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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 [ H]-ESTRADIOL J  BINDING PROTEINS TO HORMONAL AND DRUG MODULATION OF HEPATIC MICROSOMAL ARYL HYDROCARBON HYDROXYLASE IN THE RAT by •MALCOLM JOHN PAUL FINLAYSON B.A., San J o s e S t a t e  University,  1976  M . S c , U n i v 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 h e s i s as conforming  to the required  standard  THE UNIVERSITY OF BRITISH COLUMBIA August, 1983 ©  MALCOLM JOHN PAUL FINLAYSON, 1983  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  requirements f o r an advanced degree a t the  the  University  of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make it  f r e e l y a v a i l a b l e f o r reference  and  study.  I further  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may department or by h i s or her  be granted by the head o f representatives.  my  It is  understood t h a t copying o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l gain  s h a l l not be  allowed without my  permission.  Department of  Pharmaceutical Sciences  The  U n i v e r s i t y of B r i t i s h Columbia  1956  Main Mall  Vancouver, Canada V6T 1Y3  DE-6  (3/81)  written  ABSTRACT  P i t u i t a r y hormones are known to a l t e r sex steroid receptor levels i n the l i v e r , and possibly the actions of the steroids as well.  Recently,  two classes of estrogen binding proteins have  been characterized i n male r a t 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 , capacity sex steroid binding component  higher  (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 i n r a t hepatic cytosol.  Therefore, we have investigated the relationship of the  moderate a f f i n i t y component to sex steroid modulation of hepatic a r y l hydrocarbon hydroxylase (AHH) a c t i v i t y as a possible control mechanism.  Because of the sexual dimorphism for hepatic drug  and steroid metabolism known to occur i n rat l i v e r , we chose this model to study. We have shown that no sex difference exists f o r the binding of pH]-estradiol to the estrogen receptor from either immature or adult r a t s .  However, the moderate a f f 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 i n adult female or immature rats of either sex. s i t e could normally only be measured i n the adult male.  This  These  findings were consistent with the age and sex dependent elevation of male AHH a c t i v i t y .  We have also observed that gonadectomy of  iii  the male reduced the l e v e l s o f AHH a c t i v i t y and t h e c a p a c i t y of the moderate a f f i n i t y component i n a t e s t o s t e r o n e r e v e r s i b l e f a s h i o n . These r e s u l t s were obtained  using  e i t h e r unlabeled  e s t r a d i o l or  3 dihydrotestosterone binding.  (DHT) as competitors f o r [ H ] - e s t r a d i o l  A d m i n i s t r a t i o n of m e s t r a n o l reduced AHH a c t i v i t y and  the c a p a c i t y of the moderate a f f i n i t y  component i n the male.  moderate a f f i n i t y component was n o t d e t e c t e d  The  i n t h e pseudoherma-  p h r o d i t i c r a t which resembled t h e female, r a t h e r than the male, with respect  to c o n t r o l and induced AHH  activity.  Hypophysectomy- of the female r e s u l t e d i n an i n c r e a s e i n AHH and  d e t e c t i o n of the moderate a f f i n i t y component.  activity  Hypophysectomy  of the male reduced b o t h t h e c a p a c i t y of the moderate a f f i n i t y component and AHH a c t i v i t y .  U n l i k e the gonadectomized male,  t e s t o s t e r o n e 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 or the c a p a c i t y of the moderate a f f i n i t y sectomized r a t . reversed  component i n the hypophy-  Continuous i n f u s i o n of r a t growth hormone  the e f f e c t of hypophysectomy on the i n c r e a s e d AHH  and c a p a c i t y of the moderate a f f i n i t y Administration  activity  (rGH) activity  component i n the female.  of rGH t o the hypophysectomized male a b o l i s h e d the  d e t e c t i o n of the moderate a f f i n i t y component and reduced AHH to c o n t r o l female l e v e l s .  activity  T h i s suggested rGH may be the p i t u i t a r y  hormone i n v o l v e d i n p r o d u c t i o n  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 m o d u l a t i o n of AHH a c t i v i t y by p e r i p h e r a l sex s t e r o i d s , and the r e g u l a t i o n of these parameters by rGH. shown, the c a p a c i t y of the moderate a f f i n i t y  We have  component t o vary- i n  iv  a manner t h a t p a r a l l e l e d changes i n h e p a t i c AHH d i f f e r e n t p h y s i o l o g i c a l models. were not found to be  activity  in  Changes i n the estrogen  c o n s i s t e n t w i t h changes i n AHH  receptor  activity in  these models. We  conclude that the moderate a f f i n i t y component i s comparable  t o the male h e p a t i c c y t o s o l i c DHT-binding p r o t e i n .  Furthermore,  t h i s component i s a s s o c i a t e d w i t h sex s t e r o i d a c t i o n on AHH  hepatic  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  the estrogen  have a l s o shown t h i s component as w e l l as  r e c e p t o r , to bind p o l y c y c l i c aromatic  hydrocarbons.  Both 3-methylcholanthrene and benzo[a]pyrene competed f o r  3 [ H ] - e s t r a d i o l b i n d i n g to the estrogen component.  r e c e p t o r and moderate a f f i n i t y  In a d d i t i o n , d i o x i n congeners demonstrated  f o r the e s t r o g e n r e c e p t o r i n the female.  specificity  However, t h i s was  not  observed f o r the estrogen r e c e p t o r 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 p r e s e n t l y u n c l e a r .  Gail. D, Bell,ward, Supervisor  Ph.D.  V  TABLE OF CONTENTS  PAGE  ABSTRACT  i i  LIST OF TABLES  x  LIST OF FIGURES LIST OF ABBREVIATIONS AND TRIVIAL  xiv CHEMICAL NAMES  ACKNOWLEDGEMENT  .xvii  INTRODUCTION 1.  xv  1  Sex dependent d i f f e r e n c e s i n h e p a t i c mixed function oxidase a c t i v i t y  1  2.  Neonatal androgen induced i m p r i n t i n g  3  3.  Involvement o f t h e hypophyseal-hypothalamic a x i s i n t h e maintenance o f sex dependent d i f f e r e n c e s i n h e p a t i c metabolism  5  a.  Pituitary effects  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 h e p a t i c metabolism  5.  I n t e r a c t i o n of sex s t e r o i d s and h e p a t i c  9  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 o f p i t u i t a r y hormones on h e p a t i c sex s t e r o i d b i n d i n g  20  8.  Hepatic sex s t e r o i d - b i n d i n g p r o t e i n s  22  9.  Hypothesis  24  vi  PAGE  MATERIALS AND  METHODS  26  1.  Chemical  2.  Animals  27  3.  Animal treatments  28  4.  H e p a t i c microsomal enzyme assays  29  a.  H e p a t i c microsomal p r o t e i n p r e p a r a t i o n  29  b.  A r y l hydrocarbon  30  c.  T e s t o s t e r o n e A4 r e d u c t a s e a c t i v i t y  5.  and r e a g e n t s  26  hydroxylase a c t i v i t y  31  H e p a t i c sex s t e r o i d b i n d i n g assays  32  a.  Preliminary studies  32  b.  H e p a t i c e s t r o g e n r e c e p t o r and moderate a f f i n i t y b i n d i n g component assay  34  A n a l y s i s of b i n d i n g d a t a  36  6.  D e t e r m i n a t i o n of p r o t e i n content  38  7.  Serum hormone l e v e l s  38  8.  Statistical  39  c.  analysis  RESULTS 1.  4  I n i t i a l c h a r a c t e r i z a t i o n of h e p a t i c e s t r o g e n b i n d i n g components a.  Comparison of d i f f e r e n t  0  ^0  estrogen r e c e p t o r  assays b.  Time, temperature  c.  and pH  studies  ^  4  Freezing studies  4  8  d.  Competitor  4  8  e.  B i n d i n g t o u t e r u s , b o v i n e serum and plasma  studies albumin  51  vii  PAGE  2.  E f f e c t s of p h y s i o l o g i c a l manipulation h e p a t i c estrogen b i n d i n g components a. b.  3.  b.  Age and sex r e l a t e d m o d u l a t i o n of h e p a t i c estrogen binding  51  P i t u i t a r y modulation o f h e p a t i c binding  55  estrogen  70  Sex r e l a t e d m o d u l a t i o n o f h e p a t i c AHH and testosterone A 4 reductase a c t i v i t i e s Endocrine  manipulation  70  o f h e p a t i c enzyme  activity  76  i.  E f f e c t o f p e r g o l i d e i n a d u l t female r a t s  76  E f f e c t o f p e r g o l i d e i n pre-pubescent female r a t s  80  E f f e c t o f pimozide i n a d u l t male r a t s  82  ii. iii.  5.  51  C h a r a c t e r i z a t i o n o f h e p a t i c 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.  4.  on  S t u d i e s r e l a t i n g h e p a t i c AHH a c t i v i t y to h e p a t i c c y t o s o l i c e s t r o g e n b i n d i n g components  84  a.  E f f e c t of p e r g o l i d e  84  b.  Effect  86  c.  E f f e c t o f continuous hormones  of pimozide i n f u s i o n of p i t u i t a r y  88  In v i v o and i n v i t r o e f f e c t s o f x e n o b i o t i c s on h e p a t i c AHH a c t i v i t y and c y t o s o l i c estrogen b i n d i n g parameters  9  3  a.  Induction  9  3  b.  Inhibition  9  8  c.  The e f f e c t o f p h y s i o l o g i c a l m a n i p u l a t i o n i n v i t r o b i n d i n g o f 3-MC  d.  R e l a t i o n s h i p o f estrogen t o Ah-receptor  binding  on 1  0  0  components 100  viii  PAGE  106  DISCUSSION 1.  2.  Estrogen b i n d i n g s t u d i e s : C h a r a c t e r i z a t i o n and comparison o f h e p a t i c c y t o s o l i c estrogen b i n d i n g components w i t h p r e v i o u s l y p u b l i s h e d r e s u l t s E f f e c t s of p h y s i o l o g i c a l m a n i p u l a t i o n estrogen a.  i. ii. b.  3.  on h e p a t i c m  binding  Gonadectomy  106  e f f e c t s on h e p a t i c e s t r o g e n  binding  m  I m p r i n t i n g o f the moderate a f f i n i t y component Sucrose d e n s i t y g r a d i e n t v e r s u s binding studies  E f f e c t s of hypophysectomy on h e p a t i c binding  competitive estrogen  Comparison o f h e p a t i c enzyme a c t i v i t y t o h e p a t i c estrogen b i n d i n g components a.  General  c o n c l u s i o n s from p u b l i s h e d o b s e r v a t i o n s  b.  Age and sex dependency  c.  Androgen dependency  d. e.  E f f e c t of estrogens Pseudohermaphroditic r a t model  f.  Hypophysectomy model  ion  122  1 o/  4.  R e l a t i o n s h i p of p i t u i t a r y hormones t o h e p a t i c a c t i v i t y and e s t r o g e n b i n d i n g  AHH  a.  Background  b.  E f f e c t s of pimozide  c.  E f f e c t s of p e r g o l i d e  128  d.  P i t u i t a r y hormone replacement: E f f e c t s on h e p a t i c 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  Hormone replacement i n female r a t s  133  ii.  126 ,  127  ix  PAGE  5.  The e f f e c t s of x e n o b i o t i c s on h e p a t i c AHH a c t i v i t y and c y t o s o l i c e s t r o g e n b i n d i n g parameters  135  a.  In v i t r o e f f e c t s  135  i.  Ah r e c e p t o r  135  Hepatic  137  ii. b.  c y t o s o l i c BP/3-MC b i n d i n g p r o t e i n  I n v i v o e f f e c t s of x e n o b i o t i c s  140  6.  M u l t i p l i c i t y of Cytochromes P-450  142  7.  Proposed  144  8.  P h y s i o l o g i c a l relevance  experiments  147  SUMMARY  152  BIBLIOGRAPHY  155  APPENDIX I  I  1)  6  8  A d d i t i o n a l s t u d i e s o f [ H ] - e s t r a d i o l b i n d i n g to the e s t r o g e n r e c e p t o r and moderate a f f i n i t y component i n r a t h e p a t i c c y t o s o l  168  a)  Determination  of optimal  168  b)  Determination  of l i g a n d s p e c i f i c i t y  i n c u b a t i o n time a t 4°C i n female  rat hepatic cytosol  173  (Assays performed by Dr. B. Warren) 2)  Determination (Assays  o f serum PRL l e v e l s by b i o a s s a y  performed by Mr. P. Gout)  173  X  LIST OF TABLES  PAGE  Table 1  The apparent Kd and c a p a c i t y of [ H ] - e s t r a d i o l b i n d i n g i n d i f f e r e n t f r a c t i o n s of a d u l t male and female r a t l i v e r u s i n g v a r i o u s c o m p e t i t o r s ... 42  Table 2  The apparent Kd and c a p a c i t y o f [ H ] - e s t r a d i o l b i n d i n g t o d i f f e r e n t f r a c t i o n s of a d u l t male r a t l i v e r  44  Time course of s p e c i f i c b i n d i n g a t 25°C  46  Table 3  Table 4  T a b l e 5,  Table 6  Table 7  Table 8  Table 9  T a b l e 10  3  [ H]-estradiol  The e f f e c t o f pH on the apparent Kd and c a p a c i t y of t h e h e p a t i c estrogen r e c e p t o r and moderate a f f i n i t y component  47  The e f f e c t of f r e e z i n g on the apparent Kd and c a p a c i t y o f the estrogen r e c e p t o r 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 b i n d i n g to the e s t r o g e n r e c e p t o r and moderate a f f i n i t y component i n the a d u l t male r a t  50  A comparison of t h e apparent Kd and c a p a c i t y of t h e h e p a t i c c y t o s o l i c e s t r o g e n r e c e p t o r and moderate a f f i n i t y component i n male, female, and pseudohermaphroditic r a t s  53  The e f f e c t of age and gonadectomy on the apparent Kd and c a p a c i t y of h e p a t i c c y t o s o l i c e s t r o g e n r e c e p t o r and moderate a f f i n i t y b i n d i n g component i n male and female r a t s ....  . 54  The e f f e c t o f gonadectomy and t e s t o s t e r o n e replacement on the apparent Kd and c a p a c i t y of t h e h e p a t i c c y t o s o l i c e s t r o g e n r e c e p t o r and moderate a f f i n i t y b i n d i n g component i n male and female r a t s  56  The e f f e c t of hypophysectomy on the b i n d i n g c h a r a c t e r i s t i c s of t h e h e p a t i c c y t o s o l i c e s t r o g e n r e c e p t o r i n male and female r a t s ..  59  xi:_  PAGE  T a b l e 11  Table 12  Table  Table  Table  Table  13  14  15  16  The e f f e c t of hypophysectomy on the b i n d i n g c h a r a c t e r i s t i c s o f the moderate a f f i n i t y component i n male and female rats  61  The e f f e c t of hypophysectomy on t h e apparent Kd and c a p a c i t y of the h e p a t i c c y t o s o l i c estrogen r e c e p t o r and moderate a f f i n i t y b i n d i n g component a t f o u r weeks p o s t - s u r g e r y i n male and female r a t s  64  The e f f e c t o f t e s t o s t e r o n e and growth hormone on the b i n d i n g c h a r a c t e r i s t i c s of the h e p a t i c c y t o s o l i c estrogen r e c e p t o r i n the hypophysectomized r a t s .  65  The e f f e c t of t e s t o s t e r o n e and growth hormone on the b i n d i n g c h a r a c t e r i s t i c s of the h e p a t i c 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  67  The e f f e c t of p h e n o b a r b i t a l and s p i r o n o l a c t o n e on h e p a t i c AHH a c t i v i t y i n male, female and psudohermaphroditic r a t s  ^1  The e f f e c t o f 3-methylcholanthrene on h e p a t i c AHH a c t i v i t y i n male, female, and pseudohermophrodite  Table  Table  17  18  rats  The e f f e c t of hypophysectomy on h e p a t i c a c t i v i t y i n male and female r a t s  '  3  AHH  The e f f e c t of hypophysectomy and t e s t o s t e r o n e on h e p a t i c AHH a c t i v i t y i n male and female rats  Table  Table  19  20  T a b l e 21  7 ^  ^  4  7 s / J  The e f f e c t of 3-methylcholanthrene and p h e n o b a r b i t a l on h e p a t i c 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, p e r g o l i d e and t e s t o s t e r o n e on h e p a t i c AHH and t e s t o s t e r o n e A4 r e d u c t a s e a c t i v i t y i n female r a t s  79  The e f f e c t of pre-pubescent gonadectomy and p r o l a c t i n d e p l e t i o n on the a c t i o n o f t e s t o s t e r o n e on h e p a t i c AHH and t e s t o s t e r o n e A4 r e d u c t a s e a c t i v i t i e s i n t h e a d u l t female r a t  XXI  PAGE  Table 22  Table  Table  23  24  T a b l e 25  Table  Table  Table  26  27  28  T a b l e 29  The e f f e c t of gonadectomy, p i m o z i d e and t e s t o s t e r o n e on h e p a t i c AHH and t e s t o sterone A4 r e d u c t a s e a c t i v i t y i n a d u l t male r a t s  83  The e f f e c t of p e r g o l i d e and t e s t o s t e r o n e on h e p a t i c AHH a c t i v i t y and t h e apparent Kd and c a p a c i t y of t h e h e p a t i c c y t o s o l i c estrogen r e c e p t o r and moderate a f f i n i t y component i n a d u l t gonadectomized and sham-operated female r a t s  85  The e f f e c t of pimozide and t e s t o s t e r o n e on h e p a t i c estrogen b i n d i n g and h e p a t i c AHH a c t i v i t y i n sham and gonadectomized male r a t s Comparison of t h e e f f e c t of hypophysectomy and hormone replacement on h e p a t i c c y t o s o l i c estrogen b i n d i n g components and AHH a c t i v i t y i n male and female r a t s  89  The e f f e c t of 3-methylcholanthrene and s p i r o n o l a c t o n e on the apparent Kd and c a p a c i t y o f t h e h e p a t i c c y t o s o l i c estrogen r e c e p t o r and the moderate a f f i n i t y b i n d i n g component i n male and female r a t s  95  The e f f e c t o f 4 days treatment w i t h phenob a r b i t a l on the apparent Kd and c a p a c i t y of the h e p a t i c c y t o s o l i c estrogen r e c e p t o r and moderate a f f i n i t y b i n d i n g component i n male and female r a t s  96  The e f f e c t o f 10 days treatment w i t h m e s t r a n o l , p h e n o b a r b i t a l and 3-methylcholanthrene on h e p a t i c c y t o s o l i c estrogen b i n d i n g c h a r a c t e r i s t i c s and h e p a t i c AHH a c t i v i t y i n male and female rats  99  The e f f e c t of gonadectomy and t e s t o s t e r o n e replacement on l i g a n d s p e c i f i c i t y of the h e p a t i c c y t o s o l i c estrogen r e c e p t o r and moderate a f f i n i t y b i n d i n g component i n male and female r a t s  101  xiii  PAGE  T a b l e 30  Ligand s p e c i f i c i t y of t h e h e p a t i c c y t o s o l i c e s t r o g e n r e c e p t o r and t h e moderate a f f i n i t y s i t e f o r various competitors f o r [ H ] - e s t r a d i o l b i n d i n g i n the male and female r a t 103 3  T a b l e 31  T a b l e 32  T a ^ l e 33  Table A l  The apparent Kd and c a p a c i t y o f the e s t r o g e n r e c e p t o r i n a d u l t female r a t s as determined w i t h d i f f e r e n t d i o x i n congeners  104  Summary o f c o n t r o l v a l u e s f o r apparent Kd and c a p a c i t y o f the e s t r o g e n r e c e p t o r and moderate a f f i n i t y component  105  Summary o f r e l a t i v e changes i n c a p a c i t y of the e s t r o g e n r e c e p t o r , moderate a f f i n i t y b i n d i n g component and AHH a c t i v i t y r e l a t i v e t o t h e a d u l t male ( u n l e s s otherwise noted)  121  3 Ligand s p e c i f i c i t y o f [ H ] - e s t r a d i o l b i n d i n g 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 o f l i v e r from female r a t s  174  Ligand s p e c i f i c i t y of [ H] - e s t r a d i o l b i n d i n g s i t e s i n t h e whole c y t o s o l f r a c t i o n of l i v e r from female r a t s  175  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  3  T a b l e A2  T a b l e A3  xiv  LIST OF FIGURES  PAGE  Figure 1  Figure 2  E f f e c t o f p h y s i o l o g i c a l m a n i p u l a t i o n on the s p e c i f i c [ % ] - e s t r a d i o l b i n d i n g t o the moderate a f f i n i t y component  58  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 b i n d i n g t o the moderate a f f i n i t y component  60  E f f e c t o f hypophysectomy on the s p e c i f i c [ H ] - e s t r a d i o l binding to the estrogen receptor  69  3  Figure 3  3  Figure A l  F i g u r e A2  Time c o u r s e o f 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 s u l f a t e f r a c t i o n  170  Time c o u r s e o f s p e c i f i c [ H ] - e s t r a d i o l b i n d i n g t o the moderate a f f i n i t y component a t 4°C i n male r a t l i v e r , whole c y t o s o l f r a c t i o n  172  3  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  aldosterone  3,20-diketo-llf3,18-oxido-4-pregnene-18 , 2 1 - d i o l  androstenedione  hydroxylase  A4-androsten-3,17-dione  bGH  bovine growth hormone  BP  benzo [a]pyrene  2,7-DCDD DES dexamethasone  2,7-dichlorodibenzo-p-dioxin diethylstilbestrol 9-fluoro-llg,17,21-trihydroxy-16a-methylp r e g n a - l ,4-diene-3 ,20-dione  DCC  d e x t r a n coated c h a r c o a l  DHT  dihydrotestosterone  E2  estradiol  EDTA  ethylenediamine t e t r a a c e t i c  estradiol  1 3 5(10) A ' ' -estratrien-3,17g-diol  FSH  f o l l i c l e s t i m u l a t i n g hormone  GH  growth hormone  Gx  gonadectomized  hGH Hx  acid  human growth hormone hypophysectomized  Imm  immature ( l e s s than 30 days of age)  i.p.  intraperitoneally  methyltrienolone  17g-hydroxy-17-methylestra-4,9,ll-trien-3-one  xvi  mestranol  3-methoxy-19-nor-17a-pregna-1,3,5(10)-t r i e n e 2 0-yn-17-ol  3-MC  3-methylcholanthrene  OCDD  octachlorodibenzo-p-dioxin  oPRL  ovine  PAH  p o l y c y c l i c aromatic hydrocarbon  Perg  pergolide  Pim  pimozide  PRL  prolactin  progesterone  A4-pregnene-3,20-dione  Pseudo  pseudohermaphrodite  rGH  r a t growth hormone  rPRL  rat  prolactin  prolactin  s. c.  subcutaneously  T4  thyroxine  Test  testosterone  testosterone  &4-androsten-17p-ol-3-one  triamcinolone acetonide  9q-fluoro-llB,16a,17,21-tetra-ol-pregnal,4-diene-3,20-dione c y c l i c 16,17 a c e t a l w i t h acetone  xvii  ACKNOWLEDGEMENT  I w i s h t o express my deepest for  her i n s i g h t and guidance  graduate  training.  g r a t i t u d e t o Dr. G a i l  throughout  Bellward  t h e course o f my  I would a l s o l i k e t o thank Dr. B e t t y Warren  for  her c o l l a b o r a t i o n i n t h e s e s t u d i e s , and Dr. Edward Keenan  for  h i s i n t e r e s t and i n v a l u a b l e s u g g e s t i o n s .  indebted  t o Mrs.  assistance.  