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The influence of peripubertal testosterone on hepatic microsomal erythromycin demethylase in prepubertally… Cadario, Barbara Jane 1989

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THE INFLUENCE OF PERIPUBERTAL TESTOSTERONE ON HEPATIC MICROSOMAL ERYTHROMYCIN DEMETHYLASE IN PREPUBERTALLY OVARIECTOMIZED FEMALE RATS by BARBARA JANE CADARIO B.Sc.(Hon), U n i v e r s i t y of T o r o n t o , 1975 B.Sc. Phm., U n i v e r s i t y of T o r o n t o , 1981 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES ( D i v i s i o n of Pharmacology and T o x i c o l o g y of the F a c u l t y of P h a r m a c e u t i c a l S c i e n c e s ) We ac c e p t t h i s t h e s i s as c o n f o r m i n g t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA November 1989 © BARBARA JANE CADARIO, 1989 \ 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 PHARMACEUTICAL SCIENCES The University of British Columbia Vancouver, Canada Date DECEMBER 23, 1989 DE-6 (2/88) ABSTRACT The i n f l u e n c e of p e r i p u b e r t a l exposure to p h y s i o l o g i c a l l e v e l s of testosterone on the a d u l t androgen responsiveness of the cytochrome P450 enzyme a c t i v i t y , hepatic microsomal erythromycin demethylase a c t i v i t y , was i n v e s t i g a t e d . Rats were i n j e c t e d subcutaneously with testosterone enanthate 5 Mmoles/kg/day e i t h e r p e r i p u b e r t a l l y , during adulthood or i n both time periods. In adult untreated r a t s , hepatic microsomal erythromycin demethylase a c t i v i t y was higher i n males than i n females. I n t a c t adult male r a t s , but not i n t a c t adult female r a t s , responded to adult testosterone treatment with an increase i n hepatic microsomal erythromycin demethylase a c t i v i t y . Female and male r a t s were gonadectomized before the onset of puberty. In the adult female r a t s which had been p r e p u b e r t a l l y ovariectomized, exposure to testosterone p e r i p u b e r t a l l y r e s u l t e d i n an a d u l t androgen responsiveness for hepatic microsomal erythromycin demethylase a c t i v i t y . This i n d i c a t e d that the p o t e n t i a l i s present i n the p r e p u b e r t a l l y ovariectomized female rat f o r the pubertal i m p r i n t i n g by testosterone of an a d u l t androgen responsiveness for hepatic microsomal erythromycin demethylase a c t i v i t y . ii Prepubertal c a s t r a t i o n of male r a t s reduced hepatic microsomal erythromycin demethylase a c t i v i t y and plasma testosterone l e v e l s from c o n t r o l l e v e l s . Hepatic microsomal erythromycin demethylase a c t i v i t y was found to be p a r t i a l l y c o r r e l a t e d with plasma testosterone l e v e l s . The higher hepatic microsomal erythromycin demethylase a c t i v i t y i n the adult male rat may t h e r e f o r e be r e l a t e d to high adult male l e v e l s of c i r c u l a t i n g t e s t o s t e r o n e . The a d m i n i s t r a t i o n of testosterone to adult male r a t s which had been p r e p u b e r t a l l y c a s t r a t e d r e s u l t e d i n hepatic microsomal erythromycin demethylase a c t i v i t y which was lower than that of i n t a c t males and of i n t a c t males t r e a t e d with testosterone i n adulthood. These r e s u l t s i n d i c a t e d that a d u l t androgen responsiveness of hepatic microsomal erythromycin demethylase a c t i v i t y i s not completely imprinted i n male r a t s i n the neonatal p e r i o d . This study provided evidence i n support of the hypothesis that the p e r i p u b e r t a l p e r i o d i s a time during which i m p r i n t i n g by testosterone of a d u l t androgen responsiveness of hepatic P450 enzymes can occur. TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES v i i LIST OF FIGURES v i i i LIST OF ABBREVIATIONS X ACKNOWLEDGEMENTS x i 1. INTRODUCTION 1 1.1. Theory of Endocrine C o n t r o l of Hepatic Microsomal Cytochrome P450 2 1.1.1. Gustafsson's Theory 2 1.1.2. The E v o l u t i o n of Gustafsson's Theory 5 1.1.2.1. M u l t i p l e Hepatic Cytochrome P450 Enzymes 5 1.1.2.2. Sexual D i f f e r e n t i a t i o n of the C e n t r a l Nervous System 14 1.1.2.3. The Hypothalamo-P i t u i t a r y - L i v e r A x i s . 16 1.1.2.4. Androgenic C o n t r o l of Growth Hormone Secretion Patterns .... 18 1.1.2.5. Current Theory 19 1.1.2.6. Unanswered Questions and A l t e r n a t i v e Hypotheses 22 1.2. Evidence of Imprinting during Puberty of P450 Adult Androgen Responsiveness 26 1.2.1. Neonatal Imprinting Is Not Always Required 26 1.2.1.1. Benzo[aJpyrene Hydroxylase . 26 1.2.2. Which Cytochrome P450s? 29 1.2.2.1. P450g 30 1.2.2.2. P450h 30 1.2.2.3. P450 I I I Family 33 1.2.2.4. P450 RLM2 40 1.3. Proposed Research 40 1.3.1. I n i t i a l Focus on P450 I I I 40 1.3.2. Animal Model 41 1.3.3. Treatment P r o t o c o l 43 1.3.4. S p e c i f i c Hypothesis 43 2. MATERIALS AND METHODS 45 2.1. Chemicals 45 2.2. Animals 45 i v 2.3. Treatments 46 2.4. Microsome Preparation 47 2.5. Erythromycin Demethylase A c t i v i t y 48 2.6. Protein — 49 2.7. Total Cytochrome P450 50 2.8. Plasma Preparation 50 2.9. Plasma Testosterone 51 2.10. Plasma E s t r a d i o l 51 2.11. S t a t i s t i c a l Analysis 51 2.12. Other Analysis 52 3. RESULTS 53 3.1. ERYTHROMYCIN DEMETHYLASE ASSAY CONDITIONS ... 53 3.1.1. Protein concentration 53 3.1.2. Incubation Time 54 3.1.3. NADPH 54 3.1.4. Substrate Concentration 55 3.1.5. Phosphate Buffer 59 3 .1 .6. Formaldehyde Standard Curve 59 3.1.7. F i n a l conditions 60 3.2. ERYTHROMYCIN DEMETHYLASE STABILITY 100 3.3. THE PILOT STUDY 103 3.3.1. Pubertal Testosterone Treatment in Ovariectomized Females 103 3.3.1.1. Hepatic Microsomal Erythromyc in Demethylase A c t i v i t y . 103 3.3.1.2. Cytochrome P450 and Protein 104 3.3.2. A l l Treatment Groups 104 3.3.2.1. Hepatic Microsomal Erythromycin Demethylase A c t i v i t y . 104 3.3.2.2. Liver Cytochrome P450 and Liver Protein .... 106 3.3.2.3. Body and Liver Weights . 106 3.4. THE MAJOR STUDY 119 3.4.1. Prepubertally Ovariectomized Female Rats 119 3.4.1.1. Hepatic Microsomal Erythromyc in Demethylase A c t i v i t y . 119 3.4.1.2. Cytochrome P450 and Protein 120 3.4.1.3. Body and Liver Weights . 121 3.4.1.4. Plasma E s t r a d i o l and Testosterone 121 3.4.2. A l l Treatment Groups 122 3.4.2.1. Erythromycin Demethylase A c t i v i t y 122 v 3.4.2.2. Microsomal P450, Liver P450 and Protein 126 3.4.2.3. Body and Liver Weights . 127 3.4.2.4. Plasma E s t r a d i o l 127 3.4.2.5. Plasma Testosterone .... 128 3.4.2.6. Erythromycin Demethylase A c t i v i t y versus Plasma Testosterone 130 4. DISCUSSION 156 4.1. Study Results 156 4.1.1. Non-gonadectomized Animals 156 4.1.2. Effe c t of Prepubertal Gonadectomy . 157 4.1.3. Peripubertal Testosterone 159 4.1.4. Correlation with Plasma Testosterone .. 162 4.1.5. Comparison with BPH Studies 163 4.1.6. Relation to Gustafsson's Theory ... 166 4.2. Potential Study Limitations 168 4.2.1. Erythromycin Demethylase Assay .... 169 4.2.1.1. S p e c i f i c i t y 169 4.2.1.2. S e n s i t i v i t y and Detection Limits ..... 172 4.2.1.3. D e f i n i t i o n of Puberty .. 174 4.2.1.4. Testosterone Dose 175 4.2.1.5. Calculations of Erythromycin Demethylase A c t i v i t y . 176 4.3. Speculation on Relevance to Humans 179 4.4. Proposals for Future Research 182 4.5. Summary and Conclusions 184 5. REFERENCES 187 6. APPENDIX 202 vi list of tables Table I. Predominantly Female P450 Forms in Liver Microsomes 8 Table II. Predominantly Male P450 Forms in Liver Microsomes 10 Table III. Approximately Equal P450 Forms in Liver Microsomes 12 Table IV. P450III Forms in Rat Liver Microsomes 34 Table V . Estimated Michaelis-Menten constants in untreated male Sprague Dawley ratss for erythromycin demethylase in hepatic microsomes 82 Table V I . Estimated Michaelis-Menten constants for erythromycin demethylase activity in hepatic microsomes from untreated adult female Sprague Dawley rats 88 Table VII. Pilot study: The influence of pubertal testosterone on ovariectomized female rat liver hepatic microsomal erythromycin demethylase activity 108 Table VIII. Pilot study: hepatic microsomal erythromycin demethylase activity in all treatment groups 113 Table IX. Major Study Results in Prepubertally Ovariectomized Female Sprague Dawley Rats 131 Table X . Results of Major Study, all treatment groups 139 Table X I . Major study: statistical analysis for erythromycin demethylase activity calculated per mg protein 141 Table XII.Major study: statistical analysis for erythromycin demethylase activity calculated per nmole P450 142 Table XIII. Major study: statistical analysis for body weights 143 Table X I V . Major study: statistical analysis for liver weights 144 Table X V . Major study: statistical analysis for plasma testosterone concentrations. 153 vii list of figures Figure 1. Gustafsson's Theory .• 24 Figure 2. Erythromycin demethylase assay: absorbance versus protein concentration in untreated male Sprague Dawley rat hepatic microsomes. 62 Figure 3. Erythromycin demethylase assay: absorbance versus final protein concentration in untreated female Sprague Dawley rat hepatic microsomes 64 Figure 4. Erythromycin demethylase assay: absorbance versus incubation time in control male Sprague Dawley rat hepatic microsomes 66 Figure 5. Erythromycin demethylase assay: absorbance versus incbation time in untreated female Sprague Dawley rat hepatic microsomes 68 Figure 6. Erythromycin demethylase assay: absorbance versus incubation time, showing the effect of additional N A D P H in untreated male Sprague Dawley rat hepatic microsomes 70 Figure 7. Erythromycin demethylase assay: absorbance versus incubation time, showing the effect of additional N A D P H in untreated female Sprague Dawley rat hepatic microsomes 72 Figure 8. Erythromycin demethylase activity versus Final N A D P H concentration in untreated male Sprague Dawley rat hepatic microsomes. 74 Figure 9. Erythromycin demethylase activity versus final N A D P H concentration in untreated female Sprague Dawley rat hepatic microsomes. 76 Figure 10. Erythromycin demethylase activity versus substrate concentration in untreated adult male Sprague Dawley rat hepatic microsomes 78 Figure 11. Erythromycin demethylase activity versus substrate concentration in untreated adult female Sprague Dawley rat hepatic microsomes 80 Figure 12. Eadie Hofstee plot for erythromycin demethylase activity in untreated adult female Sprague Dawley rat hepatic microsomes 84 Figure 13, Lineweaver-Burk plot for erythromycin demethylase activity in untreated adult female Sprague Dawley rat hepatic microsomes. 86 Figure 14. Eadie Hofstee plot for erythromycin demethylase activity in hepatic microsomes from untreated adult male Sprague Dawley rats 90 viii Figure 15. Lineweaver-Burk plot for erythromycin demethylase activity in hepatic microsomes from untreated adult male Sprague Dawley rats 92 Figure 16. Erythromycin demethylase activity with varied potassium phosphate buffers .94 Figure 17. Formaldehyde standard curve in untreated male Sprague Dawley rat liver microsomes 96 Figure 18. Formaldehyde standard curve in untreated female Sprague Dawley rat liver microsomes 98 Figure 19. Erythromycin demethylase activity: stability in untreated adult female Sprague Dawley rat hepatic microsomes 101 Figure 20. Pilot study: erythromycin demethylase calculated on the basis of microsomal protein in ovariectomized female rats 109 Figure 21. Pilot study: erythromycin demethylase calculated on the basis of cytochrome P450 content in ovariectomized females I l l Figure 22. Pilot study: Hepatic microsomal erythromycin demethylase calculated on the basis of microsomal protein in all groups 115 Figure 23. Pilot study: hepatic microsomal erythromycin demethylase activity calculated on the basis of P450 content in all groups 117 Figure 24. Major Study: erythromycin demethylase activity calculated per protein in prepubertally ovariectomized female Sprague Dawley rats 133 Figure 25. Major study: erythromycin demethylase per P450 in prepubertally ovariectomized female Sprague Dawley rats 135 Figure 26. Comparison of erythromycin demethylase activity in the pilot study and major study in prepubertally ovariectomized female rats 137 Figure 27.Major study: erythromycin demethylase activity calculated per protein for all treatment groups 145 Figure 28. Major study: erythromycin demethylase activity calculated per P450 in all treatment groups 147 Figure 29. Major study: plasma estradiol levels 149 Figure 30. Major study: plasma testosterone concentrations 151 Figure 31. Erythromycin demethylase activity versus plasma testosterone 154 ix ANOVA BPH Dex EDTA HPLC K 3-MC Na NADPH Pb PCN LIST OF ABBREVIATIONS A n a l y s i s of variance Benzo[a]pyrene hydroxylase Dexamethasone Ethylenediamine-t e t r a a c e t i c a c i d High performance (pressure) l i q u i d chromatography Potassium 3-methylcholanthrene Sodium /3-nicot inamide adenine-dinucleotide phosphate, reduced Phenobarbital Pregnenolone-1 6 a - c a r b o n i t r i l e ACKNOWLEDGEMENTS This t h e s i s i s dedicated to Paul Warwick, who endured so much, and survive d . I wish to thank Dr. G a i l Bellward f o r her wise counsel and inv a l u a b l e support i n a l l phases of t h i s challenge. I am indebted to E d i t h Lemieux for her constant help i n many areas r e l a t e d to t h i s study. I a l s o acknowledge Bruce A l l e n , Roland Burton and Tom Chang for t h e i r constant encouragement. xi 1. INTRODUCTION The term cytochrome P450 refers to a . c o l l e c t i o n of hemoprotein enzymes which, when reduced, form a complex with carbon monoxide with absorption peaks at approximately 450 nm (Omura & Sato, 1962); the "P" stands for pigment (Garfinkel, 1958; Klingenberg, 1958). These enzymes f are of s c i e n t i f i c interest due to their a b i l i t y to catalyze the biotransformation of both endogenous compounds, notably steroids, fatty acids and prostaglandins, as well as xenobiotics, including therapeutically-active drugs and po t e n t i a l l y toxic chemicals. Although our knowledge of the cytochrome P450 enzymes in humans is rapidly expanding, the majority of studies in t h i s area have used the rat model. Cytochrome P450 is ubiquitous, the highest concentration being found in l i v e r c e l l endosplasmic reticulum (Guengerich, 1987). Therefore most studies, including the one presented here, investigate cytochrome P450 in microsomes prepared from rat l i v e r . It i s currently known that the rat hepatic P450 family contains at least 20 d i f f e r e n t enzymes (Nebert et a l . , 1987; Guengerich, 1987). The degree to which each of these P450 enzymes i s expressed varies between individual animals, tThe term isozyme is not used, since these enzymes catalyze different reactions. 1 INTRODUCTION / 2 depending on genetic predisposition, for example a genetic polymorphism; on environmental factors, such as exposure to inducing chemicals; and on physiological factors such as sex, age and disease (Gonzalez, 1989). Research is currently directed at discovering the factors and mechanisms involved in the regulation of the expression of the d i f f e r e n t P450 enzymes. The observed sexual and developmental variations suggest that for certain P450s, in p a r t i c u l a r those which are present in untreated rats, regulation may involve hormones. This study investigates, in simplest terms, the relationship between exposure to androgenic hormone during a discrete developmental period and a c a t a l y t i c a c t i v i t y representative of P450 enzyme expression. As background, i t w i l l f i r s t be necessary to review b r i e f l y the th e o r e t i c a l framework within which such research has been conducted in the past. 1.1. THEORY OF ENDOCRINE CONTROL OF HEPATIC MICROSOMAL CYTOCHROME P450 1.1.1. Gustafsson's Theory It has been known for some time that the rat l i v e r i s a sexually dimorphic organ (Roy & Chatterjee, 1983). Sexual differences exist in l i v e r p r o l a c t i n receptor lev e l s (Kelly INTRODUCTION / 3 et al, 1974), in the hepatic synthesis of urinary male-specific a2-microglobul in (Roy et al., 1983), and in l i v e r enzymes involved in the metabolism of drugs and steroids (Yates et al, 1958). That hormones are involved in the control of hepatic steroid metabolism has been evident for at least 30 years. It was observed that neonatal male castration could . remove sex differences in steroid metabolism whereas androgen treatment returned the a c t i v i t y to the normal male l e v e l (Yates et al, 1958; Denef & DeMoor, 1968a, 1968b, 1972). The timing of these manipulations was also important; C o r t i s o l metabolism in male rats was "feminized" by neonatal gonadectomy but r e l a t i v e l y unaffected by postpubertal castration (Denef & DeMoor, 1968a, 1968b, 1972). Einarsson, Gustafsson and Stenberg (1973) extended this work and formulated a theory r e l a t i n g sex hormones and hepatic microsomal metabolism. Based on their studies of the e f f e c t s of castration or testosterone treatment at various developmental times (Berg & Gustafsson, 1973; Einarsson et al., 1 973), they postulated that microsomal enzymes could be grouped into at least three categories (Figure 1A, page 25). Group I enzymes, represented by 2a-hydroxylation of 4-pregnene-3,20-dione and 5a-androstane-3a, 17j3~diol, were en t i r e l y dependent on androgens for i r r e v e r s i b l e "imprinting" or "programming" prepubertally and reversible stimulation by androgens INTRODUCTION / 4 postpubertally. Group II, exemplified by 6/3-hydroxylation of 4-androstene-3,17-dione and 4-pregnene-3,20-diol, were only p a r t i a l l y dependent on androgen, having a basal l e v e l of a c t i v i t y determined by nongonadal factors, as seen in females, but a reversible i n d u c i b i l i t y by androgens. The d i s t i n c t i o n between Groups I and I I thus lay in the presence of Group I I enzyme a c t i v i t y in females, and could be shown by the e f f e c t s of neonatal castration of 'the male, which abolished Group I levels but did not abolish a Group I I basal l e v e l . Group I I I enzymes, for example 7a-hydroxylation of 4-androstene-3,17-dione and c h o l e s t e r o l , were not dependent on androgens, being unaffected by gonadectomies and testosterone treatment. Two important concepts were thus introduced. "Adult androgen responsiveness" was described as a reversible stimulation by androgens in the adult period. The word "imprinting" derived from i t s use by Konrad Lorenz (1971) within a behavioral context. As described by Lorenz (1971), "imprinting" involved an i r r e v e r s i b l e process which was limited to a "circumscribed ontogenetic phase". It has since been applied to other b i o l o g i c a l phenomena (Skett, 1979). Gustafsson applied "imprinting" to hepatic steroid metabolism as an effect of neonatal androgen on enzyme l e v e l and androgen responsiveness of the enzyme in the adult period (Gustafsson INTRODUCTION / 5 & Stenberg, 1973; Gustafsson et al, 1974a, 1974b). Therefore, according to the o r i g i n a l theory, adult androgen responsiveness in Group I and II enzymes was imprinted neonatally, and in Group I a basal l e v e l was also imprinted neonatally by androgens (Gustafsson et al, 1983). Gustafsson and coworkers observed that the 2a-hydroxylation of 5a-androstene-3a-17/3-diol and the 6/3-hydroxylat ion of 4-androstene-3,17-dione responded to adult testosterone in male rats castrated at 14 days of age, but not in neonatally castrated males (Gustafsson & Stenberg, 1974b), indicating imprinting of adult androgen responsivity within the f i r s t 14 days of l i f e . 1.1.2. The Evolution of Gustafsson's Theory Since 1973, our understanding of the endocrine control of hepatic steroid metabolism has benefited from several directions of research. 1.1.2.1. Multiple Hepatic Cytochrome P450 Enzymes It should be realized that when Gustafsson's theory was f i r s t proposed in 1973, i t was not widely accepted that hepatic P450 was composed of more than one enzyme. Indeed, at that time the idea that "the commonly-used term cytochrome P450 may actually embrace a group of cytochromes INTRODUCTION / 6 with i d e n t i c a l hemes bound to d i f f e r e n t proteins" was a matter of controversy (Einarsson et al., 1 973). It i s now clear that rat hepatic P450 consists of multiple enzymes (Gonzalez, 1989), and there has been a great deal of research on the expression of s p e c i f i c P450 enzymes in untreated male and female rat s . Tables I, II and III summarize current information regarding the levels of P450 enzymes in hepatic microsomes from untreated rats, and their responses to hormonal manipulation. Only those forms shown to be d i s t i n c t enzymes on the basis of t h e i r - spectral a c t i v i t y , electrophoretic mobility, substrate s p e c i f i c i t y and amino-terminal sequences, and p u r i f i e d to electrophoretic homogeneity, are presented. For other forms detected in rat l i v e r microsomes (for reviews see Guengerich, 1987; Gonzalez, 1989), only minimal information is a vailable on influence by hormonal factors. For s i m p l i c i t y , the nomenclature of Ryan & Levin (Ryan et al., 1979, 1980, 1982, 1984, 1985) w i l l be used for the majority of P450 forms, however in some cases nomenclature based on P450 gene families (Nebert et al., 1987, 1989) w i l l be used. Several generalizations are apparent from these studies of s p e c i f i c P450 enzymes in untreated adult rat l i v e r (Tables I - I I I ) . F i r s t l y , rat hepatic microsomal P450 enzymes can be divided into those detected only in females or at greater INTRODUCTION / 7 levels in females than in males (P450a, P450f, P450i), those detected only in males or at greater lev e l s in adult males than females (P450g, P450h, RLM2 and the P450 III family), and those present at approximately equivalent levels (defined here as a less than two-fold difference) in untreated adults of both sexes (P450b, P450c, P450d, P450e, P450j, db1 and P450k). Secondly, the sexually d i f f e r e n t i a t e d P450 enzymes do not appear to be coordinately regulated, as indicated by their varied responses to gonadectomy, hypophysectomy and hormonal treatment. Rather, each sex-specific P450 appears to be regulated in a unique manner, and influenced by hormones. Thirdly, testosterone i s oxidized in a regio- and stereo-selective manner which i s ch a r a c t e r i s t i c of certain P450 forms. And fourthly, developmental changes are observed in the expression of many of the P450 enzymes, with many of the sex differences becoming manifest around puberty. The present state of s c i e n t i f i c investigation involves further work to id e n t i f y the c r i t i c a l factors, in pa r t i c u l a r hormones, involved in the regulation of each s p e c i f i c P450, and to delineate the c r i t i c a l developmental time periods during which these factors exert their e f f e c t s . INTRODUCTION / 8 Table I. Predominantly Female P450 Forms in Liver Microsomes from Adult Rats. Animals were untreated, except as indicated. Values indicate immunoquantitated leve l s of protein, which may vary since d i f f e r e n t methods were used in di f f e r e n t studies. Abbreviations: NC, no change; ty, increase; decrease; Gx, gonadectomy; Hx, hypophysectomy; neo, neonatal; ND, not detectable; PR, p a r t i a l l y restored to pre-surgical adult l e v e l ; FR, f u l l y restored to pre-surgical adult l e v e l ; Pb, phenobarbital; 3-MC, 3-methylcholanthrene; /3NF, /3naphthof lavone; Dex, dexamethasone; PCN, pregnenolone-16a-carbonitrile; Neg, ne g l i g i b l e ; T,testosterone. References: P450a: Jacobson & Kuntzman, 1969; Thomas et al., 1981; Parkinson et al., 1983; Waxman et al., 1 985; Dannan et al, 1986; Nagata et al., 1 987; Ar l o t t o & Parkinson, 1989; Arlot t o et al., 1989. P450f: Ryan et al., 1984; Bandiera et al., 1986. P450i: Kamataki et al., 1983; MacGeoch et al., 1984; Ryan et al., 1984; Kamataki et al., 1985; Waxman et al., 1985. INTRODUCTION / 9 P450a P450f P450i % of Total P450: Male: 3% 7% ND Female: 6% 1 4% 45% Testosterone Hydroxylation Sites 6a,7 a 1 6a -Developmental Changes: A week 1:male: V female: / -A at puberty:male: y/ female: / y/ $ at puberty:male: \J - \f female: -Hormonal E f f e c t s : Gx n a„:male: NC A neo T female: V50% - V67% Gx n e o+T:male: - - ND female: -G x l a t e r : m a l e : ~ " N C female: smallV - NC/V G x l a t e r + T : m a l e : " " female: - - ND H x a d u l t : m a l e : " " N D female: - - ND Thyroid Hormones: V -Inducers:Pb: / NC NC 3-MC/0NF: / s m a l l i NC Dex/PCN: - smallv A INTRODUCTION / 10 Table I I . Predominantly Male P450 Forms in Liver Microsomes from Adult Rats. Animals were untreated, except as indicated. Values indicate immunoquantitated levels of protein, which may vary since d i f f e r e n t methods were used in di f f e r e n t studies. Abbreviations: NC, no change; ^, increase; ^ f decrease; Gx, gonadectomy; Hx, hypophysectomy; neo, neonatal; ND, not detectable; PR, p a r t i a l l y restored to pre-surgical adult l e v e l ; FR, f u l l y restored to pre-surgical adult l e v e l ; Pb, phenobarbital; 3-MC, 3-methylcholanthrene; j3NF, j3naphthof lavone; Dex, dexamethasone; PCN, pregnenolone-16a-carbonitrile; Neg, ne g l i g i b l e ; T, testosterone; @, PCN2; f, PCN1; *, 2a/PCN-E; #, Pb-1. References: P450g: Cheng & Schenkman, 1982;. 