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Studies relating hepatic cytosolic [|H]-estradiol binding proteins to hormonal and drug modulation of… Finlayson, Malcolm John Paul 1983

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STUDIES RELATING HEPATIC CYTOSOLIC [JH]-ESTRADIOL BINDING PROTEINS TO HORMONAL AND DRUG MODULATION OF HEPATIC MICROSOMAL ARYL HYDROCARBON HYDROXYLASE IN THE RAT by •MALCOLM JOHN PAUL FINLAYSON B.A., San Jose State University, 1976 M.Sc, Univ e r s i t y of B r i t i s h Columbia, 1980 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES FACULTY"OFfPHARMACEUTICAL SCIENCES UNIVERSITY OF BRITISH COLUMBIA We accept t h i s t hesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1983 © MALCOLM JOHN PAUL FINLAYSON, 1983 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available f o r reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Pharmaceutical Sciences The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6 (3/81) ABSTRACT Pituitary hormones are known to alter sex steroid receptor levels in the li v e r , and possibly the actions of the steroids as well. Recently, two classes of estrogen binding proteins have been characterized in male rat hepatic cytosol: a high a f f i n i t y , low capacity estrogen receptor, and a lower a f f i n i t y , higher capacity sex steroid binding component (moderate a f f i n i t y component). It i s of interest that the moderate a f f i n i t y component binds both androgens and estrogens. A high a f f i n i t y , low capacity androgen receptor has not been convincingly demonstrated in rat hepatic cytosol. Therefore, we have investigated the relationship of the moderate aff i n i t y component to sex steroid modulation of hepatic aryl hydrocarbon hydroxylase (AHH) activity as a possible control mechanism. Because of the sexual dimorphism for hepatic drug and steroid metabolism known to occur in rat l i v e r , we chose this model to study. We have shown that no sex difference exists for the binding of pH]-estradiol to the estrogen receptor from either immature or adult rats. However, the moderate aff i n i t y component does exhibit a sex difference. We did not detect binding to the moderate a f f i n i t y component in adult female or immature rats of either sex. This site could normally only be measured in the adult male. These findings were consistent with the age and sex dependent elevation of male AHH activity. We have also observed that gonadectomy of i i i the male reduced the l e v e l s of AHH a c t i v i t y and the capacity of the moderate a f f i n i t y component i n a testosterone r e v e r s i b l e fashion. These r e s u l t s were obtained using either unlabeled e s t r a d i o l or 3 dihydrotestosterone (DHT) as competitors f or [ H ] - e s t r a d i o l binding. Administration of mestranol reduced AHH a c t i v i t y and the capacity of the moderate a f f i n i t y component i n the male. The moderate a f f i n i t y component was not detected i n the pseudoherma-phroditic rat which resembled the female, rather than the male, with respect to control and induced AHH a c t i v i t y . Hypophysectomy- of the female resulted i n an increase i n AHH a c t i v i t y and detection of the moderate a f f i n i t y component. Hypophysectomy of the male reduced both the capacity of the moderate a f f i n i t y component and AHH a c t i v i t y . Unlike the gonadectomized male, testosterone had no r e s t o r a t i v e e f f e c t on the l e v e l s of AHH a c t i v i t y or the capacity of the moderate a f f i n i t y component i n the hypophy-sectomized r a t . Continuous i n f u s i o n of rat growth hormone (rGH) reversed the ef f e c t of hypophysectomy on the increased AHH a c t i v i t y and capacity of the moderate a f f i n i t y component i n the female. Administration of rGH to the hypophysectomized male abolished the detection of the moderate a f f i n i t y component and reduced AHH a c t i v i t y to c o n t r o l female l e v e l s . This suggested rGH may be the p i t u i t a r y hormone involved i n production of the female l e v e l of metabolism. The e f f e c t s of p r o l a c t i n were not as c l e a r . Therefore, we have demonstrated the modulation of AHH a c t i v i t y by peripheral sex stero i d s , and the reg u l a t i o n of these parameters by rGH. We have shown, the capacity of the moderate a f f i n i t y component to vary- i n i v a manner that p a r a l l e l e d changes i n hepatic AHH a c t i v i t y i n d i f f e r e n t p h y s i o l o g i c a l models. Changes i n the estrogen receptor were not found to be consistent with changes i n AHH a c t i v i t y i n these models. We conclude that the moderate a f f i n i t y component i s comparable to the male hepatic c y t o s o l i c DHT-binding protein. Furthermore, t h i s component i s associated with sex steroid action on hepatic AHH a c t i v i t y i n the male r a t . I n t e r e s t i n g l y , we have also shown t h i s component as well as the estrogen receptor, to bind p o l y c y c l i c aromatic hydrocarbons. Both 3-methylcholanthrene and benzo[a]pyrene competed for 3 [ H ] - e s t r a d i o l binding to the estrogen receptor and moderate a f f i n i t y component. In addition, dioxin congeners demonstrated s p e c i f i c i t y for the estrogen receptor i n the female. However, th i s was not observed for the estrogen receptor or moderate a f f i n i t y component i n the male. The s i g n i f i c a n c e of t h i s i s presently unclear. Gail. D, Bell,ward, Ph.D. Supervisor V TABLE OF CONTENTS PAGE ABSTRACT i i LIST OF TABLES x LIST OF FIGURES x i v LIST OF ABBREVIATIONS AND TRIVIAL CHEMICAL NAMES xv ACKNOWLEDGEMENT .x v i i INTRODUCTION 1 1. Sex dependent differences i n hepatic mixed function oxidase a c t i v i t y 1 2. Neonatal androgen induced imprinting 3 3. Involvement of the hypophyseal-hypothalamic axis i n the maintenance of sex dependent differences i n hepatic metabolism 5 a. P i t u i t a r y e f f e c t s 5 b. Hypothalamic e f f e c t s 7 c. Feminotropin 8 4. E f f e c t s of p i t u i t a r y hormones on hepatic metabolism 9 5. Interaction of sex steroids and hepatic lactogen binding 14 6. P i t u i t a r y modulation of lactogen binding 17 7. E f f e c t s of p i t u i t a r y hormones on hepatic sex s t e r o i d binding 20 8. Hepatic sex steroid-binding proteins 22 9. Hypothesis 24 v i PAGE MATERIALS AND METHODS 26 1. Chemical and reagents 26 2. Animals 27 3. Animal treatments 28 4. Hepatic microsomal enzyme assays 29 a. Hepatic microsomal protein preparation 29 b. A r y l hydrocarbon hydroxylase a c t i v i t y 30 c. Testosterone A4 reductase a c t i v i t y 31 5. Hepatic sex s t e r o i d binding assays 32 a. Preliminary studies 32 b. Hepatic estrogen receptor and moderate a f f i n i t y binding component assay 34 c. Analysis of binding data 36 6. Determination of protein content 38 7. Serum hormone l e v e l s 38 8. S t a t i s t i c a l a n alysis 39 RESULTS 4 0 1. I n i t i a l c h a r a c t e r i z a t i o n of hepatic estrogen binding components ^0 a. Comparison of d i f f e r e n t estrogen receptor assays b. Time, temperature and pH studies 4 ^ c. Freezing studies 4 8 d. Competitor studies 4 8 e. Binding to uterus, bovine serum albumin and plasma 51 v i i PAGE 2. E f f e c t s of p h y s i o l o g i c a l manipulation on hepatic estrogen binding components 51 a. Age and sex related modulation of hepatic estrogen binding 51 b. P i t u i t a r y modulation of hepatic estrogen binding 55 3. Characterization of hepatic enzyme a c t i v i t y i n d i f f e r e n t p h y s i o l o g i c a l models 70 a. Sex related modulation of hepatic AHH and testosterone A 4 reductase a c t i v i t i e s 70 b. Endocrine manipulation of hepatic enzyme a c t i v i t y 76 i . E f f e c t of pergolide i n adult female r a t s 76 i i . E f f e c t of pergolide i n pre-pubescent female r a t s 80 i i i . E f f e c t of pimozide i n adult male ra t s 82 4. Studies r e l a t i n g hepatic AHH a c t i v i t y to hepatic c y t o s o l i c estrogen binding components 84 a. E f f e c t of pergolide 84 b. E f f e c t of pimozide 86 c. E f f e c t of continuous infusion of p i t u i t a r y hormones 88 5. In vivo and i n v i t r o e ffects of xenobiotics on hepatic AHH a c t i v i t y and c y t o s o l i c estrogen binding parameters 9 3 a. Induction 9 3 b. I n h i b i t i o n 9 8 c. The e f f e c t of p h y s i o l o g i c a l manipulation on in v i t r o binding of 3-MC 1 0 0 d. Relationship of estrogen binding components to Ah-receptor 100 v i i i DISCUSSION PAGE 106 1. Estrogen binding studies: Characterization and comparison of hepatic c y t o s o l i c estrogen binding components with previously published r e s u l t s 106 2. E f f e c t s of p h y s i o l o g i c a l manipulation on hepatic estrogen binding m a. Gonadectomy e f f e c t s on hepatic estrogen binding m i . Imprinting of the moderate a f f i n i t y component i i . Sucrose density gradient versus competitive binding studies b. E f f e c t s of hypophysectomy on hepatic estrogen binding 3. Comparison of hepatic enzyme a c t i v i t y to hepatic estrogen binding components a. General conclusions from published observations ion b. Age and sex dependency c. Androgen dependency 122 d. E f f e c t of estrogens e. Pseudohermaphroditic rat model 1 o / f. Hypophysectomy model 4. Relationship of p i t u i t a r y hormones to hepatic AHH a c t i v i t y and estrogen binding a. Background 126 b. E f f e c t s of pimozide , 127 c. E f f e c t s of pergolide 128 d. P i t u i t a r y hormone replacement: E f f e c t s on hepatic AHH a c t i v i t i e s and c y t o s o l i c estrogen binding 129 i . Hormone replacement i n male r a t s 131 i i . Hormone replacement in female r a t s 133 i x PAGE 5. The e f f e c t s of xenobiotics on hepatic AHH a c t i v i t y and c y t o s o l i c estrogen binding parameters 135 a. In v i t r o e f f e c t s 135 i . Ah receptor 135 i i . Hepatic c y t o s o l i c BP/3-MC binding protein 137 b. In vivo e f f e c t s of xenobiotics 140 6. M u l t i p l i c i t y of Cytochromes P-450 142 7. Proposed experiments 144 8. P h y s i o l o g i c a l relevance 147 SUMMARY 152 BIBLIOGRAPHY 155 APPENDIX I I 6 8 1) Additional studies of [ H ] - e s t r a d i o l binding to the estrogen receptor and moderate a f f i n i t y component i n rat hepatic c y t o s o l 168 a) Determination of optimal incubation time at 4°C 168 b) Determination of ligand s p e c i f i c i t y i n female rat hepatic cytosol 173 (Assays performed by Dr. B. Warren) 2) Determination of serum PRL l e v e l s by bioassay 173 (Assays performed by Mr. P. Gout) X LIST OF TABLES PAGE Table 1 Table 2 Table 3 Table 4 Table 5, Table 6 The apparent Kd and capacity of [ H ] - e s t r a d i o l binding i n d i f f e r e n t f r a c t i o n s of adult male and female rat l i v e r using various competitors ... 42 The apparent Kd and capacity of [ 3 H ] - e s t r a d i o l binding to d i f f e r e n t f r a c t i o n s of adult male rat l i v e r 44 Time course of s p e c i f i c [ H ] - e s t r a d i o l binding at 25°C 46 The e f f e c t of pH on the apparent Kd and capacity of the hepatic estrogen receptor and moderate a f f i n i t y component 47 The e f f e c t of freezing on the apparent Kd and capacity of the estrogen receptor and moderate a f f i n i t y component 49 Ligand s p e c i f i c i t y of [ H ] - e s t r a d i o l binding to the estrogen receptor and moderate a f f i n i t y component i n the adult male rat 50 Table 7 A comparison of the apparent Kd and capacity of the hepatic c y t o s o l i c estrogen receptor and moderate a f f i n i t y component i n male, female, and pseudohermaphroditic r a t s 53 Table 8 The e f f e c t of age and gonadectomy on the apparent Kd and capacity of hepatic c y t o s o l i c estrogen receptor and moderate a f f i n i t y binding component i n male and female r a t s .... . 54 Table 9 The e f f e c t of gonadectomy and testosterone replacement on the apparent Kd and capacity of the hepatic c y t o s o l i c estrogen receptor and moderate a f f i n i t y binding component in male and female r a t s 56 Table 10 The e f f e c t of hypophysectomy on the binding c h a r a c t e r i s t i c s of the hepatic c y t o s o l i c estrogen receptor i n male and female r a t s .. 59 xi:_ PAGE Table 11 The e f f e c t of hypophysectomy on the binding c h a r a c t e r i s t i c s of the moderate a f f i n i t y component in male and female rat s 61 Table 12 The e f f e c t of hypophysectomy on the apparent Kd and capacity of the hepatic c y t o s o l i c estrogen receptor and moderate a f f i n i t y binding component at four weeks post-surgery i n male and female ra t s 64 Table 13 Table 14 Table 15 Table 16 Table 17 Table 18 Table 19 Table 20 The e f f e c t of testosterone and growth hormone on the binding c h a r a c t e r i s t i c s of the hepatic c y t o s o l i c estrogen receptor i n the hypophysectomized r a t s . 65 The e f f e c t of testosterone and growth hormone on the binding c h a r a c t e r i s t i c s of the hepatic c y t o s o l i c moderate a f f i n i t y component in hypophysectomized rats 67 The e f f e c t of phenobarbital and spirono-lactone on hepatic AHH a c t i v i t y i n male, female and psudohermaphroditic r a t s ^1 The effect of 3-methylcholanthrene on hepatic AHH a c t i v i t y i n male, female, and 7 ^ pseudohermophrodite rats ' 3 The e f f e c t of hypophysectomy on hepatic AHH a c t i v i t y i n male and female rats ^ 4 The effect of hypophysectomy and testosterone on hepatic AHH a c t i v i t y i n male and female 7 s r a t s / J The e f f e c t of 3-methylcholanthrene and phenobarbital on hepatic AHH a c t i v i t y i n hypophysectomized male and female r a t s 77 The e f f e c t of gonadectomy, pergolide and testosterone on hepatic AHH and testosterone A4 reductase a c t i v i t y i n female ra t s 79 Table 21 The e f f e c t of pre-pubescent gonadectomy and p r o l a c t i n depletion on the action of testosterone on hepatic AHH and testosterone A4 reductase a c t i v i t i e s i n the adult female rat XXI PAGE Table 22 The e f f e c t of gonadectomy, pimozide and testosterone on hepatic AHH and te s t o -sterone A4 reductase a c t i v i t y i n adult male rats 83 Table 23 Table 24 The ef f e c t of pergolide and testosterone on hepatic AHH a c t i v i t y and the apparent Kd and capacity of the hepatic c y t o s o l i c estrogen receptor and moderate a f f i n i t y component in adult gonadectomized and sham-operated female ra t s The e f f e c t of pimozide and testosterone on hepatic estrogen binding and hepatic AHH a c t i v i t y i n sham and gonadectomized male r a t s 85 Table 25 Comparison of the effect of hypophysectomy and hormone replacement on hepatic c y t o s o l i c estrogen binding components and AHH a c t i v i t y in male and female ra t s 89 Table 26 Table 27 The e f f e c t of 3-methylcholanthrene and spironolactone on the apparent Kd and capacity of the hepatic c y t o s o l i c estrogen receptor and the moderate a f f i n i t y binding component i n male and female r a t s The e f f e c t of 4 days treatment with pheno-b a r b i t a l on the apparent Kd and capacity of the hepatic c y t o s o l i c estrogen receptor and moderate a f f i n i t y binding component in male and female r a t s 95 96 Table 28 The e f f e c t of 10 days treatment with mestranol, phenobarbital and 3-methylcholanthrene on hepatic c y t o s o l i c estrogen binding c h a r a c t e r i s t i c s and hepatic AHH a c t i v i t y i n male and female rats 99 Table 29 The e f f e c t of gonadectomy and testosterone replacement on ligand s p e c i f i c i t y of the hepatic c y t o s o l i c estrogen receptor and moderate a f f i n i t y binding component i n male and female ra t s 101 x i i i PAGE Table 30 Ligand s p e c i f i c i t y of the hepatic c y t o s o l i c estrogen receptor and the moderate a f f i n i t y s i t e for various competitors for [ 3 H ] - e s t r a d i o l binding i n the male and female rat 103 Table 31 The apparent Kd and capacity of the estrogen receptor i n adult female r a t s as determined with d i f f e r e n t dioxin congeners 104 Table 32 Summary of c o n t r o l values for apparent Kd and capacity of the estrogen receptor and moderate a f f i n i t y component 105 Ta^le 33 Summary of r e l a t i v e changes in capacity of the estrogen receptor, moderate a f f i n i t y binding component and AHH a c t i v i t y r e l a t i v e to the adult male (unless otherwise noted) 121 3 Table A l Ligand s p e c i f i c i t y of [ H] - e s t r a d i o l binding s i t e s i n the 50% ammonium s u l f a t e (AS) f r a c t i o n of l i v e r from female r a t s 174 3 Table A2 Ligand s p e c i f i c i t y of [ H] - e s t r a d i o l binding s i t e s i n the whole cytosol f r a c t i o n of l i v e r from female r a t s 175 Table A3 S p e c i f i c a t i o n s of p i t u i t a r y hormones used i n the continuous i n f u s i o n studies 177 x i v LIST OF FIGURES PAGE Figure 1 E f f e c t of p h y s i o l o g i c a l manipulation on the s p e c i f i c [ % ] - e s t r a d i o l binding to the moderate a f f i n i t y component 58 Figure 2 E f f e c t of hypophysectomy on the s p e c i f i c [ 3 H ] - e s t r a d i o l binding to the moderate a f f i n i t y component 60 Figure 3 E f f e c t of hypophysectomy on the s p e c i f i c [ 3 H ] - e s t r a d i o l binding to the estrogen receptor 69 Figure A l Time course of s p e c i f i c [. H ] - e s t r a d i o l binding to the estrogen receptor at 4°C i n male and female r a t l i v e r , 50% ammonium sul f a t e f r a c t i o n 170 Figure A2 Time course of s p e c i f i c [ 3 H ] - e s t r a d i o l binding to the moderate a f f i n i t y component at 4°C i n male rat l i v e r , whole cytosol f r a c t i o n 172 XV LIST OF ABBREVIATIONS ACTH a d r e n o c o r t i c o t r o p i c hormone AHH a r y l hydrocarbon hydroxylase aldosterone 3,20-diketo-llf3,18-oxido-4-pregnene-18 ,21-diol androstenedione A4-androsten-3,17-dione bGH bovine growth hormone BP benzo [a]pyrene 2,7-DCDD 2,7-dichlorodibenzo-p-dioxin DES d i e t h y l s t i l b e s t r o l dexamethasone 9-fluoro-llg,17,21-trihydroxy-16a-methyl-pregna-l ,4-diene-3 ,20-dione DCC dextran coated charcoal DHT dihydrotestosterone E2 e s t r a d i o l EDTA ethylenediamine t e t r a a c e t i c acid 1 3 5(10) e s t r a d i o l A ' ' -estratrien-3,17g-diol FSH f o l l i c l e stimulating hormone GH growth hormone Gx gonadectomized hGH human growth hormone Hx hypophysectomized Imm immature (less than 30 days of age) i. p . i n t r a p e r i t o n e a l l y methyltrienolone 17g-hydroxy-17-methylestra-4,9,ll-trien-3-one x v i mestranol 3-MC OCDD oPRL PAH Perg Pim PRL progesterone Pseudo rGH rPRL s. c. T4 Test testosterone triamcinolone acetonide 3-methoxy-19-nor-17a-pregna-1,3,5(10)-t rien e -2 0-yn-17-ol 3-methylcholanthrene octachlorodibenzo-p-dioxin ovine p r o l a c t i n p o l y c y c l i c aromatic hydrocarbon pergolide pimozide p r o l a c t i n A4-pregnene-3,20-dione pseudohermaphrodite rat growth hormone rat p r o l a c t i n subcutaneously thyroxine testosterone &4-androsten-17p-ol-3-one 9q-fluoro-llB,16a,17,21-tetra-ol-pregna-l,4-diene-3,20-dione c y c l i c 16,17 a c e t a l with acetone x v i i ACKNOWLEDGEMENT I wish to express my deepest gratitude to Dr. G a i l Bellward for her insight and guidance throughout the course of my graduate t r a i n i n g . I would also l i k e to thank Dr. Betty Warren fo r her c o l l a b o r a t i o n i n these studies, and Dr. Edward Keenan for his i n t e r e s t and invaluable suggestions. F i n a l l y , I am indebted to Mrs. Christine Lee for her expert s e c r e t a r i a l assistance. 1 INTRODUCTION 1) Sex dependent differences i n hepatic mixed function oxidase a c t i v i t y It i s w e l l known that the a c t i v i t i e s of c e r t a i n rat hepatic microsomal cytochrome P-450 dependent mixed function oxidases exhibit a sex differ e n c e i n the adult rat (Conney, 1967; Kato, 1974; Kato and G i l l e t t e , 1965 ; Quinn, 1958). These differences are a t t r i b u t e d to changes in the hormonal m i l i e u of the adult animal. Studies to be discussed have indicated the important influences exerted by gonadal and p i t u i t a r y hormones toward expression of these differences i n metabolic a c t i v i t y . However, the exact mechanism by which these differences are expressed i s s t i l l unclear. E l Defrawy E l Masry and Mannering (1974) and E l Defrawy E l Masry et a l . , (1974) have confirmed the sexual dimorphism for c e r t a i n hepatic microsomal mixed function oxidase a c t i v i t i e s i n the r a t . Using animals ranging i n development from pre-pubescent to adult i t was shown that hepatic N-demethylation of ethylmorphine and aminopyrine were increased i n males r e l a t i v e to females as a function of age. This age and sex dependent difference has also been demonstrated for the metabolism of the p o l y c y c l i c aromatic hydrocarbon benzo(a)pyrene (BP) by the cytochrome P-450 dependent a c t i v i t y a r y l hydrocarbon hydroxylase (AHH). Oesch et a l . , (1976) reported no sex difference at day f i v e f o r AHH a c t i v i t y . However, a f t e r day ninety, male AHH a c t i v i t y increased r e l a t i v e to female. 2 Similarly, studies from our laboratory (Bellward et a l . , 1981; Gontovnick and Bellward, 1981) have shown no sex differ e n c e f o r AHH a c t i v i t y between the immature male and female both of which are s i g n i f i c a n t l y lower than the adult male. The sexual dimorphism observed i n the mature animal was suggested to be due to hormonal influences i n the male at puberty. E l Defrawy E l Masry and Mannering (1974) observed a reduction i n the l e v e l s of ethylmorphine N-demethylase i n the adult male following c a s t r a t i o n . Castration produced no e f f e c t on the female ethylmorphine N-demethylase a c t i v i t y . E l Defrawy E l Masry and Mannering (1974) also demonstrated that administration of testosterone to adult castrated males resulted i n the r e s t o r a t i o n of N-demethylase a c t i v i t y to c o n t r o l male l e v e l s . In addition, i t was shown that not only was testosterone able to reverse the e f f e c t s of c a s t r a t i o n i n the male, testosterone also increased N-demethylase a c t i v i t y i n the female. These investigators subsequently demonstrated that the administration of e s t r a d i o l to adult male rats would reduce ethylmorphine N-demethylase a c t i v i t y to a l e v e l s i m i l a r to that seen following c a s t r a t i o n . Kato and Onoda (1970) have also noted a testosterone r e v e r s i b l e decrease i n hepatic microsomal metabolism of aminopyrine and hexobarbital following gonadectomy of the male. Kramer et a l . , (1975a) have shown AHH a c t i v i t y to respond s i m i l a r l y . These workers observed a testosterone r e v e r s i b l e decrease i n adult male AHH a c t i v i t y following c a s t r a t i o n . Gurtoo and Parker (1977) confirmed these findings and further noted that gonadectomy of the female did not r e s u l t i n any change in AHH a c t i v i t y . In addition, the administration of testosterone to 3 female r a t s was shown to increase AHH a c t i v i t y to con t r o l male l e v e l s . Results from our laboratory have demonstrated s i m i l a r e f f e c t s on AHH a c t i v i t y following gonadectomy of the adult male and female rat (Gontovnick et a l . , 1979). The sexual dimorphism expressed for hepatic metabolism of xenobiotics has been described for st e r o i d substrates as w e l l . Einarsson et a l . , (1973) have reported greater 2g-hydroxylation of 5a-androstane-3cx,17g-diol, 68-hydroxylation of 5a-androstane-3a, 178-diol and 4-pregnene-3,20-dione and 18 hydroxylation of 5a-androstane-3a,17B_diol i n adult male r a t s . In addition, c a s t r a t i o n of the male was shown to abolish these differences i n a testosterone r e v e r s i b l e manner. Similar e f f e c t s have been reported by Kato et al.»(1969) for the hydroxylation of testosterone and progesterone. I t i s of course i n t u i t i v e l y obvious that an age and sex differ e n c e would be present for s t e r o i d metabolism as the p h y s i o l o g i c a l requirements for steroids change with age and gender. 2) Neonatal androgen induced imprinting The importance of androgens to the expression of sex dependent differences i n hepatic drug and st e r o i d metabolism lead to further study of metabolic d i f f e r e n t i a t i o n . In the f i e l d of drug metabolism, very few workers have published papers r e l a t i n g to d i f f e r e n t i a t i o n . Chung et a l . , (1975) reported that male rats castrated at b i r t h respond l e s s at maturity to androgen stimulation of aminopyrine, ethylmorphine and hexobarbital metabolism than i f exposed to androgens as a neonate. 4 However, a number of workers have studied sex dependent expression of normal s t e r o i d metab'o,li s m. Denef and DeMoor (1968) demonstrated that a s i n g l e dose of testosterone on day one of l i f e would r e s u l t i n a masculine pattern of C o r t i s o l metabolism i n neonatally castrated male or female r a t s . Subsequently, i t was shown that normal d i f f e r e n t i a t i o n of C o r t i s o l metabolism was blocked in male ra t s castrated before day ten of l i f e (Denef and DeMoor, 1972). Castration a f t e r day ten did not i n h i b i t normal metabolic d i f f e r e n -t i a t i o n . In addition, these workers demonstrated that the androgen antagonist, cyproterone acetate, i n h i b i t e d the e f f e c t of testosterone on the d i f f e r e n t i a t i o n of C o r t i s o l metabolism in the female and prevented the normal d i f f e r e n t i a t i o n i n the male (Denef and DeMoor, 1972). Einarsson et a l . , (1973) reported that the 2ct-hydroxylation of 4-pregnene-3,20-dione and 5a-androstane-3a,17B-diol, the 16 16a-hydroxylation of 4-androstene-3,17-dione, the A -C s t e r o i d i y epoxidation Q f 4,16-androstadine-3-one and the 178-hydroxy s t e r o i d reduction of 4-androstene-3,17-dione were more active i n male than female r a t s . Unlike the previously mentioned ste r o i d hydroxylases, only c a s t r a t i o n of the neonate removed the sex differences i n these a c t i v i t i e s . Post-pubescent gonadectomy resulted in only a p a r t i a l reduction of a c t i v i t y . From these studies i t was concluded that the e a r l i e r the male rat i s i s o l a t e d from androgens, the les s d i f f e r e n t i a t e d c e r t a i n enzyme a c t i v i t i e s w i l l be at maturity. Furthermore, i t was suggested that the l i v e r i s i n i t i a l l y "feminine", and that the c h a r a c t e r i s t i c d i f f e r e n t i a t i o n of metabolism seen in the adult was "organized" by 5 t e s t i c u l a r .laetor(9)within the f i r s t ten to twelve days of l i f e (Chung, 1975; DeMoor and Denef, 1968; Gustafsson and Stenberg, 1974a). This e f f e c t i s referred to as neonatal androgen induced imprinting (Chung, 1974; Einarsson et a l . , 1973; McEwen et a l . , 1975), and i n c e r t a i n instances i t i s i r r e v e r s i b l e . That i s , neonatally castrated males are non-responsive to testosterone stimulation of enzyme a c t i v i t y at maturity. This has been demonstrated f or the 6g-hydroxylation of 4-androstene-3,17-dione (Gustafsson and Stenberg, 1974a). 3) Involvement of the hypophyseal-hypothalamic axis i n the maintenance of sex dependent differences i n hepatic metabolism a) P i t u i t a r y e f f e c t s The importance of neonatal androgens to the d i f f e r e n t i a t i o n of hepatic metabolism lead to study of the e f f e c t s of the hypophyseal-hypothalamic axis on t h i s process. The r a t i o n a l e was that the p i t u i t a r y and ulti m a t e l y the hypothalamus regulate the l e v e l s of sex steroids i n vivo . Colby et a l . , (1973) demonstrated that hypophysectomy reduced corticosterone A4 reductase a c t i v i t y i n adult gonadectomized male r a t s . In addition, following hypo-physectomy, e s t r a d i o l f a i l e d to stimulate A4 reductase a c t i v i t y i n adult gonadectomized male r a t s . Kramer et a l . , (1975) demonstrated that the reduction of ethylmorphine N-demethylase a c t i v i t y following hypophysectomy of adult female r a t s could not be stimulated by administration of testosterone. 6 In addition, Kramer and co-workers (1979) have shown that reduction of hepatic ethylmorphine N-demethylase and a r y l hydrocarbon hydroxylase a c t i v i t i e s following hypophysectomy would not increase i n response to the administration of testosterone or dihydrotestosterone (DHT) to adult gonadectomized male r a t s . Gustafsson and Stenberg (1974a) reported s i m i l a r findings for s t e r o i d substrates. These investigators described hypophysectomy producing an o v e r a l l masculinization of hepatic s t e r o i d metabolism i n female r a t s : increasing 2a-, 2g-, 7g-and 18-hydroxylation of 5a-androstane-3a,17g d i o l ; and 6$-, 16a-hydroxylation, and 3g-17a-hydroxysteroid reduction of 4-androstene-3,17-dione. A feminization of 5a-reductase was noted following hypophysectomy of the male. Furthermore, treatment with testosterone and e s t r a d i o l , which normally masculinize and feminize st e r o i d metabolism r e s p e c t i v e l y , had no e f f e c t on these s t e r o i d hydroxylase and reductase a c t i v i t i e s i n the hypophysectomized male or female r a t . These findings are s i m i l a r to those observed i n our laboratory which indi c a t e that hypophysectomy produces an equalization or d e - d i f f e r e n t i a t i o n of hepatic drug metabolism (Bellward et a l . , 1982). Denef (1974) has shown that hypophysectomy removes the normal d i f f e r e n t i a t i o n of testosterone metabolism i n male r a t s . However, i f these same hypophysectomized males received autonomous p i t u i t a r y implants from e i t h e r male or female donors, then a feminization of testosterone metabolism would r e s u l t . Gustafsson and Stenberg (1976a) also examined the e f f e c t s of autonomous p i t u i t a r y implants on hepatic s t e r o i d metabolism. P i t u i t a r i e s from female donors were implanted under the re n a l capsule of adult hypophysectomized castrated male 7 r a t s . This procedure resulted i n a "feminization" of hepatic s t e r o i d metabolism which was not responsive to the administration of t e s t o -sterone. In addition, Skett et a l . , (1978a) demonstrated that p i t u i t a r i e s donated from r a t s younger than 35 days of age would not feminize the hepatic 5a-reductase a c t i v i t y of hypophysectomized male r e c i p i e n t s . The investigators concluded from these studies that an unknown factor was released from the pubescent and post-pubescent p i t u i t a r y of either sex that r e s u l t e d i n the feminization of meta-bolism. Since both male and female p i t u i t a r y donors produced a feminization of metabolism, although the male does not exhibit female l e v e l s of metabolism, the influence of the hypothalamus was investigated. b) Hypothalamic e f f e c t s To characterize the regulatory r o l e of the hypothalamus, Gustafsson e t _ a l . , (1976b) produced electrothermic lesions i n the hypothalamic area. Lesioning resulted i n an o v e r a l l feminization of hepatic s t e r o i d metabolism i n the male, but produced no s i g n i f i c a n t e f f e c t s i n the female. In addition, i t was shown that f r o n t a l de-afferentation at the retrochiasmatic and supra-chiasmatic l e v e l resulted i n complete feminization of hepatic st e r o i d metabolism i n the male r e l a t i v e to co n t r o l . Again*no e f f e c t was noted following de-afferentation of the female (Gustafsson et a l . , 1978). I t was postulated, therefore, that a feminizing factor 8 secreted by the female p i t u i t a r y at puberty i s i n h i b i t e d by a hypothalamic re l e a s i n g i n h i b i t o r y factor i n the male. This i n h i b i t o r y f a c t o r , which i s not present i n the female, may be the r e s u l t of neonatal t e s t i c u l a r androgen induced imprinting of the male hypo-thalamus causing i t to d i f f e r e n t i a t e at puberty and express a male metabolic pattern. Furthermore, the action of e s t r a d i o l i n the male was suggested to be at the l e v e l of the hypothalamus to reduce the secretion of the release i n h i b i t o r y factor. This would allow for an increased release of the p i t u i t a r y feminizing factor r e s u l t i n g i n a female pattern of metabolism (Gustafsson and Stenberg, 1976a). The p i t u i t a r y factor responsible for the observed e f f e c t s on hepatic metabolism was i n i t i a l l y r e f e r r e d to as "feminotropin". c) Feminotropin Characterization of feminotropin was attempted by Gustafsson et a l . , (1975a) and Mode et a l ^ , (1978). i n each case feminizing a c t i v i t y was assessed by increases i n 5a-reductase a c t i v i t y of hepatoma (HTC) c e l l s i n t i s s u e c u l t u r e . I n i t i a l l y hypophyseal extracts from male and female r a t s were administered to the t i s s u e c u l t u r e . The macromolecular f r a c t i o n of the female p i t u i t a r y extract was shown to increase 5a _reductase a c t i v i t y , whereas neither the male or low molecular weight female extract were observed to have any e f f e c t . In addition, known p u r i f i e d p i t u i t a r y hormones were added to the HTC culture but also shown to have no e f f e c t , (Gustafsson et a l . , 1975a). Thus the authors concluded that feminotropin was a novel hormone. However, several c r i t i c i s m s 9 can be r a i s e d concerning t h i s study because the extract contained p i t u i t a r y hormones, and no quantitative analysis was attempted, i . e . the e f f e c t s observed may be due to differences i n these hormone-concentrations. A l t e r n a t i v e l y , since p u r i f i e d hormones were added i n d i v i d u a l l y and not i n combination, e f f e c t s may be due to the in t e r a c t i o n of two or more hormones present i n the extract. Further ch a r a c t e r i z a t i o n using extracts derived from C011RAP p i t u i t a r y tumor t i s s u e (as well as female rat p i t u i t a r y o for comparative purposes) revealed that p r o l a c t i n (PRL) eluted i n the area that corresponded to the "low" (below 8,000) molecular weight f r a c t i o n of feminotropin following gel chromatography (Mode et a l . , 1978). More recent studies by Gustafsson's group have suggested that feminotropin may be an i d e n t i f i e d p i t u i t a r y hormone. P i t u i t a r y hormones are known to have pronounced e f f e c t s on hepatic metabolic a c t i v i t y . 