C h r i s t i n e Lee f o r her expert  F i n a l l y , I am secretarial  1  INTRODUCTION  1)  Sex dependent d i f f e r e n c e s i n h e p a t i c mixed f u n c t i o n  oxidase  activity  I t i s w e l l known t h a t t h e a c t i v i t i e s of c e r t a i n r a t h e p a t i c microsomal cytochrome P-450 dependent mixed f u n c t i o n  oxidases  e x h i b i t a sex d i f f e r e n c e i n t h e a d u l t r a t (Conney, 1967; Kato, 1974; Kato and G i l l e t t e , 1965  ; Quinn, 1958).  These d i f f e r e n c e s a r e  a t t r i b u t e d t o changes i n the hormonal m i l i e u of t h e a d u l t have i n d i c a t e d t h e important  animal.  Studies  t o be d i s c u s s e d  exerted  by gonadal and p i t u i t a r y hormones toward e x p r e s s i o n  these d i f f e r e n c e s i n m e t a b o l i c a c t i v i t y .  influences of  However, t h e exact  mechanism by which these d i f f e r e n c e s a r e 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 s e x u a l  dimorphism f o r  c e r t a i n h e p a t i c microsomal mixed f u n c t i o n o x i d a s e a c t i v i t i e s i n the r a t . to adult  U s i n g a n i m a l s r a n g i n g i n development from pre-pubescent i t was  shown t h a t h e p a t i c  and aminopyrine were f u n c t i o n o f age.  increased  N-demethylation of e t h y l m o r p h i n e  i n males r e l a t i v e t o females as a  T h i s age and sex dependent d i f f e r e n c e has a l s o  been demonstrated f o r t h e metabolism of the p o l y c y c l i c aromatic hydrocarbon b e n z o ( a ) p y r e n e (BP) by t h e cytochrome P-450 dependent a c t i v i t y a r y l hydrocarbon h y d r o x y l a s e (AHH). reported  no sex d i f f e r e n c e at day f i v e f o r AHH  a f t e r day n i n e t y , male AHH  a c t i v i t y increased  Oesch e t a l . , (1976) activity.  However,  r e l a t i v e to female.  2  S i m i l a r l y , s t u d i e s from our l a b o r a t o r y Gontovnick and B e l l w a r d ,  (Bellward  et a l . , 1981;  1981) have shown no sex d i f f e r e n c e f o r  AHH a c t i v i t y between t h e immature male and female b o t h of which are  s i g n i f i c a n t l y lower than t h e a d u l t male. The sexual  dimorphism observed i n t h e mature animal  was  suggested t o be due to hormonal i n f l u e n c e s i n the male at puberty. E l Defrawy E l Masry and Mannering (1974) observed a r e d u c t i o n i n the l e v e l s o f ethylmorphine N-demethylase i n t h e a d u l t male castration.  following  C a s t r a t i o n produced no e f f e c t on t h e female ethylmorphine  N-demethylase a c t i v i t y .  E l Defrawy E l Masry and Mannering  a l s o demonstrated t h a t a d m i n i s t r a t i o n o f t e s t o s t e r o n e  (1974)  to adult  c a s t r a t e d males r e s u l t e d 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 t o c o n t r o l male l e v e l s . was t e s t o s t e r o n e  a b l e to r e v e r s e  male, t e s t o s t e r o n e female.  In a d d i t i o n , i t was  shown t h a t not o n l y  t h e e f f e c t s of c a s t r a t i o n i n t h e  also increased  N-demethylase a c t i v i t y i n t h e  These i n v e s t i g a t o r s subsequently demonstrated t h a t t h e  administration  o f e s t r a d i o l t o a d u l t male r a t s would r e d u c e  ethylmorphine N-demethylase a c t i v i t y t o a l e v e l s i m i l a r to t h a t seen f o l l o w i n g c a s t r a t i o n . a testosterone  Kato and Onoda (1970) have a l s o noted  r e v e r s i b l e decrease i n h e p a t i c microsomal metabolism  of aminopyrine and h e x o b a r b i t a l  f o l l o w i n g gonadectomy o f t h e male.  Kramer et a l . , (1975a) have shown AHH a c t i v i t y to respond similarly.  These workers observed a t e s t o s t e r o n e  reversible  decrease i n a d u l t male AHH a c t i v i t y f o l l o w i n g c a s t r a t i o n . and  Gurtoo  Parker (1977) confirmed t h e s e f i n d i n g s and f u r t h e r noted t h a t  gonadectomy activity.  o f t h e female d i d not r e s u l t i n any change i n AHH In a d d i t i o n , t h e a d m i n i s t r a t i o n o f t e s t o s t e r o n e  to  3  female r a t s was shown t o i n c r e a s e AHH a c t i v i t y t o c o n t r o l male l e v e l s . R e s u l t s from our l a b o r a t o r y 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 f o l l o w i n g gonadectomy o f t h e a d u l t male and female rat  (Gontovnick e t a l . , 1979). The  s e x u a l dimorphism expressed  f o r h e p a t i c metabolism of  x e n o b i o t i c s has been d e s c r i b e d f o r s t e r o i d s u b s t r a t e s as w e l l . E i n a r s s o n e t a l . , (1973) have r e p o r t e d g r e a t e r 2 g - h y d r o x y l a t i o n 5a-androstane-3cx,17g-diol, 6 8 - h y d r o x y l a t i o n  of  o f 5a-androstane-3a,  1 7 8 - d i o l and 4-pregnene-3,20-dione and 18 h y d r o x y l a t i o n o f 5a-androstane3a,17B_diol  i n a d u l t male r a t s .  In a d d i t i o n , c a s t r a t i o n o f t h e male  was shown t o a b o l i s h these d i f f e r e n c e s i n a t e s t o s t e r o n e r e v e r s i b l e manner.  S i m i l a r e f f e c t s have been r e p o r t e d by Kato et al.»(1969)  f o r t h e h y d r o x y l a t i o n o f t e s t o s t e r o n e and progesterone. of course be present  Iti s  i n t u i t i v e l y obvious t h a t an age and sex d i f f e r e n c e would f o r s t e r o i d metabolism as t h e p h y s i o l o g i c a l requirements  f o r s t e r o i d s change w i t h age and gender.  2)  N e o n a t a l androgen induced  The  imprinting  importance o f androgens t o t h e e x p r e s s i o n o f sex dependent  d i f f e r e n c e s i n h e p a t i c drug and s t e r o i d metabolism l e a d t o f u r t h e r study o f m e t a b o l i c  differentiation.  In t h e f i e l d o f drug metabolism,  v e r y few workers have p u b l i s h e d papers r e l a t i n g t o d i f f e r e n t i a t i o n . Chung e t a l . , (1975) r e p o r t e d t h a t male r a t s c a s t r a t e d a t b i r t h respond l e s s a t m a t u r i t y  t o androgen s t i m u l a t i o n o f aminopyrine,  ethylmorphine and h e x o b a r b i t a l metabolism than i f exposed t o androgens as a neonate.  4  However, a number of workers have s t u d i e d sex e x p r e s s i o n of normal s t e r o i d metab'o,li . sm  dependent  Denef and DeMoor (1968)  demonstrated t h a t a s i n g l e dose o f t e s t o s t e r o n e on day one o f would r e s u l t  life  i n a m a s c u l i n e p a t t e r n of C o r t i s o l metabolism i n  n e o n a t a l l y c a s t r a t e d male or female r a t s .  Subsequently, i t was  shown t h a t normal d i f f e r e n t i a t i o n o f C o r t i s o l metabolism was b l o c k e d i n male r a t s c a s t r a t e d b e f o r e day t e n of l i f e  (Denef and DeMoor, 1972).  C a s t r a t i o n a f t e r day t e n d i d not i n h i b i t normal m e t a b o l i c d i f f e r e n tiation.  In a d d i t i o n , t h e s e workers demonstrated t h a t the androgen  a n t a g o n i s t , c y p r o t e r o n e a c e t a t e , i n h i b i t e d t h e e f f e c t of t e s t o s t e r o n e 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 i n 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). E i n a r s s o n et a l . , (1973) r e p o r t e d t h a t t h e 2ct-hydroxylation of  4-pregnene-3,20-dione  and 5 a - a n d r o s t a n e - 3 a , 1 7 B - d i o l , t h e 16  1 6 a - h y d r o x y l a t i o n of 4-androstene-3,17-dione, the A  -C  steroid iy  epoxidation  Q  f 4,16-androstadine-3-one  and t h e 178-hydroxy  steroid  r e d u c t i o n of 4-androstene-3,17-dione were more a c t i v e i n male than female r a t s .  U n l i k e t h e p r e v i o u s l y mentioned  steroid hydroxylases,  o n l y c a s t r a t i o n o f the neonate removed t h e sex d i f f e r e n c e s i n t h e s e activities.  Post-pubescent gonadectomy r e s u l t e d  in only a p a r t i a l  reduction of a c t i v i t y . From t h e s e s t u d i e s i t was  concluded t h a t t h e e a r l i e r t h e  male r a t i s i s o l a t e d from androgens, the l e s s d i f f e r e n t i a t e d  certain  enzyme a c t i v i t i e s w i l l be a t m a t u r i t y .  suggested  t h a t the l i v e r  is initially  Furthermore, i t was  " f e m i n i n e " , and t h a t t h e 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 i n t h e a d u l t was  " o r g a n i z e d " by  5  t e s t i c u l a r .laetor(9)within t h e f i r s t  t e n t o twelve days o f l i f e  (Chung, 1975; DeMoor and Denef, 1968; G u s t a f s s o n and Stenberg, 1974a). T h i s e f f e c t i s r e f e r r e d t o as n e o n a t a l androgen induced (Chung, 1974; E i n a r s s o n in  c e r t a i n instances  et a l . , 1973; McEwen et a l . , 1975), and  i t i s irreversible.  That i s , n e o n a t a l l y  c a s t r a t e d males a r e n o n - r e s p o n s i v e t o t e s t o s t e r o n e of enzyme a c t i v i t y a t m a t u r i t y . the  imprinting  stimulation  T h i s has been demonstrated f o r  6 g - h y d r o x y l a t i o n o f 4-androstene-3,17-dione (Gustafsson and  Stenberg, 1974a).  3)  Involvement o f t h e hypophyseal-hypothalamic a x i s i n t h e maintenance o f sex dependent d i f f e r e n c e s i n h e p a t i c  a)  metabolism  Pituitary effects  The  importance o f n e o n a t a l androgens t o t h e d i f f e r e n t i a t i o n  of h e p a t i c metabolism l e a d t o study o f t h e e f f e c t s o f t h e hypophyseal-hypothalamic a x i s on t h i s p r o c e s s .  The r a t i o n a l e  was t h a t t h e p i t u i t a r y and u l t i m a t e l y t h e hypothalamus the l e v e l s o f sex s t e r o i d s i n v i v o .  Colby e t a l . , (1973) demonstrated  t h a t hypophysectomy reduced c o r t i c o s t e r o n e in  adult  gonadectomized male r a t s .  gonadectomized male r a t s .  that the reduction  of  activity  A4 r e d u c t a s e  activity  Kramer e t a l . , (1975) demonstrated  o f ethylmorphine N-demethylase a c t i v i t y  hypophysectomy o f a d u l t administration  A4 r e d u c t a s e  In a d d i t i o n , f o l l o w i n g hypo-  physectomy, e s t r a d i o l f a i l e d t o s t i m u l a t e in adult  regulate  female r a t s c o u l d not be s t i m u l a t e d  testosterone.  following by  6  In a d d i t i o n , Kramer and co-workers  (1979) have shown t h a t  r e d u c t i o n o f h e p a t i c ethylmorphine N-demethylase h y d r o x y l a s e a c t i v i t i e s f o l l o w i n g hypophysectomy  and a r y l hydrocarbon would not  i n response t o t h e a d m i n i s t r a t i o n o f t e s t o s t e r o n e (DHT)  to adult  increase  or d i h y d r o t e s t o s t e r o n e  gonadectomized male r a t s .  G u s t a f s s o n and Stenberg (1974a) r e p o r t e d s i m i l a r for steroid substrates.  These i n v e s t i g a t o r s d e s c r i b e d  findings hypophysectomy  p r o d u c i n g an o v e r a l l m a s c u l i n i z a t i o n o f h e p a t i c s t e r o i d metabolism i n female r a t s : i n c r e a s i n g 2a-, 2g-, 7g-and 1 8 - h y d r o x y l a t i o n of 5a-androstane-3a,17g  d i o l ; and 6$-, 1 6 a - h y d r o x y l a t i o n , and  1 7 a - h y d r o x y s t e r o i d r e d u c t i o n of 4-androstene-3,17-dione. f e m i n i z a t i o n of 5a-reductase was noted f o l l o w i n g of the male.  3gA  hypophysectomy  Furthermore, treatment w i t h t e s t o s t e r o n e and  estradiol,  which n o r m a l l y m a s c u l i n i z e and f e m i n i z e s t 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 t h e s e s t e r o i d h y d r o x y l a s e and r e d u c t a s e a c t i v i t i e s i n the hypophysectomized male or female r a t .  These f i n d i n g s a r e  s i m i l a r t o t h o s e observed i n our l a b o r a t o r y which i n d i c a t e t h a t hypophysectomy  produces an e q u a l i z a t i o n o r d e - d i f f e r e n t i a t i o n  of h e p a t i c drug metabolism Denef  (Bellward  et a l . ,  1982).  (1974) has shown t h a t hypophysectomy  removes the normal  d i f f e r e n t i a t i o n o f t e s t o s t e r o n e metabolism i n male r a t s . if  However,  these same hypophysectomized males r e c e i v e d autonomous p i t u i t a r y  implants from e i t h e r male or female donors, then a f e m i n i z a t i o n of t e s t o s t e r o n e metabolism would r e s u l t . a l s o examined  G u s t a f s s o n and Stenberg  (1976a)  t h e e f f e c t s of autonomous p i t u i t a r y i m p l a n t s on h e p a t i c  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 r e n a l c a p s u l e of a d u l t hypophysectomized  c a s t r a t e d male  7  rats.  T h i s procedure r e s u l t e d i n a " f e m i n i z a t i o n " of h e p a t i c  steroid  metabolism which was not r e s p o n s i v e t o t h e a d m i n i s t r a t i o n o f t e s t o sterone.  In a d d i t i o n , Skett  e t 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 o f hypophysectomized  male r e c i p i e n t s . The i n v e s t i g a t o r s concluded from these s t u d i e s t h a t an unknown f a c t o r was r e l e a s e d  from t h e pubescent and post-pubescent  p i t u i t a r y o f e i t h e r sex t h a t r e s u l t e d i n t h e f e m i n i z a t i o n o f metabolism.  S i n c e b o t h male and female p i t u i t a r y donors produced a  f e m i n i z a t i o n o f metabolism, a l t h o u g h t h e male does n o t e x h i b i t female l e v e l s o f metabolism, the i n f l u e n c e o f the hypothalamus  was  investigated.  b)  Hypothalamic e f f e c t s  To c h a r a c t e r i z e t h e r e g u l a t o r y r o l e o f t h e hypothalamus, Gustafsson e t _ a l . , hypothalamic a r e a . of h e p a t i c  (1976b) produced e l e c t r o t h e r m i c Lesioning  l e s i o n s i n the  r e s u l t e d i n an o v e r a l l f e m i n i z a t i o n  s t e r o i d metabolism i n t h e male, but produced no  s i g n i f i c a n t e f f e c t s i n t h e female.  In a d d i t i o n , i t was shown  that f r o n t a l d e - a f f e r e n t a t i o n at the retrochiasmatic chiasmatic  and s u p r a -  l e v e l r e s u l t e d i n complete f e m i n i z a t i o n o f h e p a t i c  s t e r o i d metabolism i n t h e male r e l a t i v e t o c o n t r o l .  Again*no  e f f e c t was n o t e d f o l l o w i n g d e - a f f e r e n t a t i o n o f t h e female ( G u s t a f s s o n e t a l . , 1978). I t was p o s t u l a t e d ,  therefore, that a feminizing  factor  8  s e c r e t e d by t h e female p i t u i t a r y a t puberty i s i n h i b i t e d by a hypothalamic r e l e a s i n g  i n h i b i t o r y f a c t o r i n the male.  f a c t o r , which i s not p r e s e n t i n the female, may  This inhibitory  be the r e s u l t  of  n e o n a t a l t e s t i c u l a r androgen induced i m p r i n t i n g of the male hypothalamus  c a u s i n g i t t o d i f f e r e n t i a t e a t puberty and express a male  metabolic pattern. male was  Furthermore,  the a c t i o n o f e s t r a d i o l i n t h e  suggested t o be a t the l e v e l of the hypothalamus to reduce  t h e s e c r e t i o n of the r e l e a s e i n h i b i t o r y f a c t o r .  T h i s would a l l o w  f o r an i n c r e a s e d r e l e a s e o f t h e p i t u i t a r y f e m i n i z i n g f a c t o r r e s u l t i n g i n a female p a t t e r n of metabolism  ( G u s t a f s s o n and Stenberg, 1976a).  The p i t u i t a r y f a c t o r r e s p o n s i b l e f o r t h e observed on h e p a t i c metabolism  c)  was  effects  i n i t i a l l y r e f e r r e d t o as " f e m i n o t r o p i n " .  Feminotropin  C h a r a c t e r i z a t i o n o f f e m i n o t r o p i n was et a l . ,  (1975a) and Mode et a l ^ ,  a c t i v i t y was  (1978).  attempted  by G u s t a f s s o n  i n each case  feminizing  a s s e s s e d by i n c r e a s e s i n 5a-reductase a c t i v i t y of  hepatoma (HTC)  cells in tissue culture.  Initially  hypophyseal  e x t r a c t s from male and female r a t s were a d m i n i s t e r e d t o t h e t i s s u e culture.  The macromolecular  e x t r a c t was  f r a c t i o n o f the female  pituitary  shown t o i n c r e a s e 5 a r e d u c t a s e a c t i v i t y , whereas n e i t h e r _  the male o r low m o l e c u l a r weight have any e f f e c t .  In a d d i t i o n , known p u r i f i e d p i t u i t a r y hormones  were added t o t h e HTC (Gustafsson e t a l . , f e m i n o t r o p i n was  female e x t r a c t were observed to  c u l t u r e but a l s o shown t o have no  1975a).  Thus t h e a u t h o r s concluded  a n o v e l hormone.  However, s e v e r a l  effect, that  criticisms  9  can be r a i s e d c o n c e r n i n g t h i s study because  the e x t r a c t contained  p i t u i t a r y hormones, and no q u a n t i t a t i v e a n a l y s i s was  attempted,  i . e . the e f f e c t s observed may be due t o d i f f e r e n c e s i n these hormoneconcentrations.  A l t e r n a t i v e l y , s i n c e 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 t o t h e i n t e r a c t i o n o f two o r more hormones p r e s e n t i n t h e e x t r a c t . F u r t h e r c h a r a c t e r i z a t i o n u s i n g e x t r a c t s d e r i v e d from C 11RAP p i t u i t a r y tumor t i s s u e o 0  (as w e l l as female r a t p i t u i t a r y  f o r comparative purposes) r e v e a l e d t h a t p r o l a c t i n  (PRL) e l u t e d i n  the a r e a t h a t corresponded t o t h e "low" (below 8,000) m o l e c u l a r weight  f r a c t i o n o f f e m i n o t r o p i n f o l l o w i n g g e l chromatography  (Mode et a l . ,  1978).  More r e c e n t s t u d i e s by Gustafsson's  have suggested t h a t f e m i n o t r o p i n may be an i d e n t i f i e d hormone.  4)  pituitary  P i t u i t a r y hormones a r e known t o have pronounced  on h e p a t i c m e t a b o l i c  group  effects  activity.  E f f e c t s o f p i t u i t a r y hormones on h e p a t i c metabolism  It  i s important t o n o t e f o r t h e f o l l o w i n g d i s c u s s i o n  p i t u i t a r y hormone p r e p a r a t i o n s a r e not c o m p l e t e l y pure.  that  These  p r e p a r a t i o n s g e n e r a l l y c o n t a i n s m a l l amounts ( l e s s than 2%) o f more than one o t h e r a n t e r i o r p i t u i t a r y hormone. the r a t p r o l a c t i n  (rPJAL) used  i n our study c o n t a i n e d 1.5% growth hormone  c o n t a m i n a t i o n as determined by radioimmunoassay. p i t u i t a r y hormones e x h i b i t  For example,  cross r e a c t i v i t y .  (hGH) was shown t o have both somatogenic  Furthermore,  Human growth hormone  and l a c t o g e n i c  properties  10  ( K l e i n b e r g and Todd, 1980; Mode et a l . , 1981).  The b o v i n e growth  hormone (bGH) used i n t h i s study was r e p o r t e d t o c o n t a i n 62% lactogenic a c t i v i t y . origin  Therefore, while  species v a r i a t i o n  i n hormone  i s apparent, t h e l a c k o f s p e c i f i c i t y o f some o f these  p r e p a r a t i o n s must be c o n s i d e r e d ,  o r i n c o r r e c t c o n c l u s i o n s may be  reached. The  initial  candidates  f o r p i t u i t a r y hormones a c t i v e on  h e p a t i c metabolism a r e t h e gonadotropins l u t e i n i z i n g hormone (LH) and  follicle  s t i m u l a t i n g hormone (FSH) s i n c e they a r e a s s o c i a t e d  w i t h t h e p r o d u c t i o n and r e l e a s e o f sex s t e r o i d s . (1977) i n v e s t i g a t e d t h e d i r e c t  Kramer et a l . ,  e f f e c t s o f LH and FSH on h e p a t i c  drug metabolism u s i n g gonadectomized male and female r a t s .  They  r e p o r t e d t h a t FSH or LH i n c r e a s e d ethylmorphine N-demethylase a c t i v i t y i n males'and females., whereas FSH i n c r e a s e d aminopyrine N-demethylase a c t i v i t y i n females o n l y . any  Neither  FSH or LH produced  e f f e c t s on t h e sex-independent a n i l i n e h y d r o x y l a s e  animals o f e i t h e r sex. Kramer and co-workers (1977)  activity in concluded  t h a t t h e gonadotropins had a c t i o n s on h e p a t i c mixed f u n c t i o n oxidase  a c t i v i t y which were independent o f gonadal hormones.  Gustafsson  (1975b) has r e p o r t e d t h a t FSH but n o t LH w i l l  increase  t h e a c t i v i t y o f androgen dependent and not androgen independent s t e r o i d hydroxylase is  important  dotropins  activity  i n gonadectomized r a t s .  However, i t  t o c o n s i d e r t h a t when s t u d y i n g t h e e f f e c t s of gona-  i n gonadectomized r a t s w i t h  i n t a c t p i t u i t a r i e s , an  e l e v a t i o n of gonadotropins w i l l a l r e a d y e x i s t due t o t h e removal o f feedback i n h i b i t i o n by sex s t e r o i d s .  Therefore,  a d m i n i s t r a t i o n of  11  gonadotropin t o an e l e v a t e d system makes any c o n c l u s i o n The major c r i t e r i o n f o r the f e m i n i z i n g f a c t o r e s t r o g e n mimetic  difficult.  i s that  i t be  ( e i t h e r d i r e c t l y or i n d i r e c t l y ) w i t h r e s p e c t to  h e p a t i c microsomal metabolism.  W i l s o n (1969) had demonstrated  that  GH reduced h e p a t i c ethylmorphine and aminopyrine N-demethylase a c t i v i t y i n a d u l t male r a t s 48 hours a f t e r a d m i n i s t r a t i o n .  These  e f f e c t s were not observed f o l l o w i n g PRL or a d r e n o c o r t i c o t r o p i c hormone (ACTH) treatment. that t h i s e f f e c t days of age  In a d d i t i o n , W i l s o n (1970) has shown  cannot be produced  (pre-pubescent).  E l Defrawy E l Masry et a l . ,  i n male r a t s l e s s than t h i r t y  T h i s supports t h e o b s e r v a t i o n s o f (1974) t h a t no sex d i f f e r e n c e i s  p r e s e n t i n t h e immature animal and, to an e x t e n t , S k e t t et a l . , ( 1 9 7 8 ) t h a t pre—pubescent donor p i t u i t a r i e s would not f e m i n i z e s t e r o i d metabolism.  T h i s a l s o i n d i c a t e s t h a t the e f f e c t s of  GH  a r e not simply d i r e c t l y  inhibitory.  t h a t h e p a t i c metabolism  of h e x o b a r b i t a l c o u l d be reduced by GH i n  gonadectomized, rats.  