites at the four concentrations prepared. Coefficients were calculated from the standard deviation divided by the mean of the six replications. An acceptable level of variation is less than 15%. 59 Table 3.5. Inter-assay variation of the testosterone hydroxylase assay. Testosterone Metabolites 6(3 7a 16a 160 2a A 25 u M 35.8 24.4 28.2 31.9 18.3 19.3 Stock a 0.1 2.0 1.2 2.3 1.7 3.3 Stock b 13.5 9.7 13.2 12.9 11.0 19.2 50 u M 7.9 7.6 6.9 7.4 9.2 15.7 100 u M 5.6 5.5 5.7 4.8 4.7 5.2 250 u M 10.2 1.05 10.3 9.6 13.8 8.9 Standard mixtures of six testosterone metabolites (60-, 7a-, 160-, 2a-, 16a-hydroxytestosterone and androstenedione [A]) were prepared in methanol at the amounts indicated. These mixtures, in duplicate, were extracted into dichloromethane and separated by reverse-phase HPLC on six separate days. The peak areas were measured relative to an 110-hydroxytestosterone internal standard. Coefficients of variation (in percentages) of the peak area ratios are tabulated above for the six testosterone metabolites at the four concentrations prepared. Coefficients were calculated from the standard deviation divided by the mean of the six replications. Two separate stock solutions of standards were used over the six days and account for the unacceptably (>15%) high coefficients at the lowest concentration. 60 The inter-assay variability was determined from six standard curves constructed over six different days. Inter-assay coefficients of variation are presented in Table 3.5. The coefficients of variation were below the accepted limit of 15% with the exception of the lowest standard of 25 uM. Two different stock mixtures of standards were used to construct six separate standard curves. When the results from the two stock mixtures were separated, the coefficients of variation were within the accepted limits. Again, there was no significant difference in variability between the metabolites. Without separating the data from the two stock metabolite mixtures, an overall average inter-assay variability of 12.8%) was calculated. The limits of detection (LOD) and limits of quantitation (LOQ) for the testosterone hydroxylase assay were determined by diluting the standard metabolite mixture until the metabolites could no longer be detected. The LOQ is defined as the smallest amount of metabolite that can be quantified by this assay with a coefficient of variation less than 20%. The LOD is the smallest amount of metabolite that can be detected by the assay with a signal that is three times the baseline noise. According to this definition, any peak with a peak area of 100 counts or larger was considered to be testosterone metabolite and not background noise. The L O D and the LOQ values are equal for each metabolite (Table 3.6.). The LOD/LOQ amounts for this assay were converted to corresponding units of activity to make them more applicable to the data obtained for this study. This was accomplished by dividing the amount of metabolite by the reaction time (ten minutes) and total CYP amount (0.3 nmol) present in the incubation mixture. The LOD/LOQ value for testosterone 6p-hydroxylase activity was 61 Table 3.6. Limits of quantitation (LOQ) and detection (LOD) of the testosterone hydroxylase assay expressed as both the amount of metabolite and the corresponding enzyme activity. Testosterone Metabolites 60 7a 16a 16B 2a A nmol 0.2 0.05 0.05 0.1 0.1 0.05 nmol formed/min/ 0.067 0.017 0.017 0.033 0.033 0.017 nmol total C Y P The LOQ and LOD were the same for each metabolite. Standard mixtures of six testosterone metabolites (60-, 7a-, 160-, 2a-, 16a-hydroxytestosterone and androstenedione [A]) were serially diluted in methanol. These mixtures (six replicates) were extracted into dichloromethane and analyzed by HPLC in a single day. The peak areas were measured relative to an 110-hydroxytestosterone internal standard. The LOQ was defined as the amount of metabolite that can be detected with a coefficient of variation less than 20%. The LOD was defined as the amount of metabolite that could still be detected in every sample by the assay with an area greater than 100. 0.067 nmol/min/nmol total CYP, the LOD/LOQ value for testosterone 160- and 2a-hydroxylase activities was 0.033 nmol/min/nmol total C Y P , and the LOD/LOQ value for the formation of 7a- and 16a-hydroxytestosterone or androstenedione was 0.017 nmol/min/nmol total C Y P . Metabolite analysis and quantitation is difficult with microsomal samples from female rats because very low metabolite levels are detected as compared to samples from male rats. Conventionally, testosterone metabolites formed by male microsomes are analyzed after a five-minute incubation period. To maximize the amounts of metabolites formed by our samples, a ten-minute incubation period was used in this study. Therefore, the assay had to be validated for a ten-minute reaction period. Figure 3.3. illustrates the linearity of formation of 7a-hydroxytestosterone over ten minutes. This is the primary 62 metabolite formed in microsome samples prepared from gonadectomized female rats and it can be assumed that the formation of the other metabolites is linear with respect to time because the other metabolites are formed at much lower rates. Four microsome samples were chosen to test the linearity of metabolite formation with respect to time, two samples from gonadectomized female rats, and two samples from testosterone enanthate-treated female rats. The formation of 7a-hydroxytestosterone is linear with respect to time between two and a half and ten minutes for three of the four samples. For sample B2, a testosterone enanthate-treated female rat, 7oc-hydroxytestosterone formation was not linear due to an unexplained high activity at the 2.5 minute time point. However, the ten minute value was similar to the other samples. > TJ 0 5 10 15 < Incubation time (minutes) A1 - Gx female —•— A2 - Gx female •B1 - TE treated female O B2 - TE treated female Figure 3.3. Formation of 7a-hydroxytestosterone with respect to time. The amount of 7a-hydroxytestosterone present is measured as the nmol of 7ct-hydroxytestosterone formed per minute per nmol total C Y P present in the reaction mixture. Four microsome samples, as indicated by the sample number in the legend, were analyzed for 2.5, 5, and 10-minute reaction incubations each. Each point is the mean of two incubations. 63 3.1.3. Immunoblots Solutions containing known concentrations of purified C Y P enzymes were used to construct standard curves for immunoblot assay validation. The measured amount of a particular C Y P was determined from the standard curves and replicate values were used to calculate variability of the assay. Inter-assay variation was determined from separate standard curves included on separate immunoblots. Intra-assay variation was calculated by quantifying each purified C Y P standard loaded on a single blot in replicate. 3.1.3.1. CYP2C11 immunoblots The immunoblot method was validated with purified CYP2C11 standards. The mean standard curve for CYP2C11 is shown in Figure 3.4. The optical density of each standard was corrected for variations between blot intensities with an internal standard. Relative optical density increased linearly to 0.6 pmol. The levels of CYP2C11 detected in microsomal samples analyzed in this study were within the linear portion of the standard curve. The results of the validation procedure are presented in Table 3.7. The LOQ was found to be 0.2 pmol of CYP2C11 standard loaded onto the gel, which is equivalent to 0.01 nmol/nmol total C Y P when 0.02 nmol total C Y P is loaded onto the gel. The variability at the lowest concentration tested is too high (>20% C.V.) to quantify but a signal was still detected above background. Therefore, the L O D was defined to be 0.06 pmol, which is equivalent to 0.003 nmol/nmol total C Y P . 64 o 2 -, ty rat 1.5 : densi 1 : CO o 0.5 : Q . O 0 :-0 pmolof CYP2C11 Figure 3.4. Mean standard curve for CYP2C11 immunoblots. Purified CYP2C11 standards were prepared at the concentrations indicated and analyzed in the same manner as microsome samples, discussed in the Experimental section. CYP2C11 bands were quantified by densitometry and optical density (OD x mm) was measured. The ratio of the OD x mm of the sample band to the OD x mm of an internal standard was calculated for each standard. Each point is the mean ± S E M of seven determinations. Table 3.7. Inter-assay and intra-assay variation of the CYP2C11 irnmunoblot assay. Amount of CYP 2C11 Inter-assay CV Intra-assay CV 0.06 pmol** 44.7 23.4 0.2 pmol* 23.9 11.2 0.6 pmol 11.2 1.2 1.2 pmol 10.7 3.1 Purified CYP2CT1 was prepared at the amounts indicated and analyzed by the western blotting method. Inter-assay variability was determined by seven separate determinations over seven different days. Intra-assay variability was determined by quantifying each standard in quadruplicate on a single blot. Coefficients (expressed as percentages) are within accepted limits i f less than 15%. LOQ: defined as the lowest standard concentration with an intrassay C V of less than 20%. LOD: defined as the lowest standard concentration that could still be detected at three times background level. 65 3.1.3.2. CYP3A immunoblots The immunoblot method was validated with purified CYP3A1 standards. This standard was prepared from microsomes of dexamethasone-treated female rats, which contain mostly CYP3A1 and little CYP3A2, and defined to be pure by SDS-PAGE. The specific content was estimated to be 10 nmol/mg protein and CYP3A1 standards were prepared to known C Y P concentrations with this value. Control male CYP3A1 levels were calculated using these standards and were found to be consistent with other studies (Cooper et al. 1993). Therefore, the assumption regarding the specific content was valid. The mean standard curve is shown in Figure 3.5. The optical density of each standard was corrected for variations between blot intensities with an internal standard. The assay was determined to be linear up to 0.5 pmol CYP3A1. The optical densities of the microsome samples were not more intense than the highest standard concentration. The inter-assay variability ranged from 1.2% to 6.4% over the concentration range tested. These values were determined from four standard curves analyzed over four separate days. Because the CYP3A1 standard was available in limited quantity, the intra-assay variability was determined by quadruplicate samples at 0.5 pmol only. The intra-assay variability at 0.5 pmol was found to be 10.0%. The LOQ and LOD was found to be 0.03 pmol of CYP3A1 standard loaded onto the gel. This is equivalent to 0.0015 nmol/nmol total C Y P when 0.02 nmol C Y P is loaded onto the gel. 66 3 i 0 0.2 0.4 0.6 pmol of CYP3A Figure 3.5. Mean standard curve for CYP3A immunoblots. Purified CYP3A1 standards were prepared at the concentrations indicated and analyzed in the same manner as microsome samples, discussed in the Experimental. CYP3A1 bands were quantified by densitometry and optical density (OD x mm) was measured. The ratio of the OD x mm of the sample band to the OD x mm of an internal standard was calculated for each standard. Each point is the mean ± S E M of four determinations. 67 3.1.3.3. CYP2A1 immunoblots The immunoblot method was validated with purified CYP2A1 standards. Figure 3.6. shows the mean standard curve used to quantify the CYP2A1 levels in the microsome samples. The optical density of each standard was corrected for variations between blot intensities with an internal standard. Relative optical density increased linearly to 0.5 pmol CYP2A1. The microsomal samples analyzed in this study were within the linear range of this standard curve. The inter-assay variability ranged from 3.42% to 11.7% for the four standards tested. These values were determined from four standard curves analyzed on four separate days. The intra-assay variability was determined with quadruplicate samples at 0.375 and 0.5 pmol and found to be 2.76% and 2.60%, respectively. The LOQ was found to be 0.125 pmol, which is equivalent to 0.006 nmol/nmol total C Y P when 0.02 nmol C Y P is loaded onto the gel. The lowest standard tested to be detectable was 0.0625 pmol. This band was still easily visible so it is likely that the actual L O D is less than 0.0625 pmol. 68 0 -I—, , , r—, r—n 1 r—, 0 0.2 0.4 0.6 pmol of CYP2A1 Figure 3.6. Mean standard curve for CYP2A1 immunoblots. Purified CYP2A1 standards were prepared at the concentrations indicated and analyzed in the same manner as microsome samples, discussed in the Experimental. CYP2A1 bands were quantified by densitometry and optical density (OD x mm) was measured. The ratio of the OD x mm of the sample band to the OD x mm of an internal standard was calculated for each standard. Each point is the mean ± S E M of four determinations. 69 3.2. Effect of Pubertal Androgen Treatment on Hepatic CYP Enzymes Forty-four ovariectomized females Sprague-Dawley rats were divided into five experimental treatment groups and one control group (Table 2.1.). A second control group consisted of eight intact adult male Sprague-Dawley rats. One of the female controls had to be removed from the study because of an error in microsome preparation. Pubertal imprinting of hepatic constitutive C Y P enzymes was determined by measuring protein levels and associated activities of selected enzymes. Expression of a male-specific enzyme and a male-dominant enzyme were monitored for increases following pubertal androgen treatment. A female-dominant C Y P enzyme was included in the study to determine if the observed effects were indicative of defeminization of C Y P expression or due to general induction. Immunoblots were used to measure the protein levels of male-specific CYP2C11, male-dominant CYP3A, and female-dominant enzyme, CYP2A1 enzymes. The testosterone hydroxylase assay provided activity data about these C Y P isoforms. Testosterone 2a- and 16a-hydroxylase activities were used as a measure of CYP2C11 expression while testosterone 6p-hydroxylase activity was used to indicate CYP3A expression. Testosterone 7a-hydroxylase activity was a marker for CYP2A1 expression. Levels of plasma testosterone were monitored with the [125I]-testosterone RIA kit following pubertal androgen treatment in the same rats used for microsome preparation of analysis of C Y P activities and levels. The data are organized into subsections that relate to the first three objectives separately. In each subsection, the objective being addressed is restated along with the results of the experiments. For each objective, the C Y P protein levels and corresponding 70 activities are presented for each enzyme separately. Plasma testosterone concentrations and testosterone elimination data are included for the treatment groups addressed by the first and second objectives. Additional data that are not central to the objectives were obtained during the course of this study and are included in the Appendix. Liver and body weights of all experimental groups are included in Table I. Protein and C Y P levels of all experimental groups are listed in Table II. Rates of 16p-hydroxytestosterone and androstenedione formation are found in Table III. 3.2.1. Effect of Pubertal Testosterone Enanthate Treatment (1) To confirm, in prepubertally gonadectomized female rats, the increased expression of male-specific enzymes and suppression of female-specific enzymes during adulthood by androgen treatment administered pubertally. Protein levels and associated activities of microsomes from testosterone enanthate-treated and gonadectomized female rats were compared to determine i f pubertal testosterone enanthate treatment was able to increase expression of CYP2C11 and CYP3A and decrease expression of CYP2A1. 