4) E f f e c t s of p i t u i t a r y hormones on hepatic metabolism It i s important to note for the following discussion that p i t u i t a r y hormone preparations are not completely pure. These preparations generally contain small amounts (less than 2%) of more than one other anterior p i t u i t a r y hormone. For example, the rat p r o l a c t i n (rPJAL) used i n our study contained 1.5% growth hormone contamination as determined by radioimmunoassay. Furthermore, p i t u i t a r y hormones exhibit cross r e a c t i v i t y . Human growth hormone (hGH) was shown to have both somatogenic and lactogenic properties 10 (Kleinberg and Todd, 1980; Mode et a l . , 1981). The bovine growth hormone (bGH) used i n t h i s study was reported to contain 62% lactogenic a c t i v i t y . Therefore, while species v a r i a t i o n i n hormone o r i g i n i s apparent, the lack of s p e c i f i c i t y of some of these preparations must be considered, or incorrect conclusions may be reached. The i n i t i a l candidates for p i t u i t a r y hormones active on hepatic metabolism are the gonadotropins l u t e i n i z i n g hormone (LH) and f o l l i c l e stimulating hormone (FSH) since they are associated with the production and release of sex steroids. Kramer et a l . , (1977) investigated the d i r e c t e f f e c t s of LH and FSH on hepatic drug metabolism using gonadectomized male and female r a t s . They reported that FSH or LH increased ethylmorphine N-demethylase a c t i v i t y i n males'and females., whereas FSH increased aminopyrine N-demethylase a c t i v i t y i n females only. Neither FSH or LH produced any e f f e c t s on the sex-independent a n i l i n e hydroxylase a c t i v i t y i n animals of eit h e r sex. Kramer and co-workers (1977) concluded that the gonadotropins had actions on hepatic mixed function oxidase a c t i v i t y which were independent of gonadal hormones. Gustafsson (1975b) has reported that FSH but not LH w i l l increase the a c t i v i t y of androgen dependent and not androgen independent st e r o i d hydroxylase a c t i v i t y i n gonadectomized r a t s . However, i t i s important to consider that when studying the e f f e c t s of gona-dotropins i n gonadectomized r a t s with intact p i t u i t a r i e s , an elevation of gonadotropins w i l l already exist due to the removal of feedback i n h i b i t i o n by sex ster o i d s . Therefore, administration of 11 gonadotropin to an elevated system makes any conclusion d i f f i c u l t . The major c r i t e r i o n for the feminizing factor i s that i t be estrogen mimetic (either d i r e c t l y or i n d i r e c t l y ) with respect to hepatic microsomal metabolism. Wilson (1969) had demonstrated that GH reduced hepatic ethylmorphine and aminopyrine N-demethylase a c t i v i t y i n adult male rats 48 hours a f t e r administration. These e f f e c t s were not observed following PRL or adrenocorticotropic hormone (ACTH) treatment. In addition, Wilson (1970) has shown that t h i s e f f e c t cannot be produced i n male r a t s l e s s than t h i r t y days of age (pre-pubescent). This supports the observations of E l Defrawy E l Masry et a l . , (1974) that no sex diffe r e n c e i s present i n the immature animal and, to an extent, Skett et a l . , (1978) that pre—pubescent donor p i t u i t a r i e s would not feminize ster o i d metabolism. This also indicates that the e f f e c t s of GH are not simply d i r e c t l y i n h i b i t o r y . Wilson (1971) has also shown that hepatic metabolism of hexobarbital could be reduced by GH i n gonadectomized, adrenalectomized or hypophysectomized adult male r a t s . However, no e f f e c t s were observed when i n t a c t females were used, nor were the e f f e c t s of GH noted with a l l substrates. Wilson concluded that the e f f e c t s of GH were sex dependent, age dependent, and not dependent upon gonadal, adrenal or p i t u i t a r y f a c t o r s . Kramer et a l . , (1975a,1978) have demonstrated that the e f f e c t s of e s t r a d i o l on drug metabolizing enzymes i n the castrated male rat are s i m i l a r to those produced by GH. In addition, i t was observed that the combined administration of GH and e s t r a d i o l did not reduce ethylmorphine N-demethylase a c t i v i t y i n the castrated male to any greater extent than did GH or e s t r a d i o l alone, suggesting 1 2 a common mechanism of action. Evidence to t h i s point, including the observations of Lloyd et a l . , (1971) that e s t r a d i o l produces an increase in serum GH l e v e l s i n adult male r a t s , suggests that GH may be the feminizing f a c t o r . I f GH i s the feminotropin that Gustafsson describes, then one would expect i t , unlike e s t r a d i o l , to act in the hypophysectomized animal. However, Colby et a l . , (1974) have shown that GH i n the presence of ACTH w i l l reduce coricosterone metabolism i n hypo-physectomized male rats, i . e . to "masculinize". Furthermore, PRL, LH, FSH, FSH + LH, GH and ACTH given i n d i v i d u a l l y produced no change in corticosterone metabolism d i f f e r e n t from hypophysectomy alone. Administration of GH to hypophysectomized males produced an increase (masculinization) of ethylmorphine N-demethylase and benzo(a)pyrene hydroxylase a c t i v i t i e s (Kramer et a l . , 1975b). These r e s u l t s were opposite to what was expected. I n t e r e s t i n g l y , Rumbaugh and Colby (1980) have shown that GH i n the presence of thyroxine (T^) and ACTH w i l l decrease ethylmorphine N-demethylase and benzo(a)pyrene hydroxylase, and increase A4-hydrogenase a c t i v i t i e s i n the hypo-physectomized male r a t . These s h i f t s in a c t i v i t y are i n d i c a t i v e of a feminization; however, as with the corticosterone metabolism discussed e a r l i e r , GH was unable to produce t h i s feminization alone. Colby concluded that GH contributes to the feminization and mediates e s t r a d i o l action on hepatic metabolism. Unfortunately the e f f e c t s of ACTH and/or T^ plus GH i n the presence of e s t r a d i o l or testosterone in the hypophysectomized rat were not investigated. Further evidence supporting the hypothesis that GH i s involved with feminization was obtained with extracts from p i t u i t a r y tumors. 13 These extracts which contain GH were shown to feminize the st e r o i d metabolism of hepatocyte and hepatoma c e l l culture (Skett et a l . , 1978). However, the tumor l i n e (Cgll-RAP) from which the extracts were produced was found to secrete large amounts of PRL (Skett et a l . , 1977). This tumor c e l l l i n e was shown to produce the most complete feminization of hepatic s t e r o i d metabolism i n the intact male r a t . Eneroth et a l . , (1977) have shown that the feminization of 5a-reductase a c t i v i t y produced by autonomous p i t u i t a r y transplants c o r r e l a t e s with an increase i n serum PRL l e v e l s following implantation. Skett et a l . , (1978a) have shown that p i t u i t a r i e s donated from r a t s younger than 35 days of age cannot produce a feminization of hepatic 5a-reductase a c t i v i t i e s i n hypophysectomized adult male r e c i p i e n t s . In addition, feminization was correlated with increased serum PRL l e v e l s . This observation i s in keeping with the age dependency associated with metabolic d i f f e r e n t i a t i o n . Results discussed to t h i s point suggest that growth hormone i s not the only hormone to be considered i n the feminization of metabolism. Although Colby was not able to produce a feminization of metabolism with PRL, Lax et a l . ,(.197 6). have shown PRL to feminize hepatic s t e r o i d metabolism i n hypophysectomized male and female r a t s . Similar r e s u l t s have been reported by Schriefers et a l . , (1975). Thus the r o l e of PRL i s not yet resolved. Mode et a l . , (.1981) demonstrated that continuous infusion of hGH resu l t e d i n a feminization of hepatic s t e r o i d metabolism of the hypophysectomized male r a t . However, as indicated,hGH possesses both somatogenic and lactogenic q u a l i t i e s (Kleinberg and Todd, 1980; Mode et a l . , 1981). Further studies with rat growth hormone (rGH) 14 produced a feminization of ste r o i d hydroxylase a c t i v i t y as well. However, rPRL also resulted i n a p a r t i a l feminization of s t e r o i d metabolism. This study was not consistent with the r e s u l t s of Colby et a l . , (1974) and Rumbaugh and Colby (1980), which reported that PRL was without e f f e c t and that GH produced a "masculinization" of hepatic drug metabolism. A second inconsistency between the two previously c i t e d studies involved adrenal and thyroid hormone, modulation of GH actio n . According to Rumbaugh and Colby (1980) a "feminization" of drug metabolism i n the hypophysectomized male could only be produced by bGH i f T^ and ACTH were administered concurrently. This suggested adrenal and/or thyr o i d involvement i n the hepatic enzyme regulation. However, neither of these hormones, i n d i v i d u a l l y or i n combination produced any e f f e c t on AHH a c t i v i t y i n the hypo-physectomized male. In addition, Mode et a l . , (1981) demonstrated that adrenalectomy and thyroidectomy d i d not a l t e r the response to continuous i n f u s i o n of hGH on hepatic s t e r o i d metabolism i n hypophysectomized male r a t s . This discrepancy i s quite conceivably due to diff e r e n c e s i n hormone, administration and/or species o r i g i n of GH (Mode et a l . , 1981). In any case, the i d e n t i t y of the "feminotropin" was s t i l l very unclear. 5) Interaction of sex steroids and hepatic lactogen binding If we presume that GH or PRL i s acting at the l i v e r to produce a "feminization" of metabolism, then i t i s reasonable to assume t h i s action involves a receptor mediated pathway. K e l l y et a l . , (1974) have shown an age and sex dependent increase i n PRL and GH binding 125 to hepatic t i s s u e i n the r a t . The s p e c i f i c binding of I-oPRL 125 and I-hGH increased between day 20 and 40 of l i f e i n the female but remained low throughout l i f e in the male. This can be r e l a t e d to the i n a b i l i t y of immature p i t u i t a r y implants to feminize (Skett et a l . , 1978a) and the expression of d i f f e r e n t i a t e d metabolism at maturity ( E l Defrawy E l Masry et a l . , 1974a). Since sex steroids are known to modulate hepatic metabolism i t would be reasonable to assume that sex steroids might modulate hepatic lactogenic receptor l e v e l s . The i n t e r a c t i o n of sex steroids with GH and PRL has been well documented. Posner et a l . , (1974) have shown that the administration of estrone induced a 10 f o l d increase i n oPRL arida 30 fold increase in hGH binding to the male hepatic lactogen receptor. Similar e f f e c t s were seen with e s t r a d i o l . Treatment with estrone resulted i n an approximate two f o l d increase in oPRL and hGH binding i n the adult female. Hypophysectomy reduced the l e v e l s of lactogen receptor and abolished the inductive e f f e c t s of estrogen i n both males and females. Furthermore, an increase in hepatic oPRL binding has been demonstrated following gonadectomy of the male (Aragona et a l . , 1976). The increase i n PRL binding was shown to be completely i n h i b i t e d following the administration of testosterone. Adrenalectomy did not influence t h i s increased binding following gonadectomy (Aragona et a l . , 1976). The testosterone r e v e r s i b l e post-castration increase i n male hepatic PRL binding was confirmed by Barkey et a l . (1979). In addition, Barkey and co-workers (1979) demonstrated that gonadectomy resulted in a decrease i n PRL binding to the prostate. Subsequent testosterone replacement increased PRL binding to the prostate, reversing the e f f e c t s of gonadectomy. Thus, a r e l a t i o n s h i p between testosterone l e v e l s and PRL binding does e x i s t . The r e l a t i o n s h i p between hepatic PRL binding and testosterone may be secondary to the e f f e c t s observed at the accessory sex gland l e v e l where binding depends on testosterone. This i s a reasonable assumption as the presence of the t e s t i s are an i n t e g r a l part of a masculine hepatic metabolic p r o f i l e . The in t e r a c t i o n of PRL and testosterone on the accessory sex glands "'can be described as a synergism. Keenan et a l . , (1975) have shown that concurrent administration of PRL and testosterone increased prostate and seminal v e s i c l e weights to a greater extent than did testosterone alone. Furthermore, administration of PRL and testosterone to rat v e n t r a l prostate c e l l culture stimulates RNA and protein synthesis whereas testosterone alone produced no ef f e c t (Johansson, 1975). PRL has also been shown to stimulate nuclear 3 uptake of. [ H]^testosterone i n rat v e n t r a l prostate (Slaunwhite and Sharma, 1977). The i n t e r a c t i o n between PRL and testosterone may r e s u l t from the involvement of PRL with the regulation of LH receptor l e v e l s i n the t e s t i s as w e l l . Bohnet et a l . , (1975) have shown that PRL stimulates t e s t i c u l a r LH binding i n r a t s . This would, of course, lead to an increase in testosterone l e v e l s and according to Barkey's group a decrease i n hepatic PRL binding. The ultimate r e s u l t of t h i s increased testosterone would be a decrease i n LH receptors v i a feedback i n h i b i t i o n . Belanger et a l . , (1979) have shown that, when LH receptors have been induced by LHRH agonists, PRL p a r t i c i p a t e s i n the down regulation of these 17 receptors. In addition, i t has been shown that PRL i s i n h i b i t o r y to gonadotropin release. Hyperprolactinemia induced by the transplantation of p i t u i t a r i e s or by PRL priming r e s u l t s in a reduction of basal serum LH l e v e l s as well as a decrease in LHRH stimulated LH release i n adult male ra t s (Greeley et a l . , 1981). Therefore, the r e l a t i o n between PRL, testosterone, and LH receptors suggest that PRL i s involved in the regulation of hepatic metabolic p r o f i l e s observed in post-pubertal r a t s . In support of t h i s hypothesis, Ferland et a l . , (1979) have demonstrated that e s t r a d i o l reverses the i n h i b i t o r y e f f e c t s of dopamine on PRL release r e s u l t i n g i n increased serum PRL l e v e l s . Furthermore, Posner and co-workers (1974) have shown that e s t r a d i o l stimulates hepatic oPRL binding i n adult male and female r a t s . The increase in hepatic PRL binding observed i n the male correlates with the e s t r a d i o l feminization of hepatic drug metabolism previously discussed. 6) P i t u i t a r y modulation of lactogen binding Recalling that the p i t u i t a r y axis must be present for the expression of testosterone and e s t r a d i o l e f f e c t s on hepatic metabolism, i t i s of i n t e r e s t to note that the stimulating e f f e c t of e s t r a d i o l or estrone on oPRL and hGH binding i s not demonstrable in the hypophysectomized r a t . Hypophysectomy produces a decrease i n hepatic lactogen receptor in the female which can be p a r t i a l l y reversed by an autonomous p i t u i t a r y implant (Posner et a l . , 1975). Furthermore, Bohnet et a l . , (1976) have shown administration of oPRL,and to a lesser extent hGH,to hypophysectomized females 18 incfr eased hepatic PRL specific binding. ACTH, estradiol, and bGH had no effect. However, when oPRL was combined with ACTH, hGH or estradiol but not bGH even greater induction of hepatic PRL binding was seen in the hypophysectomized female. In addition, the Combination of hGH and ACTH also produced a dramatic increase in PRL binding. Interestingly, the synergistic effect of ACTH was not observed following adrenalectomy. The investigators suggested that ACTH stimulation of adrenal estrogen secretion potentiated the up regulation of hepatic PRL binding, (Bohnet et a l . , 1976). However, unlike the female,the administration of oPRL to the hypophysectomized male was not able to increase hepatic PRL binding (Aragona et a l . , 1976). Induction of PRL binding to the liver required hGH or ACTH. The greatest increase in specific PRL binding (33%) was achieved by concurrent administration of hGH and ACTH (without oPRL). A similar increase was produced in rats bearing a GH secreting tumor. The addition of estradiol to the oPRL-ACTH combination increased PRL binding greater than oPRL-ACTH alone. Concurrent administration of .-testosterone and"oPRL-ACTH reduced the oPRL-ACTH synergistic effect on binding. This was of interest as adrenalectomy was shown not to affect the testosterone reversal of gonadectomy induced increases in hepatic PRL binding in the male. Unfortunately, this study did not include an adrenalectomized model for comparative purposes. The preceeding two investigations are of interest when considering the work of Rumbaugh and Colby (1980) regarding their inability to feminize hepatic drug metabolism without a combination of bGH, ACTH, and T,. 19 In contrast to the implied importance these studies lend to thyroid and adrenal hormones, Norstedt et a l . , (1981a) have shown that adrenalectomy and thyroidectomy have no e f f e c t on hepatic PRL binding or hGH stimulation of PRL binding. This r e s u l t may be due to a difference i n hormone de l i v e r y . However, Norstedt et a l . , (1981a) did confirm some hormonal e f f e c t s on hepatic PRL binding reported by Aragona et a l . , (197 6) and Bohnet et a l . , (1976). Continuous infusion of hGH and oPRL produced increases i n hepatic PRL binding in hypophysectomized female r a t s . In addition, hGH and rGH increased hepatic PRL s i t e s i n hypophysectomized males. Int e r e s t i n g l y , rPRL had no e f f e c t on increasing PRL binding i n e i t h e r male or female r a t s following hypophysectomy. This again.- indicates the species differences between p i t u i t a r y hormones as oPRL was shown to be e f f e c t i v e . The e f f e c t of sex steroids on modulation of p i t u i t a r y hormone induction of lactogen binding in the hypophysectomized animal i s of importance. These r e s u l t s indicate that sex s t e r o i d s are acting i n concert with p i t u i t a r y hormones at the l i v e r to increase or decrease hepatic PRL binding. Previous studies suggested the actions of e s t r a d i o l and testosterone were mediated c e n t r a l l y due to their lack of e f f e c t following hypophysectomy. However, t h i s does not appear to be the case following p i t u i t a r y hormone replacement. Local i n t e r a c t i o n of sex steroids and p i t u i t a r y hormones at the l i v e r to modulate p r o l a c t i n binding has also been suggested by p i t u i t a r y implant studies. K e l l y et a l . , (1977) have shown autonomous p i t u i t a r y implants e l i c i t e d induction of hepatic PRL binding i n hypophysectomized female r a t s . This was i n h i b i t e d 20 following the administration of DHT. It i s possible that DHT may be acting on the p i t u i t a r y implant, but taken with the p i t u i t a r y hormone replacement studies, peripheral regulation by sex steroids cannot be discounted. 7) E f f e c t s of p i t u i t a r y hormones on hepatic sex s t e r o i d binding Estrogens have been shown to mediate various hepatic functions. Increases i n hepatic plasma renin substrate and l i p o p r o t e i n synthesis have been correlated with hepatic estrogen binding (Eisenfeld et a l . , 1977; Snow et a l . , 1978), and t h i s suggested a receptor mediated process. Although the l i v e r i s not generally considered a target t i s s u e for sex steroids, estrogen receptors have been i d e n t i f i e d and characterized (Aten et a l . , 1978; Dickson et a l . , 1978; E i s e n f e l d et a l . , 1977). Therefore i f PRL or GH are not acting d i r e c t l y to "feminize" hepatic metabolism but in connection with "peripheral f a c t o r s " as suggested by hormone replacement studies, and, i f we assume sex steroids are acting d i r e c t l y on the l i v e r by a receptor mediated pathway, then an a l t e r n a t i v e p o s s i b i l i t y for the actions of p i t u i t a r y hormones involves t h e i r modulation of hepatic sex st e r o i d receptor l e v e l s . This would explain why sex steroids do not a f f e c t hepatic metabolism in hypophysectomized r a t s . In t h i s regard, Chamnes et a l . , (1975) have shown that oPRL p a r t i a l l y restores hepatic estrogen receptor l e v e l s reduced by hypophysectomy in the adult female r a t . Moreover, Lucier et a l . , (1981) demonstrated a complete r e v e r s a l of hypophysectomy induced 21 reduction of hepatic estrogen receptor s i t e s following the adminis-t r a t i o n of bGH. In addition, no e f f e c t was noted with rPRL, bovine LH, or -rat FSH. •• Unlike, the study of -Norstedt--- et' a l . , (1931b) these investigators i n j e c t e d r e l a t i v e l y large amounts of hormone. Using continuous infusion of low concentrations of hormone, Norstedt and co-workers (1981b) reported that hGH produced an increase in s p e c i f i c binding of e s t r a d i o l i n the hypophysectomized, ovariectomized female r a t . I n t e r e s t i n g l y , Lucier et a l . , (1981) have reported that other than hypophysectomy only adrenalectomy produced a s i g n i f i c a n t reduction in hepatic e s t r a d i o l binding i n the ovariectomized female. No e f f e c t was seen in the male. The reduction of hepatic e s t r a d i o l binding following adrenalectomy of the female was confirmed by Norstedt et a l . , (1981b). These investigators have also found that the estrogen receptor did not respond to a p i t u i t a r y implant in the hypophysectomized-ovariectomized female. However, when dexamethasone was administered a s i g n i f i c a n t increase in hepatic s p e c i f i c e s t r a d i o l binding was seen. Dexamethasone alone had no e f f e c t on e s t r a d i o l binding i n the hypophysectomized, ovariectomized female. A s y n e r g i s t i c e f f e c t was also observed when dexamethasone was combined with hGH. In t h i s case s p e c i f i c e s t r a d i o l binding of the:hypophysectomized, ovariectomized female was completely restored to the ovariectomized l e v e l (Norstedt et a l . , 1981b). In summary, p i t u i t a r y hormones are intimately linked to maintenance of hepatic estrogen receptor l e v e l s although gluco-c o r t i c o i d s do appear to modulate these l e v e l s . 22 8) Hepatic sex steroid binding proteins To this point we have discussed the interaction between pituitary hormones and the hepatic estrogen receptor. It is recognized that androgens modulate both hepatic lactogen binding, and drug and steroid metabolism in the rat. However, to date a high affinity,low capacity androgen receptor that posseses the classic cytosolic receptor characteristics established for the estrogen receptor (Jensen and DeSombre, 197 6) has not been convincingly demonstrated. Indirect evidence by Gustafsson et a l . , (1975) has suggested the presence of such a receptor following in vivo nuclear uptake of radiolabeled testosterone. However, no cytosolic characterization was done and the highest avidity nuclear binding was produced by the metabolite androstenedione. Putative cytosolic androgen receptors have been characterized by Roy et a l . , (1973). This binding protein sedimented at 3.5S on a sucrose density gradient, was present in the adult male, and was correlated to the hepatic synthesis of a urinary protein, c^^globulin. The presence of this site as well as c^-globulin were shown to be androgen dependent. The levels of both proteins were reduced by castration or treatment with estradiol. Interestingly, this binding protein specifically bound DHT, testosterone and estradiol. In fact, examination of the binding data from Roy et a l . , (1973) indicates that 17g-estradiol is probably the correct ligand for this protein and not DHT due to the inconsistency of the DHT Scatchard plot. This is despite the fact that the apparent Kd for —8 —7 DHT (Kd = 4.5 x 10 M) is less than that for estradiol (Kd = 3.5 x 10~ M) . 23 It i s also questionable what p h y s i o l o g i c a l r o l e t h i s sex s t e r o i d binding protein plays as the ligand a f f i n i t y i s well below that of the estrogen receptor (Kd = 0.1 nM). It i s c e r t a i n l y questionable what p h y s i o l o g i c a l ligand normally occupies t h i s s i t e . Sato et a l . , (1980a, 1980b) and Ota et a l . , (1980) have detected the presence of a hepatic c y t o s o l i c androgen binding protein that sedimented at 10S following sucrose density gradient a n a l y s i s . The presence of t h i s protein was shown to be androgen dependent. The apparent Kd for testosterone was reported to be 1.37 x 10 which was s i m i l a r to that reported by Roy et a l . , (1974) for e s t r a d i o l . Unlike the binding protein described by Roy et a l . , (1974) neither e s t r a d i o l or DHT were e f f e c t i v e competitors 3 for [ H]-testosterone binding. These r e s u l t s are suggestive of a hepatic androgen receptor; however, s i m i l a r data had not been reported by other i n v e s t i g a t o r s . A l t e r n a t i v e l y these binding proteins may be associated with a second c l a s s of hepatic c y t o s o l i c estrogen binding proteins i d e n t i f i e d and characterized by Dickson et a l . , (1978), Eagon et a l . , (1980), and Powell-Jones et a l . , (1980,1981). These inv e s t i g a t o r s have demonstrated the presence of a male-specific estrogen binding protein that sedimented i n the 4S region following sucrose density gradient a n a l y s i s . This protein bound estrogens and androgens with an apparent Kd between 0.1 and 1 x 10 M^, and i n t e r e s t i n g l y did not bind d i e t h y l s t i l b e s t r o l (DES), which i s commonly used to detect the estrogen receptor, nor did i t bind g l u c o c o r t i c o i d s nor progestins. 24 Certain aspects of t h i s male s p e c i f i c binding protein p a r a l l e l the DHT binding protein described by Roy et a l . , (1974). The male s p e c i f i c sex ster o i d binding protein i s only detected i n the adult male rat; l i t t l e or none i s found i n the immature male or female or adult female. The presence of t h i s protein has been shown to be androgen dependent. Castration of the adult male reduced the capacity i n a testosterone r e v e r s i b l e fashion, (Eagon et al.,(1980). However, t h i s e f f e c t was only observed by Powell-Jones et a l . , (1980,1981) following neonatal gonadectomy. Another i n t e r e s t i n g c h a r a c t e r i s t i c of t h i s binding protein noted by Powell-Jones et al.,(1980) was that hypophysectomy produced a decrease in 4S binding i n the male but increased 4S binding i n the female.. Therefore, t h i s male s p e c i f i c s i t e that binds androgens and estrogens has been shown to be age and sex dependent, require androgens and the hypophyseal-hypothalamic axis f o r d i f f e r e n t i a t i o n in the male, and may be imprinted neonatally i n the male by androgens. 9) Hypothesis The regulatory aspects of the hepatic male s p e c i f i c sex ste r o i d binding protein p a r a l l e l those that have been established for hepatic drug and st e r o i d metabolism previously discussed. Powell-Jones et a l . , (1980,1981) have shown no ontogenetic differences for the estrogen receptor between male and female r a t s , and evidence for the existence of a high a f f i n i t y , l o w capacity 25 androgen receptor i s doubtful. We hypothesized that the modulation of hepatic sex dependent enzyme a c t i v i t i e s by sex steroids i s mediated by the male s p e c i f i c sex steroid binding protein, and that the presence of t h i s binding protein i s regulated by the hypophyseal-hypothalamic axis. Therefore, we investigated the binding of e s t r a d i o l to the hepatic male s p e c i f i c binding protein, and also to the estrogen receptor, in r e l a t i o n to hepatic a r y l hydrocarbon hydroxylase and testosterone A4 reductase a c t i v i t i e s . Various p h y s i o logical models were studied to determine i f a c o r r e l a t i o n existed. These included the following: adult male versus female, adult versus immature, male and female versus pseudohermaphrodite, sham-operated versus gonadectomized or hypophysectomized. In addition, to gain an understanding of the regulatory mechanisms involved we investigated the e f f e c t s of hormone replacement with testosterone enanthate, mestranol, bGH, rGH, oPRL, rPRL, and induction by 3-methylcholanthrene, pheno-b a r b i t a l or spironolactone i n some of these models. 26 MATERIALS AND METHODS 1) Chemicals and reagents Activated charcoal (untreated), benzo(a)pyrene (BP) ( r e c r y s t a l l i z e d from methanol following d i s s o l u t i o n i n benzene), bovine serum albumin (BSA), Coomassie b r i l l i a n t blue G, D-l d i t h i o -t h r e i t o l (DTT), disodium ethylenediamine.tetraacetic acid (EDTA), D-glucose-6-phosphate monosodium s a l t (G6P), glucose-6-phosphate dehydrogenase type XII (G6PD), 3-methylcholanthrene (3-MC), sodium molybdate,nicotinamide adenine d i n u l c l e o t i d e (reduced form) (NADH), nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH), nicotinamide adenine dinucleotide phosphate (NADP), quinine s u l f a t e , (R) spironolactone, Trizma^base along with the following steroids: aldosterone, androstenedione, dihydrotestosterone (DHT), d i e t h y l -s t i l b e s t r o l (DES), dexamethasone, 173-estradiol, progesterone, testosterone, and triamcinolone were obtained from Sigma Chemical Company (St. Louis, MO.) 3 Labeled, steroids, [6,7- H(N)] e s t r a d i o l , 40-60 Ci/mmol, [2,4,6,7,16,17-3H(N)] e s t r a d i o l 130-170 Ci/mmol and Biofluor® s c i n t i l l a t i o n c o c k t a i l were purchased from New England Nuclear, 3 (Boston, "MA. ). In addition, [6,7- H] e s t r a d i o l , 40-60 Ci/mmol, was also obtained from Amersham Ltd. (Oakville, Ont.). Unlabeled synthetic steroids promogestone (R5020), methyltrienolone (R1881), and moxestrol (R2858) were obtained from New England Nuclear under l i c e n s e agreement from Roussel-Uclaf (Romainville, France). A l l 27 steroids were checked for p u r i t y by manufacturer recommended chromatography procedures upon r e c e i p t . Dextran T-70 was supplied by Pharmacia/Fine Chemicals AB (Uppsala, Sweden). Ammonium s u l f a t e as well as sodium phenobarbitone were purchased from B r i t i s h Drug House (Vancouver, B.C.). Mestranol was obtained from Ortho Pharmaceuticals (Don M i l l s , Ont.) S i l i c a gel TLC plates were supplied by Eastman Kodak Co. (Rochester, NY.,). Pimozide (McN-JR-6238) was a g i f t from McNeil Laboratories (Spring House, PA.) as was pergolide from E l i L i l l y Co. (Indianapolis, IN.) The following p i t u i t a r y hormones were a g i f t of the National Hormone and P i t u i t a r y Program (Baltimore, MP..)': bovine growth hormone (NIH-bGH-B18) (bGH), ovine p r o l a c t i n (NIAMDD-oPRL-15) (oPRL), rat growth hormone (NIAMDD-rGH-B7) (rGH), rat p r o l a c t i n . (NIADDK-rPRL-B4) (rPRL). 