W i l s o n (1971) has a l s o shown  a d r e n a l e c t o m i z e d o r hypophysectomized  a d u l t male  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 o f GH noted w i t h a l l  substrates.  concluded t h a t the e f f e c t s of GH were sex dependent,  age  and not dependent  factors.  upon gonadal, a d r e n a l or p i t u i t a r y  Kramer et a l . ,  (1975a,1978) have demonstrated  Wilson  dependent,  t h a t the e f f e c t s  of e s t r a d i o l on drug m e t a b o l i z i n g enzymes i n the c a s t r a t e d male r a t a r e s i m i l a r to t h o s e produced by GH.  In a d d i t i o n , i t was  observed t h a t the combined a d m i n i s t r a t i o n o f GH and not reduce ethylmorphine N-demethylase a c t i v i t y  estradiol did  i n the c a s t r a t e d  male t o any g r e a t e r extent than d i d GH or e s t r a d i o l a l o n e , s u g g e s t i n g  12  a common mechanism of a c t i o n . the o b s e r v a t i o n s an  increase  GH may  be  one  i n serum GH  produces  l e v e l s i n a d u l t male r a t s , suggests t h a t factor.  i s the f e m i n o t r o p i n  that  Gustafsson d e s c r i b e s ,  would expect i t , u n l i k e e s t r a d i o l , to a c t  animal.  including  of L l o y d et a l . , (1971) that e s t r a d i o l  the f e m i n i z i n g  I f GH  Evidence to t h i s p o i n t ,  then  i n the hypophysectomized  However, Colby et a l . , (1974) have shown t h a t GH  i n the  presence of ACTH w i l l reduce c o r i c o s t e r o n e metabolism i n hypophysectomized male r a t s , i . e . to "masculinize". Furthermore, LH,  FSH,  FSH  + LH,  GH and  ACTH g i v e n  i n d i v i d u a l l y produced no  i n c o r t i c o s t e r o n e metabolism d i f f e r e n t Administration  of GH  (masculinization)  from hypophysectomy  of ethylmorphine N-demethylase and  t o what was  (Kramer et a l . , 1975b).  expected.  (1980) have shown t h a t GH  benzo(a)pyrene  I n t e r e s t i n g l y , Rumbaugh and  i n the presence of t h y r o x i n e  (T^)  Colby and  benzo(a)pyrene  i n c r e a s e A4-hydrogenase a c t i v i t i e s  physectomized male r a t .  increase  These r e s u l t s were  ACTH w i l l decrease ethylmorphine N-demethylase and h y d r o x y l a s e , and  change  alone.  t o hypophysectomized males produced an  hydroxylase a c t i v i t i e s opposite  PRL,  i n the  These s h i f t s i n a c t i v i t y a r e  hypo-  indicative  of a f e m i n i z a t i o n ; however, as w i t h the c o r t i c o s t e r o n e metabolism discussed  e a r l i e r , GH was  Colby concluded t h a t GH  unable to produce t h i s f e m i n i z a t i o n  c o n t r i b u t e s t o the f e m i n i z a t i o n and  e s t r a d i o l a c t i o n on h e p a t i c metabolism. of ACTH and/or T^ p l u s GH  evidence supporting  w i t h f e m i n i z a t i o n was  the  i n the presence o f e s t r a d i o l or  i n the hypophysectomized r a t were not Further  Unfortunately  obtained  alone.  mediates effects testosterone  investigated.  the h y p o t h e s i s t h a t GH  i s involved  w i t h e x t r a c t s from p i t u i t a r y tumors.  13  These e x t r a c t s which c o n t a i n  GH were shown t o f e m i n i z e  metabolism o f h e p a t o c y t e and hepatoma c e l l 1978).  However, the tumor l i n e  the steroid  c u l t u r e (Skett et a l . ,  (Cgll-RAP) from which t h e e x t r a c t s  were produced was found t o s e c r e t e l a r g e amounts o f PRL (Skett et  a l . , 1977).  T h i s tumor c e l l l i n e was shown t o produce t h e most  complete f e m i n i z a t i o n o f h e p a t i c male r a t .  s t e r o i d metabolism i n t h e i n t a c t  E n e r o t h et a l . , (1977) have shown t h a t t h e f e m i n i z a t i o n  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 w i t h an i n c r e a s e  implantation.  Skett  i n serum PRL l e v e l s f o l l o w i n g  e t a l . , (1978a) have shown t h a t p i t u i t a r i e s donated from r a t s  younger than 35 days o f age cannot produce a f e m i n i z a t i o n o f h e p a t i c 5a-reductase a c t i v i t i e s  i n hypophysectomized a d u l t male r e c i p i e n t s .  In a d d i t i o n , f e m i n i z a t i o n was c o r r e l a t e d w i t h i n c r e a s e d levels.  This observation  serum PRL  i s i n keeping w i t h t h e 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 t h a t growth hormone  i s n o t t h e o n l y hormone t o be c o n s i d e r e d metabolism.  i n the feminization of  A l t h o u g h Colby was not a b l e t o produce a f e m i n i z a t i o n  of metabolism w i t h PRL, Lax e t a l . ,(.197 6). have shown PRL t o f e m i n i z e hepatic rats. (1975).  s t e r o i d metabolism i n hypophysectomized male and female S i m i l a r r e s u l t s have been r e p o r t e d  by S c h r i e f e r s et a l . ,  Thus t h e r o l e o f PRL i s n o t y e t r e s o l v e d . Mode et a l . , (.1981) demonstrated t h a t continuous i n f u s i o n of  hGH r e s u l t e d i n a f e m i n i z a t i o n o f h e p a t i c the hypophysectomized male r a t .  However, as indicated,hGH possesses  both somatogenic and l a c t o g e n i c q u a l i t i e s Mode et a l . , 1981).  Further  s t e r o i d metabolism o f  ( K l e i n b e r g and Todd, 1980;  s t u d i e s w i t h r a t growth hormone (rGH)  14  produced a f e m i n i z a t i o n o f s t e r o i d h y d r o x y l a s e a c t i v i t y as w e l l . However, rPRL a l s o r e s u l t e d i n a p a r t i a l of s t e r o i d metabolism.  feminization  T h i s study was n o t c o n s i s t e n t w i t h t h e  r e s u l t s o f Colby et a l . , (1974) and Rumbaugh and Colby which r e p o r t e d  (1980),  t h a t PRL was without e f f e c t and t h a t GH produced a  " m a s c u l i n i z a t i o n " of h e p a t i c  drug metabolism.  A second i n c o n s i s t e n c y between t h e two p r e v i o u s l y studies involved adrenal action.  According  cited  and t h y r o i d hormone, m o d u l a t i o n o f GH  t o Rumbaugh and Colby (1980) a " f e m i n i z a t i o n "  of drug metabolism i n t h e hypophysectomized male c o u l d o n l y be produced by bGH i f T^ and ACTH were a d m i n i s t e r e d T h i s suggested a d r e n a l enzyme r e g u l a t i o n . or  concurrently.  and/or t h y r o i d involvement i n t h e h e p a t i c  However, n e i t h e r o f these hormones, i n d i v i d u a l l y  i n combination produced any e f f e c t on AHH a c t i v i t y  physectomized male.  i n t h e hypo-  In a d d i t i o n , Mode et a l . , (1981) demonstrated  t h a t adrenalectomy and thyroidectomy d i d not a l t e r t h e response t o continuous i n f u s i o n o f hGH on h e p a t i c hypophysectomized male r a t s . due  This discrepancy  t o d i f f e r e n c e s i n hormone, a d m i n i s t r a t i o n  of GH (Mode e t a l . , 1981). "feminotropin"  5)  s t e r o i d metabolism i n i s quite  conceivably  and/or s p e c i e s  origin  I n any case, t h e i d e n t i t y of t h e  was s t i l l v e r y  unclear.  I n t e r a c t i o n o f sex s t e r o i d s and h e p a t i c  lactogen  binding  I f we presume t h a t GH or PRL i s a c t i n g a t t h e l i v e r t o produce a " f e m i n i z a t i o n " o f metabolism, then i t i s r e a s o n a b l e t o assume t h i s a c t i o n i n v o l v e s a r e c e p t o r mediated pathway.  K e l l y et a l . , (1974)  have shown an age  and  sex dependent i n c r e a s e i n PRL  and  GH  binding  125 to  h e p a t i c t i s s u e i n the r a t .  The  s p e c i f i c b i n d i n g of  I-oPRL  125 and  I-hGH i n c r e a s e d between day  but remained low throughout l i f e  20 and  40 of l i f e  i n the male.  female  T h i s 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  et  a l . , 1978a) and  maturity  i n the  to f e m i n i z e  (Skett  t h e e x p r e s s i o n o f d i f f e r e n t i a t e d metabolism a t  ( E l Defrawy E l Masry e t a l . , 1974a).  S i n c e sex s t e r o i d s a r e known t o modulate h e p a t i c metabolism i t would be r e a s o n a b l e  t o assume t h a t sex s t e r o i d s might modulate  hepatic lactogenic receptor l e v e l s . w i t h GH and  PRL  The  i n t e r a c t i o n of sex s t e r o i d s  has been w e l l documented. Posner  have shown t h a t the a d m i n i s t r a t i o n of e s t r o n e i n c r e a s e i n oPRL arida 30 f o l d i n c r e a s e i n hGH hepatic lactogen receptor.  et a l . , (1974)  induced  oPRL and  hGH  S i m i l a r e f f e c t s were seen w i t h  b i n d i n g i n the a d u l t female.  reduced the l e v e l s of l a c t o g e n r e c e p t o r and e f f e c t s of e s t r o g e n  i n b o t h males and  fold  b i n d i n g to the male  Treatment w i t h e s t r o n e r e s u l t e d i n an approximate two in  a 10  fold  estradiol. increase  Hypophysectomy abolished the i n d u c t i v e  females.  Furthermore, an  i n c r e a s e i n h e p a t i c oPRL b i n d i n g has been demonstrated f o l l o w i n g gonadectomy of the male (Aragona e t a l . , 1976). PRL  b i n d i n g was  shown t o be completely  administration of  The  increase i n  i n h i b i t e d f o l l o w i n g the  testosterone.  Adrenalectomy d i d not  influence t h i s increased  f o l l o w i n g gonadectomy (Aragona et a l . , 1976).  The  binding  testosterone  r e v e r s i b l e p o s t - c a s t r a t i o n i n c r e a s e i n male h e p a t i c PRL was  confirmed  by Barkey et a l . (1979).  binding  In a d d i t i o n , Barkey  and  co-workers (1979) demonstrated t h a t gonadectomy r e s u l t e d i n a  d e c r e a s e i n PRL b i n d i n g t o t h e p r o s t a t e .  Subsequent  testosterone  replacement i n c r e a s e d PRL b i n d i n g t o t h e p r o s t a t e , r e v e r s i n g t h e e f f e c t s o f gonadectomy.  Thus, a r e l a t i o n s h i p between t e s t o s t e r o n e  l e v e l s and PRL b i n d i n g does e x i s t .  The r e l a t i o n s h i p between h e p a t i c  PRL b i n d i n g and t e s t o s t e r o n e may be secondary to the e f f e c t s observed at t h e a c c e s s o r y  sex g l a n d l e v e l where b i n d i n g depends on t e s t o s t e r o n e .  This i s a reasonable  assumption as t h e presence o f t h e t e s t i s a r e  an i n t e g r a l p a r t o f a masculine h e p a t i c m e t a b o l i c  profile.  i n t e r a c t i o n o f PRL and t e s t o s t e r o n e on the a c c e s s o r y "'can be d e s c r i b e d as a synergism. that concurrent  sex glands  Keenan e t a l . , (1975) have shown  a d m i n i s t r a t i o n o f PRL and t e s t o s t e r o n e  increased  p r o s t a t e and seminal v e s i c l e weights t o a g r e a t e r extent testosterone alone.  The  than d i d  Furthermore, a d m i n i s t r a t i o n o f PRL and  testosterone to r a t v e n t r a l prostate c e l l culture stimulates and  RNA  p r o t e i n s y n t h e s i s whereas t e s t o s t e r o n e a l o n e produced no e f f e c t  (Johansson, 1975).  PRL has a l s o been shown t o s t i m u l a t e  nuclear  3 uptake of. [ H ] ^ t e s t o s t e r o n e i n r a t v e n t r a l p r o s t a t e Sharma, 1977).  (Slaunwhite  and  The i n t e r a c t i o n between PRL and t e s t o s t e r o n e may  r e s u l t from t h e involvement o f PRL w i t h t h e r e g u l a t i o n o f LH receptor  l e v e l s i n the t e s t i s as w e l l .  Bohnet e t a l . , (1975)  have shown t h a t PRL s t i m u l a t e s t e s t i c u l a r LH b i n d i n g i n r a t s . T h i s would, o f course,  l e a d t o an i n c r e a s e i n t e s t o s t e r o n e  levels  and  a c c o r d i n g t o Barkey's group a decrease i n h e p a t i c PRL b i n d i n g .  The  u l t i m a t e r e s u l t of t h i s i n c r e a s e d t e s t o s t e r o n e would be a  decrease i n LH r e c e p t o r s v i a feedback i n h i b i t i o n .  Belanger e t a l . ,  (1979) have shown t h a t , when LH r e c e p t o r s have been induced by LHRH a g o n i s t s , PRL p a r t i c i p a t e s i n the down r e g u l a t i o n o f these  17  receptors.  In a d d i t i o n , i t has been shown t h a t PRL  to gonadotropin  release.  Hyperprolactinemia  t r a n s p l a n t a t i o n of p i t u i t a r i e s or by PRL  is inhibitory  induced  by  the  priming r e s u l t s i n a  r e d u c t i o n of b a s a l serum LH l e v e l s as w e l l as a d e c r e a s e i n LHRH s t i m u l a t e d LH r e l e a s e i n a d u l t male r a t s Therefore,  t h e r e l a t i o n between PRL,  suggest t h a t PRL  t e s t o s t e r o n e , and  LH  receptors  i s i n v o l v e d i n t h e r e g u l a t i o n of h e p a t i c  p r o f i l e s observed i n p o s t - p u b e r t a l r a t s . hypothesis,  (Greeley et a l . , 1981).  In support  of  this  F e r l a n d et a l . , (1979) have demonstrated t h a t  r e v e r s e s t h e i n h i b i t o r y e f f e c t s of dopamine on PRL i n i n c r e a s e d serum PRL  levels.  female r a t s .  Furthermore, Posner and  The  estradiol  release resulting co-workers  (1974) have shown t h a t e s t r a d i o l s t i m u l a t e s h e p a t i c oPRL i n a d u l t male and  metabolic  binding  i n c r e a s e i n h e p a t i c PRL  binding  observed i n t h e male c o r r e l a t e s w i t h t h e e s t r a d i o l f e m i n i z a t i o n of h e p a t i c drug metabolism p r e v i o u s l y d i s c u s s e d .  6)  P i t u i t a r y m o d u l a t i o n of l a c t o g e n  binding  R e c a l l i n g t h a t the p i t u i t a r y a x i s must be present expression  of t e s t o s t e r o n e and  e s t r a d i o l e f f e c t s on  for  the  hepatic  metabolism, i t i s of i n t e r e s t to n o t e t h a t t h e s t i m u l a t i n g e f f e c t of e s t r a d i o l o r e s t r o n e on oPRL and hGH i n the hypophysectomized r a t . in hepatic lactogen receptor reversed  b i n d i n g i s not  demonstrable  Hypophysectomy produces a decrease i n the female which can be  by an autonomous p i t u i t a r y implant  partially  (Posner et a l . , 1975).  Furthermore, Bohnet e t a l . , (1976) have shown a d m i n i s t r a t i o n of oPRL,and t o a l e s s e r extent  hGH,to hypophysectomized females  18  incfr eased hepatic PRL s p e c i f i c binding. no e f f e c t .  ACTH, estradiol, and bGH had  However, when oPRL was combined with ACTH, hGH or  e s t r a d i o l but not bGH even greater induction of hepatic PRL binding was seen i n the hypophysectomized female. of hGH and ACTH also produced a dramatic  In addition, the Combination increase i n 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). l i v e r required hGH or ACTH.  Induction of PRL binding to the  The greatest increase i n s p e c i f i c  PRL binding (33%) was achieved by concurrent administration of hGH and ACTH (without oPRL).  A similar increase was produced i n  rats bearing a GH secreting tumor.  The addition of e s t r a d i o l 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 i n hepatic PRL binding in the male.  Unfortunately, t h i s study did not include an  adrenalectomized  model f o r comparative purposes.  The preceeding two investigations are of interest when considering the work of Rumbaugh and Colby (1980) regarding their i n a b i l i t y to feminize hepatic drug metabolism without of bGH, ACTH, and T,.  a combination  19  In c o n t r a s t t o the i m p l i e d importance these to  t h y r o i d and a d r e n a l hormones, Norstedt  shown t h a t adrenalectomy and h e p a t i c PRL r e s u l t may Norstedt  b i n d i n g or hGH be due  studies lend  e t a l . , (1981a) have  thyroidectomy have no  s t i m u l a t i o n of PRL  effect  binding.  t o a d i f f e r e n c e i n hormone d e l i v e r y .  on  This However,  et a l . , (1981a) d i d c o n f i r m some hormonal e f f e c t s  h e p a t i c PRL  b i n d i n g r e p o r t e d by Aragona et a l . , (197 6)  Bohnet et a l . , (1976).  Continuous i n f u s i o n of hGH  produced i n c r e a s e s i n h e p a t i c PRL female r a t s .  In a d d i t i o n , hGH  i n hypophysectomized males. i n c r e a s i n g PRL  and  and oPRL  b i n d i n g i n hypophysectomized rGH  i n c r e a s e d h e p a t i c PRL  sites  I n t e r e s t i n g l y , rPRL had no e f f e c t  on  b i n d i n g i n e i t h e r male or female r a t s f o l l o w i n g  hypophysectomy.  T h i s again.- i n d i c a t e s the s p e c i e s d i f f e r e n c e s  between p i t u i t a r y hormones as oPRL was The  and  on  effect  shown to be  effective.  of sex s t e r o i d s on modulation of p i t u i t a r y hormone  i n d u c t i o n of l a c t o g e n b i n d i n g i n the hypophysectomized a n i m a l i s of importance.  These r e s u l t s i n d i c a t e t h a t sex s t e r o i d s a r e a c t i n g  i n c o n c e r t w i t h p i t u i t a r y hormones at the l i v e r t o i n c r e a s e or d e c r e a s e h e p a t i c PRL  binding.  a c t i o n s o f e s t r a d i o l and  Previous  s t u d i e s suggested  the  t e s t o s t e r o n e were mediated c e n t r a l l y  t o their l a c k of e f f e c t f o l l o w i n g hypophysectomy.  due  However, t h i s does  not appear t o be the case f o l l o w i n g p i t u i t a r y hormone replacement. L o c a l i n t e r a c t i o n of sex s t e r o i d s and  p i t u i t a r y hormones a t  l i v e r t o modulate p r o l a c t i n b i n d i n g has a l s o been suggested p i t u i t a r y implant  studies.  the by  K e l l y et a l . , (1977) have shown  autonomous p i t u i t a r y implants  elicited  i n d u c t i o n of h e p a t i c  b i n d i n g i n hypophysectomized female r a t s .  T h i s was  PRL  inhibited  20  f o l l o w i n g the a d m i n i s t r a t i o n be a c t i n g on the p i t u i t a r y hormone replacement cannot be  7)  of DHT.  implant,  I t i s p o s s i b l e t h a t DHT but  taken w i t h the  s t u d i e s , p e r i p h e r a l r e g u l a t i o n by  pituitary sex  steroids  discounted.  E f f e c t s of p i t u i t a r y hormones on h e p a t i c  sex  s t e r o i d binding  E s t r o g e n s have been shown to mediate v a r i o u s h e p a t i c Increases i n hepatic  plasma r e n i n s u b s t r a t e and  et a l . , 1977;  Snow et a l . , 1978), and  mediated p r o c e s s .  Although the l i v e r  a t a r g e t t i s s u e f o r sex i d e n t i f i e d and  (Eisenfeld  t h i s suggested a  receptor  i s not  considered  generally  s t e r o i d s , estrogen receptors  characterized  (Aten et a l . , 1978;  have been  Dickson et a l . ,  E i s e n f e l d et a l . , 1977). Therefore  i f PRL  h e p a t i c metabolism but  or GH  are not  i n connection  a c t i n g d i r e c t l y to  "feminize"  w i t h " p e r i p h e r a l f a c t o r s " as  suggested by hormone replacement s t u d i e s , and,  i f we  assume  s t e r o i d s a r e a c t i n g d i r e c t l y on the l i v e r by a r e c e p t o r  p i t u i t a r y hormones i n v o l v e s t h e i r modulation of h e p a t i c steroid receptor  levels.  T h i s would e x p l a i n why  sex  sex  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 f o r the a c t i o n s  not  functions.  l i p o p r o t e i n synthesis  have been c o r r e l a t e d w i t h h e p a t i c e s t r o g e n b i n d i n g  1978;  may  of sex  steroids  do  a f f e c t h e p a t i c metabolism i n hypophysectomized r a t s . In t h i s r e g a r d ,  Chamnes et a l . , (1975) have shown t h a t oPRL  p a r t i a l l y restores hepatic estrogen receptor hypophysectomy i n the a d u l t female r a t .  l e v e l s reduced  by  Moreover, L u c i e r et a l . ,  (1981) demonstrated a complete r e v e r s a l of hypophysectomy  induced  21  r e d u c t i o n of h e p a t i c e s t r o g e n r e c e p t o r s i t e s f o l l o w i n g the t r a t i o n of bGH.  In a d d i t i o n , no e f f e c t was  adminis-  noted w i t h rPRL, bovine  LH,  or -rat FSH. •• U n l i k e , the study of -Norstedt--- et' a l . , (1931b) t h e s e i n v e s t i g a t o r s i n j e c t e d r e l a t i v e l y l a r g e amounts of hormone. continuous  Using  i n f u s i o n of low c o n c e n t r a t i o n s of hormone, N o r s t e d t  co-workers (1981b) r e p o r t e d t h a t hGH b i n d i n g of e s t r a d i o l  produced  and  an i n c r e a s e i n s p e c i f i c  i n the hypophysectomized, o v a r i e c t o m i z e d  female  rat. I n t e r e s t i n g l y , L u c i e r et a l . , (1981) have r e p o r t e d t h a t o t h e r than hypophysectomy o n l y adrenalectomy  produced  a  significant  r e d u c t i o n i n h e p a t i c e s t r a d i o l b i n d i n g i n the o v a r i e c t o m i z e d female.  No  e f f e c t was  seen  i n the male.  The r e d u c t i o n of h e p a t i c  e s t r a d i o l b i n d i n g f o l l o w i n g adrenalectomy by N o r s t e d t et a l . , (1981b).  of the female was  These i n v e s t i g a t o r s have a l s o  t h a t the e s t r o g e n r e c e p t o r d i d not respond i n the hypophysectomized-ovariectomized dexamethasone was  female. was  female.  administered a s i g n i f i c a n t  s p e c i f i c e s t r a d i o l b i n d i n g was effect  to a p i t u i t a r y  seen.  confirmed found  implant  However, when  increase in hepatic  Dexamethasone a l o n e had  no  on e s t r a d i o l b i n d i n g i n t h e hypophysectomized, o v a r i e c t o m i z e d A s y n e r g i s t i c e f f e c t was  combined w i t h hGH.  the:hypophysectomized,  a l s o observed when dexamethasone  In t h i s case s p e c i f i c e s t r a d i o l b i n d i n g of  o v a r i e c t o m i z e d female was  to t h e o v a r i e c t o m i z e d l e v e l  completely r e s t o r e d  (Norstedt et a l . , 1981b).  In summary, p i t u i t a r y hormones a r e i n t i m a t e l y l i n k e d to maintenance of h e p a t i c e s t r o g e n r e c e p t o r l e v e l s a l t h o u g h g l u c o c o r t i c o i d s do appear to modulate these  levels.  22  8)  Hepatic sex steroid binding proteins To t h i s point we have discussed the interaction between  p i t u i t a r y hormones and the hepatic estrogen receptor.  