1983; Ryan et al, .1984; Jansson et al., 1985c; Bandiera et al, 1985; Bandiera et al., 1 986; McClellan-Green et al., 1989. P450h: Cheng & Schenkman, 1982; 1983; Guengerich et al, 1982; Kato & Kamataki, 1982; Kamataki et al, 1983; Kamataki et al., 1984; Ryan et al., 1984; Waxman et al., 1984; Kamataki et al., 1985; Morgan et ah, 1985; Waxman et al., 1985; C r e s t e i l et al., 1986; Dannan et al, 1986; Kato et al, 1986; Shimada et al., 1987; Mode et al., 1988; Shimada et al, 1988. P450III: Gozukara et al, 1984; Jansson et al, 1985c; Waxman et al., 1985; C r e s t e i l et al, 1986; Dannan et al, 1986; Gonzalez et al., 1986; Waxman et al, 1986; Imaoka et al, 1988; Yamazoe et al, 1988. P450 RLM2: Jansson et al, 1985a; Jansson et al., 1985b; Jansson et al., 1 985c; Waxman et al., 1986. INTRODUCTION / 1 1 P450g P450h P450RLM2 P450III % of Total P450: Male: Female: <1%/>10% ND 33% ND ND 25%# <2%# Testosterone Hydroxylation Sites 6/3, la, 1 5a 2a, 2/3, 6/3 16a,17a, 1 5-6/3,7a, 7/3, 1 5a 1 5/3 6/3#§ Developmental Changes: ^ in week 1:male: female: ^ at puberty:male: female: V at puberty:male: female: • v/ • • NC* Hormonal E f f e c t s : Gx„,:male: neo ND ND ND* female: Gx +T:male: neo PR PR -PR* female: G x l a t e r : m a l e : female: G x l a t e r + T : m a l e : female: H x a d u l t : m a l e : female: Thyroid Hormones: NC T t -V FR PR present NC T t NC* t t Inducers:Pb: 3-MC//3NF: Dex/PCN: NC small^ smallv V 1 1 @^*# NC@ f t INTRODUCTION / 12 Table I I I . Approximately Equal P450 Forms in Liver Microsomes in Adult Male and Female Rats. Animals were untreated, except as indicated. Values indicate immunoquantitated leve l s of protein, which may vary, since d i f f e r e n t methods were used in d i f f e r e n t studies. Abbreviations: NC, no change; ^, increase; V, decrease; Gx, gonadectomy; Hx, hypophysectomy; neo, neonatal; ND, not detectable; PR, p a r t i a l l y restored to pre-surgical adult l e v e l ; FR, f u l l y restored to pre-surgical adult l e v e l ; Pb, phenobarbital; 3-MC, 3-methylcholanthrene; 0NF, /Jnaphthof lavone; Dex, dexamethasone; PCN, pregnenolone-16a-carbonitrile; Neg, ne g l i g i b l e ; T, testosterone. References: P450 b+e: Ryan et al., 1979; Thomas et al., 1981; Parkinson et al., 1983, Waxman et al., 1983; Wood et al, 1983; Waxman et al, 1985. P450 c+d: Thomas et al., 1981; Parkinson et al, 1983; Waxman et al, 1985; G i a c h e l l i & Omiecinski, 1987. P450j: Song et al, 1986; Thomas et al., 1987; Williams & Simonet, 1988. P450k: Waxman & Walsh, 1983; Waxman et al., 1985; Gonzalez et al., 1986 P450 db1 : Al-dabbagh et al, 1981; Larrey et al., 1984; Gonzalez et al, 1 987 . INTRODUCTION / 13 P450b+e P450c+d P450J P450k P450db1 % of T o t a l P450: Male: Female: 1-2% 1-2% 1% 1% <8% 1 0% 6% 0.3/6% Testosterone Hydroxylation S i t e s 1 6a, 1 6/3 2/3,6/3 - 1 6a, 16/3 Neg Developmental Changes: A i n week 1:male: female: A at puberty:male: female: V at puberty:male: female: V / / / V • / / • Hormonal E f f e c t s : Gx„,:male: neo NC _ NC _ female: Gx +T:male: neo — NC NC — smallV NC — female: G x l a t e r : m a l e : female: G x l a t e r + T : m a l e : female: H x a d u l t : m a l e : female: Thyroid Hormones: -NC T ! NC — Inducers:Pb: 3-MC//3NF: Dex/PCN: 4* t V(male) f(male) t NC NC neg neg neg INTRODUCTION / 14 1.1.2.2. Sexual Differentiation of the Central Nervous System Recent years have a l s o brought an expansion of our knowledge concerning many s e x u a l l y d i f f e r e n t i a t e d phenomona, i n c l u d i n g behavior and gonadotropin s e c r e t i o n . Several important conclusions have been reached. As reviewed by MacLusky & N a f t o l i n (1981), the general hypothesis for sexual d i f f e r e n t i a t i o n of the CNS presumes that the CNS i s i n t r i n s i c a l l y organized for the homogametic sex, t h i s being females i n mammals. A change away from t h i s inherent p a t t e r n i n males re q u i r e s exposure to gonadal hormones (Go r s k i , 1971). In mammals, e a r l y gonadal s e c r e t i o n s appear to be important. I t i s known that the s e n s i t i v i t y of the CNS to the permanent o r g a n i z a t i o n a l e f f e c t s of hormones v a r i e s with age. For r a t s , a " c r i t i c a l p e r i o d " of CNS sexual d i f f e r e n t i a t i o n of reproductive f u n c t i o n s and sex behavior has been determined as approximately 18-27 days a f t e r conception, with b i r t h u s u a l l y o c c u r r i n g on days 20-22 (MacLusky & N a f t o l i n , 1981). E a r l y hormonal e f f e c t s on the CNS are u s u a l l y permanent, and d i s t i n c t from r e v e r s i b l e hormonal e f f e c t s observed in adulthood. In many animals the adul t s e x - r e l a t e d reproductive behavior depends on the presence of c i r c u l a t i n g hormone l e v e l s both during the e a r l y " c r i t i c a l p e r i o d " and INTRODUCTION / 15 also during later periods, since adult gonadectomy removes the behavior and hormone treatment restores i t (Goy & McEwen, 1980; MacLusky & Na f t o l i n , 1981). In order to achieve masculine CNS functions two modification processes have been hypothesized as necessary: (i) a "defeminization" involving suppression of female patterns, and ( i i ) a "masculinization" which produces masculine c h a r a c t e r i s t i c s (Beach, 1975). Interestingly, i t i s currently thought that androgen acts on the developing rat brain via aromatization to estrogen and interaction with estrogen receptors. This i s indicated by studies showing a block of neonatal testosterone e f f e c t s by the estrogen antagonist MER-25 (McDonald & Doughty, 1972), and the demonstrated a b i l i t y of the brain to convert testosterone to estrogen (Naftolin et al., 1975). Endogenous estrogen does not enter the brain and masculinize female rats because i t i s bound in the c i r c u l a t i o n by a-fetoprotein, a product synthesized by the yolk sac and f e t a l l i v e r which disappears over the f i r s t few weeks of l i f e (Raynaud et al., 1971; U r i e l et al., 1972; Aussel et a l , 1973). Neonatal female rats have s i g n i f i c a n t l e v e l s of testosterone INTRODUCTION / 16 in the plasma during the apparent " c r i t i c a l period" (Weisz & Ward, 1980), and yet are obviously not masculinized. It i s possible that prior hormonal exposure has in some way changed the response to subsequent testosterone (Weisz & Ward, 1980). As well, i t has been shown that the presence of ovaries can i n h i b i t defeminization of sexual behavior in female rats by neonatal testosterone (Blizard & Denef, 1973). Thus, current concepts of sexual d i f f e r e n t i a t i o n of the CNS are, as might be expected, similar to those related to hepatic enzymes, including a " c r i t i c a l " imprinting period and a response to adult hormones. It is not known i f the " c r i t i c a l " period i s the same for a l l sexually d i f f e r e n t i a t e d phonomena, such as l i v e r enzymes. 1.1.2.3. The Hypothalamo- Pituitary- Liver Axis Meanwhile, a relat i o n s h i p between the l i v e r and the hypothalamo-pituitary complex was becoming apparent. Early studies by Denef (1974) and Gustafsson & Ingelman-Sundberg (1975) had indicated a role for the p i t u i t a r y gland in sexually d i f f e r e n t i a t e d steroid metabolism. Hypophysectomy removed the sex difference in A4-3-keto reduction of testosterone (Denef, 1974) and in 15j3-hydroxylase a c t i v i t y INTRODUCTION / 17 (Gustafsson & Ingelman-Sundberg, 1975). Recently, the most markedly sex-specific P450 enzymes present in l i v e r microsomes from noninduced rats, P450h and P450i (Tables I and I I ) , have been shown to be influenced by the pattern of growth hormone secretion from the p i t u i t a r y (MacGeoch et al, 1 984; Morgan et al, 1985; MacGeoch et al, 1987; Strom et al, 1987) . In the rat, plasma growth hormone level s are high in the fetus and newborn (Rieutort et al, 1974). Levels then decrease, to reach a low at days 18-22 of age. Growth hormone l e v e l s r i s e during the late prepubertal and pubertal period to attain a higher and se x u a l l y - d i f f e r e n t i a t e d pattern of secretion by 30 days of age (Eden, 1979). In the adult male rat, growth hormone i s secreted from the p i t u i t a r y in bursts at 3-4 hour in t e r v a l s , and levels are low or undetectable between bursts (Eden, 1979). In the adult female, growth hormone secretion i s more continuous, with higher basal levels and a lack of the high peaks of the adult male. Using the male-specific P450h as an example, i t has been shown that adult hypophysectomy reduces the normally high levels of P450h in male l i v e r microsomes (Morgan et al, 1985; Kamataki et al, 1985). Administration of growth hormone by INTRODUCTION / 18 continuous infusion, similar to the normal female pattern (Eden, 1979), reduces P450h in normal and hypophysectomized males (Kato et al, 1986). Intermittent growth hormone, however, mimics the male secretion pattern and returns P450h and associated mRNA levels to those of a normal adult male in hypophysectomized males (Kato et al, 1 986; Shimada et al., 1988; Mode et al, 1988). Therefore, P450h expression in the adult male l i v e r microsomes requires a p u l s a t i l e pattern of growth hormone secretion. 1.1.2.4. Androgenic Control of Growth Hormone Secretion Patterns Concurrently, a connection has been established between gonadal steroids and the pattern of growth hormone secretion by the p i t u i t a r y . It has been shown that growth hormone secretion i s under moment-to-moment control from the hypothalamus, with growth hormone releasing factor being stimulatory and somatostatin in h i b i t o r y (Brazeau et al., 1973; Jansson et al., 1985). The sexually d i f f e r e n t i a t e d pattern, however, has been shown by Jansson and co-workers (1984, 1985a, 1985b, 1985c, 1987), in an elegant series of experiments, to be organized by androgenic hormones. Neonatal castration, but not prepubertal castration, lowered the pulse height of growth hormone secretion in males (Jansson et al, 1984). Females INTRODUCTION / 19 treated with testosterone neonatally displayed t y p i c a l high peaks of growth hormone secretion only i f they were neonatally ovariectomized (Jansson & Frohman, 1987). Therefore, peak growth hormone levels were permanently set by neonatal testosterone, and the ovaries were inhibitory to this neonatal imprinting by testosterone. Both neonatal and prepubertal castration increased growth hormone baseline levels in male rats, an effect reversed by testosterone therapy (Jansson et al., 1984). Therefore the t y p i c a l low valleys in males appeared to be dependent on the continued presence of testosterone. In males treated with estrogen in adulthood, the basal growth hormone l e v e l was elevated, again suggesting a feminizing e f f e c t for estrogens which was antagonistic to the eff e c t s of testosterone (Mode et al., 1982). Therefore, i t currently appears that neonatal testosterone imprints a pattern of high growth hormone peaks in the male, and c i r c u l a t i n g testosterone in the male i s required for the low basal l e v e l between peaks. Ovarian factors are inhib i t o r y to both of these e f f e c t s . 1.1.2.5. Current Theory Given that gonadal hormones (Tables I and II) and growth hormone (Jansson et al, 1983, 1984, 1985a, 1 985b, 1985c) can influence the expression of certain hepatic microsomal P450 enzymes, i t remained to be shown whether gonadal hormones INTRODUCTION / 20 exert their effect by direct action on the l i v e r , or i n d i r e c t l y by c o n t r o l l i n g growth hormone secretion patterns. To date, a direc t e f f e c t by androgens and estrogens on the l i v e r remains a p o s s i b i l i t y . Androgen receptors are found in the l i v e r , however a sex-related difference has not been shown (Gustafsson et al., 1975). Estrogen receptors are also found in both male and female rat l i v e r s in approximately equal concentrations (Wrange et al, 1980). However, the p o s s i b i l i t y does exist that one sex could possess another steroid-binding protein of high capacity and low a f f i n i t y . It has been shown that, for certain P450s at least, gonadal steroids may regulate their expression v i a effects on p i t u i t a r y growth hormone. Consider again, for example, P450h. The most markedly male-specific P450 enzyme i s P450h (Ryan et al, 1984) which has been isolated by various groups as P450-male (Kamataki et al., 1983), RLM5 (Cheng & Schenkman, 1982), P450-2c (Waxman & Walsh, 1983) and UT-A (Guengerich et al, 1982). P450h has not been reported in microsomes from female rats of any age (Cheng & Schenkman, 1982; Kamataki et al., 1983; Waxman et al., 1984; Waxman et al., 1 985). P450h i s strongly influenced by androgen, since castration of males at b i r t h completely removed detectable lev e l s in the adult (Kamataki et al., 1 983; Waxman et al, 1985; Dannan et al., 1986; INTRODUCTION / 21 Shimada et al, 1987). P a r t i a l male levels of expression of P450h in the neonatally castrated male could be produced by neonatal testosterone therapy (Kamataki et al, 1983; Waxman et al., 1985; Dannan et al, 1986; Shimada et al., 1987). Hypophysectomy also reduced the normally high P450h levels in adult male l i v e r microsomes, an ef f e c t which was not reversed by testosterone therapy neonatally (Kamataki et al., 1985; Kato et al., 1986). Consistent with t h i s was the observation that female rats, normally lacking P450h, would p a r t i a l l y express P450h when hyphophysectomized, despite a r e l a t i v e lack of androgen (Kamataki et al, 1985). These studies showed that, at least for P450h, growth hormone pattern i s the main factor in sex-specific expression. These findings are consistent with the hypothesis that androgens influence P450s i n d i r e c t l y via growth hormone. These results were similar to those reported for the gonadal control of the female-specific P450i (MacGeoch et al., 1984; Kamataki et al, 1985): hypophysectomy of females removed the expression of P450i, while P450i expression was found in males treated with continuous growth hormone, mimicking the female pattern. Therefore, considerable evidence now exists for regulation of P450i and P450h by the gonadal-hypothalamo-pituitary-liver axis. On the other hand, as can be seen from the varied e f f e c t s of INTRODUCTION / 22 hypophysectomy on the levels of the various P450 forms shown in Tables I and II, i t i s unlikely that i d e n t i c a l regulatory mechanisms exist for a l l of the P450s. For male-specific P450g and the P450 III family, for example, p u l s a t i l e growth hormone exerts a suppressive e f f e c t , the opposite to i t s effec t on P450h (McClellan-Green et al, 1989; Waxman et al. , 1986). The only c l e a r l y expressed theory of gonadal regulation of sex-related hepatic microsomal P450 expression has been that of Gustafsson, as described above. What o r i g i n a l l y consisted "of a grouping of l i v e r steroid metabolizing enzymes into at least three categories on the basis of gonadal regulation, with an emphasis on neonatal imprinting of basal levels and testosterone responsiveness, has evolved to include a role for a gonadal-hypothalamo-pituitary-liver axis in the regulation of some s p e c i f i c forms (Figure 1). 1.1.2.6. Unanswered Questions and Alternative Hypotheses Many questions s t i l l ' remain to be addressed. The o r i g i n a l groupings of steroid-metabolizing enzymes of Gustafsson (Einarsson et al, 1973) have never been repudiated or improved, and s t i l l provide a theoreti c a l framework (Waxman et al, 1985). It i s not clear how our current knowledge that multiple forms of P450 are involved in steroid INTRODUCTION / 23 hydroxylations (Tables I & II) f i t s with the o r i g i n a l groupings. The role of the gonadal-hypothalamo- p i t u i t a r y - l i v e r axis in the regulation of forms other than P450h and P450i i s not known. The mechanisms involved in "imprinting" and "adult androgen responsiveness" are not known. We do not know i f imprinting is necessary for both a basal l e v e l of expression and for adult androgen responsiveness for d i f f e r e n t P450 forms. And f i n a l l y , and of importance to the present study, i s the neonatal period the only " c r i t i c a l period" for imprinting? The present study stems from recent evidence that P450 responsiveness to androgens in adult males may be "imprintable" after the neonatal period, at around the time of puberty. INTRODUCTION / 24 Figure 1A. Gustafsson's Theory. A. Grouping of microsomal steroid-metabolizing enzyme a c t i v i t i e s , as o r i g i n a l l y defined (Einarsson et al, 1973). GROUP DEFINITION I enzymes i r r e v e r s i b l y "imprinted" or "programmed" by androgens during the prepubertal period and reversibly stimulated by androgens postpubertally;• II enzymes with a basal a c t i v i t y regulated by nongonadal factors but reversibly inducible by androgens; III enzymes primarily regulated by nongonadal factors and only s l i g h t l y affected by androgens. INTRODUCTION / 25 Figure I B . Current h y p o t h e t i c a l scheme of growth hormone r e g u l a t i o n of c e r t a i n hepatic microsomal P450 enzymes ( Zaphi ropoulos et al.r 1989). Hypothetical scheme for GH regulation of sexually different steroid metabolism in the rat liver. In males (left) CHis released in a pulsatile fashion that induces a male type of steroid metabolism. The male type of GH secretion is regulated by hypothalamic peptides (somatostatin and GHRH) where somatostatin (produced in the anterior periventricular area) is likely to cause the troughs in mate GH secretory pattern that masculinize the liver. In females (right) GH is released continuously, causing a feminized liver. The inhibition from hypothalamic centres in females is less marked, resulting in a more continuous GH reiease and a feminized liver. The postulated sites of action for gonadal steroids are indicated: E 2 . estrogens: and T. androgens. INTRODUCTION / 26 1.2. EVIDENCE OF IMPRINTING DURING PUBERTY OF P450 ADULT ANDROGEN RESPONSIVENESS 1.2.1. Neonatal Imprinting Is Not Always Required 1.2.1.1. Benzo[a]pyrene Hydroxylase The only si t u a t i o n in which the p o s s i b i l i t y of post-neonatal imprinting has been d i r e c t l y tested i s the hydroxylation of the carcinogen benzo[a]pyrene. Evidence has been presented by Pak and • co-workers (1984) that the androgen responsiveness of the 3-hydroxylation of benzofa]pyrene (BPH) a c t i v i t y can be imprinted postneonatally, at around the time of puberty. Hepatic microsomal BPH a c t i v i t y i s approximately four-fold higher in adult males than females (Wiebel & Gelboin, 1975; Pak et al., 1984). Part of thi s sex-related difference i s due to a responsiveness to c i r c u l a t i n g testosterone in the adult male, since castration of adult males reduces BPH a c t i v i t y , and testosterone treatment restores i t (Kramer et al, 1979; Al-Turk et al., 1981). For BPH, adult responsiveness to testosterone appeared to require imprinting but was not neonatally imprinted, since neonatally castrated males did not respond to adult testosterone even i f treated neonatally with testosterone (Pak, unpublished). This suggested a post-neonatal stage for imprinting of androgen INTRODUCTION / 27 responsiveness of BPH a c t i v i t y . Adult females appear to lack a testosterone response system for BPH, being unaffected by ovariectomy or adult testosterone administration (Pak et al., 1984). However, i t was possible to produce a responsiveness to testosterone for BPH in the adult female rat i f testosterone treatment was given peripubertally, days 35-50 of age (Pak et al., 1984), reaching approximately 40% of a normal male l e v e l of BPH a c t i v i t y . Further, the presence of ovaries was in h i b i t o r y to this peripubertal imprinting of androgen responsiveness by testosterone for BPH. Females gonadectomized pr i o r to puberty, administered testosterone peripubertally, and tested for a response to androgen as adults, expressed BPH a c t i v i t y at a higher l e v e l than non-ovariectomized females, approximately 75% of a normal adult male. Pubertal imprinting of testosterone responsiveness for BPH has also been demonstrated in male rats (Pak, unpublished). Male rats castrated prepubertally on day 26-28 of age, dropped to 50% of the hepatic microsomal BPH a c t i v i t y of the adult male, and did not respond to adult testosterone treatment unless treated with testosterone over days 35-50 of age. INTRODUCTION / 28 While no other d i r e c t i n v e s t i g a t i o n s of pu b e r t a l i m p r i n t i n g by testosterone of androgen responsiveness for BPH a c t i v i t y have been published, i t should be r e a l i z e d that not a l l information on BPH a c t i v i t y i s c o n s i s t e n t with the observations of Pak and colle a g u e s . In a study by Shimada (1987), neonatal c a s t r a t i o n reduced BPH a c t i v i t y , as expected. However, i n c o n t r a s t to the previous reports by Pak et al, treatment of neo n a t a l l y c a s t r a t e d r a t s with testosterone i n adulthood (at 19 weeks of age) r e s u l t e d i n a 100% increase i n microsomal BPH a c t i v i t y . An adult androgen response f o r BPH was ther e f o r e observed i n male r a t s despite a lack of testosterone both neonatally and p e r i p u b e r t a l l y . However, t h i s discrepancy i n d i c a t e s the importance of using a p h y s i o l o g i c a l dose of testosterone to t e s t f or androgen responsiveness. In the study by Shimada et al. (1987), where an adult androgen response f o r BPH was observed, the adult testosterone dose (58 ymoles/kg of testosterone propionate on a l t e r n a t e days for 4-5 doses) was roughly 10-fold higher than the p h y s i o l o g i c a l dose used in the s t u d i e s of Pak and colleagues (testosterone enanthate 2.5 nmoles/kg/day f o r 9 doses). INTRODUCTION / 29 1.2.2. Which Cytochrome P450s? In order to further investigate the p o s s i b i l i t y of post-neonatal imprinting of an androgen responsiveness, i t i s necessary to determine which s p e c i f i c hepatic microsomal P450 enzymes are involved. Based on the work of Pak et al. (1984; unpublished), i t seems probable that one or more predominantly male P450 enzyme(s) which can catalyze the hydroxylation of BPH at the 3 position may be androgen responsive following peripubertal testosterone. At least seven d i f f e r e n t rat l i v e r microsomal P450 forms, p u r i f i e d from untreated animals or animals treated with phenobarbital, PCN or /3-naphthof lavone, have a demonstrated c a t a l y t i c a b i l i t y for BPH hydroxylation: P450s a, b, c, d, e, h (untreated), and Pb/PCN-E from the P450 III family (Guengerich et al., 1982; Ryan et al., 1984). P450c i s the major form involved in BPH hydroxylation in 3-methylcholanthrene-induced rat l i v e r microsomes (Ryan et al., 1979), however in noninduced animals l i t t l e P450c contribution i s l i k e l y , since i t constitutes only approximately 1% of the t o t a l microsomal P450 (Thomas et al., 1981). P450 forms which are present in higher quantities in adult male than female hepatic microsomes are P450g, P450h, RLM2 and the P450 III family (Table I I ) . What i s the evidence INTRODUCTION / 30 that any of these are possibly androgen responsive due to peripubertal testosterone imprinting? 