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was supplied by KOR Isotopes, (Cambridge, MA..). Octachlorodibenzo-p-dioxin and 2,7-dichlorodibenzo-p-dioxin were obtained from U l t r a (R) S c i e n t i f i c (Hope, RT.) . A l z e t ~ model 2002 osmotic minxpumps were_: purchased from Alza Corp. (Palo A l t o , CA.). A l l other chemicals and reagents used were of the best grade and p u r i t y a v a i l a b l e . 2) Animals Male and female Wistar r a t s were obtained from Canadian Breeding Farms (Montreal, Que.). Pseudohermaphrodite ra t s along with the li t t e r m a t e control males and females were supplied by the International Foundation for Study of Rat Genetics and Rodent Control (Oklahoma City , OK:.-') . Pseudohermaphrodite ra t s as well as the l i t t e r m a t e controls were treated with an anthelmintic on alternate weeks 28 for 3 weeks during quarantine. Hypophysectomized and sham-operated controls were supplied through Canadian Breeding Farms; however, a l l s u r g i c a l procedures were conducted by Charles River Breeding Laboratories (Stone Ridge, NY'Y'.) . A l l animals used in the hypophysectomy study were operated or sham-operated between 50-60 days of age, and received 5% sucrose upon a r r i v a l for one week. The completeness of hypophysectomy was determined by v i s u a l inspection at the time of s a c r i f i c e as well as decrease i n body weight. Animals were housed i n a separate animal room on Lobund® bedding (Paxton Processing Ltd., Paxton, IL. ) under co n t r o l l e d temperature (22°C) and l i g h t (on 0600-2000 hrs . ) . They were allowed free access to food (Purina Laboratory Chow, Ralston Purina of Canada Ltd., Woodstock, Ont.) and water ad lib i t u m . 3) Animal treatments Pimozide was administered (0.6 mg/kg/day, i n 40% propylene-glycol) subcutaneously (s.c.) for ten days to sham-operated or gonadectomized adult male r a t s . Adult female sham-operated or gonadectomized r a t s received pergolide (0.1 mg/kg/day, in corn o i l ) subcutaneously for eleven days. Intact adult male and female r a t s were treated with mestranol (1.0 mg/kg/day, i n corn o i l ) subcutaneously for ten days.' Spironolactone was administered ..(100 mg/kg/day, in,corn o i l ) by the i n t r a p e r i t o n e a l (i.p.) route for four days. Unless otherwise s p e c i f i e d , 3-MC was administered once d a i l y , (20 mg/kg i . p . , i n corn o i l ) for two days, and sodium phenobarbitone once d a i l y (80 mg/kg i . p . , i n normal 29 saline) f o r four days. Rats treated with testosterone enanthate received 1.0 mg/kg/day s.c. i n corn o i l for ten days. P i t u i t a r y (R) hormones were administered by continuous infusion using A lzetS^ model 2002 osmotic minipumps. The hormones were dissolved i n normal s a l i n e (pH 8.5); pumps were weighed before and a f t e r loading to ensure complete f i l l i n g . The pumps had a l i f e expectancy of fourteen days at a pumping rate of 0.4'58 yl/ h r ± 0.026 yl/hr (S.D.). Pumps were implanted subcutaneously in the r o s t r a l half of ether anesthetized hypophysectomized and sham-operated r a t s . Animals were s a c r i f i c e d 10 days following implantation; six to eight weeks post-hypophysectomy. Certain groups received testosterone enanthate (1.0 mg/kg/d s.c.) concurrently f o r ten days. The doses of the p i t u i t a r y hormones were as follows:. bGH, 0.01 I.U./hr; rGH, 0.017 I.U./hr; oPRL, 0.20 I.U./hr; and rPRL, 0.20 I.U./hr, according to Mode et a l . , (1982). In a separate study, bGH was administered by i n j e c t i o n (2.0 I.U./kg, in normal s a l i n e pH 8.5) twice d a i l y for ten days. 4) Hepatic microsomal enzyme assays a) Hepatic microsomal protein preparation Animals were s a c r i f i c e d between 0900-1000 hours. They were stunned by a blow to the head and k i l l e d by c e r v i c a l d i s l o c a t i o n . I n f e r i o r • vena cava were cut and l i v e r s were.'perfused with 10 ml of ice cold 1.15% KC1 v i a the p o r t a l v e i n . Li v e r s were excised into ice cold KC1 1.15%, blotted dry and weight recorded. They were then minced and homogenized 1:4 (w/v) in 1.15% KC1 using a Potter-30 Elvejhem t i s s u e homogenizer. Homogenates were centrifuged at 10,000g using an International Equipment Company (TEC) (Needham l i t s . , MA.) model B-20 r e f r i g e r a t e d centrifuge f or 10 minutes at 4°C. The supernatants were centrifuged at 100,000'g", for 60 minutes at 4°C using a Beckman L5-50 or L2-65B u l t r a c e n t r i f u g e with either a type 40 or type 65 fixed angle rotor (Beckman Instruments Corp., Palo Alto,OA.',) . Following centrifugation each p e l l e t was washed with 3 volumes of ice cold 0.1M' NaK2P04 buffer pH 7.2, resuspended and d i l u t e d 1:3.75 i n the same buffer. b) A r y l hydrocarbon hydroxylase a c t i v i t y Hepatic a r y l hydrocarbon hydroxylase (AHH)activity was determined according to the method of Nebert and Gelboin (1968) as modified by Alvares (1970) using benzo(a)pyrene as a substrate. The amount of 3-hydroxy-benzo(a)pyrene produced was quantitated f l u o r o m e t r i c a l l y - r e l a t i v e to a quinine su l f a t e standard. The f i n a l volume of the incubation mixture (1.0 ml) contained 0.36 ymol NADH; 0.36 ymol NADPH; 3 ymol MgCl 2.6H 20; 8.7 nmol BSA; 150 u l microsomal protein; 25 ymol T r i s buffer pH 7.5; and 80 nmol BP i n 40 y l acetone. The assay was i n i t i a t e d by the addition of BP following a one minute and f i f t e e n second enzyme preincubation period. The incubation was continued for 2.5 minutes at 37°C. The reaction was terminated by the addition of 1.0 ml of ice cold acetone followed by 3.3 ml of hexane. Enzyme and substrate blanks were 31 c a r r i e d through the incubation and the appropriate component was added a f t e r termination of the reaction . The samples were mixed and the phases allowed to separate. A 2i..-0 ml aliquot of the hexane phase was transferred to 4.0 ml of 1.0N NaOH and mixed. The NaOH phase was frozen i n a dry i c e -acetone bath and the hexane was discarded. The NaOH phase was thawed i n a warm water bath and fluorescence was measured with a Turner Spectrofluorometer Model 430. The e x c i t a t i o n was set at 396 nm and emission at 522 nm. The instrument was zeroed with 1.0N NaOH and c a l i b r a t e d with quinine s u l f a t e (0.1 g in 1.0£ ^ SO^ 0.1N). A l l determinations were done i n duplicate. c) Testosterone A4 reductase a c t i v i t y Hepatic testosterone A4 reductase was assayed according to the method of McGuire and Tomkins (1959),. The a c t i v i t y of the reductase was determined spectrophotometrically by the disappearance of substrate r e l a t i v e to a standard curve. A c t i v i t y was expressed as nmoles testosterone reduced per minute per mg microsomal protein. The f i n a l volume of the incubation mixture (1.78 ml) contained 12.5 ymol MgCl 2.6H 20; 1.0 ymol NADP; 20.0 ymol G6P; 1 unit G6PD; 200 y l protein and 0.3 ymol testosterone in 20 y l methanol. The enzyme suspension was preincubated for one minute and f i f t e e n seconds at 37°C at which time the assay was i n i t i a t e d by the addition of testosterone. The reaction was terminated at 10 minutes by the addition of 4.0 ml dichloromethane. Testosterone 32 or enzyme were added to the appropriate blank after the addition of dichloromethane. The samples were mixed and the emulsion centrifuged to expedite separation. The aqueous phase was aspirated and the remaining testosterone was measured spectrophotometrically by scanning from 290 nm to 210 nm in the U.V. region using a Perkin Elmer Model 124 double beam spectrophotometer, (absroption maximum ^240 nm). Each activity was the result of a duplicate determination. 5) Hepatic sex steroid binding assays a) Preliminary studies I n i t i a l studies to examine hepatic estrogen binding were conducted by the method of Powell-Jones et a l . , (1980,1981). Details of this procedure are outlined below. The assay conditions were as follows. Rats were sacrificed by cervical dislocation and livers were perfused with ice cold 10 mM Tris-HCl (TED) buffer that contained 1.0 mM DTT , and 1.0 mM disodium-EDTA, pH 7.4 at 4°C. Livers were homogenized 1:10 in buffer and cytosol prepared by centrifugation at 100,000g, for 60 minutes. The resulting supernatant was removed leaving the superficial l i p i d coat, and divided into two aliquots. The f i r s t was diluted with TED buffer to a protein content of 5-7 mg/ml and placed on ice. The second aliquot was partially purified by a 30% ammonium sulfate fractionation. An equal volume of ammonium sulfate was added drop-wise to cytosol. This was mixed 33 for 30 minutes on i c e , and centrifuged for 30 minutes at 15,000g The r e s u l t i n g p e l l e t was resuspended to the o r i g i n a l volume with buffer. Incubations were carried out by adding 100 y l of protein 3 to 100 y l of [ H] - e s t r a d i o l , 0.2-10 nM, ( i n buffer) i n the presence and absence of a 100 f o l d excess of unlabeled • d i e t h y l s t i l b e s t . r o l (DES) ( i n b u f f e r ) . Protein and ster o i d were incubated for 2 hours at 4 ° C Bound was separated from free by the addition of dextran (0.05%) coated charcoal (0.5%) (DCC) i n TED buffer. Protein was incubated with DCC for 10 minutes and DCC was removed by c e n t r i -fugation at 800g f or 10 minutes. Supernatants were counted for bound e s t r a d i o l using standard s c i n t i l l a t i o n counting techniques. The estrogen receptor assay described by Powell-Jones et a l . , (1980,1981) was compared to the estrogen receptor assay employed at the Hormone Receptors Laboratory, University of Oregon (Portland). The second method, normally used for quantitation of estrogen receptors i n uterine and mammary t i s s u e , had the following modifications: TED buffer contains 50 mM Tr i s - H C l ; 1.5 mM EDTA; 0.5 mM DTT; 20 mM sodium molybdate; 10% g l y c e r o l ; pH 7.5 at 4°C. Furthermore, following homogenization using a Brinkman polytron, samples were centrifuged at 800g'; for 10 minutes. The r e s u l t i n g supernatant was then centrifuged for 30 minutes at 100,000g' to obtain the c y t o s o l i c f r a c t i o n . Protein was d i l u t e d to 1-3 mg/ml i n TED buffer. Incubation mixture contained 500 y l protein and 3 10 y l [ H] - e s t r a d i o l 0.2-10 nM (in 95% ethanol) i n the presence and absence of 10 y l of a 100 f o l d excess of unlabeled ' DES (in 95% ethanol). Separation of bound from free was achieved by the 34 a d d i t i o n of 500 u l of DCC for 5 c e n t r i f u g a t i o n at 1500g for 10 was counted as before. minutes. DCC was removed by minutes. The r e s u l t i n g supernatant b) Hepatic estrogen receptor and moderate a f f i n i t y binding component assay For purposes of detection and e f f i c i e n c y we adopted an estrogen receptor assay protocol that combined several aspects of the two previously described methods. This assay was used for a l l p h y s i o l o g i c a l and pharmacological studies and i s described below. TED buffer contained 50 mil T r i s - H C l , 1.5 mM EDTA, 0.5 mM DTT, 20 mM sodium molybdate, and 10% g l y c e r o l , pH 7.5 at 4°C. Livers were minced and homogenized 1:10 (w/v) in TED buffer using a Potter-Elvehjem homogenizer. Following homogenization samples were centrifuged at 800g ; for 10 minutes at 4°C using a Beckman (Palo A l t o , OA.) J6-B r e f r i g e r a t e d centrifuge with a type JS 4.2 rotor. The r e s u l t i n g supernatant was centrifuged at 100,000g for 30 minutes at 4°C to obtain the c y t o s o l i c f r a c t i o n using e i t h e r a Beckman (Palo A l t o , CA.) L5-50 or L65-B u l t r a c e n t r i f u g e with a type 40 or type 65 fixed angle r o t o r . The supernatant was removed, leaving the s u p e r f i c i a l l i p i d coat, and divided into two a l i q u o t s . The f i r s t a liquot was d i l u t e d to a protein content of 1-3 mg/ml with TED buffer and placed on i c e . This represented the whole cyotosol f r a c t i o n . The second aliquot was p a r t i a l l y p u r i f i e d by a 50% ammonium sulf a t e f r a c t i o n a t i o n . An equal volume of saturated (at room temperature) ammonium s u l f a t e (in 10 mM T r i s ; 1.0 mM EDTA; 1.0 mM DTT pH 7.5) was added dropwise to the cytosol f r a c t i o n . This was mixed for 30 minutes i n an i c e bath, and centrifuged at 15,000g for 30 minutes at 4°C using an IEC model -B-20refrigerated centrifuge. The r e s u l t i n g p e l l e t was washed and resuspended to the o r i g i n a l volume of cytosol with TED buffer. The binding c h a r a c t e r i s t i c s of e s t r a d i o l were determined by Scatchard (1949) analysis of" competitive binding curves-using both the whole cytosol and p a r t i a l l y p u r i f i e d f r a c t i o n s . The high a f f i n i t y , low capacity hepatic estrogen receptor was assessed in the ammonium s u l f a t e f r a c t i o n . The incubation mixture contained 3 500 p i protein, 10 y l 0.05-2.5 nM [ H ] - e s t r a d i o l (140 Ci/mmol) (in 95% ethanol) i n the presence or absence of 10 y l of a 100 f o l d excess unlabeled e s t r a d i o l ( i n 95% ethanol). The moderate a f f i n i t y , higher capacity component was measured in the whole cytosol f r a c t i o n . The incubation mixture contained 500 y l protein, 10 y l [ 3 H ] - e s t r a d i o l (7-10 Ci/mmol) 10-200 nM (in 95% ethanol) i n the presence or absence of a 100 f o l d excess of unlabeled e s t r a d i o l or a 1000 f o l d excess of unlabelled DHT. Incubations were c a r r i e d out for 90 minutes at 4°C. Bound ster o i d was separated from free by the a d d i t i o n of 500 y l dextran T-70 (0.05% w/v) coated charcoal (0.5% w/v) (DCC) in TED buffer (prepared as previously described minus molybdate). Following the addition of DCC, samples were mixed and DCC was sedimented by centrifugation at 1500g'" for 10 minutes at 4°C using a Beckman J6-B r e f r i g e r a t e d centrifuge with a type JS 4.2 rotor. The r e s u l t i n g supernatants were sampled (0.5 ml) and combined with 10 ml Biofluor® 36 l i q u i d s c i n t i l l a t i o n c o c k t a i l . Samples were counted for bound e s t r a d i o l using a Searle Mark III Liquid S c i n t i l l a t i o n System-Model 6880 (Searle A n a l y t i c Inc., Des P l a i n s , I L . ) . A l l points were assayed in.duplicate. The tubes which contained 3 [ H]-estradiol without competitor were a measure of t o t a l binding 3 (TB). Tubes containing both [ H]-estradiol and competitor represented the non-specific binding (NSB) present. c) Analysis of binding data Ca l c u l a t i o n of s p e c i f i c bound was done as follows. The counts 3 per minute (cpm) recorded for [ H]-estradiol bound were converted to d i s i n t i g r a t i o n s per minute (dpm) based on the percent e f f i c i e n c y of the instrument. E f f i c i e n c y for t r i t i u m varied between 45-48% based on the degree of quenching for each sample. Thus dpms were calculated on the basis of e f f i c i e n c y , which was determined f o r each sample from previously prepared quench standards. The dpms were converted to Curies for each sample on the basis that 12 1 C i = 2.2 x 10 dpm. The s p e c i f i c a c t i v i t i e s of the ligands were employed to cal c u l a t e the femtomoles of binding which was normalized for protein content. At t h i s point; the N.S.B. tube values were subtracted from T.B. tube values to give s p e c i f i c binding (S.B.) at any point. Duplicates were then averaged. The t o t a l ligand ( t o t a l counts) added, less the S.B., represents the free concentration (F) used i n c a l c u l a t i o n of s p e c i f i c bound over free (B/F). In cases where displacement was observed the B/F was graphed 37 as a function of S.B. to y i e l d a curve-linear plot with a negative slope. The inverse of the slope of the l i n e a r portion of the plot represented the d i s s o c i a t i o n constant (Kd), and the x-intercept the 1 uncorrected measure of capacity. Points deviating from l i n e a r i t y at the low and high end of the curve (due to non-optimal ligand-protein concentration and other binding s i t e s of lower a f f i n i t y respectively) were not used for analysis of binding data. In addition, binding data were subject to non-linear regression analysis using the curve f i t - m o d e l l i n g program Ligand developed by Munson and Rodbard (1980). This was done to confirm goodness of f i t , as well as values for the apparent Kd and capacity of the binding components. However, t h i s program was designed for i n h i b i t i o n studies that employ a f i x e d concentration of tracer and increasing amounts of unlabeled competitor. Therefore, the program was written to consider non-specific binding to be a constant value for each concentration, and does not normalize data for protein content. The assay we have employed v a r i e s both the unlabeled competitor and tracer together. As such, non-specific binding was measured at each concentration because the r a t i o of N.S.B.tT.B. i s not constant. Furthermore, we have normalized our data to protein concentration because we are interested in the r e l a t i v e change in binding capacity following p h y s i o l o g i c a l manipula-t i o n . Because of these features of the Ligand program, we obtained reasonable values for the apparent Kd of the estrogen binding proteins, but the c a p a c i t i e s were unreasonably high. 38 We were also not able to combine the male estrogen receptor with the moderate a f f i n i t y binding data to model the o v e r a l l curve. The reason for t h i s was that the assays were conducted i n d i f f e r e n t f r a c t i o n s and used tracers of d i f f e r e n t s p e c i f i c a c t i v i t i e s . Therefore, the Ligand program had a l i m i t e d a p p l i c a t i o n to our studies, and was not extensively used. 6) Determination of protein content Microsomal and c y t o s o l i c protein concentrations were determined by the method of Bradford (1976). The assay i s l i n e a r f o r protein concentrations from 0.1-0.5 mg/ml using BSA as a standard. Protein content was assessed spectrophotometrically at 595 nm using a Perkin-Elmer Model 124 double beam spectrophotometer. Samples were read f i v e minutes a f t e r the addition of 5.0 ml of Bradford reagent which contained 100 mg Coomassie-brilliant blue-G; 50 ml absolute ethanol; 100 ml phosphoric acid 85%; d i l u t e d to 1.0 % and f i l t e r e d using a Buschner suction apparatus. 7) Serum hormone l e v e l s Blood samples were c o l l e c t e d by cardiac puncture when animals were s a c r i f i c e d . Whole bloods were allowed to c l o t then centrifuged at 1500g for 10 minutes using a Beckman (Palo Al t o , CA.) J6-B r e f r i g e r a t e d centrifuge with a type JS 4.2 r o t o r . Serum was aspirated and frozen at -20°C for l a t e r a n a l y s i s . Procedures for analysis are described i n Appendix 1. 39 8) S t a t i s t i c a l analysis Differences were considered s i g n i f i c a n t from c o n t r o l at. p < 0.05 using a two-tailed Students t-Test when only one condition was.altered. However, the majority of the studies introduced several v a r i a b l e s . As such, comparisons between experimental groups were accomplished using the Newman-Keuls or Duncan multiple range t e s t s at p < 0.05, A summary of the c o n t r o l values for female and male estrogen receptor and male moderate a f f i n i t y apparent Kd and capacity are presented i n Table 32. In addition, the mean and standard error of the mean are reported. It i s evident from this- t able that a wide v a r i a b i l i t y e x i s t s for these binding parameters. Because of t h i s , i n d i v i d u a l experiments were not combined but were considered only in the study in which they were c a r r i e d out and r e l a t i v e to the c o n t r o l values established at that time. Individual studies were conducted on s i n g l e shipments of animals, within short time periods, using the same stock solutions of buffer and s t e r o i d throughout each study. 40 RESULTS 1) I n i t i a l c h aracterization of hepatic estrogen binding components a) Comparison of d i f f e r e n t estrogen receptor assays The developmental studies e s t a b l i s h i n g c h a r a c t e r i s t i c s and conditions for estrogen binding were done i n conjunction with Dr. B. Warren. Studies to determine estrogen binding to rat hepatic c y t o s o l i c proteins were i n i t i a l l y c a r r i e d out by the method of Powell-Jones et a l . , (1980,1981) as described i n the methods section. Scatchard analysis of the r e s u l t s from the ammonium sul f a t e f r a c t i o n from adult male hepatic cytosol yielded a saturable binding curve with an apparent Kd of 1.8 nM and a capacity of 11 fmol/mg protein 3 (0.2-10 nM [ H ] - e s t r a d i o l ) . Having confirmed the method of Powell-Jones et a l . , (1980,1981) for the detection of a high a f f i n i t y , low capacity estrogen binding component in r a t hepatic c y t o s o l , we compared the technique to that employed f or detection of uterine and mammary high a f f i n i t y estrogen binding s i t e s . This second method, employed at the Hormone Receptors Laboratory, University of Oregon, (Portland) had c e r t a i n modifications (see materials and methods for d e t a i l s ) . The most notable d i f f e r e n c e between these two estrogen binding assays was that: the TED buffer of the l a t t e r contained g l y c e r o l and molybdate. Furthermore, cytosol was not subjected to an ammonium s u l f a t e f r a c t i o n a t i o n and steroids were added to the incubation mixture i n 95% ethanol rather than buffer. 41 Scatchard analysis of t h i s cytosol f r a c t i o n from adult male ra t s yielded an apparent Kd of 0.14 uM, and a capacity of 3.3 pmol/mg protein. However, when measured i n females, the apparent Kd and capacity were si m i l a r to those obtained by the Powell-Jones et a l . , (1980,1981) method (Kd = 2.2 nM, capacity = 30 fmol/mg pr o t e i n ) . The s p e c i f i c binding curve for the male suggested that more than one binding s i t e was present i n the adult male whole c y t o s o l , one below 2.5 nM and one above. This lower a f f i n i t y , higher capacity binding s i t e , the moderate a f f i n i t y , moderate capacity s i t e described by Powell-Jones et al.(1980, 1981), e f f e c t i v e l y masked the higher a f f i n i t y s i t e i n the male. This accounted for the observation of the lower a f f i n i t y higher capacity s i t e . To detect the higher a f f i n i t y s i t e i n the male we used an ammonium su l f a t e f r a c t i o n of the whole cy t o s o l . S p e c i f i c binding (determined by the l a t t e r method) yielded an apparent Kd = 1.8 nM, and a capacity of 50 fmol/mg. Therefore, i t was apparent that ammonium s u l f a t e f r a c t i o n a t i o n was necessary for detection of the high a f f i n i t y s i t e i n male cy t o s o l . Subsequently, we employed t h i s method f or determination of the high a f f i n i t y component in both male and female r a t s . In order to characterize these f r a c t i o n s (ammonium s u l f a t e vs whole cytosol) and the binding components present, a series of experiments was c a r r i e d out using various st e r o i d competitors (Table 1). The high a f f i n i t y component was apparent i n the 3 ammonium su l f a t e f r a c t i o n of the male when [ H ] - e s t r a d i o l was competed with e s t r a d i o l and DES. The moderate a f f i n i t y component 3 was apparent i n the male whole cytosol f r a c t i o n when [ H ] - e s t r a d i o l TABLE 1 The apparent Kd and capacity of [ H]-estradiol binding in different fractions of adult male and female rat liver using various competitors. Incubations were carried out for 90 minutes at 4°C. Competitor [ 3H]-estradiol concentration range Male Female Ammonium Sulfate Fract ion Whole Cytosol Fraction Ammonium Sulfate Fract ion Whole Cytosol Fraction Apparent Kd (xlO 9 M) Capacity (per mg protein) Apparent Kd (xlO 9 M) Capacity (per mg protein) Apparent Kd (xlO 9 M) Capacity (per mg protein) Apparent Kd (xlO 9 M) Capacity (per mg protein) DES (0.05 - 15 nM) 1.8 49 fmol NS NS 0.46 34 fmol 2.2 29 fmol DES (25 - 1000 nM) NS NS NS NS 290 0.4 pmol 230 0.4 pmol DHT (0.05 - 15 nM) NS NS NS NS NS NS NS NS DHT (25 - 1000 nM) NS NS 250 13 pmol NS NS NS NS Estradiol 1 (0.05 - 15 nM) 1.8 158 fmol NS NS 2.2 89 fmol 1.5 33 fmol Estradiol 1 (25 - 1000 nM) 120 1.7 pmol 340 7.5 pmol NS NS NS NS Data provided by Dr. B. Warren DES = Diethylstilbestrol DHT = Dihydrotestosterone NS = No Scatchard 43 was competed with e s t r a d i o l and DHT and i n the ammonium su l f a t e when e s t r a d i o l was used. The female cytosol contained high a f f i n i t y binding i n both f r a c t i o n s using e s t r a d i o l and DES, (not DHT) as competitors. Furthermore, we detected a small amount of lower a f f i n i t y binding i n the female when DES was used as a competitor. It was apparent that the ammonium su l f a t e f r a c t i o n a t i o n would not completely remove the moderate a f f i n i t y component from male whole cy t o s o l . When the male ammonium sul f a t e f r a c t i o n was studied over a wide ligand concentration range the moderate a f f i n i t y component was detected. We therefore compared that d i f f e r e n c e between a 30% and a 50% ammonium s u l f a t e f r a c t i o n a t i o n i n an attempt to separate the components. Table 2 i l l u s t r a t e s the reduction i n capacity of the moderate a f f i n i t y component by ammonium s u l f a t e without producing major e f f e c t s on the high a f f i n i t y component. Although we could not completely abolish moderate a f f i n i t y binding, we employed the 50% ammonium su l f a t e f r a c t i o n i n further studies. Since we were interested i n the c h a r a c t e r i s t i c s of both the high and moderate a f f i n i t y binding components and t h e i r i n t e r a c t i o n s , we measured binding i n both f r a c t i o n s . The high a f f i n i t y component was measured i n the ammonium s u l f a t e f r a c t i o n between 0.05-2.5 nM 3 [ H] - e s t r a d i o l , and the moderate a f f i n i t y component was measured 3 i n whole cytosol between 10-200 nM I H] - e s t r a d i o l . Furthermore, for future mathematical modeling c a p a b i l i t i e s , we u t i l i z e d e s t r a d i o l as our competior since i t displaced bound e s t r a d i o l i n both f r a c t i o n s . 44 TABLE 2. 3 The apparent Kd and capacity of [ H ] - e s t r a d i o l binding to d i f f e r e n t f r a c t i o n s of adult male rat l i v e r . Incubations were c a r r i e d out for 90 minutes at 4°C. A 100 f o l d excess of unlabeled e s t r a d i o l was used as a competitor. Fraction Apparent Kd Capacity [ 3 H ] - e s t r a d i o l (nM) ( f m o l / m g p r o t e i n ) concentration range 30% Ammonium Sulfate 1.1 118.5 (0.05-2.5 nM) 50% Ammonium Sulfate 1.2 89.2 (0.05-2.5 nM) Whole Cytosol NS NS (0.05-2.5 nM) 30% Ammonium Sulfate 700 0.78 (10-200 nM) 50% Ammonium Sulfate 220 1.00 (10-200 nM) Whole Cytosol 180 6.60 (10-200 nM) NS = No Scatchard b) Time, Temperature and pH Studies Because the assay had been modified we conducted time, temperature and pH studies to e s t a b l i s h optimal assay conditions. The optimal incubation time was determined to be 90 minutes at 4°C for both high and moderate a f f i n i t y components (Appendix 1, Figure A1,A2).To further investigate the binding c h a r a c t e r i s t i c s for both s i t e s , incubation time courses were done at 25°C. S p e c i f i c binding was determined for incubation times ranging from 15 minutes to 24 hours. Results i n Table 3 indicate maximum binding for the high a f f i n i t y s i t e was reached by 60 minutes i n both immature male and female ammonium sul f a t e f r a c t i o n s , and by 30 minutes i n adult male. Moderate a f f i n i t y binding i n male whole cytosol was maximal at 15 minutes with e s t r a d i o l as a competitor and 30 minutes with DHT. Thereafter a l l binding decreased. The difference i n s p e c i f i c binding at 4 versus 25°C was not s i g n i -f i c a n t at 60 minutes. The apparent Kd and capacity at 4°C were 1.14 nM and 124 fmol/mg as compared to 1.16 nM and 117 fmol/mg at 25°C, The binding of e s t r a d i o l was stable to small changes i n pH. The optimum pH range f o r the moderate a f f i n i t y component i n male whole cytosol was determined to be pH 7.0-8.0. At higher pH, no binding occurred. Increasing the pH produced a s l i g h t decrease i n a f f i n i t y and increase i n capacity for the male high a f f i n i t y s i t e , even smaller e f f e c t s were noted f o r the female (Table 4). We selected pH 7.5 to be p h y s i o l o g i c a l l y relevant and sim i l a r to previously published studies. TABLE 3 Time course of s p e c i f i c [ H]-estradiol binding. Incubations were car r i e d out for 90 minutes at 25°C using a 100 f o l d excess of unlabeled e s t r a d i o l as a competitor for 2 nM radio-labeled e s t r a d i o l in the ammonium sulfate f r a c t i o n and 200 nM radio-labeled e s t r a d i o l in the whole cytosol f r a c t i o n . Time BINDING (CPM) (minutes) Male Female Adult Immature Adult Ammonium Sulfate Whole Cytosol Ammonium Sulfate Whole Cytosol Ammonium Sulfate Fract ion Fraction Fraction Fraction Fraction 15 3355 790 N.D. N.D. 4352 30 3998 453 5227 0 4562 60 2621 487 5719 0 6381 90 2876 216 3781 0 5122 120 N.D. N.D. 4347 0 5204 24 hours 610 0 1213 0 170 N.D. = Not determined TABLE 4 The effect of pH on the apparent Kd and capacity of the hepatic estrogen receptor and moderate a f f i n i t y component. Incubations were conducted at the pH values l i s t e d for 90 minutes at 4°C. S p e c i f i c [ 3 H ] - e s t r a d i o l binding was determined using a 100 f o l d excess of unlabeled e s t r a d i o l as a competitor i n adult male and female rat hepatic preparations. pH Estrogen Receptor Moderate A f f i n i t y Component Male Female Male Apparent Kd Capacity Apparent Kd Capacity Apparent Kd Capacity IQ (fmol/mg 1 Q (fmol/mg _ (pmol/mg (xlO M) protein) (xlO M) protein) (xlO M) protein) 7.0 14.0 16.7 7.5 8.5 160 16.8 215 11.0 10.7 8.0 15.0 13.3 8.5 14.9 226 18.5 298 0 0 9.0 0 0 48 c) Freezing Studies To determine the s t a b i l i t y of these binding components to storage the following study was done. A large pool of l i v e r was c o l l e c t e d from male and female r a t s and divided into several samples. Both high and moderate a f f i n i t y components were assessed from each pool immediately. A l l other samples were frozen i n l i q u i d nitrogen and stored at -80°C for l a t e r a n a l y s i s . Results i n Table 5 indicated that the moderate a f f i n i t y component i s stable for one week, whereas the high a f f i n i t y component i s stable at two months in both males and females. d) Competitor Studies We examined the effectiveness of various competitors i n di s p l a c i n g e s t r a d i o l from the high a f f i n i t y and moderate a f f i n i t y s i t e i n the male l i v e r f r a c t i o n s . The r e s u l t s of t h i s study are 3 shown in Table 6. [ H ] - e s t r a d i o l was e f f e c t i v e l y displaced from the high a f f i n i t y s i t e by estrogens i n both male (Table 6) and female (Appendix 1, Table '-AlO ammonium sul f a t e f r a c t i o n s . However, both estrogens and androgens (testosterone and DHT) were able to displace e s t r a d i o l from the moderate a f f i n i t y s i t e i n male whole cy t o s o l . Progestins, g l u c o c o r t i c o i d s and mineratotorticoids were not e f f e c t i v e in e i t h e r f r a c t i o n i n either sex. Since DHT and testosterone were e f f e c t i v e i n d i s p l a c i n g e s t r a d i o l from the moderate a f f i n i t y s i t e i n male whole c y t o s o l , 3 we further examined the binding of these androgens. Both [ H]-DHT 3 and [ H]-testosterone were employed as ligands (separately) i n male TABLE 5 The effect of freezing on the apparent Kd and capacity of the estrogen receptor and moderate a f f i n i t y component. Tissue pools were divided and frozen at -80°C. Assays were conducted on the unfrozen t i s s u e pool and at various time points on thawed tissue pool samples. Time (weeks) Estrogen Receptor Male Female Moderate A f f i n i t y Component Male Apparent Kd ( x l O 1 0 M) Capacity (fmol/mg protein) Apparent Kd ( x l O 1 0 M) Capacity (fmol/mg protein) Apparent Kd (xlO? M) Capacity (pmol/mg protein) 0 0.6 65 0.7 203 11.9 8.5 1 0.8 99 1.7 200 7.9 10.2 2 1.8 178 1.3 141 NS NS 4 0.7 96 0.8 89 NS NS 8 1.3 80 1.1 130 NS NS NS = No Scatchard TABLE 6 Ligand s p e c i f i c i t y of [ H]-estradiol binding to the estrogen receptor and moderate a f f i n i t y component i n the adult male r a t . Incubations were carried out for 90 minutes at 4°C. S p e c i f i c binding was determined using a 10,100, or 1000 f o l d excess of unlabeled competitor for 0.5 nM [ 3 H ] - e s t r a d i o l bound to the estrogen receptor i n the ammonium s u l f a t e f r a c t i o n or 50 nM [ 3H]-e s t r a d i o l bound to the moderate a f f i n i t y component i n the whole cytosol f r a c t i o n . Percent of t o t a l binding i n the presence of excess competitor Steroid Estrogen Receptor Moderate A f f i n i t y Component Competitor 10-fold 100-fold 1000-fold 10-fold 100-fold 1000-fold Control 100 100 100 100 100 100 E s t r a d i o l 64 54 47 100 67 66 D i e t h y l s t i l b e s t r o l 62 60 58 100 100 100 Moxestrol 68 61 58 88 76 66 Testosterone 94 97 100 100 100 78 Dihydrotestosterone 94 95 91 100 100 77 Androstenedione 96 97 93 100 90 85 Methyltrienolone 96 92 89 91 100 100 Progesterone 91 90 90 88 89 91 Promegestone 75 95 91 92 100 100 Triamcinolone 90 92 91 70 95 100 Dexamethasone 91 90 89 100 95 85 Aldosterone 100 96 95 100 100 100 * The binding of [ H]- e s t r a d i o l i n the presence of competitor i s given as % of control values (control value = binding in the absence of competitor) 51 whole cytosol i n a concentration range of 10-200 nM and were competed with unlabeled DHT and testosterone (respectively) or e s t r a d i o l . Although displacable binding was observed, the r e s u l t s from using androgens as ligands did not generate a Scatchard p l o t . e) Binding to uterus, bovine serum albumin and plasma The binding of e s t r a d i o l i n l i v e r was compared to that i n uterus, a .classic target t i s s u e . An ammonium s u l f a t e f r a c t i o n of u t e r i was prepared i n p a r a l l e l with l i v e r and assayed under si m i l a r conditions. We determined the apparent Kd and cap a c i t i e s of l i v e r and u t e r i to be 0.9 nM; 128 fmol/mg and 0.5 nM; 438 fmol/mg res p e c t i v e l y . In addition, the percent displacement at 0.5 nM 3 [ H ] — e s t r a d i o l was found to be 64% i n l i v e r and 91% in uterus at 100 f o l d excess e s t r a d i o l . In a s i m i l a r study the binding of e s t r a d i o l to bovine serum albumin (BSA) or plasma was investigated. We detected no binding to BSA or to plasma of male or female r a t s i n either the high or moderate a f f i n i t y range. 