3.2.1.1. Elimination of testosterone following pubertal testosterone enanthate treatment A true imprinting effect requires an undetectable level of testosterone between puberty and adulthood. Following daily subcutaneous injections of testosterone 71 enanthate between 35 and 49 days of age, the length of time required to reach undetectable levels of plasma testosterone was determined by monitoring testosterone concentrations for 72 hours following the final injection of testosterone enanthate (figure 3.7.). Testosterone was eliminated very slowly from the rats with the elimination half-life calculated to be 2.48 days. However, this analysis was not continued for a sufficient period to allow accurate determination of the half-life and the length of time required to eliminate testosterone. Another group of prepubertally gonadectomized female rats was treated pubertally with testosterone enanthate in the same manner as the previous group. The level of plasma testosterone was monitored for 40 days following the final injection of testosterone enanthate (figure 3.8.). The mean plasma testosterone concentration fell below the detection limit after ten days. The apparent half-life of testosterone in these rats was calculated to be 2.85 days. There was a large degree of interindividual variability in the elimination rate of testosterone because detectable levels of testosterone were still present in three of the eight rats at forty days though they were below the limit of quantitation. 3.2.1.2. Effect of pubertal testosterone enanthate treatment on CYP2C11 protein levels and testosterone 2a- and 16a -hydroxylase activities The effect of pubertal treatment with testosterone enanthate on testosterone 2a -and 16a -hydroxylase activities is shown in figure 3.9. As observed, testosterone 2a-hydroxylase activity was undetectable in liver microsomes from gonadectomized female microsomes at 90 days of age. In contrast, testosterone 2a-hydroxylase activity in 72 100 i 0 20 40 60 80 Time (hours) Figure 3.7. Elimination of testosterone following pubertal testosterone enanthate treatment in prepubertally ovariectomized female rats. Rats (n=7) were injected subcutaneously on days 35-49 with 5 umol/kg testosterone enanthate daily. Blood samples were drawn from the tail vein at selected time intervals following the final injection of testosterone enanthate. Plasma samples were analyzed for testosterone with a radioimmunoassay kit as outlined in the Experimental section. Values are the mean ± standard error of the mean. 0 10 20 30 40 Time (days) Figure 3.8. Elimination of testosterone in ovariectomized female rats following pubertal testosterone enanthate treatment. Rats (n=9) were injected subcutaneously on days 35-49 with 5 umol/kg testosterone enanthate daily. Blood samples were drawn from the tail vein at selected time intervals following the final injection of testosterone enanthate. Open diamonds indicate measurements that were below the limit of detection of the RIA. Plasma samples were analyzed for testosterone with a radioimmunoassay as outlined in the Experimental section. Values are the mean ± standard error of the mean. 73 (A) c E cu E o E c >» •> < 1.2 1 0.8 0.6 0.4 0.2 0 Gx Females TE 5umol/kg 90 Intact Males days (B) c E T5 o E o D_ >-O o 1.6 1.2 < 0.8 0.4 0 * T Gx Females TE 5umol/kg 90 days Intact Males Figure 3.9. Effect of pubertal testosterone enanthate treatment on (A) testosterone 2a-hydroxylase activity and (B) testosterone 16a-hydroxylase activity in hepatic microsomes prepared from prepubertally gonadectomized female rats. Results are expressed as mean ± standard error of the mean. (n=7 for gonadectomized and TE-treated females, n=8 for control males) "Below the limit of detection. *Significantly different from gonadectomized female values (p=0.002 for testosterone 2a-hydroxylase activity and p=0.004 for testosterone 16a-hydroxylase activity). 74 microsomes from control male rats was 0.88 ± 0.12 nmol 2a-hydroxytestosterone formed/minute/nmol total CYP. Pubertal testosterone enanthate treatment increased testosterone 2a-hydroxylase activity five-fold above the LOQ of the assay to 0.18 ±0.08 nmol 2cc-hydroxytestosterone formed/minute/nmol total CYP. This value was significantly different than that of gonadectomized female rats (p=0.002) and agrees with previous results (Cadario et al. 1992). Testosterone 16a-hydroxylase activities paralleled the testosterone 2a-hydroxylase activities. As shown in figure 3.9.B, this activity was 16-fold higher in liver microsomes from testosterone enanthate-treated female rats than from gonadectomized female rats (p=0.004). Testosterone 2a- and 16a- hydroxylase activities are catalyzed by CYP2C11 in microsomes of untreated male rats (Sonderfan 1987, Ryan and Levin 1990). Therefore, the levels of CYP2C11 protein in these same microsomes were measured by irnmunoblot analysis to confirm the enzyme activity data. Figure 3.10. illustrates a representative blot that was probed with anti-CYP2Cl 1 IgG. The antibody recognized a single protein band that is identified as CYP2C11. CYP2C11 expression was undetectable in gonadectomized female microsomes at 90 days. Stained bands that migrated identically to CYP2C11 appeared in lanes containing microsomal samples from testosterone enanthate-treated female rats. However, the staining is not as intense as the bands from microsomes of control male rats. Data from the quantitation of the stained bands is shown in figure 3.11. The level of CYP2C11 in liver microsomes from male control rats is 0.131 ± 0.006 nmol/nmol total CYP. Pubertal testosterone enanthate treatment increased CYP2C11 expression to a level 7 5 GxF TE 90d Purified CYP2C11 T(1X) T(2X) M Figure 3.10. Representative immunoblot of hepatic microsomes from adult rats probed with polyclonal antibody against rat CYP2C11. Primary antibody (rabbit anti-CYP2Cl 1 polyclonal IgG) was used at a concentration of 50 pg/ml, secondary antibody, goat anti-rabbit IgG was used at a dilution of 1:3000, and the alkaline phosphatase reaction was stopped after four minutes. Abbreviations used in the figure include: microsomes from gonadectomized female rats at 90 days of age (Gx F), microsomes from female rats at 90 days of age treated with testosterone enanthate pubertally (TE 90d), microsomes from female rats at 90 days of age treated with testosterone once daily during puberty (T (IX)), microsomes from female rats at 90 days of age treated with testosterone twice daily during puberty (T (2X)), and microsomes from control male rats at 90 days of age (M). Microsomal samples were applied to the gel at a final concentration of 0.02 nmol total C Y P per lane except for M samples that were loaded at 0.01 nmol total C Y P per lane. Purified CYP2C11 was included on the immunoblot at concentrations of 0.06 pmol, 0.2 pmol, internal standard (unknown concentration), 0.6 pmol, and 1.2 pmol per lane. 76 0.16 O 0.12 0) 75 > * -CD P hi = 0.08 o 0.04 E 0 * T Gx Females TE 5umol/kg 90 days Intact Males Figure 3.11. Effect of pubertal testosterone enanthate treatment on CYP2C11 protein levels in hepatic microsomes prepared from prepubertally gonadectomized female rats. Results are expressed as mean ± standard error of the mean. "Below the limit of detection. * Significantly different from gonadectomized female values (p=0.0003). 77 three times above the LOQ of the assay to 0.033 ± 0.008 nmol/nmol total C Y P (p=0.0003). 3.2.1.3. Effect of pubertal testosterone enanthate treatment on CYP3A protein levels and testosterone 613 -hydroxylase activities Microsomes from control male rats contained nine times greater levels of testosterone 6p-hydroxylase activity than microsomes from gonadectomized female rats. However, in this study, testosterone 6p-hydroxylase activity was not increased significantly at 90 days (p=0.10) by pubertal testosterone enanthate treatment (figure 3.12.A) compared to gonadectomized female rats. Because testosterone 6p-hydroxylase activity can be correlated to C Y P 3 A levels (Imaoka 1988), CYP3A protein was quantitated. Figure 3.13. shows a representative immunoblot probed with polyclonal anti-CYP3A IgG. The antibody recognizes CYP3A1 and CYP3A2 and produces a single protein band on immunoblots that migrates identically with purified CYP3 A l . However, stained bands are visible in lanes loaded with microsomes from gonadectomized female rats. The band in each of the lanes containing microsomes from testosterone enanthate-treated female rats does not appear to be more intensely stained than in lanes containing microsomes from gonadectomized female rats. When CYP3A protein levels were quantified by densitometric analysis, a significant increase (p=0.006) in CYP3A protein levels was observed (figure 3.12.B). The microsomal CYP3A level was 0.037 ± 0.004 nmol /nmol total C Y P for gonadectomized female and 0.078 ± 0.007 nmol /nmol total C Y P for control male rats. 78 (A) c E •8 CD E o E c Q_ >-O ro •4—* O o < 1.4 1.2 1 0.8 0.6 0.4 0.2 0 Gx Females TE 5umol/kg 90 Intact Males days (B) co _ i Q_ >-O 0.1 YP) 0.08 o I total 0.06 mol 0.04 c "o E 0.02 c 0 Gx Females TE 5umol/kg 90 Intact Males days Figure 3.12. Effect of pubertal testosterone enanthate treatment on (A) testosterone 6(3-hydroxylase activity and (B) CYP3A protein levels in hepatic microsomes prepared from prepubertally gonadectomized female rats. Results are expressed as mean ± standard error of the mean. (n=7 for gonadectomized and TE-treated females, n=8 for control males) * Significantly different from gonadectomized female values (p=0.006). Gx F TE 90d Purified T(1X) T(2X) CYP3A1 Figure 3.13. Representative irnmunoblot of hepatic microsomes from adult rats probed with polyclonal antibody against rat CYP3A. Primary antibody (rabbit anti-CYP3A polyclonal IgG) was used at a concentration of 50 pg/ml, secondary antibody, goat anti-rabbit IgG was used at a dilution of 1:3000, and the alkaline phosphatase reaction was stopped after five minutes. The same abbreviations were used as in figure 3.10. Microsomal samples were applied to the gel at a final concentration of 0.02 nmol total C Y P per lane, with the exception of the male control samples that were loaded at 0.01 nmol. Purified CYP3A1 was included on the blot at concentrations of 0.0625 pmol, 0.125 pmol, 0.200 pmol, 0.250 pmol per lane. 80 To determine which CYP3A form was increased by the pubertal androgen treatment, additional immunoblots were probed with monoclonal CYP3A1 and monclonal CYP3A2 antibodies. No CYP3A1 was detected in randomly chosen microsome samples from intact male or gonadectomized female rats or from testosterone enanthate-treated rats (data not shown). This was confirmed by developing the same blot with an enhanced chemiluminescence (ECL) kit, which is a more sensitive detection system. However, in those same microsome samples, CYP3A2 was detected in the sample from male control rats and one of the samples from the testosterone enanthate-treated rats (figure 3.14.). The testosterone enanthate-treated rat with detectable CYP3A2 levels was sample B2, the same sample that did not display linear testosterone 7a-hydroxylase activity over ten minutes. However, this was the only sample from the testosterone enanthate treated rats with detectable CYP3A2 levels. No CYP3A2 was detected with the E C L detection system when the other testosterone enanthate-treated rats were tested. 