1.2.2.1. P450g From data currently a v a i l a b l e , P450g i s an unlikely candidate for pubertal imprinting of adult androgen responsiveness. P450g p u r i f i e d from untreated male rats does not catalyze BPH hydroxylation in a reconstituted system (Ryan et al, 1984), and therefore may not be a contributor to the phenomena described by Pak et al. ( 1984) for BPH imprinting. P450g expression i s male-specific, immunochemical levels of protein being detected only in l i v e r microsomes from mature male rats, and i s regulated in part by a genetic polymorphism (Ryan et al., 1 984; Bandiera et al, 1985; Bandiera et al., 1 986). As well, castuation at 35 days of age does not a l t e r P450g expression in 70-day old adult male rats, therefore a dependence on adult levels of c i r c u l a t i n g testosterone i s not seen (McClellan-Green et al., 1989). 1.2.2.2. P450h Several l i n e s of evidence suggest that P450h might contribute to the component of BPH a c t i v i t y in males shown to be related to pubertally-imprintable androgen responsiveness by Pak et al. (1984). INTRODUCTION / 31 P450h has been isolated by d i f f e r e n t laboratories under the names P450-male (Kamataki et al, 1983), RLM5 (Cheng & Schenkman, 1982), P450-2c (Waxman & Walsh, 1983) and UT-A (Guengerich et al., 1982). Like BPH a c t i v i t y , hepatic microsomal P450h content (Waxman et al, 1985) and testosterone 16a-hydroxylase a c t i v i t y (Jac'obson & Kuntzman, 1969) increase dramatically in males around the time of puberty. Both P450h leve l s and BPH a c t i v i t y depend in the adult male on a response to c i r c u l a t i n g testosterone (Kamataki et al., 1983; Waxman et al., 1985). A correlation (r=0.850) between hepatic microsomal BPH a c t i v i t y and P450-male content has been demonstrated (Kato, 1987); the observed axis intercept suggests that as much as 75% of BPH a c t i v i t y correlates with P450-male, while approximately 25% i s provided by other enzyme a c t i v i t y . Recently, a P450 enzyme named P450/B[a]P has been p u r i f i e d from untreated male rat l i v e r microsomes (Ohgiya et al., 1989). Based on immunoinhibition studies, P450/B[a]P i s responsible for 80% of BPH a c t i v i t y in l i v e r microsomes from untreated males. Further, P450/B[a]P is similar to P450h, based on amino-terminal sequence analysis, molecular weight and spectral properties. Hypophysectomy of adult males decreases both BPH a c t i v i t y (Kamataki et al, 1985; Yamazoe et al, 1988; Lemoine et al, 1988) and P450-male content INTRODUCTION / 32 (Kamataki et al., 1985; Kato et al., 1986; Shimada et al., 1988). Most importantly, f u l l expression of P450h at normal adult male leve l s can be produced without neonatal exposure to testosterone. This has been observed in both male and female rats which were neonatally gonadectomized, and exposed to physiological levels of testosterone over days 35-70 of age (Dannan et al., 1986). Similar results have been reported by Shimada et al. (1987) following testosterone administration over days 56 to 63 of age. However, i t should be noted that in that study r e l a t i v e l y high testosterone doses were used; 58 jumoles/kg/day of testosterone propionate on alternate days for 4-5 doses would have provided a dose approximately 10-fold higher than that used in the studies by Pak (1984; unpublished). These observations suggest that, as for BPH a c t i v i t y (Pak et al., 1984), an adult responsiveness to testosterone for P450h does not depend on neonatal testosterone imprinting, and might be influenced by peripubertal testosterone. The treatment groups necessary to demonstrate a necessity for imprinting of an adult androgen responsiveness for P450h have not been . included in these studies. However, i t i s known that neonatal testosterone treatment of neonatally castrated males restores only 40-50% of f u l l adult expression of P450h (Kamataki et al., 1983; Kamataki et al, 1984; Waxman et al., 1985; Dannan et al., 1986; INTRODUCTION / 33 Shimada et al, 1987). Whether t h i s deficiency involves a lack of the response system to testosterone or simply a lack of c i r c u l a t i n g testosterone in the adult i s unknown. 1.2.2.3. P450 III Family Our knowledge of the cytochrome P450 III family has suffered from the confusion produced by the i s o l a t i o n of similar yet not necessarily i d e n t i c a l forms by many di f f e r e n t laboratories. True to the history of P450, what was o r i g i n a l l y thought to be one enzyme has since been shown in rats to consist of two s t r u c t u r a l l y similar subfamilies (Nebert et al, 1987; Nebert et al., 1989) (Table IV). This family i s complex and not yet f u l l y understood. INTRODUCTION / 34 Table IV. P450III Forms in Rat Liver Microsomes. Immunochemically-related members of the P450III gene family in rats have been separated into subgroups IIIA1 and IIIA2 on the basis of amino-terminal sequences (Halpert, 1988). The forms l i s t e d under these headings are considered i d e n t i c a l or very similar in structure and expression, however i t i s possible that further d i s t i n c t i o n s w i l l be made within these groups in future. Abbreviations: UN, untreated; IND, induced with phenobarbital, PCN or trioleandomycin. References: Elshourbagy & Guzelian, 1980; Guengerich et al, 1982; Waxman et al., 1985; Gonzalez et al., 1986; Waxman et al, 1986; Halpert et al, 1988; Imaoka et al., 1988; Yamazoe et al., 1988. IIIA1 IIIA2 LABORATORY ANIMAL SOURCE PCN1 PCN2 Gonzalez UN,IND Pb/PCN-E Guengerich/ UN,IND Pb-2a Waxman P Guzelian UN,IND PCNa PCNb,PCNc Halpert IND Pb-1 Kato UN INTRODUCTION / 35 The IIIA1 subfamily i s induced by steroids in both sexes, however one form, PCN1, i s not present at detectable levels in untreated male or female rats (Gonzalez et al, 1986). Enzymes allocated by Halpert (1988) to this IIIA1 group include the pregnenolone-16a-carbonitrile (PCN)-inducible P450p (Elshourbagy & Guzelian, 1980), PCN1 (Gonzalez et al., 1986), and PCNa (Graves et al., 1987; Halpert et al., 1988), The second subfamily has been designated IIIA2. Three IIIA2 forms, which may be i d e n t i c a l based on amino-terminal sequences, have been named P450PCN2, which i s not steroid-inducible (Gonzalez et al, 1986), P450PCNb (Graves et al., 1987; "Halpert et al., 1988), and P450 Pb-1 (Imaoka et al., 1988). The laboratories of Waxman and Guengerich have reported a P450 enzyme in untreated rats which they have designated Pb-2a/PCN-E (Waxman et al., 1985) or 2a (Waxman et al., 1986). Halpert (1988) has reported P450 PCN-E as indistinguishable from PCNb, and thus Pb-2a/PCN-E can be tentatively allocated to the IIIA2 family, although sequencing on 2a has not been reported. The terms IIIA1 and IIIA2 therefore do not necessarily refer to single enzymes. Nor i s i t clear that t r i v i a l names such as P450p necessarily refer to a single enzyme. For the purposes of t h i s discussion, the name given by the investigators w i l l be used in order to avoid any incorrect INTRODUCTION / 36 assumptions. Despite the present confusion, the following information i s available based on data from untreated rats. Adult male hepatic microsomes contain higher levels of th i s P450 family with respect to P450 PCN2 (Gonzalez et al., 1986), Pb-2a/PCN-E (Gozukara et al., 1984; Waxman et al., 1985; C r e s t e i l et al, 1986), and P450 Pb-1 (Imoaka et al., 1988), with adult female levels being low or undetectable. It i s not clear i f the developmental changes for these enzymes are p a r a l l e l to those for BPH a c t i v i t y . In female rats, PCN2 mRNA and protein are present at b i r t h , increase in the f i r s t week and subsequently decrease between 2 to 4 weeks of age (Gonzalez et al, 1986). These results are consistent with reports for P450 Pb-1 (Imaoka et al., 1988, Yamazoe et al., 1988) and P450 Pb-2a/PCN-E (Waxman et al., 1985), which also describe a decrease in immunochemically detectable enzyme levels around the time of puberty in the female. BPH levels in females, however, slowly increase to reach adult levels by the time of puberty (Wiebel & Gelboin, 1975; C r e s t e i l et al., 1986). Reports on the age-related changes in P450III forms in the male have been contradictory. Both a lack of change with age INTRODUCTION / 37 and an increase with age have been reported. Both P450PCN2 mRNA and protein increase in the f i r s t week of l i f e in male rats, and increase further between 4 to 12 weeks of age (Gonzalez et al, 1986). In contrast, P450 Pb-1 has been reported to gradually increase over days 1-30 of age and then remain high but r e l a t i v e l y unchanging after day 30 of age (Yamazoe et al, 1988). Waxman et al. ( 1985) have reported a lack of change in P450 Pb-2a/PCN-E levels over the period of of 2-12 weeks of age. It therefore appears that some forms of P450 III increase in the male rat around the age of puberty, as seen for BPH a c t i v i t y (Wiebel & Gelboin, 1975; C r e s t e i l et al., 1986), while others do not. These discrepancies are based on level s of ummunochemically-detectable protein and therefore may be due to differences in the s p e c i f i c i t y of the antibodies employed. It i s possible that the early r i s e in the f i r s t week or so of l i f e in males and females reported by Gonzalez et al. (1986), Yamazoe et al. (1988) and perhaps by Waxman et al. (1985) may be due to detection of an immunochemically-similar but di f f e r e n t enzyme which i s present only during early l i f e . The detection of t h i s enzyme may hide the r i s e of a second enzyme around the time of puberty which i s then present as the male-specific form in the adult. Several investigators have reported a related INTRODUCTION / 38 protein which i s present early in l i f e and then disappears ( C r e s t e i l et al, 1986; Kitada et al, 1987a, 1987b, 1988). A l t e r n a t i v e l y , there may be two s t r u c t u r a l l y - s i m i l a r P450s involved which are both expressed in the adult male, one which does not change at puberty, as described by Waxman et al. ( 1985), and one which does increase per ipubertally (Gonzalez et al., 1986). Hostetler et al. (1987) have reported two electrophoretically-separable proteins which were immunochemically-related to P450p in untreated adult males. 60-Hydroxytestosterone i s the major monohydroxylated testosterone metabolite formed by l i v e r microsomes from immature male rats and a major metabolite in those from mature males (Lee & Park, 1989). P450III appears to be the major catalyst of testosterone 60-hydroxylase in untreated animals, since t h i s a c t i v i t y in l i v e r microsomes i s completely inhibited by antibodies against P450PCN1 (Gonzalez et al., 1986) or against P450 Pb-1 (Imaoka et al., 1988). It i s of p a r t i c u l a r interest that testosterone 6/3-hydroxylase a c t i v i t y in l i v e r microsomes from untreated male rats increases peripubertally (Jacobson & Kuntzman, 1969).. It i s not clear whether adult male leve l s of P450 III are a product of a responsiveness to c i r c u l a t i n g testosterone. INTRODUCTION / 39 S t u d i e s of sex hormonal i n f l u e n c e s have been conducted o n l y f o r P450 Pb-2a/PCN-E and 2a/PCN-E. E x p r e s s i o n of these forms i n the male shows dependence on androgen exposure, s i n c e c a s t r a t i o n a t b i r t h c o m p l e t e l y removes immunochemically d e t e c t a b l e P450Pb-2a/PCN-E and 2a/PCN-E i n the a d u l t male r a t , and n e o n a t a l t e s t o s t e r o n e p a r t i a l l y r e s t o r e s the enzyme l e v e l s (Waxman et al., 1985; Dannan et al, 1986). N e o n a t a l i m p r i n t i n g of a b a s a l l e v e l t h e r e f o r e o c c u r s . Waxman et al. (1985) r e p o r t e d t h a t c a s t r a t i o n of males a t 35 days of age d i d not a l t e r immunoquantitated l e v e l s of P450 Pb-2a/PCN-E, i n d i c a t i n g a l a c k of dependence on c i r c u l a t i n g androgens. In c o n t r a s t , s t e r o i d 6/3-hydroxylase i s androgen r e s p o n s i v e i n males, b e i n g d e c r e a s e d by a d u l t c a s t r a t i o n and r e s t o r e d by t e s t o s t e r o n e t r e a t m e n t ( E i n a r s s o n et al, 1973). In f e m a l e s , t h e r e i s a s m a l l and v a r i a b l e response t o a d u l t t e s t o s t e r o n e w i t h r e s p e c t t o s t e r o i d 6/3-hydroxylase, s u g g e s t i n g a l a c k of androgen r e s p o n s i v e n e s s ( E i n a r s s o n et al., 1973) as seen f o r BPH (Pak et al, 1984) . W h i l e hypophysectomy of a d u l t male r a t s d e c r e a s e s BPH a c t i v i t y (Kamataki et al, 1985; Lemoine et al, 1988, Yamazoe et al, 1988), t h i s s u r g i c a l p r o c e d u r e i n c r e a s e s P450 2a c o n t e n t (Waxman et al., 1 986). The c o m p l e x i t y of the P 4 5 0 I I I f a m i l y and the l a c k of INTRODUCTION / 40 information on di f f e r e n t forms renders d i f f i c u l t any assessment of a role for hormonal regulation and imprinting. However, in neonatally gonadectomized males or females treated with physiological doses of testosterone from days 35 to 70 of age, immunochemically detectable P450 2a/PCN-E reaches the l e v e l of a normal adult male, irrespective of neonatal testosterone treatment (Dannan et al., 1986). This c l e a r l y shows that neonatal testosterone i s not required for complete expression of P450 2a/PCN-E. It i s therefore possible, as for P450h, that testosterone exposure peripubertally, days 35-50 of age, can imprint a response to subsequent testosterone for a member of the P450III family. 1.2.2.4. P450 RLM2 I n s u f f i c i e n t evidence i s available to provide an indication of the relationship between th i s male-specific form and. androgen exposure (Table I I ) . 1.3. PROPOSED RESEARCH 1.3.1. Initial Focus on P450 III From the above discussion i t i s clear that both P450h and P450' III forms, and possibly other P450 forms, may be influenced by peripubertal testosterone in a manner similar to BPH (Pak et al., 1984). We chose to i n i t i a l l y investigate INTRODUCTION / 41 P450 III, since (i) the most s p e c i f i c P450h analysis requires either the use of s p e c i f i c antibodies for immunochemical quantitation, or HPLC analysis of associated testosterone hydroxylations (Waxman et al, 1 985; Sonderfan et al, 1987). Neither of these methodologies were available at the beginning of the study, nor r e a l i s t i c a l l y accessible within the time l i m i t s of the study; and ( i i ) a r e l a t i v e l y simple method, suitable for an i n i t i a l investigative study, existed for P450 I I I . The demethylation of erythromycin i s a c a t a l y t i c a c t i v i t y linked to P450p (Wrighton et al, 1 985a; 1985b; Watkins et al., 1 986; A r l o t t o et al, 1987). We decided to investigate the response of erythromycin demethylase a c t i v i t y to testosterone in adulthood following peripubertal testosterone exposure. The following methodology was proposed. 1.3.2. Animal Model Female rats were chosen as the preferred animal model for the investigation of testosterone effects during a s p e c i f i c developmental period, since they would lack male leve l s of pre- and post-natal testosterone, other than that administered. Further, prepubertal ovariectomy of these female rats was required to remove possible influences of ovarian secretions INTRODUCTION / 42 on testosterone imprinting. Previous studies have reported an i n h i b i t i o n by estrogen or the presence of ovaries on imprinting by neonatal testosterone in female rats of an adult male pattern of l i v e r C o r t i s o l metabolism (Denef & DeMoor, 1972), open f i e l d and sexual behavior (Blizard & Denef, 1973), hepatic estrogen-binding proteins (Sloop et al, 1983), hepatic demethylated epoxide metabolism (Finnen & Hassall, 1984), and growth hormone secretion pattern (Jansson & Frohman, 1987). It has been suggested that this i s a mechanism by which masculinization of females by endogenous testosterone is avoided (MacLusky & Naftolin, 1981). Further, the study which has demonstrated a pubertal imprinting by testosterone of an androgen responsiveness for BPH a c t i v i t y c l e a r l y showed an i n h i b i t i o n of t h i s imprinting in non-ovariectomized females (Pak et al, 1984). And f i n a l l y , the strongest evidence for a possible peripubertal imprinting for P450h and P450III forms in females comes from a study using neonatally ovariectomized animals which therefore lacked ovaries during the putative imprinting period (Dannan et al., 1986). As well as ovariectomized females, i t was decided that male rats would be included in the study as a physiological comparison. INTRODUCTION / 43 1.3.3. Treatment Protocol An experimental design similar to that used by Pak et al. (1984) was adopted. Thus, prepubertally ovariectomized females were to be treated with physiological doses of testosterone over a peripubertal period, defined as days 35-50 of age. A test for subsequent adult responsiveness to testosterone would be made 30 days l a t e r . As well, a control, non-ovariectomized group would be tested for adult response to testosterone, indicative of the normal adult responsiveness to androgen. 1.3.4. Specific Hypothesis S p e c i f i c a l l y , we hypothesized that exposure to testosterone Of during puberty would influence the responsiveness of hepatic microsomal erythromycin demethylase a c t i v i t y \to testosterone in adult prepubertally ovariectomized female rats. Corollary one: There would be a resulting increase in erythromycin demethylase a c t i v i t y in females exposed to testosterone in both periods, compared to females exposed to testosterone during either period alone; Corollary two: That the pattern seen in the above females would not be i d e n t i c a l to that in s i m i l a r l y treated males, since they would have been exposed to prepubertal testosterone and neonatally imprinted. INTRODUCTION / 44 * "influence" s p e c i f i e s that there must be an interaction between the two time periods of testosterone, not merely an additive e f f e c t . 2. MATERIALS AND METHODS 2.1. CHEMICALS The following chemicals were obtained from Sigma Chemical Co. (St. Louis, MO): acetyl acetone (2,4-pentanedione) , ammonium acetate, erythromycin base, ethylenediaminetetraacetic acid (EDTA), semicarbazide, testosterone enanthate, and trizma base. Preservative-free corn o i l was obtained from Lifestream Natural Foods, Ltd. (Richmond, B.C.). NADPH was purchased from Boehringer-Mannheim (W. Germany). Formaldehyde, magnesium chloride, potassium chloride, and sodium d i t h i o n i t e were obtained from BDH Chemicals Ltd. (Toronto, Canada). Sodium phosphate dibasic, sodium phosphate monobasic, and potassium phosphate monobasic were purchased from Fisher S c i e n t i f i c Co. (Fair Lawn, NJ). A Bradford Protein Assay Kit was obtained from Bio-Rad Laboratories (Richmond, CA). A l l other chemicals used were a n a l y t i c a l reagent q u a l i t y . 2.2. ANIMALS Male and female Sprague Dawley rats were purchased from Charles River Co. (Montreal, Canada). The animals were housed in plastic-bottomed cages with corncob bedding free of a n t i b a c t e r i a l s (Paxton Processing Co., Inc., Paxton, IL) under conditions of controlled temperature (23°C) and 45 MATERIALS AND METHODS / 46 l i g h t i n g ( l i g h t s on 0800 t o 2200 h o u r s ) . Ad lib a c c e s s t o food ( P u r i n a Rodent L a b o r a t o r y Chow 5001, R a l s t o n P u r i n a of Canada L t d . , Woodstock, Canada) and water was p r o v i d e d . Gonadectomy or sham gonadectomy was performed by the breeder under e t h e r a n e s t h e s i a when the a n i m a l s were 25 days of age ± one day. The a n i m a l s were a l l o w e d t o e q u i l i b r a t e f o r a t l e a s t 8 days f o l l o w i n g s u r g e r y and 3 days f o l l o w i n g t r a v e l b e f o r e t r e a t m e n t s were s t a r t e d . Each t r e a t m e n t group i n i t i a l l y c o n t a i n e d f o u r a n i m a l s i n the p i l o t s t u d y , and 9 an i m a l s i n the major s t u d y . 2.3. TREATMENTS The t r e a t m e n t s c h e d u l e s a r e shown i n Appendix F i g u r e 1 . A s o l u t i o n of t e s t o s t e r o n e enanthate 4.0 mg/mL i n c o r n o i l was pr e p a r e d e v e r y 5 days d u r i n g the tr e a t m e n t p e r i o d s . P e r i p u b e r t a l i n j e c t i o n s were g i v e n from 35 t o 49 days of age. A d u l t t e s t o s t e r o n e t r e a t m e n t s were a d m i n i s t e r e d d a i l y from 81 t o 89 days of age. In the p i l o t s t u d y , p e r i p u b e r t a l i n j e c t i o n s c o n t a i n e d e i t h e r t e s t o s t e r o n e e n a n t h a t e 5 Mmoles/kg/day, e s t r a d i o l benzoate 1.5 /nmoles/kg on a l t e r n a t e days, or c o r n o i l , w h i l e a d u l t i n j e c t i o n s were e i t h e r t e s t o s t e r o n e e n a nthate 2.5 Mmoles/kg/day or c o r n o i l . In the major s t u d y , a n i m a l s r e c e i v e d i n j e c t i o n s once d a i l y of e i t h e r t e s t o s t e r o n e e n a nthate 5 Mmoles/kg/day o r an eq u a l volume of c o r n o i l , i n both p e r i p u b e r t a l and a d u l t p e r i o d s . MATERIALS AND METHODS / 47 A l l injections were subcutaneous and administered at the same time of day ± two hours. Animals were s a c r i f i c e d at 90 days of age, at approximately 24 hours aft e r their l a s t i n j e c t i o n . One animal was removed from the major study due to an error in i n j e c t i o n . 2.4. MICROSOME PREPARATION When the animals reached 90 days of age, they were s a c r i f i c e d by decapitation. Livers were quickly removed, placed in ice-cold 0.05 M T r i s , 1.15% KC1, pH 7.5, blotted, and minced. A l l subsequent steps were performed at 4°C. The entire l i v e r was added to 20.0 mL of the Tris/KCl buffer, and homogenized using a Potter-Elvehjem glass mortar and a motor-driven pestle. The homogenate was centrifuged at 10,000 x g for 20 minutes. The supernatant was f i l t e r e d through four layers of cheesecloth and centrifuged at 100,000 x g for 60 minutes. The resulting microsomal p e l l e t was resuspended in 10 mM EDTA, 1.15% KC1, pH 7.4, and centrifuged again at 100,000 x g for 60 minutes. The p e l l e t was resuspended in 4.0 mL of 0.25 M sucrose, resulting in a f i n a l microsomal protein concentration of 20-40 mg/mL. Aliquots were frozen in cryotubes at -80°C. Two animals were removed from the major study due to an error in microsome preparation. M A T E R I A L S A N D M E T H O D S / 48 2.5. ERYTHROMYCIN DEMETHYLASE ACTIVITY Erythromycin demethylase a c t i v i t y in hepatic microsomes was determined according to the method of Arlot t o et al. ( 1987), using the spectrophotometric measurement of formaldehyde formation- from the method of Nash (1953). Frozen hepatic microsomes were thawed rapidly at 37°C and di l u t e d with 0.25 M sucrose to a concentration that would give a f i n a l protein concentration in the incubation mixtures in the major study of 0.8 mg/mL (females) or 0.35 mg/mL (males). In the p i l o t study, f i n a l protein concentrations in the incubation mixtures were 1.1 mg/mL (females) and 0.48 mg/mL (males). The t o t a l incubation mixture contained the following components in 1.5 mL: 100 al 6 mM erythromycin base in 30% ethanol, 100 y l 45 mM magnesium chloride, 100 ul 75 mM semicarbazide, 10 ul 150 mM NADPH in 200 mM Na,K-phosphate buffer, pH 7.4, 990 ul Na, K-phosphate buffer, and 200 y l dilut e d hepatic microsomes. For each sample, two blanks were assayed, one which lacked substrate during the reaction, and one which lacked microsomes. A l l samples were assayed in duplicate. The incubation mixtures were pre-incubated at 37°C for 75 seconds in a water bath. The reaction was started by the addition of the NADPH. After 20.0 minutes at 37°C, the reaction was stopped by the addition of 0.6 mL of ice-cold 17% perchloric acid. The tubes were vortexed and placed on ice. The components lacking in the blank tubes MATERIALS AND METHODS / 49 were added. The tubes were centrifuged at 1000 x g for 15 muinutes at 4°C. Double Nash Reagent (ammonium acetate 7.5 g, acetyl acetone 0.1 mL, d i s t i l l e d water to 25 mL) was freshly prepared and 0.5 mL was added to 1.5 mL of each supernatant. These solutions were incubated at 60°C in a water bath for 15 minutes, then allowed to cool for 5 minutes. Absorbance was measured at 412 nm with a Hewlett Packard 8452A Diode Array spectrophotometer. For each sample, the net absorbance was calculated by subtracting the average absorbance of the two blanks, and formaldehyde formation was determined from a formaldehyde standard curve. Erythromycin demethylase a c t i v i t y was calculated as both nmoles formaldehyde/ min/ mg protein and nmoles formaldehyde/ min/ nmole P450. 