2) E f f e c t s of p h y s i o l o g i c a l manipulation on hepatic estrogen binding components a) Age and sex re l a t e d modulation of hepatic estrogen binding Having established the conditions for determination of hepatic estrogen binding, as well as some of the binding c h a r a c t e r i s t i c s , the assay was applied to various p h y s i o l o g i c a l models. In so doing 52 the v a l i d i t y of the assay was confirmed and p h y s i o l o g i c a l relevance was established. Preliminary studies aimed at es t a b l i s h i n g physio-l o g i c a l relevance were conducted i n the following animal models. The binding a f f i n i t i e s for the estrogen receptor, high a f f i n i t y , low capacity ammonium sul f a t e component, were found to be sim i l a r i n the male, female and pseudohermaphroditic rat (Table 7). The capacity of the estrogen receptor i n the adult l i t t e r m a t e c o n t r o l male was lower than the female and pseudohermaphrodite; however, generally there were no major differences i n capacity between sexes. As previously described the moderate a f f i n i t y s i t e was not detected i n the female. Furthermore, no moderate a f f i n i t y binding was detected i n the pseudohermaphrodite rat (Table 7). The e f f e c t of age and gonadectomy on the binding c h a r a c t e r i s t i c s of the estrogen receptor are presented i n Table 8. The capacity of the estrogen receptor was lower i n the immature animal (25-30 days of age) as compared to adult controls. Similar to the adult, there was no sex difference i n the capacity of the estrogen receptor in the immature r a t . However, the apparent Kd for the estrogen receptor was reduced i n the immature female r e l a t i v e to the immature male or mature animal. I n t e r e s t i n g l y , gonadectomy s l i g h t l y reduced the apparent Kd of the estrogen receptor i n both males and females, but the capacity only i n the male. As with the adult female no moderate a f f i n i t y binding was observed i n the immature male or female (Table 8). Gonadectomy of the male reduced the capacity of the moderate a f f i n i t y component approximately six f o l d without a l t e r i n g the apparent Kd. In addition, s i m i l a r r e s u l t s were obtained when unlabeled DHT was used as a competitor f o r 53 TABLE 7 A comparison of the apparent Kd and capacity of the hepatic c y t o s o l i c estrogen receptor and moderate a f f i n i t y component i n male, female, and pseudohermaphroditic r a t s . Incubations were c a r r i e d out f o r 90 minutes at 4°C as described i n the methods section on a sample pool from 4 animals. Model Male Female P seudohermaphrod i t e Estrogen Receptor 1.1 261 1.1 368 1.0 406 Moderate A f f i n i t y Component Capacity (pmol/mg protein) 1.4 6.6 0 0 0 0 Kd Capacity R d g (fmol/mg 7 (xlO M) protein) (xlO M) TABLE 8 The e f f e c t of age and gonadectomy on the apparent Kd and capacity of hepatic c y t o s o l i c estrogen receptor and moderate a f f i n i t y binding component i n male and female r a t s . Adult male and female animals were gonadectomized (Gx) or sham-operated 11 days p r i o r to s a c r i f i c e . Immature (Imm.) rats were s a c r i f i c e d between days 25 and 30 of l i f e . A pool of 4 l i v e r s was used. Model Estrogen Receptor Moderate A f f i n i t y Component Ammonium Sulfate Fraction Whole Cytosol Fraction Kd (x l O 9 M) Capacity (fmol/mg protein) Kd (xlO 7 M) Capacity (pmol/mg protein) Kd* (xlO? M) Capacity* (pmol/mg protein) Male 2.8 620 1.4 31.4 1.0 23.5 Imm. Male 2.5 305 0 0 0 0 Gx Male 1.1 353 1.5 5.1 0.8 3.1 Female 3.7 641 0 0 0 0 Imm. Female 1.5 267 0 0 0 0 Gx Female 1.1 544 0 0 0 0 * Indicates values obtained using a 1000 f o l d excess of unlabeled DHT as a competitor for [ H] - e s t r a d i o l a l l others are a 100 f o l d excess of unlabeled 'estradiol. 55 [ J H ] - e s t r a d i o l binding. Testosterone was administered to gonadectomized males to confirm that the male s p e c i f i c moderate a f f i n i t y binding protein was androgen dependent. The r e s u l t s of t h i s study are reported i n Table 9, with either unlabeled e s t r a d i o l or DHT as competitors 3 for [ H ] - e s t r a d i o l binding. A f i v e f o l d decrease in the capacity of the moderate a f f i n i t y component was observed following gonadectomy confirming the r e s u l t s seen in Table 9. Administration of t e s t o -sterone enanthate (1.0 mg/kg/day subcutaneously for ten days) reversed the e f f e c t s of gonadectomy on the capacity of t h i s s i t e (Figure 1A). Neither gonadectomy nor the administration of testosterone to the gonadectomized female res u l t e d in detection of a moderate a f f i n i t y component. Studies with testosterone replacement were also c a r r i e d out on the estrogen receptor. Testosterone did not a f f e c t the binding c h a r a c t e r i s t i c s of the estrogen receptor i n either gonadectomized male or female r a t s (Table 9). Furthermore, unlike the previous study (Table 8), gonadectomy i t s e l f produced only minor e f f e c t s on the apparent Kd and capacity of the estrogen receptor i n the male or female. b) P i t u i t a r y modulation of hepatic estrogen binding Hypothalamic-hypophyseal influences on the hepatic estrogen binding proteins were examined i n adult male and female r a t s . As shown in Table 10,capacity of the estrogen receptor decreased to below 50% of the control l e v e l s two weeks following hypophysectomy in both male and female r a t s . Hypophysectomy did not a l t e r the Kd of the estrogen receptor. Hypophysectomy also greatly reduced the TABLE 9 The e f f e c t of gonadectomy and testosterone replacement on the apparent Kd and capacity of the hepatic c y t o s o l i c estrogen receptor and moderate a f f i n i t y binding component in male and female r a t s . Adult male and female rats were gonadectomized 11 days p r i o r to s a c r i f i c e . Where indicated animals received testosterone enanthate (Test) (1.0 mg/kg/day subcutaneously i n corn o i l ) for 10 days p r i o r to s a c r i f i c e . Animals were s a c r i f i c e d 24 hours a f t e r the l a s t i n j e c t i o n . Assays were conducted as described in the methods section on a t i s s u e pool of 4 l i v e r s . Model Estrogen Receptor Moderate A f f i n i t y Component Ammonium Sulfate Fraction Kd .9 Capacity (xlO M) (fmol/mg protein) Whole Cytosol Fraction Kd Capacity Kd Capacity Q Q (xl0° M) (pmol/mg protein) (xlO M) (pmol/mg protein) Male 1.05 Gx Male 0.73 Gx Male + 0.75 Test Female 0.82 Gx Female 0.62 Gx Female + 0.89 Test 255 202 207 188 225 224 6.4 4.1 3.9 0 0 0 11.0 2.2 10.0 0 0 0 4.2 1.5 6.3 0 0 0 8.5 1.1 13.9 0 0 0 * Indicates values obtained using a 1000 f o l d excess of unlabeled DHT as a competitor for [ H ] - e s t r a d i o l binding instead of the 100 f o l d excess of unlabeled ."estradiol -used.'-in, a l l other• experiments. 57 Figure 1 3 E f f e c t of p h y s i o l o g i c a l manipulation on the s p e c i f i c [ML]^estradiol binding to the moderate a f f i n i t y component. Scatchard analyses was -performed on t i s s u e pools from four animals over a 3 concentration range of 10-200 nM [ H ] - e s t r a d i o l . S p e c i f i c binding was determined using a 100 f o l d excess of unlabeled e s t r a d i o l as a competitor. Incubations were c a r r i e d out for 90 minutes at 4°C. a) Adult male r a t s were sham-operated (O), or gonadectomized ( ® ) eleven days p r i o r to s a c r i f i c e . One group of gonadectomized males received testosterone enanthate, (1.0 mg/kg/day s.c. for t en days) ( • ) . b) Adult male r a t s were sham-operated ( • ) , or hypophysectomized (O) two weeks p r i o r to s a c r i f i c e . B Ln 00 To" 4.0 8.0 10.0 BOUND(pmol/mg protein) 59 TABLE 10 The e f f e c t of hypophysectomy on the binding c h a r a c t e r i s t i c s of the hepatic c y t o s o l i c estrogen receptor i n male and female r a t s . Adult male and female r a t s were hypophysecto-mized (Hx) or sham-operated between 50 and 60 days of age. Animals were s a c r i f i c e d two weeks post-hypophysectomy. A pool of 4 l i v e r s was used. Model (x l O y M) Capacity (fmol/mg protein) Sham Male 2.2 516 Hx Male 2.5 190 Sham Female 2.5 506 Hx Female 3.4 250 60 capacity of the moderate a f f i n i t y binding component i n the male, (Figure IB), with either unlabeled e s t r a d i o l or DHT as competitors (Table 11) (to 16.1% and 12.2% of control r e s p e c t i v e l y ) . The decrease in capacity of the moderate a f f i n i t y component was not accompanied by a change i n apparent Kd. Of p a r t i c u l a r interest was the appearance of a moderate a f f i n i t y binding s i t e i n the female two weeks following hypophysectomy (Table 11, Figure 2k). Although the capacity was only one tenth that of the hypophysecto-mized male, the s i t e was detectable for the f i r s t time in the female, and had an apparent Kd s i m i l a r to that of the male. This appearance of a moderate a f f i n i t y s i t e i n the hypo-physectomized female was studied at a -longer time period. The capacity of the hypophysectomized female moderate a f f i n i t y s i t e was found to be nearly equal that of the hypophysectomized male at four and s i x weeks post-surgery (Tables 12, 13; Figure 2B).. The apparent Kd-was approximately two/fold greater i n the female than the male at four weeks following surgery (Table 12). The e f f e c t of hypophysectomy at four weeks was otherwise s i m i l a r to the e f f e c t of hypophysectomy at two weeks. That i s , there was a decrease i n the capacity of the estrogen receptor and the moderate a f f i n i t y component, with no e f f e c t on the apparent Kd (Table 12). The e f f e c t of testosterone on estrogen binding i n the hypophysectomized animal was studied to determine ce n t r a l involvement of sex st e r o i d a c t i o n . Results of t h i s study are presented i n Tables 13 and 14. These studies were conducted six weeks following hypo-physectomy. At t h i s time point the estrogen receptor was not detectable i n the hypophysectomized female, and was dramatically reduced in the male (Table 13). Testosterone enanthate, previously 61 TABLE 11 The effect of hypophysectomy on the binding c h a r a c t e r i s t i c s of the moderate a f f i n i t y component i n male and female r a t s . Adult male and female r a t s were hypophysectomized (Hx) between days 50 and 60 of- l i f e . Animals were s a c r i f i c e d two weeks following hypophysectomy. Moderate a f f i n i t y s p e c i f i c binding was detected using a 100 f o l d excess of unlabeled e s t r a d i o l or a 1000 f o l d excess of dihydrotestosterone as competitors. A t i s s u e pool of 4 animals was used f o r each group. Model Competitor E s t r a d i o l Dihydrotestosterone ^ Capacity 1 X 1 1 Capacity (xlO' M) (pmol/mg (xlO 7 M) (pmol/mg protein) protein) Sham Male Hx Male Sham Female Hx Female 0.9 0.7 0 0.5 14.9 2.4 0 0.2 0.6 0.5 0 0 11.4 1.4 0 0 62 Figure 2 3 E f f e c t of hypophysectomy on the s p e c i f i c [ H ] - e s t r a d i o l binding to the moderate a f f i n i t y component. Scatchard analyses was • performed on t i s s u e pools from four animals over a concentration 3 range of 10-200 nM [ H ] - e s t r a d i o l . S p e c i f i c binding was determined using a 100 f o l d excess of unlabeled e s t r a d i o l as a competitor. Incubations were c a r r i e d out for 90 minutes at 4°C. a) Adult male (•) and female ()( ) r a t s were hypophysectomized two weeks p r i o r to s a c r i f i c e . b) Adult male (•) and female (O) ra t s were hyophysectomized s i x weeks p r i o r to s a c r i f i c e . BOUND/FREE £9 TABLE 12 The e f f e c t of hypophysectomy on the apparent Kd and capacity of the hepatic c y t o s o l i c estrogen receptor and moderate a f f i n i t y binding component at four weeks post-surgery i n male and female rats . Adult male and female rats were hypophysectomized (Hx) or sham-operated between days 50 and 60 of l i f e . Animals were s a c r i f i c e d four weeks following surgery. A pool of 4 l i v e r s were used for each group. Model Estrogen Receptor Moderate A f f i n i t y Component Ammonium Sulfate Fraction Whole Cytosol Fraction Kd Capacity . Kd Capacity Kd Capacity ( x l 0 y M) (fmol/mg protein) (xlO M) (pmol/mg protein) (xlO 8 M) (pmol/mg protein) Sham Male 1.11 Hx Male 1.32 Sham Female 1.5 Hx Female 1.47 260 156 447 98 8.8 9.2 0 17.9 19.3 12.2 0 7.0 6.9 5.5 0 16.9 15.4 8.3 0 5.9 * Indicates values obtained using a 1000 f o l d excess of unlabeled DHT as a competitor f o r [ H ] - e s t r a d i o l instead of a 100 f o l d excess of unlabeled .estradiol. 65 TABLE 13 The e f f e c t of testosterone and growth hormone' on the binding c h a r a c t e r i s t i c s of the hepatic c y t o s o l i c estrogen receptor in the hypophysectomized r a t . Adult male and female r a t s were hypophysectomized (Hx) between days 50 and 60 of l i f e . Where indicated animals received testosterone enanthate (Test) (1.0 mg/kg/day s.c. in corn o i l ) or bovine growth hormone (bGH) (2 I.U./kg/day s.c. twice d a i l y i n saline) for 10 days p r i o r to s a c r i f i c e . Animals were s a c r i f i c e d 24 hours following the l a s t i n j e c t i o n , s i x weeks post-surgery. Tissue from 4 animals was pooled f o r each group. Model Kd Capacity (xlO 9 M) (fmol/mg protein) Hx Male 1.21 84.9 Hx Male + Test 1.06 82.3 Hx Male + bGH 0.76 169.0 Hx Female 0 0 Hx Female + Test 0 0 Hx Female + bGH 0 0 66 shown not to a f f e c t the capacity of the estrogen receptor, had no e f f e c t on the apparent Kd or capacity of the estrogen receptor in the hypophysectomized male or female r a t . In addition, the e f f e c t s of growth hormone on hepatic estrogen binding were examined. -Following the administration of bGH (4.0 I.U./kg/day, s.c.) in a divided dose twice d a i l y for ten days, we'observed a s l i g h t decrease in apparent Kd along with an increase i n capacity ;of,the.estrogen-receptor, i n the male,(Tabl:e 13). Administration',of. bGH -clearly'had/no r e s t o r a t i v e e f f e c t on the estrogen receptor i n the hypophysectomized female. The e f f e c t of testosterone and bGH on binding of the moderate a f f i n i t y component was also studied. Unlike the gonadectomized male, testosterone had no r e s t o r a t i v e e f f e c t on the capacity of the moderate a f f i n i t y component in the hypophysectomized animal, (Table 14). However, bGH (2. I.U./kg/day s.c. twice d a i l y ) d i d r e s u l t i n a 50-80% decrease i n the capacity of the moderate a f f i n i t y component i n hypophysectomized male and female r a t s , r e s p e c t i v e l y . Neither testosterone nor bGH had any e f f e c t on the apparent Kd of the moderate a f f i n i t y s i t e i n the hypophysecto-mized r a t . Furthermore, since the apparent Kd and capacity of the moderate a f f i n i t y component were s i m i l a r i n the male and female at six weeks following hypophysectomy, a d d i t i o n a l estrogen binding studies involving hypophysectomized animals were conducted at least s i x weeks post-surgery. In addition, at t h i s time point the estrogen receptor was often absent from male as well as female r a t s (Figure 3A, B). 67 TABLE 14 The e f f e c t of testosterone and growth hormone on the binding charac-t e r i s t i c s of the hepatic c y t o s o l i c moderate a f f i n i t y component i n hypophysectomized r a t s . Adult male and female r a t s were hypophysecto-mized (Hx) between days 50 and 60 of l i f e . Where indicated animals received testosterone enanthate (Test) (1.0 mg/kg/day s.c. i n corn o i l ) or bovine growth hormone (bGH) (2 I.U./kg/day twice d a i l y (s.c. i n saline) for 10 days p r i o r to s a c r i f i c e . Animals were s a c r i f i c e d 24 hours a f t e r the l a s t i n j e c t i o n , s i x weeks following hypophysectomy. Apparent Kd and capacity were determined using a 100 f o l d excess of unlabeled e s t r a d i o l or 1000 f o l d excess of dihydrotestosterone-as competitors. A pool of 4 l i v e r s was used per group. Model Competitor E s t r a d i o l Dihydrotestosterone Kd Capacity Kd Capacity (xlO M) (pmol/mg (xlO 8 M) (pmol/mg protein) protein) Hx Male 4.8 4.7^ 4.7 3.9 Hx Male + Test 3.5 4.3 2.9 3.1 Hx Male + bGH 6.1 2.1 3.6 1.3 Hx Female 6.7 6.1 4.1 3.4 Hx Female + Test 2.7 4.3 4.2 4.5 Hx Female + bGH 7.2 1.3 3.5 0.6 68 Figure 3 3 E f f e c t of hypophysectomy on the s p e c i f i c [ H]-estradiol binding to the estrogen receptor. Scatchard analyses was performed on tis s u e pools from four animals over a concentration range 3 of 0.05-2.5 nM [ H ] - e s t r a d i o l . S p e c i f i c binding was determined using a 100 f o l d excess unlabeled e s t r a d i o l as a competitor. Incubations were c a r r i e d out for 90 minutes at 4°C. a) Adult sham-operated (•) and hypophysectomized (O) male ra t s eight weeks following surgery. b) Adult sham-operated (•) and hypophysectomized (O) female r a t s eight weeks following surgery. 70 3) Characterization of hepatic enzyme a c t i v i t y i n d i f f e r e n t p h y s i o l o g i c a l models a) Sex related modulation of hepatic AHH and testosterone A4 reductase a c t i v i t i e s . As has been extensively documented, the male l e v e l s of hepatic AHH a c t i v i t y observed i n the r a t are l a r g e l y modulated by t e s t i c u l a r androgens. To further characterize t h i s androgen dependency, hepatic AHH a c t i v i t y was determined i n Stanley-Gumbreck pseudohermaphroditic r a t s . The control l e v e l s of AHH a c t i v i t y of the pseudohermaphrodite r e l a t i v e to the l i t t e r m a t e c o n t r o l males and females, as well as the response to phenobarbital and spirono-lactone are presented in Table 15. The AHH a c t i v i t y of the c o n t r o l pseudohermaphrodite was the same as that of the control female, both of which were s t a t i s t i c a l l y lower than the control male. Administration of phenobarbital (80 mg/kg/day, i n t r a p e r i t o n e a l l y ) for four days s i g n i f i c a n t l y induced AHH a c t i v i t y i n both the female and pseudohermaphrodite r e l a t i v e to t h e i r c o n t r o l s . Phenobarbital did not a f f e c t AHH a c t i v i t y i n the male. Following treatment with spironolactone (100 mg/kg/day, i n t r a p e r i t o n e a l l y ) for four days, 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 reduction i n male AHH a c t i v i t y was observed compared to control (Table 19). However, spironolactone, l i k e phenobarbital, s i g n i f i c a n t l y induced hepatic AHH a c t i v i t y i n the female and pseudohermaphrodite. In a second study the e f f e c t s of 3-MC were examined. The c o n t r o l l e v e l s of the pseudohermaphrodite were again s i m i l a r to 7 1 TABLE 15 The e f f e c t of phenobarbital and spironolactone on hepatic AHH a c t i v i t y i n male, female and pseudohermaphroditic r a t s . Adult male, female and pseudohermaphroditic (pseudo) r a t s were treated with e i t h e r phenobarbital (80 mg/kg/d x 4d i . p . i n saline) or spironolactone (100 mg/kg/d x 4d i . p . i n s a l i n e ) . Control animals received v e h i c l e alone. Animals were s a c r i f i c e d 24 hours following the l a s t i n j e c t i o n . Differences between groups were considered s i g n i f i c a n t at p < 0.05 using the Duncan multiple range t e s t . Model AHH A c t i v i t y (pmol/min/mg protein)±S.E.M., (n) Control Phenobarbital Spironolactone Male 1986 ± 154 1604 ± 78 1275 ± 62* (5) (4) (4) Female 217 ± 6 + 694 ± 53* 741 ± 97* (4) (4) (4) i ± 10 + 658 ± 43* 1135 ± : (5) (4) (4) Pseudo 295 152* * S i g n i f i c a n t l y d i f f e r e n t from respective c o n t r o l groups, t S i g n i f i c a n t l y d i f f e r e n t from male control group. 72 that of the female (Table 16). Following administration of 3-MC (20 mg/kg/day, i n t r a p e r i t o n e a l l y ) f o r two days, AHH was induced i n a l l three animal models to a common l e v e l . Studies on the e f f e c t of hypophysectomy on hepatic AHH a c t i v i t y were also conducted. The requirement of the hypophyseal-hypothalamic axis for the maintenance of the sex difference was demonstrated as shown in Table 17. Hypophysectomy produced a decrease i n AHH a c t i v i t y i n the male and an increase i n AHH a c t i v i t y i n the female at two weeks post-surgery. The reduction in d i f f e r e n t i a t i o n of AHH a c t i v i t y noted following hypophysectomy was a consistent observation (Tables 17, 18, 19). However, when the number of treatment groups was increased (as in Table 18) requ i r i n g analysis by multiple range t e s t i n g , and a comparison with the much greater a c t i v i t y i n the male, the s i g n i f i c a n c e of the increase i n the female was no longer apparent. In a d d i t i o n , as shown in Table 18, the i n a b i l i t y of t e s t o -sterone (2.0 mg/kg/day, subcutaneously for fourteen days) to reverse the decrease i n AHH a c t i v i t y i n the male at four weeks post-hypophysectomy was demonstrated. In f a c t , testosterone produced a s l i g h t reduction i n AHH a c t i v i t y i n the sham-operated 'male. However, the increase i n AHH a c t i v i t y i n the female observed at two weeks following hypophysectomy (Table 17) 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 by the Newman-Keuls test at four weeks a f t e r surgery (Table 18). In addition to the differences i n s t a t i s t i c a l techniques used, hypophysectomy i s an invasive procedure that not a l l animals survive. As such, the sample group assayed at four weeks may represent a d i f f e r e n t population from those assayed at two weeks 73 TABLE 16 The e f f e c t of 3-methylcholanthrene on hepatic AHH a c t i v i t y i n male, female, and pseudohermophrodite r a t s . Adult male, female, and pseudohermaphrodite (pseudo) r a t s were treated with 3-methylcholanthrene (3-MC) (20 mg/kg/d x 2d i . p . i n corn o i l ) . Control animals received v e h i c l e alone. Animals were s a c r i f i c e d 24 hours following the l a s t i n j e c t i o n . Differences between groups were considered s i g n i f i c a n t at p < 0.05 using the Newman-Keuls multiple range te s t (n=4). Model AHH A c t i v i t y pmol/min/mg protein ± S.E.M. Control 3-Methylcholanthrene Male 1695 ± 133 4555 ± 300* + Female 475 ± 24 5037 ± 334 Pseudo 407 ± 70 T 5251 ± 278 * S i g n i f i c a n t l y d i f f e r e n t from respective control groups, t S i g n i f i c a n t l y d i f f e r e n t from male control group. TABLE 17 The ef f e c t of hypophysectomy on hepatic AHH a c t i v i t y i n male and female r a t s . Adult male and female r a t s were hypophysectomized (Hx) or sham-operated between 50 to 60 days of age. Animals were s a c r i f i c e d two weeks following surgery. Differences were considered s i g n i f i -cant at p < 0.05 using the two t a i l e d Students t-Test. (n=6) . Model AHH A c t i v i t y pmol/min/mg protein ± S.E.M. Sham Hx Male 2030 ± 243 782 ± 114 Female 184 ± 7 312 ± 49 * Indicates s i g n i f i c a n t differences between Hx and .sham-operated c o n t r o l . TABLE 18 The e f f e c t of hypophysectomy and testosterone on hepatic AHH a c t i v i t y i n male and female r a t s . Adult male and female r a t s were hypophysectomized (Hx) or sham-operated between days 50 to 60 of l i f e . Males received testosterone enanthate (Test) (2.0 mg/kg/d, s.c. i n corn o i l ) f or 14 days p r i o r to s a c r i f i c e . Animals were s a c r i f i c e d 24 hours following the l a s t i n j e c t i o n , and four weeks post-surgery. Control animals received v e h i c l e alone. Model AHH A c t i v i t y pmol/min/mg protein ± S.E.M., (n) Sham Hx Male 2788 ± 1 6 3 a b c d 744 ± 105 d (8) (6) Male + Test 1306 ± 2 2 0 a b c d 389 ± 40 a (6) b ( 6 ) c Female 578 ± 25 734 ± 36 (9) (6) Differences between groups were considered s i g n i f i c a n t at p < 0.05 using the Newman-Keuls multiple range t e s t . S i g n i f i c a n t d ifferences between groups were indicated by common l e t t e r s . following hypophysectomy. The effectiveness of known inducers of mixed function oxidase a c t i v i t y i n control male and female ra t s was examined in hypo-physectomized animals. Phenobarbital (80 mg/kg/day, i n t r a p e r i t o n e a l l y for four days) was unable to a f f e c t AHH a c t i v i t y i n the hypophysecto-mized male (Table 19). This was predicted based on r e s u l t s from Table 15. I n t e r e s t i n g l y though, phenobarbital was capable of producing an increase in AHH a c t i v i t y i n the hypophysectomized female r e l a t i v e to the hypophysectomized c o n t r o l . Again we observed the reduction i n d i f f e r e n t i a t i o n (as seen in Table 17) at 2 weeks post-hypophysectomy. Like phenobarbital, 3-MC had no e f f e c t on AHH a c t i v i t y i n the hypophysectomized male (Table 19). The e f f e c t of 3-MC on AHH a c t i v i t y i n the hypophysectomized female was not determined. b) Endocrine manipulation of hepatic enzyme a c t i v i t y i ) E f f e c t of pergoiide i n adult female ra t s I t had been hypothesized that PRL may represent the p i t u i t a r y factor involved i n the sex dependent d i f f e r e n t i a t i o n of hepatic drug metabolism. P r o l a c t i n was shown to be present i n r e l a t i v e l y higher amounts i n the adult female than male (Posner et a l . , 1974). As such, the elevated l e v e l s of PRL in the female might account for the sex diffe r e n c e i n metabolism seen at maturity. To assess the e f f e c t s of PRL, pergoiide (0.1 mg/kg/day) was administered subcutaneously to adult sham-operated or gonadectomized- female rats f o r eleven days. Pergoiide, a dopamine agonist, was found to reduce TABLE 19 The e f f e c t of 3-methylcholanthrene and phenobarbital on hepatic AHH a c t i v i t y i n hypophysecto-mized male and female r a t s . Adult male and female rats were hypophysectomized (Hx) or sham-operated between days 50 to 60 of l i f e . Where indicated Hx—rats received 3-methylcholan-threne (3-MC) (20 mg/kg/d i.p. in corn o i l ) for 2 days, or phenobarbital (PB) (80 mg/kg/d i . p . in saline) f o r 4 days p r i o r to s a c r i f i c e . Animals were s a c r i f i c e d two weeks following surgery, and 24 hours af t e r the l a s t i n j e c t i o n . Differences between groups were considered s i g n i f i c a n t at p < 0.05 using the Newman-Keuls multiple range t e s t . Model AHH A c t i v i t y (pmol/min/mg protein) ± S.E.M., (n) Sham Hx Hx + 3-MC Hx + PB Male 3550.8 ± 318.8 1900.3 ± 195.2* 2224,0 ± 121.4* 2456.8 ± 109,2* (5) (7) (4) (4) : ± 20.9 + 1196.3 ± 121.8* + N.D. 2073.0 ± : (5) (8) (3) * S i g n i f i c a n t l y d i f f e r e n t from respective control groups, t S i g n i f i c a n t l y d i f f e r e n t from male control group. N.D. = not determined. 78 serum PRL l e v e l s 90% i n the adult female rat (Appendix 1). To test the hypothesis, the response of hepatic AHH and testosterone testosterone A 4 reductase a c t i v i t y i n the, administration of testosterone enanthate (1.0 mg/kg/day subcutaneously for ten days) were determined. The dose of testosterone had been previously determined i n our laboratory to maintain at control l e v e l s the weights of androgen s e n s i t i v e t i s s u e s and sex-dependent hepatic enzyme a c t i v i t y i n adult gonadectomized males. The r e s u l t s of t h i s study are presented i n Table 20. Female AHH a c t i v i t y , unlike the male, was not a f f e c t e d by gonadectomy, nor was the a c t i v i t y of A4 reductase. Administration of testosterone to the sham-operated female produced an increase i n AHH a c t i v i t y that was i n h i b i t e d by concurrent administration of pergolide. These differences were found not to be s i g n i f i c a n t following a n a l y s i s by the Newman-Keuls multiple range t e s t ; however, they were s i g n i f i c a n t when analyzed by the Duncan multi p l e range test (not shown). This trend was clearer in the gonadectomized model where administration of testosterone increased AHH a c t i v i t y i n the gonadectomized female, and pergolide p a r t i a l l y blocked t h i s e f f e c t (Table 20). Similar to AHH a c t i v i t y , A4 reductase was not affected by gonadectomy. The l e v e l s of A4 reductase are normally greater i n the adult female as compared to the adult male (Table 22). Following the administration of testosterone the hepatic A4 reductase a c t i v i t y was decreased i n both the sham-^operated and •gonadectomized .female. However unlike AHH a c t i v i t y , concurrent administration of pergolide did not i n h i b i t the action of testosterone (Table 20). 79 TABLE 20 The e f f e c t of gonadectomy, pergoiide and testosterone on hepatic AHH and testosterone A4 reductase a c t i v i t y i n female r a t s . Adult female r a t s were gonadectomized (Gx) or sham-operated 15 days, p r i o r to s a c r i f i c e . Where indicated animals received testosterone' enanthate (Test) (1.0 mg/kg/d s.c. i n corn o i l ) f o r 10 days or pergoiide (Perg.) (0.1 mg/kg/d s.c. i n corn o i l ) for 11 days p r i o r to s a c r i f i c e between days 55 to 65 of l i f e . Animals were s a c r i f i c e d 24 hours a f t e r the l a s t i n j e c t i o n . Differences between groups were considered s i g n i f i c a n t at p < 0.05 using the Newman-Keuls multiple range t e s t . S i g n i f i c a n t d ifferences between groups were indicated by common l e t t e r s . Model AHH A c t i v i t y (pmol/min/mg protein) ± S.E.M., (n) A 4 Red. A c t i v i t y (nmol/min/mg protein) ± S.E.M., (n) Sham Gx Gx + Perg Sham + Test Gx + Test Sham + Test + Perg Gx + Test + Perg 423.1 ± 28.8 (9) 390.2 ± 64.7° ( 4 ) a 311.5 ± 26.1 (4) 645.0 ± 167 e (4) 1489.0 ± 91.9 (5) 353.6 ± 34.1* (5) 822.8 ± 82.8 (5) abcde abed cf i 31.3 ± 1.3 (9) 31.3 ± 0 . 