It i s  recognized that androgens modulate both hepatic lactogen binding, and drug and steroid metabolism in the r a t .  However, to date a  high a f f i n i t y , l o w capacity androgen receptor that posseses the c l a s s i c cytosolic receptor c h a r a c t e r i s t i c s 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 i n vivo nuclear uptake of radiolabeled testosterone.  However, no cytosolic  characterization was done and the highest a v i d i t y 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  sucrose density gradient, was present in the adult male, and  on a  was  correlated to the hepatic synthesis of a urinary protein, c^^globulin.  The presence of t h i s s i t e as well as c ^ - g l o b u l i n  were shown to be androgen dependent.  The l e v e l s of both proteins  were reduced by castration or treatment with e s t r a d i o l . t h i s binding protein s p e c i f i c a l l y bound DHT,  Interestingly,  testosterone and  estradiol. In fact, examination of the binding data from Roy et a l . , (1973) indicates that 17g-estradiol i s probably the correct ligand for t h i s protein and not DHT Scatchard plot. DHT  due to the inconsistency of the  DHT  This i s despite the fact that the apparent Kd for  (Kd = 4.5 x 10  —8  —7 M) i s less than that for e s t r a d i o l (Kd = 3.5 x 10~ M) .  23  It  i s a l s o q u e s t i o n a b l e what p h y s i o l o g i c a l r o l e t h i s sex  b i n d i n g p r o t e i n p l a y s as the l i g a n d a f f i n i t y the e s t r o g e n  receptor  (Kd = 0.1  nM).  what p h y s i o l o g i c a l l i g a n d n o r m a l l y  Sato et a l . , (1980a, 1980b) and detected  analysis.  The  dependent. x 10  site.  Ota et a l . , (1980) have  The  f o l l o w i n g sucrose d e n s i t y  presence of t h i s p r o t e i n was apparent Kd  which was  (1974) f o r e s t r a d i o l . Roy  this  questionable  the presence of a h e p a t i c c y t o s o l i c androgen b i n d i n g  p r o t e i n t h a t sedimented a t 10S  1.37  i s w e l l below t h a t of  It i s certainly  occupies  steroid  gradient  shown to be androgen  f o r t e s t o s t e r o n e was  r e p o r t e d to  s i m i l a r to t h a t r e p o r t e d by Roy  be  et a l . ,  U n l i k e the b i n d i n g p r o t e i n d e s c r i b e d  et a l . , (1974) n e i t h e r e s t r a d i o l or DHT  were e f f e c t i v e  by competitors  3 for  [ H]-testosterone  binding.  These r e s u l t s a r e s u g g e s t i v e of a  h e p a t i c androgen r e c e p t o r ; however, s i m i l a r data had not  been  r e p o r t e d by o t h e r 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 b i n d i n g p r o t e i n s may a second c l a s s of h e p a t i c c y t o s o l i c e s t r o g e n i d e n t i f i e d and  c h a r a c t e r i z e d by Dickson  et a l . , (1980), and  be a s s o c i a t e d  binding  with  proteins  et a l . , (1978), Eagon  Powell-Jones et a l . , (1980,1981).  These  i n v e s t i g a t o r s have demonstrated the presence of a m a l e - s p e c i f i c estrogen  b i n d i n g p r o t e i n t h a t sedimented i n the 4S r e g i o n f o l l o w i n g  sucrose d e n s i t y g r a d i e n t a n a l y s i s . and  T h i s p r o t e i n bound  androgens w i t h an apparent Kd between 0.1  i n t e r e s t i n g l y d i d not  bind d i e t h y l s t i l b e s t r o l  commonly used t o d e t e c t the e s t r o g e n g l u c o c o r t i c o i d s nor  progestins.  and  estrogens  1 x 10 ^M,  and  (DES), which i s  r e c e p t o r , nor d i d i t b i n d  24  C e r t a i n a s p e c t s o f t h i s male s p e c i f i c b i n d i n g p r o t e i n p a r a l l e l the DHT b i n d i n g p r o t e i n d e s c r i b e d by Roy e t a l . , (1974). s p e c i f i c sex s t e r o i d  binding protein i s only detected  The male  i n the a d u l t  male r a t ; l i t t l e o r none i s found i n the immature male or female or a d u l t female.  The presence of t h i s p r o t e i n has been shown t o  be androgen dependent.  C a s t r a t i o n o f the a d u l t male reduced the  c a p a c i t y i n a t e s t o s t e r o n e r e v e r s i b l e f a s h i o n , (Eagon et a l . , ( 1 9 8 0 ) . However, t h i s e f f e c t  was o n l y observed by Powell-Jones e t a l . ,  (1980,1981) f o l l o w i n g n e o n a t a l Another i n t e r e s t i n g  gonadectomy.  characteristic  of t h i s binding protein  n o t e d by Powell-Jones e t al.,(1980) was t h a t hypophysectomy produced a decrease i n 4S b i n d i n g i n the male but i n c r e a s e d 4S b i n d i n g i n t h e female.. Therefore, estrogens  t h i s male s p e c i f i c  s i t e t h a t b i n d s androgens and  has been shown t o be age and sex dependent, r e q u i r e  androgens and t h e hypophyseal-hypothalamic a x i s f o r d i f f e r e n t i a t i o n i n the male, and may be i m p r i n t e d n e o n a t a l l y i n the male by androgens.  9)  Hypothesis  The r e g u l a t o r y a s p e c t s o f the h e p a t i c male s p e c i f i c sex steroid  b i n d i n g p r o t e i n p a r a l l e l those  t h a t have been e s t a b l i s h e d  f o r h e p a t i c drug and s t e r o i d metabolism p r e v i o u s l y d i s c u s s e d . Powell-Jones e t a l . , (1980,1981) have shown no d i f f e r e n c e s f o r the estrogen and  evidence  ontogenetic  r e c e p t o r between male and female r a t s ,  f o r the existence of a high a f f i n i t y , l o w  capacity  25  androgen r e c e p t o r of h e p a t i c  i s doubtful.  We  h y p o t h e s i z e d t h a t the m o d u l a t i o n  sex dependent enzyme a c t i v i t i e s by  mediated by the male s p e c i f i c sex  sex  steroid binding  steroids i s protein,  t h a t the presence of t h i s b i n d i n g p r o t e i n i s r e g u l a t e d hypophyseal-hypothalamic a x i s . binding and  Therefore,  hydrocarbon h y d r o x y l a s e and  i n r e l a t i o n to hepatic  testosterone  the  investigated  of e s t r a d i o l t o the h e p a t i c male s p e c i f i c b i n d i n g  a l s o t o the e s t r o g e n r e c e p t o r ,  Various  we  by  and  A4 r e d u c t a s e  the protein, aryl  activities.  p h y s i o l o g i c a l models were s t u d i e d to determine i f a  correlation existed.  These i n c l u d e d  the f o l l o w i n g : a d u l t male  v e r s u s female, a d u l t v e r s u s immature, male and  female v e r s u s  pseudohermaphrodite, sham-operated v e r s u s gonadectomized or hypophysectomized.  In a d d i t i o n , to g a i n an u n d e r s t a n d i n g of  r e g u l a t o r y mechanisms i n v o l v e d we  i n v e s t i g a t e d the e f f e c t s of  hormone replacement w i t h t e s t o s t e r o n e rGH,  oPRL, rPRL, and  the  enanthate, m e s t r a n o l ,  bGH,  i n d u c t i o n by 3-methylcholanthrene, pheno-  b a r b i t a l or s p i r o n o l a c t o n e  i n some of these models.  26  MATERIALS AND  1)  METHODS  Chemicals and r e a g e n t s  Activated charcoal  ( u n t r e a t e d ) , benzo(a)pyrene  ( r e c r y s t a l l i z e d from methanol bovine serum albumin threitol  following dissolution  (BSA), Coomassie  (BP)  i n benzene),  b r i l l i a n t b l u e G, D - l d i t h i o -  (DTT), disodium e t h y l e n e d i a m i n e . t e t r a a c e t i c a c i d  D-glucose-6-phosphate dehydrogenase  monosodium s a l t  (G6P),  glucose-6-phosphate  t y p e XII (G6PD), 3-methylcholanthrene  m o l y b d a t e , n i c o t i n a m i d e adenine d i n u l c l e o t i d e n i c o t i n a m i d e adenine d i n u c l e o t i d e phosphate n i c o t i n a m i d e adenine d i n u c l e o t i d e phosphate  (EDTA),  (3-MC), sodium  (reduced form) (NADH), (reduced form) (NADPH),  (NADP), q u i n i n e s u l f a t e ,  (R) spironolactone, T r i z m a ^ b a s e along with the f o l l o w i n g  steroids:  a l d o s t e r o n e , a n d r o s t e n e d i o n e , d i h y d r o t e s t o s t e r o n e (DHT), stilbestrol  diethyl-  (DES), dexamethasone, 1 7 3 - e s t r a d i o l , p r o g e s t e r o n e ,  t e s t o s t e r o n e , and t r i a m c i n o l o n e were o b t a i n e d from Sigma Chemical Company ( S t . L o u i s ,  MO.)  3 Labeled, s t e r o i d s ,  [6,7- H(N)]  e s t r a d i o l , 40-60 Ci/mmol,  [2,4,6,7,16,17- H(N)] e s t r a d i o l 130-170 Ci/mmol and 3  scintillation  c o c k t a i l were purchased from New  Biofluor®  England N u c l e a r ,  3 (Boston, "MA.  ).  In a d d i t i o n ,  [6,7- H] e s t r a d i o l , 40-60 Ci/mmol,  was a l s o o b t a i n e d from Amersham L t d . ( O a k v i l l e , Ont.).  Unlabeled  s y n t h e t i c s t e r o i d s promogestone (R5020), m e t h y l t r i e n o l o n e (R1881), and m o x e s t r o l (R2858) were o b t a i n e d from New  England N u c l e a r under  l i c e n s e agreement from R o u s s e l - U c l a f ( R o m a i n v i l l e , F r a n c e ) . A l l  27  s t e r o i d s were checked f o r p u r i t y by manufacturer recommended chromatography  procedures upon r e c e i p t .  Dextran T-70 was (Uppsala, Sweden).  s u p p l i e d by Pharmacia/Fine Chemicals AB  Ammonium s u l f a t e as w e l l as sodium  phenobarbitone  were purchased from B r i t i s h Drug House (Vancouver, B.C.). was  o b t a i n e d from Ortho P h a r m a c e u t i c a l s (Don M i l l s ,  gel  TLC p l a t e s were s u p p l i e d by Eastman Kodak Co. Pimozide  (McN-JR-6238) was a g i f t  (Spring House, PA.) IN.)  as was  Ont.)  Mestranol Silica  (Rochester, NY.,).  from McNeil L a b o r a t o r i e s  p e r g o l i d e from E l i L i l l y  The f o l l o w i n g p i t u i t a r y hormones were a g i f t  Co.  of the N a t i o n a l  Hormone and P i t u i t a r y Program ( B a l t i m o r e , MP..)': bovine hormone (NIH-bGH-B18) (bGH), o v i n e p r o l a c t i n  (Indianapolis,  growth  (NIAMDD-oPRL-15)  (oPRL), r a t growth hormone (NIAMDD-rGH-B7) (rGH), r a t p r o l a c t i n . (NIADDK-rPRL-B4) (rPRL). was  s u p p l i e d by KOR  2,3,7,8-tetrachlorodibenzo-p-dioxin  I s o t o p e s , (Cambridge, MA..).  (TCDD)  Octachlorodibenzo-p-  d i o x i n and 2 , 7 - d i c h l o r o d i b e n z o - p - d i o x i n were o b t a i n e d from  Ultra  (R) Scientific  (Hope,  RT.) .  A l z e t ~ model 2002 osmotic minxpumps were_:  purchased from A l z a Corp. (Palo A l t o ,  CA.).  A l l other chemicals  and r e a g e n t s used were o f the b e s t grade and p u r i t y  2)  available.  Animals  Male and female W i s t a r r a t s were o b t a i n e d from Canadian B r e e d i n g Farms ( M o n t r e a l , Que.).  Pseudohermaphrodite  r a t s a l o n g w i t h the  l i t t e r m a t e c o n t r o l males and females were s u p p l i e d by the I n t e r n a t i o n a l Foundation f o r Study o f Rat G e n e t i c s and Rodent C o n t r o l C i t y , OK:.-') .  Pseudohermaphrodite  (Oklahoma  r a t s as w e l l as the l i t t e r m a t e  c o n t r o l s were t r e a t e d w i t h an a n t h e l m i n t i c on a l t e r n a t e weeks  28  f o r 3 weeks d u r i n g q u a r a n t i n e .  Hypophysectomized  and  sham-operated c o n t r o l s were s u p p l i e d through Canadian Farms; however, a l l s u r g i c a l procedures were conducted R i v e r B r e e d i n g L a b o r a t o r i e s (Stone Ridge, NY'Y'.) .  Breeding by C h a r l e s  A l l animals used  i n t h e hypophysectomy study were operated o r sham-operated between 50-60 days o f age, and r e c e i v e d 5% s u c r o s e upon a r r i v a l f o r one week.  The completeness  o f hypophysectomy was determined by v i s u a l  i n s p e c t i o n a t t h e time o f s a c r i f i c e as w e l l as decrease i n body weight. bedding  Animals were housed i n a s e p a r a t e animal room on L o b u n d ® (Paxton P r o c e s s i n g L t d . , Paxton, I L . ) under c o n t r o l l e d  temperature  (22°C) and l i g h t  (on 0600-2000 h r s . ) .  They were a l l o w e d  f r e e a c c e s s t o food ( P u r i n a L a b o r a t o r y Chow, R a l s t o n P u r i n a o f Canada L t d . , Woodstock, Ont.) and water ad l i b i t u m .  3)  Animal  treatments  Pimozide was a d m i n i s t e r e d (0.6 mg/kg/day, i n 40% p r o p y l e n e g l y c o l ) subcutaneously  ( s . c . ) f o r t e n days t o sham-operated or  gonadectomized  a d u l t male r a t s .  gonadectomized  r a t s r e c e i v e d p e r g o l i d e (0.1 mg/kg/day, i n corn  oil)  A d u l t female sham-operated o r  subcutaneously f o r e l e v e n days.  r a t s were t r e a t e d w i t h m e s t r a n o l subcutaneously  f o r t e n days.'  I n t a c t a d u l t male and female  (1.0 mg/kg/day, i n c o r n o i l )  S p i r o n o l a c t o n e was a d m i n i s t e r e d  ..(100 mg/kg/day, i n , c o r n o i l ) by t h e i n t r a p e r i t o n e a l ( i . p . ) r o u t e f o r f o u r days. a d m i n i s t e r e d once d a i l y , and sodium phenobarbitone  Unless o t h e r w i s e s p e c i f i e d , 3-MC was  (20 mg/kg i . p . , i n corn o i l ) f o r two days, once d a i l y  (80 mg/kg i . p . , i n normal  29  s a l i n e ) f o r f o u r days.  Rats t r e a t e d w i t h t e s t o s t e r o n e enanthate  r e c e i v e d 1.0 mg/kg/day s . c . i n corn o i l f o r t e n days.  Pituitary (R)  hormones were a d m i n i s t e r e d by c o n t i n u o u s i n f u s i o n u s i n g model 2002 osmotic minipumps. normal s a l i n e to  AlzetS^  The hormones were d i s s o l v e d i n  (pH 8.5); pumps were weighed b e f o r e and a f t e r  ensure complete f i l l i n g .  loading  The pumps had a l i f e expectancy of  f o u r t e e n days a t a pumping r a t e o f 0.4'58 y l / h r ± 0.026 y l / h r (S.D.).  Pumps were implanted subcutaneously i n the r o s t r a l  h a l f of e t h e r rats. six  a n e s t h e t i z e d hypophysectomized and  Animals were s a c r i f i c e d 10 days f o l l o w i n g  t o e i g h t weeks post-hypophysectomy.  sham-operated implantation;  C e r t a i n groups r e c e i v e d  t e s t o s t e r o n e enanthate (1.0 mg/kg/d s.c.) c o n c u r r e n t l y f o r t e n days.  The doses of t h e 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, a c c o r d i n g t o Mode e t a l . ,  (1982).  s e p a r a t e study, bGH was a d m i n i s t e r e d by i n j e c t i o n  In a  (2.0 I.U./kg,  i n normal s a l i n e pH 8.5) t w i c e d a i l y f o r t e n days.  4)  H e p a t i c microsomal enzyme a s s a y s  a)  H e p a t i c microsomal p r o t e i n  preparation  Animals were s a c r i f i c e d between 0900-1000 hours. stunned by a blow t o t h e head and k i l l e d by c e r v i c a l Inferior  They were  dislocation.  • vena cava were cut and l i v e r s were.'perfused w i t h 10 ml of  ice  c o l d 1.15% KC1 v i a t h e p o r t a l v e i n .  ice  c o l d KC1 1.15%, b l o t t e d dry and weight r e c o r d e d .  then minced and homogenized  1:4  L i v e r s were e x c i s e d  into  They were  (w/v) i n 1.15% KC1 u s i n g a P o t t e r -  30  Elvejhem t i s s u e homogenizer. 10,000g  Homogenates were c e n t r i f u g e d a t  u s i n g an I n t e r n a t i o n a l Equipment  l i t s . , MA.)  model B-20 r e f r i g e r a t e d  Company (TEC)  (Needham  c e n t r i f u g e f o r 10 minutes  at 4°C. The s u p e r n a t a n t s were c e n t r i f u g e d a t 100,000'g", f o r 60 minutes a t 4°C u s i n g a Beckman L5-50 or L2-65B u l t r a c e n t r i f u g e w i t h e i t h e r a t y p e 40 o r type 65 f i x e d a n g l e r o t o r  (Beckman Instruments  Corp., P a l o Alto,OA.',) . F o l l o w i n g c e n t r i f u g a t i o n each p e l l e t was washed w i t h 3 volumes of i c e c o l d  0.1M' NaK2P04 b u f f e r pH 7.2, resuspended and d i l u t e d  1:3.75 i n t h e same b u f f e r .  b)  A r y l hydrocarbon h y d r o x y l a s e a c t i v i t y  H e p a t i c a r y l hydrocarbon h y d r o x y l a s e ( A H H ) a c t i v i t y was determined a c c o r d i n g t o t h e method o f Nebert and G e l b o i n (1968) as m o d i f i e d by A l v a r e s  (1970) u s i n g benzo(a)pyrene as a s u b s t r a t e .  The amount o f 3-hydroxy-benzo(a)pyrene fluorometrically  produced was q u a n t i t a t e d  - r e l a t i v e to a quinine s u l f a t e standard.  The  f i n a l volume o f t h e i n c u b a t i o n m i x t u r e (1.0 ml) c o n t a i n e d 0.36 ymol NADH; 0.36 ymol NADPH; 3 ymol MgCl .6H 0; 8.7 nmol BSA; 150 u l 2  2  microsomal p r o t e i n ; 25 ymol T r i s b u f f e r 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 t h e a d d i t i o n o f BP f o l l o w i n g a one minute and f i f t e e n second enzyme p r e i n c u b a t i o n p e r i o d . i n c u b a t i o n was c o n t i n u e d f o r 2.5 minutes a t 37°C. was  The r e a c t i o n  t e r m i n a t e d by t h e a d d i t i o n o f 1.0 ml o f i c e c o l d  f o l l o w e d by 3.3 ml o f hexane.  The  acetone  Enzyme and s u b s t r a t e b l a n k s were  31  c a r r i e d through t h e i n c u b a t i o n and t h e a p p r o p r i a t e component was added a f t e r t e r m i n a t i o n of t h e r e a c t i o n . The  samples were mixed and t h e phases a l l o w e d t o s e p a r a t e .  A 2i..-0 ml a l i q u o t of t h e hexane phase was t r a n s f e r r e d t o 4.0 ml of 1.0N NaOH and mixed. acetone  The NaOH phase was f r o z e n i n a d r y i c e -  bath and t h e hexane was d i s c a r d e d .  The NaOH phase was  thawed i n a warm water b a t h and f l u o r e s c e n c e was measured w i t h a Turner  S p e c t r o f l u o r o m e t e r Model 430.  396 nm and e m i s s i o n a t 522 nm. 1.0N  The instrument was zeroed  NaOH and c a l i b r a t e d w i t h q u i n i n e s u l f a t e  0.1N).  c)  The e x c i t a t i o n was s e t a t with  (0.1 g i n 1.0£ ^ S O ^  A l l d e t e r m i n a t i o n s were done i n d u p l i c a t e .  T e s t o s t e r o n e A4 r e d u c t a s e  activity  H e p a t i c t e s t o s t e r o n e A4 r e d u c t a s e was assayed  according to  t h e method o f McGuire and Tomkins (1959),. The a c t i v i t y o f t h e r e d u c t a s e was determined  s p e c t r o p h o t o m e t r i c a l l y by t h e disappearance  of s u b s t r a t e r e l a t i v e t o a standard curve. as nmoles t e s t o s t e r o n e reduced protein.  A c t i v i t y was expressed  per minute per mg microsomal  The f i n a l volume o f t h e i n c u b a t i o n m i x t u r e  (1.78 ml)  c o n t a i n e d 12.5 ymol MgCl .6H 0; 1.0 ymol NADP; 20.0 ymol G6P; 2  1 unit  2  G6PD; 200 y l p r o t e i n and 0.3 ymol t e s t o s t e r o n e i n 20 y l  methanol. The enzyme suspension was p r e i n c u b a t e d f o r one minute and f i f t e e n seconds a t 37°C a t which time t h e assay was i n i t i a t e d by the a d d i t i o n o f t e s t o s t e r o n e .  The r e a c t i o n was t e r m i n a t e d a t  10 minutes by t h e a d d i t i o n o f 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  a c t i v i t y 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 t h i s procedure are outlined below. The assay conditions were as follows.  Rats were s a c r i f i c e d  by c e r v i c a l dislocation and l i v e r s were perfused with i c e 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.  homogenized at  Livers were  1:10 i n buffer and cytosol prepared by centrifugation  100,000g, for 60 minutes.  The r e s u l t i n g 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 aliquots. The f i r s t was diluted with TED buffer to a protein content of 5-7 mg/ml and placed on i c e .  The second aliquot was p a r t i a l l y p u r i f i e d  by a 30% ammonium sulfate fractionation.  An equal volume of  ammonium s u l f a t e was added drop-wise to c y t o s o l .  This was mixed  33  f o r 30 minutes on i c e , and c e n t r i f u g e d f o r 30 minutes a t 15,000g The r e s u l t i n g p e l l e t was resuspended  t o t h e o r i g i n a l volume w i t h  buffer. I n c u b a t i o n s were c a r r i e d out by adding  100 y l o f p r o t e i n  3 t o 100 y l o f [ H ] - e s t r a d i o l ,  0.2-10 nM, ( i n b u f f e r ) i n t h e presence  and absence o f a 100 f o l d excess o f u n l a b e l e d • d i e t h y l s t i l b e s t . r o l (DES)  (in buffer).  at 4 ° C  P r o t e i n and s t e r o i d were i n c u b a t e d f o r 2 hours  Bound was separated from f r e e by t h e a d d i t i o n o f dextran  (0.05%) coated c h a r c o a l (0.5%) (DCC) i n TED b u f f e r .  P r o t e i n was  i n c u b a t e d w i t h DCC f o r 10 minutes and DCC was removed by c e n t r i f u g a t i o n a t 800g f o r 10 minutes.  Supernatants  f o r bound e s t r a d i o l u s i n g standard s c i n t i l l a t i o n  were  counted  counting techniques.  The e s t r o g e n r e c e p t o r assay d e s c r i b e d by Powell-Jones et a l . , (1980,1981) was compared t o t h e e s t r o g e n r e c e p t o r assay employed a t t h e Hormone Receptors L a b o r a t o r y , U n i v e r s i t y o f Oregon (Portland).  The second method, n o r m a l l y used  f o r q u a n t i t a t i o n of  e s t r o g e n r e c e p t o r s i n u t e r i n e and mammary t i s s u e , had t h e f o l l o w i n g m o d i f i c a t i o n s : TED b u f f e r c o n t a i n s 50 mM T r 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 a t 4°C. Furthermore,  f o l l o w i n g homogenization  u s i n g a Brinkman p o l y t r o n ,  samples were c e n t r i f u g e d at 800g'; f o r 10 minutes.  The r e s u l t i n g  supernatant was then c e n t r i f u g e d f o r 30 minutes a t 100,000g' t o obtain the cytosolic f r a c t i o n . i n TED b u f f e r .  P r o t e i n was d i l u t e d t o 1-3 mg/ml  Incubation mixture  c o n t a i n e d 500 y l p r o t e i n and  3 10 y l [ H ] - e s t r a d i o l 0.2-10 nM ( i n 95% ethanol) i n t h e presence and absence o f 10 y l o f a 100 f o l d excess o f u n l a b e l e d ' DES ( i n 95% e t h a n o l ) .  S e p a r a t i o n o f bound from f r e e was a c h i e v e d by t h e  34  a d d i t i o n of 500  u l of DCC  c e n t r i f u g a t i o n at 1500g  f o r 5 minutes. for  10 minutes.  was  counted as  b)  Hepatic estrogen receptor  DCC  was  removed  by  The  r e s u l t i n g supernatant  before.  