2.6. PROTEIN Hepatic microsomal protein was measured spectrophotometrically by the method of Bradford (1976). Bovine serum albumin was used as the standard. A l l samples were measured in duplicate. Protein content per gram wet weight of l i v e r was estimated as (hepatic microsomal protein (mg/mL))/((liver weight(g))/(20+liver weight(mL)). MATERIALS AND METHODS / 50 2.7. TOTAL CYTOCHROME P450 Hepatic microsomal t o t a l cytochrome P450 was measured spectrophotometrically according to the method of Omura and Sato (1964a). Hepatic microsomal samples were thawed rapidly at 37°C, and diluted 1:20 in 100 mM sodium phosphate, 20% gly c e r o l , 0.1 mM EDTA, pH 7.4. The microsomes were reduced with a few mg sodium d i t h i o n i t e , and then saturated with carbon monoxide. The difference spectrum was recorded on an SLM-Aminco DW-2 spectrophotometer. Total hepatic microsomal cytochrome P450 concentration was calculated using a molar extinction c o e f f i c i e n t of 0.091 cm 1 nM 1 (Omura & Sato, 1964b), and was expressed as nmoles P450/mg protein. Total P450 per gram wet weight of l i v e r was estimated as (nmoles P450/mL microsome)/((liver weight(g))/(20+liver weight(mL)). 2.8. PLASMA PREPARATION Blood was col l e c t e d in test tubes containing heparin immediately following decapitation of each animal. After centrifugation at 1000 x g for 15 minutes at 4°C, the plasma was transferred into Eppendorf polypropylene micro test tubes and stored at -80°C. MATERIALS AND METHODS / 51 2.9. PLASMA TESTOSTERONE T o t a l unconjugated plasma testosterone concentration was 125 measured using a double-antibody [I ] radioimmunoassay k i t (ICN Biomedicals, Inc., Carson, CA). The d e t e c t i o n l i m i t of the assay was reported by the manufacturer as 0.1 ng/mL. 2.10. PLASMA ESTRADIOL T o t a l unconjugated plasma 17/3-estradiol c o n c e n t r a t i o n was 125 determined with a double-antibody [I ] radioimmunoassay k i t (ICN Biomedicals, Inc., Carson, CA). The d e t e c t i o n l i m i t reported by the manufacturer f o r t h i s assay was 10 pg/mL. 2.11. STATISTICAL ANALYSIS Comparisons of mean values i n the treatment groups were performed using a n a l y s i s of variance (ANOVA), followed by the Student Newman Keuls m u l t i p l e comparison t e s t . One-way ANOVA was used i n the p i l o t study to examine the e f f e c t of treatments. Two-way ANOVA was used i n the p i l o t and major st u d i e s to i n v e s t i g a t e the e f f e c t s of the f a c t o r s sex and treatment, or pubertal testosterone and adul t t e s t o s t e r o n e , and t h e i r i n t e r a c t i o n s . An i n t e r a c t i o n was i n d i c a t e d i f the e f f e c t of one f a c t o r depended upon the presence of another f a c t o r i n a non-additive manner. The c o r r e l a t i o n between hepatic microsomal erythromycin demethylase a c t i v i t y and plasma testosterone l e v e l s was examined using l i n e a r regression. Differences s i g n i f i c a n t when p<0.05. MATERIALS AND METHODS / 52 were considered s t a t i s t i c a l l y 2.12. OTHER ANALYSIS Enzyme kinetic analysis was done using the non-linear regression program E n z f i t t e r (Leatherbarrow, 1987). Value estimates were determined from Eadie Hofstee pl o t s . Weightings used were robust and simple, based on study of the residuals. 3. RESULTS 3.1. ERYTHROMYCIN DEMETHYLASE ASSAY CONDITIONS The conditions used by previous investigators (Arlotto et a l , 1987; Wrighton et at., 1985a) for the determination of erythromycin demethylase a c t i v i t y in rat hepatic microsomes were v e r i f i e d to insure that product formation was linear with respect to both protein concentration and incubation time. Assay conditions were investigated using hepatic microsomes from both female and male untreated Sprague Dawley rats. The samples were composed of a pooled mixture of hepatic microsomes from four individual animals of the same sex. The data shown represent the results of at least one t y p i c a l experiment. A l l experiments were repeated at least once. 3.1.1. Protein concentration The erythromycin demethylase-related product formation in untreated male Sprague Dawley rat l i v e r microsomes was found to be line a r over the f i n a l protein concentration range in the incubation mixture of 0.11 to 0.68 mg/mL, as shown in Figure 2. In untreated female Sprague Dawley rat hepatic microsomes (Figure 3) the reaction was linear over the range 0.56 to 1.12 mg protein per mL incubation mixture. Even at thi s r e l a t i v e l y high protein concentration, the product 53 RESULTS / 54 formation indicated by absorbance from the untreated female microsomes was lower than that of the untreated male. 3.1.2. Incubation Time Although previous investigators have used an incubation time of ten minutes (Arlotto et al., 1987; Wrighton et al, 1985a), the p o s s i b i l i t y of increasing the incubation time in order to increase product formation and thus absorbance was investigated. It was observed in the untreated male hepatic microsomes that, at a f i n a l protein concentration of 0.34 mg/mL, the reaction remained linear with respect to time for at least 30 minutes (Figure 4). This experiment was repeated twice at a f i n a l protein concentration of 0.34 mg/mL. The reaction was also linear over 5 to 25 minutes at a f i n a l protein concentration of 0.43 mg/mL. In the untreated female microsomes (Figure 5) the reaction remained linear in two experiments over the f i r s t 24 minutes at a f i n a l protein concentration of 0 . 9 1 mg/mL. 3.1.3. NADPH To insure that NADPH was not being depleted or inactivated during prolonged incubation i n t e r v a l s , additional NADPH equal to the amount used to i n i t i a t e the reaction was added during some of the preliminary test incubations. A lack of RESULTS / 55 NADPH would be indicated by an increase in net absorbance when additional NADPH was added. As shown in Figures 6 and 7, additional NADPH midway through the incubation period decreased rather than increased the product formation, therefore depletion of NADPH was not indicated. Additional NADPH appeared to i n h i b i t product formation. That the assay conditions provided an excess of NADPH in the incubation mixture was further investigated in studies of erythromycin demethylase a c t i v i t y versus f i n a l NADPH concentration. Curvilinear plots were obtained using both untreated male (Figure 8) and untreated female (Figure 9) hepatic microsomes. In both cases, the erythromycin demethylase a c t i v i t y was r e l a t i v e l y independent of NADPH at a f i n a l concentration of 1 mM. Doubling the f i n a l NADPH concentration resulted in a lower erythromycin demethylase a c t i v i t y , consistent with the observation made above that at high NADPH the erythromycin demethylase reaction i s inhi b i t e d . 3.1.4. Substrate Concentration The e f f e c t of varied substrate concentration on erythromycin demethylase was investigated, using f i n a l substrate concentrations ranging from 0.005 to 8 mM. At substrate concentrations below th i s the signal-to-noise r a t i o was RESULTS / 56 excessively small, while at higher substrate concentrations the r e l a t i v e l y poor water s o l u b i l i t y of the substrate, erythromycin base, resulted in p r e c i p i t a t i o n . Direct plots of erythromycin demethylase a c t i v i t y versus substrate concentration are shown in Figures 10 and 11 for untreated male and female hepatic microsomes. Based on these results, a f i n a l substrate concentration of 0.4 mM was chosen for subsequent assays because (1) t h i s concentration r e f l e c t s a point on or past the shoulder of the curve, thus the substrate concentration i s not l i m i t i n g the rate of erythromycin demethylase a c t i v i t y ; (2) the use of a higher substrate concentration presents the p o s s i b i l i t y of p r e c i p i t a t i o n of the substrate in the incubation mixture; and (3) t h i s i s the substrate concentration used in previous reports of erythromycin demethylase a c t i v i t y (Arlotto et al., 1987; Wrighton et al., 1985a), and using the same substrate concentration allows us to compare re s u l t s . It has been reported that at least two enzymes contribute to erythromycin demethylase a c t i v i t y in untreated adult female Long Evans rat hepatic microsomes (Arlotto et al, 1987). Representative data for untreated adult female Sprague Dawley rat hepatic microsomes are presented in Table VI and Figures 11 to 13. These experiments were performed a t o t a l of 14 times, involving from 6 to 12 points per experiment RESULTS / 57 and a substrate range of 0.005 to 4 mM. Based on the experimental data, estimated values of Michaelis-Menten constants were produced by the E n z f i t t e r program using non-linear regression (Leatherbarrow, 1987) for both a one enzyme and a two enzyme model (Tables V-VI). The f o l l o w i n g c r i t e r i a were considered: (1) g r a p h i c a l a n a l y s i s (Figures 11 to 13) suggested the presence of more than one enzyme; (2) the reduced chi-squared values, c a l c u l a t e d as the weighted sum of the squared d e v i a t i o n s of c a l c u l a t e d from experimental values, and i n d i c a t i n g the f i t of the data (Leatherbarrow, 1987), were s i m i l a r f or the one component and for the two component model f o r the ma j o r i t y of data sets analyzed (Table V I ) ; (3) negative values for Vmax2 and Km2 were generated i n the two enzyme model, but not for the one enzyme model (Table V I ) ; (4) f o r each a n a l y s i s , standard e r r o r s were c a l c u l a t e d for the estimated parameters (Table V I ) . These i n d i c a t e the accuracy of the estimates, and for a good set of experimental data should g e n e r a l l y not be greater than approximately 10% of the value of the parameter (Leatherbarrow, 1987). Standard e r r o r s were higher, up to 109% of the parameter estimates, i n the two enzyme model, compared with a maximum 47% i n the one enzyme model. The regression a n a l y s i s d i d not support a conclusion of RESULTS / 58 m u l t i p l e enzymes, and suggested the presence of one enzyme with Vmax 0.42 nanomoles formaldehyde/min/mg p r o t e i n and Km 0.04 mM c a t a l y z i n g the erythromycin demethylase r e a c t i o n i n adult female Sprague Dawley r a t hepatic microsomes. Since the g r a p h i c a l representations suggested m u l t i p l e enzymes, a d e f i n i t e c o n c l u s i o n could not be made from the data. S i m i l a r l y , the k i n e t i c s of erythromycin demethylase i n hepatic microsomes from untreated adult male Sprague Dawley r a t s was i n v e s t i g a t e d i n a t o t a l of 6 experiments, i n v o l v i n g 6 to 12 p o i n t s per run and a substrate concentration range of 0.0125 to 8 mM. Representative r e s u l t s are shown i n Table V and Figures 10,14 and 15. I t was observed t h a t : (1) g r a p h i c a l p r e s e n t a t i o n suggested the presence of more than one enzyme (Figures 14 to 15); (2) reduced chi-squared values were smaller for the one enzyme model than f o r the two enzyme model (Table V); (3) negative values f o r Vmax2 and Km2 were generated i n the two enzyme model but not i n the one enzyme model (Table V); (4) small standard e r r o r s for the parameter estimates were observed f o r both the one enzyme model and the two enzyme model, i n some cases (Table V). Therefore, while the most conservative i n t e r p r e t a t i o n of the data from untreated adult male Sprague Dawley r a t s i n d i c a t e d the presence of a s i n g l e enzyme (Vmax=l.59 nanomoles RESULTS / 59 formaldehyde/min/mg protein, Km=0.12 mM) catalyzing the erythromycin demethylase reaction in hepatic microsomes, the p o s s i b i l i t y of multiple enzymes could not be discarded. 3.1.5. Phosphate Buffer Previous investigators (Arlotto et al., 1987) have sp e c i f i e d a "potassium phosphate buffer 0.1 M, pH 7.4" for use in the erythromycin demethylase assay. The effe c t of di f f e r e n t buffers on erythromycin demethylase a c t i v i t y i s shown in Figure 16. It was observed that higher erythromycin demethylase a c t i v i t i e s were produced using a phosphate buffer composed of a mixture of 0.2 M potassium phosphate monobasic and 0.2 M sodium phosphate dibasic in a r a t i o of 19/81 (v/v) respectively, at pH 7.4 (Na,K-phosphate buffer 0.2 M), in comparison with either the same mixture at 0.1 M or a mixture of potassium monobasic and dibasic phosphate s a l t s at 0.1 M. 3.1.6. Formaldehyde Standard Curve In a l l assays measuring erythromycin demethylase a c t i v i t y performed in this study, a standard curve of formaldehyde in Na,K-phosphate buffer 0.2 M, pH 7.4, was employed to measure product formation. Since t h i s standard curve lacked most components of the incubation mixtures, a comparison of the absorbance produced by formaldehyde in the presence and RESULTS / 60 absence of the missing components was made. Assay c o n d i t i o n s used were those decided upon for the measurement of erythromycin demethylase a c t i v i t y i n hepatic microsomes as described below. I t was observed that the presence of hepatic microsomes, s u b s t r a t e , NADPH and c o f a c t o r s decreased net absorbance due to formaldehyde (Figures 17 and 18). Using l i n e a r r e g r e s s i o n , slopes of net absorbance versus formaldehyde concentration were c a l c u l a t e d and c o r r e c t i o n f a c t o r s f o r the male assay c o n d i t i o n s (corrected absorbance=1.18 x net absorbance) and for the female assay c o n d i t i o n s (corrected absorbance= 1.26 x net absorbance) were determined. 3.1.7. Final conditions Based on these p r e l i m i n a r y s t u d i e s , the f i n a l incubation mixture concentrations chosen for the measurement of erythromycin demethylase a c t i v i t y i n rat hepatic microsomes were: microsomal p r o t e i n 0.35 mg/mL (male) or 0.8 mg/mL (female), incubation time 20 minutes, erythromycin base 0.4 mM, NADPH 1 mM, and a phosphate b u f f e r composed of a mixture of sodium phosphate d i b a s i c 0.2 M plus potassium phosphate monobasic 0.2 M i n a r a t i o of 81/19 (v/v) r e s p e c t i v e l y (Na,K-phosphate 0.2 M), pH 7.4. The concentrations of magnesium c h l o r i d e (3 mM) and semicarbazide (5 mM) were i d e n t i c a l to those used i n previous p u b l i c a t i o n s f or the RESULTS / 61 erythromycin demethylase assay (Arlotto et al., 1987; Wrighton et al, 1 985a) . The c o e f f i c i e n t of variation of the assay, indicating r e l a t i v e error in separate experiments, was calculated as 14%, from eight experiments done on d i f f e r e n t days using male microsomes. RESULTS / 62 Figure 2. Erythromycin demethylase assay: absorbance (412 nm) versus protein concentration in untreated male Sprague Dawley rat hepatic microsomes (pool of 4 l i v e r s ) . Assay conditions: incubation time 20 minutes, f i n a l erythromycin concentration 0.4 mM, f i n a l NADPH concentration 1 mM. 0.08 Final Prote in concen t ra t i on ( m g / m L ) K cn C r H o CO RESULTS / 64 Figure 3. Erythromycin demethylase assay: absorbance (412 nm) versus f i n a l p r o t e i n concentration i n untreated female Sprague Dawley rat hepatic microsomes (a pool of 4 l i v e r s ) . Assay c o n d i t i o n s : incubation time 20 minutes, f i n a l erythromycin concentration 0.4 mM, f i n a l NADPH concentration 1 mM. RESULTS / 66 Figure 4. Erythromycin demethylase assay: absorbance (412 nm) versus incubation time in untreated male Sprague Dawley rat hepatic microsomes (a pool of 4 l i v e r s ) . Assay conditions: f i n a l protein concentration 0.34 mg/mL, f i n a l erythromycin concentration 0.4 mM, f i n a l NADPH concentration 1 mM. (J ^ -s 1| CD L " or CN O r -CD v — ' < 10 2 0 3 0 INCUBATION TIME ( m i n u t e s ) 4 0 R E S U L T S / 68 Figure 5. Erythromycin demethylase assay: absorbance (412 nm) versus incubation time in untreated female Sprague Dawley rat hepatic microsomes ( a pool of 4 l i v e r s ) . Assay conditions: f i n a l protein concentration 0 . 9 1 mg/mL, f i n a l erythromycin concentration 0.4 mM, f i n a l NADPH concentration 1 mM. 0 . 0 4 INCUBATION TIME ( m i n u t e s ) RESULTS / 70 Figure 6. Erythromycin demethylase assay: absorbance (412 nm) versus incubation time, showing the e f f e c t of a d d i t i o n a l NADPH midway through the incubation p e r i o d , i n untreated male Sprague Dawley r a t hepatic microsomes (a pool of 4 l i v e r s ) . Assay c o n d i t i o n s : f i n a l p r o t e i n c o n c e n t r a t i o n 0.43 mg/mL, f i n a l - erythromycin concentration 0.4 mM, f i n a l NADPH concentration 1 mM. 0.08 INCUBATION TIME ( m i n u t e s ) RESULTS / 72 Figure 7. Erythromycin demethylase assay: absorbance (412 nm) versus incubation time, showing the e f f e c t of a d d i t i o n a l NADPH midway through the incubation p e r i o d , i n untreated female Sprague Dawley r a t hepatic microsome (a pool of 4 l i v e r s ) . Assay c o n d i t i o n s : f i n a l p r o t e i n concentration 1.12 mg/mL, f i n a l erythromycin concentration 0.4 mM, f i n a l NADPH concentration 1 mM. 0.08 0.07 0 .06 0 .05 0.04 0 .03 0 .02 0.01 0 .00 O o-o" O" •O" .O' • o - Q •o-o- •o- •O' plus NADPH m i d w a y 0 10 20 30 40 50 INCUBATION TIME (m inu tes ) 60 R E S U L T S / 74 Figure 8. Erythromycin demethylase a c t i v i t y versus f i n a l NADPH concentration in untreated Sprague Dawley male rat hepatic microsomes (a pool of 4 livers'). Assay conditions: f i n a l protein concentration 0.34 mg/mL, f i n a l erythromycin concentration 0.4 mM, incubation time 20 minutes. ERYTHROMYCIN DEMETHYLASE ( n a n o m o l e s f o r m a l d e h y d e / m i n / m g p ro ie in ) 9L i s j / i n s a y RESULTS / 76 Figure 9. Erythromycin demethylase a c t i v i t y versus f i n a l NADPH concentration i n untreated Sprague Dawley female rat hepatic microsomes (a pool of 4 l i v e r s ) . Assay c o n d i t i o n s : f i n a l p r o t e i n concentration 0.73 mg/mL, f i n a l erythromycin concentration 0.4 mM, incubation time 20 minutes. 2 L J if) < _ J >-X CJ >-o or x t— >-or L J UJ I— o cc 0_ o 2 Q >-I U J Q _ J < cc: O u_ m U l _j o o 2 < 1 2 F ina l NADPH concen t ra t i on (mM) 93 W CO a r 02 RESULTS / 78 Figure 10. Erythromycin demethylase a c t i v i t y versus substrate concentration in untreated adult male Sprague Dawley rat hepatic microsomes (a pool of 4 l i v e r s ) . Assay conditions: f i n a l protein concentration 0.28-0.35 mg/mL, f i n a l NADPH concentration 1 mM, incubation time 20 minutes. Symbols indicate four separate experiments. In one of these experiments, the a c t i v i t y measured at 8 mM substrate did not d i f f e r from that at 1.6 mM substrate. 6Z. / s j / i n s a u RESULTS I 80 Figure 11. Erythromycin demethylase a c t i v i t y versus substrate concentration in untreated adult female Sprague Dawley rat hepatic microsomes (a pool of 4 l i v e r s ) . Assay conditions: f i n a l protein concentration 0.8-0.9 mg/mL, f i n a l NADPH concentration 1 mM, incubation time 20 minutes. Symbols indicate three separate experiments. "o V-C L OO E _) c x E Ld U J _C O <D ? O O E >- ^ O oo X o >-or 0.6 0.4H 0.2-ti 0 . 0 A A O 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 E R Y T H R O M Y C I N (mM) ?3 a CO a r H CO CO R E S U L T S / 82 Table V. Estimated Michaelis-Menten constants in untreated male Sprague Dawley rats for erythromycin demethylase in hepatic microsomes. Data i s shown as the estimated value ± S.E.M. (S.E.M. as a percentage of the estimated value). V EXPT ONE COMPONENT MODEL Vmax Km Red Chi -Square Vmax 1 TWO COMPONENT MODEL Kml Vmax2 Km2 Red Chi Square 1 1 .64+0 .02 0.18+0 .01 0 ,0015 3.53+0.0 6.9510.0 - 1.9110.0 -0.005910.0 0.0 ( 1 ) (6) 2 1.7 1+0 .05 0.09810 .01 0. .0036 0.69+2.9 0.07510.52 1.0212.86 0.12+0.44 0.0060 (3) ( 12> (420) (280) ( 367 ) 3 1 .46 + 0 .06 0.08310 ,01 0 ,0049 0.71010.001 0.0210.0003 0.90±0.002 0.3 110.002 0.0056 (4) (17) (0.1) (1) (0.2) (0.5) 4 1 . 54+0. 06 0.11+0. .02 0. 0053 0. 58±0,.001 0.022±0.0004 1.12±0.0017 0. 36±0.0017 0.0061 A VG 1 .59±0. 02 0.12+0. 02 0.65±0.10 0.02210.0 n CO G r H CO 00 CO RESULTS / 84 F i g u r e 12. E a d i e H o f s t e e p l o t f o r e r y t h r o m y c i n demethylase a c t i v i t y i n u n t r e a t e d a d u l t female Sprague Dawley r a t h e p a t i c microsomes. Assay c o n d i t i o n s , as f o r F i g u r e 11. RESULTS / 85 if) > > RESULTS / 86 Figure 13. Lineweaver-Burk plot for erythromycin demethylase a c t i v i t y in untreated adult female Sprague Dawley rat hepatic microsomes. Assay conditions, as for Figure 11. RESULTS / 8 7 ( u i a j o j d 6 u j / u i u j / s a | o u j o u D u ) A / I RESULTS / 88 T a b l e V I . E s t i m a t e d M i c h a e l i s - M e n t e n c o n s t a n t s f o r e r y t h r o m y c i n demethylase a c t i v i t y i n h e p a t i c microsomes from u n t r e a t e d a d u l t female Sprague Dawley r a t s . Data i s shown as the e s t i m a t e d v a l u e ± S.E.M. (S.E.M. as a p e r c e n t a g e of the e s t i m a t e d v a l u e ) . VI EXPT ONE Vmax COMPONENT MODEL Km Red Ch1 -Square Vmax 1 TWO Km1 COMPONENT MODEL Vmax2 Km2 Red Chi Square 1 0.31+0.013 0.027+0.005 0.00052 0.30±0.03 0.04010.009 0.02 10.03 -0.005 0.00029 (4) C 19) (8) (24) ( 109) (92) 2 0.29+0.007 0.010+0.002 0.00025 0.29+0.0002 0.0111 -0.00056+ -0 .0410.006 0.00029 0.00004 7 0.00012 (2) ( 15> (0. 1) (0.4) (21) ( 16) 3 0.46+0.04 0.09+0.02 0.001 2 0.3310.05 0. 1 110.05 0. 1410.05 0 .07+0.05 0.0015 (9) (28) ( 14) (43) (30) (79) 4 0.62+0.05 0.04±0.02 0.0050 0.74+0.004 0.022+0.001 -0.1110.003 •0.0491 0.0088 0.002 AVG 0.42+0.08 0.04+0.02 0.42±0.11 0.0510.02 0 0 C CO CO (5 RESULTS / 90 Figure 14. Eadie Hofstee plot for erythromycin demethylase a c t i v i t y in hepatic microsomes from untreated adult male Sprague Dawley rats. Assay condition, as in Figure 10. RESULTS / 91 O o o .O' .o o o LO o > + m o o CN m o m o o o > RESULTS / 92 Figure 15. Lineweaver-Burk p l o t f o r erythromycin demethylase a c t i v i t y i n hepatic microsomes from untreated adult male Sprague Dawley r a t s . Assay c o n d i t i o n s , as i n Figure 10. R E S U L T S / 93 ( U l 9 | 0 j d 6 U J / U I U J / S 8 | 0 L U 0 U D U ) A / I R E S U L T S / 94 F i g u r e 16. E r y t h r o m y c i n demethylase a c t i v i t y w i t h v a r i e d p o t a s s i u m phosphate b u f f e r s i n u n t r e a t e d male and female Sprague Dawley r a t h e p a t i c microsomes ( p o o l of 4 l i v e r s ) . Assay c o n d i t i o n s : f i n a l p r o t e i n c o n c e n t r a t i o n 0.34 mg/mL (male) or 0.8 mg/mL ( f e m a l e ) , f i n a l e r y t h r o m y c i n c o n c e n t r a t i o n 0.4 mM, f i n a l NADPH c o n c e n t r a t i o n 1 mM, i n c u b a t i o n t ime 20 mi n u t e s . B u f f e r s used: Na,K-phosphate 0.2 M = p o t a s s i u m phosphate monobasic, sodium phosphate d i b a s i c 0.2 M, pH 7.4; K,K-phosphate 0.1 M= p o t a s s i u m phosphate monobasic and d i b a s i c 0.1 M, pH 7.4; Na,K-phosphate 0.1 M= p o t a s s i u m phosphate monobasic, sodium phosphate d i b a s i c 0.2 M, pH 7.4. RESULTS / 95 ( u j a j . o j d 6 u j / u i u j / a p A u , 9 p | D U J J O j s a | o u j o u o u ) 3SV~1AH13W3Q NIOAWOcIHlAc!3 RESULTS / 96 Figure 17. Formaldehyde standard curve i n untreated male Sprague Dawley r a t l i v e r microsomes (a pool of 4 l i v e r s ) . Standard curves: Formaldehyde + buffer = formaldehyde i n Na,K-phosphate b u f f e r , 0.2 M, pH 7.4; Formaldehyde + a l l components = formaldehyde i n a t y p i c a l incubation mixture * c o n t a i n i n g untreated male hepatic microsomes ( f i n a l p r o t e i n c o n c e n t r a t i o n 0.35 mg/mL), f i n a l erythromycin concentration 0.4 mM, f i n a l NADPH concentration 1 mM, incubation time 20 minutes, magnesium c h l o r i d e 3 mM, semicarbazide 5 mM, i n Na,K-phosphate buffer 0.2 M, pH 7.4. Data shown represent the r e s u l t s of l i n e a r r e g r e s s i o n a n a l y s i s of d u p l i c a t e experiments. 0.1 8 L d <i CC O N O r -(/) CD ^ < 0.1 6 0 . 1 4 0.1 2 0.1 0 -0 . 0 8 -0 . 0 6 0 . 0 4 0 . 0 2 0 . 0 0 A f o r m a l d e h y d e + b u f f e r , C = 7 . 2 8 A 4 1 2 © f o r m a l d e h y d e + a l l c o m p o m C = 8 . 6 0 A 4 1 2 0 . 0 0 . 2 0 .4 0 .6 0 . 