8 b e h <4> »A0 30.8 ± 1.8 S (4) 21.3 ± 3 . 6 g h l (4) 15.3 ± 0.7' (5) 22.9 ± 1.4 a d (5) ,abc 15.4 ± 1.2 (5) def 80 i i ) E f f e c t of perg©l-ide in. pre-pubescent female, rats Female ra t s were sham-operated and gonadectomized pre-pubertally (approximately 25-30 days of age) and treated with pergoiide from day 30 to day 60 of l i f e to examine the e f f e c t s of p r o l a c t i n elevation during puberty on the response of AHH and A4 reductase a c t i v i t y to testosterone. This study i s summarized i n Table 21. Treatment with pergoiide did not reduce hepatic AHH a c t i v i t y i n the sham-• operated female. Similar to the previous study gonadectomy, a l b e i t pre-pubescent, had no e f f e c t on AHH a c t i v i t y . Furthermore, prolonged administration of pergoiide did not produce any e f f e c t on AHH a c t i v i t y i n either the sham-operated or gonadectomized-female. The adminis t r a t i o n of testosterone in the presence or absence of the concurrent administration of pergoiide was also without e f f e c t i n the sham-operated female. However, sim i l a r to the previous r e s u l t s , testosterone produced an increase i n AHH a c t i v i t y i n the gonadectomized female. Of i n t e r e s t was the pergoiide i n h i b i t i o n of the testosterone induced increase of AHH a c t i v i t y i n the pre-pubescently gonadectomized female (Table 21). This e f f e c t was also observed in females gonadectomized as adults (Table 20) r e c e i v i n g pergoiide at maturity. With respect to A4 reductase a c t i v i t y , both pergoiide and testosterone i n d i v i d u a l l y or in combination were without effect in the s ham- o p e r a t ed female ^  (Table : 21)., ' The'-reduction" produced by testo-sterone in pre-pubescently gonadectomized females was s i m i l a r to that observed i n the previous study following adult gonadectomy. Furthermore, t h i s decrease was not affected by concurrent administration of pergoiide i n 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 manner 81 TABLE 21 The e f f e c t of pre-pubescent gonadectomy and p r o l a c t i n depletion on the action of testosterone on hepatic AHH and testosterone A4 reductase a c t i v i t i e s i n the adult female r a t . Immature female r a t s were gonadectomized (Gx) or sham-operated between days 25 and 30 of l i f e . Where indicated r a t s received pergolide (Perg ) (0.1 mg/kg/day s.c. i n corn o i l ) between days 25 to 60 of l i f e . In a ddition, testosterone enanthate (Test) was administered (1.0'mg/kg/day s.c. i n corn o i l ) for 10 days between days 50 to 60 of l i f e . Animals were s a c r i f i c e d 24 hours a f t e r the l a s t i n j e c t i o n . Differences between groups were considered s i g n i f i c a n t at p < 0.05 using the Newman-Keuls multiple range t e s t . S i g n i f i c a n t differences between groups were indicated by common l e t t e r s . Female Model AHH A c t i v i t y (pmol/min/mg protein) ± S.E.M., (n) A4 Red. A c t i v i t y (nmol/min/mg protein) ± S.E.M., (n) Sham Sham + Perg Gx Gx + Perg 666.3 ± 7.3 (3) 537.5 ± 24.3* (4) 576.2 ± 57.4C (5) 449.6 ± 14.2£ (5) 33.8 ± 1.5 (3) 33.7 ± 0.9 (4) ag bh 33.4 ± 2.1 (5) 41.0 ± 1.5 (5) ek f l Sham + Test Gx + Test, Sham + Test + Perg Gx + Test + Perg 709.0 ± 83.5 (4) 2567.8 ± 263.2 (5) abcdef 744.0 ± 22.5 (5) f , 1508.6 ± 259.1' (5) abcdef 31.4 ± 2.5 (4) 14.9 ± 1.3 (5) 37.2 ± 1.7 (5) 22.6 ± 2.V (5) cx abcdef ghij k l 82 (Table 21). i i i ) E f f e c t of pimozide i n adult male rats A second approach to the involvement of PRL and a c o r o l l a r y to the pergoiide studies investigated the e f f e c t s of elevating PRL i n male r a t s . Adult sham-operated or gonadectomized male ra t s were treated with pimozide to produce an increase in PRL l e v e l s . Pimozide, a dopamine antagonist has been shown to increase serum PRL as well as stimulate PRL synthesis i n male and female r a t s (Ojeda et a l . , 1974; Maurer and Gorski, 1977). The r a t i o n a l e was that an elevation in PRL would be postulated to reduce AHH a c t i v i t y and increase A4 reductase a c t i v i t i e s and/or reduce the response of t h e s e - a c t i v i t i e s to testosterone r e l a t i v e to c o n t r o l . To a s c e r t a i n the e f f e c t s of elevated PRL, hepatic AHH and A4 reductase a c t i v i t i e s were determined following administration of pimozide (0.6 mg/kg/day, subcutaneously) for ten days. In addition, the response to testosterone was also determined i n pimozide treated animals. The r e s u l t s of t h i s study are shown in Table 22. Gonadectomy of the/male res u l t e d i n a testosterone r e v e r s i b l e decrease in AHH a c t i v i t y . However, administration of pimozide did not a l t e r AHH a c t i v i t y i n sham-operated\or gonadectomized males. Nor did pimozide influence the e f f e c t of testosterone in either sham-operated or gonadectomized male r a t s (Table 22). Testosterone A4 reductase, normally low i n the adult male,was increased following gonadectomy. This increase was i n h i b i t e d by the administration of testosterone. 83 TABLE 22 The e f f e c t of gonadectomy, pimozide and testosterone on hepatic AHH and testosterone A4 reductase a c t i v i t y i n adult male r a t s . Adult male ra t s were gonadectomized (Gx) or sham-operated 11 days p r i o r to s a c r i f i c e . Where indicated animals received pimozide (Pirn ) (0.6 mg/kg/day i n t a r t a r i c acid) for 10 days or testosterone enanthate (Test ) (1.0 mg/kg/day i n corn o i l ) f o r 10 days p r i o r to s a c r i f i c e . Animals were s a c r i f i c e d 24 hours a f t e r the l a s t i n j e c t i o n . Differences between groups were considered s i g n i f i c a n t at p < 0.05 using the Newman-Keuls multiple range t e s t . S i g n i f i c a n t differences between groups were indicated by common l e t t e r s . Male Model AHH A c t i v i t y (pmol/min/mg protein) ± S.E.M., (n) A4 Red. A c t i v i t y (nmol/min/mg protein ± S.E.M., (n) Sham Sham + Pirn Gx Gx + Pirn Sham + Test Gx + Test Sham + Test. + Pirn Gx + Test + Pirn, 3971.5 ± 205.0 (4) f h 3848.2 ± 271.5 (5) 1107.2 ± 95.7 (5) eg cdgh 761.4 ± 100.2 (5) 4147.5 ± 236.5 abef j l (4) 3478.6 ± 243.3 (5) 4435.8 ± 193.6 (5) bdkl i k 3647.8 ± 209.7 a c l : j (5) 13.9 ± 1.2 (4) 18.7 ± l . l a c f g i (5) 28.1 ± 1 . 0 a c s i (5) 29.9 ± 0 . 6 b d e f h j (5) gh 13.3 ± 0.9 (4) 11.5 ± 0.5 (5) 10.5 ± 1.1 (5) 15.5 ± 1.0' (5) cd ab ace 84 In addition, pimozide was found to be i n h i b i t o r y to the actions of testosterone in the gonadectomized male (Table 22). Furthermore, pimozide while having no e f f e c t s alone in the gonadectomized male, produced an increase i n A4 reductase a c t i v i t y i n the sham-operated male. Although t h i s increase was not as great as that produced by gonadectomy i t could be reversed by testosterone. Testosterone, by i t s e l f , had no e f f e c t on A4 reductase a c t i v i t y in the sham-operated male. 4) Studies r e l a t i n g hepatic AHH a c t i v i t y to hepatic c y t o s o l i c estrogen binding components a) E f f e c t of pergoiide In the following studies hepatic AHH a c t i v i t y and hepatic c y t o s o l i c estrogen binding c h a r a c t e r i s t i c s were determined con-currently. The binding studies were conducted on a t i s s u e pool comprised of the treatment group, whereas hepatic AHH a c t i v i t y was determined for the i n d i v i d u a l animals in the treatment group. In an e f f o r t to detect a moderate a f f i n i t y component i n a non-hypophysectomized female, pergoiide was administered to reduce PRL l e v e l s as previously described, to sham-operated and gonadectomized females i n the presence or absence of testosterone. However, as shown in Table 23, t h i s regimen did not r e s u l t i n the detection of a moderate a f f i n i t y component. Pergoiide increased the apparent Kd of the estrogen receptor i n the shamr-ope rated female, and reduced the capacity of the estrogen receptor in the gonadectomized animal. TABLE 23 The e f f e c t of pergolide and testosterone on hepatic AHH a c t i v i t y and the apparent Kd and capacity of the hepatic c y t o s o l i c estrogen receptor and moderate a f f i n i t y component in adult gonadectomized and sham-operated female r a t s . Adult female r a t s were gonadectomized (Gx) or sham-operated 11 days p r i o r to s a c r i f i c e . Where indicated animals received testosterone enanthate (Test ) (1.0 mg/kg/day s.c. in corn o i l ) f o r 10 days or pergolide (Perg ) (0.1 mg/kg/day s.c. in corn o i l ) for 11 days p r i o r to s a c r i f i c e . Animals were s a c r i f i c e d 24 hours a f t e r the l a s t i n j e c t i o n . Differences were considered s i g n i f i c a n t at p < 0.05 using the Newman-Keuls multiple range te s t ; none were found. Tissue from each experimental group was pooled for binding assays. Model Estrogen Receptor Moderate A f f i n i t y Component Kd Capacity Kd Capacity AHH A c t i v i t y ( x l O 1 0 M) (fmol/mg protein) (xlO 8 M) (pmol/mg) (pmol/min/mg protein) protein + s.E.M. (n=4) Sham 8.1 487 0 0 196 + 15 Gx 7.5 490 0 0 203 + 18 Sham + Test 7.9 414 0 0 231 + 30 Gx + Test 5.5 362 0 0 186 + 22 Sham + Perg 15.3 445 0 0 211 + 34 Gx + Perg 5.5 272 0 0 322 + 85 Sham + Test + Perg 8.9 341 0 0 285 + 85 Gx + Test + Perg 6.2 235 0 0 270 + 22 86 These r e s u l t s support the d i r e c t r e l a t i o n s h i p between PRL, estrogens and hepatic estrogen receptor l e v e l s . We did not observe any pergolide i n h i b i t o r y e f f e c t on the action of testosterone to increase AHH a c t i v i t y as testosterone i t s e l f was without e f f e c t . b) E f f e c t of pimozide The e f f e c t ; of pimozide on the testosterone r e s p o n s i v i t y of AHH a c t i v i t y was also studied. The r e s u l t s .of t h i s study are summarized i n Table 24. Adult sham-operated and gonadectomized animals were treated with pimozide as previously described to increase serum PRL. Pimozide produced a s l i g h t increase in the capacity of the estrogen receptor which i s consistent with PRL regulation of hepatic estrogen binding. A s l i g h t increase i n apparent Kd was also observed. These increases were reversed by testosterone. However, as with the changes noted in capacity of the estrogen receptor following pergolide, i t should be emphasized that these d i f f e r e n c e s are within experimental v a r i a t i o n . Thus, t h e i r s i g n i f i c a n c e i s unknown. Pimozide did not a f f e c t the binding c h a r a c t e r i s t i c s of the moderate a f f i n i t y component in the sham^ -operated male. Following gonadectomy an increase in the capacity of the estrogen receptor was observed which was reversed following testosterone administration (Table 24). Furthermore,pimozide did not a f f e c t the apparent Kd or capacity of the estrogen receptor i n the gonadectomized male or the gonadectomized male re c e i v i n g testosterone r e l a t i v e to t h e i r respective controls. However, administration of pimozide abolished the moderate a f f i n i t y binding TABLE 24 The e f f e c t of pimozide and testosterone on hepatic estrogen binding and hepatic AHH a c t i v i t y i n sham and gonadectomized male r a t s . Adult male rats were gonadectomized (Gx) or sham-operated 11 days p r i o r to s a c r i f i c e . Where indicated animals received pimozide (Pirn) (0.6 mg/kg/day s.c. i n propylene glycol) or testosterone enanthate (Test-) (1.0 mg/kg/day s.c. i n corn o i l ) f or 10 days p r i o r to s a c r i f i c e . Animals were s a c r i f i c e d 24 hours a f t e r the l a s t i n j e c t i o n . Differences were considered s i g n i f i c a n t at p < 0.05 using the Newman-Keuls multiple range t e s t . S i g n i f i c a n t differences between groups were indicated by common l e t t e r s . Tissue from each group was pooled for binding assays. Model Estrogen Receptor (Ammonium Sulfate Fraction) Moderate A f f i n i t y Component (Whole Cytosol Fraction) AHH A c t i v i t y (pmol/min/mg protein) K d Capacity ( x l O 1 0 M) (fmol/mg protein) Kd Capacity (xlO^ M) (pmol/mg protein) iS.E.M., (n) Sham 7.5 274 5.3 10.3 689 ± 1 0 7 C 1 (3) Sham + Pirn 10.3 395 4.5 8.9 617 ± 64 dJ Sham + Test• 6.6 271 5.9 8.2 687 ± 1 1 7 e k (4) 652 ± 6 9 ^ Sham + Test - + Pirn 6.0 241 6.7 11.5 Gx 8.8 455 5.2 2.5 (4) 413 ± 4 3 a b c d e f Gx + Pirn 7.7 507 0 0 (4) 302 + 3 5 S h i j k l (4) Gx + Test 9.9 359 7.1 13.2 757 ± 18 a§ (4) 560 ± 1 0 5 b h (4) Gx + Test + Pirn 5.8 309 6.7 4.1 88 component i n the gonadectomized male, and i n h i b i t e d the action of testosterone i n r e s t o r i n g the capacity of t h i s component following gonadectomy (Table 24). These changes were not r e f l e c t e d by s i m i l a r changes in AHH a c t i v i t y ; although the trends i n AHH a c t i v i t y are s i m i l a r to those seen in Table 27. c) E f f e c t of continuous i n f u s i o n of p i t u i t a r y hormones The e f f e c t of continuous i n f u s i o n of p i t u i t a r y hormones on hepatic AHH a c t i v i t y and hepatic c y t o s o l i c estrogen binding components i s summarized i n Table 25. P i t u i t a r y hormones were administered by continuous infusion using Alzet model 2002 osmotic minipumps. Administration of bGH (0.01 I.U./hr for ten days) to shamnoperated males did not a l t e r binding c h a r a c t e r i s t i c s of the estrogen receptor or the moderate a f f i n i t y component (Table 25). However, a decrease i n AHH a c t i v i t y was observed (Table 25). Similar e f f e c t s have been previously described by Wilson (1969) and Colby (1980). The e f f e c t s of oPRL were also examined since the bGH used i n t h i s study contained 62% lactogenic a c t i v i t y . Ovine PRL, l i k e bGH, did not a l t e r the apparent Kd or capacity of the estrogen receptor or moderate a f f i n i t y component, but did produce a decrease i n AHH a c t i v i t y i n the sham-operated male (Table 25). As described e a r l i e r (and i l l u s t r a t e d i n Table 25), hypo-physectomy reduced moderate a f f i n i t y binding and abolished the estrogen receptor in the male. Following continuous infusion of bGH a further decrease in AHH a c t i v i t y was observed in the hypo-TABLE 25 Comparison of the effect of hypophysectomy and hormone replacement on hepatic cytosolic estrogen binding components and AHH activity in male and female rats. Adult male and female rats were hypophysectomized (Hx) or sham-operated between 50 and 60 days of age. Where Indicated animals received bovine growth hormone (bGH) (0.01 I.U./hr); rat growth hormone (rGH) (0.017 I.U./hr); ovine prolactin (oPRL) (0.2 I.U./hr); and rat prolactin (rPRL) (0.2 I.U./hr) in saline by continuous infusion for 10 days using osmotic minipumps or testosterone enanthate (Test) (1.0 mg/kg/day s.c. in corn oil) for 10 days prior to sacrifice. Animals were sacrificed 24 hours after the last injection, six to eight weeks following hypophysectomy. Binding studies were done on a tissue pool from 2 animals. Estrogen Receptor: Moderate Affinity Component AHH Activity Ammonium Sulfate Fraction Whole Cytosol Fraction (pmol/min/mg protein S.E.M., (n=4) Kd Capacity Kd Capacity (xlO 1 0 M) (fmol/mg protein) (xlO M) (pmol/mg protein) Sham Male 7.8 263 14.3 17.9 1568 ± 94 6.6 234 6.0 11.5 adghijklmn Hx Male 0 0 4.4 4.0 399 ± 32 0 0 3.0 5.2 cf ghijklm Sham Male + bGH 12.0 269 5.3 11.3 833 ± 43 7.8 226 4.8 15.0 adghijklmn Sham Male + oPRL 8.2 220 3.3 12.5 642 ± 62 10.6 289 3.9 19.4 adghijklmn Hx Male + rGH 0 0 0 0 218 ± 18 0 0 0 0 g Hx Male + bGH 0 0 4.9 5.9 163 ± 13 0 0 3.1 2.4 def Hx Male + rPRL 0 0 4.4 5.1 302 ± 34 0 0 7.7 6.1 ad Hx Male + bGH + Test 0 0 2.9 3.3 242 + 35 0 0 4.7 1.5 1 Hx Male + oPRL + Test 0 0 4.2 4.1 227 ± 29 0 0 10.1 3.3 j oo cont Sham Female 8.2 330 0 0 233 + 9 8.7 448 0 0 h ® Hx Female 0 0 3.7 1.6 312 + 30 0 0 1.7 1.5 ben @ Hx Female + rGH 0 0 0 0 232 + 24 @ 0 0 0 0 k ® Hx Female + rPRL 0 0 * * 225 + 30 0 0 * * 1 © Hx Female + bGH + Test 0 0 2.8 0.6 162 + 22 0 0 3.8 0.4 abc © Hx Female + oPRL + Test 0 0 8.3 1.9 255 + 28 0 0 5.7 1.4 m © * Although negative relationship detected between bound/free v.s. bound, correlation coefficient of linear regression too low to predict slope or intercept. Differences between groups were considered significant at p < 0.05 using the Duncan multiple-range test. Significant differences between groups were indicated by common letters. Circled letters indicate separate Duncan analysis on female model. 91 physectomized male. It i s • of i n t e r e s t that when rGH was administered continuously (0.017 I.U./hr) not only was AHH a c t i v i t y reduced but the moderate a f f i n i t y binding component was abolished i n the hypo-physectomized male. Furthermore, the AHH a c t i v i t y produced by growth hormone (bGH or rGH) i n the hypophysectomized male was the only instance when male AHH a c t i v i t y was completely reduced to cont r o l female l e v e l s . It i s also important to note that neither bGH or rGH were able to restore the estrogen receptor binding s i t e abolished by hypophysectomy. To delineate between the somato- and l a c t o t r o p i c e f f e c t s of growth hormone, rPRL was also used.. Following infusion of rPRL' no e f f e c t s • were observed on estrogen receptor or moderate, a f f i n i t y binding,-'or AHH a c t i v i t y i n the hypophysectomized male. In the female hypophysectomy has been shown to r e s u l t i n a lo s s of the high a f f i n i t y component as i s seen in the male. In addition, following hypophysectomy the moderate a f f i n i t y component (not normally present) was detected i n the female. Furthermore, normally accompanying the appearance of the moderate a f f i n i t y component i s an increase i n AHH a c t i v i t y . These e f f e c t s have been described i n previous studies. However, the increase i n AHH a c t i v i t y was s i g n i f i c a n t only when the females were considered separately from the males. The administration of rGH to the hypophysectomized female abolished the moderate a f f i n i t y component produced by hypophysectomy. The l o s s of moderate a f f i n i t y binding was accompanied by a decrease i n AHH a c t i v i t y to a l e v e l s i m i l a r to the sham-operated female. 92 rPRL (0.2 I.U./hr) did not produce the same consistent e f f e c t i n r e l a t i o n to AHH a c t i v i t y as did rGH. Continuous infusion of rPRL i n the hypophysectomized male did not a f f e c t the changes produced by hypophysectomy on the apparent Kd and capacity of the moderate a f f i n i t y component, or AHH a c t i v i t y (Table 25). However, rPRL did reduce AHH a c t i v i t y of the hypophysectomized female to a sham value, but did not e n t i r e l y remove moderate a f f i n i t y binding. S p e c i f i c a l l y , displaceable binding was s t i l l present but a Scatchard plot could not be generated. Similar to growth hormone, rPRL did not restore the high a f f i n i t y binding s i t e which was abolished by hypophysectomy. Testosterone enanthate (1.0 mg/kg/day, subcutaneously for ten days) was administered to examine the re s p o n s i v i t y of the hypophysectomized rat i n the presence of somatotropic or l a c t o -t r o p i c hormones. Administration of testosterone to hypophysecto-mized males r e c e i v i n g either bGH or oPRL did not produce any marked changes i n binding c h a r a c t e r i s t i c s or AHH a c t i v i t y (Table 25). Nor was testosterone able to induce AHH a c t i v i t y i n the hypophysecto-mized female r e c e i v i n g bGH or oPRL. The combination of either bGH or oPRL with testosterone had no r e s t o r a t i v e action on the high a f f i n i t y s i t e i n e i t h e r hypophysectomized males or females. 9 3 5) In vivo and i n v i t r o e f f e c t s of xenobiotics on hepatic AHH a c t i v i t y and c y t o s o l i c estrogen binding parameters a) Induction Since induction of AHH a c t i v i t y by testosterone in the gonadectomized male i s associated with an increase in moderate a f f i n i t y binding, the e f f e c t of other inducing agents both i n vivo 3 and i n v i t r o on the binding of [ H ] - e s t r a d i o l to the hepatic c y t o s o l i c estrogen receptor and moderate a f f i n i t y component was investigated. The e f f e c t s of 3-MC and spironolactone on estrogen binding parameters are i l l u s t r a t e d i n Table 26. In addition, the 3 effectiveness of 3-MC as a competitor for [ H ] - e s t r a d i o l binding to these components was also investigated. Administration of 3-MC (20 mg/kg/day, i n t r a p e r i t o n e a l l y ) for two days resulted in a decrease i n the capacity of the estrogen receptor in male and female r a t s with no observable e f f e c t on the apparent Kd for either sex. Spironolactone (100 mg/kg/day, i n t r a p e r i t o n e a l l y ) for four days reduced the capacity of the estrogen receptor in the female but not i n the male. 3 When 3-MC was employed as a competitor for [ H ] - e s t r a d i o l binding, the apparent Kd was s i m i l a r to that obtained using unlabeled e s t r a d i o l as a competitor in control male and female r a t s . This s i m i l a r i t y was noted in males following induction by 3-MC as w e l l . However, a Scatchard could not be generated i n the female following induction by 3-MC and using 3-MC as a competitor 3 for [ H ] ^ e s t r a d i o l binding to the estrogen receptor. This discrepancy was not observed following the administration of spironolactone to the female. However, the apparent Kd obtained 94 by 3-MC competition i n spironolactone treated males and females was somewhat lower than those values derived by estradiol" competition (Table 26). The capacities generated following 3-MC competition were generally one-half those obtained with unlabeled e s t r a d i o l . However, following treatment with spironolactone an approximate four f o l d decrease was detected with 3-MC. I n t e r e s t i n g l y , these differences i n apparent Kd and capacity between 3-MC and e s t r a d i o l competition were not seen for the male moderate a f f i n i t y component. Of greater interest were the e f f e c t s of in vivo administration of 3-MC and spironolactone, which produced an increase i n s p e c i f i c binding to the moderate a f f i n i t y component. This was ureflected as an increase in capacity, e s p e c i a l l y evident following treatment with 3-MC (Table 26). However, neither 3-MC nor spironolactone t r e a t -ment r e s u l t e d i n detection of a moderate a f f i n i t y binding component in the female. In a r e l a t e d study the e f f e c t s of phenobarbital (80 mg/kg/day, i n t r a p e r i t o n e a l l y for four days), on estrogen receptor and moderate a f f i n i t y binding components were investigated. In t h i s study unlabeled e s t r a d i o l or BP were used as competitors 3 for [ H ] - e s t r a d i o l binding. The r e s u l t s , reported i n Table 27, indicated small decreases i n the capacity and the apparent Kd of the estrogen receptor i n both male and female r a t s following administration of phenobarbital, using e s t r a d i o l as a competitor. In a l l cases, lower estrogen receptor c a p a c i t i e s were observed when t h i s parameter was measured using BP as a competitor. Further-i more, unlike 3-MC, BP competition .produced a-Scatchard p l o t for the TABLE 26 The effect of 3-inethylcholanthrene and spironolactone on the apparent Kd and capacity of the hepatic cytosolic estrogen receptor and the moderate affinity binding component in male and female rats. Where indicated adult male and female rats received either 3-methylcholanthrene (3-MC) (20 mg/kg/day i.p. in corn oil) for 2 days or spironolactone (Spir ) (100 mg/kg/day l.p. in corn oil) for 4 days prior to sacrifice. Animals were sacrificed 24 hours following the last injection. Binding parameters were determined using either a 100 fold excess of unlabeled estradiol (E2) or a 1000 fold excess of unlabeled 3-methylcholanthrene (3-MC) as competitors for [-*H]-estradiol binding. Competitors are indicated in parenthesis. Each group represents a tissue pool from 4 animals. Model: (Competitor) Estrogen Receptor Moderate Affinity Component (Ammonium Sulfate Fraction) (Whole Cytosol Fraction) "7- Capacity Capacity ( x l 0 l u M) (fraol/mg protein) (xlO 8 M) (pmol/mg protein) Male (E2) 9.1 352 6.25 9.47 Male (3-MC) 8.9 149 4.55 6.5 3-MC Male (E2) 8.7 206 7.87 17.2 3-MC Male (3-MC) 7.2 113 7.02 12.5 Spir. Male (E2) 13.9 290 6.25 16.2 Spir. Male (3-MC) 5.9 75 5.84 12.0 Female (E2) 7.8 437 0 0 Female (3-MC) 8.8 202 0 0 3-MC Female (E2) 7.0 228 0 0 3-MC Female (3-MC) No Scatchard 0 0 Spir. Female (E2) 9.1 232 0 0 Spir. Female (3-MC) 5.5 38 0 0 I TABLE 27 The e f f e c t of 4 days treatment with phenobarbital on the apparent Kd and capacity of the hepatic c y t o s o l i c estrogen receptor and moderate a f f i n i t y binding component i n male and female r a t s . Where indicated adult male and female r a t s received phenobarbital (Phenobarb) (80 mg/kg/day i. p . i n saline) f o r 4 days p r i o r to s a c r i f i c e . Animals were s a c r i f i c e d 24 hours af t e r the l a s t i n j e c t i o n . Binding parameters were determined using e i t h e r a 100 f o l d excess of unlabeled e s t r a d i o l (E2) or a 1000 f o l d excess of benzo(a)pyrene (BP) as competitors for [ 3H]-estradiol binding. A. tis s u e pool from 4 animals was used for eacb/fg Model: Competitor Estrogen Receptor Moderate A f f i n i t y Component (Ammonium Sulfate Fraction) (Whole Cytosol Fraction) K q Capacity K d Capacity (xlO M) (fmol/mg protein) (xl0 yM) (pmol/mg protein) . VD — — — _ ^ Male (E2) 2.25 549 6.99 6.2 Male (BP) 0.98 190 3.15 3.0 Phenobarb Male (E2) 1.11 198 5.19 11.3 Phenobarb Male (BP) 0.88 97 4.06 6.4 Female (E2) 2.17 496 0 0 Female (BP) 0.67 115 0 0 Phenobarb Female (E2) 1.13 278 0 0 Phenobarb Female (BP) 0.73 91 0 0 97 estrogen receptor i n the female. Competition of [ H ] - e s t r a d i o l binding to the estrogen receptor by BP resulted i n lower c a p a c i t i e s than those obtained by e s t r a d i o l competition i n control males and females. Although treatment with phenobarbital d i d reduce the apparent Kd of the estrogen receptor by e s t r a d i o l competition, t h i s reduction was not seen using BP as a competitor. Similar to 3-MC, phenobarbital increased the capacity of the male moderate a f f i n i t y component. Benzo(a)pyrene was an e f f e c t i v e 3 competitor for [ H ] - e s t r a d i o l binding to the moderate a f f i n i t y component as w e l l . However, unlike 3-MC, the capacity that resulted from BP competition was approximately one-half that obtained using e s t r a d i o l as a competitor. As with the estrogen receptor, the apparent Kd of the moderate a f f i n i t y component was reduced i n the co n t r o l male when BP was used instead of e s t r a d i o l to compete for 3 I H ] - e s t r a d i o l binding. However, as with the estrogen receptor, t h i s difference was l o s t following phenobarbital treatment. These changes were small, and t h e i r p h y s i o l o g i c a l s i g n i f i c a n c e i s unclear, Administration of phenobarbital did not produce any detectable moderate a f f i n i t y component i n the female (Table 27). To evaluate the duration of induction, 3-MC and phenobarbital were administered to male and female rats for ten days. In addition, the e f f e c t of a synthetic estrogen, mestranol, was also examined. The e f f e c t of phenobarbital (80 mg/kg/day, i n t r a p e r i t o n e a l l y ) and 3-MC (20 mg/kg/day, i n t r a p e r i t o n e a l l y ) on hepatic AHH and estrogen binding are presented i n Table 28. Neither 3-MC nor phenobarbital treatment for ten days s i g n i f i c a n t l y a l t e r e d the binding c h a r a c t e r i s t i c s of the estrogen receptor. Furthermore, administration of phenobarbital 98 for t h i s time period did not a f f e c t the apparent Kd or capacity of the moderate a f f i n i t y component i n the male. However, following ten days of 3-MC, the capacity of the moderate a f f i n i t y component i n the male decreased amount 20 f o l d , with a near 50% reduction i n apparent Kd (Table 28). No moderate a f f i n i t y component was detected i n the female following long term administration of inducers. The e f f e c t of these agents on AHH a c t i v i t y was as expected. No change, or only s l i g h t i n h i b i t i o n , and several f o l d induction of AHH a c t i v i t y were seen following phenobarbital treatment of male and female rats r e s p e c t i v e l y . As expected, 3-MC induced AHH a c t i v i t y i n males and females to comparable l e v e l s (Table 28). b) I n h i b i t i o n As previously shown testosterone was e f f e c t i v e at reversing the decrease i n AHH a c t i v i t y and moderate a f f i n i t y binding in the male following gonadectomy. Estrogens are also known to reduce AHH a c t i v i t y (Gurtoo and Parker, 1979). The i n h i b i t o r y e f f e c t of estrogens on AHH a c t i v i t y i n the male was demonstrated following administration of mestranol (1.0 mg/kg/day subcutaneously for ten days) as shown i n Table 28. Mestranol d i d not produce any e f f e c t on AHH a c t i v i t y i n the female. Furthermore, mestranol abolished s p e c i f i c binding to the estrogen receptor i n males and females as the moderate a f f i n i t y component i n males. TABLE 28 The e f f e c t of 10 days treatment with mestranol, phenobarbital and 3-methylcholanthrene on hepatic c y t o s o l i c estrogen binding c h a r a c t e r i s t i c s and hepatic AHH a c t i v i t y i n male and female r a t s . Adult male and female ra t s received either mestranol (1.0 mg/kg/day s.c. i n corn o i l ) for 14 days, phenobarbital (Phenobarb) (80 mg/kg/day i.p. i n saline) for 10 days or 3-methylcholanthrene (3-MC) (20 mg/kg/day i.p. i n corn o i l ) for 10 days p r i o r to s a c r i f i c e . Animals were s a c r i f i c e d 24 hours a f t e r the l a s t i n j e c t i o n f or phenobarb and 48 hours a f t e r the l a s t i n j e c t i o n for mestranol and 3-MC. Tissue from 4 animals was pooled for each group. Differences between groups were considered s i g n i f i c a n t at p < 0.05 using the Newman-Keuls multiple range t e s t . S i g n i f i c a n t d ifferences were indicated- by common l e t t e r s . Model Estrogen Receptor (Ammonium Sulfate Fraction) Moderate A f f i n i t y Component (Whole Cytosol Fraction) AHH A c t i v i t y (pmol/min/mg protein) ± S.E.M., (n=4) Kd ( x l O 1 0 M) Capacity (fmol/mg protein) Kd (xlO 8 M) Capacity (pmol/mg protein) Control Male 5.1 199 14.2 19.8 801 ± 2 5 a c e i Mestranol Male 0 0 0 0 132 ± 1 6 e f Phenobarb Male 5.4 177 12.5 19.4 542 ± 2 5 a c e h 3-MC Male 9.4 153 7.4 0.9 1201 ± 6 7 a c e Control Female 5.3 145 0 0 99 ± l l a b Mestranol Female 0 0 0 0 124 ± 1 3 c d Phenobarb Female 7.5 217 0 0 405 ± 3 ? a c e g 3-MC Female 7.8 140 0 0 1290 ± g 3 b d f g h i 100 c) The e f f e c t of p h y s i o l o g i c a l manipulation on ^ n v i t r o binding of 3-MC To examine the ph y s i o l o g i c a l c o r r e l a t i o n of p o l y c y c l i c 3 aromatic hydrocarbon (PAH) competition for [ H ] - e s t r a d i o l binding, testosterone replacement i n gonadectomized r a t s was investigated. As shown i n Table 29, administration of testosterone increased the binding of the moderate a f f i n i t y component as determined by either 3-MC or e s t r a d i o l competition. Administration of t e s t o -sterone to the gonadectomized female, shown not to r e s u l t i n any e s t r a d i o l competed moderate a f f i n i t y binding, did not r e s u l t i n 3-MC competed binding e i t h e r . As indicated previously, 3-MC 3 competed for approximately one-half the [ H]- e s t r a d i o l binding to the estrogen receptor as did e s t r a d i o l . With the exception of the gonadectomized female r e c e i v i n g testosterone, 3-MC and e s t r a d i o l competition for binding to the estrogen receptor resulted i n s i m i l a r apparent Kd values (Table 29). d) Relationship of estrogen binding components to Ah-receptor 3 To probe any association to the Ah-receptor, [ H ] - e s t r a d i o l binding was competed with a 100 f o l d excess of tetrachlorodibenzo-p-dioxin (TCDD), 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD), or octachlorodibenzo-p-dioxin (OCDD). A l l three dioxin congeners 3 competed for [ H ] - e s t r a d i o l binding to the estrogen receptor i n males and females. However, only OCDD and 2,7-DCDD were e f f e c t i v e 3 competitors for [ H]-estradiol,binding to the moderate a f f i n i t y TABLE 29 The e f f e c t of gonadectomy and testosterone replacement on li g a n d s p e c i f i c i t y of the hepatic c y t o s o l i c estrogen receptor and moderate a f f i n i t y binding component in male and female r a t s . Adult male and female rats were gonadectomized (Gx) 11 days p r i o r to s a c r i f i c e . Where indicated animals received testosterone enanthate (Test ) (1.0 mg/kg/day s.c. i n corn o i l ) for 10 days. Animals were s a c r i f i c e d 24 hours a f t e r the l a s t i n j e c t i o n . Binding parameters were determined using a 100 fo l d excess of unlabeled e s t r a d i o l (E2) or a 1000 f o l d excess of unlabeled 3-methylcholanthrene (3-MC) as competitors for [ 3 H ] - e s t r a d i o l . Studies were done on ti s s u e pooled from 4 animals. Model: Competitor Estrogen Receptor Moderate A f f i n i t y Component (Ammonium Sulfate Fraction) (Whole Cytdsdl Fraction) K d Capacity K d Capacity (xlO 9 M) (fmol/mg protein) (xlO 8 M) (pmol/mg protein) Gx Male (E2) 1.22 566 5.67 3.1 Gx Male (3-MC) 1.06 271 5.36 2.3 Gx Male + Test (E2) 1.34 319 16.06 29.7 Gx Male + Test (3-MC) 0.95 189 5.4 8.4 Gx Female (E2) 1.54 496 0 0 Gx Female (3-MC) 1.12 201 0 0 Gx Female + Test (E2) 1.01 345 0 0 Gx Female + Test (3-MC) 0.59 161 0 0 102 component i n the male (Table 30). Furthermore, acceptable Scatchard plo t s could only be generated for the female estrogen receptor following competition with these dioxin congeners. The apparent Kd and capacity for a l l dioxin congeners were sim i l a r but lower than that obtained by e s t r a d i o l competition (Table 31). Unlike 3-MC or BP, s p e c i f i c binding that re s u l t e d i n good Scatchard analysis could not be produced with these dioxin congeners for the male estrogen receptor or moderate a f f i n i t y component, even though displaceable binding was observed (Table 30). A summary of c o n t r o l apparent Kd values and ca p a c i t i e s for the adult female and male estrogen receptor and male moderate a f f i n i t y component are presented i n Table 32. 