3.2.1.4. Effect of pubertal testosterone enanthate treatment on CYP2A1 protein levels and testosterone 7 a -hydroxylase activities The female-dominant testosterone 7a-hydroxylase activity is 2.5 times greater in female rats than male rats (figure 3.15.A.). In this study, pubertal testosterone enanthate treatment did not significantly decrease (p=0.13) testosterone 7a-hydroxylase activity. CYP2A1 is solely responsible for 7a-hydroxytestosterone formation (Ryan and Levin 1990). CYP Gx TE T T M 3A1 F 90d (IX) (2X) Figure 3.14. Representative irnmunoblot of hepatic microsomes from adult rats probed with monoclonal antibody against rat CYP3A2. Primary antibody (mouse anti-CYP3A2 monoclonal IgG) was used at a concentration of 1 pg/ml, secondary antibody (goat anti-mouse IgG) was used at a dilution of 1:3000, and the alkaline phosphatase reaction was stopped after seven minutes. The same abbreviations were used as in figure 3.10. Microsomal samples were applied to the gel at a final concentration of 0.02 nmol total C Y P per lane. 82 (A) c E CD E o E c >. "> o < Q_ > O " r o -*—• o o E c 0.6 0.5 0.4 0.3 0.2 0.1 0 Gx Females TE 5umol/kg 90 days Intact Males (B) 0.05 > 0.04 _ O CD O i 0.03 5: E 0.02 | 0.01 0 Gx Females TE 5umol/kg 90 days Intact Males Figure 3.15. Effect of pubertal testosterone enanthate treatment on (A) testosterone 7a-hydroxylase activity and (B) CYP2A1 protein levels in hepatic microsomes prepared from prepubertally gonadectomized female rats. Results are expressed as mean ± standard error of the mean. (n=7 for gonadectomized and TE-treated females, n=8 for control males) GxF TE 90d Purified CYP2 A1 T(1X) T(2X) M Figure 3.16. Representative immunoblot of hepatic microsomes from adult rats probed with polyclonal antibody against rat CYP2A1. Primary antibody (rabbit anti-CYP2Al polyclonal IgG) was used at a concentration of 10 u,g/ml, secondary antibody, goat anti-sheep IgG was used at a dilution of 1:250, and the alkaline phosphatase reaction was stopped after three minutes. The same abbreviations were used as in figure 3.10. A l l microsome samples were applied to the gel at a concentration of 0.02 nmol total C Y P per lane. Purified CYP2A1 was included on the blot at concentrations of 0.125 pmol, 0.250 pmol, 0.375 pmol, and 0.500 pmol per lane. 84 Figure 3.16. shows a representative blot probed with anti-CYP2Al. This antibody recognizes a single protein band that migrates with purified CYP2A1. There was no qualitative difference in the intensity of the stained bands from all female experimental groups. When the protein levels of CYP2A1 were quantified, it was observed that CYP2A1 is expressed at three-fold higher levels in gonadectomized female rats than in control male rats (figure 3.15.B.). Pubertal testosterone enanthate treatment did not significantly decrease CYP2A1 protein levels relative to gonadectomized female rats. 3.2.2. Effect of Dosing Frequency during Pubertal Androgen Treatment (2) To determine the effects of more frequent testosterone injections, a treatment protocol more accurately simulating physiological testosterone secretion patterns, on the increased expression of male-specific enzymes and suppression of female-specific enzymes during adulthood Protein levels and associated activities of microsomes from rats treated once or twice daily with testosterone were compared to microsomes of gonadectomized female rats to determine if the a more frequent dosing protocol was able to increase expression of CYP2C11 and CYP3A and decrease expression of CYP2A1. 3.2.2.1. Elimination of a single subcutaneous androgen injection Pubertal treatment using a single daily dose of testosterone enanthate was not sufficient to completely masculinize the C Y P profile in female microsomes. It is 85 hypothesized that a pattern of serum testosterone peaks and inter-peak valleys of undetectable testosterone regulate the masculine expression of C Y P enzymes. Physiologically, plasma testosterone levels peak three times in a 24-hour period. It is conceivable that the frequency of the testosterone peaks and valleys during puberty is important for effective imprinting of C Y P enzyme expression during adulthood. To determine i f a more physiological treatment protocol would be more effective, the effect of multiple daily injections was investigated. First, the elimination profile following a single subcutaneous injection of testosterone enanthate needed to be characterized to ensure a valley of undetectable testosterone would be achieved between injections. The plasma concentration of testosterone in gonadectomized female rats was monitored following a single subcutaneous injection of testosterone enanthate at a dose of 5 umol/kg or 10 umol/kg (figure 3.17.). A subcutaneous injection of testosterone enanthate at a dose of 5 umol/kg produces physiological plasma concentrations of testosterone (Pak et al. 1984). A larger dose of testosterone enanthate (10 umol/kg) was included to determine the feasibility of investigating the effects of higher testosterone peak concentrations on the pubertal imprinting effect. The results shown in figure 3.17. indicate that plasma testosterone levels increased to a maximum at 10 to 12 hours after the injection. The apparent half-lives of testosterone were found to be 4 - 14.5 hours and 10-35 hours after 5 umol/kg and 10 umol/kg doses of testosterone enanthate, respectively. A range of values for the half-lives was necessary because the terminal points of the elimination phases are estimates. The lower number in the range reflects the half-life calculated i f it is assumed that testosterone was not present at the time the unquantifiable measurements of plasma testosterone were made. The upper number in 86 (A) (B) as E CO D_ a> c o CD - • — ' CO o -*—' CO CO r-1 t 0.1 0.01 0.001 CD c 0 T — i — i — i — i — r 25 50 75 Time (hours) 100 (C) CD c 1 CO o i _ E CO CO oste E 0.1 CO Q_ Test £-0.01 0.001 "T 1 1 1 1 1 1 1 25 50 75 T i m e (hours ) 100 cu 100 CO o — 10 E co CO oste E 1 ^ 0.1 Q . Test ~ 0.01 0.001 0 25 50 75 T i m e (hours ) 100 Figure 3.17. Elimination of testosterone in ovariectomized female rats. Rats were injected subcutaneously with a single dose of (A) 5 u.mol/kg testosterone enanthate, (B) 10 u.mol/kg testosterone enanthate, or (C) 5 umol/kg testosterone. Blood samples were drawn from the tail vein at selected time intervals. Plasma samples were analyzed for testosterone by a radioimmunoassay as outlined in the Experimental section. Results are shown as the mean of four rats. Open diamonds are measurements that were below the limit of quantitation. 87 the range represents the half-life calculated when the values estimated by the gamma counter was used. The increase in half-life with an increase in dose indicates that the elimination of testosterone at these concentrations is not first order and that there is a rate-limiting step in the elimination process that is being saturated. Therefore, larger daily doses of testosterone enanthate were not possible because the elimination of testosterone would be too slow. Subsequent doses would only further increase plasma concentrations to nonphysiological levels and not produce the desired peak and valley pattern. Multiple daily dosing with testosterone enanthate was not possible either because additional doses of testosterone enanthate within 12 hours of the previous dose would also further increase plasma concentrations to nonphysiological levels. A n androgen formulation that is more rapidly removed is required to avoid accumulation of testosterone and produce the rise and fall of androgen concentrations similar to that observed physiologically. Therefore, elimination of the nonesterified form of testosterone was examined because it is eliminated more quickly than testosterone enanthate and could possibly be used to study the effects of multiple daily injections. As shown in figure 3.17., a single dose of testosterone (5 umol/kg) was eliminated much more quickly than a single dose of testosterone enanthate. The apparent half-life of testosterone following testosterone administration was calculated to be between two and three hours. The plasma testosterone concentration falls below the limit of detection twenty hours after injection. The results suggest that two daily injections of testosterone at a dose of 2.5 umol/kg (net daily dose is kept constant with this treatment) would be able to produce valleys of undetectable testosterone that are required to investigate the effect of the pattern of androgen secretion on pubertal imprinting of C Y P . 88 C o CO o -#-» CO 0 I-ro E CO CD CO c g co —^' c CD O c o 100 10 1 0.1 0.01 0.001 0 20 40 60 Time (hours) 80 Figure 3.18. Elimination of testosterone in ovariectomized female rats. Rats were injected subcutaneously on days 35-49 of age with 2.5 umol/kg testosterone twice daily (squares) and 5 pmol/kg testosterone once daily (diamonds). Blood samples were drawn at selected time intervals for 72 hours following the final injection of androgen. Samples that were below the limit of detection of the assay were not included in the figure. Plasma samples were analyzed for testosterone with a radioimmunoassay as outlined in the Experimental section. Values are shown as mean + standard error of the mean (n=8 for two daily doses and n=7 for one daily dose). Open symbols are measurements that were below the limit of quantitation. 89 3.2.2.2. Elimination of testosterone following pubertal testosterone treatment Having decided to use unesterified testosterone, rats were injected with testosterone either once or twice daily during the pubertal period. Rats were either given a single dose of 5 pmol/kg testosterone every 24 hours or a dose of 2.5 pmol/kg testosterone every 12 hours. The concentration of plasma testosterone was monitored testosterone were monitored for 72 hours following the final injection of androgen (figure 3.18.). The rate of testosterone elimination could only be measured in the rats administered testosterone once daily because plasma testosterone concentrations fell below the limit of quantitation aftetr the first time point in the rats injected twice daily. The half-life of testosterone in rats treated with testosterone once daily was estimated to be between 0.9 and 2.7 hours. 3.2.2.3. Effect of dosing frequency during pubertal androgen treatment on adult CYP2C11 protein levels and testosterone 2 a -and 16a -hydroxylase activities The effect of pubertal treatment with testosterone once or twice daily on testosterone 2a -and 16a -hydroxylase activities is shown in figure 3.19. Testosterone 2a-hydroxylase activity was undetectable in microsomes from gonadectomized female rats and was increased 1.5-fold above the LOQ of this assay (to 0.052 ± 0.025 2a-hydroxytestosterone nmol formed/minute/nmol total CYP) following two daily doses of 2.5 pmol/kg testosterone (figure 3.19.A). Microsomes from rats treated with a single daily 5 pmol/kg dose of testosterone had undetectable levels of testosterone 2a-hydroxylation. Two daily doses of testosterone was not significantly more effective than a single daily dose of testosterone (p=0.14) and a large standard error was associated with the mean value of this treatment group. 90 (A) c E CD D_ >-O o E S o E S o s i o < 1.2 1 0.8 0.6 0.4 0.2 0 1 Gx Females T 5 umol/kg T 2.5 Intact Males 90 days umol/kg (2X) 90 days (B) c E CO E o E c o ^ I o < 2 1.6 1.2 0.8 0.4 0 I Gx Females T 5 umol/kg T 2.5 Intact Males 90 days umol/kg (2X) 90 days Figure 3.19. Effect of dosing frequency on (A) testosterone 2a-hydroxylase activity and (B) testosterone 16a-hydroxylase activity in hepatic microsomes prepared from prepubertally gonadectomized female rats. Results are expressed as mean + standard error of the mean. (n=7 for gonadectomized and T-treated females, n=8 for control males) aBelow the limit of detection. 91 Testosterone 16a-hydroxylase activities parallel the results observed with testosterone 2a-hydroxylase activity (figure 3.19.B.). Two daily doses of testosterone increased testosterone 16ct-hydroxylase activity 2.6-fold above the gonadectomized female level. But this increase was not significantly different than the activity observed in microsome samples from rats treated with a single dose of testosterone. The highly variable effect of two daily injections of testosterone was also observed with CYP2C11 protein levels. As shown on the representative blot probed with polyclonal anti-CYP2Cl 1 IgG (figure 3.10.), CYP2C11 was undetectable in female rats treated once daily but was detected in two of the three rats treated twice daily with testosterone. From the quantitation data reported in figure 3.20., it was observed that a twice-daily treatment protocol increased CYP2C11 protein levels to 0.010 ± 0.004 nmol/nmol total CYP, which is just above the LOQ of 0.003 nmol/nmol total C Y P . Again, a large standard error was associated with the mean value and the mean value was not significantly different (p=0.12) from the undetectable level observed in microsome samples from rats treated with a single daily dose. Yet, the fact that CYP2C11 expression was increased above detection limits is scientifically important although the statistical test did not attribute this increase to be significant. 3.2.2.4. Effect of dosing frequency during pubertal androgen treatment on adult CYP 3A protein levels and testosterone 6(3 -hydroxylase activities Neither two daily injections of testosterone nor a single injection of testosterone during puberty were effective in increasing the activity of hepatic testosterone 6p~ 92 0.16 £ o . 12 CD I D_ l o ~o E0. c .08 04 0 i i • • i — , Gx Females T 5 umol/kg T 2.5 Intact Males 90 days umol/kg (2X) 90 days Figure 3.20. Effect of dosing frequency on CYP2C11 protein levels in hepatic microsomes prepared from prepubertally gonadectomized female rats. Results are expressed as mean ± standard error of the mean. (n=7 for gonadectomized and T-treated females and n=8 for control males). "Below the limit of detection. 93 (A) c JE CD E o E s i o < 1.4 1.2 1 0.8 0.6 0.4 0.2 0 Gx Females T 5 umol/kg T 2.5 Intact Males 90 days umol/kg (2X) 90 days (B) CD > CD I Q_ >-O _ 0.1 cjO.08 ! o . 0 6 | 0.04 c o 0.02 -I E ^ 0 Gx Females T 5 umol/kg T 2.5 Intact Males 90 days umol/kg (2X) 90 days Figure 3.21. Effect of dosing frequency on (A) testosterone 6p-hydroxylase activity and (B) CYP3A protein levels in hepatic microsomes prepared from prepubertally gonadectomized female rats. Results are expressed as mean ± standard error of the mean. (n=7 for gonadectomized and T-treated females and n=8 for control males). 94 hydroxylase (figure 3.21.A) in microsome samples from ovariectomized female rats (p=0.48). The same trend was observed with CYP3 A protein levels. In the representative blot shown in figure 3.13., both treatment protocols with testosterone appeared to decrease the staining intensity of immunologically reactive CYP3 A band below that of gonadectomized females. The quantitation data confirms that two daily injections of testosterone during puberty were unable to significantly increase CYP3A levels (p=0.06) above those observed following pubertal treatment with a single daily injection of testosterone (figure 3.21.B). The monoclonal antibodies were not able to detect any CYP3A1 or CYP3A2 in two randomly chosen samples from either the rats treated once daily or twice daily with testosterone (figure 3.14.). 3.2.2.5. Effect of dosing frequency during pubertal androgen treatment on adult CYP2A1 protein levels and testosterone 7 a -hydroxylase activities Two daily injections of testosterone during puberty were not able to decrease testosterone 7ct-hydroxylase activity (figure 3.22.A.). In the representative blot shown in figure 3.16., the protein levels of CYP2A1 do not appear to be affected by either treatment protocol with testosterone. This is confirmed with the quantified protein levels of CYP2A1 (figure 3.22.B). 