8 1 .0 .2 F O R M A L D E H Y D E C O N C E N T R A T I O N g ( M g / 2 m L ) g r H CO RESULTS / 98 Figure 18. Formaldehyde standard curve in untreated female Sprague Dawley rat l i v e r microsomes (a pool of 4 l i v e r s ) . Standard curves: Formaldehyde + buffer = formaldehyde in Na,K-phosphate buffer, 0.2 M, pH 7.4; Formaldehyde + a l l components = formaldehyde in a t y p i c a l incubation mixture containing untreated female hepatic microsomes ( f i n a l protein concentration 0.80 mg/mL), f i n a l erythromycin concentration 0.4 mM, f i n a l NADPH concentration 1 mM, incubation time 20 minutes, magnesium chloride 3 mM, semicarbazide 5 mM, in Na,K-phosphate buffer 0.2 M, pH 7.4. Data shown represent the results from li n e a r regression analysis of duplicate experiments. A f o r m a l d e h y d e + b u f f e r , C = 6 . 9 6 A 4 1 2 © f o r m a l d e h y d e + a l l c o m p o n e n t s , C = 8 . 7 6 A 4 1 2 0.0 1 .2 F O R M A L D E H Y D E C O N C E N T R A T I O N C a g / 2 m L ) RESULTS / 100 3.2. ERYTHROMYCIN DEMETHYLASE STABILITY Since erythromycin demethylase a c t i v i t y would be assayed in hepatic microsomes which had been stored at -80°C for several months, the s t a b i l i t y of this enzyme a c t i v i t y over time was investigated; Hepatic microsomes from untreated adult female Sprague Dawley rats were assayed for erythromycin demethylase a c t i v i t y immediately after preparation, then stored at -80°C. As shown in Figure 19, the erythromycin demethylase a c t i v i t y in aliquots sampled after being frozen, and then thawed at various time points, was unchanged for up to 300 days (10 months). R E S U L T S / 101 Figure 19. Erythromycin demethylase a c t i v i t y : s t a b i l i t y i n adul t female Sprague Dawley r a t hepatic microsomes stored at -80°C (a pool of 4 l i v e r s ) . Assay c o n d i t i o n s : f i n a l p r o t e i n c o n c e n t r a t i o n 0.86 mg/mL, erythromycin 0.4 mM, NADPH 1mM, incubation time 20 minutes. , c a l c u l a t e d average; , ± one standard d e v i a t i o n from the average. E R Y T H R O M Y C I N D E M E T H Y L A S E (nanomoles formaldehyde/min/mg protein) O O O O O O Q ET1 0 1 o CO NJ O O _ o NO O O O O O O O o o cn 20i / sxmsay RESULTS / 103 3.3. THE PILOT STUDY 3.3.1. Pubertal Testosterone Treatment in Ovariectomized Females Since the research question was primarily concerned with the effe c t of pubertal testosterone on the adult androgen responsiveness of hepatic microsomal erythromycin demethylase a c t i v i t y in prepubertally ovariectomized female rats, the data from only the four groups s p e c i f i c a l l y related to thi s question were examined separately (Table VII, Appendix Table A, Figures 20 and 21). As well, other comparisons were of interest when a l l study groups were considered (Table VIII, Appendix Table B, Figures 20 and 21 ). 3.3.1.1. Hepatic Microsomal Erythromycin Demethylase Activity Hepatic microsomal erythromycin demethylase a c t i v i t y was calculated on the basis of both microsomal protein and microsomal P450 concentration. The results are presented in Table VII and Figures 20 and 21. A two-way ANOVA (2x2) was performed (Appendix Figure 2), with pubertal testosterone and adult testosterone as the two factors. A s t a t i s t i c a l l y s i g n i f i c a n t interaction between these two factors was not detected (p=0.09l (protein), p=0.058 (P450), however each factor alone had a s i g n i f i c a n t e f f e c t (p=0.0l2 and 0.024 (protein), p=0.0l5 and 0.014 (P450). By multiple comparison RESULTS / 104 test, erythromycin demethylase a c t i v i t y was s i g n i f i c a n t l y higher in prepubertally ovariectomized female rats which were exposed to testosterone both peripubertally and in adulthood, when compared to ovariectomized females treated with testosterone during either period alone. 3.3.1.2. Cytochrome P450 and Protein The e f f e c t s of peripubertal and adult testosterone on the content of P450 and of protein in the l i v e r were examined for the four prepubertally ovariectomized female groups, as shown in Appendix Table A. Neither peripubertal testosterone, adult testosterone nor the combination of the two treatments s i g n i f i c a n t l y altered microsomal P450 (nmoles P450/mg protein), the hepatic P450 (P450/g wet weight of li v e r ) or the hepatic protein content (mg protein/g wet weight of l i v e r ) in these groups. 3.3.2. Al l Treatment Groups 3.3.2.1. Hepatic Microsomal Erythromycin Demethylase Activity Erythromycin demethylase a c t i v i t y was measured in individual hepatic microsomal samples from study animals in a l l treatment groups. These results are presented based on microsomal protein concentration as well as based on cytochrome P450 concentration (Table VIII, Figures 22 and RESULTS / 105 23). By either method of c a l c u l a t i o n , mean hepatic microsomal erythromycin demethylase a c t i v i t y was higher, approximately 2 - fol d , in control males than in control females. Oneway ANOVA indicated a s i g n i f i c a n t difference between female treatment groups for both c a l c u l a t i o n s . By either c a l c u l a t i o n , ovariectomy on day 25 of age resulted in a 38% decrease in mean hepatic microsomal erythromycin demethylase a c t i v i t y from control female l e v e l s , however this decrease was not s t a t i s t i c a l l y s i g n i f i c a n t , either by ANOVA or by Student's T-test. Neither e s t r a d i o l nor testosterone administration in any single time period s i g n i f i c a n t l y altered mean erythromycin demethylase a c t i v i t y compared with control females. The combination of peripubertal testosterone and adult testosterone treatments in ovariectomized females s i g n i f i c a n t l y increased erythromycin demethylase a c t i v i t y compared to ovariectomized females not given testosterone by 103% (by protein) and by 106% (by P450). The hepatic microsomal erythromycin demethylase a c t i v i t y in ovariectomized females given both peripubertal and adult testosterone did not d i f f e r s t a t i s t i c a l l y from that of control females. The hepatic microsomal erythromycin demethylase a c t i v i t y in prepubertally ovariectomized females given both testosterone treatments reached 57% (by protein) RESULTS / 106 and 68% (by P450) of average control male a c t i v i t y . The mean hepatic microsomal erythromycin demethylase a c t i v i t y in the group treated with peripubertal e s t r a d i o l and adult testosterone did not d i f f e r s t a t i s t i c a l l y from that of the ovariectomized female group. 3.3.2.2. Liver Cytochrome P450 and Liver Protein Both protein and P450 concentrations in the l i v e r s from study animals were assessed. These results were analysed by one-way ANOVA. As shown in Appendix Table B, no s i g n i f i c a n t differences were found between the d i f f e r e n t study groups with respect to hepatic microsomal P450 content (nmoles cytochrome P450/mg microsomal protein). S i m i l a r l y , the amount of P-450 or protein per gram wet weight of l i v e r did not d i f f e r between the treatment groups. 3.3.2.3. Body and Liver Weights The mean body and l i v e r weights at the conclusion of the study are shown in Appendix Tables B and C. One-way ANOVA and multiple comparison tests revealed many differences between the female treatment groups for these parameters. Of interest was the observation that the average body weight and l i v e r weight in the prepubertally ovariectomized female group treated with both pubertal and adult testosterone were RESULTS / 107 s i g n i f i c a n t l y h i g h e r than observed in the c o n t r o l female group , a l t h o u g h the average l i v e r weight as a percentage of t o t a l body weight was not d i f f e r e n t . RESULTS / 108 Table VII. P i l o t study: the influence of pubertal testosterone on ovariectomized female rat l i v e r hepatic microsomal erythromycin demethylase a c t i v i t y . Data i s presented as mean ±SEM (n=4), and i s also included in Table VIII. Abbreviations: EDM, hepatic microsomal erythromycin demethylase a c t i v i t y ; per protein, nmoles formaldehyde/min/mg protein; per P450, nmoles formaldehyde/min/nmole P450; Fern, female; Gx, prepubertally ovariectomized; Ta, adult testosterone treatment; Tp, peripubertal testosterone treatment; "*" indicates a s i g n i f i c a n t difference from a l l other groups in that row (p<0.05, two-way ANOVA). TREATMENT GROUP Fern Fern Fern Fern Gx GxTa GxTp GxTpTa EDM * per protein 0.29±0.05 0.33±0.04 0.35±0.04 0.59±0.08 EDM per P450 * 0 .36±0 .08 0 . 4 1 ± 0 . 0 4 0 . 4 1 ± 0 . 0 4 0 . 7 4 ± 0 . 1 0 RESULTS / 109 Figure 20. P i l o t study: erythromycin demethylase calculated on the basis of microsomal protein in ovariectomized female rats (mean ± S.E.M., n=4). These results were also included in Figure 22. "*" indicates a s i g n i f i c a n t difference from a l l other treatment groups (p<0.05). Animals received the following treatments: Gx Fern: gonadectomy day 25, corn o i l days 35-50, corn o i l days 81-90. GxTa Fern: gonadectomy day 25, corn o i l days 35-50, testosterone enanthate in corn o i l 2.5 jumoles/kg/day days 81-90. GxTp Fern: gonadectomy day 25, testosterone enanthate in corn o i l 5 Mmoles/kg/day days 35-50, corn o i l days 81-90. GxTpTa Fern: gonadectomy day 25, testosterone enanthate in corn o i l 5 Mmoles/kg/day days 35-50, testosterone enanthate in corn o i l 2.5 jimoles/kg/day days 81-90. E R Y T H R O M Y C I N D E M E T H Y L A S E ( n a n o m o l e s f o r m a l d e h y d e / m i n / m g protein) O i l / SJ / inS3H R E S U L T S / 111 Figure 21. P i l o t study: erythromycin demethylase calculated on the basis of cytochrome P450 content in ovariectomized females, mean +S.E.M., n=4) "*" indicates a s i g n i f i c a n t difference from a l l other treatment groups (p<0.05). These results were also included in Figure 23. Animals received the following treatments: Gx Fern: gonadectomy day 25, corn o i l days 35-50, corn o i l days 81-90. GxTa Fern: gonadectomy day 25, corn o i l days 35-50, testosterone enanthate in corn . o i l 2.5 Mmoles/kg/day days 81-90. GxTp Fern: gonadectomy day 25, testosterone enanthate in corn o i l 5 Mmoles/kg/day days 35-50, corn o i l days 81-90. GxTpTa Fern: gonadectomy day 25, testosterone enanthate in corn o i l 5 Mmoles/kg/day days 35-50, testosterone enanthate in corn o i l 2.5 Mmoles/kg/day days 81-90. E R Y T H R O M Y C I N D E M E T H Y L A S E n a n o m o l e s f o r m a l d e h y d e / m i n / n m o l e c y t o c h r o m e P - 4 5 0 ) CD CL 3 o o o rO O O CO O CO O 211 / sxmsay RESULTS / 113 Table VIII. P i l o t study: hepatic microsomal erythromycin demethylase a c t i v i t y in a l l treatment groups. Data i s presented as mean ±S.E.M. (n=4). Abbreviations: EDM, erythromycin demethylase; per protein, nmoles formaldehyde/min/mg protein; per P450, nmoles formaldehyde/min/nmole P450; Fern, female; Gx, prepubertal ovariectomy; Ep, pubertal e s t r a d i o l ; Ta, adult testosterone; Tp, peripubertal testosterone. "*" indicates a s i g n i f i c a n t difference from the mean for the Fern Gx, FemGxTa, FemGxTp and FemGxEp groups in that row (one-way ANOVA, female groups, p<0.05). Tao 1 e v III . CON CON F EM FEM FEM F EM FEM FEM MALE FEM Gx GxTa GxTp GxTpTa GxEp GxEpTa E OM * per 1.03±0.13 0.47+0.07 0.29±0.05 0.33+0.04 0.35+0.04 0.59±0.08 0.36+0.04 0.46+0.05 p r o t e i n EDM per P450 1.09+0.16 0.58+0.10 0.36+0.08 0.41+0.04 0.41+0.04 0.74+0.10 0.42+.0.03 0.57+0.04 70 PJ Co C r GO RESULTS / 115 Figure 22. P i l o t study: Hepatic microsomal erythromycin demethylase calculated on the basis of microsomal protein in a l l groups, (mean ±S.E.M., n=4). "*" indicates a s i g n i f i c a n t difference from the mean for groups FemGx, FemGxTa, FemGxTp, and FemGxEp (one-way ANOVA, female groups, p<0.05). Animals received the following treatments: Con Male: sham gonadectomy day 25, corn o i l days 35-50, corn o i l days 81-90. Con Fern: sham gonadectomy day 25, corn o i l days 35-50, corn o i l days 81-90. Gx Fern: gonadectomy day 25, corn o i l days 35-50, corn o i l days 81-90. GxTa Fern: gonadectomy day 25, corn o i l days 35-50, testosterone enanthate in corn o i l 2.5 Mmoles/kg/day days 81-90. GxTp Fern: gonadectomy day 25, testosterone enanthate in corn o i l 5 Mmoles/kg/day days 35-50, corn o i l days 81-90. GxTpTa Fern: gonadectomy day 25, testosterone enanthate in * corn o i l 5 Mmoles/kg/day days 35-50, testosterone enanthate in corn o i l 2.5 Mmoles/kg/day days 81-90. GxEp Fem: gonadectomy day 25, e s t r a d i o l benzoate in corn o i l 1.5 Mmoles/kg on alternate days, days 35-50, corn o i l days 81-90. GxEpTa Fem: gonadectomy day 25, e s t r a d i o l benzoate in corn o i l 1.5 Mmoles/kg on alternate days, days 35-50, testosterone enanthate in corn o i l 2.5 Mmoles/kg/day days 81-90. E R Y T H R O M Y C I N D E M E T H Y L A S E ( n a n o m o l e s f o r m a l d e h y d e / m i n / m g p r o t e i n ) 9 i i / s a / i n s a y RESULTS / 117 Figure 23. P i l o t study: hepatic microsomal erythromycin demethylase a c t i v i t y calculated on the basis of P450 content in a l l groups. Values are expressed as mean ±S.E.M. (n=4). "*" indicates a s i g n i f i c a n t difference from the means for groups FemGx, FemGxTa, FemGxTp and FemGxEp (one-way ANOVA, female groups, p<0.05). Animals received the following treatments: Con Male: sham gonadectomy day 25, corn o i l days 35-50, corn o i l days 81-90. Con Fern: sham gonadectomy day 25, corn o i l days 35-50, corn o i l days 81-90. Gx Fern: gonadectomy day 25, corn o i l days 35-50, corn o i l days 81-90. GxTa Fern: gonadectomy day 25, corn o i l days 35-50, t e s t o s t e r o n e enanthate in corn o i l 2.5 M m o l e s/kg/day days 81-90. GxTp Fern: gonadectomy day 25, t e s t o s t e r o n e enanthate in corn o i l 5 M m o l e s/kg/day days 35-50, corn o i l days 81-90. GxTpTa Fern: gonadectomy day 25, testosterone enanthate in corn o i l 5 M m o l e s/kg/day days 35-50, t e s t o s t e r o n e enanthate in corn o i l 2.5 M m o l e s/kg/day days 81-90. GxEp Fern: gonadectomy day 25, e s t r a d i o l benzoate in corn o i l 1.5 M i n o l e s/kg on alternate days, days 35-50, corn o i l days 81-90. GxEpTa Fern: gonadectomy day 25, e s t r a d i o l benzoate in corn o i l 1.5 M m o l e s / k g on alternate days, days 35-50, t e s t o s t e r o n e enanthate in corn o i l 2.5 M m o l e s/kg/day days 81-90. E R Y T H R O M Y C I N D E M E T H Y L A S E ( n a n o m o l e s f o r m a l d e h y d e / m i n / n m o l e c y t o c h r o m e P — 4 5 0 ) RESULTS / 119 3.4. THE MAJOR STUDY 3.4.1. Prepubertally Ovariectomized Female Rats Since the research question primarily concerned the influence of peripubertal testosterone on adult androgen responsiveness in prepubertally ovariectomized females, only those four groups s p e c i f i c a l l y addressing t h i s question were considered f i r s t . 3.4.1.1. Hepatic Microsomal Erythromycin Demethylase Activity Erythromycin demethylase a c t i v i t y was calculated on the basis of both microsomal protein and microsomal P450 in the incubation mixture. The results are shown in Table IX and Figures 24 and 25. A two-way ANOVA (2 by 2, pubertal testosterone by adult testosterone, Appendix Figure 3) indicated a s i g n i f i c a n t interaction (p=0.003, power=0.99) between pubertal testosterone and the response to adult testosterone in erythromycin demethylase a c t i v i t y in ovariectomized females when a c t i v i t y was calculated on the basis of protein. A s i g n i f i c a n t interaction was not indicated (p=0.217, power<0.3) when erythromycin demethylase a c t i v i t y was calculated on the basis of microsomal P450. By either c a l c u l a t i o n , both pubertal testosterone and adult testosterone s i g n i f i c a n t l y (p=0.000) affected erythromycin demethylase a c t i v i t y , and multiple comparison testing RESULTS / 120 indicated that the combination of both treatments resulted in a s i g n i f i c a n t l y higher erythromycin demethylase a c t i v i t y compared with the other three groups (Figures 24 and 25). Erythromycin demethylase a c t i v i t y was 77% higher (by either calculation) -in ovariectomized females which received both adult and peripubertal testosterone than in ovariectomized females which were not given testosterone. The pattern of erythromycin demethylase a c t i v i t y within these four groups was similar to that seen in the p i l o t study (Figure 26). 3.4.1.2. Cytochrome P450 and Protein Since hepatic microsomal erythromycin demethylase a c t i v i t y results d i f f e r e d s l i g h t l y depending on the method of calc u l a t i o n used, the effects of peripubertal and adult testosterone on hepatic microsomal P450 and on l i v e r P450 and protein content in the four peripubertally-ovariectomized groups were examined. As shown in Table IX, the protein content of the l i v e r was unaffected by testosterone treatment in ovariectomized females. Testosterone treatments did have an effect on the P450 content of the l i v e r . By two-way ANOVA (2 by 2, adult testosterone and peripubertal testosterone), a s i g n i f i c a n t interaction was indicated. The combined pubertal and adult testosterone treatment resulted in a l i v e r P450 content which was s i g n i f i c a n t l y higher than that following either RESULTS / 121 testosterone treatment alone. Hepatic microsomal P450 content (nmoles P450/mg p r o t e i n , Table IX) r e f l e c t e d the l i v e r P450 con c e n t r a t i o n s , however no s i g n i f i c a n t d i f f e r e n c e s were detected. These r e s u l t s w i l l t h e r e f o r e a f f e c t the c a l c u l a t i o n of hepatic microsomal erythromycin demethylase a c t i v i t y per nmole P450. 3.4.1.3. Body and Liver Weights Body and l i v e r weights i n the four ovariectomized female r a t groups at the conclusion of the study are shown i n Table IX. By two-way ANOVA, adult testosterone s i g n i f i c a n t l y increased both body weight and l i v e r weight i n the p r e p u b e r t a l l y ovariectomized female animals. When l i v e r weight was considered as a percentage of body weight, however, no s i g n i f i c a n t e f f e c t s of testosterone treatments were i n d i c a t e d . 3.4.1.4. Plasma Estradiol and Testosterone The plasma e s t r a d i o l and testosterone concentrations at the conc l u s i o n of the study f o r the four main study groups, the pr e p u b e r t a l l y ovariectomized females, are shown i n Table IX. Within these ovariectomized female groups, the plasma e s t r a d i o l l e v e l s d i d not d i f f e r according to treatment. Mean plasma testosterone was high i n groups which had been t r e a t e d with testosterone i n adulthood, j u s t p r i o r to the RESULTS / 122 end of the study, indicating high c i r c u l a t i n g levels as expected. Plasma testosterone l e v e l s were s i g n i f i c a n t l y higher in the group which received both peripubertal and adult testosterone than in the group which received only adult testosterone. Plasma testosterone l e v e l s were not detectable in those groups which did not receive adult testosterone. 3.4.2. All Treatment Groups 3.4.2.1. Erythromycin Demethylase Activity Erythromycin demethylase a c t i v i t y in a l l twelve treatment groups i s presented in Table X and Figures 27 and 28, calculated on the basis of both microsomal protein and microsomal P450. S t a t i s t i c a l analysis was done using two-way ANOVA (2 x 6, sex by treatment) followed by multiple comparison te s t i n g . Group variances were found to d i f f e r , however the ANOVA procedure i s considered to be "robust" and r e l a t i v e l y unaffected by nonhomogeneous variances when the number of cases per group is approximately equal, as in thi s study (Norusis, 1988). Consistent with t h i s i s the observation that when the data were transformed to reduce variance inequality, as shown in Appendix Tables D and E, the s t a t i s t i c a l results were not greatly d i f f e r e n t from the untransformed r e s u l t s . By a l l methods of cal c u l a t i o n used, RESULTS / 123 both sex and treatment were s i g n i f i c a n t and interactive factors, thus the erythromycin demethylase response to treatment depended upon the sex of the animal. The results of multiple comparison testing are shown in Tables XI and XII. The following comparisons were of inter e s t . Erythromycin demethylase a c t i v i t y was s i g n i f i c a n t l y higher in control males than in control females, 1.6 fo l d (by protein) and 1.5 f o l d (by P450). Nongonadectomized males and females responded d i f f e r e n t l y to adult testosterone administration: erythromycin demethylase a c t i v i t y increased in the males but not in the females. This indicated the presence of- an androgen response system in the adult males which was not observed in the adult female. Further, the increase in the males showed that the control males were not expressing erythromycin demethylase a c t i v i t y at their maximum potential l e v e l . In the female rats, prepubertal ovariectomy appeared to decrease erythromycin demethylase a c t i v i t y by 37% (by protein) and 40% (by P450) from control female l e v e l s , however t h i s difference was not s t a t i s t i c a l l y s i g n i f i c a n t . This decrease became s t a t i s t i c a l l y s i g n i f i c a n t when the group variances were made more homogeneous by data transformation (Appendix Tables D & E) , The administration RESULTS / 124 of both peripubertal and adult testosterone to prepubertally-ovariectomized females resulted in an erythromycin demethylase a c t i v i t y which did not d i f f e r from that of control females, and reached 31% (by protein) and 29% (by P450) of control male l e v e l s . In the male rats, prepubertal castration reduced erythromycin demethylase a c t i v i t y by 58% (by protein) and 51% (by P450), a s t a t i s t i c a l l y s i g n i f i c a n t difference. The erythromycin demethylase a c t i v i t y in- .the prepubertally castrated males was the same as the erythromycin demethylase a c t i v i t y in the prepubertally ovariectomized females and in control females. Administration of testosterone to castrated males around the time of puberty resulted in an increase in erythromycin demethylase a c t i v i t y by 52% (by protein) and 31% (by P450) over castrated l e v e l s , however these lev e l s were not s i g n i f i c a n t l y d i f f e r e n t . S i m i l a r l y , adult testosterone administration alone in prepubertally castrated males increased mean erythromycin demethylase a c t i v i t y (45% by protein, 41% by P450) but the le v e l s were not s t a t i s t i c a l l y d i f f e r e n t . The l e v e l of hepatic microsomal erythromycin demethylase a c t i v i t y in prepubertally castrated male rats treated with adult testosterone was s i g n i f i c a n t l y lower than that of control males and of control males treated with adult testosterone. The administration of both RESULTS / 125 peripubertal and adult testosterone to castrated males resulted in a s i g n i f i c a n t increase in erythromycin demethylase a c t i v i t y over that of castrated males which did not receive testosterone, by 125% (by protein) and 83% (by P450). The mean erythromycin demethylase a c t i v i t y in those animals treated with both periods of testosterone did not d i f f e r from that of the control males, or with that of castrated males treated with adult testosterone. The combination treatment produced s i g n i f i c a n t l y higher mean erythromycin demethylase a c t i v i t y than seen in the group treated with pubertal testosterone only, when calculated on the basis of microsomal protein. The combination treatment also produced higher hepatic microsomal erythromycin demethylase a c t i v i t y in the prepubertally castrated males than in the prepubertally ovariectomized females. The results for the prepubertally castrated male rats were analyzed s t a t i s t i c a l l y by two-way ANOVA (2x2, pubertal testosterone by adult testosterone) in order to compare these results with those of the ovariectomized females. By either c a l c u l a t i o n , the combined testosterone treatments did not have an interactive e f f e c t on hepatic microsomal erythromycin demethylase a c t i v i t y in the castrated male rats (p=0.499 (protein), p=0.732 (P450)), however each testosterone treatment alone had a s i g n i f i c a n t e f f e c t RESULTS / 126 (p=0.004 and 0.007 (protein), p=0.023 and 0.004 (p450)). 