1 0 3 TABLE 30 Ligand s p e c i f i c y of the hepatic c y t o s o l i c estrogen receptor and the moderate a f f i n i t y s i t e for various competitors of [ 3 H ] - e s t r a d i o l binding i n the male and female r a t . Unless otherwise noted competitors were added i n a 100 f o l d excess to 0.5 nM [ 3 H ] - e s t r a d i o l i n the ammonium su l f a t e f r a c t i o n f or determination of competition f o r binding to the estrogen receptor, or to 50 nM p H ] - e s t r a d i o l in the whole cytosol f r a c t i o n f or determination of competition for binding to the moderate a f f i n i t y component. Incubations were c a r r i e d out for 90 minutes at 4 °C. Tissue pooled from 4 animals were used f o r each group. Percent of Tot a l [ H ] - e s t r a d i o l Bound Unlabeled Competitor Estrogen Receptor Moderate A f f i n i t y S i t e Female Male Male Control 100 100 100 E s t r a d i o l 47 54 66 TCDD OCDD 2,7-DCDD 59 T 75+ 72+ 70+ 100 58 67 * 3-MC BP* 57 66 62 .57-40 52 * 1000 f o l d excess unlabeled competitor t ligand concentration = 0 . 1 nM TCDD Tetrachlorodibenzo-p-dioxin OCDD Octachlorodibenzo-p-dioxin 2,7-DCDD 2,7-Dichlorodibenz o-p-d i o x i n 3-MC 3-Methylcholanthrene BP Benzo(a)pyrene 1 0 4 TABLE 31 The apparent Kd and capacity of the estrogen receptor i n adult female r a t s as determined with d i f f e r e n t dioxin eongenefs. A l l competitors were added in a 100 f o l d excess. Studies were conducted on t i s s u e pooled from 4 animals. Apparent Kd Capacity Competitor ( x l O 9 M) (fmol/mg protein) E s t r a d i o l 2.4 438 Tetrachlorodibenzo-p-dioxin 1.0 117 Octachlorodibenzo-p-dioxin 1.2 142 2,7-Dichlorodibenzo-p-dioxin 0.9 120 TABLE 32 Summary of control values for apparent Kd and capacity of the estrogen receptor and moderate a f f i n i t y component. Female Estrogen Recept or Male Estrog en Receptor Male Moderate A f f i n i t y Kd(M) Capacity Kd (M) Capacity Kd(M.) Capacity (fmol/mg protein) (fmol/mg protein) (pmol/mg protein' 1.1 x IO" 9 368 1.1 x 10" 9 261 14.3 x IO" 8 6.6 2.5 x 10" 9 506 2.2 x 10" 9 516 14.0 x IO" 8 31.4 3.7 x 10" 9 641 2.8 x 10~ 9 620 6.4 x IO" 8 11.0 0.82 x 10" 9 188 1.05 x 10" 9 555 9.0 x IO" 8 14.9 1.52 x 10" 9 477 1.11 x 10" 9 260 8.8 x IO" 8 19.3 8.1 x 1 0 " 1 0 487 1.9 x 10" 9 405 17.5 x IO" 8 15.0 2.07 x 10" 9 489 7.5 x 1 0 " 1 0 274 5.3 x IO" 8 10.3 8.2 x 1 0 " 1 0 ,330' 7.8 x 1 0 " 1 0 263 14.3 x IO" 8 17.9 8.7 x 1 0 " 1 0 448 6.6 x 1 0 " 1 0 234 6.0 x IO" 8 11.5 9.6 x 1 0 " 1 0 337 8.4 x 10" 1 0 251 8.9 x IO" 8 19.6 5.3 x 10 145 5.1 x 10" 1 0 199 14.2 x IO" 8 19.8 7.8 x 1 0 " 1 0 437 9.1 x I O " 1 0 352 6.25x 10 8 9.47 2.17 x 10" 9 496 2.25x 10" 9 549 6.99x 10" 8 6.2 1 .43 ± 0.25xl0~ 9 411.5 ± 37.7 1.29 ± 0.20xl0" 9 341.5 ± 38.1 10.15 ± 1.14x10" ! 8 14.8 ± 1.9 (3 .7 - 0.53xl0- 9) (145 - 641) (2.8 - 0.5] x ,10"9) (199 - ( 520) (14.3 • - 5.3xl0 _ £ S) (6.2 - 31.4) 106 DISCUSSION 1) Estrogen binding studies: characterization and comparison of hepatic c y t o s o l i c estrogen binding components with previously published r e s u l t s I n i t i a l c h aracterization of the hepatic c y t o s o l i c binding proteins provided data s i m i l a r to that reported by other investigators in t h i s area. The developmental studies established an optimal incubation time of ninety minutes at 4°C for both the estrogen receptor and the moderate a f f i n i t y component. This time was long enough to allow for completion of ligand complex formation yet not so long as to observe exchange or degradation. These binding data were provided by Dr. B. Warren and are presented in Appendix 1, Figures A l , A2. A p a r a l l e l study presented in Table 3 examined the time course of binding at 25°C. Comparison of these temperature-time studies indicates that completion of ligand-complex formation occurred e a r l i e r at 25°C, than at 4°C, as expected. The 90 minute incubation time was used for subsequent studies and was comparable to times employed by other investigators (Eagon et a l . , 1980; Powell-Jones et^_al., 1980, 1981; and Sloop et a l . , 1983). However, unlike the previously c i t e d studies, molybdate was included i n the trisma-EDTA-dithiothreitol buffer system. Sodium molybdate has been shown to prevent receptor aggregation and increase s t a b i l i t y to heat a c t i v a -t i o n thus making the measurement of s p e c i f i c binding more probable (Moncharmont et a l . , 1982; Noma et a l . , 1980). In addition, 107 contrary to the methodology of Powell-Jones et a l . , (1980, 1981) and Sloop et a l . , (1983), exposure to DCC was reduced from t h i r t y to f i v e minutes to avoid s t r i p p i n g o f f bound ste r o i d as described by Peck and Clark (1977). Studies pertaining to ligand s p e c i f i c i t y of the estrogen receptor and the moderate a f f i n i t y protein were examined for comparison with published c h a r a c t e r i s t i c s to confirm the i d e n t i t y of these binding components. These competitor studies indicated 3 that estrogens were e f f e c t i v e i n h i b i t o r s of [ H ] - e s t r a d i o l binding in v i t r o i n male (Table 6) and female (Table A2, Appendix 1) r a t s . However, as reported by Eagon et a l . , (1980), Powell-Jones et a l . , (1980, 1981), and Sloop et a l . , (1983) DES does not compete for binding to the moderate a f f i n i t y component. Similar r e s u l t s are reported i n Table 5. Furthermore, androgens, which do not compete for binding to the estrogen receptor (Table 6; Table A2, Appendix 1) 3 are e f f e c t i v e i n h i b i t o r s of [ H ] - e s t r a d i o l binding in v i t r o to the moderate a f f i n i t y component i n male whole cytosol (Table 6). 3 Other s t e r o i d hormones are not e f f e c t i v e competitors for [ H ] - e s t r a d i o l binding to the estrogen receptor or the moderate a f f i n i t y component. A l l of these r e s u l t s are in agreement with the previously c i t e d i n v e s t i g a t o r s . It was concluded,therefore,that the 50% ammonium sulf a t e f r a c t i o n i n both male and female r a t s contained the estrogen receptor and the male whole cytosol f r a c t i o n contained the moderate a f f i n i t y component as defined by previous reports (Aten et al.,1978; Dickson et a l . , 1978; Eagon et a l . , 1980; Powell-Jones et a l . , 1980, 1981; Sloop et a l . , 1983). 108 Further confirmation of the i d e n t i t y of the hepatic c y t o s o l i c binding components involved examination of e s t r a d i o l binding in d i f f e r e n t p h y s i o l o g i c a l models. In so doing, p h y s i o l o g i c a l relevance as well as the v a l i d i t y of the binding assays were established. Previous studies have demonstrated that the presence of the moderate a f f i n i t y s i t e was age and sex-dependent (Eagon et a l . , 1980; Powell-Jones et a l . , 1980, 1981; Sloop et a l . , 1983). Furthermore, Powell-Jones et a l . , (1981) have reported that no ontogenic difference can be demonstrated for the estrogen receptor in r a t l i v e r c y t o s o l . The sex-dependency of the moderate a f f i n i t y component as well as the non-sex-dependency of the estrogen receptor has been demon-strated i n the course of our assay development. There are, however, c e r t a i n minor discrepancies between previous reports and the r e s u l t s presented here. Powell-Jones et a l . , (1980, 1981) describe the presence of small amounts of the 4S protein i n adult female, and pre-pubescent male and female r a t s . However, i t i s incorrect to assume that the 4S peak of the immature animal or adult female i s the moderate a f f i n i t y component as these authors suggest without further supporting evidence. Eagon et a l . , (1980) have demonstrated that a sex d i f f e r e n c e i s present for not only the capacity but the ligand s p e c i f i c i t y of the moderate a f f i n i t y component. Unlike Powell-Jones et a l . , (1980,1981) Eagon's group have conducted competitive i n h i b i t i o n studies on the 4S protein in both male and female c y t o s o l . They have demonstrated that binding to the 4S protein can be p a r t i a l l y i n h i b i t e d by e s t r a d i o l but not DES in the male. However,, in the female whole cytosol f r a c t i o n , both e s t r a d i o l and 3 DES i n h i b i t e d - [ H ] - e s t r a d i o l binding to the 4S peak. Therefore, 109 the 4S peak i n the female was probably representative of the 4S species of the estrogen receptor and not the moderate a f f i n i t y component. These r e s u l t s are consistent with observations presented here. That i s , no moderate a f f i n i t y binding component could be detected in the adult female or i n immature male or female r a t hepatic cytosol (Tables 7-9, 11, 12, 23, 25-28). Thus, in general, the trends observed for the sucrose gradient studies which have characterized these estrogen binding proteins were reproducible by Scatchard a n a l y s i s . Moreover, the binding components described by previous workers possessed s i m i l a r c h a r a c t e r i s t i c s to those we have demonstrated. Eagon et a l . , (1980) reported a sex difference in the quantity of the estrogen receptor present in the adult. Following 3 the incubation of 2 nM [ H ] - e s t r a d i o l , sucrose density gradient separation revealed 70% of t o t a l r a d i o a c t i v i t y bound to the 8-9S region i n the female, but only 15% bound to the 8-9S peak i n the male. Powell-Jones et a l . , (1980) have also demonstrated a sex difference i n binding to the 8-9S region. They found that the 3 8-9S region was saturated at 4 nM [ H ] - e s t r a d i o l i n the female, 3 but that concentrations in excess of 16 nM [ H ] - e s t r a d i o l were required to saturate the 8-9S protein i n the male. At ligand concentrations greater than 16 nM, no sex difference in binding to the 8-9S region was observed. Therefore, both Eagon et a l . , (1980) and Powell-Jones et a l . , 3 (1980) reported the sex d i f f e r e n c e i n t o t a l [ H ] - e s t r a d i o l bound to the 8-9S region at lower ligand concentrations. However, when the ontogeny of the estrogen receptor was investigated, Powell-Jones 110 e t a l . , (1981) found no sex difference in specific binding of 4.0 nM 3 [ H]-estradiol between male and female rats using a 100 fold excess of DES as a competitor. Therefore, the sex difference cited by Eagon et a l . , (1980) and Powell-Jones et a l . , (1980) for the total 3 binding of lower concentrations of [ H]-estradiol to the 8-9S region was probably a result of ligand binding to the moderate a f f i n i t y component in the male. The results of the ontogeny study (Powell-Jones et a l . , 1981) indicated a concomitant time dependent increase in DES sensitive 3 [ H]-estradiol binding from day 20 to day 42. The most rapid increase occurred between days 30 and 40. After day 42 levels were reported to plateau, with no consistent sex differences being evident. However, examination of the results presented by Powell-Jones et a l . , (1981)showed that the concentration of estrogen receptor varied at least two fold after day 42. Therefore, the v a r i a b i l i t y between control animals that we have noted in Table 32 is not uncommon. The observation of Powell-Jones et a l . , (1981) that there is no sex difference in the ontogeny of the estrogen receptor was consistent with our findings. Scatchard analysis demonstrated that the capacities of the estrogen receptor in the immature male and female were equivalent, and one-half the capacity of the adult of either sex (Table 8). Furthermore, the apparent Kd of the adult and immature animals .was-- found to be comparable (Table 8). However, although the capacity of the adult male and female was similar, i t was noted that the capacity of the male was usually slightly less than that of the female. 2) E f f e c t s of p h y s i o l o g i c a l manipulation on hepatic estrogen binding a) Gonadectomy e f f e c t s on hepatic estrogen binding P h y s i o l o g i c a l manipulation of t h i s system has further confirmed the relevance of the moderate a f f i n i t y component. We have shown that gonadectomy of the male reduces the capacity of the moderate a f f i n i t y component with a s l i g h t concomitant increase i n the capacity of the estrogen receptor (Tables 8, 9,24). However, the increase in capacity of the estrogen receptor was not always seen, and may be within normal v a r i a b i l i t y . It i s of int e r e s t that Powell-Jones 3 et a l . , (1980,1981) observed an increase i n [ H ] - e s t r a d i o l binding to the 8-9S region following gonadectomy which was consistent with our r e s u l t s . These data are i n agreement with sucrose density gradient a n a l y s i s of male cytosol by Eagon et a l . , (1980), who demonstrated a testosterone r e v e r s i b l e increase i n DES-sensitive 3 [ H ] - e s t r a d i o l binding to the 8-9S region following gonadectomy. As with the increase i n the capacity of the estrogen receptor, we did not con s i s t e n t l y observe a testosterone r e v e r s a l of a gona-dectomy induced increase i n capacity. Powell-Jones et a l . , (1980,1981) did not detect a decrease 3 i n [ H ] - e s t r a d i o l binding to the 4S region following gonadectomy of the male. This i s i n marked contrast to the consistent testosterone r e v e r s i b l e decrease we have noted f or the moderate a f f i n i t y component in the gonadectomized male (Tables 8, 9, 24). In t e r e s t i n g l y , Eagon et a l . , (1980) have shown that while gona-3 dectomy does not a f f e c t [ H ] - e s t r a d i o l binding to the 4S region 112 per se, i t does change the ligand s p e c i f i c i t y of the 4S protein. R e c a l l i n g that the control male 4S protein i s not DES-sensitive, t h i s group has demonstrated a 60% decrease i n [ H ] - e s t r a d i o l binding to the 4S protein i n the presence of a 100 f o l d excess of unlabeled. DES following gonadectomy. Normally, DES i s only competitive for [ H ] - e s t r a d i o l binding to the 8-9S protein (estrogen receptor) i n males and females and the 4S protein i n the female (Aten et a l . , 1978; Dickson et a l . , 1978; Eagon et a l . , 1980; E i s e n f e l d et a l . , 1977b; Powell-Jones et a l . , 1980, 1981). However, Eagon et a l (;1980) c l e a r l y •demonstrated .av'DESv-sensitive . binding to.the 4S protein i n the gonadectomized.male. In add i t i o n , following gel f i l t r a t i o n , Eagon and co-workers (1980) have shown the DES-insensitive, male s p e c i f i c 4S binding protein to have been reduced 16 f o l d . Furthermore, the e f f e c t s of gonadectomy on DES-s e n s i t i v e and - i n s e n s i t i v e binding i n the 4S region of male hepatic cytosol were shown to be DHT r e v e r s i b l e (Eagon et a l . , 1980). These observations support our findings that gonadectomy of the male r e s u l t s i n decreased capacity of the moderate a f f i n i t y component, which i s reversed by testosterone. The preceeding observations are of interest i n l i g h t of the recent report by Sloop and co-workers (1983) who were not 3 able to demonstrate DES competition for [ H ] - e s t r a d i o l in female or neonatally gonadectomized male whole cytosol f r a c t i o n s . Our r e s u l t s (Table 1) and those of Aten et a l . , (1978), Eagon et al..," (1980) and E i s e n f e l d et a l . , (1977), c l e a r l y demonstrated'''DES 3 i n h i b i t i o n of [ H]- e s t r a d i o l binding in female whole cytosol f r a c t i o n s . However, the study by Sloop et a l . , (1983) employed 113 a concentration of 30 nM [ H ] - e s t r a d i o l whereas the previously 3 c i t e d investigators used between 2 and 5 nM [ H ] - e s t r a d i o l . At the higher concentration of radio-labeled_ tracer the p o s s i b i l i t y e x i s t s that the competition f o r the estrogen receptor would be masked because a l l s i t e s would be saturated. Dickson et a l . , (1978) —6 3 have shown t h i s to be the case. At 1 x 10 M [ H ] - e s t r a d i o l i n female hepatic c y t o s o l , no reduction i n binding was observed using a 1000 f o l d excess of unlabeled e s t r a d i o l as a competitor. In male cytosol an 80% decrease i n binding was reported under these conditions. This i s in p a r t i a l agreement with data presented in Table 1. Similar to Dickson et a l . , (1978) we were unable to generate a Scatchard plot i n female whole cytosol using unlabeled e s t r a d i o l as a competitor at a 100 f o l d excess between 3 25 and 1000 nM [ H ] - e s t r a d i o l . Furthermore, a Scatchard plot was generated under s i m i l a r conditions i n the male. However, unlike Sloop et a l . , (1983), we observed displaceable binding and were able to produce a Scatchard plot using a 100 f o l d excess 3 of DES in female whole cytosol between 25 and 1000 nM [ H ] - e s t r a d i o l . Therefore the decrease i n moderate a f f i n i t y binding we observed i n the male following gonadectomy, not reported by other groups (Powell-Jones et a l . , 1980, 1981; Sloop et a l . , 1983), 3 may be the r e s u l t of the concentration of [ H ] - e s t r a d i o l used i n t h e i r sucrose gradient analysis studies. The higher concentration 3 of [ H ] - e s t r a d i o l may have saturated the DES-sensitive s i t e and masked the decrease in the DES-insensitive s i t e . This would r e s u l t i n no noticeable change in t o t a l binding to the 4S peak as the DES-sensitive protein would bind e s t r a d i o l as well as the male 114 s p e c i f i c 4S DES-insensitive protein. This must be the case 3 regardless of the concentration of [ H ] - e s t r a d i o l used as neither Eagon et a l . , (1980) nor Powell-Jones et a l . , (1980,1981) reported 3 gross changes in t o t a l binding of [ H ] - e s t r a d i o l to the 4S peak of sucrose gradients following gonadectomy. The gonadectomized male resembles the female with respect to binding parameters more so than the intact male. Therefore, i t i s possible that the DES-s e n s i t i v e 4S peak was saturated and could not be demonstrated at 3 30 nM [ H ] - e s t r a d i o l j u s t as the estrogen receptor could not be demonstrated at elevated tracer l e v e l s described above. This p o s s i b i l i t y becomes probable when one considers that the apparent Kd and capacity of the DES-sensitive 4S protein were reported to be 0.36 nM and 0.16 fmol/mg protein r e s p e c t i v e l y (Eagon et a l . , 1980). Therefore the discrepancy between our observation that gonadectomy reduces the capacity of the moderate a f f i n i t y component and those of Powell-Jones et a l . , (1980,1981) are possibly the r e s u l t of measuring only t o t a l binding i n sucrose density gradient a n a l y s i s . However, t h i s too was contrary to our r e s u l t s as we c o n s i s t e n t l y observed a decrease in t o t a l as well as s p e c i f i c 3 binding of [ H ] - e s t r a d i o l to the moderate a f f i n i t y component following gonadectomy of the adult male. i ) Imprinting, of the ^ moderate a f f i n i t y ' component 3 Powell-Jones and co-workers (1981) have reported that [ H]-e s t r a d i o l binding to the 4S protein could be abolished only by. neonatal gonadectomy of the male. This observation was confirmed by Sloop et a l . , (1983). They further demonstrated that the 115 decrease i n [ H ] - e s t r a d i o l binding to the HCLA s i t e ( t h e i r term for moderate a f f i n i t y component) in adult male whole cytosol following neonatal gonadectomy could be reversed following administration of a large dose of testosterone(57.5 mg/kg) on days 2, 6, 9 and 13 of l i f e . In addition, the ligand s p e c i f i c i t y of the testosterone restored HCLA s i t e was i d e n t i c a l to the intact adult male. As a r e s u l t of these studies Sloop et a l . , (1983) have suggested that the moderate'affinity component or HCLA s i t e i s neonatally imprinted by t e s t i c u l a r androgens in the male r a t . Sloop and co-workers (1983) have shown a f i v e f o l d increase 3 in t o t a l [ H ] - e s t r a d i o l binding in adult gonadectomized female whole cytosol following the administration of testosterone (57.5 mg/kg) on days 2, 6, 9 and 13 of l i f e . However, t h i s was not seen when i n t a c t females were treated s i m i l a r l y . Conversely, Dickson et a l . , (1978) have demonstrated a four f o l d increase in s p e c i f i c 3 [ H ] - e s t r a d i o l binding to i n t a c t female whole cytosol following the administration of DHT (0.3 yg/g/day) for seven days. However, 3 t h i s increase in [ H ] - e s t r a d i o l binding was shown to be DES-sensitive so i t does not represent a moderate a f f i n i t y or HCLA s i t e . Further-more, a 30% ammonium su l f a t e f r a c t i o n a t i o n of the whole cytosol 3 revealed DHT treatment decreased [ H ] - e s t r a d i o l binding by 50%. These r e s u l t s , when compared with those of Sloop et a l . , (1983) , show not only the misinterpretation that can a r i s e from studies that do not investigate s p e c i f i c binding and ligand s p e c i f i c i t y , but the discrepancy between ligand binding in crude verses p a r t i a l l y p u r i f i e d c y t o s o l . Our study did not demonstrate changes of the magnitude reported by Dickson et a l . , (1978) or Sloop et a l . , (1983) 116 for t o t a l or s p e c i f i c binding of [ H ] - e s t r a d i o l to the ammonium sulf a t e or whole cytosol f r a c t i o n s following administration of testosterone to sham-operated or• gonadectomized females> (Tables-9_, 23, 2 The differences in the methodology employed by various investigators make comparisons between studies d i f f i c u l t . One may conclude, however, that imprinting of the moderate a f f i n i t y component i s highly suspect. This i s e s p e c i a l l y true in regard to the differences in ligand s p e c i f i c i t y of the 4S protein that occurred following p h y s i o l o g i c a l manipulation as shown by Eagon et a l . , (1980). Before the HCLA s i t e i s considered to be imprinted, competition studies should be conducted at lower concentrations 3 of [ H ] - e s t r a d i o l to determine i f t h i s binding s i t e r e t a i n s the same binding c h a r a c t e r i s t i c s . i i ) Sucrose density gradient versus competitive binding studies A p o t e n t i a l confounding factor e x i s t s with regard to the comparison of r e s u l t s from sucrose density gradient analysis and competitive binding studies. This involves the p r a c t i c e of l a b e l i n g the cytosol p r i o r to gradient separation. A l l the sucrose density gradient studies described were conducted on pre-labeled gradients. While t h i s technique i s acceptable for measurement of the estrogen receptor, i t poses a problem in consideration of moderate a f f i n i t y binding. This has been demonstrated for a s i m i l a r lower a f f i n i t y higher capacity 4S estrogen binding protein (type II s i t e ) i s o l a t e d from rat uterus (Clark et a l . , 1978) and mouse mammary tumor (MXT-3590) (Watson and Clark, 1980). These workers have demonstrated 1 1 7 that s i g n i f i c a n t under-estimation of the type II s i t e occurs i f 3 gradients are pre-labeled with [ H ] - e s t r a d i o l . The under-estimation i s a r e s u l t of the d i s s o c i a t i o n that occurs during u l t r a c e n t r i f u g a t i o n . The authors point out that as the centrifugation i s us u a l l y 16-18 hours, the h a l f - l i f e of the ligand receptor complex, e s p e c i a l l y a lower a f f i n i t y binding s i t e , would be accommodated. As such, the authors suggest post - l a b e l i n g gradients and have demonstrated an increase i n estrogen receptor assay s e n s i t i v i t y for both the 8-9S and i n p a r t i c u l a r the 4-5S region. Thus the question p e r s i s t s as to what the ligand s p e c i f i c i t y of the protein sedimenting in the 4S region might-be. •'In addition., .one may. speculate that the lac k of dif f e r e n c e i n 4S.binding following gonadectomy .reported - by Powell-Jone& et a l . ,- (1980) , was a function of d i s s o c i a t i o n p r i o r to measurement. b) E f f e c t s of hypophysectomy on hepatic estrogen binding The androgen requirement f o r maintaining the capacity of the moderate a f f i n i t y component i n males was confirmed (Tables 9, 24, 29). Following t h i s , the degree of p i t u i t a r y involvement was investigated. We have found that hypophysectomy reduced the capacity of the.estrogen receptor i n male and female r a t s i n a time-dependent fashion (Tables 10, 12, 13, 25). Furthermore, the capacity of the moderate a f f i n i t y component i n the male was also decreased post-hypophysectomy (Tables 11, 12, 14, 25; Figure 3). However, the moderate a f f i n i t y s i t e , which was normally not present i n the female, was detected for the f i r s t time by Scatchard analysis two weeks following hypophysectomy (Table 11). At t h i s time the capacity of the female moderate a f f i n i t y s i t e was one-tenth that of the hypo-118 physectomized male. At four weeks post-hypophysectomy the capacit of t h i s s i t e was approximately one half, and by s i x weeks equal to, that of the hypophysectomized male (Tables 11, 12, 14, 25; Figure These r e s u l t s agree with those reported by Powell-Jones et a l . , (1980) . This report was the f i r s t demonstration of hypophyseal-hypothalamic influence on the binding of e s t r a d i o l to the hepatic c y t o s o l i c estrogen receptor and the moderate a f f i n i t y s i t e . Using pre-labeled sucrose gradients, Powell-Jones and co-workers (1980) 3 have shown that following hypophysectomy [ H ] - e s t r a d i o l binding to the 8-9S region was abolished in both males and females. Furthermore, binding to the 4S region was reduced i n the male and increased i n the female to approximately one half the hypo-physectomized male l e v e l at three weeks post-surgery. However, Powell-Jones et a l . , (1980) reported that at f i v e weeks the ,4S binding p r o f i l e s of the hypophysectomized male and female were the same as that observed at three weeks. The moderate a f f i n i t y component i n the hypophysectomized male and female appear equivalent on the basis of t h e i r binding c h a r a c t e r i s t i c s . However, the binding c h a r a c t e r i s t i c s of t h i s protein may change following hypophysectomy as Eagon et a l . , (1980) demonstrated for the 4S protein following gonadectomy. Therefore, i t would be of in t e r e s t to address the ligand s p e c i f i c i t y of the post-hypophysectomy 4S protein (moderate a f f i n i t y binding component) at 2-5 nM and 30 nM 3 [ H ] - e s t r a d i o l . This i s of importance as the apparent Kd of both male and female moderate a f f i n i t y protein appears to decrease by s i x to eight weeks a f t e r hypophysectomy. 119 3) Comparison of hepatic enzyme a c t i v i t y to hepatic estrogen binding components a) General conclusions from published observations We have observed a c o r r e l a t i o n with p h y s i o l o g i c a l manipulation between- AHH' a c t i v i t y , and the binding -of estrogens to c y t o s o l i c proteins i n the l i v e r . Although changes in the capacity of the estrogen receptor were observed, the binding of the moderate a f f i n i t y component was p a r t i c u l a r l y i n t e r e s t i n g as a point of modulation of AHH a c t i v i t y . To r e i t e r a t e , .a sexual dimorphism e x i s t s with respect to several hepatic mixed function oxidase a c t i v i t i e s i n the r a t , (Kato and G i l l e t t e , 1965", Quinn et a l . , 1958). E l Defrawy E l Masry et a l . , (1974a, 1974b) have demonstrated that rat hepatic cytochrome P-450 dependent N-demethylase a c t i v i t y increased with age i n males r e l a t i v e to females. Furthermore, the differences in these a c t i v i t i e s between adult male and female r a t s were reduced by gonadectomy or estrogen treatment of the male. Gonadectomy did not a f f e c t these a c t i v i t i e s i n the female. Furthermore, the e f f e c t of gonadectomy of the male was shown to be testosterone r e v e r s i b l e . This demonstrated the involvement of androgens i n the expression of sex differences for c e r t a i n hepatic mixed function oxidase a c t i v i t i e s . Rat hepatic AHH a c t i v i t y i s known to respond s i m i l a r l y . An age and sex dependency has been described (Oesch et a l . , 1976) as have requirements for t e s t i c u l a r androgens (Gontovnick et a l . , 1979; Gurtoo and Parker, 1977; Kramer et a l . , 1975). Moreover, 120 the testosterone r e v e r s i b i l i t y of the decrease i n AHH a c t i v i t y following gonadectomy and the non-responsiveness of the adult female to gonadectomy have been previously reported (Kramer et a l . , 1979;. Gurtoo and Parker, 1977). We have confirmed these observations 3 and have noted that the binding of [ H ] - e s t r a d i o l to the moderate a f f i n i t y component v a r i e d i n a p a r a l l e l manner following physio-l o g i c a l manipulation (Table 33). b) Age and sex dependency We have shown that the l e v e l s of AHH a c t i v i t y were greater in the adult control male than female (Tables 15-19, 25, 28). The capacity of the estrogen receptor was not d i f f e r e n t between the adult male and female (Tables 7-10, 12, 25-28). However, the moderate a f f i n i t y component could only be detected i n the adult c o n t r o l male. I t was not found i n the c o n t r o l female (Tables 7-9, 23, 25-28). The AHH a c t i v i t y of the immature male or female was shown to be s i m i l a r to that of the adult female (Bellward et a l . , 1982). The capacity of the estrogen receptor, while reduced in the immature animal, did not ex h i b i t any sex difference. No_ moderate a f f i n i t y component could be detected in the immature rat (Table 8). Therefore the increase i n AHH a c t i v i t y with age in the male i s consistent with the detection of the moderate a f f i n i t y component in the male and inconsistent with the increase i n estrogen receptor i n both males- arid females, 1 2 1 TABLE 33 Summary of r e l a t i v e changes in capacity of the estrogen receptor, moderate a f f i n i t y binding component and AHH a c t i v i t y r e l a t i v e to the adult male (unless otherwise noted). Model Binding to Estrogen Receptor Binding to Moderate A f f i n i t y Component Hepatic AHH A c t i v i t y Female + 0 Pseudo f 0 Immature male 4- 0 Immature female 4- 0 Gx male + 4~> 4-Gx male + Testosterone 4-> +' Gx female -> 0 Gx female + Testosterone ->- 0 Male + Mestranol 0 0 Male + pimozide + -> Gx male + pimozide 4 4-Female + pergoiide -y 0 Gx female + pergoiide 4- 0 Hx male 0 4-Hx male + rGH 0 0 Hx Female 0 t Hx Female + rGH 0 0 Male + 3-MC 4-Male + Phenobarbital 4- + Female + 3-MC 4- 0 Female + Phenobarbital 4- 0 4-4-I t * 4-4-t t *• Relative to Gx male ** Relative to Gx female t Relative to sham-operated female t t Relative to Hx female Changes i n s p e c i f i c binding were indicated as follows: t = increase; 4- = decrease; -»- = no change; 0 = none detected. 