and moderate a f f i n i t y  binding  component a s s a y  For purposes of d e t e c t i o n and estrogen receptor of the two for  previously described  20 mM  TED  sodium molybdate, and  c e n t r i f u g e d at 800g (Palo A l t o , OA.) rotor.  The  10%  Following  first  to o b t a i n  buffer using a  b u f f e r and  cyotosol  fraction.  The  i n TED  at 4°C.  Livers Potter-  An  4.2  c e n t r i f u g e d at 100,000g  for  either  L5-50 or L65-B u l t r a c e n t r i f u g e w i t h a type  on  second a l i q u o t was  sulfate fractionation.  DTT,  u s i n g a Beckman  The  supernatant was  coat, and  removed,  d i v i d e d i n t o two  d i l u t e d to a p r o t e i n content o f 1-3  placed  mM  homogenization samples were  the c y t o s o l i c f r a c t i o n u s i n g  superficial lipid  w i t h TED  EDTA, 0.5  (w/v)  r e s u l t i n g supernatant was  a l i q u o t was  i s described  7.5  40 o r type 65 f i x e d angle r o t o r .  The  mM  used  r e f r i g e r a t e d c e n t r i f u g e w i t h a type JS  a Beckman (Palo A l t o , CA.)  l e a v i n g the  an  g l y c e r o l , pH  f o r 10 minutes a t 4°C  ;  J6-B  30 minutes a t 4°C  T h i s assay was  50 mil T r i s - H C l , 1.5  homogenized 1:10  Elvehjem homogenizer.  methods.  p h a r m a c o l o g i c a l s t u d i e s and  b u f f e r contained  were minced and  adopted  assay p r o t o c o l t h a t combined s e v e r a l a s p e c t s  a l l p h y s i o l o g i c a l and  below.  e f f i c i e n c y we  ice.  This represented  mg/ml  the whole  p a r t i a l l y p u r i f i e d by a 50%  equal volume of s a t u r a t e d  aliquots.  ammonium  (at room  temperature) ammonium s u l f a t e DTT  pH 7.5)  was  ( i n 10 mM  T r i s ; 1.0  mM  EDTA; 1.0  added dropwise to the c y t o s o l f r a c t i o n .  mixed f o r 30 minutes i n an i c e bath, and  This  mM was  c e n t r i f u g e d at 15,000g  f o r 30 minutes a t 4°C u s i n g an IEC model - B - 2 0 r e f r i g e r a t e d c e n t r i f u g e . The r e s u l t i n g p e l l e t was  washed and  resuspended t o the  original  volume of c y t o s o l w i t h TED b u f f e r . The b i n d i n g 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 by S c a t c h a r d  determined  (1949) a n a l y s i s of" c o m p e t i t i v e b i n d i n g c u r v e s - u s i n g both  the whole c y t o s o l 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  affinity,  assessed i n  low c a p a c i t y h e p a t i c e s t r o g e n r e c e p t o r was  t h e ammonium s u l f a t e f r a c t i o n .  The  incubation mixture  high  contained  3  500 p i p r o t e i n , 10 y l 0.05-2.5 nM ( i n 95% e t h a n o l )  estradiol  ( i n 95% e t h a n o l ) .  h i g h e r c a p a c i t y component was The  3  The moderate a f f i n i t y ,  c o n t a i n e d 500 y l p r o t e i n ,  Ci/mmol) 10-200 nM  o r absence of a 100  e s t r a d i o l o r a 1000  ( i n 95%  ethanol)  f o l d excess of u n l a b e l e d  f o l d excess of u n l a b e l l e d  DHT.  I n c u b a t i o n s were c a r r i e d out f o r 90 minutes a t 4°C. s t e r o i d was T-70  separated  (0.05% w/v)  Bound  from f r e e by the a d d i t i o n of 500 y l d e x t r a n  coated  c h a r c o a l (0.5% w/v)  (DCC)  i n TED b u f f e r  (prepared as p r e v i o u s l y d e s c r i b e d minus molybdate). the a d d i t i o n of DCC,  fold  measured i n the whole c y t o s o l  incubation mixture  10 y l [ H ] - e s t r a d i o l (7-10 i n the presence  Ci/mmol)  i n the presence or absence of 10 y l of a 100  excess u n l a b e l e d  fraction.  [ H ] - e s t r a d i o l (140  samples were mixed and DCC  was  Following sedimented  by c e n t r i f u g a t i o n at 1500g'" f o r 10 minutes a t 4°C u s i n g a Beckman J6-B  refrigerated  c e n t r i f u g e w i t h a type JS 4.2  s u p e r n a t a n t s were sampled  (0.5 ml)  and  rotor.  The  combined w i t h 10 ml  resulting Biofluor®  36  liquid  scintillation cocktail.  e s t r a d i o l using  Samples were counted f o r bound  a S e a r l e Mark I I I L i q u i d S c i n t i l l a t i o n  System-Model  6880 ( S e a r l e A n a l y t i c Inc., Des P l a i n s , I L . ) . A l l p o i n t s were assayed i n . d u p l i c a t e .  The tubes which  3  [ H ] - e s t r a d i o l w i t h o u t competitor were a measure o f t o t a l  binding  3  (TB).  Tubes c o n t a i n i n g b o t h [ H ] - e s t r a d i o l and competitor  the n o n - s p e c i f i c b i n d i n g  c)  A n a l y s i s of b i n d i n g  contained  represented  (NSB) p r e s e n t .  data  C a l c u l a t i o n o f s p e c i f i c bound was done as f o l l o w s .  The counts  3 per minute (cpm) recorded  f o r [ H ] - e s t r a d i o l bound were c o n v e r t e d  to d i s i n t i g r a t i o n s per minute (dpm) based on the p e r c e n t of the i n s t r u m e n t .  E f f i c i e n c y f o r t r i t i u m v a r i e d between 45-48%  based on t h e degree o f quenching f o r each sample. calculated  efficiency  Thus dpms were  on t h e b a s i s o f e f f i c i e n c y , which was determined f o r  each sample from p r e v i o u s l y prepared quench s t a n d a r d s . were c o n v e r t e d t o C u r i e s f o r each sample on the b a s i s  The dpms 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 t h e l i g a n d s  were employed t o c a l c u l a t e  the femtomoles o f b i n d i n g which was  n o r m a l i z e d f o r p r o t e i n content. values  were s u b t r a c t e d  binding  Duplicates  to g i v e  specific  were then averaged.  The  ( t o t a l counts) added, l e s s the S.B., r e p r e s e n t s the  free concentration free  from T.B. tube v a l u e s  (S.B.) a t any p o i n t .  total ligand  A t t h i s p o i n t ; the N.S.B. tube  (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  (B/F). In cases where displacement was observed t h e B/F was graphed  37  as a f u n c t i o n of S.B. slope.  The  to y i e l d a curve-linear plot with a  i n v e r s e of the s l o p e o f the l i n e a r p o r t i o n of t h e  represented  the d i s s o c i a t i o n constant  uncorrected  measure of c a p a c i t y .  (Kd), and  plot  the x - i n t e r c e p t t h e  P o i n t s d e v i a t i n g from l i n e a r i t y at the low and the c u r v e  negative  h i g h end  1  of  (due to non-optimal l i g a n d - p r o t e i n c o n c e n t r a t i o n and  other  b i n d i n g s i t e s of lower a f f i n i t y r e s p e c t i v e l y ) were not used f o r a n a l y s i s of b i n d i n g  data.  In a d d i t i o n , b i n d i n g data were s u b j e c t to n o n - l i n e a r a n a l y s i s u s i n g the curve Munson and fit,  b i n d i n g components. inhibition and  f i t - m o d e l l i n g program Ligand developed  Rodbard (1980).  as w e l l as v a l u e s  T h i s was  by  done t o c o n f i r m goodness of  f o r the apparent Kd and  However, t h i s program was  c a p a c i t y of designed  the  for  s t u d i e s t h a t employ a f i x e d c o n c e n t r a t i o n of t r a c e r  i n c r e a s i n g amounts of u n l a b e l e d  program was  competitor.  Therefore,  w r i t t e n to c o n s i d e r n o n - s p e c i f i c b i n d i n g to be a  v a l u e f o r each c o n c e n t r a t i o n , and does not n o r m a l i z e p r o t e i n content. unlabeled  regression  The  competitor  b i n d i n g was  constant  data f o r  assay we have employed v a r i e s b o t h the and  t r a c e r t o g e t h e r . As such, n o n - s p e c i f i c  measured at each c o n c e n t r a t i o n because the r a t i o  N.S.B.tT.B. i s not  the  constant.  Furthermore, we  data t o p r o t e i n c o n c e n t r a t i o n because we  of  have n o r m a l i z e d  our  a r e i n t e r e s t e d i n the  r e l a t i v e change i n b i n d i n g c a p a c i t y f o l l o w i n g p h y s i o l o g i c a l m a n i p u l a tion.  Because of t h e s e  reasonable  values  features  of the Ligand  program, we  f o r the apparent Kd o f t h e e s t r o g e n  but the c a p a c i t i e s were u n r e a s o n a b l y  high.  obtained  binding proteins,  38  We were a l s o not a b l e t o combine the male e s t r o g e n r e c e p t o r w i t h the moderate a f f i n i t y b i n d i n g data t o model the o v e r a l l c u r v e . reason f o r t h i s was  t h a t t h e assays were conducted  f r a c t i o n s and used t r a c e r s o f d i f f e r e n t  specific  The  in different  activities.  T h e r e f o r e , the L i g a n d program had a l i m i t e d a p p l i c a t i o n to our s t u d i e s , and was  6)  not e x t e n s i v e l y used.  D e t e r m i n a t i o n of p r o t e i n content  Microsomal  and c y t o s o l i c p r o t e i n c o n c e n t r a t i o n s were  by the method of B r a d f o r d (1976).  determined  The assay i s l i n e a r f o r p r o t e i n  c o n c e n t r a t i o n s from 0.1-0.5 mg/ml u s i n g BSA as a s t a n d a r d . content was  Protein  a s s e s s e d s p e c t r o p h o t o m e t r i c a l l y a t 595 nm u s i n g a  P e r k i n - E l m e r Model 124  double beam spectrophotometer.  Samples  were read f i v e minutes a f t e r the a d d i t i o n of 5.0 ml of B r a d f o r d reagent which c o n t a i n e d 100 mg  C o o m a s s i e - b r i l l i a n t blue-G;  a b s o l u t e e t h a n o l ; 100 ml phosphoric a c i d 85%; and f i l t e r e d u s i n g a Buschner s u c t i o n  7)  50 ml  d i l u t e d to 1.0  %  apparatus.  Serum hormone l e v e l s  Blood samples were c o l l e c t e d by c a r d i a c puncture when animals were s a c r i f i c e d .  Whole b l o o d s were allowed t o c l o t then c e n t r i f u g e d  at 1500g f o r 10 minutes u s i n g a Beckman (Palo A l t o , CA.)  J6-B  refrigerated  was  a s p i r a t e d and  c e n t r i f u g e w i t h a t y p e JS 4.2  rotor.  f r o z e n a t -20°C f o r l a t e r a n a l y s i s .  f o r a n a l y s i s are d e s c r i b e d i n Appendix  1.  Serum  Procedures  39  8)  Statistical  analysis  D i f f e r e n c e s were c o n s i d e r e d p < 0.05  using  was.altered.  a two-tailed  significant  Students t - T e s t when o n l y one  However, the m a j o r i t y of the s t u d i e s  several variables.  As  range t e s t s at p <  introduced  the Newman-Keuls or Duncan m u l t i p l e  0.05,  A summary of the c o n t r o l v a l u e s  presented  f o r female and  and male moderate a f f i n i t y apparent Kd and i n T a b l e 32.  In a d d i t i o n , the mean and  of the mean a r e r e p o r t e d .  It i s evident  i n d i v i d u a l experiments were not  standard  error  combined but were  e s t a b l i s h e d at t h a t time.  considered  relative  Individual  the same s t o c k s o l u t i o n s of b u f f e r and  throughout each study.  a  Because of  to  studies  were conducted on s i n g l e shipments of a n i m a l s , w i t h i n short periods, using  are  from this- t a b l e t h a t  o n l y i n the s t u d y i n which they were c a r r i e d out and the c o n t r o l v a l u e s  male e s t r o g e n  capacity  wide v a r i a b i l i t y e x i s t s f o r t h e s e b i n d i n g parameters. this,  condition  such, comparisons between e x p e r i m e n t a l  groups were accomplished u s i n g  receptor  from c o n t r o l a t .  time  steroid  40  RESULTS  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  a)  Comparison of d i f f e r e n t e s t r o g e n  receptor  binding  components  assays  The developmental s t u d i e s 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 c o n d i t i o n s f o r e s t r o g e n b i n d i n g were done i n c o n j u n c t i o n  with  Dr. B. Warren. S t u d i e s t o determine e s t r o g e n b i n d i n g t o r a t h e p a t i c  cytosolic  p r o t e i n s were i n i t i a l l y c a r r i e d out by t h e method o f Powell-Jones et a l . , (1980,1981) as d e s c r i b e d i n t h e methods s e c t i o n .  Scatchard  a n a l y s i s of t h e r e s u l t s from the ammonium s u l f a t e f r a c t i o n  from  a d u l t male h e p a t i c c y t o s o l y i e l d e d a s a t u r a b l e b i n d i n g curve  with  an apparent Kd of 1.8 nM and a c a p a c i t y o f 11 fmol/mg p r o t e i n  3 (0.2-10 nM [ H ] - e s t r a d i o l ) .  Having confirmed  t h e method o f  Powell-Jones e t a l . , (1980,1981) f o r t h e d e t e c t i o n o f a h i g h a f f i n i t y , low c a p a c i t y e s t r o g e n  b i n d i n g component i n r a t h e p a t i c  c y t o s o l , we compared the technique  t o t h a t employed f o r d e t e c t i o n  of u t e r i n e and mammary h i g h a f f i n i t y e s t r o g e n  binding s i t e s .  second method, employed a t t h e Hormone Receptors  This  Laboratory,  U n i v e r s i t y o f Oregon, ( P o r t l a n d ) had c e r t a i n m o d i f i c a t i o n s (see m a t e r i a l s and methods f o r d e t a i l s ) . between these two e s t r o g e n the l a t t e r c o n t a i n e d  The most n o t a b l e d i f f e r e n c e  b i n d i n g a s s a y s was that: the TED b u f f e r o f  g l y c e r o l and molybdate.  Furthermore, c y t o s o l  was not s u b j e c t e d t o an ammonium s u l f a t e f r a c t i o n a t i o n and s t e r o i d s were added t o t h e i n c u b a t i o n m i x t u r e i n 95% e t h a n o l r a t h e r buffer.  than  41  Scatchard  a n a l y s i s o f t h i s c y t o s o l f r a c t i o n from a d u l t  male r a t s y i e l d e d an apparent Kd of 0.14 uM, and a c a p a c i t y of 3.3 pmol/mg p r o t e i n .  However, when measured i n females, the  apparent Kd and c a p a c i t y were s i m i l a r t o those o b t a i n e d Powell-Jones et a l . , (1980,1981) method 30 fmol/mg p r o t e i n ) .  (Kd = 2.2 nM, c a p a c i t y =  The s p e c i f i c b i n d i n g curve  f o r t h e male  suggested t h a t more than one b i n d i n g s i t e was present male whole c y t o s o l , one below 2.5 nM and one above. a f f i n i t y , higher  by the  i n the adult T h i s lower  c a p a c i t y b i n d i n g s i t e , t h e moderate a f f i n i t y ,  moderate c a p a c i t y s i t e d e s c r i b e d by Powell-Jones et a l . ( 1 9 8 0 , e f f e c t i v e l y masked t h e h i g h e r a f f i n i t y  s i t e i n t h e male.  This  accounted f o r t h e o b s e r v a t i o n o f the lower a f f i n i t y h i g h e r site.  To d e t e c t t h e h i g h e r  affinity  and  Specific  by t h e l a t t e r method) y i e l d e d an apparent  a c a p a c i t y o f 50 fmol/mg.  Therefore,  s i t e i n male c y t o s o l .  t h i s method f o r d e t e r m i n a t i o n  binding  Kd = 1.8 nM,  i t was apparent t h a t  ammonium s u l f a t e f r a c t i o n a t i o n was n e c e s s a r y the h i g h a f f i n i t y  capacity  s i t e i n t h e male we used an  ammonium s u l f a t e f r a c t i o n o f t h e whole c y t o s o l . (determined  1981),  f o r d e t e c t i o n of  Subsequently, we employed  o f t h e h i g h a f f i n i t y component i n  both male and female r a t s . In o r d e r t o c h a r a c t e r i z e these f r a c t i o n s (ammonium s u l f a t e vs whole c y t o s o l ) and t h e b i n d i n g components p r e s e n t , a s e r i e s of experiments was c a r r i e d out u s i n g v a r i o u s s t e r o i d  competitors  (Table 1 ) . The h i g h a f f i n i t y component was apparent i n the  3  ammonium s u l f a t e f r a c t i o n o f the male when [ H ] - e s t r a d i o l was competed w i t h e s t r a d i o l and DES.  The moderate a f f i n i t y  3  component  was apparent i n t h e male whole c y t o s o l 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 i n different fractions of adult male and female rat l i v e r using various competitors. Incubations were carried out for 90 minutes at 4°C.  Competitor [ H]-estradiol concentration range  Female  Male  3  Ammonium Sulfate Fract ion  Whole Cytosol Fraction  Ammonium Sulfate Fract ion  Whole Cytosol Fraction  Capacity (per mg protein)  Apparent Kd (xlO M)  Capacity (per mg protein)  Apparent Kd (xlO M)  Capacity (per mg protein)  NS  NS  0.46  34 fmol  2.2  29 fmol  NS  NS  NS  290  0.4 pmol  230  0.4 pmol  NS  NS  NS  NS  NS  NS  NS  NS  DHT (25 - 1000 nM)  NS  NS  NS  NS  NS  NS  Estradiol (0.05 - 15 nM)  1.8  158 fmol  NS  Estradiol (25 - 1000 nM)  120  1.7 pmol  340  Apparent Kd (xlO M)  Capacity (per mg protein)  DES (0.05 - 15 nM)  1.8  49 fmol  DES (25 - 1000 nM)  NS  DHT (0.05 - 15 nM)  9  1  1  Apparent Kd (xlO M) 9  250  13 pmol NS 7.5 pmol  Data provided by Dr. B. Warren  9  2.2 NS  89 fmol NS  9  1.5 NS  DES = Diethylstilbestrol DHT = Dihydrotestosterone NS = No Scatchard  33 fmol NS  43  was  competed w i t h e s t r a d i o l and DHT and i n the ammonium  when e s t r a d i o l was used.  The female c y t o s o l  sulfate  contained high  a f f i n i t y b i n d i n g i n both f r a c t i o n s u s i n g e s t r a d i o l and DES, (not DHT) as c o m p e t i t o r s .  Furthermore, we d e t e c t e d a s m a l l  amount of lower a f f i n i t y b i n d i n g i n the female when DES was used as a c o m p e t i t o r . I t was apparent t h a t not  the ammonium s u l f a t e f r a c t i o n a t i o n would  c o m p l e t e l y remove the moderate a f f i n i t y  whole c y t o s o l .  component from male  When t h e male ammonium s u l f a t e f r a c t i o n was  over a wide l i g a n d  studied  c o n c e n t r a t i o n range the moderate a f f i n i t y  was  detected.  We t h e r e f o r e compared that d i f f e r e n c e  and  a 50% ammonium s u l f a t e f r a c t i o n a t i o n  the  components.  component  between a 30%  i n an attempt t o s e p a r a t e  T a b l e 2 i l l u s t r a t e s the r e d u c t i o n i n c a p a c i t y  of t h e moderate a f f i n i t y component by ammonium s u l f a t e without p r o d u c i n g major e f f e c t s on t h e h i g h a f f i n i t y component.  Although  we c o u l d n o t c o m p l e t e l y a b o l i s h moderate a f f i n i t y b i n d i n g , we employed t h e 50% ammonium s u l f a t e f r a c t i o n i n f u r t h e r Since we were i n t e r e s t e d  studies.  i n the c h a r a c t e r i s t i c s of both the  h i g h and moderate a f f i n i t y b i n d i n g components and t h e i r we measured b i n d i n g i n both f r a c t i o n s . was  interactions,  The h i g h a f f i n i t y  component  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]-estradiol,  and the moderate a f f i n i t y component was measured 3  i n whole c y t o s o l for  between 10-200 nM I H ] - e s t r a d i o l .  Furthermore,  f u t u r e mathematical modeling c a p a b i l i t i e s , we u t i l i z e d  as our competior s i n c e  i t displaced  estradiol  bound e s t r a d i o l i n both  fractions.  44  TABLE 2.  3  The apparent Kd and c a p a c i t y o f [ H ] - e s t r a d i o l b i n d i n g t o d i f f e r e n t f r a c t i o n s o f a d u l t male rat l i v e r . I n c u b a t i o n s were c a r r i e d out f o r 90 minutes a t 4°C. A 100 f o l d excess o f u n l a b e l e d e s t r a d i o l was used as a c o m p e t i t o r .  Fraction [ H]-estradiol c o n c e n t r a t i o n range 3  Apparent Kd (nM)  Capacity (fmol/mg p r o t e i n )  30%  Ammonium S u l f a t e (0.05-2.5 nM)  1.1  118.5  50%  Ammonium S u l f a t e (0.05-2.5 nM)  1.2  89.2  Whole C y t o s o l (0.05-2.5 nM)  NS  NS  30%  Ammonium S u l f a t e (10-200 nM)  700  0.78  50%  Ammonium S u l f a t e (10-200 nM)  220  1.00  180  6.60  Whole C y t o s o l (10-200 nM)  NS = No S c a t c h a r d  b)  Time, Temperature and pH S t u d i e s  Because t h e a s s a y had been m o d i f i e d we conducted time, temperature and pH s t u d i e s t o e s t a b l i s h o p t i m a l a s s a y c o n d i t i o n s . The  optimal  i n c u b a t i o n time was determined t o be 90 minutes a t  4°C  f o r b o t h h i g h and moderate a f f i n i t y components  F i g u r e A1,A2).To f u r t h e r i n v e s t i g a t e t h e b i n d i n g for  both s i t e s ,  (Appendix 1,  characteristics  i n c u b a t i o n time courses were done a t 25°C.  S p e c i f i c b i n d i n g was determined f o r i n c u b a t i o n times  ranging  from 15 minutes t o 24 hours.  R e s u l t s i n T a b l e 3 i n d i c a t e 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 s u l f a t e f r a c t i o n s , and by 30 minutes i n a d u l t male.  Moderate a f f i n i t y b i n d i n g i n male whole  c y t o s o l was maximal a t 15 minutes w i t h e s t r a d i o l as a and  30 minutes w i t h DHT.  The  d i f f e r e n c e i n s p e c i f i c binding a t 4 versus  f i c a n t a t 60 minutes.  Thereafter a l l binding  competitor  decreased.  25°C was n o t s i g n i -  The apparent Kd and c a p a c i t y a t 4°C were  1.14 nM and 124 fmol/mg as compared t o 1.16 nM and 117 fmol/mg at  25°C, The  The  b i n d i n g o f e s t r a d i o l was s t a b l e t o s m a l l changes i n pH.  optimum pH range f o r t h e moderate a f f i n i t y component i n male  whole c y t o s o l was determined t o be pH 7.0-8.0. binding occurred.  At h i g h e r  I n c r e a s i n g t h e pH produced a s l i g h t  pH, no  decrease  i n a f f i n i t y and i n c r e a s e i n c a p a c i t y f o r t h e male h i g h a f f i n i t y even s m a l l e r e f f e c t s were noted f o r the female (Table 4 ) .  We  s e l e c t e d pH 7.5 t o be p h y s i o l o g i c a l l y r e l e v a n t and s i m i l a r t o previously published  studies.  site,  TABLE 3 Time course o f s p e c i f i c [ H ] - e s t r a d i o l b i n d i n g . I n c u b a t i o n s were c a r r i e d out f o r 90 minutes a t 25°C u s i n g a 100 f o l d excess o f u n l a b e l e d e s t r a d i o l as a competitor f o r 2 nM r a d i o - l a b e l e d e s t r a d i o l i n t h e ammonium s u l f a t e f r a c t i o n and 200 nM r a d i o - l a b e l e d e s t r a d i o l i n t h e whole c y t o s o l f r a c t i o n .  