95 (A) c E co E Q_ >-co •4—• O o E S ° o < 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 Gx Females T 5 umol/kg T 2.5 Intact Males 90 days umol/kg (2X) 90 days (B) D_ >-_ O CO TO CO o a. >-o o E c o E c 0.06 0.05 0.04 0.03 0.02 0.01 0 I Gx Females T 5 umol/kg T 2.5 Intact Males 90 days umol/kg (2X) 90 days Figure 3.22. Effect of dosing frequency on (A) testosterone 7a-hydroxylase activity and (B) CYP2A1 protein levels in hepatic microsomes prepared from prepubertally gonadectomized female rats. Results are expressed as mean ± standard error of the mean. (n=7 for gonadectomized and T-treated females and n=8 for control males). 96 3.2.3. Effect of Pubertal Testosterone Enanthate Treatment at Various Ages During Adulthood (3) To determine the duration of any androgen-mediated effects on CYP expression during adulthood, to test the hypothesis of imprinting or the possibility that long-term induction is occurring. Protein levels and associated activities of microsomes from testosterone enanthate-treated rats at 129 and 169 days of age were compared to microsomes from testosterone enanthate-treated rats at 90 days of age to determine if pubertal testosterone enanthate treatment was able to increase expression of CYP2C11 and CYP3A and decrease expression of CYP2A1 permanently during adulthood. 3.2.3.1. CYP2C11 protein levels and testosterone 2a - and 16a -hydroxylase activities at various ages during adulthood following pubertal testosterone enanthate treatment. Figure 3.23. shows testosterone 2a - and 16a -hydroxylase activities at 90, 129, and 169 days of age. Testosterone 2a-hydroxylase activity was observed to decline after 90 days of age (figure 3.23. A). This activity fell below the LOQ by 129 days and is significantly decreased from the activity observed at 90 days of age (p=0.004). The same trend is observed with testosterone 16a-hydroxylase activity (figure 3.23.B.) with its activity approaching gonadectomized female levels at 169 days of age. The decline in testosterone 2a- and 16a-hydroxylase activities is reflected by a similar decline in CYP2C11 protein levels with only very lightly stained bands visible in lanes loaded with (A) 97 c E co E D_ >-O o E c > < 0.3 0.25 0.2 5 _ | 0.15 0.1 0.05 0 TE 5umol/kg 90 TE 5 umol/kg days 129 days TE 5 umol/kg 169 days (B) CO E o_ >-O TO •4—" O o o < o E c 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 TE 5umol/kg 90 days TE 5 umol/kg 129 days TE 5 umol/kg 169 days Figure 3.23. Decline in (A) testosterone 2ct-hydroxylase activity and (B) testosterone 16a-hydroxylase activity with increasing age following pubertal testosterone enanthate treatment. The dotted line represents the gonadectomized female level of testosterone 16a-hydroxylase activity. Results are expressed as the mean ± standard error of the mean (n=7). "Below the limit of quantitation and significantly different from 90 days of age (p=0.004). 98 M TE129d Purified CYP2C11 TE 169d Gx F TE 90d Figure 3.24. Representative irnmunoblot of hepatic microsomes from adult rats probed with polyclonal antibody against rat CYP2C11. Primary antibody (rabbit anti-CYP2Cl 1 polyclonal IgG) was used at a concentration of 50 pg/ml, secondary antibody, goat anti-rabbit IgG was used at a dilution of 1:3000, and the alkaline phosphatase reaction was stopped after four minutes. Abbreviations used in the figure include: microsomes from gonadectomized female rats at 90 days of age (Gx F), microsomes from control male rats at 90 days of age (M), microsomes from female rats at 129 days of age treated with testosterone enanthate pubertally (TE 129d), microsomes from female rats at 169 days of age treated with testosterone enanthate pubertally (TE 169). Microsomal samples were applied to the gel at a final concentration of 0.02 nmol total C Y P per lane except for M samples that were loaded at 0.01 nmol. Purified CYP2A1 was included on the irnmunoblot at concentrations of 0.06 pmol, 0.2 pmol, internal standard (unknown concentration), 0.6 pmol, and 1.2 pmol per lane. 99 CL 0.045 -, 0.04 -o 0.035 -> 15 0.03 CD 1 o 0.025 -D_ o 0.02 ->- E .c 0.015 o 0.01 -E c 0.005 -0 ^ I 1 , TE 5umol/kg 90 TE 5 umol/kg TE 5 umol/kg days 129 days 169 days Figure 3.25. Decline in CYP2C11 protein levels with increasing age following pubertal testosterone enanthate treatment. Results are expressed as the mean ± standard error of the mean (n=7). "Below the limit of quantitation and significantly different from 90 days of age (p=0.004). 100 microsomes from testosterone enanthate treated rats at 129 and 169 days of age (figure 3.24.). However, CYP2C11 expression was decreased significantly below the LOQ (p=0.004) by 129 days (figure 3.25.). CYP2C11 protein levels and testosterone 2a-hydroxylase activities were still detectable at 169 days of age, therefore the effect of pubertal testosterone enanthate had not completely disappeared. 3.2.3.2. CYP3A protein levels and testosterone 6fi-hydroxylase activities at various ages during adulthood following pubertal testosterone enanthate treatment. As shown in figure 3.26.A., testosterone 6(}-hydroxylase activity did not decline significantly after 90 days of age as compared with 129 days of age (p=0.06) and 169 days of age (p=0.1). The mean activity was decreased to gonadectomized female levels but the large standard error associated with the activity measured at 90 days of age prevented the decrease from being significant. On the other hand, CYP3 A protein levels appeared to decrease on the immunoblots after 90 days of age (figure 3.27.). The quantitation data revealed that CYP3A protein levels did decrease significantly after 90 days of age to gonadectomized female levels by 129 days (figure 3.26.B). 3.2.3.3. CYP2A1 protein levels and testosterone 7a-hydroxylase activities at various ages during adulthood following pubertal testosterone enanthate treatment. Testosterone 7a-hydroxylase activity did not significantly decrease after 90 days of age (figure 3.28.A.). There was no apparent visual change in the intensity of the stained bands on the immunoblots probed with anti-CYP2Al IgG (figure 3.29.) In fact, 101 the quantitation data demonstrated that CYP2A1 protein levels were not significantly altered over time (figure 3.28.B.). 102 (A) c E E - m o "5 E — S o E > ^ < 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 —\ — -!-=^-=i T. TE 5umol/kg 90 TE 5 umol/kg days 129 days TE 5 umol/kg 169 days (B) 0.07 £0.06 _ ° 0.05 co o 0.04 >-o 0.03 0 E 1 0.02 0 — — - ^ t = TE 5umol/kg 90 days TE 5 umol/kg 129 days TE 5 umol/kg 169 days Figure 3.26. Decline in (A) testosterone 6p-hydroxylase activity and (B) CYP3A protein levels with increasing age following pubertal testosterone enanthate treatment. The dotted line represents the gonadectomized female level. Results are expressed as the mean + standard error of the mean (n=7). * Significantly different from 90 days of age (p=0.01 at 129 days of age and p=0.0003 at 169 days of age). 103 M TE129d TE169d Purified GxF TE 90d CYP3A1 Figure 3.27. Representative immunoblot of hepatic microsomes from adult rats probed with polyclonal antibody against rat CYP3A. Primary antibody (rabbit anti-CYP3A polyclonal IgG) was used at a concentration of 50 fig/ml, secondary antibody, goat anti-rabbit IgG was used at a dilution of 1:3000, and the alkaline phosphatase reaction was stopped after five minutes. The same abbreviations were used as in figure 3.24. Microsomal samples were applied to the gel at a final concentration of 0.02 nmol total C Y P per lane, with the exception of the male control samples that were loaded at 0.01 nmol total C Y P per lane. Purified CYP3A1 was included on the blot at concentrations of 0.0625 pmol, 0.125 pmol, 0.200 pmol, 0.250 pmol per lane. (A) .£ 0.7 1 -0.6 CD Q_ | S 0 5 £ -5 0.4 1 2 0.3 C l 0 . 2 0.1 0 o < TE 5umol/kg 90 days TE 5 umol/kg 129 days TE 5 umol/kg 169 days (B) 0.04 Q_ O 0.03 CD O 0_ >-O = 0.02 E c o 0.01 E 0 TE 5umol/kg 90 days TE 5 umol/kg 129 days TE 5 umol/kg 169 days Figure 3.28. Lack of change in (A) testosterone 7oc-hydroxylase activity and (B) CYP2A1 protein levels with increasing age following pubertal testosterone enanthate treatment. The dotted line represents the gonadectomized female level. Results are expressed as the mean ± standard error of the mean (n=7). M TE129d TE169d Purified CYP2 A1 GxF TE 90d Figure 3.29. Representative immunoblot of hepatic microsomes from adult rats probed with polyclonal antibody against rat CYP2A1. Primary antibody (rabbit anti-CYP2Al polyclonal IgG) was used at a concentration of 10 p.g/ml, secondary antibody, goat anti-sheep IgG was used at a dilution of 1:250, and the alkaline phosphatase reaction was stopped after three minutes. The same abbreviations were used as in figure 3.24. Microsomal samples were applied to the gel at a final concentration of 0.02 nmol total C Y P per lane. Purified CYP2A1 was included on the blot at concentrations of 0.125 pmol, 0.250 pmol, 0.375 pmol, and 0.500 pmol per lane. 106 4. DISCUSSION The purpose of the present study was to further characterize the pubertal androgen imprinting phenomenon observed previously in adult sex-dependent hepatic C Y P -mediated activities and C Y P protein levels. Daily subcutaneous injections of testosterone enanthate for two weeks during puberty have been shown to increase expression of male-specific CYP2C11 and its marker activity, testosterone 2ct-hydroxylase, in ovariectomized rats for 40 days following treatment (Cadario et al. 1992). Smaller, nonsignificant increases in testosterone 6(3-hydroxylase and erythromycin N-demethylase activities, which are indicative of the male-dominant CYP3 A subfamily, were also observed with the same treatment. However, testosterone 7a-hydroxylase activity, which is catalyzed exclusively by the female-dominant CYP2A1, was unaffected by pubertal androgen treatment. A n additional androgen treatment period during adulthood further increased CYP2C11 and CYP3A to control male levels, while treatment during the adult period treatment period alone was insufficient to completely masculinize these hormonally regulated C Y P enzymes, suggesting that the pubertal treatment period imprinted these enzymes to respond to adult androgen exposure. The focus of the previous study was to demonstrate that adult male androgen responsiveness could be programmed during the pubertal period in female rats. In the current study, androgen treatment solely during the pubertal period was examined to investigate pubertal imprinting of CYP2C11, CYP3A, and CYP2A1. The present investigation also examined the effects of changing the pubertal dosing frequency on adult expression of these C Y P enzymes to determine whether the androgen secretion 107 pattern regulates adult C Y P expression. In addition, the duration of the effect of pubertal androgen treatment on expression of sex-dependent C Y P enzymes was monitored over the adult period to determine i f altered enzyme expression was a permanent effect. We chose to investigate three sexually differentiated C Y P enzymes that were also examined in the previous study. These three enzymes, CYP2C11, CYP3A, and CYP2A1, were feasible to study because a selective enzyme assay was known for each of the enzymes and antibodies against these enzymes were readily available. The female specific enzyme, CYP2C12, was not included in this study because an antibody was not available and a selective enzyme assay was not feasible. Although pubertal testosterone enanthate treatment alters adult C Y P expression, it can be argued that changes in C Y P levels are due to a combination of long-term induction and suppression resulting from continued exposure to residual androgen rather than actual imprinting. In addition to monitoring the effects of pubertal androgen on C Y P protein levels and activities over a period during adulthood, the plasma levels of testosterone were measured following the final androgen injection. This was to determine i f residual testosterone enanthate remained in the rats, slowly releasing testosterone into the vascular system. 