1 2 2 c) Androgen dependency We have shown that androgens play a r o l e in maintaining the l e v e l s of AHH a c t i v i t y in the male. Gonadectomy of the adult male reduces hepatic AHH a c t i v i t y and the capacity of the moderate a f f i n i t y component in a testosterone r e v e r s i b l e manner (Tables 8, 9, 24, 29). Gonadectomy did produce s l i g h t f l u c t u a t i o n s in the capacity of the estrogen receptor in the male which at times appeared to be reversed by testosterone. However, these small changes i n capacity of the estrogen receptor were probably not p h y s i o l o g i c a l l y relevant. Furthermore, we have shown that gona-dectomy does not a l t e r AHH a c t i v i t y i n the female (Tables 8, 9, 22), nor were any changes seen in estrogen binding c h a r a c t e r i s t i c s , (Tables 20-22). d) E f f e c t of estrogens It has been demonstrated that the administration of estrogens to males w i l l produce e f f e c t s s i m i l a r to gonadectomy with respect to sex dependent mixed function oxidase a c t i v i t y (El Defrawy E l Masry and Mannering, 1974; Sladek et a l . , 1974). We have demonstrated that following the administration of mestranol, male AHH a c t i v i t y was reduced to female l e v e l s , whereas female AHH a c t i v i t y was not affected (Table 28). The administration of mestranol 3 abolished [ H ] - e s t r a d i o l binding to the moderate a f f i n i t y component in the male whole cytosol f r a c t i o n . Mestranol also abolished 3 [ H ] - e s t r a d i o l binding to the hepatic estrogen receptor in male and female r a t s . This was not consistent with the increase in 123 s p e c i f i c binding to the 8-9S region reported by Eagon et a l . , (1980) following administration of DES or e s t r a d i o l to intact adult male r a t s . However, our observations could be the r e s u l t of the following: (1) nuclear t r a n s l o c a t i o n of a l l c y t o s o l i c estrogen receptor, (2) down regulation of the c y t o s o l i c receptor, (3) occupation of a l l unoccupied c y t o s o l i c estrogen receptor binding s i t e s by mestranol or (4) a combination of these p o s s i b i l i t i e s . Whereas most treatments were discontinued 24 hours p r i o r to s a c r i f i c e , mestranol was discontinued 48 hours p r i o r to s a c r i f i c e i n an e f f o r t to circumvent these e f f e c t s . This observation points out an inherent problem with the non-equilibrium receptor assay system. The methodology only allows for the determination of unoccupied receptor s i t e s . To f u l l y understand the mechanism(s) involved in receptor mediated pathways t o t a l binding s i t e s should also be determined -- which would permit the determination of occupied.receptors. e) Pseudohermaphroditic rat model Assays were c a r r i e d out i n pseudohermaphroditic r a t s as a further c h a r a c t e r i z a t i o n of androgen involvement in the modulation of sex dependent hepatic mixed function oxidases as well as hepatic estrogen binding. The pseudohermaphroditic rat i s genotypically a male but despite high serum testosterone l e v e l s i s phenotypically a pseudohermaphrodite, (Stanley et a l . , 1973). This i s thought to be due to a d e f i c i e n c y i n the androgen receptor pathway 124 (Bardin et a l . , 1973). The pseudohermaphrodite i s an i n t e r e s t i n g model since hepatic mixed function oxidase a c t i v i t i e s have been shown to resemble the female in both control and induced states (Sonawane et a l . , 197 9). Our r e s u l t s confirm t h i s report. Following the administration of various inducers of drug metabolism (phenobarbital, 3-MC and spironolactone) the AHH a c t i v i t y of the pseudohermaphroditic rat p a r a l l e l e d that of the female, and not of the male, i n a l l cases (Tables 15, 16). A l l three drugs increased AHH a c t i v i t y i n the female and pseudohermaphrodite to comparable l e v e l s . In the male, as expected, only 3-MC induced AHH a c t i v i t y . The s l i g h t decrease i n AHH a c t i v i t y produced by phenobarbital arid -^h:±bit;ion,' of'-AHH. a c t i v i t y by spironolactone i n the' -male 'eon.fi^med-'Cpreyious ••<repo-r;'fcs.':-(Gontovnick e t a l . ,"1981-;.. Stripp et - a l ; , 1973); .. " • I n t e r e s t i n g l y , as with the female, no moderate a f f i n i t y binding was detected in the pseudohermaphrodite (Table 7). Further-more, while the capacity of the estrogen receptor was reduced in the l i t t e r m a t e control male, there were e s s e n t i a l l y no differences between the female and pseudohermaphrodite. Thus, the pseudo-hermaphrodite not only resembled the female with respect to estrogen binding but in response to inducers of drug metabolism as well. f) Hypophysectomy model The e f f e c t s of hypophysectomy on hepatic mixed function 125 oxidase a c t i v i t y have been well documented (Colby et a l . , 1973; Kramer et a l . , 1975, 1979; Rumbaugh and Colby, 1980). Furthermore, androgens and estrogens have been found to produce no e f f e c t on hepatic drug (Colby et a l . , 1973; Kramer et a l . , 1975, 1979) or st e r o i d (Gustafsson and Stenberg, 1974, 1976; Lax et a l . , 1974) metabolizing a c t i v i t i e s . This was in agreement with our r e s u l t s . Hypophysectomy reduced the d i f f e r e n t i a t i o n i n AHH a c t i v i t y between the adult male and female r a t . We observed a decrease in AHH a c t i v i t y i n the male and a small but c o n s i s t e n t l y s i g n i f i c a n t increase i n the female (Tables 17-19, 25). The e f f e c t of hypophysectomy on estrogen binding i n hepatic cytosol was of i n t e r e s t . As i l l u s t r a t e d i n Table 25, the e f f e c t of hypophysectomy on the estrogen receptor did not p a r a l l e l changes in AHH a c t i v i t y of male and female r a t s . 3 The binding of [ H ] - e s t r a d i o l to the estrogen receptor was abolished i n both males and females following hypophysectomy (Tables 13, 25). We also observed the capacity of the moderate a f f i n i t y component i n the male to be reduced by t h i s procedure (Tables 11, 12, 14, 25). Most s t r i k i n g was the appearance of the moderate a f f i n i t y component in the hypophysectomized female (Tables 11, 12, 14, 25). This was consistent with the increase in AHH a c t i v i t y and inconsistent with the decrease i n estrogen receptor. Furthermore, unlike the gonadectomy model, administration of testosterone did not have any e f f e c t on AHH a c t i v i t y (Table 18). 3 In addition, testosterone did not a f f e c t [ H ] - e s t r a d i o l binding of e i t h e r the estrogen receptor or the moderate a f f i n i t y component i n the hypophysectomized r a t s of either sex, (Tables 13, 14). 126 These results also demonstrated that the moderate a f f i n i t y component was not solely responsible for mediating the actions of testosterone. The results of the hypophysectomy study were consistent with the hypothesis that the pituitary mediates the expression of the sex differences in hepatic metabolism (Colby, 1980; Skett et a l . , 1981) and the capacity of the estrogen receptor (Posner et a l . , 1974). 4) Relationship of pituitary hormones to hepatic AHH activity and estrogen binding a) Background We had i n i t i a l l y hypothesized that PRL may be the pituitary hormone responsible for the observed levels of hepatic enzyme activity in the female for the following reasons: PRL regulates i t s own (Bohnet et a l . , 1976) as well as estrogen (Chamness et a l . , 1975) receptor levels in the li v e r ; castration results in a testosterone reversible increase in hepatic PRL receptors(Aragona et a l . , 1976); and estrogens induce hepatic PRL receptors and hypophysectomy reduces hepatic PRL binding in a non-estrogen responsive manner (Posner et a l . , 1974). Therefore, PRL was modulating hepatic estrogen as well as lactogenic receptors. Several recent reports have strongly suggested that GH is the pituitary factor involved in the production of female levels of drug and steroid metabolism (Kramer and Colby, 1976; Mode et a l . , 1981; Norstedt et a l . , 1983; Rumbaugh and Colby, 1980). However, as indicated earlier i t was not clear whether these effects were due to the somato- or lacto-tropic properties of the 127 hormones used. b) E f f e c t s of Pimozide As an i n i t i a l method of p i t u i t a r y hormone manipulation adult male sham-operated and gonadectomized r a t s were treated with the dopamine antagonist pimozide. The dose of pimozide used reportedly produces an approximate 6 f o l d increase i n plasma PRL l e v e l s (Ojeda et a l . , 1974). In addition, following a single i n j e c t i o n of pimozide, PRL synthesis was induced for four days and GH synthesis s l i g h t l y i n h i b i t e d f o r two days (Maurer and Gorski, 1977). These workers further demonstrated that adminis-t r a t i o n of e s t r a d i o l to male and female ra t s increased PRL synthesis while GH synthesis was unaffected. In addition, pimozide has been reported to reduce diurnal f l u c t u a t i o n s and stress-induced release of GH (Schwinn et a l . , 1976). We observed that pimozide, while not a f f e c t i n g AHH a c t i v i t y , did increase A4 reductase a c t i v i t y of the sham-operated male. In addition, pimozide i n h i b i t e d the action of testosterone on A4 reductase in the gonadectomized male (Table 22). Subsequent binding studies were conducted in conjunction with AHH determinations, and s i m i l a r e f f e c t s were observed (Table 24). Pimozide did not a l t e r AHH a c t i v i t y in any model but did a b o l i s h the detection of the moderate a f f i n i t y component i n the gonadectomized male. It also i n h i b i t e d the r e s t o r a t i o n of the moderate a f f i n i t y component by testosterone i n t h i s model". This suggests that i n the gonadectomized male the elevation 1 2 8 i n PRL, and/or reduction of GH, i s i n h i b i t o r y to the moderate a f f i n i t y component. The action of testosterone in t h i s case may be to anta-gonize the elevation i n PRL produced by pimozide (Giguere et a l . , 1982) thus i n h i b i t i n g the reduction of the moderate a f f i n i t y component. However, the reduced release of GH must be considered. While we did observe p a r a l l e l e f f e c t s with A4 reductase as previously discussed, the action of pimozide was not r e f l e c t e d by s t a t i s t i -c a l l y s i g n i f i c a n t changes in AHH a c t i v i t y . It i s of i n t e r e s t , however, that pimozide did decrease the numerical value of the AHH a c t i v i t y i n the gonadectomized male to a l e v e l s i m i l a r to that found i n females. c) E f f e c t s of pergoiide The second approach to the i n vivo manipulation of PRL u t i l i z e d the dopamine agonist pergoiide to reduce PRL l e v e l s . The r a t i o n a l e was that i f PRL were responsible for the sex d i f f e r e n c e in enzyme a c t i v i t y , t h e n reduction of PRL i n the female may reduce the sex differences in basal a c t i v i t y or enhance the a b i l i t y of testosterone to e l i c i t a response. However, we found the action of pergoiide to be contrary to our p r e d i c t i o n . Pergoiide i n h i b i t e d the e f f e c t of testosterone on AHH a c t i v i t y i n adult gonadectomized females (Table 20) and i n adult females gonadectomized pre-pubescently (Table 21) receiving pergoiide throughout the pubertal period. However, when hepatic estrogen binding and AHH a c t i v i t y were studied concurrently (Table 23) testosterone did not increase AHH a c t i v i t y . Therefore, the e f f e c t of pergoiide to i n h i b i t testosterone induction was not demonstrated. 1 2 9 Furthermore, no moderate a f f i n i t y binding was detected following the administration of testosterone. Pergolide did tend to reduce the capacity of the estrogen receptor in the gonadectomized male, which i s consistent with the d i r e c t involvement of PRL with hepatic estrogen binding (Norstedt et a l . , 1981b), Unlike pimozide, pergolide d i d not a f f e c t the. action of testosterone on A4 reductase. If PRL were the hormone responsible for the observed l e v e l s of female enzyme a c t i v i t y , t h e n one would expect the actions of testosterone to be potentiated in a hypoprolactinemic female. However, pergolide i s a dopamine agonist. Although i t i s purported to be s e l e c t i v e f or PRL depletion, elevations i n GH would be expected. Therefore i f , as several previously c i t e d studies indicate, GH (and not PRL) i s responsible for female l e v e l s of a c t i v i t y , then we would expect pergolide-induced elevations of GH to i n h i b i t the action of testosterone. As such, we would not expect to detect a moderate a f f i n i t y binding component i n t h i s model and t h i s was confirmed experimentally. d) P i t u i t a r y hormone replacement: E f f e c t s on hepatic AHH a c t i v i t i e s and c y t o s o l i c estrogen binding I t was clear that the use of pergolide and pimozide did not produce conclusive r e s u l t s . Therefore, to determine the r o l e of p i t u i t a r y hormones on enzyme and estrogen binding parameters, various hormones were administered to hypophysectomized animals. While t h i s hormone replacement model was more invasive, and produced changes i n larger numbers of hormones, i t d i d provide 130 more conclusive r e s u l t s . The e f f e c t of bGH on hepatic mixed function oxidase a c t i v i t i e s has been well characterized. Kramer and Colby (1976), arid Wilson (1970,1973) have shown GH to reduce sex dependent a c t i v i t i e s i n the intact and gonadectomized male r a t . However, GH has a "paradoxical" e f f e c t . Rumbaugh and Colby (1980) reported that bGH reduced AHH a c t i v i t y i n castrated males, yet increased AHH a c t i v i t y i n hypo-physectomized males. To i n i t i a l l y characterize the e f f e c t s of t h i s hormone, bGH was administered according to the protocol of Rumbaugh and Colby (1980). This regimen s p e c i f i e d i n j e c t i o n of bGH twice d a i l y achieving 4.0 I.U./kg/day. Following t h i s treatment schedule we observed a pronounced decrease in the capacity of the moderate a f f i n i t y component (Table 14). If GH i s the p i t u i t a r y f actor responsible for female l e v e l s of a c t i v i t y , and we hypothesize that the moderate a f f i n i t y component i s r e l a t e d to male l e v e l s of a c t i v i t y , then the decrease i n the moderate a f f i n i t y component following bGH i s consistent with the hypothesis. However, t h i s i s not consistent with the increase i n AHH a c t i v i t y reported by Rumbaugh and Colby (1980). Subsequent studies by Mode et a l . , (1981) conclusively demonstrated feminization of hepatic s t e r o i d metabolism following continuous infusion of GH i n adult i n t a c t , hypophysectomized, and gonadectomized r a t s . , I n t e r e s t i n g l y , there i s a sex d i f f e r e n c e i n the secretory pattern of GH . i n the r a t . Males exhibit a p u l s a t i l e secretory pattern with a 131 high peak to trough r a t i o at regular three to four hour i n t e r v a l s . Females have an i r r e g u l a r p u l s a t i l e release with higher l e v e l s between peaks than males ( Eden, 1979; Terry, et al., 1977) Therefore, i t i s l o g i c a l to suggest that differences between the observations of Rumbaugh and Colby (1980) and those of Mode et a l . , (1981) were due to the regimen mimicking the i n vivo release pattern of the male and female r e s p e c t i v e l y . Because of t h i s , we c a r r i e d out subsequent replacement studies according to the protocol established by Mode et a l . , (1981), using Alzet® osmotic minipumps. The v a l i d i t y of t h i s technique has been confirmed by Norstedt et a l . , (1981a, 1981b, 1983). i ) Hormone replacement i n male rats We found that bGH replacement by continuous infusion (0.01 I.U./hour) reduced AHH a c t i v i t y in sham-operated and-hypophysectomized male rats.(Table 25). This observation was consistent with the feminization of hepatic s t e r o i d metabolism reported by Mode et a l . , (1981) and i n disagreement with the r e s u l t s described by Rumbaugh and Colby (1980). As previously mentioned t h i s i s probably due to the method of hormone de l i v e r y : I n t e r e s t i n g l y , although continuous infusion of bGH reduced AHH a c t i v i t y i n the sham-operated and hypophysectomized male, no obvious changes i n capacity of the moderate a f f i n i t y component were detected as they had been following i n j e c t i o n of hypophysectomized males with bGH. However, the bGH administered possessed 62% lactogenic a c t i v i t y . Furthermore, 132 when oPRL was administered to the sham-operated male using osmotic minipumps, we observed an even greater reduction of AHH a c t i v i t y without any change i n the capacity of the moderate a f f i n i t y component. Therefore, the l a c t o t r o p i c properties of these hormones may produce the reduction i n AHH a c t i v i t y . The i n t e r p l a y between somatotropic and l a c t o t r o p h i c a c t i v i t y was more pronounced i n the hypophysectomized rat (Table 25). Continuous i n f u s i o n of GH or PRL d i d not restore binding to the estrogen receptor in the hypophysectomized r a t . rGH, which has n e g l i g i b l e l a c t o t r o p i c a c t i v i t y , not only reduced AHH a c t i v i t y to c o n t r o l female l e v e l s , but abolished the detection of the moderate a f f i n i t y component on both male and female r a t s . Therefore, i t was of i n t e r e s t to examine the e f f e c t s of rPRL. Continuous infusion of "rPRL did not reduce AHH a c t i v i t y or the capacity of the moderate a f f i n i t y component. Furthermore, the rPRL as well as oPRL used had n e g l i g i b l e somatotropic a c t i v i t y . Therefore, one must conclude that the l a c t o t r o p i c action i s not modulating AHH a c t i v i t y or the capacity of the moderate a f f i n i t y component. This i s in agreement with the r e s u l t s of Mode et a l . , (1981) who demonstrated that continuous infusion of rPRL did not a f f e c t hepatic st e r o i d metabolism i n hypophysectomized male r a t s . The e f f e c t of oPRL in the sham-operated animal might be due to increased PRL modulation of other p i t u i t a r y f a c t o r s , or an inherent d i f f e r e n c e between the two lactotrophs. To examine the e f f e c t s of oPRL i n the hypophysectomized animal, the concurrent administration of testosterone must be taken into account. 1 3 3 Testosterone was administered concurrently with bGH and oPRL to determine i f i n the presence of these p i t u i t a r y hormones AHH a c t i v i t y could be increased. However, the presence of te s t o -sterone d i d not a l t e r the effect of bGH on AHH a c t i v i t y i n the hypophysectomized male. This might be expected since testosterone by i t s e l f produced no r e s t o r a t i v e e f f e c t on AHH a c t i v i t y or hepatic estrogen binding i n the hypophysectomized animal. This does not mean that GH and PRL are not the p i t u i t a r y hormones involved i n the action of testosterone. Rather, i t indicated that at the l e v e l s administered, bGH and oPRL did not modify the binding components s u f f i c i e n t l y to permit a testosterone response. It i s obvious that the l e v e l s of GH and PRL are low as demonstrated by the i n a b i l i t y to detect any unoccupied estrogen receptor s i t e s i n the hypophysectomized rat re c e i v i n g hormone replacement. The i n a b i l i t y to detect estrogen receptor s i t e s may be the r e s u l t of the non-equilibrium assay, or the requirement of an adrenal factor as suggested by Lucier et a l . , (1981), and Norstedt et a l . , (1981b). However, these hormones did not a l t e r the capacity of the estrogen receptor i n the sham-operated male either (Table 25). i i ) Hormone replacement in female r a t s The e f f e c t s of hormone replacement in the female were analyzed separately (Table 25). I n i t i a l grouping of the female and male models from Table 25 for s t a t i s t i c a l analysis indicated that hormone replacement, and even hypophysectomy, did not r e s u l t i n s i g n i f i c a n t changes i n female AHH a c t i v i t y according to the Duncan 1 3 4 multiple range t e s t . However, we have con s i s t e n t l y demonstrated that hypophysectomy increases AHH a c t i v i t y i n the female. Therefore the hypophysectomized female model was analyzed separately. As a r e s u l t we have demonstrated, as expected, hypophysectomy produces an increase i n AHH a c t i v i t y concomitant with the appearance of a moderate a f f i n i t y binding component. Continuous infusion of rGH reduced AHH a c t i v i t y to sham-operated female l e v e l s and abolished the moderate a f f i n i t y component in the hypophysectomized female. This was the same e f f e c t observed in the hypophysectomized male following rGH i n f u s i o n . However, the e f f e c t of rPRL was not as c l e a r . Unlike the male model, rPRL reduced AHH a c t i v i t y i n the hypophysectomized female to sham-operated female values. Furthermore, s p e c i f i c binding to the moderate a f f i n i t y component was evident, and although a negative r e l a t i o n s h i p between bound/-free versus bound was present, l i n e a r regression did not provide an acceptable c o r r e l a t i o n c o e f f i c i e n t . The concurrent administration of testosterone with ei t h e r bGH or oPRL did not induce AHH a c t i v i t y i n the hypophysectomized female. On the contrary AHH a c t i v i t y was reduced i n both models. However,as. the appropriate controls were not a v a i l a b l e i t i s d i f f i c u l t to resolve the e f f e c t s of the combined treatment. 135 5) The e f f e c t s of xenobiotics on hepatic AHH a c t i v i t y and c y t o s o l i c estrogen binding parameters a) In v i t r o e f f e c t s i ) Ah receptor In addition to sex steroids and p i t u i t a r y hormones, a v a r i e t y of xenobiotics are known to induce hepatic mixed function oxidase a c t i v i t i e s (Conney, 1967). The p o l y c y c l i c aromatic hydrocarbons (PAH) " are predominently metabolized by AHH, and administration of PAH such as 3-MC w i l l induce AHH a c t i v i t y . Induction of AHH a c t i v i t y i s considered to be mediated by the Ah (ar y l hydrocarbon) receptor (Nebert et a l . , 1982; Okey et a l . , 197 9; Poland and Glover, 1976). This receptor i s often r e f e r r e d to as the TCDD receptor because i t s most potent agonist i s tetrachlorodibenzo-p-dioxin (TCDD). Characterization of the Ah receptor has revealed an 8-9S species on hypotonic sucrose density gradient a n a l y s i s . However, binding of BP and;TCDD i n the 4§ region has'been -observed (Okey.:et a l . , 1.979.). To assess the r e l a t i o n s h i p between the Ah receptor, the estrogen receptor, and the moderate a f f i n i t y binding s i t e , three dioxin congeners of d i f f e r i n g Ah binding c h a r a c t e r i s t i c s (Poland 3 and Glover, 1976) were used as competitors f o r [ H] - e s t r a d i o l binding. We have demonstrated that a l l three dioxin congeners were 3 e f f e c t i v e competitors of [ H ] - e s t r a d i o l binding to the estrogen receptor i n male and female r a t s (Table 30). The Scatchard plots 136 of the female estrogen receptor produced an apparent Kd and capacity that were lower than that obtained by unlabeled e s t r a d i o l (Table 31). Scatchard p l o t s y i e l d i n g l i n e a r regression of acceptable c o r r e l a t i o n could not be generated for the male with any of the dioxin congeners. This occurred despite the fact that a l l congeners competed for [ H.]-estradiol binding to the estrogen receptor. A s i m i l a r r e s u l t was noted for the moderate a f f i n i t y component. Unlike the estrogen receptor, only 2,7-DCDD and OCDD competed for ["^H]-estradiol binding to the moderate a f f i n i t y component i n the male. However, l i k e the estrogen receptor i n the male, acceptable Scatchard p l o t s could not be generated with these congeners. Furthermore, as with a l l other competitors, none of the dioxin congeners led to detection of moderate a f f i n i t y binding i n the female. The i n a b i l i t y to generate Scatchard plots for the male estrogen receptor with dioxin was not due to d i f f i c u l t i e s associated with a dextran-coated charcoal assay (Okey et a l . , 1979) as evidenced by the r e s u l t s f or the female estrogen receptor. We do not f e e l the binding i n the female represents the Ah receptor as i t would have been detected i n the male as well. Poland and Glover, (1976) have shown 17B-estradiol does not compete f o r TCDD binding to the Ah receptor. However, t h i s was probably a r e f l e c t i o n of the a v i d i t y f or the binding s i t e rather than ligand s p e c i f i c i t y . In any case, the r e s u l t s of the dioxin competition for [ 3 H ] - e s t r a d i o l binding i n male c y t o s o l i c f r a c t i o n s further demonstrate the problems with r e l y i n g on sing l e point competition analysis to detect s p e c i f i c binding components. 137 i i ) Hepatic c y t o s o l i c BP./S^ MC• binding ;prot,e,ih. In addition to the Ah receptor, a 4S BP binding component has been i d e n t i f i e d i n hepatic cytosol (Weaver et a l . , 1980). In a d d i t i o n , 3-MC has been shown to interact non-covalently with a 4S binding protein i n hepatic cytosol as well (Tierney et a l . , 1980) . C o l l i n s and Marietta (1983) have demonstrated the c y t o s o l i c 4S BP binding protein i s not r e l a t e d to the Ah receptor or glutathione S-transferase B. However, 3-MC and BP do bind to these s i t e s as well (Banderia et a l . , 1982; D i x i t et a l . , 1982, 1983; Guenther et a l . , 1977). The 4S hepatic c y t o s o l i c protein that binds 3-MC and BP i s capable of nuclear translocation (Holder et a l . , 1981; Tierney et a l . , 1980, 1981; Weaver et a l . , 1980). This t r a n s l o c a t i o n did not require an a c t i v a t i o n step (Tierney et a l . , 1980; Weaver et a l . , 1980), as i s seen with the estrogen receptor (Jensen and DeSombre, 1973) . The values reported for the apparent Kd and capacity of the 3-MC binding protein,2.8 nM, 770 fmol/mg (Tierney et a l . , 1980) and the BP binding protein, 2.5 nM, 530 fmol/mg (Holder et a l . . 1981) , are below those values observed for the moderate a f f i n i t y component. Tierney et a l . . (1980) have demonstrated that i n addition to an array of PAH's, 17g-estradiol i s an e f f e c t i v e competitor for 3 [ H]3-MC binding to the 4S protein. A 100 f o l d excess of unlabeled 3 17 3-estradiol competed for 55% of [ H]3-MC binding i n male r a t hepatic c y t o s o l . The r e l a t i o n s h i p of t h i s c y t o s o l i c 4S 3-MC and BP binding protein to the moderate a f f i n i t y s i t e i s presently unclear. 138 The estrogen receptor has been shown to bind a v a r i e t y of xenobiotics: 3-diethylaminoethyl-2,2-diphenylpentanoate (SKF 525-A) (Bulger and Kupfer, 1981), o,p'-DDT (Kupfer and Bulger, 1976), Kepone (Bulger et a l . , 1979) and 2-acetylaminofluorene (Farley and Coogan, 1983). Since we are interested in the modulation of AHH a c t i v i t y by PAH's as well as sex steroids,the r e l a t i o n s h i p of the 3-MC/BP binding s i t e and the moderate a f f i n i t y component was investigated. unlabeled 3-MC and BP were used as competitors for 3 [ H ] - e s t r a d i o l binding i n hepatic cytosol f r a c t i o n s in male and female r a t s . 3 We have shown that both 3-MC and BP competed for [ H ] - e s t r a d i o l binding to the estrogen receptor i n male and female and for the moderate a f f i n i t y component i n the male r a t . (Table 30). Furthermore, the competition data produced good l i n e a r p l o t s when analyzed by the Scatchard technique. This was true i n both the males and females, un l i k e the data from the dioxin study. The apparent Kd f o r 3-MC competition was s i m i l a r i n both male and female, and similar, to the apparent Kd obtained by e s t r a d i o l competition (Table 26). There was no sex d i f f e r e n c e in the capacity, which was approximately 50% that detected by e s t r a d i o l competition. With respect to BP competition, (Table 27) both the apparent Kd and capacity were s i m i l a r but reduced i n both male and female r e l a t i v e to e s t r a d i o l competition. Following induction with 3-MC or phenobarbital we observed a decrease in the capacity of the estrogen receptor when e s t r a d i o l was used as a competitor. But t h i s decrease was not observed following induction using either 3-MC or BP as competitors. Therefore, i t appears that the PAH's are competing for e s t r a d i o l binding to a 139 subclass of receptor sites. Competition for binding to the moderate af f i n i t y component by 3-MC and BP resulted in similar shifts of apparent Kd and capacity relative to estradiol competition as were reported for the estrogen receptor. It may be argued that 3-MC and BP are displacing [ 3H.]-estradiol bound to sites other than the moderate a f f i n i t y component. However, following gonadectomy (Table 29) or induction (Table 26) we observed parallel shifts in displacement of labeled estradiol using unlabeled estradiol or 3-MC as competitors indicating physiological relevance. Moreover, the similar a f f i n i t i e s that result from 3-MC or estradiol competition suggest that the difference in capacities may be due to a difference in ligand avidity for the moderate a f f i n i t y binding site. We have demonstrated an interaction between PAH's, the estrogen receptor, and the moderate a f f i n i t y component. It would be of interest to examine the ontogenetic profile of the BP/3-MC binding protein in relation to the moderate a f f i n i t y site. This has yet to be addressed. In fact, investigation of the 4S BP/3-MC binding protein has so far been conducted in adult males only. On the basis of the similarity of ligand specificity that we have shown, and the similarity of sucrose density gradient sedimentation, one can postulate that the moderate a f f i n i t y component and the PAH binding protein possess functional identity. Alternatively, there may be a family of heterogeneous proteins with partially overlapping binding a f f i n i t i e s . Heterogeneity of hepatic estrogen binding sites has been reported (Thompson et al.., 1981; Thompson and Lucier, 1982). Certainly a heterogeneity of the estrogen receptor has been demonstrated (Greene and Jensen, 1982). This may account for the differences in capacity noted with various competitors. 