Time  BINDING (CPM)  (minutes)  Male  Female  Adult Ammonium S u l f a t e F r a c t ion  Immature Whole C y t o s o l Fraction  Ammonium S u l f a t e Fraction  Adult  Whole C y t o s o l Fraction  Ammonium S u l f a t e 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  0  1213  0  170  24 hours  610  N.D. = Not determined  TABLE 4 The e f f e c t of pH on t h e apparent Kd and c a p a c i t y of t h e h e p a t i c e s t r o g e n r e c e p t o r and moderate a f f i n i t y component. I n c u b a t i o n s were conducted at t h e pH v a l u e s l i s t e d f o r 90 minutes at 4°C. Specific [ H]-estradiol binding was determined u s i n g a 100 f o l d excess of u n l a b e l e d e s t r a d i o l as a competitor i n a d u l t male and female r a t h e p a t i c p r e p a r a t i o n s . 3  pH  Estrogen  Receptor  Moderate A f f i n i t y  Male Apparent Kd IQ (xlO M)  Female Capacity (fmol/mg protein)  Apparent Kd 1  Q  (xlO  M)  Male Capacity (fmol/mg protein)  7.0 7.5  8.5  160  16.8  215  8.0 8.5 9.0  14.9  226  18.5  Component  298  Apparent Kd _ (xlO M)  Capacity (pmol/mg protein)  14.0  16.7  11.0  10.7  15.0  13.3  0  0  0  0  48  c)  Freezing  Studies  To determine t h e s t a b i l i t y storage  o f these b i n d i n g components to  the f o l l o w i n g 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 d i v i d e d i n t o s e v e r a l samples.  Both h i g h and moderate a f f i n i t y  from each p o o l immediately.  A l l other  components were  samples were f r o z e n  n i t r o g e n and s t o r e d at -80°C f o r l a t e r a n a l y s i s . i n d i c a t e d t h a t t h e moderate a f f i n i t y week, whereas t h e high a f f i n i t y  assessed  component  component  Results  in liquid  i n Table 5  i s s t a b l e f o r one  i s s t a b l e at two months  i n b o t h males and females.  d)  Competitor  Studies  We examined t h e e f f e c t i v e n e s s of v a r i o u s competitors  in  d i s p l a c i n g e s t r a d i o l from t h e h i g h a f f i n i t y and moderate a f f i n i t y site  i n t h e male l i v e r  fractions.  The r e s u l t s o f t h i s study a r e  3 shown i n T a b l e  6.  the h i g h a f f i n i t y  [ H ] - e s t r a d i o l was e f f e c t i v e l y d i s p l a c e d from s i t e by e s t r o g e n s i n b o t h male ( T a b l e 6) and female  (Appendix 1, T a b l e '-AlO ammonium s u l f a t e f r a c t i o n s .  However, both  e s t r o g e n s and androgens ( t e s t o s t e r o n e and DHT) were a b l e to d i s p l a c e e s t r a d i o l from t h e moderate a f f i n i t y Progestins,  site  i n male whole c y t o s o l .  g l u c o c o r t i c o i d s and m i n e r a t o t o r t i c o i d s were n o t e f f e c t i v e  i n e i t h e r f r a c t i o n i n e i t h e r sex. S i n c e DHT and t e s t o s t e r o n e 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 t h e moderate a f f i n i t y  site  i n male whole c y t o s o l ,  3 we f u r t h e r examined t h e b i n d i n g o f t h e s e androgens. and  3  [ H]-testosterone  were employed as l i g a n d s  Both [ H]-DHT  ( s e p a r a t e l y ) i n male  TABLE 5 The e f f e c t of f r e e z i n g on the apparent Kd and c a p a c i t y of t h e e s t r o g e n r e c e p t o r and moderate a f f i n i t y component. T i s s u e pools were d i v i d e d and f r o z e n a t -80°C. Assays were conducted on the unfrozen t i s s u e p o o l and at v a r i o u s time p o i n t s on thawed t i s s u e p o o l samples.  Time  Estrogen  (weeks)  Receptor  Moderate A f f i n i t y  Male Apparent (xlO  1 0  Kd M)  Female Capacity (fmol/mg protein)  Apparent (xlO  1 0  Kd M)  Component  Male 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 o f [ H ] - e s t r a d i o l b i n d i n g t o the e s t r o g e n r e c e p t o r and moderate a f f i n i t y component i n the a d u l t male r a t . I n c u b a t i o n s were c a r r i e d out f o r 90 minutes a t 4°C. Specific b i n d i n g was determined u s i n g a 10,100, or 1000 f o l d excess of u n l a b e l e d c o m p e t i t o r f o r 0.5 nM [ H ] - e s t r a d i o l bound t o the estrogen r e c e p t o r i n the ammonium s u l f a t e f r a c t i o n o r 50 nM [ H ] e s t r a d i o l bound t o t h e moderate a f f i n i t y component i n t h e whole c y t o s o l f r a c t i o n . 3  3  Percent of t o t a l b i n d i n g i n the presence of excess Steroid Competitor Control  10-fold  Estrogen Receptor 100-fold 1000-fold  competitor  Moderate A f f i n i t y Component 10-fold 100-fold 1000-fold  100  100  100  100  100  100  Estradiol Diethylstilbestrol Moxestrol  64 62 68  54 60 61  47 58 58  100 100 88  67 100 76  66 100 66  Testosterone Dihydrotestosterone Androstenedione Methyltrienolone  94 94 96 96  97 95 97 92  100 91 93 89  100 100 100 91  100 100 90 100  78 77 85 100  Progesterone Promegestone  91 75  90 95  90 91  88 92  89 100  91 100  90 91 100  92 90 96  91 89 95  70 100 100  95 95 100  100 85 100  Triamcinolone Dexamethasone Aldosterone  * The b i n d i n g of [ H ] - e s t r a d i o l i n the presence of competitor i s g i v e n as % o f c o n t r o l v a l u e s ( c o n t r o l v a l u e = b i n d i n g i n t h e absence o f competitor)  51  whole c y t o s o l i n a c o n c e n t r a t i o n range o f 10-200 nM and were competed with unlabeled Although  DHT and t e s t o s t e r o n e ( r e s p e c t i v e l y ) o r e s t r a d i o l .  d i s p l a c a b l e b i n d i n g was observed,  androgens as l i g a n d s d i d n o t generate  e)  B i n d i n g t o u t e r u s , bovine  t h e r e s u l t s from u s i n g  a Scatchard  plot.  serum albumin and plasma  The b i n d i n g o f e s t r a d i o l i n l i v e r was compared t o t h a t i n uterus, a .classic target t i s s u e . u t e r i was prepared conditions. and  An ammonium s u l f a t e f r a c t i o n o f  i n p a r a l l e l w i t h l i v e r and assayed  under  similar  We determined t h e apparent Kd and c a p a c i t i e s o f l i v e r  u t e r i t o be 0.9 nM; 128 fmol/mg and 0.5 nM; 438 fmol/mg  respectively.  I n a d d i t i o n , t h e percent  displacement  a t 0.5 nM  3 [ H ] — e s t r a d i o l was found 100  f o l d excess  t o be 64% i n l i v e r and 91% i n u t e r u s a t  estradiol.  In a s i m i l a r study the b i n d i n g o f e s t r a d i o l t o bovine albumin (BSA) o r plasma was i n v e s t i g a t e d .  serum  We d e t e c t e d no b i n d i n g  t o BSA o r t o plasma o f male o r female r a t s i n e i t h e r t h e h i g h 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 h e p a t i c  estrogen  b i n d i n g components  a)  Age and sex r e l a t e d modulation o f h e p a t i c e s t r o g e n  binding  Having e s t a b l i s h e d t h e c o n d i t i o n s f o r d e t e r m i n a t i o n e s t r o g e n b i n d i n g , as w e l l as some o f t h e b i n d i n g  of h e p a t i c  characteristics,  the assay was a p p l i e d t o v a r i o u s 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 t h e assay was confirmed and p h y s i o l o g i c a l r e l e v a n c e  was e s t a b l i s h e d . logical  P r e l i m i n a r y s t u d i e s aimed a t e s t a b l i s h i n g p h y s i o -  r e l e v a n c e were conducted  i n t h e f o l l o w i n g animal models.  The b i n d i n g a f f i n i t i e s f o r t h e e s t r o g e n r e c e p t o r , h i g h a f f i n i t y , low c a p a c i t y ammonium  s u l f a t e component, were found t o be s i m i l a r  i n t h e male, female and pseudohermaphroditic  r a t (Table 7 ) . The  capacity of the estrogen receptor i n the adult l i t t e r m a t e male was lower than t h e female and pseudohermaphrodite; g e n e r a l l y t h e r e were no sexes.  control  however,  major d i f f e r e n c e s i n c a p a c i t y between  As p r e v i o u s l y d e s c r i b e d t h e moderate a f f i n i t y  not d e t e c t e d i n t h e female.  Furthermore,  s i t e was  no moderate a f f i n i t y  b i n d i n g was d e t e c t e d i n t h e pseudohermaphrodite r a t ( T a b l e 7 ) . The e f f e c t o f age and gonadectomy on t h e b i n d i n g c h a r a c t e r i s t i c s of  t h e e s t r o g e n r e c e p t o r a r e p r e s e n t e d i n T a b l e 8.  of  t h e e s t r o g e n r e c e p t o r was lower  The c a p a c i t y  i n t h e immature animal  days o f age) as compared t o a d u l t c o n t r o l s .  (25-30  S i m i l a r to the a d u l t ,  t h e r e was no sex d i f f e r e n c e i n t h e c a p a c i t y o f t h e e s t r o g e n r e c e p t o r i n t h e immature r a t . r e c e p t o r was reduced  However, t h e apparent Kd f o r t h e e s t r o g e n i n t h e immature female r e l a t i v e t o t h e  immature male or mature a n i m a l .  I n t e r e s t i n g l y , gonadectomy  slightly  reduced t h e apparent Kd o f t h e e s t r o g e n r e c e p t o r i n both males and females, but t h e c a p a c i t y o n l y i n t h e male.  As w i t h t h e a d u l t  female no moderate a f f i n i t y b i n d i n g was observed male o r female  (Table 8 ) .  i n t h e immature  Gonadectomy o f t h e male reduced t h e  c a p a c i t y o f t h e moderate a f f i n i t y component a p p r o x i m a t e l y without a l t e r i n g t h e apparent Kd.  In a d d i t i o n , s i m i l a r  six fold  results  were o b t a i n e d when u n l a b e l e d DHT was used as a competitor f o r  53  TABLE 7 A comparison o f the apparent Kd and c a p a c i t y of t h e h e p a t i c c y t o s o l i c e s t r o g e n r e c e p t o r 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 a t 4°C as d e s c r i b e d i n t h e methods s e c t i o n on a sample p o o l from 4 a n i m a l s .  Estrogen Receptor Kd g  Model  (xlO  M)  Moderate A f f i n i t y Component  Capacity (fmol/mg protein)  R  d  7  (xlO  M)  Capacity (pmol/mg protein)  Male  1.1  261  1.4  6.6  Female  1.1  368  0  0  P seudohermaphrod i t e  1.0  406  0  0  TABLE 8 The e f f e c t o f age and gonadectomy on t h e apparent Kd and c a p a c i t y o f h e p a t i c c y t o s o l i c e s t r o g e n r e c e p t o r and moderate a f f i n i t y b i n d i n g component i n male and female r a t s . A d u l t male and female animals were gonadectomized (Gx) o r sham-operated 11 days p r i o r t o s a c r i f i c e . Immature (Imm.) r a t s were s a c r i f i c e d between days 25 and 30 o f l i f e . A p o o l of 4 l i v e r s was used.  Model  Estrogen Receptor  Moderate A f f i n i t y Component  Ammonium S u l f a t e F r a c t i o n  Whole C y t o s o l F r a c t i o n Kd  Kd*  Kd ( x l O M)  Capacity (fmol/mg p r o t e i n )  Male  2.8  620  1.4  31.4  1.0  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.  1.5  267  0  0  0  0  1.1  544  0  0  0  0  9  Gx  Female  Female  (xlO  7  M)  Capacity (pmol/mg p r o t e i n )  (xlO? M)  * I n d i c a t e s v a l u e s obtained u s i n g a 1000 f o l d excess of u n l a b e l e d DHT as a competitor a l l o t h e r s a r e a 100 f o l d excess of u n l a b e l e d ' e s t r a d i o l .  Capacity* (pmol/mg p r o t e i n )  23.5  for [ H]-estradiol  55  [ H]-estradiol J  binding.  Testosterone  was  administered  t o gonadectomized males to  c o n f i r m t h a t the male s p e c i f i c moderate a f f i n i t y b i n d i n g p r o t e i n was  androgen dependent.  i n Table  The  r e s u l t s of t h i s study a r e  9, w i t h e i t h e r u n l a b e l e d  e s t r a d i o l or DHT  as  reported competitors  3 for  [ H]-estradiol binding.  A f i v e f o l d decrease i n the  of t h e moderate a f f i n i t y component was c o n f i r m i n g the r e s u l t s seen i n T a b l e  capacity  observed f o l l o w i n g gonadectomy  9.  A d m i n i s t r a t i o n of t e s t o -  sterone enanthate (1.0 mg/kg/day subcutaneously f o r ten days) r e v e r s e d the e f f e c t s of gonadectomy on t h e c a p a c i t y of t h i s ( F i g u r e 1A).  site  N e i t h e r gonadectomy nor the a d m i n i s t r a t i o n of  t e s t o s t e r o n e t o the gonadectomized female r e s u l t e d i n d e t e c t i o n of a moderate a f f i n i t y component.  Studies with  testosterone  replacement were a l s o c a r r i e d out on the e s t r o g e n Testosterone estrogen  d i d not a f f e c t  receptor  ( T a b l e 9).  receptor.  t h e b i n d i n g c h a r a c t e r i s t i c s of  the  i n e i t h e r gonadectomized male or female r a t s  Furthermore, u n l i k e t h e p r e v i o u s  study  (Table 8 ) ,  gonadectomy i t s e l f produced o n l y minor e f f e c t s on the apparent Kd and  b)  c a p a c i t y of the e s t r o g e n  receptor  i n the male or  P i t u i t a r y m o d u l a t i o n of h e p a t i c e s t r o g e n  female.  binding  Hypothalamic-hypophyseal i n f l u e n c e s on t h e h e p a t i c b i n d i n g p r o t e i n s were examined i n a d u l t male and As  shown i n T a b l e  to below 50%  10,capacity  of the e s t r o g e n  of the c o n t r o l l e v e l s two  i n both male and  female r a t s .  of the e s t r o g e n r e c e p t o r .  estrogen  female r a t s .  receptor  decreased  weeks f o l l o w i n g hypophysectomy  Hypophysectomy d i d not a l t e r the  Hypophysectomy a l s o g r e a t l y reduced  Kd the  TABLE 9 The e f f e c t of gonadectomy and t e s t o s t e r o n e replacement on the apparent Kd and c a p a c i t y of the h e p a t i c c y t o s o l i c e s t r o g e n r e c e p t o r and moderate a f f i n i t y b i n d i n g component i n male and female r a t s . Adult male and female r a t s were gonadectomized 11 days p r i o r to s a c r i f i c e . Where i n d i c a t e d animals r e c e i v e d t e s t o s t e r o n e enanthate (Test) (1.0 mg/kg/day subcutaneously i n c o r n 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 injection. Assays were conducted as d e s c r i b e d i n the methods s e c t i o n on a t i s s u e p o o l of 4 l i v e r s .  Model  Estrogen  Receptor  Moderate A f f i n i t y  Ammonium S u l f a t e F r a c t i o n Kd .9 (xlO M)  Capacity  Component  Whole C y t o s o l F r a c t i o n Kd Q  (fmol/mg p r o t e i n )  (xl0° M)  Capacity (pmol/mg p r o t e i n )  Kd (xlO  Q  Capacity M)  (pmol/mg p r o t e i n )  Male  1.05  255  6.4  11.0  4.2  8.5  Gx Male  0.73  202  4.1  2.2  1.5  1.1  Gx Male + Test  0.75  207  3.9  10.0  6.3  13.9  Female  0.82  188  0  0  0  0  Gx Female  0.62  225  0  0  0  0  Gx Female + Test  0.89  224  0  0  0  0  * I n d i c a t e s v a l u e s obtained u s i n g a 1000 f o l d excess of u n l a b e l e d DHT as a c o m p e t i t o r f o r [ H ] - e s t r a d i o l b i n d i n g i n s t e a d of t h e 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 m a n i p u l a t i o n on the s p e c i f i c b i n d i n g t o the moderate a f f i n i t y component. was  [ML]^estradiol  Scatchard analyses  -performed on t i s s u e p o o l s from f o u r a n i m a l s over a  3 c o n c e n t r a t i o n range o f 10-200 nM  [ H]-estradiol.  Specific  b i n d i n g was determined u s i n g a 100 f o l d excess o f u n l a b e l e d e s t r a d i o l as a c o m p e t i t o r .  I n c u b a t i o n s were c a r r i e d out f o r  90 minutes at 4°C. a)  A d u l t male r a t s were sham-operated e l e v e n days p r i o r to s a c r i f i c e . males r e c e i v e d  ( O ) , or gonadectomized  (®)  One group of gonadectomized  t e s t o s t e r o n e enanthate, (1.0 mg/kg/day s.c. f o r  t en days) ( • ) . b)  A d u l t male r a t s were sham-operated (O)  two weeks p r i o r t o s a c r i f i c e .  ( • ) , or hypophysectomized  B  Ln 00  4.0  To"  8.0  BOUND(pmol/mg protein)  10.0  59  TABLE 10 The e f f e c t o f hypophysectomy on t h e b i n d i n g c h a r a c t e r i s t i c s of t h e h e p a t i c c y t o s o l i c e s t r o g e n r e c e p t o r i n male and female r a t s . A d u l t male and female r a t s were hypophysectomized (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 p o o l o f 4 l i v e r s was used.  Model  (xlO  y  M)  Capacity (fmol/mg p r o t e i n )  Sham Male  2.2  516  Hx Male  2.5  190  Sham Female  2.5  506  Hx  3.4  250  Female  60  c a p a c i t y of the moderate a f f i n i t y b i n d i n g component i n the male, ( F i g u r e I B ) , w i t h e i t h e r u n l a b e l e d e s t r a d i o l or DHT ( T a b l e 11) decrease  (to 16.1%  and  12.2%  competitors  of c o n t r o l r e s p e c t i v e l y ) .  The  i n c a p a c i t y of the moderate a f f i n i t y component was  accompanied by a change i n apparent Kd. was  as  Of p a r t i c u l a r  interest  the appearance of a moderate a f f i n i t y b i n d i n g s i t e i n the  female two Although  weeks f o l l o w i n g hypophysectomy (Table 11,  the c a p a c i t y was  mized male, the s i t e was female, and  had  o n l y one  d e t e c t a b l e f o r the f i r s t  an apparent Kd  physectomized female was  Figure  time i n the  s i m i l a r to that of the male. s i t e i n the hypo-  s t u d i e d at a -longer time p e r i o d . The  c a p a c i t y of the hypophysectomized female moderate a f f i n i t y found  2k).  t e n t h t h a t of the hypophysecto-  T h i s appearance of a moderate a f f i n i t y  was  not  site  t o be n e a r l y e q u a l t h a t of the hypophysectomized male  at f o u r and  s i x weeks p o s t - s u r g e r y  The apparent Kd-was approximately  ( T a b l e s 12,  13;  F i g u r e 2B)..  two/fold greater i n  t h e female than the male a t f o u r weeks f o l l o w i n g surgery  (Table  The  similar  e f f e c t of hypophysectomy at f o u r weeks was  t o the e f f e c t of hypophysectomy at two a decrease  of sex s t e r o i d a c t i o n . 14.  physectomy.  e f f e c t on the apparent Kd  the moderate  (Table  s t u d i e d to determine c e n t r a l  12).  involvement  R e s u l t s of t h i s study a r e p r e s e n t e d  i n Tables  These s t u d i e s were conducted s i x weeks f o l l o w i n g hypoAt t h i s time p o i n t the e s t r o g e n r e c e p t o r was  d e t e c t a b l e i n the hypophysectomized female, and was reduced  was  e f f e c t o f t e s t o s t e r o n e on e s t r o g e n b i n d i n g i n the  hypophysectomized animal was  13 and  That i s , t h e r e  i n t h e c a p a c i t y of the e s t r o g e n r e c e p t o r and  a f f i n i t y component, w i t h no The  weeks.  otherwise  12).  i n the male ( T a b l e 13).  Testosterone  not  dramatically  enanthate, p r e v i o u s l y  61  TABLE 11 The e f f e c t of hypophysectomy on t h e b i n d i n g c h a r a c t e r i s t i c s o f t h e moderate a f f i n i t y component i n male and female r a t s . A d u l t 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 f o l l o w i n g hypophysectomy. Moderate a f f i n i t y s p e c i f i c b i n d i n g was d e t e c t e d u s i n g a 100 f o l d excess o f u n l a b e l e d e s t r a d i o l o r a 1000 f o l d excess o f d i h y d r o t e s t o s t e r o n e a s c o m p e t i t o r s . A t i s s u e p o o l of 4 animals was used f o r each group.  Model  Competitor Estradiol ^ (xlO' M)  Dihydrotestosterone Capacity (pmol/mg protein)  1X11  (xlO  7  M)  Capacity (pmol/mg protein)  Sham Male  0.9  14.9  0.6  11.4  Hx Male  0.7  2.4  0.5  1.4  0  0  0  0  0.5  0.2  0  0  Sham Female Hx Female  62  Figure 2 3  E f f e c t of hypophysectomy on t h e s p e c i f i c to t h e moderate a f f i n i t y component.  [ H]-estradiol binding  Scatchard  analyses was •  performed on t i s s u e p o o l s from f o u r animals over a c o n c e n t r a t i o n 3  range o f 10-200 nM  [ H]-estradiol.  determined u s i n g a 100 f o l d a competitor.  Incubations  Specific binding  excess of u n l a b e l e d  was  e s t r a d i o l as  were c a r r i e d out f o r 90 minutes  a t 4°C. a)  A d u l t male (•)  and female ()( ) r a t s were hypophysectomized  two weeks p r i o r t o s a c r i f i c e . b)  A d u l t male (•)  and female (O)  s i x weeks p r i o r t o s a c r i f i c e .  r a t s were hyophysectomized  BOUND/FREE  £9  TABLE 12 The e f f e c t of hypophysectomy on t h e apparent Kd and c a p a c i t y of t h e h e p a t i c c y t o s o l i c e s t r o g e n r e c e p t o r and moderate a f f i n i t y b i n d i n g component a t f o u r weeks p o s t - s u r g e r y i n male and female r a t s . A d u l t male and female r a t s were hypophysectomized (Hx) or shamoperated between days 50 and 60 of l i f e . Animals were s a c r i f i c e d f o u r weeks f o l l o w i n g surgery. A p o o l of 4 l i v e r s were used f o r each group.  Model  E s t r o g e n Receptor  Moderate A f f i n i t y  Ammonium S u l f a t e F r a c t i o n  Component  Whole C y t o s o l F r a c t i o n  Kd ( x l 0 M)  Capacity (fmol/mg p r o t e i n )  . Kd (xlO M)  Sham Male  1.11  260  8.8  19.3  6.9  15.4  Hx Male  1.32  156  9.2  12.2  5.5  8.3  Sham Female  1.5  447  0  0  0  Hx Female  1.47  y  98  17.9  Capacity (pmol/mg p r o t e i n )  0 7.0  Kd ( x l O M) 8  16.9  * I n d i c a t e s v a l u e s obtained u s i n g a 1000 f o l d excess of u n l a b e l e d DHT as a c o m p e t i t o r i n s t e a d o f a 100 f o l d excess o f u n l a b e l e d . e s t r a d i o l .  Capacity (pmol/mg p r o t e i n )  5.9  for [ H]-estradiol  65  TABLE 13 The e f f e c t o f t e s t o s t e r o n e and growth hormone' on t h e b i n d i n g 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 t h e hypophysectomized r a t . A d u l t male and female r a t s were hypophysectomized (Hx) between days 50 and 60 of l i f e . Where i n d i c a t e d a n i m a l s r e c e i v e d t e s t o s t e r o n e 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 s.c. t w i c e d a i l y i n s a l i n e ) 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 f o l l o w i n g the l a s t i n j e c t i o n , s i x weeks p o s t - s u r g e r y . T i s s u e from 4 a n i m a l s was pooled f o r each group.  Model  Kd ( x l O M) 9  Hx Male  Capacity (fmol/mg p r o t e i n )  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 +  0  0  bGH  66  shown not t o a f f e c t effect  the c a p a c i t y of the e s t r o g e n  r e c e p t o r , had  on the apparent Kd or c a p a c i t y of t h e e s t r o g e n  the hypophysectomized male or female r a t .  receptor in  In a d d i t i o n , the  e f f e c t s of growth hormone on h e p a t i c estrogen examined.  