108 4.1. Effect of Pubertal Androgen Treatment on Hepatic CYP Enzymes 4.1.1. The Effect of Pubertal Testosterone Enanthate Treatment on Adult C Y P Expression. Increases in CYP2C11 protein levels and in testosterone 2cc-hydroxylase and testosterone 16a-hydroxylase activities are comparable to those observed previously (Cadario et al. 1992, Chang and Bellward 1996). In the present study, C Y P expression, reported as protein levels and associated activities, increased to 20% of control male levels. Similar increases in CYP2C11 protein levels and testosterone 16ct-hydroxylase activities were observed in neonatally sham-operated female rats that were implanted at five weeks of age with a subcutaneous capsule packed with testosterone proprionate (Dannan et al. 1986). In these rats, plasma testosterone concentrations at 10 weeks of age were still within physiological levels. Implanting a capsule containing 210 mg testosterone proprionate at five weeks of age in neonatally castrated rats was able to increase ethylmorphine demethylase activity at ten weeks of age to control male levels (Virgo 1991). Ethylmorphine demethylase is a male-dominant activity that is known to be catalyzed by purified CYP2C11 (Guengerich et al. 1982). Another study using neonatally ovariectomized rats did not observe an increase in CYP2C11 protein levels after daily injections of testosterone proprionate at a dose of 5 umol/kg for two weeks during puberty unless the rats were first pretreated with the androgen neonatally (Bandiera and Dworschak, 1992). Differences in the results of these studies are likely due to different testosterone esters (proprionate versus enanthate), doses, and mode of administration (capsule versus daily injections) used. 109 In the present study, testosterone 6p-hydroxylase activity increased, but not significantly, although the magnitude of the increase is similar to that reported previously (Cadario et al. 1992). However, erythromycin TV-demethylase activity did not increase following pubertal testosterone enanthate treatment (Cadario 1990). As shown in figure 3.12., CYP3A protein levels, a parameter that was not measured before, increased significantly. This new information suggests that at least one member of the CYP3 A subfamily is responsive to pubertal androgen. Dannan et al. (1986) claimed that CYP3A levels increased at ten weeks of age in control female rats to 30% of control male levels after a subcutaneous capsule of testosterone proprionate was implanted at five weeks of age. Yet, CYP3 A2 was detected in only one of the seven animals treated with pubertal testosterone enanthate in the present study. Therefore, the increase in CYP3A observed in the present study is predominantly a result of an increase of the unidentified CYP3 A form(s) not resolved by SDS-PAGE from CYP3A1/3A2. A n unknown CYP3A enzyme present in female rats may be responding to pubertal testosterone enanthate treatment, while contributing to constitutive testosterone 6p-hydroxylase or erythromycin Af-demethylase activities. The antibody used in the present study recognizes a female CYP3A-related enzyme. There is a degree of uncertainty regarding the number of forms in the CYP3 A subfamily. For many years, it has been believed that there were two major members of this subfamily in rat microsomes, CYP3A1 and CYP3A2. New advances have shown that there may be as many as four different CYP3A-related enzymes with testosterone 6p-hydroxylase activity (Nagata et al. 1990) and that there are female-dominant CYP3A enzymes as well (Mahnke et al. 1997). The regulation of these isoforms and their contributions to the 110 standard marker activities is unknown at this time. Therefore, the interpretation of the enzyme activities and protein levels presented here are clouded by the individual contributions of separately regulated CYP3A enzymes. The results presented herein show that testosterone 7oc-hydroxylase activity and CYP2A1 protein levels were not significantly altered by pubertal testosterone enanthate treatment in agreement with the previous report of Cadario et al. (1992). A slight trend is observed in the protein level and enzyme activity in the present and previous study that suggests CYP2A1 expression is decreased, however, the effect is too small to be observed statistically. Dannan et al. (1986) also noted that exogenous androgen administration had a small effect (20% to 25% decrease) on CYP2A1 protein levels and testosterone 7a-hydroxylase activity in neonatally ovariectomized rats. Androgen regulation of CYP2A1 expression in males is complicated because neonatal and adult castration had relatively small effects on CYP2A1 expression in one study (Waxman et al. 1985), yet expression of CYP2A1 was doubled in another study (Waxman et al. 1989). The fact that CYP2A1 is less affected by androgen imprinting in this study is consistent with a model in which sex-dependent C Y P are regulated by the pattern of G H secretion which is, in turn, regulated by sex steroids. CYP2A1 expression is largely unaffected by the pattern of G H secretion because protein levels of CYP2A1 increased by only a relatively small amount after hypophysectomy in both male and female rats (Kato and Yamazoe, 1993). Many studies emphasize the importance of neonatal imprinting of CYP2C11 and CYP3A. Castration at 5 weeks of age decreases CYP2C11 and CYP3A-mediated activities to 75% of control male levels while neonatal castration decreases the activities I l l to control female levels (Waxman et al. 1990). Testosterone proprionate treatment of rats one and three days after birth was able to restore CYP3A activity to approximately 80% of control male levels. But the same injections only restored CYP2C11 to approximately 47%) of control male levels. These results indicate that CYP2C11 is more dependent upon androgen production than CYP3 A between three and thirty-five days of age. Our results reflect this observation because larger, more significant effects of pubertal testosterone enanthate were observed in CYP2C11 than CYP3A. Unlike CYP2A1, both CYP2C11 and CYP3A2 are highly dependent upon G H regulation. Hypophysectomy increases CYP3A2 by more than 500% in male and female rats, decreases CYP2C11 in male rats to half its control male level, and increases CYP2C11 to this intermediate level in female rats. Plasma testosterone levels decreased very slowly following pubertal testosterone enanthate treatment. Mean testosterone concentrations did not fall below the limit of quantitation of the radioimmunoassay until ten days after the final androgen injection. This average elimination rate could be interpreted to mean that the rats had undetectable levels of testosterone for thirty days prior to measurement of C Y P levels and activities although, some of the individual rats still had detectable levels of testosterone at 90 days of age, which is the time when microsomes were prepared and C Y P protein levels and activities were measured. Unfortunately, different rats were used to measure plasma testosterone levels for forty days and C Y P expression so it was impossible to see i f there was a direct relationship between the length of exposure to testosterone and increase in CYP2C11 and CYP3A expression. This data questions the validity of using the term imprinting to describe the effect of testosterone enanthate treatment on adult C Y P 112 expression because there may have been residual testosterone present in some of the rats that was able to directly induce CYP2C11 and CYP3A levels. 4.1.2. The Effect of Dosing Frequency of Pubertal Androgen Treatment on Adult C Y P Expression Single daily doses of testosterone enanthate during puberty were not able to increase adult C Y P expression to control male levels. A subcutaneous implantation of a testosterone enanthate capsule, which created a continuous release of testosterone during 35 to 49 days of age, feminized adult C Y P expression (Chang and Bellward, 1996) so it was hypothesized that an oscillating plasma level of testosterone was required to regulate masculine C Y P expression. It was decided to see i f increasing the dosage frequency of unesterified testosterone to twice daily at 0800 and 2000h but maintaining the same total daily dose would be more effective in imprinting adult C Y P expression to control male levels. Physiologically, rats at 40 days of age have an innate trimodal rhythmicity of serum testosterone secretion with three pulses of testosterone daily at 0200, 1200, and 1800h (Mock etal. 1978). Increasing the dosage frequency of unesterified testosterone from once every 24 hours to once every twelve hours did not increase the response of C Y P enzymes to express control male levels. In fact, the effect of a twice-daily dosage protocol on CYP2C11, CYP3A, or CYP2A1 expression was not significantly different from a single dose of testosterone. However, CYP2C11 protein levels and testosterone 2oc-hydroxylase and 16cc-hydroxylase activities were slightly greater in rats that were treated twice daily than rats that only received'a single daily injection. CYP2C11 protein levels and 113 testosterone 2a-hydroxylase activity were below the limit of detection in all of the microsomes prepared from rats treated once daily with unesterified testosterone. In some individual rats treated with two daily injections of testosterone, both CYP2C11 protein levels and testosterone 2cc-hydroxylase activities were detectable above control. Interpreting the results of CYP3A expression was more complicated because more than one C Y P enzyme is detected. Quantitation of immunoblots detected net changes in CYP3 A levels that are the sum of increases and decreases of individual enzymes. Testosterone treatment, once or twice daily, appeared to suppress the expression of the CYP3A forms that were detected by the polyclonal antibody. It is possible that we were detecting a female form of CYP3A that is decreased by pubertal androgen treatment. Unfortunately, it is impossible at this stage to determine the sex-difference in the constitutive expression of this unidentified CYP3A form because the level of this enzyme is overwhelmed by the presence of CYP3 A2 in control male samples. Testosterone was not as effective as testosterone enanthate at increasing expression of CYP2C11 and CYP3A. However, the study was not designed to investigate the difference between the effects of testosterone and testosterone enanthate and therefore, no statistical analysis was conducted to determine if the differences were statistically significant. It seems that it is not the pattern of androgen administration that is as important as the formulation of androgen. This may be a manifestation of the pharmacokinetic differences between the two androgen formulations. The elimination of unesterified testosterone was much more rapid than the elimination of testosterone enanthate. Plasma testosterone was below the limit of quantitation after ten hours 114 following the final injection of unesterified testosterone, regardless of the dosing frequency. The elimination was rapid in all of the individual rats and therefore rats were essentially not exposed to circulating testosterone for 40 days prior to measuring C Y P levels and activities. 4.1.3. Changes in Adult C Y P Expression over Time Following Pubertal Testosterone Enanthate Treatment. Two observations in this study questioned the validity of using the term imprinting to describe the effect of pubertal testosterone enanthate treatment on adult C Y P expression. The slow elimination of testosterone enanthate and the decreased effectiveness of the more quickly eliminated unesterified testosterone both suggest that the observed effects of testosterone enanthate on CYP2C11 and CYP3A result from long-term induction. Although this outcome could not have been known prior to the design of the experiment, we decided to investigate whether the effect of pubertal testosterone treatment on adult C Y P expression is permanent throughout adulthood. A permanent effect would be considered more support for the hypothesis that pubertal androgen imprinting of C Y P expression is possible. On the other hand, a temporary effect that diminishes over time suggests that testosterone enanthate causes induction of certain C Y P enzymes that depend upon continued androgen presence for expression. In this study, dramatic decreases in mean CYP2C11 and CYP3A expression were observed after 129 days. The decline is variable because certain individual animals maintain higher levels of expression of these enzymes at 129 days, but all of the samples had decreased levels by 169 days. We cannot dispute these results because there is more 115 than just a partial decline of the levels of these enzymes. CYP2C11 and CYP3A protein levels and their respective marker activities return to control female values within the time span examined. Similarly, a decrease in ethylmorphine demethylase activity, a male-dominant activity, was observed after a pubertal implant of testosterone proprionate was removed on day 71 (Virgo 1991). In this instance, ethylmorphine demethylase activity returned to control female levels in two weeks. The time span examined by Virgo and in the present study was too short to consider the decrease in C Y P expression to be a result of normal physiological decreases that occur with age (Imaoka 1991). This is unequivocal proof that the previous observations reported by Cadario were not due to pubertal androgen imprinting of adult C Y P expression. Rather, an unusual situation of long-term induction because of a prolonged period of testosterone exposure is likely to have increased CYP2C11 and CYP3A expression for over a month. 4.2. Potential Limitations of the Study 4.2.1. Relevance of the Doses In order to establish pubertal imprinting as a possible physiological phenomenon, it was important to ensure that the doses of testosterone enanthate used were producing physiological levels of testosterone in the plasma. The dose of testosterone enanthate used in this study, 5 pmol/kg, "was found to maintain normal levels of sex accessory tissue weights and sex-dependent drug metabolizing enzyme activities in gonadectomized animals" (Pak et al. 1984). Normal control male levels of plasma testosterone were measured to be 1.31 ± 0.31 ng/ml in our study. However, the rhythmicity of testosterone secretion makes it difficult to determine the physiological range of testosterone 116 concentrations. Different studies have measured from less than 1 to 7 ng/ml testosterone in male rats (Ddhler and Wuttke 1975, Mock et al. 1978, Forest 1979). Pubertal treatment with testosterone enanthate at 5 pmol/kg/day was found to elevate plasma levels of testosterone above control male levels measured in the present study but plasma concentrations were within the range reported in the literature. Decreasing the dose of pubertal testosterone enanthate to 2.5 umol/kg did not increase testosterone 2a-hydroxylase activity as effectively as 5 umol/kg (Chang and Bellward, 1996). Thus, it was felt that a dose of 5 pmol/kg/day was required to achieve the maximal imprinting effect. Daily subcutaneous injections of testosterone enanthate produce a continuous, non-pulsatile level of testosterone in the circulation. It was argued that the effects of testosterone enanthate could not be considered physiological because this treatment does not mimic physiological events in the developing rat. In adult male rats, a trimodal rhythm of testosterone secretion is observed with peak levels of testosterone measured daily at 0200, 1200, and 1800 hours when a twelve-hour light, twelve-hour darkness cycle is used (Mock et al. 1978). We administered unesterified testosterone twice daily in an attempt to mimic the circadian rhythm of physiological testosterone within practical limits. Unfortunately, this pattern is not entirely physiological because the highest testosterone concentrations occur early in the morning and in the evening but Mock et al. (1978) noticed that testosterone levels were generally higher during the day than during the night. Furthermore, the same total daily dose of unesterified testosterone as testosterone enanthate produced peak plasma levels of testosterone that were well above physiological male levels. 