140 However, we have observed s i g n i f i c a n t discrepancies with the c o r r e l a t i o n between the l e v e l s of AHH a c t i v i t y and the presence of the moderate a f f i n i t y component. These inconsistencies were f i r s t described in the hormone models, and have been observed following treatment with xenobiotics as w e l l . b) In vivo e f f e c t s of xenobiotics Duvivier et a l . , (1981) have demonstrated a c o r r e l a t i o n between 3-MC induction and the capacity of the estrogen receptor. Following the administration of 3-MC t h i s group observed a decrease i n binding to the estrogen receptor i n ovariectomized female r a t s . We have observed e i t h e r a decrease or no change i n capacity of the estrogen receptor i n intact male and female r a t s . These authors also reported that phenobarbital produced a s l i g h t increase in estrogen binding. Our data indicates that s i m i l a r to 3-MC, phenobarbital produced a decrease or no change in the capacity of the estrogen receptor. When AHH a c t i v i t y was induced with 3-MC i n the male, an increase i n the capacity of the moderate a f f i n i t y component was also noted. However, phenobarbital, which was shown not to induce AHH a c t i v i t y in the adult male, also increased the capacity of the moderate a f f i n i t y component. Furthermore, following the administration of 3-MC, spironolactone or phenobarbital, (which were shown to induce AHH a c t i v i t y i n the female), no moderate a f f i n i t y binding component could be detected in the female. Administration of 3-MC and phenobarbital for ten days (instead of the customary 2 and 4 days respectively) s t i l l did not r e s u l t in detection of a moderate a f f i n i t y component i n the female.even:though AHH a c t i v i t y was induced. Interesting 141 the prolonged administration of 3-MC to males nearly abolished the moderate a f f i n i t y component, although AHH a c t i v i t y remained induced. The reason f o r the decrease i n capacity of the moderate a f f i n i t y component in t h i s instance may be much the same as that observed following administration of mestranol. We were measuring unoccupied s i t e s and, as was discussed e a r l i e r , the 3-MC binding to these s i t e s may have masked detection. Thus, i n the female, the only procedure that produced an increase i n AHH a c t i v i t y and moderate a f f i n i t y binding was hypophysectomy. Furthermore, we have noted procedures that increased the capacity of the moderate a f f i n i t y component i n the male without a f f e c t i n g AHH a c t i v i t y (phenobarbital) or which reduced AHH a c t i v i t y (spironolactone). We have also observed decreases i n AHH a c t i v i t y i n the male that have not .produced decreases the capacity of t h i s binding component (oPRL, bGH). Therefore, the c o r r e l a t i o n of AHH a c t i v i t y with the capacity of the moderate a f f i n i t y component i s not consistent. We have suggested that t h i s may be due to differences i n the form of the moderate a f f i n i t y component following p h y s i o l o g i c a l manipulation. Our l i m i t e d c h aracterization did not detect t h i s but others have reported the p o s s i b i l i t y (Eagon et a l . , 1980). A l t e r n a t i v e l y , these discrepancies may be due to the m u l t i p l i c i t y of cytochromes P-450. 142 6) M u l t i p l i c i t y of Cytochromes P-450 Cytochromes P-450 comprise a group of c o n s t i t u t i v e and inducible isozymes e x h i b i t i n g optimal and overlapping substrate s p e c i f i c i t i e s , as well as re g i o - and s t e r e o - s e l e c t i v i t y (Johnson and Schwab, 1983; Lu and West, 1979). Although only a f i n i t e number of P-450 isozymes have been p u r i f i e d to homogeneity, the d i v e r s i t y of substrate s p e c i f i c i t y observed for the P-450 system has lead Nebert et a l . , (1982) to compare i t to the immune system. They suggest that i n response to xenobiotics, P-450 isozymes may be synthesized that are s p e c i f i c for metabolism of that xenobiotic, i . e . a s i m i l a r process to antigen stimulation of s p e c i f i c antibody production occurs. Recently a microheterogeneity has been reported f o r the phenobarbital inducible form of P-450 i n the rat (Waxman and Walsh et a l . , 1982) that v a r i e s between species and stra i n s or colonies (Vlasuk et a l . , 1982). Moreover, c a t a l y t i c a l l y d i s t i n c t subclasses of rabbit P-450 3b act i v e towards hydroxylation of progesterone have been characterized (Johnson et a l . , 1983). These differences may be due to p o s t - t r a n s l a t i o n a l modification such as a l l o s t e r i c regulation (Johnson et a l . , 1983) rather than genomic v a r i a t i o n . Or, p o s t - t r a n s l a t i o n a l modification may occur s i m i l a r to that demonstrated f o r synthesis of f e t a l hemoglobin by chick blastoderm. Mainwaring and Irving (1980) have reported that 53- reduced steroids enhance globin synthesis following binding to cytoplasmic proteins not found i n the yolk. This binding increased t r a n s l a t i o n of s p e c i f i c mRNA coded f o r globin synthesis. The 143 cytoplasmic proteins are recognized to be cap binding proteins which increase t r a n s l a t i o n of capped but not uncapped mRNA (Sonenberg et a l . j 1980). This means that the amount of enzyme present i n vivo i s subject to modification at several points, which greatly enhances both the complexity and f i n e control of t h i s system. It i s of interest that the forms of P-450 present i n female r a t s have been shown to be d i f f e r e n t from those i n males with respect to substrate a c t i v i t y (Kamataki et a l . , 1981, 1982). In addition to a sex dif f e r e n c e , the presence of the P-450 isozyme responsible f o r the 16a-hydroxylation of testosterone i n the adult male was shown to be a function of neonatal imprinting by androgens (Chao and Chung, 1982; Chung and Chao, 1980). These inve s t i g a t o r s also noted that no q u a l i t a t i v e differences were evident i n the column e l u t i o n p r o f i l e s of P-450 from neonatally imprinted or non-imprinted r a t s . It was suggested that these differences i n substrate s p e c i f i c i t y between si m i l a r forms of P-450 in male and female r a t s are responsible for sex differences i n mixed function oxidase a c t i v i t i e s i n the adult. Based on t h i s type of data, one may speculate that the moderate a f f i n i t y component may be associated with a P-450 isozyme that i s imprinted and s p e c i f i c to the male, and also that BP i s included in i t s substrate p r o f i l e . This i s of int e r e s t considering the reports of Sloop et a l . , (1983) that the HCLA s i t e i s neonatally imprinted. In t h i s case, we would not expect to f i n d the moderate a f f i n i t y component i n the d i f f e r e n t i a t e d female. S i m i l a r l y the response to inducing agents may be d i f f e r e n t with respect to P-450 isozyme 144 substrate s p e c i f i c i t y i n male and female r a t s . Therefore, an induction of AHH a c t i v i t y i n the female may not be r e l a t e d to the male imprinted form of P-450. The p l e i o t r o p i c response of the female would not be expected to include production of the moderate a f f i n i t y component. As a r e s u l t , we would expect to observe an increase i n AHH a c t i v i t y without a concomitant detection of the moderate a f f i n i t y component, i n c e r t a i n circumstances. 7) Proposed experiments We have hypothesized that the moderate a f f i n i t y protein i s associated with the l e v e l s of c e r t a i n hepatic enzyme a c t i v i t i e s i n the adult male. Studies by Eagon et a l . , (1980) have demonstrated a change in the ligand s p e c i f i c i t y of the 4S protein following gonadectomy of the male. This suggests that the moderate a f f i n i t y protein may not simply be reduced i n capacity following gonadectomy but that a conformational change may occur. Therefore, the ligand s p e c i f i c i t y of the moderate a f f i n i t y component should be investigated following procedures such as gonadectomy and hypophysectomy to determine i f the binding s i t e i s representative of the same component present i n the sham-operated animal. I f as Eagon et a l . , (1980) report the moderate a f f i n i t y component becomes DES-sensitive, then i t would necessitate re-evaluation of the biochemical and p h y s i o l o g i c a l c h a r a c t e r i s t i c s of the protein. It would also be of int e r e s t to determine i f a neonatally gonadectomized adult male re t a i n s the testosterone r e v e r s a l of the reduction i n capacity of the moderate a f f i n i t y component 145 observed following gonadectomy of the adult. This would examine the p o s s i b i l i t y of imprinting suggested by Sloop et a l . , (1983). If neonatal gonadectomy does i r r e v e r s i b l y a b o lish the male moderate a f f i n i t y component, then administration of testosterone to a neonatally gonadectomized female during the f i r s t week of l i f e should be attempted. This would be done in an e f f o r t to induce production of the moderate a f f i n i t y component in the adult female following testosterone challenge. If the female did respond in t h i s fashion then i t would confirm the imprinting process. The e f f e c t s of glucocorticoids and thyroxine should be studied to further c l a r i f y the hormonal m i l i e u required for the maintenance of the estrogen receptor and the moderate a f f i n i t y component following hypophysectomy.; These..hormones., in./addition to GH, have been reported to be necessary for feminization of hepatic metabolism and maintenance of the estrogen receptor. With. . ' the addition of DHT, these hormones are-required for the maintenance of the DHT binding protein. This i s inconsistent with a feminization of metabolism, although, as previously discussed, t h i s discrepancy could be due to the hormone regimen. In any case Roy et a l . , (1973) reported that t h i s combination of hormones restored the DHT binding protein. If t h i s e f f e c t could be repeated for the moderate a f f i n i t y component then i t may be possible to observe an increase in AHH a c t i v i t y following administration of testosterone to the hypophysectomized animal. We have also presented preliminary r e s u l t s on the i n t e r a c t i o n of sex s t e r o i d binding proteins with dioxin congeners. Using dioxin congeners, we have noted differences between the male and female i n v i t r o binding c h a r a c t e r i s t i c s . 146 This suggests that the moderate a f f i n i t y component may be i n t e r f e r i n g with dioxin binding to the estrogen receptor in the male. Examination of in v i t r o binding of dioxins i n the gona-dectomized male should resolve t h i s question. The r a t i o n a l e behind t h i s proposal i s that gonadectomy reduces the capacity of the moderate a f f i n i t y component but does not a f f e c t the estrogen receptor .torancappreclable.-degree. TCDD i s a very potent inducer of the Ah receptor and AHH a c t i v i t y . We have established that TCDD binds to the estrogen receptor. It would be of interest to induce both male and female r a t s with TCDD and measure the hepatic c y t o s o l i c estrogen binding parameters as TCDD may well induce the-estrogen receptor. We have suggested that the moderate a f f i n i t y component i s rel a t e d to the c y t o s o l i c 3-MC/BP binding protein. I t would be of i n t e r e s t to conduct incubations using 3-MC and e s t r a d i o l 3 (separately) as competitors to [ H]-3—MC binding. P a r a l l e l incubations should be conducted using 3-MC and e s t r a d i o l (separately) 3 as competitors to [ H] - e s t r a d i o l binding. By examining the changes i n the binding parameters, following p h y s i o l o g i c a l manipulation of the animals, one could determine the r e l a t i o n s h i p of these proteins. Ultimately, i s o l a t i o n , p u r i f i c a t i o n and amino acid sequencing of the various binding proteins must be ca r r i e d out. S p e c i f i c monoclonal antibody techniques would prove invaluable for further b i o l o g i c a l studies. 147 8. P h y s i o l o g i c a l relevance We have suggested that the moderate a f f i n i t y component i s a male s p e c i f i c sex st e r o i d binding protein that i s regulated by both hypothalamic-hypophyseal and gonadal f a c t o r s . While the moderate a f f i n i t y component may possess "functional i d e n t i t y with c y t o s o l i c 3-MC/BP binding p r o t e i n s , i t does appear to require the presence of other factor(s) to exhibit a response to testosterone. As demonstrated in the hypophysectomy model which res u l t e d i n the loss of the estrogen receptor, testosterone was unable to produce a response even though the moderate a f f i n i t y component was present. Therefore, a high a f f i n i t y , low capacity receptor may be required for t e s t o -sterone to e l i c i t a response, and, l i k e the estrogen receptor, i t may be l o s t following hypophysectomy. This p o s s i b i l i t y i s supported by recent reports that have indicated the presence of a high a f f i n i t y , low capacity androgen receptor i n the l i v e r (Eagon et a l . , 1983; E i s e n f e l d et a l . , 1983; Decker et a l . , 1983). Therefore, the hepatic moderate a f f i n i t y component may be si m i l a r i n function to a binding protein found in the uterus. Clark et a i . , . (19.78) , and Watson and Clark (1980) have characterized a 4S estrogen binding component i n rat uterus and mouse mammary tumor, r e s p e c t i v e l y . This binding protein (type II) was present i n both c y t o s o l i c and nuclear f r a c t i o n s and was shown to be of higher capacity and lower a f f i n i t y than the type I-estrogen receptor s i t e . The type II s i t e was not capable of nuclear translocation. Markaverich and Clark (1979) reported that these s i t e s act to increase the l o c a l concen-t r a t i o n of e s t r a d i o l a v a i l a b l e for translocation or nuclear i n t e r a c t i o n . Furthermore, the type II s i t e was correlated with the 1 4 8 delayed and prolonged a c t i v a t i o n of RNA polymerase I and II as well as uterine hypertrophy and hyperplasia (Hardin et a l . , 1976). These authors reported that a single i n j e c t i o n of e s t r a d i o l produced an elevation of the nuclear type I (estrogen receptor) s i t e and a sustained elevation of the type II s i t e . This correlated with increased uterine wet weight. However, e s t r i o l administered s i m i l a r l y , did not produce any elevation in type II binding nor did i t stimulate uterine growth or produce a prolonged elevation in RNA polymerase I and I I . Thus i t was concluded that uterine growth was associated with sustained elevations in type II binding. This i s of i n t e r e s t when considering the actions of estrogens on the hypothalamus and p i t u i t a r y . Kelner and Peck J r . (1981) , have reported that neither the hypothalmus or p i t u i t a r y contain the type II s i t e . Furthermore, administration of e s t r a d i o l did not produce an a c t i v a t i o n of RNA polymerase I. RNA polymerase II exhibited a transient a c t i v a t i o n with a time course that correlated with nuclear accumulation of e s t r a d i o l (Kelner et a l . , 1980). The authors speculate that the type II s i t e acts to prolong nuclear retention of e s t r a d i o l thus increasing genetic i n t e r a c t i o n . This would allow for prolonged a c t i v a t i o n of RNA polymerase I and IT r e s u l t i n g in the end p l e i o t r o p i c responses: increased protein synthesis, hypertrophy and hyperplasia. Furthermore, the type II s i t e s i n rat uterine cytosol (Eriksson et a l . , 1978) may act in a similar fashion" that i s , to prolong elevated estrogen l e v e l s , creating an estrogen r i c h environment for nuclear t r a n s l o c a t i o n by the estrogen receptor. 149 That the function of the hepatic moderate a f f i n i t y component may be s i m i l a r to that suggested for the uterine, type.11 srt;e i s supported by the s t r i k i n g s i m i l a r i t y (both p h y s i o l o g i c a l l y and biochemically) between the moderate a f f i n i t y component and the male hepatic DHT and e s t r a d i o l binding protein previously described. This DHT binding protein i s correlated with synthesis of the urinary protein c ^ - g l o b u l i n . M i l i n and Roy(1973)and Roy et al.,(l974)reported t h i s binding protein to be absent in immature male and female, adult female and pseudohermaphrodite r a t s . This protein was shown to be androgen dependent, reduced by gonadectomy in a testosterone r e v e r s i b l e manner. P a r a l l e l changes were seen for urinary output of o ^ - g l o b u l i n . Administration of e s t r a d i o l to intact adult male r a t s resulted in a decrease in DHT binding as well as urinary output of c ^ - g l o b u l i n . Roy et a l . , (1976) have correlated the administration of DHT to a r i s e in urinary o ^ ^ g l o b u l i n and an increase in hepatic trans-l a t a b l e mRNA for o ^ - g l o b u l i n . Furthermore, Roy et a l . , (1973) have observed that hypophysectomy reduced o ^ ^ g l o b u l i n synthesis which could only be restored following simultaneous administration of GH, thyroxine, C o r t i s o l and DHT. In conclusion, there i s a s i m i l a r i t y between ligand s p e c i f i c i t y , apparent Kd, sedimentation c o e f f i c i e n t , hormonal regulation, and p h y s i o l o g i c a l c o r r e l a t i o n between the moderate a f f i n i t y component and the DHT binding protein. This leads .one to;speculate that they are the same binding s i t e . This s i t e has been shown by others to be involved i n the t r a n s l a t i o n process and, therefore, may provide a function i n the l i v e r s i m i l a r to that described for the 150 uterine s i t e II component. A l t e r n a t i v e l y , binding to the moderate a f f i n i t y component may retard the metabolic degradation of s t e r o i d and s t e r o i d - l i k e compounds. This may allow them to act i n the cytosol at the l e v e l of p o s t - t r a n s l a t i o n a l modification of protein synthesis as suggested by Mainwaring and Irving (1980) for 5a-reduced steroids i n chick blastoderm, i . e . , to increase t r a n s l a t i o n of s p e c i f i c mRNA coded for s p e c i f i c proteins. We cannot p r e c i s e l y define the r o l e of the hepatic moderate a f f i n i t y component-in the male. However, a reasonable p o s s i b i l i t y can be suggested. The hepatic androgen receptor recently described by Eagon et a l . , (1983), Ei s e n f e l d et a l . , (1983), and Decker et a l . , (1983) was detected in both male and female r a t s and not a l t e r e d by gonadectomy. These are unexpected c h a r a c t e r i s t i c s f or a receptor involved i n maintenance of a sex d i f f e r e n c e . However, we have shown the hepatic moderate a f f i n i t y component to be sex dependent and responsive to gonadectomy. As such, the androgen receptor does not have to exhibit a sex d i f f e r e n c e i f the r o l e of the moderate a f f i n i t y component i s to provide a stable pool of androgen a v a i l a b l e for binding to a higher a f f i n i t y , lower capacity androgen receptor for subsequent nuclear t r a n s l o c a t i o n . The action of e s t r a d i o l i n t h i s s i t u a t i o n may simply be to compete for binding to t h i s protein thereby reducing i n t r a c e l l u l a r concentration of androgen a v a i l a b l e for nuclear t r a n s l o c a t i o n by the androgen receptor. Whether binding to t h i s component prevents metabolic degradation of the steroid to bring the s t e r o i d into the proximity of a nuclear t r a n s l o c a t i o n 151 protein, or for p o s t - t r a n s c r i p t i o n a l modification, or both, i s unclear and- warrents further study. SUMMARY Despite inconsistencies with the female we have observed a c o r r e l a t i o n with the l e v e l s of AHH a c t i v i t y i n the male r a t and the capacity of the moderate a f f i n i t y component. These r e s u l t s were based on Scatchard a n a l y s i s , and not sucrose density gradient determinations as were many of the studies we have c i t e d . These include the following points: 1) an age dependent sex difference i n the capacity of the moderate a f f i n i t y component was observed. 2) a sex d i f f e r e n c e was also reported for AHH a c t i v i t y (and i s known to be age r e l a t e d , Bellward et a l . , 1982) 3) gonadectomy produced a testosterone r e v e r s i b l e decrease i n both AHH a c t i v i t y and the capacity of the moderate a f f i n i t y component i n the male 4) hypophysectomy abolished binding to the estrogen receptor, and reduced both AHH a c t i v i t y and the capacity of the moderate a f f i n i t y component i n the male 5) hypophysectomy abolished binding to the estrogen receptor, and increased both AHH a c t i v i t y and the capacity of the moderate a f f i n i t y component i n the female 6) the administration of rGH to both hypophysectomized male or female rats reduced AHH a c t i v i t y and abolished moderate a f f i n i t y binding; i n addition, bGH and oPRL reduced AHH a c t i v i t y i n the sham-operated male without 153 a l t e r i n g hepatic estrogen binding parameters the administration of mestranol to the adult intact male reduced AHH a c t i v i t y and abolished binding to both the estrogen receptor and the moderate a f f i n i t y component the administration of phenobarbital, spironolactone and 3-MC to the adult male r a t increased the capacity of the moderate a f f i n i t y component, although only 3-MC produced an increase i n AHH a c t i v i t y following administration of phenobarbital, 3-MC or spironolactone, the changes in AHH a c t i v i t y of the pseudohermaphrodite p a r a l l e l e d those of the s i m i l a r l y treated female and not the male no moderate a f f i n i t y component was detected in the p seu dohermaphr od i t i c rat 3 pimozide abolished [ H ] - e s t r a d i o l binding to the moderate a f f i n i t y component in the gonadectomized male no procedure, other than hypophysectomy, resulted in detection of the moderate a f f i n i t y component in the female both 3-MC and BP were shown to compete for s p e c i f i c e s t r a d i o l binding to the estrogen receptor i n males and females and the moderate a f f i n i t y component i n the male rat 154 dioxin congeners did not compete for s p e c i f i c e s t r a d i o l binding to the male estrogen receptor or moderate a f f i n i t y component as did 3-MC and BP a l l dioxin congeners demonstrated s p e c i f i c i t y for the hepatic estrogen receptor i n the female 155 BIBLIOGRAPHY Alvares, A.P., S c h i l l i n g , G., Garbut, A., and Kuntzman, R., Studies on the hydroxylation of 3,4-benzpyrene by hepatic microsomes, Biochem. 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The data contained in t h i s section were p a r a l l e l temperature time course and ligand s p e c i f i c i t y studies to those presented in the Results section. These studies were included because of t h e i r importance i n defining optimal assay conditions and hepatic e s t r a d i o l binding c h a r a c t e r i s t i c s . a) Determination of optimal incubation time at 4°C An incubation time course was conducted at 4°C to determine s p e c i f i c [ H ] - e s t r a d i o l binding to the hepatic c y t o s o l i c estrogen receptor i n male and female r a t s , and the moderate a f f i n i t y binding componenf.:in:.the:;male. As shown i n Figure A l , maximum binding to the estrogen receptor was reached by two hours in the female, and ninety minutes i n the male. Binding to the.estrogen receptor was stable for at l e a s t four hours i n both male and female r a t s . S p e c i f i c e s t r a d i o l binding to the male moderate a f f i n i t y component was found to be maximal by one hour (Figure A2). Therefore, for convenience a l l subsequent assays were incubated at 4°C for ninety minutes. 169 Figure A l . 3 Time course of s p e c i f i c [ H ] - e s t r a d i o l binding to the estrogen receptor at 4°C i n male and female rat l i v e r , 50% ammonium s u l f a t e f r a c t i o n . The binding i n each sex was determined on 3 a s i n g l e t i s s u e pool at 2 nM [ H ] - e s t r a d i o l ligand concentration and 100-fold excess e s t r a d i o l competitor. Points joined by a dashed l i n e were determined i n more than 1 t i s s u e pool. 170 MCUBATON "nME (hours) 171 Figure A2. 3 Time course of s p e c i f i c [ H ] - e s t r a d i o l binding to the moderate a f f i n i t y component at 4°C i n male rat l i v e r , whole cytosol f r a c t i o n . The binding was determined on a single t i s s u e pool 3 at 200 nM [ H ] - e s t r a d i o l ligand concentration and 100-fold excess e s t r a d i o l competitor. Points joined by a dashed l i n e were determined i n more than 1 t i s s u e pool. 172 173 b) Determination of ligand s p e c i f i c i t y i n female r a t hepatic cytosol The a b i l i t y of various unlabeled ligands to compete f o r 3 [ H ] - e s t r a d i o l binding to the hepatic c y t o s o l i c estrogen receptor in the ammonium su l f a t e f r a c t i o n and the moderate a f f i n i t y component in the whole cytosol f r a c t i o n were investigated i n the female r a t . As shown in Table A l , e s t r a d i o l was the most e f f e c t i v e competitor 3 f o r [ H ] - e s t r a d i o l binding to the estrogen receptor i n the ammonium sul f a t e f r a c t i o n . Moxestrol and DES competed s i m i l a r l y though l e s s e f f e c t i v e l y than e s t r a d i o l . Androgens, progestins and glucocorticoids 3 did not compete for [ H ] - e s t r a d i o l binding to the hepatic c y t o s o l i c estrogen receptor in the female r a t (Table A l ) . Competition for 3 s p e c i f i c [ H ] - e s t r a d i o l binding in the moderate a f f i n i t y range i s shown in Table A2. With the exception of e s t r a d i o l , none of the unlabeled steroids competed for radio-labeled e s t r a d i o l binding to female r a t whole c y t o s o l . 2) Determination of serum PRL l e v e l s by bioassay *Work was c a r r i e d out by Mr. P. Gout, Department of Biochemistry, University of B r i t i s h Columbia. Serum PRL l e v e l s were determined by bioassay in f i v e c o n t r o l and f i v e pergolid (0.1 mg/kg/day, i. p . for eleven days) treat female rats according to the method of Tanaka et a l . , (1980)."'' This assay Tanaka, T., Shiu, R.P., Gout, P.W., Beer, C.T., Noble, R.L., and Friesen, H.G., A new s e n s i t i v e and s p e c i f i c bioassay for lactogenic hormones: measurement of p r o l a c t i n and growth hormone in human serum, J. C l i n . Endocrinol. Metab. 51: 1058-1063, 1980. 174 TABLE A l o Ligand s p e c i f i c i t y of [ H ] - e s t r a d i o l binding s i t e s i n the 50% ammonium s u l f a t e (AS) f r a c t i o n of l i v e r from female r a t s . Samples of 50% f r a c t i o n were incubated f o r 90 minutes at 4°C with [ 3 H ] - e s t r a d i o l (0.5 nM) alone or i n the presence of 10, 100 or 1000-fold excess of competing ligand. % of control binding"'" in the presence of Competitor e x c e s s competitor 10-fold 100-fold 1000-fold co n t r o l 100 100 100 e s t r a d i o l 63 36 24 d i e t h y l s t i l b e s t r o l 61 61 63 moxestrol 79 64 64 testosterone 100 100 100 dihydrotestosterone 100 100 100 androstenedione 100 100 100 methyltrienelone 100 100 100 progesterone 100 99 96 promegestone 98 100 100 triamcinolone 100 100 100 dexamethasone 100 100 100 The binding of [ H ] - e s t r a d i o l in the presence of competitor i s given as % of control values (control = binding in the absence of competitor). TABLE A2 Ligand s p e c i f i c i t y of [ H ] - e s t r a d i o l binding s i t e s i n the whole cytosol f r a c t i o n of l i v e r from female r a t s . Samples of whole cytosol were incubated f o r 90 minutes at 4°C with [ 3 H ] - e s t r a d i o l (50 nM) alone or i n the presence of 10, 100, or 1000-fold excess of competing ligand. % of control b i n d i n g 1 in the presence of excess competitor Competitor 10-fold 100-fold 1000-fold c o n t r o l 100 100 100 e s t r a d i o l 79 76 100 d i e t h y l s t i l b e s t r o l 80 81 100 moxestrol 90 89 92 testosterone 100 100 100 dihydrotestosterone 100 100 100 an dr o s t en e d i on e 100 100 100 methyltrienelone 99 97 100 progesterone 95 99 100 promegestone 91 98 100 triamcinolone 99 98 100 dexamethasone 98 100 100 The binding of [ H ] - e s t r a d i o l in the presence of competitor i s given as % of control values (control = binding in the absence of competitor). 176 assay u t i l i z e s c e l l s from a transplantable lymphoma produced in lymph nodes of estrogenized male Noble r a t s (and so designated Nb2 Node c e l l s ) . The basis of t h i s bioassay i s that the growth of the Nb 2 Node lymphoma c e l l culture i s s p e c i f i c a l l y stimulated by 2 lactogenic hormones, (Gout et a l . , 1980). Serum was obtained as described in the methods section. D e t a i l s of the bioassay were as follows. Approximately 24 hours before use in bioassay, Nb 2 Node lymphoma c e l l s were transferred to Fischers medium which was supplemented with f e t a l c a l f serum (1%) , horse serum (10%), 2-mercaptoethanol (.0.1 mM), p e n i c i l l i n (50 U/ml) , streptomycin (50 pg/ml) and incubated f o r 24 hours at 37°C. C e l l s were then c o l l e c t e d by.centrifugation and resuspended to 1-2 x 10^ c e l l s / m l in Fischers medium supplemented as above with the exception of the f e t a l c a l f serum. Aliquots (2 ml) were transferred to culture dishes for determination of growth promoting a c t i v i t y . Sera to be assayed for growth promoting a c t i v i t y were added to the culture d i s h in 50 p i phosphate-buffered s a l i n e that contained bovine serum albumin (0.1%). Cultures were incubated in a CO2 incubator for 72 hours at 37°C. C e l l numbers were determined using a Coulter counter (Counter E l e c t r o n i c s , Inc., Hialeah, FL.). Growth i n control culture was zero following 72 hour incubat ion. Results from t h i s assay indicated a greater than 90% reduction i n serum PRL l e v e l s (from 23.5 ± 2.7 to 1.6 ± 0.3 ng/ml PRL) following the administration of pergoiide. 2 Gout, P.W., Beer, C.T., and Noble, R.L., Prolactin-stimulated growth of c e l l cultures established from malignant Nb rat lymphomas, Cancer Res., 40: 2433-2436, 1980. 17-7 TABLE A3 Spe c i f i c a t i o n s of p i t u i t a r y hormones used i n the continuous infusion studies. PERCENT HORMONE CONTAMINATION Bovine Growth Hormone (bGH) NIH-bGH-B18 (.81 I.U./mg) Rat Growth Hormone (rGH) NIAMDD-rGH-B-7 (1.6 I.U./mg) Rat P r o l a c t i n (rPRL) NIADDK-rPRL-B-4 (20.0 I.U./mg) Ovine P r o l a c t i n (oPRL) NIAMDD-oPRL-15 (30.5 I.U./mg) (by weight) TSH LH 6 3 ** .1% .0%** .0%** PRL 62 TSH 0 .1%* LH 0 .5%* FSH 0 .5%* PRL 0 .1%* TSH 0 .1%* LH 0 .5%* FSH 0.5%* GH 1 .5% TSH 0 .17* LH 0 .57* FSH 0 .5%* GH 0 .5%* * Determined by radioimmunoassay **Determined by bioassay Papers M.J. Finlayson* and G.D. Bellward, Introductory Remarks: Cytochrome P-450 Mixed Function Oxidases, Proc. West. Pharmacol. Soc. 23, 1-2 (1980). G.D. Bellward, R.C.K. Pak, L.S. Gontovnick, B.L. Warren, M.J.P. Finlayson, and G.I. Sunahara, Endogenous Mechanisms for the Regulation of Hepatic Microsomal Drug and Steroid Mixed Function Oxidases, in "The Combined Effect of Xenobiotics, NRC, pubs." pg 97-126 (1981). M.J. Finlayson and G.D. Bellward, Effects of Perinatal Exposure of Wistar Rats to l-tx-Acetylmethadol-HCl on Hepatic Microsomal Mixed Function Oxidase Enzyme A c t i v i t y in the Adult, Drug Metab. Dispos., 10, 74-76 (1982). Betty L. Warren, Raphael Pak, Malcolm Finlayson, Larry Contovnick, Geoffrey Suna-hara and Gall D. Bellward, Different Effects of Diabetes on Microsomal Metabolism of Various Substrates: Comparison of Streptozotocln and Spontaneously Diabetic Wistar Rats, Biochem. Pharmacol., 3 2 , 3 2 7 - 3 3 5 ( 1 9 8 3 ) . M.J. Finlayson and G.D. Bellward, Comparison of Sex Steroid Binding ln Hepatic Cytosol with Changes in Hepatic Monooxygenase Activity in the Rat, ( S u b m i t t e d , M o l . P h a r m a c o l .) Abstracts G.D. Bellward and M.J. Finlayson*, The Effects of 1-oc-Acetylmethadol (LAAM) on Neonatal Androgen Induced Imprinting in the Rat, Pharmacologist 22, 243 (1980). B.L. Warren, M. Finlayson, L. Gontovnick, R. Pak, G. Sunahara and G.D. Bellward, Microsomal Monooxygenase (MO) Activities in Genetically (BB) Diabetic Wistar Rats, Pharmacologist 23, 165 (1981). Raphael Pak, Malcolm Finlayson, Larry Gontovnick, Geoffery Sunahara, Betty Warren and Gail Bellward, Effects of Hypophysectomy on Hepatic Microsomal Drug and Steroid Metabolizing Enzymes in Rats, Pharmacologist 23, 168 (1981). G.D. Bellward, M.J. Finlayson, L.S. Gontovnick, R.C. Pak, G.I. Sunahara and B.L. Warren, Rat Hepatic Microsomal Enzyme Activities in Various Physiological Models, Can. Fed. Biol. Sci. (Proc.) 25, 391 (1982). B.L. Warren, M.J. Finlayson* and G.D. Bellward, Characterization of High and Moderate Affinity Binding of Sex Hormones in Rat Hepatic Cytosol, Can. Fed. Biol.  Sci. (Proc.) 25, 392 (1982). M.J. Finlayson* and G.D. Bellward, Hormonal Modulation of Rat Hepatic Monooxygen-ase Activity: Possible Control Mechanisms, Fed. Am. Soc. Exp. Bio l . (Proc), Chicago, April (1983). * Presented by M.J. Finlayson 

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