no  b i n d i n g were  -Following the a d m i n i s t r a t i o n of bGH  (4.0  I.U./kg/day,  i n a d i v i d e d dose t w i c e d a i l y f o r ten days, we'observed a s l i g h t i n apparent Kd along w i t h an i n c r e a s e i n i n the male,(Tabl:e 13). e f f e c t on the e s t r o g e n The affinity  capacity of,the.estrogen-receptor, ;  i n the hypophysectomized female.  e f f e c t of t e s t o s t e r o n e and also studied.  bGH  on b i n d i n g of the moderate  U n l i k e the gonadectomized  male, t e s t o s t e r o n e had no r e s t o r a t i v e e f f e c t the moderate a f f i n i t y ( T a b l e 14). result  decrease  Administration',of. bGH -clearly'had/no r e s t o r a t i v e receptor  component was  s.c.)  on the c a p a c i t y of  component i n the hypophysectomized  However, bGH  animal,  (2. I.U./kg/day s.c. t w i c e d a i l y ) d i d  i n a 50-80% d e c r e a s e i n the c a p a c i t y of t h e moderate  affinity  component i n hypophysectomized male and  respectively.  N e i t h e r t e s t o s t e r o n e nor bGH  the apparent Kd of the moderate a f f i n i t y  had  site  female r a t s , any  i n the  effect  on  hypophysecto-  mized r a t . Furthermore, s i n c e the apparent Kd and moderate a f f i n i t y at  c a p a c i t y of  component were s i m i l a r i n the male and  the female  s i x weeks f o l l o w i n g hypophysectomy, a d d i t i o n a l e s t r o g e n  binding  s t u d i e s i n v o l v i n g hypophysectomized animals were conducted a t s i x weeks p o s t - s u r g e r y . r e c e p t o r was B).  In a d d i t i o n , at t h i s time p o i n t t h e  least estrogen  o f t e n absent from male as w e l l as female r a t s ( F i g u r e  3A,  67  TABLE 14 The e f f e c t o f t e s t o s t e r o n e and growth hormone on t h e b i n d i n g c h a r a c t e r i s t i c s of t h e h e p a t i c 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 . A d u l t male and female r a t s were hypophysectomized (Hx) between days 50 and 60 o f l i f e . Where i n d i c a t e d animals r e c e i v e d t e s t o s t e r o n e 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 t w i c e d a i l y ( s . c . i n s a l i n e ) f o r 10 days p r i o r t o 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 t h e l a s t i n j e c t i o n , s i x weeks f o l l o w i n g hypophysectomy. Apparent Kd and c a p a c i t y were determined u s i n g a 100 f o l d excess of u n l a b e l e d e s t r a d i o l or 1000 f o l d excess of d i h y d r o t e s t o s t e r o n e - a s competitors. A p o o l of 4 l i v e r s was used per group.  Model  Competitor Estradiol  Dihydrotestosterone  Kd (xlO M)  Capacity (pmol/mg 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 +  7.2  1.3  3.5  0.6  bGH  Kd (xlO  8  M)  Capacity (pmol/mg p r o t e i n )  68  Figure 3  3 E f f e c t o f hypophysectomy on the s p e c i f i c estrogen  receptor.  Scatchard  [ H ] - e s t r a d i o l b i n d i n g t o the  a n a l y s e s was  performed on  t i s s u e p o o l s from f o u r animals over a c o n c e n t r a t i o n  range  3 of 0.05-2.5 nM [ H ] - e s t r a d i o l . u s i n g a 100 f o l d Incubations a)  Adult  S p e c i f i c b i n d i n g was determined  excess u n l a b e l e d  e s t r a d i o l as a  were c a r r i e d out f o r 90 minutes a t 4°C.  sham-operated (•)  and hypophysectomized  male r a t s e i g h t weeks f o l l o w i n g b)  Adult  competitor.  sham-operated  (•)  (O)  surgery.  and hypophysectomized  female r a t s e i g h t weeks f o l l o w i n g  surgery.  (O)  70  3)  C h a r a c t e r i z a t i o n of h e p a t i c enzyme a c t i v i t y  in different  p h y s i o l o g i c a l models  a)  Sex  r e l a t e d m o d u l a t i o n of h e p a t i c AHH  A4 r e d u c t a s e  and  testosterone  activities.  As has been e x t e n s i v e l y documented, the male l e v e l s h e p a t i c AHH  of  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. dependency, h e p a t i c AHH  To f u r t h e r c h a r a c t e r i z e t h i s androgen a c t i v i t y was  pseudohermaphroditic r a t s .  The  determined i n Stanley-Gumbreck  c o n t r o l l e v e l s of AHH  activity  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 w e l l as the response to p h e n o b a r b i t a l  lactone are presented  i n T a b l e 15.  pseudohermaphrodite was  AHH  a c t i v i t y of the c o n t r o l  lower than the c o n t r o l male.  AHH  a c t i v i t y i n both the female  pseudohermaphrodite r e l a t i v e to t h e i r c o n t r o l s . d i d not a f f e c t AHH  and  Phenobarbital  a c t i v i t y i n the male.  treatment w i t h  spironolactone  (100 mg/kg/day,  i n t r a p e r i t o n e a l l y ) f o r f o u r days, a s t a t i s t i c a l l y r e d u c t i o n i n male AHH (Table 19).  Administration  (80 mg/kg/day, i n t r a p e r i t o n e a l l y ) f o r f o u r days  s i g n i f i c a n t l y induced  Following  spirono-  the same as t h a t of the c o n t r o l female, both  of which were s t a t i s t i c a l l y of p h e n o b a r b i t a l  The  and  a c t i v i t y was  observed compared to c o n t r o l  However, s p i r o n o l a c t o n e , l i k e  s i g n i f i c a n t l y induced  h e p a t i c AHH  significant  phenobarbital,  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.  c o n t r o l l e v e l s of the pseudohermaphrodite were a g a i n  similar  The to  71  TABLE 15 The e f f e c t o f p h e n o b a r b i t a l and s p i r o n o l a c t o n e on h e p a t i c 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 t r e a t e d w i t h e i t h e r p h e n o b a r b i t a l (80 mg/kg/d x 4d i . p . i n s a l i n e ) or s p i r o n o l a c t o n e (100 mg/kg/d x 4d i . p . i n s a l i n e ) . C o n t r o l animals received v e h i c l e alone. Animals were s a c r i f i c e d 24 hours f o l l o w i n g the l a s t i n j e c t i o n . D i f f e r e n c e s between groups were c o n s i d e r e d s i g n i f i c a n t a t p < 0.05 u s i n g t h e Duncan m u l t i p l e range t e s t .  Model  Male  AHH A c t i v i t y (pmol/min/mg protein)±S.E.M.,  (n)  Control  Phenobarbital  Spironolactone  1986 ± 154 (5)  1604 ± 78 (4)  1275 ± 62* (4)  Female  217 ± 6 (4)  Pseudo  295i ± 1 0 (5)  +  +  694 ± 53* (4)  741 ± 97* (4)  658 ± 43* (4)  1135 ± : 152* (4)  * S i g n i f i c a n t l y d i f f e r e n t from r e s p e c t i v e c o n t r o l groups, t  Significantly different  from male c o n t r o l group.  72  t h a t o f t h e female 3-MC  ( T a b l e 16).  F o l l o w i n g a d m i n i s t r a t i o n of  (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 t h r e e animal models t o a common l e v e l . S t u d i e s on t h e e f f e c t a c t i v i t y were a l s o conducted.  of hypophysectomy on h e p a t i c AHH The requirement o f t h e h y p o p h y s e a l -  hypothalamic a x i s f o r t h e maintenance demonstrated  a s shown i n T a b l e 17.  o f t h e sex d i f f e r e n c e was  Hypophysectomy produced a  decrease i n AHH a c t i v i t y i n t h e male and an i n c r e a s e i n AHH activity  i n t h e female a t two weeks p o s t - s u r g e r y .  The r e d u c t i o n  i n d i f f e r e n t i a t i o n o f AHH a c t i v i t y noted f o l l o w i n g hypophysectomy was a c o n s i s t e n t o b s e r v a t i o n ( T a b l e s 17, 18, 1 9 ) . However, when t h e number o f treatment groups was i n c r e a s e d  (as i n T a b l e 18)  r e q u i r i n g a n a l y s i s by m u l t i p l e range t e s t i n g , and a comparison w i t h t h e much g r e a t e r a c t i v i t y i n the male, t h e s i g n i f i c a n c e of the i n c r e a s e i n t h e female was no l o n g e r apparent. In a d d i t i o n , as shown i n T a b l e 18, t h e i n a b i l i t y of t e s t o s t e r o n e (2.0 mg/kg/day, subcutaneously f o r f o u r t e e n days) t o r e v e r s e the decrease i n AHH a c t i v i t y  i n the male a t f o u r weeks p o s t -  hypophysectomy was demonstrated.  In f a c t , t e s t o s t e r o n e produced  a s l i g h t r e d u c t i o n i n AHH a c t i v i t y i n the sham-operated the i n c r e a s e i n AHH a c t i v i t y  'male.  i n t h e female observed a t two weeks  f o l l o w i n g hypophysectomy ( T a b l e 17) was n o t s t a t i s t i c a l l y by t h e Newman-Keuls  However,  t e s t a t f o u r weeks a f t e r  significant  surgery (Table 18).  In a d d i t i o n t o t h e d i f f e r e n c e s i n s t a t i s t i c a l t e c h n i q u e s used, hypophysectomy i s an i n v a s i v e procedure t h a t n o t a l l animals survive.  As such, t h e sample group assayed a t f o u r weeks may  r e p r e s e n t a d i f f e r e n t p o p u l a t i o n from those assayed a t two weeks  73  TABLE 16 The e f f e c t of 3-methylcholanthrene on h e p a t i c AHH a c t i v i t y i n male, female, and pseudohermophrodite r a t s . A d u l t male, female, and pseudohermaphrodite (pseudo) r a t s were t r e a t e d w i t h 3-methylcholanthrene (3-MC) (20 mg/kg/d x 2d i . p . i n corn o i l ) . C o n t r o l a n i m a l s r e c e i v e d v e h i c l e a l o n e . Animals were s a c r i f i c e d 24 hours f o l l o w i n g t h e l a s t i n j e c t i o n . D i f f e r e n c e s between groups were c o n s i d e r e d s i g n i f i c a n t a t p < 0.05 u s i n g t h e Newman-Keuls m u l t i p l e range t e s t (n=4).  Model  AHH A c t i v i t y pmol/min/mg p r o t e i n ± S.E.M. Control  Male  3-Methylcholanthrene  1695 ± 133  4555 ± 300*  + Female  475 ± 24  Pseudo  407 ± 7 0  5037 ± 334 T  5251 ± 278  * Significantly different  from r e s p e c t i v e c o n t r o l  t  from male c o n t r o l  Significantly different  group.  groups,  TABLE 17 The e f f e c t o f hypophysectomy on h e p a t i c AHH a c t i v i t y i n male and female r a t s . A d u l t male and female r a t s were hypophysectomized (Hx) o r sham-operated between 50 t o 60 days o f age. Animals were s a c r i f i c e d two weeks f o l l o w i n g surgery. D i f f e r e n c e s were c o n s i d e r e d s i g n i f i cant a t p < 0.05 u s i n g t h e two t a i l e d Students t - T e s t . (n=6) .  Model  AHH A c t i v i t y pmol/min/mg p r o t e i n ± S.E.M. Sham  Male Female  2030 ± 243 184 ± 7  Hx  782 ± 114 312 ±  * I n d i c a t e s s i g n i f i c a n t d i f f e r e n c e s between Hx and .sham-operated c o n t r o l .  49  TABLE 18 The e f f e c t o f hypophysectomy and t e s t o s t e r o n e on h e p a t i c 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) o r sham-operated between days 50 t o 60 o f l i f e . Males r e c e i v e d t e s t o s t e r o n e enanthate (Test) (2.0 mg/kg/d, s.c. i n c o r n o i l ) f o r 14 days p r i o r t o s a c r i f i c e . Animals were s a c r i f i c e d 24 hours f o l l o w i n g t h e l a s t i n j e c t i o n , and f o u r weeks p o s t - s u r g e r y . Control animals r e c e i v e d v e h i c l e alone.  Model  AHH A c t i v i t y pmol/min/mg p r o t e i n ± S.E.M., (n) Sham  Hx  Male  2788 ± 1 6 3 (8)  a b c d  744 ± 1 0 5 (6)  Male + Test  1306 ± 2 2 0  a b c d  389 ± 4 0  Female  (6) 578 ± 25 (9)  b  d  a  c 734 ± 36 (6) ( 6 )  D i f f e r e n c e s between groups were c o n s i d e r e d s i g n i f i c a n t at p < 0.05 u s i n g t h e Newman-Keuls m u l t i p l e range test. S i g n i f i c a n t d i f f e r e n c e s between groups were i n d i c a t e d by common l e t t e r s .  following  hypophysectomy.  The e f f e c t i v e n e s s of known i n d u c e r s of mixed activity  i n c o n t r o l male and female r a t s was  physectomized a n i m a l s .  T a b l e 15.  examined  i n hypo-  P h e n o b a r b i t a l (80 mg/kg/day, i n t r a p e r i t o n e a l l y  f o r f o u r days) was unable t o a f f e c t AHH mized male ( T a b l e 19).  function oxidase  a c t i v i t y i n t h e hypophysecto-  T h i s was p r e d i c t e d based on r e s u l t s from  I n t e r e s t i n g l y though, p h e n o b a r b i t a l was  p r o d u c i n g an i n c r e a s e i n AHH  activity  i n the hypophysectomized  female r e l a t i v e t o t h e hypophysectomized the reduction i n d i f f e r e n t i a t i o n  capable of  control.  A g a i n we observed  (as seen i n T a b l e 17) a t 2 weeks  post-hypophysectomy. L i k e p h e n o b a r b i t a l , 3-MC  had no e f f e c t  the hypophysectomized male (Table 19). activity  b)  E n d o c r i n e m a n i p u l a t i o n of h e p a t i c enzyme a c t i v i t y  on  female was not determined.  E f f e c t o f p e r g o i i d e i n a d u l t female r a t s  I t had been h y p o t h e s i z e d t h a t PRL may factor  activity in  The e f f e c t of 3-MC  AHH  i)  i n t h e hypophysectomized  on AHH  i n v o l v e d i n t h e sex dependent  drug metabolism.  P r o l a c t i n was  r e p r e s e n t the p i t u i t a r y  d i f f e r e n t i a t i o n of h e p a t i c  shown t o be p r e s e n t i n r e l a t i v e l y  h i g h e r amounts i n the a d u l t female than male (Posner et a l . , As such, the e l e v a t e d l e v e l s of PRL  i n the female might account  f o r the sex d i f f e r e n c e i n metabolism seen a t m a t u r i t y . t h e e f f e c t s o f PRL,  To a s s e s s  p e r g o i i d e (0.1 mg/kg/day) was a d m i n i s t e r e d  subcutaneously to a d u l t sham-operated e l e v e n days.  1974).  o r gonadectomized- female r a t s f o r  P e r g o i i d e , a dopamine a g o n i s t , was  found t o reduce  TABLE 19 The e f f e c t of 3-methylcholanthrene and p h e n o b a r b i t a l on h e p a t i c AHH a c t i v i t y i n hypophysectomized male and female r a t s . A d u l t male and female r a t s were hypophysectomized (Hx) o r shamoperated between days 50 t o 60 of l i f e . Where i n d i c a t e d H x — r a t s r e c e i v e d 3-methylcholant h r e n e (3-MC) (20 mg/kg/d i . p . i n corn o i l ) f o r 2 days, o r p h e n o b a r b i t a l (PB) (80 mg/kg/d i . p . i n s a l i n e ) f o r 4 days p r i o r t o s a c r i f i c e . Animals were s a c r i f i c e d two weeks f o l l o w i n g s u r g e r y , and 24 hours a f t e r t h e l a s t i n j e c t i o n . D i f f e r e n c e s between groups were c o n s i d e r e d s i g n i f i c a n t a t p < 0.05 u s i n g t h e Newman-Keuls m u l t i p l e range t e s t .  Model  AHH A c t i v i t y (pmol/min/mg p r o t e i n ) ± S.E.M., (n)  Sham  Male  Hx  3550.8 (5) ± 318.8 : ± 20.9 (5)  +  Hx + 3-MC  1900.3 (7) ± 195.2* 1196.3 ± 121.8* (8)  2224,0 (4) ± 121.4* +  * Significantly different  from r e s p e c t i v e c o n t r o l groups,  t Significantly different  from male c o n t r o l group.  N.D. = n o t determined.  N.D.  Hx + PB  2456.8 (4) ± 109,2* 2073.0 ± : (3)  78  serum PRL  l e v e l s 90%  i n the a d u l t female r a t (Appendix 1 ) .  t e s t the h y p o t h e s i s ,  the response of h e p a t i c AHH  testosterone A 4 reductase t e s t o s t e r o n e enanthate were determined.  The  (1.0 mg/kg/day subcutaneously f o r ten days) dose of t e s t o s t e r o n e had  weights of androgen s e n s i t i v e t i s s u e s and  A4  reductase.  produced an  i n T a b l e 20.  at c o n t r o l l e v e l s sex-dependent The  Female AHH  hepatic  r e s u l t s of  the a c t i v i t y  a c t i v i t y t h a t was  i n h i b i t e d by  to  f o l l o w i n g a n a l y s i s by the Newman-Keuls m u l t i p l e range  m u l t i p l e range t e s t  (not shown).  T h i s t r e n d was  by the Duncan  c l e a r e r i n the  gonadectomized model where a d m i n i s t r a t i o n of t e s t o s t e r o n e a c t i v i t y i n t h e gonadectomized female, and this effect S i m i l a r to AHH gonadectomy.  concurrent  These d i f f e r e n c e s were found not  t e s t ; however, they were s i g n i f i c a n t when a n a l y z e d  blocked  of  A d m i n i s t r a t i o n of t e s t o s t e r o n e to the sham-operated female  a d m i n i s t r a t i o n of p e r g o l i d e .  AHH  the  activity, unlike  not a f f e c t e d by gonadectomy, nor was  i n c r e a s e i n AHH  be s i g n i f i c a n t  been p r e v i o u s l y  i n a d u l t gonadectomized males.  t h i s study a r e presented the male, was  testosterone  a c t i v i t y i n the, a d m i n i s t r a t i o n of  determined i n our l a b o r a t o r y to m a i n t a i n  enzyme a c t i v i t y  and  To  The  (Table  pergolide  increased partially  20).  a c t i v i t y , A4 r e d u c t a s e  l e v e l s of A4 r e d u c t a s e  was  not a f f e c t e d by  are normally  greater i n  the a d u l t female as compared to the a d u l t male (Table 22).  Following  the a d m i n i s t r a t i o n of t e s t o s t e r o n e the h e p a t i c A4 r e d u c t a s e  activity  was  decreased  u n l i k e AHH  i n b o t h the sham-^operated and •gonadectomized .female. However  activity,  concurrent  a d m i n i s t r a t i o n of p e r g o l i d e d i d not  i n h i b i t the a c t i o n of t e s t o s t e r o n e  (Table  20).  79  TABLE 20 The e f f e c t o f gonadectomy, p e r g o i i d e and t e s t o s t e r o n e on h e p a t i c AHH and t e s t o s t e r o n e A4 r e d u c t a s e 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 t o sacrifice. Where i n d i c a t e d animals r e c e i v e d t e s t o s t e r o n e ' enanthate (Test) (1.0 mg/kg/d s.c. i n corn o i l ) f o r 10 days or p e r g o i i d e (Perg.) (0.1 mg/kg/d s.c. i n corn o i l ) f o r 11 days p r i o r t o s a c r i f i c e between days 55 t o 65 o f l i f e . Animals were s a c r i f i c e d 24 hours after the last injection. D i f f e r e n c e s between groups were c o n s i d e r e d s i g n i f i c a n t a t p < 0.05 u s i n g t h e Newman-Keuls m u l t i p l e range t e s t . S i g n i f i c a n t d i f f e r e n c e s between groups were i n d i c a t e d by common l e t t e r s .  Model  AHH A c t i v i t y (pmol/min/mg p r o t e i n ) ± S.E.M., (n)  A 4 Red. A c t i v i t y (nmol/min/mg p r o t e i n ) ± S.E.M., (n) cf i  Sham  423.1 ± 28.8 (9)  31.3 ± 1.3 (9)  Gx  390.2 ± 64.7°  31.3 ± 0 . 8  Gx + Perg  a 311.5 ± 26.1 (4)  30.8 ± 1.8 (4)  ( 4 )  Sham + Test Gx + Test Sham + Test + Perg Gx + Test + Perg  <> 4  b e h  »A  0  S  645.0 ± 1 6 7 (4) abcde 1489.0 ± 91.9 (5)  21.3 ± 3 . 6 (4) ,abc 15.3 ± 0.7' (5)  353.6 ± 34.1* (5) abed 822.8 ± 82.8 (5)  22.9 ± 1 . 4 (5) def 15.4 ± 1.2 (5)  e  g h l  a d  80  ii)  E f f e c t of perg©l-ide i n . pre-pubescent female, rats  Female r a t s were sham-operated and (approximately day  30 t o day  25-30 days of age)  and  gonadectomized p r e - p u b e r t a l l y  t r e a t e d w i t h p e r g o i i d e from  60 of l i f e t o examine the e f f e c t s of  e l e v a t i o n d u r i n g puberty  on the response of AHH  a c t i v i t y to t e s t o s t e r o n e .  T h i s study  and A4  female.  S i m i l a r to the p r e v i o u s  pre-pubescent, had no  e f f e c t on AHH  reductase  i s summarized i n T a b l e  Treatment w i t h p e r g o i i d e d i d not reduce h e p a t i c AHH • operated  prolactin  21.  a c t i v i t y i n the sham-  study gonadectomy, a l b e i t  activity.  Furthermore,  a d m i n i s t r a t i o n of p e r g o i i d e d i d not produce any  e f f e c t on  prolonged  AHH  a c t i v i t y i n e i t h e r t h e sham-operated or gonadectomized-female. The t r a t i o n of t e s t o s t e r o n e i n the presence or absence of the a d m i n i s t r a t i o n of p e r g o i i d e was female.  effect  However, s i m i l a r t o the p r e v i o u s r e s u l t s ,  produced an Of  a l s o without  i n c r e a s e i n AHH  i n t e r e s t was  i n c r e a s e of AHH  activity  concurrent  i n the  sham-operated  testosterone  i n t h e gonadectomized female.  the p e r g o i i d e i n h i b i t i o n of the t e s t o s t e r o n e activity  female ( T a b l e 21).  i n the p r e - p u b e s c e n t l y  T h i s e f f e c t was  induced  gonadectomized  a l s o observed i n females  gonadectomized as a d u l t s ( T a b l e 20)  r e c e i v i n g p e r g o i i d e at  With r e s p e c t to A4 r e d u c t a s e  maturity.  a c t i v i t y , both p e r g o i i d e  t e s t o s t e r o n e i n d i v i d u a l l y or i n combination were without  and  effect  i n t h e s ham- o p e r a t ed female ^ ( T a b l e 21)., ' The'-reduction" produced by :  sterone  i n pre-pubescently  gonadectomized females was  t h a t observed i n the p r e v i o u s Furthermore, t h i s decrease was  adminis  similar  to  study f o l l o w i n g a d u l t gonadectomy. not a f f e c t e d by  a d m i n i s t r a t i o n of p e r g o i i d e i n a s t a t i s t i c a l l y  concurrent s i g n i f i c a n t manner  testo-  81  TABLE 21 The e f f e c t o f pre-pubescent gonadectomy and p r o l a c t i n d e p l e t i o n on t h e a c t i o n o f t e s t o s t e r o n e on h e p a t i c AHH and t e s t o s t e r o n e A4 r e d u c t a s e a c t i v i t i e s i n t h e a d u l t female r a t . Immature female r a t s were gonadectomized (Gx) o r sham-operated between days 25 and 30 o f l i f e . Where i n d i c a t e d r a t s r e c e i v e d p e r g o l i d e (Perg ) (0.1 mg/kg/day s.c. i n corn o i l ) between days 25 t o 60 o f l i f e . In a d d i t i o n , t e s t o s t e r o n e enanthate (Test) was a d m i n i s t e r e d (1.0'mg/kg/day s.c. i n corn o i l ) f o r 10 days between days 50 t o 60 o f l i f e . Animals were s a c r i f i c e d 24 hours a f t e r t h e l a s t i n j e c t i o n . Differences between groups were c o n s i d e r e d s i g n i f i c a n t a t p < 0.05 u s i n g t h e Newman-Keuls m u l t i p l e range t e s t . S i g n i f i c a n t d i f f e r e n c e s between groups were i n d i c a t e d by common l e t t e r s .  AHH A c t i v i t y (pmol/min/mg p r o t e i n ) ± S.E.M., (n)  Female Model  A4 Red. A c t i v i t y (nmol/min/mg p r o t e i n ) ± S.E.M., (n)  Sham  666.3 ± 7.3 (3)  33.8 ± 1.5 ag (3)  Sham + Perg  537.5 ± 24.3* (4)  33.7 ± 0.9 (4)  Gx  576.2 ± 57.4 (5)  C  ek 33.4 ± 2.1 (5)  Gx + Perg  449.6 ± 14.2 (5)  £  Sham + Test  709.0 ± 83.5 (4)  Gx + Test,  Gx + Test  + Perg  744.0 ± 22.5 (5)  fl  31.4 ± 2.5 cx (4)  2567.8 ± 263.2 (5)  Sham + Test + Perg  41.0 ± 1.5 (5)  bh  abcdef  f ,  abcdef 1508.6 ± 259.1' (5)  14.9 ± 1.3 (5)  abcdef  37.2 ± 1.7 (5) 22.6 ± 2.V (5) ghij k l  82  (Table  21).  