117 4.2.2. Definition of puberty Puberty is defined as the time during development when sexual maturation occurs and the reproductive organs become functional. It is marked by the appearance of secondary sexual characteristics and is brought about by the increase in sex steroid production due to stimulation of the ovaries and testes by pituitary hormones. In rats, plasma testosterone levels are observed to rise after 35 days of age (Forest 1979). This time coincides with the development of sexual differentiation of certain hepatic drug-metabolizing enzymes as well. We chose to administer at 35 to 49 days of age to simulate the pubertal period of sex steroid production. This treatment protocol allowed us to be consistent with previous studies (Pak et al. 1984, Cadario 1990, Cadario et al. 1992, Chang and Bellward 1996). However, we have not simulated the conditions of puberty exactly because many other hormones such as L H and FSH are elevated during puberty and may play a role in sexual differentiation of the drug metabolizing enzymes. 4.2.3. Sensitivity of radioimmunoassay Mean plasma levels of testosterone fell below the limit of detection ten days after pubertal testosterone enanthate treatment. Unfortunately, we could not tell i f low levels of testosterone were still present in all of the rats at 90 days of age. It is possible that testosterone was present in the rats for a longer period of time than we measured and that low levels were still able to regulate C Y P expression. A few examples of low hormone levels have been reported in the literature. Low levels of G H (less than 5% of control levels) have been shown to regulate sex-dependent expression of C Y P as long as the 118 sexually differentiated patterns of G H secretion are maintained (Bullock et al. 1991). In addition, neonatal imprinting occurs during the first few days of life when testosterone levels are one tenth of adult levels (Forest 1979). Obviously very low concentrations of hormone can have profound effects on C Y P expression. 4.3. Speculation on the Results 4.3.1. Pubertal imprinting or long-term induction ? The results of the present study do not support the hypothesis that pubertal imprinting of hepatic C Y P enzymes is possible. The clear decrease in CYP2C11 expression and CYP3A expression to control female levels with increasing age following pubertal testosterone enanthate treatment is conclusive evidence that the pubertal testosterone enanthate treatment does not permanently alter the pattern of C Y P expression. The increase in expression of these same enzymes following pubertal testosterone enanthate treatment is an unusual case of induction because the duration of the effect persisted long after treatment ended. Long-term induction can explain the variable responses to the pubertal treatment that were observed. For example, some individual rats displayed greater CYP2C11 and CYP3 A expression as measured by the protein levels and marker activities. In addition, the rate of decline of expression was slower in individual rats as well because the protein levels and activities of CYP2C11 and CYP3A were variable at 129 days of age. This may be linked to the variable elimination of testosterone following testosterone enanthate treatment in these rats. It is conceivable that the increase in C Y P expression is still detectable at 90 days of age because the rats were exposed to testosterone for an extended 119 period. It was unfortunate that we could not determine if the rats with noticeably greater sensitivity to pubertal testosterone enanthate treatment and those individuals whose elevated C Y P levels were slowest to decline were the same rats exposed to testosterone for the longest period of time following testosterone enanthate treatment. The observation that testosterone was not as effective as testosterone enanthate in increasing C Y P expression supports the idea that extended exposure to androgen is responsible for the elevated expression of male C Y P enzymes. Testosterone levels in the circulation decreased very rapidly and was completely eliminated within a day after the final injection of testosterone. Testosterone elimination following testosterone enanthate administration was more gradual (figure 3.11.). It is generally accepted that the more fat-soluble testosterone enanthate is absorbed more slowly than testosterone and daily subcutaneous injections would produce a sustained and relatively unfluctuating level of testosterone (Goodman and Gilman, 1995). The rate-limiting step of testosterone elimination is the retention of androgen ester in the oil depot, which is governed by the oil/water partition coefficient (Minto et al. 1997). Once the ester appears in the extracellular fluid, it is rapidly hydrolyzed by esterases to the biologically active androgen that then can be eliminated at the same rate as pure testosterone. Other factors that may influence testosterone enanthate pharmacokinetics include the injection volume, the injection site (i.e. tissue composition and blood flow), and the fat mass of the subject (Minto et al. 1997). These factors may be contributing to the interindividual variability observed in testosterone elimination after treatment with testosterone enanthate because, in the current study, injection volume changed with the weight of the animal and the injection site was different every day to avoid skin irritation. 120 Two daily injections of testosterone were not significantly more effective than a single injection of a larger dose of testosterone in increasing expression of CYP2C11 and CYP3A. The twice daily treatment protocol was more similar to the physiological trimodal pattern of testosterone secretion and therefore the frequency of plasma testosterone pulses is probably not responsible for the observed increases in C Y P expression. This interpretation is only valid within the limits of using just two injections of testosterone per day rather than the more physiological three injections per day. Yet, this treatment protocol was able to increase CYP2C11 expression to detectable levels in some individual rats while no CYP2C11 was detected in rats dosed once daily. When taken in conjunction with the above considerations, it is conceivable that the duration of exposure to testosterone is responsible for the small effect. The rate of elimination of testosterone is the same for both doses of testosterone and thus the length of time for the testosterone contained in each injection to be eliminated is very similar (figure 3.18.). Yet, the rats given two daily doses are exposed to the androgen for twice as long. It would be interesting to see if there was a correlation between total area under the plasma testosterone curve or duration of exposure to testosterone over the pubertal period and the magnitude of the increase in CYP2C11 and CYP3A expression. There are striking differences between the effects of neonatal imprinting and the observations that we attributed to pubertal imprinting. Virgo (1991) suggested that the method of action of the sex steroids differs between the two periods. Neonatal testosterone is able to permanently alter C Y P expression to a more masculine pattern in a process he refers to as defeminization but continuous adult testosterone exposure is required to fully express C Y P enzymes at control male levels. Masculinization is a term 121 that he uses to describe the reversible process of enhancing male characteristics in adulthood. He adopts the terms defeminization and masculinization from general imprinting principles discussed by McLusky and Naftolin (1981). Virgo claims that the reversibility of the response to pubertal androgen is analogous to masculinization. The definitions of defeminization and masculinization are similar to definitions for the terms imprinting and adult androgen responsiveness. In effect, Virgo is proposing that we are merely observing adult androgen responsiveness in prepubertally ovariectomized female rats. 4.3.2. Relevance of C Y P Imprinting to Humans Gender differences in drug and xenobiotic metabolism of humans are not well documented (for a review see Bonate 1991). Generally, nonintrusive measures of metabolism combine both Phase I and Phase II metabolism and cannot provide precise information on the sex-specific differences. Analysis of enzyme activities is not possible directly, rather values such as clearance and half-life are monitored. The literature does not agree whether sex-related differences exist in humans. Often, women appear to have higher plasma concentrations of some drugs than men. For example, longer half lives for lidocaine and chlordiazepoxide have been reported in women (Bonate 1991). This is consistent with a greater occurrence of adverse drug effects in women. However, erythromycin A'-demethylation activity, a marker for CYP3 A activity is significantly higher in females (Hunt et al. 1992). CYP3A is thought to be the most abundant C Y P enzyme in human liver and is responsible for the oxidation of many pharmaceutical agents. 122 Boys are born with higher testosterone levels than girls and levels increase further in boys just hours after birth (Corbier et al. 1990). Imprinting in humans, therefore, appears to occur in utero as well as neonatally. In addition, G H secretion patterns are sexually dimorphic in humans. During adulthood, women have higher G H levels overall. At this time, both sexes secrete 13 pulses of G H per day with a steady frequency but females have higher peak amplitudes and higher valley levels (Winer et al. 1990). Studies investigating the mechanisms of imprinting may have application for humans because both drugs and disease states can affect the pattern of G H secretion. In insulin-dependent diabetes mellitus the number of pulses released per day is increased (Asplin et al. 1989). There is increase in the peak amplitude and the interpulse level even though the pulse increment remains unchanged. C Y P content is decreased in diabetic patients but CYP-related activities are decreased only in the most severe cases (Salmela et al. 1984). Tamoxifen, commonly used in the treatment and prevention of breast cancer, has been shown in animal and clinical studies to alter the pattern of G H secretion (as stated in Chang et al. 1996). Oxandrolone, a nonaromatizable androgen used to promote weight gain, increases the mean mass of G H secreted per pulse in boys with delayed puberty (Ulloa-Aguirra et al. 1990). In female rats, this androgen induces hepatic CYP3A1, CYP3A2, and CYP2C11 levels (Waskiewicz et al. 1995). The response to oxandrolone in human G H patterns is elicited also with testosterone (Zachmann 1992). The implications towards therapies that alter endocrine-functions are unknown regarding imprinting of hepatic metabolic enzymes. One could question the validity of the rat as a model of human imprinting and a suggestion that the mouse may be a more representative animal could be raised. The 123 magnitude of sex differences in C Y P protein levels is largest in the rat, while the gender-related differences in the mouse are of the same order of magnitude as humans. The female mouse has higher drug metabolizing activities and, as previously mentioned, there is some evidence that females may have higher CYP3 A levels than men. However, the rat remains the best-studied model and can still provide insight into mechanisms of regulation, regardless of its direct relationship to humans. The magnitude of sex differences in C Y P expression is much larger in the rat than any other species. This aspect of the rat model allows smaller experimental groups to be employed to detect any significant effects of pubertal androgen treatment. In the same way, smaller effects of androgen treatment on C Y P expression will be detected in these animals that would otherwise go unnoticed in another animal. 4.4. Future Areas of Research Results of the present study suggest that pubertal testosterone enanthate treatment resulted in the elevation of CYP2C11 and CYP3A expression through induction of gene expression. Future areas of research should confirm the interpretation that long-term induction is occurring. One limitation of this study is that different rats were used to profile the elimination of testosterone and the increased C Y P expression following pubertal testosterone enanthate treatment. We suggest that interindividual variability in elimination and increased C Y P expression are linked but more conclusive evidence could be derived i f the two analyses were done on the same rats. In this way, we could be able to see i f there is a relationship between the duration of exposure to testosterone and the 124 magnitude of the increase in C Y P expression at 90 days. In the same way, a study could be designed to detect a possible correlation between the duration of androgen exposure and the duration of the observable elevation of C Y P expression during adulthood. The use of other testosterone esters may be useful in this study to support the hypothesis that the presence of residual testosterone long after the pubertal treatment causes induction of C Y P enzymes in adulthood. For example, testosterone proprionate is eliminated at a rate that is intermediary between testosterone and testosterone enanthate. If the hypothesis is correct, this ester would cause an elevation of C Y P expression at 90 days that is smaller and does not last as long as testosterone enanthate. Secondly, measuring the expression of CYP2C11 and CYP3A at time points earlier than 90 days of age would provide evidence that treatment with unesterified testosterone during puberty also causes induction of these C Y P enzymes. A time course study of induction could show when peak expression occurs. If our observations do indeed indicate induction, then we are most likely not measuring the peak level of expression, but rather an already declining enzyme reserve. If this is the case, we should be able to see transiently increased expression of these enzymes even after pubertal testosterone treatment. It would also be interesting to determine which CYP3A enzymes are being affected by pubertal androgen treatment. Experiments in this area will likely have to involve sequencing the expressed genes because there are no monoclonal antibodies or enzyme activities that are selective for these unidentified enzymes. Studies investigating neonatal imprinting have examined possible mechanisms of action towards permanent alteration of C Y P expression. In particular, experiments have 125 shown the effect of neonatal androgens on G H secretion. Measuring the plasma G H levels following pubertal androgen treatment could indicate effects of testosterone at this level and determine i f testosterone is masculinizing C Y P by altering G H secretion to a male pattern. This study investigated only one aspect of androgen regulation of C Y P expression, namely imprinting. The question of adult androgen responsiveness was not included. Now that the relevance of pubertal imprinting is in question, it is necessary to confirm that increased androgen responsiveness was observed following pubertal testosterone enanthate treatment. However, we have just observed that there is residual testosterone present at that time that may be contributing to the observed effects. Adult treatment periods at later ages in adulthood would provide evidence for the involvement of residual testosterone. 