3.4.2.2. Microsomal P450, Liver P450 and Protein Since differences were observed in erythromycin demethylase results depending upon the ca l c u l a t i o n used, differences in P450 and protein concentrations were investigated. These results are presented in Table X. The microsomal P450 content was analysed by two-way ANOVA (2 x 6, sex by treatment). Both sex and treatment were s i g n i f i c a n t factors. Microsomal P450 content was s i g n i f i c a n t l y higher following adult testosterone treatment of both control males and castrated males given pubertal testosterone in comparison with adult testosterone-treated gonadectomized females and males, castrated males or ovariectomized females treated with pubertal testosterone. These microsomal differences largely r e f l e c t e d changes in l i v e r P450 content in response to both sex and treatment and the interaction of these two factors. In contrast, l i v e r protein content was affected by sex only, with control male leve l s being s i g n i f i c a n t l y lower than those of control females and ovariectomized females given peripubertal testosterone or both testosterone treatments. RESULTS / 127 3.4.2.3. Body and Liver Weights Mean animal body and l i v e r weights at the conclusion of the study are shown i n Tables X, X I I I , and XIV. Both parameters d i s p l a y e d d i f f e r e n c e s according to both sex and treatment. However, when l i v e r weight was considered as a percentage of body weight, only treatment had a s i g n i f i c a n t e f f e c t . The most c o n s i s t e n t d i f f e r e n c e of those observed was a s i g n i f i c a n t decrease i n l i v e r weight as a percentage of body weight f o l l o w i n g gonadectomy i n both males and females compared with c o n t r o l s . 3.4.2.4. Plasma Estradiol I n d i v i d u a l e s t r a d i o l l e v e l s were measured i n plasma samples obtained from female and c o n t r o l male animals at the concl u s i o n of the study, twenty-four hours a f t e r any i n j e c t i o n s . The mean plasma e s t r a d i o l l e v e l s f or these treatment groups are shown i n Figure 29. Plasma e s t r a d i o l l e v e l s i n normal Sprague Dawley female r a t s range from 20 to 330 pg/mL, p r o g r e s s i v e l y i n c r e a s i n g to proestrous (Dohler & Wuttke, 1975). C o n t r o l female l e v e l s i n t h i s study were at the lower end of t h i s normal range. Plasma e s t r a d i o l l e v e l s i n ovariectomized groups were s i g n i f i c a n t l y lower than l e v e l s i n the c o n t r o l female group, RESULTS / 128 thus confirming the expected e f f e c t s of surgery. The lower e s t r a d i o l l e v e l s i n the ovariectomized females were comparable to the l e v e l s detected i n the c o n t r o l males. The ad m i n i s t r a t i o n of testosterone i n adulthood d i d not a l t e r the plasma e s t r a d i o l l e v e l s s i g n i f i c a n t l y . 3.4.2.5. Plasma Testosterone Plasma testosterone l e v e l s from i n d i v i d u a l animals were measured i n blood samples taken 24 hours a f t e r the l a s t i n j e c t i o n . The average r e s u l t s i n each treatment group are shown i n Figure 30. Normal ad u l t male rat l e v e l s of plasma testosterone range from l e s s than 1 to approximately 7 ng/mL, and f l u c t u a t e widely; t h i s i s p a r t l y due to a d a i l y t r i m o d a l rhythm (Bartke et al., 1973; Mynson & L i u , 1973; Kalva & K a l r a , 1977; Mocta et al., 1978). Normal adult female Sprague Dawley r a t s have plasma testosterone l e v e l s on the order of 0.2-0.4 ng/mL (Dohler & Wuttke, 1975). Observed mean plasma testosterone l e v e l s i n c o n t r o l males i n t h i s study were w i t h i n the normal range, at the lower end, while female l e v e l s were below the d e t e c t i o n l i m i t s of the assay. Plasma testosterone l e v e l s were examined f o r v e r i f i c a t i o n of c a s t r a t i o n i n the male treatment groups. This data was only RESULTS / 129 useful for confirmation of surgery in some of the prepubertally castrated male groups, since in a l l groups injected with testosterone days 81-90, just prior to blood sampling, the mean plasma testosterone lev e l s were elevated, indicating the persistence of injected testosterone. Three animals were removed from the study on the basis of plasma testosterone measurements outside the range of two standard errors of the mean for their group. Other differences in plasma testosterone were of interest (Table XV). Administration of testosterone in adulthood to males or females in a l l cases resulted in s i g n i f i c a n t l y higher mean plasma testosterone levels than those of the control male group, although s t i l l within the normal range stated above. For plasma testosterone l e v e l s , the response to adult testosterone administration appeared to depend both on sex and on exposure to pubertal testosterone. Higher leve l s were detected in males and in animals which received pubertal testosterone i n j e c t i o n s . This data suggested a difference in handling of the injected testosterone related to these factors. Further, the mean plasma testosterone levels in ovariectomized females were equivalent to those in RESULTS / 130 gonadectomized males. 3.4.2.6. Erythromycin Demethylase Activity versus Plasma Testosterone The rel a t i o n s h i p between hepatic microsomal erythromycin demethylase a c t i v i t y and plasma testosterone levels was investigated, using data from a l l study animals. A si g n i f i c a n t c o r r e l a t i o n was found between erythromycin demethylase a c t i v i t y , when calculated on the basis of microsomal protein or P450, and plasma testosterone. Linear regression analysis f i t the lin e s erythromycin demethylase (per protein)=0.41 + 0.07 (±0.013 SEM) x plasma testosterone (R =0.22, p=0.000, Figure 31) and erythromycin demethylase (per P450)=0.37 + 0.045 (±0.009 SEM) x plasma testosterone (R =0.21, p=0.000). The intercepts indicate that a portion of hepatic microsomal erythromycin demethylase a c t i v i t y was not related to plasma testosterone l e v e l s . A corr e l a t i o n was not detected between the r a t i o of plasma testosterone to plasma e s t r a d i o l and hepatic microsomal erythromycin demethylase a c t i v i t y . RESULTS / 131 Table IX. Major study results in prepubertally ovariectomized female Sprague Dawley rats. Values are expressed as mean ± S.E.M. (n=9). These results are also presented in Table X. Abbreviations: EDM, erythromycin demethylase; per protein, per nmole formaldehyde/min/mg protein; per P450, per nmole formaldehyde/min/nmole P450; Fern, female; Gx, prepubertal ovariectomy; Ta, adult testosterone treatment; Tp, peripubertal testosterone treatment; ND, none detected. "*" indicates a s i g n i f i c a n t difference from a l l treatment groups in that row (p<0.05). "**" indicates a s i g n i f i c a n t difference from FemGxTa and Fern GxTp groups in that row (p<0.05). RESULTS / 132 Fem Gx TREATMENT Fem GxTa GROUPS Fem GxTp Fem GxTpTa EDM per protein 0.31±0.06 0.34±0.04 0.33±0.02 * 0.55±0.03 EDM per P450 0.26±0.02 0.32±0.03 0.33±0.03 * 0.47±0.04 mg Protein per g wet weight of l i v e r 80±4 78±3 84±3 87±2 nmoles P450 per g wet weight of l i v e r 95±5 82±4 87±6 ** 103±3 nmoles P450 per mg protein 1 .19±0.04 1.06±0.04 1 .04±0.07 1.18±0.02 Body Weight(g) 346±5 360±10 328±5 350±12 Liver Weight(g) 10.6±0.3 1 1 .5±0.3 10.5±0.2 11 .6±0.5 Liv/Body % 3.07±0.06 3. 19±0.05 3.19±0.07 3.31±0.19 Plasma Testos-terone (ng/mL) ND * 2.6±0.3 ND * 4.0±0.2 Plasma E s t r a d i o l (pg/mL) 1 9±1 18±1 1 6±1 1 5±1 RESULTS / 133 Figure 24. Major Study: Erythromycin demethylase a c t i v i t y : c a l c u l a t e d as nanomoles formaldehyde/min/mg microsomal p r o t e i n i n p r e p u b e r t a l l y ovariectomized female Sprague Dawley r a t s (mean ± standard e r r o r of the mean, n=9). "*" i n d i c a t e s a s i g n i f i c a n t d i f f e r e n c e from a l l other treatment groups shown (p<0.05) . Animals received the f o l l o w i n g treatments: Fem Gx: gonadectomy day 25, corn o i l days 35-50, corn o i l days 81-90. Fem GxTa: gonadectomy day 25, corn o i l days 35-50, testosterone enanthate i n corn o i l 5.0 Mmoles/kg/day days 81-90. Fem GxTp: gonadectomy day 25, testosterone enanthate i n corn o i l 5 Mmoles/kg/day days 35-50, corn o i l days 81-90 . Fem GxTpTa: gonadectomy day 25, testosterone enanthate i n corn o i l 5 Mmoles/kg/day days 35-50, testosterone enanthate i n corn o i l 5.0 Mmoles/kg/day days 81-90. T R E A T M E N T G R O U P RESULTS / 135 Figure 25. Major Study: Erythromycin demethylase a c t i v i t y per P450 i n pre p u b e r t a l l y - o v a r i e c t o m i z e d female Sprague Dawley r a t s (mean ± standard e r r o r of the mean, n=9). "*" i n d i c a t e s a s i g n i f i c a n t d i f f e r e n c e from a l l other treatment groups shown (p<0.05) . Animals received the f o l l o w i n g treatments: Fem Gx: gonadectomy day 25, corn o i l days 35-50, corn o i l days 81-90 . Fem GxTa: gonadectomy day 25, corn o i l days 35-50, testosterone enanthate i n corn o i l 5.0 Mmoles/kg/day days 81-90. Fem GxTp: gonadectomy day 25, testosterone enanthate i n corn o i l 5 Mmoles/kg/day days 35-50, corn o i l days 81-90 . Fem GxTpTa: gonadectomy day 25, testosterone enanthate i n corn o i l 5 Mmoles/kg/day days 35-50, testosterone enanthate i n corn o i l 5.0 Minoles/kg/day days 81-90. E R Y T H R O M Y C I N D E M E T H Y L A S E ( n a n o m o l e s f o r m a l d e h y d e / m i n / n a n o m o l e P - 4 5 0 ) 9 c i / s j / m s a u RESULTS / 137 Figure 26. Comparison of erythromycin demethylase a c t i v i t y in the p i l o t study and major study in prepubertally ovariectomized female Sprague Dawley rats (mean ± standard error of the mean, 4 (p i l o t ) or 9 (major) animals per group). Erythromycin demethylase a c t i v i t y i s expressed either per protein (nanomoles formaldehyde/min/mg mmicrosomal protein) or per P450 (nanomoles formaldehyde/min/nanomole microsomal P450). "*" indicates a s i g n i f i c a n t difference from Fem Gx, Fem GxTa, and Fem GxTp treatment groups (p<0.05). Animals received the- following treatments: Fem Gx: gonadectomy day 25, corn o i l days 35-50, corn o i l days 81-90. Fem GxTa: gonadectomy day 25, corn o i l days 35-50, testosterone enanthate in corn o i l 2.5 ( p i l o t study) or 5.0 (major study) mmoles/kg/day days 81-90. Fem GxTp: gonadectomy day 25, testosterone enanthate in corn o i l 5 Mmoles/kg/day days 35-50, corn o i l days 81-90. Fem GxTpTa: gonadectomy day 25, testosterone enanthate in corn o i l 5 Mmoles/kg/day days 35-50, testosterone enanthate in corn o i l 2.5 .(pilot study) or 5.0 (major study) Mmoles/kg/day days 81-90. E R Y T H R O M Y C I N D E M E T H Y L A S E o o o o o O M 4^ c n 00 CD x CD —I x Q + CD x X X T ? X X X X X X X X I — • + CD x + / / / /~7~ •wiimw-x / y / -r / / / / / / / 33S995BBS9—-< / / / / / / / / / / / / / -(^xxxxxxxxxxxxxx^rx^ 0 0 o c Q . -< rt> ~\ ~D I Ui O 3? o~ c Cu -< (T) -\ ~D I c n O y o c C L - < X ) n> - \ u -\ o <T> 3 RESULTS / 139 Table X. Results of major study, a l l treatment groups (mean ± S.E.M., 7-9. animals per group). Abbreviations: CON, control; Fem, female; Gx, prepubertal gonadectomy; Tp, peripubertal testosterone treatment; Ta, adult testosterone, t, for s t a t i s t i c a l analysis refer to Tables XI and XII. f t , for s t a t i s t i c a l analysis refer to Table XIII. t t t , for s t a t i s t i c a l analysis refer to Table IV. "*" indicates a s i g n i f i c a n t difference from CON MALE Ta group in that row (p<0.05); "&" indicates a s i g n i f i c a n t difference from MALE GxTpTa group in that row (p<0.05); "**" indicates a s i g n i f i c a n t difference from CON MALE group in that row (p<0.05); "+" indicates a s i g n i f i c a n t difference from Fem Gx group in that row (p<0.05); "++" indicates a s i g n i f i c a n t difference from MALE Gx group in that row (p<0.05) . T a D l e X TREATMENT GROUPS CON CON Fem Fem Fem Fem Fem CON CON MALE MALE MALE MALE MALE Fem Ta Gx GxTa GxTp GxTpTa MALE Ta Gx GxTa GxTp GxTpTa EDM t per p r o t e i n 0.49 ±0.03 0. 40 ±0.02 0.31 ±0.02 0. 34 ±0.04 0. 33 ±0.05 0.55 ±0.03 0.80 ±0.11 1 . 23 ±0.15 0. 34 ±0.03 0.49 > +0.07 0.5 1 ±0.06 0. 76 ±0.10 EDM | 0.44 ±0.04 0.3S ±0.02 0. 26 ±0.02 0. 32 ±0.03 0. 33 ±0.03 0.47 ±0.03 0. 66 +0.23 0.88 ±0. 29 0. 32 ±0. 03 0.45 ±0.13 0.42 ±0.12 0. 59 +0.06 per P450 nmo1es P450 per mg * 1.13 ±0.05 * 1.12 ±0.03 * 1.19 ±0.04 *& 1 .06 ±0.04 *& 1 .04 ±0.07 1.18 ±0.02 > » 1 . 20 ±0.06 1 . 38 ±0.04 *& 1 .06 ±0.02 1 .07 +0.07 » 1 . 20 ±0.05 1 . 29 10.03 p r o t e i n nmo1es P450 per g wet we i gn t of 97±4 93±4 9515 82±4 * 87 + 6 103±3 8717 1 10+5 * 82±3 * 85±5 93±6 10013 l i v e r mg p r o t e i n per g wet we ignt of 86±2 82±3 80±4 78±3 * * 84±4 * * 87±2 72±2 79±8 78±3 80+ 1 77±3 77+1 l i v e r Boay weight (g) T! 26415 27618 346 + 5 360110 32815 350±12 455116 45 1113 389±8 403±13 438± 1 4 458± 1 2 L i v e r we i gn t (g) 111 9.3+0.3 9.7+0.2 10.6+0.3 11.510.3 10.410.2 11.610.5 16.110.9 15.010.8 1 2 . 2+0.4 13.7+0.8 14.811 .0 1 5.8± 0 .7 L i ver/Boay •t- + + 3 . 49 ±0.08 + + + 3 . 52 10.08 3 .07 10. 06 3.19 10. 05 3.19 ±0.07 3.31 ' +0.06 + + + 3.51 10. 1 1 3.31 ±0.09 3.13 + 0.06 3 . 39 ±0.09 3 . 36 ± 0 . 1 1 + 3 . 45 ±0.08 oo 4^  O R E S U L T S / 141 Table XI. Major study: s t a t i s t i c a l a n a l y s i s f o r erythromycin demethylase a c t i v i t y c a l c u l a t e d per mg p r o t e i n . "*" i n d i c a t e s p a i r s of groups which are s i g n i f i c a n t l y d i f f e r e n t (p<0.05). Treatment groups: Grp 1: Con Fem Grp 2: Con Fem Ta Grp 3: Fem Gx Grp 4: Fem Gx Ta Grp 5: Fem Gx Tp Grp 6: Fem Gx TpTa Grp 7: Con Male Grp 8: Con Male Ta Grp 9: Male Gx Grp 10: Male Gx Ta Grp 11: Male Gx Tp Grp 12: Male Gx TpTa A b b r e v i a t i o n s : Con, c o n t r o l ; Gx, gonadectomy p r e p u b e r t a l l y ; Fem, female; Tp, p e r i p u b e r t a l t e s t o s t e r o n e ; Ta, a d u l t t e s t o s t e r o n e . Mean i i r o u p b b b b b b b b b b b r r r r r r r r r r r r P P P P P P P P P P P P 1 1 1 er 3 4 1 0 1 6 7 3 . 3 0 3 4 G r p 3 G r p 5 G r p 3 . 3 4 2 6 G r p 4 . 3 3 3 3 G r p 2 . 4 8 3 6 G r p 1 . 4 3 1 3 G r p 10 . 5 1 3 1 G r p 1 1 . 5 4 7 2 G r p 6 . 7 6 2 4 G r p 12 . 7 3 3 6 G r p 7 . 2 3 2 4 G r p 8 •* * * * #• -X- * -K- •* •* * *- *- *- * * *• *• * RESULTS / 142 Table X I I . Major study: s t a t i s t i c a l a n a l y s i s f o r erythromycin demethylase a c t i v i t y c a l c u l a t e d per nmole P450. "*" i n d i c a t e s p a i r s of groups which are s i g n i f i c a n t l y d i f f e r e n t (p<0.05). Treatment groups: Grp 1: Con Fem Grp 2 : Con Fem Ta Grp 3: Fem Gx Grp 4: Fem Gx Ta Grp 5: Fem Gx Tp Grp 6 : Fem Gx TpTa Grp 7: Con Male Grp 8: Con Male Ta Grp 9: Male Gx Grp 10: Male Gx Ta Grp 11: Male Gx Tp Grp 12: Male Gx TpTa A b b r e v i a t i o n s : Con, c o n t r o l ; Gx, gonadectomy p r e p u b e r t a l l y ; Fem, female; Tp, p e r i p u b e r t a l t e s t o s t e r o n e ; Ta, a d u l t t e s t o s t e r o n e . M e a n G r o u p . 2 6 3 2 G r p 3 . 3 2 0 4 G r p 3 r~i -—. G r p 4 . 3 3 4 2 G r p 5 . 3 5 6 9 G r p 2 . 4 1 3 6 G r p 11 . 4 4 0 1 G r p 1 . 4 5 2 8 G r p 10 . 4 6 5 6 G r p 6 . J D J O G r p 12 . 6 5 6 5 G r p 7 . 8 8 2 5 G r p 8 G G G G G G G G G G G G r r r r r r r r r r r r P P P P P P 1 P P 1 P P 1 P P *-» 9 4 t ••• 1 1 0 6 7 8 RESULTS / 143 Table X I I I . Major study: s t a t i s t i c a l a n a l y s i s f o r body weights (g). "*" i n d i c a t e s p a i r s of groups which are s i g n i f i c a n t l y d i f f e r e n t (p<0.05). Treatment groups: Grp 1 : Con Fem Grp 2 : Con Fem Ta Grp 3 : Fem Gx Grp 4 : Fem Gx Ta Grp 5: Fem Gx Tp Grp 6 : Fem Gx TpTa Grp 7 : Con Male Grp 8 : Con Male Ta Grp 9 : Male Gx Grp 1 0 : Male Gx Ta Grp 1 1 : Male Gx Tp Grp 1 2 : Male Gx TpTa A b b r e v i a t i o n s : Con, c o n t r o l ; Gx, gonadectomy p r e p u b e r t a l l y ; Fem, female; Tp, p e r i p u b e r t a l t e s t o s t e r o n e ; Ta, a d u l t t e s t o s t e r o n e . G G G G G G G G G r r r r r r r r r r r r P P P P P P P P P P P P 1 1 1 M e a n G r o u p 1 2 ^ : O 6 4 3 0 1 8 7 2 6 3 . 7 7 7 8 G r p 1 2 7 5 . 5 0 0 0 G r p 2 3 2 8 . 0 0 0 0 G r p 5 * •x-3 4 5 . 7 7 7 8 G r p 3 -x- -X-3 4 3 . 7 7 7 8 G r p 6 •x- -X-3 6 0 . 0 0 0 0 G r p 4 •x- •X-3 8 8 . 5 0 0 0 G r p 3 * -X * •*• *-4 0 3 . 1 4 2 3 G r p 10 Mr •X- -X- Mr * 4 3 8 . 2 5 0 0 G r p 11 * •X- Mr * •X- -X-4 5 0 . 8 8 8 3 G r p 8 •X- Mr •X- Mr Mi- *- •X- •X-4 5 4 . 8 8 8 3 G r p 7 -X- •X- * Mr Mr Mr * •X-4 5 8 . 0 0 0 0 G r p 12 Mr •X- •* Mr Mr Mr *• *-RESULTS / 144 Table XIV. Major study: s t a t i s t i c a l a n a l y s i s f o r l i v e r weights ( g ) . "*" i n d i c a t e s p a i r s of groups which are s i g n i f i c a n t l y d i f f e r e n t (p<0.05). Treatment groups: Grp 1: Con Fem Grp 2: Con Fem Ta Grp 3: Fem Gx Grp 4: Fem Gx Ta Grp 5: Fem Gx Tp Grp 6: Fem Gx TpTa Grp 7: Con Male Grp 8: Con Male Ta Grp 9: Male Gx Grp 10: Male Gx Ta Grp 11: Male Gx Tp Grp 12: Male Gx TpTa A b b r e v i a t i o n s : Con, c o n t r o l ; Gx, gonadectomy p r e p u b e r t a l l y ; Fem, female; Tp, p e r i p u b e r t a l t e s t o s t e r o n e ; Ta, a d u l t t e s t o s t e r o n e . G G G G G G G G G G G G r r r r r r r r r r r r P P P P P P P P P P P P 1 1 1 M e a n G r o u p 1 2 5 * > 4 6 9 0 1 8 2 7 9 . 2 2 5 7 G r p 1 9 . 6 6 6 0 G r p 2 l O . 4 4 6 1 G r p 5 1 0 . 6 3 4 3 G r p 3 1 1 . 4 7 5 1 G r p 4 1 1 . 6 0 2 6 G r p 6 -x-1 2 . 1 6 5 5 G r p 9 -x-1 3 . 7 0 8 4 G r p l O *- * •X- •X- -X-1 4 . 8 1 3 1 G r p l 1 -X- •X- -x- -X- •X- •X- * 1 4 . 9 9 5 9 G r p 8 -x- * •X- -X- •X- •X-1 5 . 8 4 5 3 G r p 12 *• * -X- •X- -X- *• 1 6 . 0 6 1 6 G r p 7 -X- •X- •X- -K- -X- -K-RESULTS / 145 Figure 27. Major study: erythromycin demethylase a c t i v i t y calculated per protein for a l l treatment groups (mean ± standard error of the mean, 7-9 animals per group). S t a t i s t i c a l analysis i s shown in Table XI. Abbreviations: Con, control; Ta, adult testosterone days 81-90; Gx, gonadectomy prepubertally on day 25 of age; Tp, peripubertal testosterone days 35-50. E R Y T H R O M Y C I N D E M E T H Y L A S E ACTIVITY ( n m o l e s f o r m a l d e h y d e / m i n / m g p r o i e i n ) 9ti / sj/insau RESULTS / 147 Figure 28. Major study: erythromycin demethylase a c t i v i t y calculated per P450 in a l l treatment groups (mean tstandard error of the mean, 7-9 animals per group). S t a t i s t i c a l analysis i s shown in Table XII. Abbreviations: Con, control; Ta, adult testosterone days 81-90; Gx, gonadectomy prepubertally on day 25 of age; Tp, peripubertal testosterone days 35-50. E R Y T H R O M Y C I N D E M E T H Y L A S E ACTIVITY ( n m o l e s f o r m a l d e h y d e / m i n / n a n o m o l e P - 4 5 0 ) O O O O O O N ) J > C O ' C O O K ) J > \_ , 1 i 1 i i i i 8*i / sxansan RESULTS / 149 Figure 29. Major study: plasma e s t r a d i o l l e v e l s (mean ± standard error of the mean, 7-9 animals per group). "*" indicates a s i g n i f i c a n t difference from Con Fem (p<0.05). "**" indicates a s i g n i f i c a n t difference from Con Fem Ta (p<0.05). Abbreviations: Fem, female; Con, control; Ta, adult testosterone days 81-90; Gx, gonadectomy prepubertally on day 25 of age; Tp, peripubertal testosterone days 35-50. T R E A T M E N T G R O U P R E S U L T S / 151 Figure 30. Major study: plasma testosterone concentrations (mean ± standard error of the mean, n=7-9 animals per group). S t a t i s t i c a l analysis i s presented in Table XIV. Abbreviations: Con, control; Ta, adult testosterone days 81-90; Gx, gonadectomy prepubertally on day 25 of age; Tp, peripubertal testosterone days 35-50, ND, not detectable. o cc U J >— ^ 00 _j ° E 0 0 \ LJ CT) H- C < ^ 00 < _ J Q_ 6 .0 5 . 0 4 . 0 3.0 2 . 0 1 .0 0 . 0 ND C o n C o n + Ta F E M A L E CXD MALE ND ND Gx Gx + Ta ND ND Gx- f Tp Gx + T p T a T R E A T M E N T G R O U P 70 W CO C r H CO RESULTS / 153 Table XV. Major study: s t a t i s t i c a l a n a l y s i s f o r plasma t e s t o s t e r o n e c o n c e n t r a t i o n s . "*" i n d i c a t e s a s i g n i f i c a n t d i f f e r e n c e between p a i r s of groups (p<0.05). Treatment groups: Grp 1: Con Fem Grp 2: Con Fem Ta Grp 3: Fem Gx ..-.«« Grp 4: Fem Gx Ta Grp 5: Fem Gx Tp Grp 6: Fem Gx TpTa Grp 7: Con Male Grp 8: Con Male Ta Grp 9: Male Gx Grp 10: Male Gx Ta Grp 11: Male Gx Tp Grp 12: Male Gx TpTa A b b r e v i a t i o n s : Con, c o n t r o l ; Gx, gonadectomy p r e p u b e r t a l l y ; Fem, female; Tp, p e r i p u b e r t a l t e s t o s t e r o n e ; Ta, a d u l t t e s t o s t e r o n e . G G G G G" G G G G G G G r r r r r r r r r r r r Mean G r o u p p p p p p p p p p p p p 1 1 1 3 9 1 5 1 7 2 4 0 6 8 2 . 02lO . 0265 . 0321 .0712 . 0921 . 9800 - ^ ' »_l 2.6257 3.2945 4.0398 5.4454 5.6978 Grp 3 Grp 9 Gr p 1 Grp 5 Gr p 1 1 Grp 7 Gr p 2 Grp 4 Grp 10 Grp 6 Grp 8 Grp 12 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * RESULTS / 154 Figure 31. Erythromycin demethylase a c t i v i t y (nanomoles formaldehyde/min/mg p r o t e i n ) versus plasma testosterone (ng/mL). RESULTS / 155 ( u i a j o j d 6 u j / u i u j / a p X q a p | o u j j o j sa|oujouou) A1IAIJLDV 3 S V 3 A H 1 3 W 3 Q NI0AWOUHJLAa3 4. DISCUSSION 4.1. STUDY RESULTS 4.1.1. Non-gonadectomized Animals Since t h i s study had as i t s base the existence of a sex-dependent hepatic microsomal P450 enzyme a c t i v i t y , erythromycin demethylase, i t was necessary to demonstrate a sex difference in non-gonadectomized animals for t h i s parameter. We observed that hepatic microsomal erythromycin demethylase a c t i v i t y was higher, approximately 1.6-fold, in the adult male rat than in the adult female. The quality and magnitude of th i s sex difference were consistent with the previous report of hepatic microsomal erythromycin demethylase a c t i v i t y by Arl o t t o et al.. (1987). These two studies do not support that of Wrighton et al. (1985a), who reported s l i g h t l y higher levels in females than in males. The existence of responsiveness to androgen in the adult male rat for hepatic microsomal erythromycin demethylase a c t i v i t y i s also required as a basis for t h i s project. We observed a clear difference between intact males and females in the response to adult testosterone for t h i s a c t i v i t y . An increased hepatic microsomal erythromycin demethylase a c t i v i t y was detected in non-gonadectomized male rats when 156 DISCUSSION / 157 they were treated with testosterone in adulthood, while the id e n t i c a l treatment did not increase erythromycin demethylase a c t i v i t y in the intact females. This indicated a second sex difference with respect to hepatic microsomal erythromycin demethylase a c t i v i t y , a lack of a b i l i t y to respond to adult androgen in the intact female rat. This sex difference in adult androgen responsiveness has not been previously reported with 'respect to hepatic microsomal erythromycin demethylase a c t i v i t y . It i s , however, consistent with our expectations, based on the observed sex difference in adult androgen responsiveness for another marker a c t i v i t y of the P450III family, steroid 6/3-hydroxylase (Einarsson et al., 1973). 