126 4.5. Summary and Conclusions 1) Prepubertally ovariectomized rats responded to pubertal testosterone enanthate treatment with an increase in expression of male-specific CYP2C11, as measured by the CYP2C11 protein levels and the marker activities, testosterone 2a-hydroxylase and 16a-hydroxylase at 90 days of age. 2) Prepubertally ovariectomized rats responded to pubertal testosterone enanthate treatment with an increase in male-dominant CYP3A expression, as measured by CYP3A protein levels and a smaller nonsignificant increase in its marker activity, testosterone 6(3-hydroxylase. Evidence from irnmunoblot analysis using monoclonal antibodies indicates that neither CYP3A1 nor CYP3A2 is responsible for the increase in CYP3A expression. 3) Pubertal testosterone enanthate treatment caused a small nonsignificant decrease in female dominant CYP2A1 expression, as measured by CYP2A1 protein levels and its marker activity, testosterone 7a-hydroxylase, suggesting that testosterone enanthate is selectively regulating the male C Y P enzymes. The three above conclusions corroborate previous findings by Cadario et al. (1992). 4) The effect of two daily injections of testosterone was not significantly different than a single dose of testosterone on expression of CYP2C11, CYP3A, and, CYP2A1, 127 suggesting the frequency of plasma testosterone pulses does not regulate adult C Y P expression. 5) Testosterone treatment was observed to be less effective than testosterone enanthate treatment at increasing expression of CYP2C11 and CYP3A, suggesting that a pharmacokinetic characteristic of testosterone enanthate is causing the above effects. 6) Testosterone was eliminated from the plasma much more slowly after pubertal testosterone enanthate treatment than testosterone treatment. Levels of plasma testosterone fell below the limit of quantitation ten hours after the final testosterone injection and ten days after the final testosterone enanthate injection. This demonstrates that there was a large difference in the period of time the two groups of rats were exposed to testosterone. 7) The elevated expression of CYP2C11 and CYP3A during adulthood after pubertal testosterone enanthate treatment was not permanent because protein levels and marker activities for these two enzymes declined to control female levels between 90 and 169 days. 8) A high degree of interindividual variability was observed in C Y P expression and testosterone plasma concentrations following testosterone enanthate treatment suggesting that the observed effect of testosterone enanthate treatment on C Y P expression results from its pharmacokinetic differences from testosterone. We suggest that there may be a 128 relationship between the duration of exposure to testosterone with the elevation of male C Y P expression. 9) This study provided evidence that questions the validity of using the term "imprinting" to describe the effect of pubertal testosterone enanthate treatment on adult C Y P expression. These results indicate that an unusual condition of long-term induction is occurring. 129 5. REFERENCES Agrawal, A . K. , Pampori, N . A . , and Shapiro, B. H. , Sex- and dose-dependent effect of neonatally administered aspartate on the ultradian patterns of circulating growth hormone regulating hexobarbital metabolism and action. Toxicology and Applied Pharmacology 108: 96-106,1991. Agrawal, A . K. , and Shapiro, B. H. , Phenobarbital induction of hepatic CYP2B1 and CYP2B2: Pretranscriptional and post-transcriptional effects of gender, adult age, and phenobarbital dose. Molecular Pharmacology 49: 523-531, 1996. Arlotto, M . P., Sonderfan, A . J., Klaassen,C. D., And Parkinson, A. , Studies on pregnenolone-16a-carbonitrile inducible form of rat liver microsomal cytochrome P-450 and UDP-glucuronosyl-transferase. Biochemical Pharmacology 36: 3859-3866, 1987. Asplin, C. M . , Faria, A . C. S., Carlsen, E. C , Vaccuaro, V . A. , Barr, R. E., Iranmanesh, A . , Lee, M . M . , Veldhuis, J. D., and Evans, W. S., Alterations in the pulsatile mode of growth hormone release in men and women with insulin-dependent diabetes mellitus. Journal of Clinical Endocrinology and Metabolism 69: 239-245, 1989. Aussel, C , Uriel, J., and Mercier-Bodard C , Rat alpha-fetoprotein: isolation, characterization, and estrogen-binding properties. Biochimie 55: 1431-1437, 1973. Bandiera, S., and Dworschak, C , Effects of Testosterone and Estrogen on Hepatic Levels of Cytochromes P450 2C7 and P450 2C11 in the Rat. Archives of Biochemistry and Biophysics 296: 286-295, 1992. Blizard, D., and Denef, C , Neonatal androgen effects on open-field activity and sexual behavior in the female rat: the modifying influence of ovarian secretions during development. Physiological Behavior 11: 65-69,1973. Bonate, P. L . , Gender-related differences in xenobiotic metabolism. Journal of Clinical Pharmacology 31: 684-690, 1991. 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 5oc-reductase. Proceedings of the National Academy of Sciences USA 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 P450 enzyme activities and cytochrome P450IIC11 by peripubertal administration of testosterone in female rats. Molecular Pharmacology 41: 981-988,1992. 130 Cadario, B. J., Pubertal Testosterone Imprinting. M . Sc. Thesis. Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, B C , 1990. Catania, V . A . , Danneberg, A . J., Luquita, M . G., Sanchez Pozzi, E. J., Tucker, J. K. , Yang, E. K. , and Mottino, A . D., Gender-related differences in the amount and functional state of rat liver UDP-glucuronosyl-transferase. Biochemical Pharmacology 50: 509-514,1995. 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 of microsomal drug activation. Journal of Pharmacology and Experimental Therapeutics 278: 1383-1391,1996. Chowen, J. A . , Garcia-Segura, L . M . , Gonzalez-Parra, S., and Argente, J. Sex steroid effects on the development and functioning of the growth hormone axis. Cellular and Molecular Neurobiology 16: 297-310. Cooper, K . O., Reik, L . M . , Jayyosi, Z., Bandiera, S., Kelley, M . , Ryan, D. E., Daniel, R., McCluskey, S. A. , Levin, W., and Thomas, P. E., Regulation of two members of the steroid-inducible cytochrome P450 subfamily (3 A) in rats. Archives of Biochemistry and Biophysics 301: 345-354,1993. Corbier, P., Dehennin, L . , Castanier, M . , Mebazaa, A. , Edwards, D. A. , and Roffi, J., Sex differences in serum luteininzing hormone and testosterone in the human neonate during the first few hours after birth. Journal of Clinical Endocrinology and Metabolism 71: 1344-1348, 1990. Crabb, D. W., Bosron, W. F., L i , T.-K., Role of the pituitary and neonatal androgenic imprinting in the hormonal regulation of liver alcohol dehydrogenase activity. Biochemical Pharmacology 35: 1527-1532, 1986. Dannan, G. A. , Guengerich, F. P., and Waxman, D. J., Hormonal regulation of rat liver microsomal enzymes: Role of gonadal steroids in programming, maintenance, and suppression of A4-steroid 5cc-reductase, flavin-containing monooxygenase, and sex-specific cytochromes P450. Journal of Biological Chemistry 261: 10728-10735, 1986. Dohler, K . D., and Wuttke, W. Changes with age in levels and serum gonadotropins, prolactin, and gonadal steroids in prepubertal male and female rats. Endocrinology 97: 898-907, 1975. Eden, S., Age- and sex-related differences in episodic growth hormone secretion in the rat. Endocrinology 105: 555-560,1979. 131 Forest, M . G., Plasma androgens (testosterone and 4-androstenedione) and 17-hydroxyprogesterone in the neonatal, prepubertal and peripubertal periods in the human and he rat: differences between species. Journal of Steroid Biochemistry 11: 543-548, 1979. Fujita, I., Sindhu, R. K. , and Kikkawa, Y. , Hepatic cytochrome P450 enzyme imprinting in adult rat by neonatal benzo[a]pyrene administration. Pediatric Research 37: 646-651, 1995. Gonzalez, F. J., The molecular biology of cytochrome P450s. Pharmacological Reviews 40: 243-288, 1989. Goodman, L . S., and Gilman, A. , The Pharmacological Basis of Therapeutics. Eighth edition. Mac Millan Publishing Co., Inc. New York, 1990. Guengerich, F. P., Reactions and significance of cytochrome P-450 enzymes. Journal of Biological Chemistry 266: 10019-10022, 1991. Guengerich F. P., Dannan, G. A. , Wright, S. T., Martin, M . V . , and Kaminsky, L . S., Purification and characterization of liver microsomal cytochromes P450: electrophoretic, spectral, catalytic, and immunochemical properties and inducibility of eight isozymes isolated from rats treated with phenobarbital or P-nappfhflavone. Biochemistry 21: 6019-6030, 1982. Gustafsson, J.-A., Mode, A. , Norstedt, G., and Skett, P., Sex steroid induced changes in hepatic enzymes. Annual Reviews in Physiology 45: 551-60, 1983. Hess, E. H , Imprinting: Early experience and the developmental psychobiology of attachment. Van Nostrand Reinhold Company, New York, 1973. Holsmer, S. L . , Effect of Tamoxifen on Hepatic Cytochrome P450 Expression in Adult Female Rats. M.Sc. Thesis. Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, B C , 1995. Hu, J. J., Lee, M.-J. , Vapiwala, M . , Reuhl, K. , Thomas, P. E., and Yang, C. S., Sex-related differences in mouse renal metabolism and toxicity of acetominophen. Toxicology and Applied Pharmacology 122: 16-26, 1993. Hunt, C. M . , Westerkam, W. R., and Stave, G. M . , Effect of age and gender on the activity of human hepatic CYP3 A . Biochemical Pharmacology 44: 275-283, 1992. Imamura, Y . , Honda, Y . , Kozono, Y. , Ryu, A. , Otagiri, M . , Combined testosterone treatment in pubertal and adult periods induces male-specific acetohexamide reductase activity in liver microsomes of female rats. Research Communications in Molecular Pathology and Pharmacology 86: 92-98, 1994. 132 Imaoka, S., Fujita, S., and Funae, Y. , Age-dependent expression of cytochrome P-450s in rat liver. Biochimica et Biophysica Acta 1097: 187-192,1991. Jansson, J. O., Eden, S., and Isaksson, O., Sexual dimorphism in the control of growth hormone secretion. Endocrine Reviews 6: 128-150, 1985a. Jansson, J. O., Ekberg, S., Isaksson, O., and Eden, S., Influence of gonadal steroids on age- and sex-related secretory patterns of growth hormone in the rat. Endocrinology 114: 1287-1294, 1984. Jansson, J. O., Ekberg, S., Isaksson, O., Mode, A. , and Gustafsson, J.-A., Imprinting of growth hormone secretin, body growth, and hepatic steroid metabolism by neonatal testosterone. Endocrinology 111: 1881-1889, 1985b. Jansson, J. O., and Frohnan, L . A. , Inhibitory effect of the ovaries on neonatal androgen imprinting of growth hormone secretion in female rats. Endocrinology 121: 1417-1423, 1987a. Jansson, J. O., and Frohnan, L . A . , Differential effects of neonatal and adult exposure on the growth hormone secretory pattern in male rats. Endocrinology 120: 1551-1557, 1987b. Jeffrey, S., Wieson, C. A. , and Carter, N . D., Hormonal manipulation and neonatal imprinting of carbonic anhydrase isozymes in rat liver. Hormones in Metabolic Research 22: 467-469, 1990. Kamataki, T., Maeda, K. , Shimada, M . , Kitani, K. , Nagai, T., and Kato, R. Age-related alteration in the activities of drug-metabolizing enzymes and contents of sex-specific forms of cytochrome P450 in liver microsomes from male and female rats. Journal of Pharmacology and Experimental Therapeutics 233: 222-228, 1985a. Kamataki, T., Maeda, K. , Yamazoe, Y . , Nagai, T., and Kato, R., Evidence for the involvement of multiple forms of cytochrome P450 in the occurrence of sex-related differences of drug metabolism in the rat. Life Sciences 31: 2603-2610, 1982. Kamataki, T., Shimada, M . , Maeda, K. , and Kato, R., Pituitary regulation of sex-specific forms of cytochrome P450 in liver microsomes of rats. Biochemical and Biophysical Research Communications 130: 1247-1253, 1985b. Kane, R. E., Lamott, J., Franklin, M . R., and Galinsky, R. E., Perinatal cimetidine exposure has no apparent effect on hepatic drug oxidative or conjugative activity in adult male rat offspring. Developmental Pharmacology and Therapeutics 12: 96-105, 1989. Karnes, H . T., Shiu, G., and Shah, V . P. Validation of bioanalytical methods. Pharmaceutical Research 8: 421 -426, 1991. 