4.1.2. Effect of Prepubertal Gonadectomy This study investigated hormonal effects in l i v e r s from prepubertally gonadectomized rats. Prepubertal ovariectomy of female rats decreased hepatic microsomal erythromycin demethylase a c t i v i t y by approximately 37 percent from intact female l e v e l s . Although t h i s l e v e l of a c t i v i t y was not s t a t i s t i c a l l y d i f f e r e n t from that of intact females, the dir e c t i o n and magnitude of the change following ovariectomy were consistent in the p i l o t (38% decrease) and major (37% decrease) studies, arguing against a random e f f e c t . This observation was unexpected, and requires v e r i f i c a t i o n . If DISCUSSION / 158 true, i t suggests that a portion of hepatic microsomal erythromycin demethylase a c t i v i t y in the adult female rat i s dependent on the presence of ovarian factors, possibly estrogens. In the males, we observed a s t a t i s t i c a l l y s i g n i f i c a n t decrease of approximately 50 percent in hepatic microsomal erythromycin demethylase a c t i v i t y following prepubertal castration. The l e v e l of hepatic microsomal erythromycin demethylase a c t i v i t y in prepubertally castrated males was equivalent to that observed in prepubertally ovariectomized females and non-ovariectomized females, indicating that the sex difference required the presence of testes in the male after day 25 of age. Plasma testosterone le v e l s were also decreased in the males by prepubertal castration, as expected. Although not s p e c i f i c a l l y tested in t h i s 'study, these observations, along with the observed sex difference in an adult androgen response potential discussed above, are also suggestive that high le v e l s of c i r c u l a t i n g testosterone plus an a b i l i t y to respond to that testosterone are required for the high hepatic microsomal erythromycin demethylase a c t i v i t y in the adult male rat. DISCUSSION / 159 4.1.3. Peripubertal Testosterone The major purpose of the present study was to investigate the a b i l i t y of peripubertal testosterone to influence the adult androgen responsiveness of an hepatic P450 enzyme a c t i v i t y in an ovariectomized female rat- l i v e r model. We observed that erythromycin demethylase a c t i v i t y in hepatic microsomes from prepubertally ovariectomized female rats increased in response to adult testosterone only i f the animals had been exposed to peripubertal testosterone. This demonstrated that, in the ovariectomized female l i v e r model used, peripubertal testosterone was capable of imprinting an adult androgen responsiveness for hepatic microsomal erythromycin demethylase a c t i v i t y . S t a t i s t i c a l analysis, when based on a c t i v i t y per mg protein, supported the conclusion that an interactive effect was present; that i s , the response of erythromycin demethylase a c t i v i t y in female rat l i v e r to adult testosterone depended on whether or not pubertal testosterone exposure had occurred. In the males, s t a t i s t i c a l analysis did not indicate an interactive e f f e c t between the two testosterone treatments. From the data, i t i s possible that each testosterone treatment had a r e l a t i v e l y small but additive e f f e c t in the male l i v e r s , however s t a t i s t i c a l l y each treatment alone had no e f f e c t . This i s an important point, since i f the DISCUSSION / 160 p o t e n t i a l f o r adult androgen responsiveness i n the male i s f u l l y imprinted p r i o r to puberty, for example n e o n a t a l l y , then males c a s t r a t e d at 25 days of age should show the same response to adult testosterone as the non-castrated r a t s . I f such a r e s u l t had been observed, then the b i o l o g i c a l relevance of pubertal i m p r i n t i n g by testosterone of adult androgen responsiveness for hepatic microsomal erythromycin demethylase a c t i v i t y would be questionable. We observed that the hepatic microsomal erythromycin demethylase a c t i v i t y l e v e l f o l l o w i n g adult testosterone i n gonadectomized males was lower than that of i n t a c t males and of i n t a c t males given a d u l t t e s t o s t e r o n e . Therefore, the magnitude of response to adult testosterone was smaller i n males which had been c a s t r a t e d at 25 days of age than i n i n t a c t males. These data i n d i c a t e d that neonatal testosterone had not permanently imprinted a complete adult androgen response p o t e n t i a l for hepatic microsomal erythromycin demethylase a c t i v i t y i n the male r a t , and i n d i c a t e d a requirement f o r t e s t i c u l a r f a c t o r s a f t e r 25 days of age i n the male f o r a f u l l androgen response f o r t h i s a c t i v i t y . In the males, the e f f e c t of p e r i p u b e r t a l testosterone f o l l o w i n g c a s t r a t i o n was s i m i l a r but not i d e n t i c a l with the e f f e c t observed i n the females. Hepatic microsomal erythromycin demethylase a c t i v i t y was equivalent i n DISCUSSION / 161 prepubertally-gonadectomized males and females, however subsequent combination testosterone treatment produced in the male case a l e v e l of hepatic microsomal erythromycin demethylase a c t i v i t y which did not d i f f e r from control male lev e l s , while in the females the i d e n t i c a l treatment resulted in an a c t i v i t y l e v e l equivalent to that of a control female, and lower than the control males. Therefore, the magnitude of the response in hepatic microsomal erythromycin demethylase a c t i v i t y to both peripubertal and adult testosterone was greater in the male than in the female l i v e r s following prepubertal gonadectomy. This greater magnitude of response suggests that peripubertal testosterone alone i s not s u f f i c i e n t to produce in females the f u l l androgen response seen in the males. This sex difference may indicate either the presence *of i n h i b i t o r y conditions, or a lack of required factors, in the ovariectomized female r a t . These results are consistent with the p o s s i b i l i t y that prepubertal or neonatal testosterone might be the required factor absent in the female. The r e s u l t s were not conclusive concerning the e f f e c t s of single testosterone treatments following prepubertal castration in the male rats. Although a small response in hepatic microsomal erythromycin demethylase a c t i v i t y was observed to both adult or peripubertal testosterone alone in DISCUSSION / 162 the males, th e s e responses d i d not d i f f e r s t a t i s t i c a l l y from t h o s e of the p r e p u b e r t a l l y o v a r i e c t o m i z e d females t o thes e s i n g l e t r e a t m e n t s w i t h t e s t o s t e r o n e . 4.1.4. Correlation with Plasma Testosterone When plasma t e s t o s t e r o n e l e v e l s were d e t e r m i n e d i n a n i m a l s of b o t h sexes and f o l l o w i n g t he v a r i o u s t r e a t m e n t s used i n t h i s s t u d y , we obs e r v e d a p o s i t i v e c o r r e l a t i o n between h e p a t i c microsomal e r y t h r o m y c i n demethylase a c t i v i t y (by e i t h e r c a l c u l a t i o n ) and a d u l t plasma t e s t o s t e r o n e l e v e l . The c o e f f i c i e n t s of d e t e r m i n a t i o n were 0.22 and 0.21, t h e r e f o r e t h e degree of c o r r e l a t i o n was not l a r g e . T h i s i n d i c a t e s t h a t o t h e r f a c t o r s a r e i n v o l v e d , or may r e l a t e t o the fact- t h a t plasma t e s t o s t e r o n e i s not a s t a t i c parameter but r a t h e r shows wide i n d i v i d u a l d i u r n a l v a r i a t i o n s ( B a r t k e et al, 1973; Ki n s o n & L i u , 1973; K a l v a & K a l r a , 1977; Mock et al, 1983). I t i s p o s s i b l e t h a t h e p a t i c m i c r o s o m a l e r y t h r o m y c i n demethylase a c t i v i t y would c o r r e l a t e d i f f e r e n t l y w i t h f r e e plasma t e s t o s t e r o n e l e v e l s , s i n c e c i r c u l a t i n g t e s t o s t e r o n e i s u s u a l l y h i g h l y bound t o sex hormone-binding g l o b u l i n (Gilman et al, 1980). The o b s e r v a t i o n t h a t h i g h e r a d u l t plasma t e s t o s t e r o n e l e v e l s c o r r e l a t e d w i t h h i g h e r h e p a t i c microsomal e r y t h r o m y c i n demethylase a c t i v i t y was i n agreement w i t h the o b s e r v a t i o n s made above which suggested t h a t h i g h h e p a t i c microsomal e r y t h r o m y c i n demethylase DISCUSSION / 163 a c t i v i t y in adult male rats i s in part due to high c i r c u l a t i n g testosterone levels in the male. We observed an intercept for hepatic microsomal erythromycin demethylase a c t i v i t y of approximately 0.41 nmoles formaldehyde/min/mg protein (± 0.04 SEM), indicating a portion of the a c t i v i t y which was not dependent on plasma testosterone. As one would expect i f thi s were so, the testosterone-independent l e v e l of hepatic microsomal erythromycin demethylase a c t i v i t y was similar to the average l e v e l observed in control adult females (0.49 ±0.03) and in castrated males (0.34 ±0.03). Therefore, there appeared to be a testosterone-independent basal l e v e l of hepatic microsomal erythromycin demethylase a c t i v i t y plus an additional testosterone-related component. 4.1.5. Comparison with BPH Studies The only other demonstration of peripubertal imprinting of testosterone responsiveness for P450 enzymes has involved another sex-differentiated a c t i v i t y , hepatic microsomal BPH (Pak et al, 1984; unpublished). It is therefore important to compare those observations with the results of the present study for hepatic microsomal erythromycin demethylase a c t i v i t y . Both a c t i v i t i e s are higher in adult male l i v e r , however BPH in males i s four times that in females (Wiebel & Gelboin, 1975; Pak et al, 1984), while only a 1.6-fold difference has been observed for hepatic microsomal DISCUSSION / 164 erythromycin demethylase a c t i v i t y (Arlotto et al., 1987). For both hepatic microsomal erythromycin demethylase a c t i v i t y and BPH, neither intact nor ovariectomized females responded to adult testosterone, indicating the lack of an adult androgen response system. For both a c t i v i t i e s , pubertal testosterone exposure has been shown to imprint an adult androgen responsiveness to testosterone in prepubertally ovariectomized female rats. However, two further differences between hepatic microsomal erythromycin demethylase a c t i v i t y and BPH were observed: (1) for BPH, combination testosterone treatment of prepubertally ovariectomized females resulted in BPH l e v e l s which were twice those of the control females, and represented 75 percent of control male", l e v e l s . For hepatic microsomal erythromycin demethylase a c t i v i t y , similar treatment in females resulted in l e v e l s which were not greater than control female l e v e l s , and only 30 percent of control male l e v e l s . This i s p a r t i c u l a r l y s t r i k i n g since a larger adult dose of testosterone was used in the present study for hepatic microsomal erythromycin demethylase a c t i v i t y , yet a smaller response was observed. (2) Prepubertal ovariectomy did not a l t e r BPH a c t i v i t y , while th i s treatment consistently reduced hepatic microsomal erythromycin demethylase a c t i v i t y . Therefore, • i t appears unlikely that the sex-dependent differences in BPH a c t i v i t y could be completely explained by the sex-related differences DISCUSSION / 165 represented by hepatic microsomal erythromycin demethylase a c t i v i t y . This was expected, since the studies of Kato et al (1987) and Ohgiya et al (1989) indicate that a large portion of BPH i s related to P450h. Since hepatic microsomal erythromycin demethylase a c t i v i t y has been linked with P450p of the P450III family (Wrighton et al, 1985a; Table IV), i t is of interest to compare the results of t h i s study with any similar P450p experiments. However, studies within the P450III family investigating androgen regulation have been conducted only for P450 Pb-2a/PCN-E (Waxman et al., 1985; Dannan et al, 1986), which i s currently not considered to be the same as P450p (Hostetler, 1987; Table IV). In a study by Waxman et al. (1985), peripubertal castration (day 35) did not a l t e r hepatic microsomal l e v e l s of P450 Pb-2a/PCN-E in adult male rats, in contrast to the 50 percent decrease in adult hepatic microsomal erythromycin demethylase a c t i v i t y observed in the present study following prepubertal castration (day 25). This suggests that either d i f f e r e n t parameters were being measured, or that the time period between days 25 and 35 of age was important. The study of Dannan et al. (1986) reported that neonatal castration completely removed detectable lev e l s of P450 2a/PCN-E in adult male rats, however subsequent treatment with testosterone continuously throughout puberty and adulthood, restored these l e v e l s . The response in neonatally DISCUSSION / 166 ovariectomized females to continuous testosterone throughout puberty and adulthood was i d e n t i c a l to that observed in the males. These results are somewhat similar to the results presented in t h i s study, in that pubertal plus adult testosterone exposure produced an increase in the ovariectomized female for the parameter measured. However, the magnitude of the response in hepatic microsomal erythromycin demethylase a c t i v i t y in females was not as large as the response in males. Again, i t i s not clear i f these differences resulted from differences in the study protocols, or from measuring d i f f e r e n t enzymes. It should also be" noted that hepatic microsomal erythromycin demethylase a c t i v i t y was detectable in female rats in the present study, while P450p (Wrighton et al., 1985a) and other P450III forms (Gozukara et al., 1984; Waxman et al, 1985? C r e s t e i l et al, 1986; Imoake et al, 1988) are not detectable or present at very low levels in female rat hepatic microsomes. It is not possible to make d e f i n i t e conclusions concerning the regulation of the P450III family based on the results of the present study. 4.1.6. Relation to Gustafsson's Theory The current concept of hormonal regulation of hepatic P450 enzymes i s strongly based upon the theories of Gustafsson (Einarsson et al, 1973; Gustafsson et al, 1974a; Gustafsson et DISCUSSION / 167 al., 1974b; Gustafsson et a l , 1983), which place an emphasis on neonatal imprinting by t e s t i c u l a r androgens. Indeed, for a subgroup of hepatic P450s evidence has been presented in support of the hypothesis that neonatal androgens influence sex-dependent hepatic steroid metabolizing P450s via an indirect effect on p i t u i t a r y growth hormone patterns (Jansson e£ al., 1984; 1985a; 1985b; 1985c; Jansson & Frohman, 1987). However, th i s proposal allows that additional control mechanisms may ex i s t , and that other P450s may be regulated in a d i f f e r e n t manner. With respect to the time of imprinting of adult androgen responsiveness for sex-dependent P450s, evidence for the neonatal period is derived primarily from a single study done by Gustafsson et al. ( 1974b). In that study, an adult androgen responsiveness for the 60-hydroxylation of 4-androstene-3,17-dione was removed in male rats castrated at b i r t h , but present i f castration was done at 14 days of age, indicating an imprinting in the f i r s t days of l i f e . Since testosterone 60-hydroxylase (Gonzalez et al., 1986; Imaoka et al.,, 1988) and hepatic microsomal erythromycin demethylase a c t i v i t y (Wrighton et al., 1 985a) are both considered as markers for members of the P450III family, the report of Gustafsson et a l (1974b) would appear to be in c o n f l i c t with finding in the present study of pubertal DISCUSSION / 168 imprinting. However, i t i s possible that t h i s discrepancy is due either to the complexity of the P450III family (Table IV), or to the use of a steroid substrate other than testosterone. Levin and co-workers (Swinney et al, 1987) have shown that steroid hydroxylations by s p e c i f i c P450s can be substrate-specific. Other studies have investigated neonatal imprinting of a basal l e v e l and not imprinting of adult androgen responsivity. It i s also possible that the potential for imprinting of adult androgen responsiveness may be present in both the neonatal and pubertal periods. It remains to be shown whether neonatal testosterone administration to female rats can result in p a r t i a l or f u l l adult androgen responsiveness for sex-differentiated P450s. Therefore, our results do not necessarily c o n f l i c t with current theory, but rather constitute an extension of the theory to include other regulatory mechanisms and other P450 forms. 4.2. POTENTIAL STUDY LIMITATIONS Several possible l i m i t a t i o n s should be considered when interpreting the results of t h i s study. DISCUSSION / 169 4.2.1. Erythromycin Demethylase Assay 4.2.1.1. Specificity Research in the area of the P450 enzymes i s currently at the stage where the properties of s p e c i f i c enzyme forms are being investigated (Tables I to I I I ) . In the present study, the erythromycin demethylase assay has been used as a marker a c t i v i t y for the P450III family. Is i t possible to link erythromycin demethylase a c t i v i t y with a single s p e c i f i c P450 form? In fact, i t is not clear which P450 form or forms are represented by hepatic microsomal erythromycin demethylase a c t i v i t y . A link with the P450III family has been made by the observation that hepatic microsomal erythromycin demethylase a c t i v i t y i s inhibited by antibodies against P450p (Wrighton et al, 1985a). In that study, polyclonal antibodies, prepared against P450p which had been p u r i f i e d from l i v e r s from PCN or dexamethasone treated female rats, and made form-specific by back-absorption against microsomes prepared from rats treated with 3-methylcholanthrene (Elshourbagy et al., 1981), were shown to block approximately 75 percent of the erythromycin demethylase a c t i v i t y in l i v e r microsomes from adult female Sprague Dawley rats which had been treated with TAO, PCN or dexamethasone. Therefore, while P450p appears to play a major role in hepatic microsomal erythromycin demethylase DISCUSSION / 170 a c t i v i t y i n the i n d u c e d female r a t , the i d e n t i t y of the enzyme(s) r e s p o n s i b l e f o r the r e m a i n i n g e r y t h r o m y c i n demethylase a c t i v i t y which was not b l o c k e d by a n t i - P 4 5 0 p a n t i b o d i e s remains unknown. I t has, as w e l l , not been proven t h a t P450p i s a s i n g l e enzyme. F u r t h e r , t h i s i m m u n o i n h i b i t i o n study c o n c e r n s o n l y the induced s i t u a t i o n i n female r a t s ; i t i s not known i f a n t i b o d i e s which r e a c t w i t h P 4 5 0 I I I forms found i n u n t r e a t e d male and female r a t s w i l l i n h i b i t h e p a t i c microsomal e r y t h r o m y c i n demethylase a c t i v i t y i n u n t r e a t e d a n i m a l s . A l t h o u g h i t has not been shown, i t i s q u i t e p o s s i b l e t h a t d i f f e r e n t P450 forms d e m e t h y l a t e e r y t h r o m y c i n i n male and female r a t s , and i n u n t r e a t e d and induced r a t s . The works of Gonzalez et al. (1986), f o r example, i d e n t i f i e d PCN/Dex-induced P 4 5 0 I I I forms which were u n d e t e c t a b l e i n u n t r e a t e d r a t s . Enzyme k i n e t i c s t u d i e s have i n d i c a t e d the presence of a t l e a s t two enzymes c a t a l y z i n g h e p a t i c microsomal e r y t h r o m y c i n demethylase a c t i v i t y i n u n t r e a t e d female Long Evans r a t s ( A r l o t t o et al, 1987). In the p r e s e n t s t u d y , a d e t a i l e d i n v e s t i g a t i o n of the enzyme k i n e t i c s of the e r y t h r o m y c i n demethylase a c t i v i t y i n Sprague Dawley r a t s was not a t t e m p t e d . The l i m i t e d d a t a were i n c o n c l u s i v e , a l l o w i n g f o r the p o s s i b i l i t y of e i t h e r s i n g l e or m u l t i p l e enzymes c a t a l y z i n g t h i s r e a c t i o n . DISCUSSION / 171 Other evidence c o r r e l a t i n g erythromycin demethylase a c t i v i t y with P450III a c t i v i t y in hepatic microsomes has included the observations that ( i) PCN induces both erythromycin demethylase a c t i v i t y (Wrighton et al, 1985a), P450Pb-2a/PCN-E (Waxman et al, 1985) and P450p (Hostetler et al, 1987); and ( i i ) both hepatic microsomal erythromycin demethylase a c t i v i t y (Arlotto et al, 1987) and immunochemically-detectable levels of P450 Pb-2a/PCN-E (Gozukara et al, 1984; Waxman et al., 1985) and P450 Pb-1 (Imaoka et al., 1988) have been reported as higher in hepatic microsomes from adult male rats than from adult female rats; and ( i i i ) in the female rat, hepatic microsomal erythromycin demethylase a c t i v i t y appears to decrease with age (Arlotto et al, 1987) as do immunochemical ly-quant i tated l e v e l s of P450 Pb-2a/PCN-E (Waxman et al., 1985) and P450 Pb-1 (Imaoka et al, 1988; Yamazoe et al, 1988). However, in the study by Wrighton et al. (1985a), P450p le v e l s did not correlate with hepatic microsomal erythromycin demethylase a c t i v i t y in untreated rats, since while P450p was detectable in hepatic microsomes from the untreated males but not from the females, the hepatic microsomal erythromycin demethylase a c t i v i t y was not only detectable in females, but higher in the- females than in the males. P450III forms, unlike erythromycin demethylase, are present at only low or undetectable levels in hepatic microsomes from adult female DISCUSSION / 172 rats (Gozukara et al, 1984; Waxman et al, 1985; C r e s t e i l et al., 1986; Imaoka etal., 1988). Therefore, although hepatic microsomal erythromycin demethylase a c t i v i t y has been linked with the P450III forms, due to the reports of multiple forms of P450 catalyzing the erythromycin demethylase reaction, and the fact that a l l of these forms have not yet been i d e n t i f i e d , i t i s not possible to conclude that changes in hepatic microsomal erythromycin demethylase a c t i v i t y reported in t h i s study were d i r e c t l y correlated with changes in a P450III form. The possible existence of multiple forms as components of erythromycin demethylase a c t i v i t y further complicated the interpretation of any re s u l t s , since, for example, i f an increase in net a c t i v i t y i s recorded, i t i s not clear whether one or more than one contributing enzyme has been affected. S i m i l a r l y , a lack of net change in such a non-specific, multi-component enzyme a c t i v i t y may indicate, rather than a lack of e f f e c t , a p o s i t i v e effect on one component balanced against a negative e f f e c t on a second contributing component. 4.2.1.2. Sensitivity and Detection Limits The s e n s i t i v i t y of an a n a l y t i c a l method can be defined as the r a t i o of the change in response ( a n a l y t i c a l signal) to the change in the quantity or concentration of analyte being DISCUSSION / 173 measured ( M a s s a r t , 1978; Ewing, 1985). In some branches of s c i e n c e , " s e n s i t i v i t y " has a l s o been used t o d e s c r i b e the d e t e c t i o n l i m i t of a method ( M a s s a r t , 1978). The e r y t h r o m y c i n demethylase assay used i n t h i s s tudy i s based on the s p e c t r o p h o t o m e t r i c measurement of formaldehyde a c c o r d i n g t o the method of Nash (1953). S p e c t r o p h o t o m e t r i c methods a r e r e l a t i v e l y i n s e n s i t i v e a n a l y t i c a l p r o c e d u r e s , i n comparison w i t h f l u o r i m e t r y ( A h u j a , 1986), r a d i o m e t r i c a s s a y s or HPLC (Smith & S t e w a r t , 1981). As w e l l , the method of Nash (1953) uses the H a n t z s c h r e a c t i o n between a c e t y l a c e t o n e , ammonia and formaldehyde. T h i s r e a c t i o n i s not s p e c i f i c , and may d e t e c t non-formaldehyde s u b s t r a t e s as w e l l as endogenous formaldehyde i n the absence of exogenously-added s u b s t r a t e or enzyme ( K l e e b e r g & K l i n g e r 1982). The r e s u l t i s a h i g h " n o i s e " ( b l a n k ) l e v e l , w hich a d v e r s e l y a f f e c t s the l i m i t s of d e t e c t i o n . In t h i s s t u d y , a t the l o w e s t o b s e r v e d e r y t h r o m y c i n demethylase a c t i v i t y ( t h e o v a r i e c t o m i z e d female r a t g r o u p ) , the s i g n a l - t o - n o i s e r a t i o (absorbance r e a d i n g / b l a n k r e a d i n g ) was a p p r o x i m a t e l y 2:1. We were t h e r e f o r e measuring v e r y c l o s e t o the l i m i t s of d e t e c t i o n of the assay ( M a s s a r t , 1978). However, a t the observ e d s i g n a l - t o - n o i s e r a t i o of 2:1, and w i t h the s t a n d a r d d e v i a t i o n of the blank r e a d i n g s which r e p r e s e n t e d 20% of the average blank r e a d i n g s i n the f e m a l e , t h e s e l o w e s t DISCUSSION / 174 measurements were outside of three standard deviations of the blank mean and therefore distinguishable s t a t i s t i c a l l y from blank readings (Massart, 1978). In terms of s e n s i t i v i t y as a measure of response to a change in the concentration being measured, the low s e n s i t i v i t y of spectrophotometric methods does not change the results in proportion to each other for hepatic microsomal erythromycin demethylase a c t i v i t y in the d i f f e r e n t treatment groups. However, i t may have decreased the a b i l i t y of the method to detect small differences between treatment groups. 4.2.1.3. Definition of Puberty Puberty has been defined as the time at which gametogenesis and gonadal hormone secretion occurs, secondary sexual characters and reproductive functions begin, and sexual dimorphism i s accentuated (Stedman, 1976). In the present study, days 35-50 of age were chosen for peripubertal testosterone treatment, in order to coincide with previous studies by Pak et al. ( 1984; unpublished) on peripubertal e f f e c t s . This time period i s consistent with statements in the l i t e r a t u r e that puberty occurs in male and female rats at between 40 to 60 days of age (Farris et al., 1950; Baker et al., 1980; Fax et al., 1984; Harkness & Wagner, 1983). Forest (1979) has c l e a r l y shown a rise in plasma testosterone DISCUSSION / 175 levels in male Sprague Dawley rats at approximately 35 days of age. Therefore, although i t cannot be ruled out that a pubertal testosterone exposure might have occurred unusually early in some of the study rats, perhaps prior to castration at 25 days of age, such an occurrence appears improbable. 4.2.1.4. Testosterone Dose It i s important to consider whether the doses of testosterone used in t h i s study were phy s i o l o g i c a l . The dose of testosterone used in the major study was that which had been shown to maintain "normal levels of sex accessory tissue weights and sex-dependent drug-metabolizing enzyme a c t i v i t i e s in gonadectomized animals" (Pak et al., 1984). Plasma testosterone levels were within the physiological range as quoted in the l i t e r a t u r e (Dohler & Wuttke, 1975), and could therefore be considered approximately phy s i o l o g i c a l . However, i t i s important to note that the pattern of plasma testosterone produced by d a i l y exogenous injections would probably have d i f f e r e d from that of the normal male rat. Large fluctuations in plasma testosterone levels in male rats have been reported (Bartke et al, 1973; Kalva & Kalra, 1977). In adult male rats, a circadian rhythm is seen; in male Sprague Dawley rats at 40-50 days of age, a trimodal rhythm has been reported with peak levels at 0200, 1200 and 1800 hours on a 12 hour l i g h t , 12 hour dark cycle DISCUSSION / 176 (Mock et al, 1978). The injections used in the present study were of testosterone enanthate ester in corn o i l , given once da i l y subcutaneously. Testosterone enanthate i s more fat-soluble than testosterone, i s more slowly absorbed and has a longer h a l f - l i f e than testosterone (Gilman et al, 1980). In humans i t i s injected every one to two weeks for replacement therapy (Gilman et al, 1980). It would therefore tend to produce a sustained, unfluctuating l e v e l of testosterone in the c i r c u l a t i o n , and c e r t a i n l y a plasma l e v e l which would not be regulated by normal physiological factors. The effects of testosterone administration observed in t h i s study may therefore not correspond to normal physiological events in the developing rat. The dose of testosterone administered to adult animals was twice as large in the major study as in the p i l o t study. In comparable treatment groups, higher erythromycin demethylase a c t i v i t y was not observed with the higher testosterone dose (Figure 26). This indicated that the e f f e c t was already maximal at the lower dose. 4.2.1.5. Calculations of Erythromycin Demethylase Activity Hepatic microsomal erythromycin demethylase a c t i v i t y was calculated both on the basis of microsomal protein and on microsomal t o t a l P450. The patterns of erythromycin DISCUSSION / 177 demethylase a c t i v i t y in the st.udy groups were similar for the two methods • of c a l c u l a t i o n , however small differences did occur which altered the s t a t i s t i c a l r e s u l t s . For example, within the ovariectomized female groups, a s i g n i f i c a n t interaction between the two testosterone treatments was detected using calculations based on protein, but not from the calculations based on t o t a l P450. Analysis of l i v e r protein and P450 content for these four study groups revealed that the treatments had affected the l i v e r P450 content but not the l i v e r protein content (Table IX). Therefore, within these four groups, i t can be argued that protein provided 0 a more stable and therefore more appropriate baseline for comparisons between groups. As well, P450 a c t i v i t i e s including erythromycin demethylase are generally presented in terms of product formation rate per mg microsomal protein (Wrighton et al., 1985a; Arlo t t o et al, 1987). Since control l i v e r microsomes generally contain approximately 1 nmole t o t a l P450 per mg protein, and P450s have molecular weights of approximately 50,000 Da, i t i s clear that only approximately 1/20 (by weight) of the protein in l i v e r microsomes i s composed of P450. It can therefore be reasoned that microsomal protein would be r e l a t i v e l y insensitive to any e f f e c t of the study treatments on the t o t a l P450 content of the l i v e r . DISCUSSION / 178 Hepatic microsomal erythromycin demethylase a c t i v i t y calculated on the basis of t o t a l P450 has a d i f f e r e n t meaning, r e f e r r i n g to the rate of product formation per nmole microsomal t o t a l P450. This c a l c u l a t i o n w i l l be very sensitive to changes in t o t a l P450 content in the l i v e r , and could give r i s e to anomolous r e s u l t s . For example, i f the study treatments i n h i b i t the synthesis of a P450 enzyme which i s not being measured in the enzyme assay, but do not affec t the P450 form which i s being measured in the assay, the resultant decrease in t o t a l P450 w i l l result in an apparent increase in the a c t i v i t y being studied. In th i s study, microsomal P450 content per mg protein decreased in the groups Fem GxTa and Fem GxTp (Table VIII) due to a r e l a t i v e decrease (8-13%) in t o t a l l i v e r P450 in these groups. Although small and not s t a t i s t i c a l l y s i g n i f i c a n t , t h i s decrease may explain the apparent increase in hepatic microsomal erythromycin demethylase a c t i v i t y in these groups when calculated on the basis of P450 as compared to ca l c u l a t i o n on the basis of protein. Therefore, these two calculations have d i f f e r e n t meanings, and can best be interpreted by consideration of the changes in l i v e r P450 and protein. In t h i s study, these effects were generally small, and did not a l t e r the pattern of re s u l t s . The largest change observed was a 15 percent increase in DISCUSSION / 179 t o t a l microsomal P450 per mg p r o t e i n i n non-gonadectomized males t r e a t e d with adult t e s t o s t e r o n e . In a d d i t i o n , these e f f e c t s were not c o n s i s t e n t between the p i l o t and major s t u d i e s , which tends to i n d i c a t e random events. There remains the question of whether changes i n hepatic microsomal erythromycin demethylase a c t i v i t y i n d i c a t e changes i n the amount of an enzyme (or .enzymes) c a t a l y z i n g the r e a c t i o n , or a change i n the enzyme forms present, with a l t e r e d product formation being due to changes i n Km and Vmax. The present study d i d not attempt to d i s t i n g u i s h between such q u a n t i t a t i v e and q u a l i t a t i v e e f f e c t s , t h e r e f o r e no f i r m conclusions can be made. I t i s p o s s i b l e that both q u a l i t a t i v e and q u a n t i t a t i v e e f f e c t s are i n v o l v e d . 4.3. SPECULATION ON RELEVANCE TO HUMANS This study would be incomplete without a b r i e f mention of i t s p o t e n t i a l relevance to the human species. Is there a sex d i f f e r e n c e i n hepatic microsomal P450s i n humans, and i f so, i s i t regulated by hormones? As i n r a t s , m u l t i p l e forms of P450 have been i d e n t i f i e d i n human l i v e r (Guengerich 1987; Gonzalez, 1989), however q u a n t i t a t i v e or q u a l i t a t i v e sex d i f f e r e n c e s i n s p e c i f i c human P450 forms have not been reported. I t i s not known i f DISCUSSION / 180 t h i s indicates a lack of sexu a l l y - d i f f e r e n t i a t e d P450 forms, or r e f l e c t s the current paucity of human data. The p o s s i b i l i t y of a sex difference in human steroid metabolism has been investigated. Thrasher et al. ( 1969) found a higher urinary 6/3-hydroxycortisol/17-hydroxycorticosteroid r a t i o in females. Pfaffenberger and Horning (1977) have also reported a sex difference in human steroid metabolism, based on an analysis of urinary steroid metabolites. However, human l i v e r studies of testosterone hydroxylation have not reported a sex difference, with 60-hydroxytestosterone being the major hydroxylated metabolite formed (Lisboa and Gustafsson, 1968; Kremers et al, 1981). As reviewed by G i u d i c e l l i & Tillement (1977) and Proksch & Lamy (1977), sex differences in drug metabolism have been reported for only a few compounds. The clearances of diazepam and prednisolone are higher in adult females (Ochs et al., 1981; Meffin et al., 1984), while the clearances of chlordiazepoxide and propranolol are higher in adult males (Roberts et al., 1979; Walle et al, 1989). These reported differences require v e r i f i c a t i o n , and have not been linked with differences in s p e c i f i c P450 forms. Information is even more sparse on regulation by hormones of sex differences, although hormonal influences on the metabolism of some drugs have been reported (Proksch & Lamy, 1977). DISCUSSION / 181 Metabolic evidence therefore suggests that se x u a l l y - d i f f e r e n t i a t e d P450 forms might eventually be i d e n t i f i e d in humans. If so, several speculations of pharmacological interest can be made. It i s possible that the appropriate dosing of drugs with low therapeutic indices might vary between the sexes. The assessment of potential ri s k from toxic chemicals may also require a consideration of gender. And f i n a l l y , "if i t is found that hormones regulate a sex difference in human P450 forms as in rats, then i t i s possible that hormonal influences could a l t e r hepatic metabolism. Diseases which a l t e r hormone l e v e l s , such as hypogonadism, Stein-Levinenthal syndrome and growth hormone secretion abnormalities, could have an influence on metabolism. Drugs which a l t e r hormone l e v e l s , for example a n t i e p i l e p t i c drugs and glucocorticoids which reduce free testosterone levels (Dana-Haeri et al., 1982; MacAdams et al., 1986) could also a l t e r metabolism. These e f f e c t s of drugs and disease on metabolism might be immediate, for example in adulthood. As well, i f present during an early and sensitive developmental period, drug or disease-related hormonal changes could permanently a l t e r a l a t e r metabolic potential, long after the disease had been ameliorated or the drug cleared from the body. DISCUSSION / 182 4.4. PROPOSALS FOR FUTURE RESEARCH In order to extend the results of the present study, several directions for future research are proposed. The interpretation of the results of this study was limited by the lack of s p e c i f i c i t y of the erythromycin demethylase assay. It i s therefore essential to conduct future research using methods which can be linked as much as possible to s p e c i f i c P450 forms. The measurement of regio- and stereo-selective testosterone hydroxylation by HPLC, or, preferably, i f available, the use of mono-specific antibodies i s required. The results of t h i s study indicated that P450III forms- should be further investigated with respect to pubertal imprinting of androgen responsiveness. As well, for the reasons outlined in the introduction, the p o s s i b i l i t y that pubertal testosterone could imprint androgen responsiveness for P450h should be investigated. Such a p o s s i b i l i t y i s p a r t i c u l a r l y exciting, since much of the research emphasizing the importance of the neonatal period for androgen imprinting has involved P450h. Therefore, a study using antibodies s p e c i f i c for P450III forms and P450h to investigate the imprinting of adult androgen responsiveness is suggested. Secondly, the timing of the " c r i t i c a l period" during which DISCUSSION / 183 adult androgen responsiveness can be imprinted by testosterone requires further c l a r i f i c a t i o n . Neonatal gonadectomy and subsequent testosterone administration at discrete intervals ranging from b i r t h through puberty and into adulthood would provide further evidence regarding the re l a t i v e importance Of the neonatal, pubertal, and later periods for the imprinting of t h i s parameter. Ultimately, the mechanisms involved in the imprinting of adult androgen responsiveness require e l u c i d a t i o n . The combination of enzyme kinetic studies with immunoquantitation of s p e c i f i c P450 levels would distinguish q u a l i t a t i v e versus quantitative e f f e c t s . Studies in both ovariectomized and non-ovariectomized female rats would indicate any possible role of the ovaries in the i n h i b i t i o n of pubertal testosterone imprinting. A possible indi r e c t effect of testosterone on the l i v e r via growth hormone should also be investigated. For example, an experiment similar to the present one, investigating the effects of intermittent, male pattern, growth hormone administration during puberty on adult androgen responsiveness could be conducted. DISCUSSION / 184 4.5. SUMMARY AND CONCLUSIONS We conclude the following: 1. Hepatic microsomal erythromycin demethylase a c t i v i t y was higher in adult male rats than in adult females, further corroborating the findings of Arlo t t o et al. (1987) that this i s an hepatic P450 enzyme a c t i v i t y which i s sexu a l l y - d i f f e r e n t i a t e d . 2. Adult male rats (non-castrated) but not adult female rats (non-ovariectomized) responded to adult testosterone treatment at physiological doses with an increase in hepatic microsomal erythromycin demethylase a c t i v i t y . Therefore, the intact adult female rat lacked the androgen responsiveness with respect to hepatic microsomal erythromycin demethylase a c t i v i t y which was present in the adult male rat. 3. Prepubertal castration of male rats reduced hepatic microsomal erythromycin demethylase a c t i v i t y and plasma testosterone levels from control l e v e l s . Hepatic microsomal erythromycin demethylase a c t i v i t y in the d i f f e r e n t treatment groups was found to be p a r t i a l l y correlated with plasma testosterone l e v e l s . These data suggested that the higher hepatic microsomal erythromycin demethylase a c t i v i t y in the adult male rat i s related to the high adult male levels of c i r c u l a t i n g testosterone. DISCUSSION / 185 4. In adult female rats which had been ovariectomized prepubertally, exposure to testosterone peripubertally resulted in an adult responsiveness to testosterone for hepatic microsomal erythromycin demethylase a c t i v i t y . This indicated that the potential is present in the prepubertally-ovariectomized female rat for the pubertal imprinting by testosterone of an adult androgen responsiveness for hepatic microsomal erythromycin demethylase a c t i v i t y . 5. The administration of testosterone to adult male rats which had been prepubertally castrated resulted in hepatic microsomal erythromycin demethylase a c t i v i t y which was lower than that of intact males and of intact males treated with testosterone in adulthood. This result indicated that adult androgen responsiveness of hepatic microsomal erythromycin demethylase a c t i v i t y i s not completely imprinted in males in the neonatal period. 6. The magnitude of the adult androgen response for hepatic microsomal erythromycin demethylase a c t i v i t y following peripubertal testosterone treatment of prepubertally-gonadectomized rats was larger in the male than in the female. These results suggested that peripubertal testosterone i s not the sole factor influencing DISCUSSION / 186 the imprinting of adult androgen responsiveness for hepatic microsomal erythromycin demethylase a c t i v i t y . 7. The results were inconclusive concerning the role of pubertal testosterone in the imprinting of an adult androgen responsiveness for hepatic microsomal erythromycin demethylase a c t i v i t y in the prepubertally-castrated male rat. This indicated that factors other than pubertal testosterone were also involved. 8. 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Waxman DJ, LeBlanc GA, Morrissey J J , Staunton J and Lapenson DP, Adult m a l e - s p e c i f i c and neonatally programmed r a t hepatic P450 forms RLM2 and 2a are not dependent on p u l s a t i l e plasma growth hormone for expression. J Biol Chem 263: 11396-1 1406, 1986. Weisz J and Ward IL, Plasma testosterone and progesterone t i t e r s of pregnant r a t s , t h e i r male and female f e t u s e s , and neonatal o f f s p r i n g . Endocrinology 106: 306-316, 1980. REFERENCES / 201 Wiebel FJ and Gelboin HV, Aryl hydrocarbon (benzo[a]pyrene) hydroxylases in l i v e r from r a t s of d i f f e r e n t age, sex and n u t r i t i o n a l S t a t u s . Biochem Pharmacol 24: 1511-1515, 1975. Williams MT and Simonet LC, Effects of growth hormone on cytochrome P450J. Biochem Biophys Res Commun 155:392-397, 1988. Wood AW, Ryan DE, Thomas PE and Levinin W, Regio- and stereoselective metabolism of two C ^ steroids by five highly p u r i f i e d and reconstituted rat hepatic cytochrome P450 isozymes. J Biol Chem 258: 8839-8847, 1983. Wrange A, Norstedt G and Gustafsson J-A, The estrogen receptor in rat l i v e r : quantitative and q u a l i t a t i v e analysis by i s o e l e c t r i c focusing in polyacrylamide gel. Endocrinology 106: 1455-1463, 1980. Wrighton SA, Maurel P, Schuetz EG, Watkins PB, Young B and Guzelian PS, I d e n t i f i c a t i o n of the cytochrome P450 induced by macrolide a n t i b i o t i c s in rat l i v e r as the glucocorticoid responsive cytochrome P450p. Biochemistry 24: 2171-2178, 1985a. Wrighton SA, Schuetz EG, Watkins PB, Maurel P, Barwick J, Bailey J, Bailey BS, Hartle HT, Young B and Guzelian P, Demonstration in multiple species of inducible hepatic cytochromes P450 and their mRNAs related to the glucocorticoid-inducible cytochrome P450 of the rat. Molec Pharmacol 28: 312-321, 1985b. Yamazoe Y, Murayama N, Shimada M, Yamauchi K, Nagata K, Imaoka S, Funae Y and Kato R, A sex-specific form of cytochrome P450 catalyzing propoxycoumarin O-depropylation and i t s identity with testosterone 6/3-hydroxylase in untreated rat l i v e r s : reconstitution of the a c t i v i t y with microsomal l i p i d s . J Biochem 104: 787-790, 1988. Yates FE, Herbst AL and Urquhart J, Sex difference in rate of ring reduction of A4-3-keto-steroids in vitro by rat l i v e r . Endocrinology 63: 887-902, 1958. Zaphiropoulos PG, Mode A, Norstedt G and Gustafsson J-A, Regulation of sexual d i f f e r e n t i a t i o n in drug and steroid metabolism. Trends in Pharmacol Sci 10: 149-153, 1989. 6. APPENDIX Table A. P i l o t study: results in ovariectomized female rats. From data obtained by RCK Pak ( l i v e r and body weights) and S. Bandiera (P450). Results are presented as mean ± S.E.M.. and are also contained in Table B. Abbreviations: Fem, Female; Gx, prepubertal gonadectomy; Ta, adult testosterone treatment; Tp, pubertal testosterone treatment; "*" indicates a s i g n i f i c a n t difference from each other in that row (p<0.05); "**" indicate a s i g n i f i c a n t difference from each other in that row (p<0.05). 202 TaD1e A. Fem Gx Fem GxTa TREATMENT GROUP Fem GxTp Fem GxTpTa mg p r o t e i n per gram wet weight 1 i ver 48.5+3.9 57.9+4.6 55 . 8 + 6 . 4 50. 1+2.9 nmoles P450 per gram wet weight 1 1 ver 41.6+5.9 47.5±4.7 48 . 2 + 4.3 44.1+7.0 nmoles P 4 5 0 per mg p r o t e i n i n hepat I c m1crosomes 0.85+0.05 0.83±0.02 0.87±0.03 0 . 8 7 i 0 . 0 9 Boay weight (9) 35 1+9 3 15 + 8 * 300±11 335+13 L i v e r Weight (9) 10.5±0.7 9.2+0.4 9.2+0.7 1 1 . 6+0.7 L(ver/Body ('/.) 3.0+0.1 2.9±0.1 * * 3.0+0. 1 3 . 4±0. 1 A P P E N D I X / 204 T a b l e B. P i l o t s t u d y : r e s u l t s i n a l l t r e a t m e n t groups. From da t a o b t a i n e d by RCK Pak ( l i v e r and body w e i g h t s ) and S. B a n d i e r a (P450). Dta i s p r e s e n t e d as mean ± S.E.M.. and i s a l s o c o n t a i n e d i n T a b l e B. A b b r e v i a t i o n s : Fem, Female; Gx, p r e p u b e r t a l gonadectomy; Ta, a d u l t t e s t o s t e r o n e t r e a t m e n t ; Tp, p u b e r t a l t e s t o s t e r o n e t r e a t m e n t ; *, f o r s t a t i s t i c s r e f e r t o T a b l e C; "**", i n d i c a t e s a s i g n i f i c a n t d i f f e r e n c e from Con Fem, Fem GxTa, Fem GxTp, Fem GxEp and Fem GxEpTa groups i n t h a t row (p<0.05); "***" i n d i c a t e s a s i g n i f i c a n t d i f f e r e n c e from Fem Gx, Fem GxTa and Fem GxTp groups i n t h a t row (p<0.05). T a b l e B TREATMENT GROUPS CON Ma 1 e CON Fem Fem Gx Fem GxTa Fem GxTp Fem GxTpTa F em GxEp Fem GxEpTa nmo1e s P450 p e r mg p r o t e i n 0 . 9 710.10 0.82+0.02 0.85+0.05 0.82+0.02 0 . 8 7 1 0 . 0 3 0 . 8 7 1 0 . 0 9 0 . 8 7 1 0 . 0 9 0.79+0.0: nmo1es P 4 5 0 p e r g wet w e i g h t o f 1 i v e r 4211 52 + 2 42±6 48±5 48±4 44 + 7 51+5 4512 mg p r o t e i n p e r g wet w e i g h t o f 1 i v e r 44+4 6414 49+4 58 + 5 5616 5013 5913 5713 B o o y W e i g h t Cg) * 5 17+18 240+13 35119 31518 300111 3 3 6 1 1 3 23319 22518 L i v e r we i g n t ( g ) 18.210.7 8.2+0.6 10.510.7 9.2+0.4 9,210.7 * * 11.610.7 8.4+0.4 8.710.6 L f v e r / B o d y 3 . 5110.04 3.3710.07 2.99+0.14 2.93+0.11 3 . 0 4 1 0 . 1 3 3.44+0. 13 » * m 3.6210.04 m * m 3.87+0.2' > W a to o APPENDIX / 206 T a b l e C. P i l o t s t u d y : s t a t i s t i c s f o r body w e i g h t s . i n d i c a t e s a s i g n i f i c a n t d i f f e r e n c e between groups ( p < 0 . 0 5 ) . Treatment groups: Grp 2: Con Fem Grp 3: Fem Gx Grp 4: Fem GxTa Grp 5: Fem GxTp Grp 6: Fem GxTpTa Grp 7: Fem GxEp Grp 8: Fem GxEpTa is <3 l i r r i r "r" r r P P P P p P P M e a n Group S 7 ~ 4 6 2 2 5 . O O O O Grp 3 2 3 3 - O O O O Grp 7 2 3 3 . 5 0 0 0 Grp 2 2 3 3 . 5 0 0 0 Grp 5 3 1 5 . O O O O Grp 4 3 3 6 . O O O O Grp G V-3 5 1 . O O O O Grp 3 -V- -V-APPENDIX / 207 Table D. Major study: s t a t i s t i c s f o r transformed erythromycin demethylase ( n a t u r a l l o garithm of nmoles formaldehyde per minute per mg p r o t e i n ) . "*" i n d i c a t e s a s i g n i f i c a n t d i f f e r e n c e between groups (p<0.05). Treatment groups: Grp 1: Con Fem Grp 2: Con Fem Ta Grp 3: Fem Gx Grp 4: Fem GxTa Grp 5: Fem GxTp Grp 6: Fem GxTpTa Grp 7: Con Male Grp 8: Con Male Ta Grp 9: Male Gx Grp 10: Male GxTa Grp 11: Male GxTp Grp 12: Male GxTpTa Mean G r o u p G G G G G G G G G G G G r r r r r r r r r r r r P P P P P P 1 P P 1 P P 1 P P «_/ 9 4 o 1 1 6 7 a -1. 1905 G r p 3 -1. 1202 G r p 9 -1. 1 173 G r p 4 -1. 1075 G r p 5 9305 G r p 2 — . 7700 G r p l O 7320 G r p 1 7311 G r p 11 6167 G r p 6 *—t--^ G r p 12 — . 3145 G r p 7 • 1325 G r p 8 * * * •X--x- * *• •X- X- •X-*- * * -X- •X- •K- •X-* * •X- •X- -X- •X- •X-APPENDIX / 208 Table E. Major study: s t a t i s t i c s f or transformed erythromycin demethylase ( n a t u r a l logarithm of nmoles formaldehyde per minute per nmole P450). "*" i n d i c a t e s a s i g n i f i c a n t d i f f e r e n c e between groups (p<0.05). Treatment groups Grp 1 : Con Fem Grp 2: Con Fem Ta Grp 3 : Fem Gx Grp 4 : Fem GxTa Grp 5: Fem GxTp Grp 6: Fem GxTpTa Grp 7: Con Male Grp 8: Con Male Ta Grp 9 : Male Gx Grp 10 : Male GxTa Grp 1 1 : Male GxTp Grp 12 : Male GxTpTa id l i i i ij ts la G 'la ta r r r r r r r r r r r r P P P P P P P P P P p P 1 1 1 Mean Group «-» 3 4 er- 2 1 1 0 6 2 7 8 -1.3577 Grp 3 -1.1781 Grp 9 -1.1741 Grp 4 -1.1223 Grp 5 -1.0383 Grp 2 -.9074 Grp 11 * -.3469 Grp 1 -.8213 Grp 10 *--.7333 Grp 6 *--.5757 Grp 12 * *• •*- *- *--.4884 Grp 7 * * •* * -.1872 Grp 8 * * * *• *• * #- *• APPENDIX / 209 F i g u r e 1. Scheme of methodology used in the major s t u d y . CD a o v_ CD •*-> CO o CO CD o 1— o o o o CO co o o o Q o X h-LU CD c o CD - 4 - * CO o -4—' CO CD o o o X O o <D CL to co CM CO CD D) CO o APPENDIX / 210 F i g u r e 2. ANOVA t a b l e f o r d a t a on gonadectomized female r a t s i n the p i l o t s t u d y . V a l u e s r e p r e s e n t the mean e r y t h r o m y c i n demethylase a c t i v i t y per mg microsomal p r o t e i n (upper t a b l e ) or per nmole P450 (lower t a b l e ) . TFLIB T A D U L T 0 1 0 . 2 3 - 3 3 ( 4) < 4 ) 1 ' . 3 5 . 5 3 ( 4 j < 4!> T A D U L T O 1 TPUB 0 . 3 6 . 4 1 '•• 4 ) < 4 ) 1 - 4 1 . 7 4 <: 4:> <: 4.i APPENDIX / 211 Figure 3. ANOVA t a b l e for data on gonadectomized female r a t s in the major study. Values represent the mean erythromycin demethylase a c t i v i t y per mg p r o t e i n (upper t a b l e ) or per nmole P450 (lower t a b l e ) . T A D U L T O 1 T P U B 0 .31 .34 ( 9) ( 9) A T - » cr cr ( 9) ( S'j T A D U L T 0 1 T P U B 0 .26 .32 ( 9.) < 9i> 1 .33 .47 ( 9.) ( 9!) 

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