133 Kato, R., and Yamazoe, Y . , Sex-specific cytochrome P450 as a cause of sex- and species-related differences in drug toxicity. Toxicology Letters 64/65: 661-667, 1992. Kato, R., and Yamazoe, Y . Hormonal regulation of cytochrome P450 in rat liver. Ch. 29 in: Cytochrome P450. Handbook of Experimental Pharmacology v. 105, Schenkman, J. B. , and Grein, H. , eds. Springer-Verlag, Berlin, pp. 447-459,1993. Kikkawa, Y . , Fujita, I., and Sindhu, R. K. , Neonatal hyperoxia and cytochrome P450 imprinting in adulthood. Pediatric Research 35: 255-258,1994. Laemmli, U . K. , Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 207: 680-685,1970. Lemkey-Johnston, N . , and Reynolds, W. A. , Nature and extent of brain lesions in mice related to ingestion of monosodium glutamate. A light and electron microscopic study. Journal of Neuropathology and Experimental Neurology 33: 74-78, 1974. Lewis, D. F. V . , Cytochrome P450: Structure, Function, and Mechanism. Taylor & Francis, London, 1996. Lowry, O. H. , Risebrough, N . J., Fair, A . C , and Randall, R. J., Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193: 265-275,1951. MacLusky, N . J., and Naftolin, F., Sexual differentiation of the central nervous system. Science 211: 1294-1311,1981. Mahnke, A . , Strotkamp, D., Roos, P. H. , Hanstein, W. G., Chabot, G.G., and Nef, P., Expression and inducibility of cytochro9me P450 3A9 (CYP3A9).and other members of the CYP3 A subfamily in rat liver. Archives of Biochemistry and Biophysics 337: 62-68, 1997. McCarthy, M . M . , Molecular aspects of sexual differentiation of the rodent brain. Psychoneuroendocrinology 19: 415-427, 1994. McEwen, B. S., Biegon, A. , Davis, P. G., Krey, L . C , Luine, V . N . , McGinnis, M . Y . , Paden, C. M . , Parsens, B. , and Rainbow, T. C , Steroid hormones: hormonal signals that alter brain properties and functions. Recent Progesses in Hormone Research 38: 41-92, 1982. McLeod, J. N . , Sorensen, M . P., And Shapiro, B. H. Strain independent elevation of hepatic monooxygenase enzymes in female mice. Xenobiotica 17: 1095-1102, 1987. Minto, C. F., Howe, C , Wishart, S., Conway, A . J., and Handelsman, D. J., Pharmacokinetics and pharmacodynamics of nandrolone esters in oil vehicle: effects of ester, injection site, and injection volume. Journal of Pharmacology and Experimental Therapeutics 281: 93-102,1997. 134 Mock, E. J., Norton, H . W., and Frankel, A . I., Daily rhythmicity of serum testosterone concentration in the male laboratory rat. Endocrinology 103: 1111-1120, 1978. Mode, A . , Wiersma-Larsson, E., Strom, A. , Zaphiropoulos, P. G., And Gustafsson, J.-A., A dual role of growth hormone as a feminizing and masculinizing factor in the control of sex-specific cytochrome P450 isozymes in rat liver. Journal of Endocrinology 120: 311-317, 1989. Nagata, K. , Gonzalez, F. J., Yamazoe, Y . , and Karo, R., Purification and characterization of four catalytically active testosterone 6p-hydroxylase P-450s from rat liver microsomes: comprison of a novel form with three structurally and functionally related forms. Journal of Biochemistry 107: 718-725,1990. Nash, T., the colorimetric estimatin of formaldehyde by means of the Hantzsch reaction. Biochemical Journal 55:416-421,1953. Nelson, D. R., Kamataki, T., Waxman, D. J., Guengerich, P., Estabrook, R. W., Feyereisen, R., Gonzales, F. J., Coon, M . F., Gunsalus, I. C , Gotoh, O., Okuda, K. , and Nebert, D. W., The P450 superfamily: Update on new sequences, gene mapping, accesssion numbers, and nomenclature. Pharmacogenetics 6: 1-42,1996. Ohgiya, N . , Yokota, FL, and Yuasa, A. , Purification and properties of cytochrome P-450 generally acting as a catalyst on benzo(a)pyrene hydroxylation from liver microsomes of untreated rats. Journal of Biochemistry 105: 234-238,1989. Omura, T., and Sato, R., The carbon monoxide binding pigment of liver microsomes: II. Solubilization and purification properties. Journal of Biological Chemistry 239: 2379-2385, 1964. Pak, R. C. K. , Tsim, K . W. K. , and Cheng, C. H. K. , Pubertal gonadal hormones in modulating the testosterone dependency of hepatic aryl hydrocarbon hydroxylase in female rats. Pharmacology 29: 121-127,1984. Pampori, N . A . , Agrawal, A . K. , Waxman, D. J., and Shapiro, B . FL, 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: 1299-1309, 1991. Pampori, N . A . , and Shapiro, B. H. , Sexual dimorphism in avian hepatic monooxygenases. Biochemical Pharmacology 46: 885-890,1993. Pampori, N . A . , and Shapiro, B. H. , Over-expression of C Y P 2C11, the major male-specific form of hepatic cytochrome P450, in the presence of nominal pulses of circulating growth hormone in adult male rats neonatally exposed to low levels of 135 monosodium glutamate. Journal of Pharmacology and Experimental Therapeutics 271: 1067-1073, 1994. Pinot, F., Grant, D. F., Spearons, J. L. , Parker, A . G., and Hammock, B . D., Differential regulation of soluble epoxide hydrolase by clofibrate and sexual hormones in the liver and kidneys of mice. Biochemical Pharmacology 50: 501-508,1995. Reyes, E. F., and Virgo, B. B. , Neonatal programming of ethylmorphine demethylase and corticosteroid 5oc-reductase by testosterone, dihydrotestosterone, and estradiol: Effects of and anti-estrogen, and anti-androgen, and an inhibitor of estrogen synthesis. Drug Metabolism and Disposition 16: 93-97, 1988. Ribeiro, V . , and Lechner, M . C , Cloning and characterization of a novel CYP3A1 allelic variant: analysis of CYP3A1 and CYP3A2 sex-hormone-dependent expression reveals that the CYP3 A2 gene is regulated by testosterone. Archives of Biochemistry and Biophysics 293: 147-152,1992. Ryan, D. E., and Levin, W., Purification and characterization of hepatic microsomal cytochrome P-450. Pharmacology and Therapeutics AS: 153-239,1990. Ryan, D. E., and Levin, W., Age- and gender-related expression of rat liver cytochrome P450. Ch. 30 in: Cytochrome P450. Handbook of experimental pharmacology v. 105, Schenkman, J., and Greim, H. , eds. Springer-Verlag, Berlin., 1993. Salmela, P. I., Associations between liver histological changes and hepatic monooxygenase activities in vitro in diabetic patients. Hormone and Metabolic Research 16: 7-10, 1984. Shah, V . P., Midha, K . K. , Dighe, S., McGilveray, I. J., Skelly, J. P., Yacobi, A . , Layloff, T., Viswanathan, C. T., Cook, C. E., McDowall, R. D., Pittman, K. A. , and Spector, S., Analytical methods validation: bioavailability, bioequivalence, and pharmacokinetic studies. Journal of Pharmaceutical Sciences 81: 309-312,1992. Shapiro, B . H. , Agrawal, A . K. , and Pampori, N . A . , Gender differences in drug metaolism regulated by growth hormone. International Journal of Biochemistry and Cell Biology 21: 9-20, 1995. Shapiro, B . H. , MacLeod, J. N . , Pampori, N . A. , Morrissey, J. J., Lapenson, D. P., and Waxman, D. )., Signalling elements in the ultradian rhythm of circulating growth hormone regulating expression of sex-dependent forms of hepatic cytochrome P450. Endocrinology 125: 2935-2944, 1989. Shimada, M . , Murayama, N . , Yamauchi, K. , Yamazoe, Y. , and Kato, R., Suppression in the expression of a male-specific cytochrome P450, P450-male: Difference in the effect of chemical inducers on P450-male mRNA and protein in rat liver. Archives of Biochemistry and Biophysics 270: 578-587,1989. 136 137 Shimada, M . , Murayama, N . Yamazoe, Y. , Hashimoto, H. , Ishidawa, H. , and Kato, R., Age- and sex-related alterations of microsomal drug- and testosterone-oxidizing cytochrome P450 in Sprague-Dawley strain-derived dwarf rats. Journal of Pharmacology and Experimental Therapeutics 275: 972-977, 1995. Skett, P., Hormonal regulation and sex differences of xenobiotic metabolism. Ch. 3 in: Progress in Drug Metabolism v. 10, Bridges, J. W., Chasseaud, L . F., and Gibson, G. G., eds. Taylor & Francis, Ltd. pp. 85-133, 1987. Sonderfan, 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: 27-41,1987. Strofkamp, D., Roos, P. H. , and Hanstein, W. G., A novel CYP3 gene from female rats. Biochemica et Biophysica Acta 1260: 341-344, 1995. Sundeth, 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 261': 3907'-3914, 1992. Tannenbaum, G. S., and Martin, J. B. , Evidence for an endogenous ultradian rhythm governing growth hormone secretion in the rat. Endocrinology 98: 562-570, 1976. Taylor, P., Enberg, B. , and Mode, A. , Growth hormone (GH) regulation of cytochrome P450IIC12, insulin-like growth factor-1 (IGF-1), and G H receptor messenger R N A expression in primary rat hepatocytes: A hormonal interplay with insulin, IGF-1, and thyroid hormone. Molecular Endocrinology 4: 1934-1942,1990. Thomas, P. E., Reik, L . M . , Ryan, D. E., and Levin, W., Regulation of three forms of cytochrome P-450 and epoxide hydrolase in rat liver microsomes. Effects of age, sex, and induction. Journal of Biological Chemistry 256: 1044-1052, 1981. Thomas, P. E., Reik, L . M . , Ryan, D. E., and Levin, W., Induction of two immunochemically related rat liver cytochrome P450 isozymes, P450c and P450d, by structurally diverse xenobiotics. Journal of Biological Chemistry 258: 4590-4598, 1983. Towbin, H. , Stachelin, T., and Gordon, J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: proceedure and some applications. Proceedings of the National Academy of Sciences USA 76: 4350-4354, 1979. Ulloa-Aguirra, A . , Blizzard, R. M . , Garcia-Rubi, E., Rogol, A . D., Link, K. , Christie, C. M . , Johnson, M . L. , and Veldhuis, J. D., Testosterone and oxandrolone, a nonaromatizable androgen, specifically amplify the mass and rate of growth hormone (GH) secreted per burst without altering G H secretory burst duration or frequency or the GHhalf-life. Journal of Clinical Endocrinology and Metabolism 71: 846-854, 1990. 138 Virgo, B . B. , The effect of peripubertal testosterone an ethylmorphine demethylase activity in adult rats testectomized neonatally. Canadian Journal of Physiology and Pharmacology 69: 459-463,1991. Waskiewicz, M . J., Choudhuri, S., Vanderbeck, S. M . , Zhang, X. -J . , and Thomas, P. E., Induction of "male-specific" cytochrome P450 isozymes in female rats by oxandrolone. Drug Metabolism and Disposition 23: 1291-1296, 1995. Waxman, D. J., and Chang, T. K . H. , Hormonal regulation of liver cytochrome P450 enzymes. Ch. 11 in: Cytochrome P450: Structure, Mechanism, and Biochemistry, 2nd ed. Ortiz de Montellano, P. R., ed. Plenum Press, New York. pp. 391-417, 1995. Waxman, D. J., Dannan, G. A. , and Guengerich, F. P., Regulation of rat hepatic cytochromeP-450. Age-dependent expression, hormonal imprinting, and xenobiotic inducibility of sex-specific isoenzymes. Biochemistry 24: 4409-4417, 1985. Waxman, D. J., Morrissey, J. J., and LeBlanc, 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: 2954-2966,1989. Waxman, D. J., Morrissey, J. J., MacLeod, J. N . , 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 5a-reductase. Endocrinology 126: 712-720,1990. Waxman, D. J., Pampori, N . A. , Ram, P. A. , Agrawal, A . K. , and Shapiro, B . H. , Interpulse interval in circulating growth hormone patterns regulates sexually dimorphic expression of hepatic cytochrome P450. Proceedings of the National Academy of Sciences, USA 88: 6868-6872, 1991. Waxman, D. J., Ram, P. A. , Pampori, N . A . , and Shaprio, B. H , Growth hormone regulation of male-specific rat liver P450s 2A2 and 3A2: Induction by intermittent growth hormone pulses in male but not female rats rendered growth hormone deficient by neonatal monosodium glutamate. Molecular Pharmacology 48: 790-797, 1995. Wehrenberg, W. B., Brazeau, P., Luben, R., Bohlen, P., and Guillemin, R., Inhibition of the pulsatile secretion of growth hormone by monoclonal antibodies to the hypothalamic growth hormone releasing factor (GRF). Endocrinology 111: 2147-2148, 1982. Wehrenberg, W., and Giustina, A. , Basic counterpoint: mechanisms and pathways of gonadal steroid modulation of growth hormone secretion. Endocrine Reviews 13: 299-308, 1986. Weisz, 1, and Ward, I. L. , Plasma testosterone and progesterone titers of pregnant rats, their male and female fetuses, and neonatal offspring. Endocrinology 106: 306-316, 1980. 139 Wells, T., Mode, A . , Floby, E., and Robinson, I. C. A . F., The sensitivity of hepatic CYP2C gene expression to baseline growth hormone (GH) bioactivity in dwarf rats: Effects of GH-binding protein in vivo. Endocrinology 134: 2135-2141, 1994. Winer, L . M . , Shaw, M . A. , and Baumann, G., Basal plasma growth hormone levels in man: New evidence for rhythmicity of growth hormone secretion. Journal of Clinical Endocrinology and Metabolism 70: 1678-1686,1990. Zachmann, M . , Interratlations between growth hormone and sex hormones: Physiology and therapeutic consequences. Hormone Research 38(suppl. 1): 1-8, 1992. Zeitler, P., Argente, J., Chowen-Breed, J. A. , Clifton, D. K. , Steiner, R. A . , Growth hormone-releasing hormone messenger ribonucleic acid in the hypothalamus of the adult more rat is increased by testosterone. Endocrinology 127: 1362-1368, 1990. 140 6. APPENDIX Table I. Final liver weights, body weights, and liver weights corrected for differences in body weight on the day of sacrifice. Treatment Age Sex (days 35-49) (days) Final Body Weight (g) Liver Weight (g) Liver Weight as % of Body Weight Female (n=7) Female (n=7) Female (n=7) Female (n=8) Female (n=7) Female (n=7) Male (n=8) Corn Oil 90 TE 5 umol/kg 90 T 2.5 umol/kg 90 (2X) TE 5 umol/kg 129 TE 5 umol/kg 169 No treatment 90 331 ± 8 320 ±11 T5umpl/kg 90 356 ± 1 0 334 ± 5 364 ± 8 a 391 ± 9 a 477 ± 1 3 c 9.5 ±0 .3 10.0 ±0 .5 10.5 ±0 .5 9.9 ±0 .3 9.1 ±0 .4 9.1 ±0 .4 16.6±0.8 C 2.9 ±0 .1 3.1 ±0 .1 3.0 ±0 .1 3.0 ±0 .1 2.5 ± 0 . 1 ' 2.3 ± o.r 3 .5±0 .1 C Each value is the mean ± standard error of the mean. A l l female rats were gonadectomized at 25 days of age. 141 Table II. Mean Cytochrome P450 concentrations, protein concentrations, and specific content values of the microsome preparations. Sex Treatment (days 35-39) Age (days) Cytochrome P450 concentration (nmol/ml) Protein concentration (mg/ml) Specific Content (nmol/mg) Female (n=7)a Corn Oil 90 35.60 ±2.22 30.66 ± 1.44 1.20 ±0.04 Female (n=7) TE 5 umol/kg 90 35.57 ±2.30 28.11 ±2.19 1.28±0.04 b Female (n=7) T 5 umol/kg 90 29.45 ± 2.59 26.24 ± 2.00 1.12 ±0.04 Female (n=8) T 2.5 umol/kg (2X) 90 29.48 ± 1.90 28.56 ±2.65 1.06±0.06 b c Female (n=7) TE 5 umol/kg 129 35.85 ±2.82 30.04 ±2.28 1.19 ±0.04 Female (n=7) TE 5 umol/kg 169 24.93 ± 2.22 19.50 ± 1.33 1.27 ±0.06° Male (n=8) No treatment 90 50.26 ± 1.69 34.47 ± 0.69 1.46±0.05 d Female rats were gonadectomized at 25 days of age. Each value is the mean + SEM. 142 Table III. Effect of testosterone and testosterone enanthate treatment during the peripubertal period on the formation of 16p-hydroxytestosterone and androstenedione. Treatment Group Age (days) 16p~hydroxytestosterone Androstenedione Female Control 90 0.105 ± 0 . 0 1 6 0.335 ± 0 . 0 4 9 T 2.5 umol/kg daily 90 0.066 ± 0.005 0.306 ± 0 . 0 5 4 T 2.5 umol/kg 2X daily 90 0.107 + 0.026 0.335 ± 0.052 T E 5 umol/kg daily 90 0.081 ± 0 . 0 1 1 0.400 ± 0.066 T E 5 umol/kg daily 129 0.087 ± 0 . 0 1 2 0.341 ± 0 . 0 4 0 T E 5 umol/kg daily 169 0.094 ± 0 . 0 1 0 0.505 ± 0.089 Male Control 90 0.063 ± 0 . 0 1 0 1.039 ± 0 . 0 9 1 Female rats were gonadectomized at 25 days of age. Activity is expressed as nmol metabolite formed/min/nmol total C Y P . Each value is the mean ± S E M . 

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.831.1-0087781/manifest

Comment

Related Items