iii)  E f f e c t of pimozide i n a d u l t male r a t s  A second approach to the to the p e r g o i i d e PRL  involvement of PRL  i n male r a t s .  Adult  sham-operated or gonadectomized male r a t s were  Pimozide, a dopamine a n t a g o n i s t as w e l l as  (Ojeda et a l . , was  s t i m u l a t e PRL  i n c r e a s e i n PRL  i n c r e a s e A4  levels.  has been shown to i n c r e a s e  s y n t h e s i s i n male and  1974; Maurer and G o r s k i , 1977).  t h a t an e l e v a t i o n i n PRL  a c t i v i t y and  a corollary  s t u d i e s i n v e s t i g a t e d the e f f e c t s of e l e v a t i n g  t r e a t e d w i t h pimozide t o produce an  PRL  and  serum  female r a t s  The r a t i o n a l e  would be p o s t u l a t e d to reduce  reductase  a c t i v i t i e s and/or reduce  AHH the  response of t h e s e - a c t i v i t i e s to t e s t o s t e r o n e 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 e l e v a t e d PRL, A4  reductase  h e p a t i c AHH  a c t i v i t i e s were determined f o l l o w i n g a d m i n i s t r a t i o n  of pimozide (0.6 mg/kg/day, subcutaneously) f o r ten days. a d d i t i o n , the response  to t e s t o s t e r o n e was  i n pimozide t r e a t e d a n i m a l s . in Table  and  The  In  a l s o determined  r e s u l t s of t h i s study a r e shown  22.  Gonadectomy of the/male r e s u l t e d i n a t e s t o s t e r o n e r e v e r s i b l e decrease i n AHH  activity.  d i d not a l t e r AHH  However, a d m i n i s t r a t i o n of pimozide  a c t i v i t y i n sham-operated\or gonadectomized males.  d i d pimozide i n f l u e n c e the e f f e c t of t e s t o s t e r o n e or gonadectomized male r a t s (Table 22). normally  low  i n e i t h e r sham-operated  Testosterone  A4  reductase,  i n the a d u l t male,was i n c r e a s e d f o l l o w i n g gonadectomy.  T h i s i n c r e a s e was  Nor  i n h i b i t e d by the a d m i n i s t r a t i o n of  testosterone.  83  TABLE 22 The e f f e c t o f gonadectomy, pimozide and t e s t o s t e r o n e on h e p a t i c AHH and t e s t o s t e r o n e A4 r e d u c t a s e a c t i v i t y i n a d u l t male r a t s . Adult male r a t s were gonadectomized (Gx) o r sham-operated 11 days p r i o r t o s a c r i f i c e . Where i n d i c a t e d animals r e c e i v e d pimozide (Pirn ) (0.6 mg/kg/day i n t a r t a r i c a c i d ) f o r 10 days or t e s t o s t e r o n e 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 t h e l a s t i n j e c t i o n . Differences between groups were c o n s i d e r e d s i g n i f i c a n t a t p < 0.05 u s i n g t h e Newman-Keuls m u l t i p l e range t e s t . S i g n i f i c a n t d i f f e r e n c e s between groups were i n d i c a t e d by common l e t t e r s .  Male Model  AHH A c t i v i t y (pmol/min/mg p r o t e i n ) ± S.E.M., (n)  A4 Red. A c t i v i t y (nmol/min/mg p r o t e i n ± S.E.M., (n)  Sham  fh 3971.5 ± 205.0 (4)  13.9 ± 1.2 (4)  Sham + Pirn  3848.2 ± 271.5 eg (5)  18.7 ± l . l (5)  Gx  1107.2 ± 95.7 (5)  cdgh  abef 761.4 ± 100.2 (5)  Gx + Pirn  Sham + Test  4147.5 ± 236.5 j l (4)  Gx + Test  3478.6 ± 243.3 (5)  Sham + T e s t . + Pirn  4435.8 ± 193.6 (5)  Gx + Test  3647.8 ± 2 0 9 . 7 (5)  + Pirn,  bdkl  ik  a c l : j  28.1 ± 1 . 0 (5) 29.9 ± 0 . 6 (5) 13.3 ± 0.9 (4) 11.5 ± 0.5 (5)  a  c  f  g  i  a c s i  b d e f h j  gh  cd  ab 10.5 ± 1.1 (5) 15.5 ± 1.0'ace (5)  84  In a d d i t i o n , pimozide was  found to be i n h i b i t o r y to the a c t i o n s of  t e s t o s t e r o n e i n t h e gonadectomized male (Table 22). pimozide w h i l e having no  e f f e c t s alone  produced an i n c r e a s e i n A4 r e d u c t a s e male.  A l t h o u g h t h i s i n c r e a s e was  i n the gonadectomized male,  a c t i v i t y i n the sham-operated  not  by gonadectomy i t c o u l d be r e v e r s e d  as great as t h a t produced  by t e s t o s t e r o n e .  by i t s e l f , had no e f f e c t on A4 r e d u c t a s e operated  4)  a c t i v i t y i n the sham-  S t u d i e s r e l a t i n g h e p a t i c AHH  Effect  binding  a c t i v i t y to h e p a t i c  of p e r g o i i d e  c y t o s o l i c estrogen currently.  cytosolic  components  In the f o l l o w i n g s t u d i e s h e p a t i c AHH  The  a c t i v i t y and  hepatic  b i n d i n g c h a r a c t e r i s t i c s were determined con-  b i n d i n g s t u d i e s were conducted on a t i s s u e  comprised of the treatment group, whereas h e p a t i c AHH was  Testosterone,  male.  estrogen  a)  Furthermore,  activity  determined f o r the i n d i v i d u a l animals i n the treatment group.  In an e f f o r t to d e t e c t a moderate a f f i n i t y component i n a hypophysectomized female, p e r g o i i d e was PRL  administered  shown i n T a b l e 23,  t h i s regimen d i d not r e s u l t  a moderate a f f i n i t y component. of the e s t r o g e n r e c e p t o r  non-  to reduce  l e v e l s as p r e v i o u s l y d e s c r i b e d , to sham-operated and  females i n the presence or absence of t e s t o s t e r o n e .  Kd  pool  gonadectomized  However, as  i n the d e t e c t i o n of  P e r g o i i d e i n c r e a s e d the apparent  i n the  c a p a c i t y of the e s t r o g e n r e c e p t o r  shamr-ope rated female, and i n t h e gonadectomized  reduced  animal.  the  TABLE 23 The e f f e c t of p e r g o l i d e and t e s t o s t e r o n e on h e p a t i c AHH a c t i v i t y and t h e apparent Kd and c a p a c i t y of t h e h e p a t i c c y t o s o l i c estrogen r e c e p t o r and moderate a f f i n i t y component i n a d u l t gonadectomized and sham-operated female r a t s . A d u l t female r a t s were gonadectomized (Gx) or sham-operated 11 days p r i o r t o s a c r i f i c e . Where i n d i c a t e d a n i m a l s r e c e i v e d t e s t o s t e r o n e enanthate (Test ) (1.0 mg/kg/day s.c. i n corn o i l ) f o r 10 days o r p e r g o l i d e (Perg ) (0.1 mg/kg/day s.c. i n corn o i l ) f o r 11 days p r i o r t o 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 t h e l a s t i n j e c t i o n . D i f f e r e n c e s were c o n s i d e r e d s i g n i f i c a n t a t p < 0.05 u s i n g t h e Newman-Keuls m u l t i p l e range t e s t ; none were found. T i s s u e from each experimental group was pooled f o r b i n d i n g a s s a y s . Model  Estrogen Receptor Kd (xlO  1 0  M)  Capacity (fmol/mg p r o t e i n )  Moderate A f f i n i t y Component Kd (xlO  8  M)  Capacity (pmol/mg) protein  AHH A c t i v i t y (pmol/min/mg p r o t e i n ) + 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  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  6.2  235  0  0  270 + 22  Sham + Perg  + Perg  86  These r e s u l t s support and  t h e d i r e c t r e l a t i o n s h i p between PRL,  hepatic estrogen receptor  pergolide inhibitory effect i n c r e a s e AHH  b)  levels.  We  estrogens  d i d not observe  any  on the a c t i o n of t e s t o s t e r o n e to  a c t i v i t y as t e s t o s t e r o n e  i t s e l f was  without  effect.  E f f e c t of pimozide  The of AHH  e f f e c t ; of pimozide on the t e s t o s t e r o n e r e s p o n s i v i t y  a c t i v i t y was  also studied.  summarized i n Table 24.  Adult  The  r e s u l t s .of t h i s study  sham-operated and  gonadectomized animals  were t r e a t e d w i t h pimozide as p r e v i o u s l y d e s c r i b e d to serum PRL.  Pimozide produced a s l i g h t  a l s o observed.  A slight  These i n c r e a s e s were r e v e r s e d by  was  testosterone. estrogen  f o l l o w i n g p e r g o l i d e , i t should be emphasized t h a t  these d i f f e r e n c e s a r e w i t h i n e x p e r i m e n t a l their  r e g u l a t i o n of  i n c r e a s e i n apparent Kd  However, as w i t h the changes n o t e d i n c a p a c i t y of the receptor  increase  i n c r e a s e i n the c a p a c i t y  of the e s t r o g e n r e c e p t o r which i s c o n s i s t e n t w i t h PRL hepatic estrogen binding.  are  s i g n i f i c a n c e i s unknown.  variation.  Thus,  Pimozide d i d not a f f e c t  the  b i n d i n g c h a r a c t e r i s t i c s of t h e moderate a f f i n i t y component i n the sham^operated  male.  of the e s t r o g e n  Following  gonadectomy an i n c r e a s e i n the  r e c e p t o r was  testosterone administration d i d not a f f e c t  observed which was (Table 24).  reversed  capacity following  Furthermore,pimozide  the apparent Kd or c a p a c i t y of the e s t r o g e n  receptor  i n t h e gonadectomized male or the gonadectomized male r e c e i v i n g t e s t o s t e r o n e r e l a t i v e to t h e i r r e s p e c t i v e c o n t r o l s .  However,  a d m i n i s t r a t i o n of pimozide a b o l i s h e d the moderate a f f i n i t y  binding  TABLE 24 The e f f e c t of pimozide and t e s t o s t e r o n e on h e p a t i c estrogen b i n d i n g and h e p a t i c AHH a c t i v i t y i n sham and gonadectomized male r a t s . Adult male r a t s were gonadectomized (Gx) o r sham-operated 11 days p r i o r t o s a c r i f i c e . Where i n d i c a t e d animals r e c e i v e d pimozide ( P i r n ) (0.6 mg/kg/day s.c. i n p r o p y l e n e g l y c o l ) or t e s t o s t e r o n e enanthate (Test-) (1.0 mg/kg/day s.c. 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 t h e l a s t i n j e c t i o n . D i f f e r e n c e s were c o n s i d e r e d s i g n i f i c a n t at p < 0.05 u s i n g the Newman-Keuls m u l t i p l e range t e s t . Significant d i f f e r e n c e s between groups were i n d i c a t e d by common l e t t e r s . T i s s u e from each group was pooled f o r b i n d i n g assays.  E s t r o g e n Receptor (Ammonium S u l f a t e F r a c t i o n )  Model  K  (xlO  d  1 0  M)  Capacity (fmol/mg p r o t e i n )  Moderate A f f i n i t y Component (Whole C y t o s o l F r a c t i o n ) Kd (xlO^ M)  Capacity (pmol/mg p r o t e i n )  7.5  274  5.3  10.3  10.3  395  4.5  8.9  Sham + T e s t •  6.6  271  5.9  8.2  Sham + Test - + Pirn  6.0  241  6.7  11.5  Gx  8.8  455  5.2  2.5  Gx + Pirn  7.7  507  0  0  Gx + Test  9.9  359  7.1  13.2  5.8  309  6.7  4.1  Sham Sham + Pirn  Gx + Test  + Pirn  AHH A c t i v i t y (pmol/min/mg p r o t e i n ) iS.E.M., (n)  689 ± 1 0 7 (3) 617 ± 6 4 J  C 1  687 ± (4) 652 ± (4) 413 ± (4) 302 + (4) 757 ± (4) 560 ± (4)  e k  d  117 69^ 43  a b c d e f  35S  h i j k l  18 § a  105  b h  88  component  i n the gonadectomized male, and i n h i b i t e d t h e a c t i o n  of t e s t o s t e r o n e i n r e s t o r i n g the c a p a c i t y of t h i s f o l l o w i n g gonadectomy  ( T a b l e 24).  These changes were not r e f l e c t e d  by s i m i l a r changes i n AHH a c t i v i t y ; although a c t i v i t y a r e s i m i l a r t o those  c)  E f f e c t of continuous  component  seen i n T a b l e  t h e t r e n d s i n AHH 27.  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  h e p a t i c AHH a c t i v i t y and h e p a t i c c y t o s o l i c e s t r o g e n components  i s summarized  administered osmotic  i n T a b l e 25.  by continuous  binding  P i t u i t a r y hormones were  infusion using Alzet  model 2002  minipumps. A d m i n i s t r a t i o n of bGH  (0.01 I.U./hr f o r t e n days) t o shamnoperated  males d i d not a l t e r b i n d i n g c h a r a c t e r i s t i c s of the e s t r o g e n r e c e p t o r o r the moderate a f f i n i t y component a decrease i n AHH a c t i v i t y was observed  (Table 25).  (Table 25).  e f f e c t s have been p r e v i o u s l y d e s c r i b e d by Wilson Colby  (1980).  Similar  (1969) and  The e f f e c t s o f oPRL were a l s o examined s i n c e the  bGH used i n t h i s study c o n t a i n e d PRL, l i k e bGH,  However,  62% l a c t o g e n i c a c t i v i t y .  Ovine  d i d not a l t e r the apparent Kd or c a p a c i t y of the  e s t r o g e n r e c e p t o r or moderate a f f i n i t y component, a d e c r e a s e i n AHH a c t i v i t y As d e s c r i b e d e a r l i e r  but d i d produce  i n the sham-operated male (Table 25). (and i l l u s t r a t e d  i n T a b l e 25), hypo-  physectomy reduced moderate a f f i n i t y b i n d i n g and a b o l i s h e d t h e estrogen receptor  i n t h e male.  Following  continuous  i n f u s i o n of  bGH a f u r t h e r decrease i n AHH a c t i v i t y was observed i n 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 o i l ) 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: Ammonium Sulfate Fraction Kd (xlO Sham Male Hx Male  10  Capacity M)  (fmol/mg protein)  Moderate A f f i n i t y Component Whole Cytosol Fraction Kd (xlO M)  AHH Activity (pmol/min/mg protein S.E.M., (n=4)  Capacity (pmol/mg protein)  7.8 6.6  263 234  14.3 6.0  17.9 11.5  0 0  0 0  4.4 3.0  4.0 5.2  1568 ± 94 adghijklmn 399 ± 32 cf ghijklm  Sham Male + bGH  12.0 7.8  269 226  5.3 4.8  11.3 15.0  833 ± 43 adghijklmn  Sham Male + oPRL  8.2 10.6  220 289  3.3 3.9  12.5 19.4  642 ± 62 adghijklmn  Hx Male + rGH  0 0  0 0  0 0  0 0  Hx Male + bGH  0 0  0 0  4.9 3.1  5.9 2.4  218 ± 18 g 163 ± 13 def  Hx Male + rPRL  0 0  0 0  4.4 7.7  5.1 6.1  302 ± 34 ad  Hx Male + bGH + Test  0 0  0 0  2.9 4.7  3.3 1.5  242 + 35 1  Hx Male + oPRL + Test  0 0  0 0  4.2 10.1  4.1 3.3  227 ± 29 j  oo  cont 233 + 9 h  8.2 8.7  330 448  0 0  0 0  Hx Female  0 0  0 0  3.7 1.7  1.6 1.5  Hx Female + rGH  0 0  0 0  0 0  0 0  @ 232 + 24 @ k ®  Hx Female + rPRL  0 0  0 0  * *  * *  225 + 30 1 ©  Hx Female + bGH + Test  0 0  0 0  2.8 3.8  0.6 0.4  Hx Female + oPRL + Test  0 0  0 0  8.3 5.7  1.9 1.4  Sham Female  ®  312 + 30 ben  162 + 22 abc  © 255 + 28 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 l e t t e r s . Circled letters indicate separate Duncan analysis on female model.  91  physectomized male. continuously  I t i s • of i n t e r e s t t h a t when rGH was a d m i n i s t e r e d  (0.017 I.U./hr) n o t o n l y was AHH a c t i v i t y reduced but  the moderate a f f i n i t y b i n d i n g component was a b o l i s h e d i n the hypophysectomized 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 o n l y i n s t a n c e when male AHH a c t i v i t y was c o m p l e t e l y reduced to c o n t r o l female l e v e l s .  I t i s a l s o important t o n o t e t h a t  neither  bGH or rGH were a b l e t o r e s t o r e t h e e s t r o g e n r e c e p t o r b i n d i n g s i t e a b o l i s h e d by hypophysectomy.  To d e l i n e a t e between t h e  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 a l s o used..  Following  i n f u s i o n of rPRL' no e f f e c t s •  were observed on e s t r o g e n r e c e p t o r or moderate, a f f i n i t y binding,-'or AHH a c t i v i t y i n t h e hypophysectomized male. In t h e female hypophysectomy has been shown to r e s u l t l o s s of the h i g h a f f i n i t y component  in a  as i s seen i n the male.  In a d d i t i o n , f o l l o w i n g hypophysectomy t h e moderate a f f i n i t y component  (not n o r m a l l y p r e s e n t ) was d e t e c t e d i n the female.  Furthermore, n o r m a l l y accompanying the appearance of the moderate a f f i n i t y component  i s an i n c r e a s e i n AHH a c t i v i t y .  have been d e s c r i b e d i n p r e v i o u s s t u d i e s .  These e f f e c t s  However, t h e i n c r e a s e i n  AHH a c t i v i t y was s i g n i f i c a n t o n l y when t h e females were c o n s i d e r e d s e p a r a t e l y from t h e males.  The a d m i n i s t r a t i o n of rGH t o the  hypophysectomized female a b o l i s h e d t h e moderate a f f i n i t y produced by hypophysectomy.  The l o s s of moderate a f f i n i t y  was accompanied by a decrease i n AHH a c t i v i t y to a l e v e l the sham-operated female.  component binding  s i m i l a r to  92  rPRL  (0.2 I.U./hr) d i d not produce the same c o n s i s t e n t  i n r e l a t i o n t o AHH  a c t i v i t y as d i d rGH.  Continuous i n f u s i o n of rPRL  i n the hypophysectomized male d i d not a f f e c t by hypophysectomy  t h e changes  activity  (Table 25).  However, rPRL  a c t i v i t y of t h e hypophysectomized female to a sham  v a l u e , but d i d not e n t i r e l y remove moderate  affinity  S p e c i f i c a l l y , d i s p l a c e a b l e b i n d i n g was  present  p l o t c o u l d not be g e n e r a t e d .  still  binding. but a  Scatchard  S i m i l a r to growth hormone, rPRL d i d  not r e s t o r e t h e h i g h a f f i n i t y b i n d i n g s i t e which was by  produced  on the apparent Kd and c a p a c i t y of the moderate  a f f i n i t y component, or AHH d i d reduce AHH  effect  abolished  hypophysectomy. Testosterone  enanthate (1.0 mg/kg/day, s u b c u t a n e o u s l y f o r  ten days) was a d m i n i s t e r e d  t o examine the r e s p o n s i v i t y of the  hypophysectomized r a t i n the presence o f somatotropic t r o p i c hormones.  A d m i n i s t r a t i o n of t e s t o s t e r o n e t o hypophysecto-  mized males r e c e i v i n g e i t h e r bGH  or oPRL d i d not produce any marked  changes i n b i n d i n g c h a r a c t e r i s t i c s o r AHH Nor was  or l a c t o -  t e s t o s t e r o n e a b l e t o induce AHH  mized female r e c e i v i n g bGH or oPRL.  activity  (Table 25).  a c t i v i t y i n the hypophysecto-  The combination of e i t h e r  or oPRL w i t h t e s t o s t e r o n e had no r e s t o r a t i v e a c t i o n on the h i g h affinity  s i t e i n e i t h e r hypophysectomized males or females.  bGH  9 3  5)  In v i v o and  i n v i t r o e f f e c t s of x e n o b i o t i c s on h e p a t i c  a c t i v i t y and  a)  c y t o s o l i c estrogen  AHH  b i n d i n g parameters  Induction  S i n c e i n d u c t i o n of AHH  a c t i v i t y by t e s t o s t e r o n e i n the  gonadectomized male i s a s s o c i a t e d w i t h an a f f i n i t y b i n d i n g , the e f f e c t of other  i n c r e a s e i n moderate  i n d u c i n g agents b o t h i n v i v o  3  and  i n v i t r o on the b i n d i n g o f  [ H ] - e s t r a d i o l to the  hepatic  c y t o s o l i c e s t r o g e n r e c e p t o r and moderate a f f i n i t y component investigated.  The  e f f e c t s o f 3-MC  b i n d i n g parameters a r e i l l u s t r a t e d  and  s p i r o n o l a c t o n e on  i n T a b l e 26.  was  estrogen  In a d d i t i o n , the  3  e f f e c t i v e n e s s of 3-MC  as a competitor  to these components was 3-MC  for [ H]-estradiol binding  also investigated.  Administration  (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  a decrease i n the c a p a c i t y of the e s t r o g e n female r a t s w i t h no sex.  Spironolactone  observable  effect  days r e s u l t e d i n  receptor  i n male and  on the apparent Kd  for either  (100 mg/kg/day, i n t r a p e r i t o n e a l l y ) f o r f o u r  days reduced the c a p a c i t y of the e s t r o g e n r e c e p t o r but not  of  i n the  female  i n the male. 3  When 3-MC  was  employed as a competitor  b i n d i n g , the apparent Kd was unlabeled rats. 3-MC  s i m i l a r to t h a t o b t a i n e d  e s t r a d i o l as a competitor  T h i s s i m i l a r i t y was as w e l l .  for [ H]-estradiol using  i n c o n t r o l male and  female  noted i n males f o l l o w i n g i n d u c t i o n by  However, a Scatchard  female f o l l o w i n g i n d u c t i o n by 3-MC  c o u l d not and  be generated i n the  u s i n g 3-MC  as a  competitor  3  for  [ H ] ^ e s t r a d i o l b i n d i n g to t h e e s t r o g e n r e c e p t o r .  d i s c r e p a n c y was  not  This  observed f o l l o w i n g the a d m i n i s t r a t i o n of  s p i r o n o l a c t o n e t o the female.  However, the apparent Kd  obtained  94  by 3-MC  competition  i n s p i r o n o l a c t o n e t r e a t e d males and females  was somewhat lower than those v a l u e s d e r i v e d by e s t r a d i o l " c o m p e t i t i o n ( T a b l e 26). The c a p a c i t i e s generated g e n e r a l l y one-half  competition  those o b t a i n e d w i t h u n l a b e l e d  However, f o l l o w i n g treatment f o u r f o l d decrease  f o l l o w i n g 3-MC  were  estradiol.  w i t h s p i r o n o l a c t o n e an approximate  was d e t e c t e d w i t h 3-MC.  I n t e r e s t i n g l y , these  d i f f e r e n c e s i n apparent Kd and c a p a c i t y between 3-MC  and e s t r a d i o l  c o m p e t i t i o n were n o t seen f o r the male moderate a f f i n i t y  component.  Of g r e a t e r i n t e r e s t were t h e e f f e c t s of i n v i v o a d m i n i s t r a t i o n of 3-MC  and s p i r o n o l a c t o n e , which produced an i n c r e a s e i n s p e c i f i c  b i n d i n g t o the moderate a f f i n i t y component.  T h i s was u r e f l e c t e d  as an i n c r e a s e i n c a p a c i t y , e s p e c i a l l y e v i d e n t f o l l o w i n g w i t h 3-MC  ( T a b l e 26).  However, n e i t h e r 3-MC  treatment  nor s p i r o n o l a c t o n e t r e a t -  ment r e s u l t e d i n d e t e c t i o n of a moderate a f f i n i t y b i n d i n g  component  i n t h e female. In a r e l a t e d  study the e f f e c t s of p h e n o b a r b i t a l  (80 mg/kg/day,  i n t r a p e r i t o n e a l l y f o r f o u r d a y s ) , on estrogen r e c e p t o r and moderate a f f i n i t y b i n d i n g components were i n v e s t i g a t e d . u n l a b e l e d e s t r a d i o l or BP were used as  In t h i s  study  competitors  3 for  [ H ] - e s t r a d i o l binding.  i n d i c a t e d s m a l l decreases  The r e s u l t s , r e p o r t e d i n T a b l e 27,  i n t h e c a p a c i t y and the apparent Kd of  the e s t r o g e n r e c e p t o r i n both male and female r a t s f o l l o w i n g a d m i n i s t r a t i o n of p h e n o b a r b i t a l , u s i n g e s t r a d i o l as a  competitor.  In a l l c a s e s , lower e s t r o g e n r e c e p t o r c a p a c i t i e s were  observed  when t h i s parameter was measured u s i n g BP as a c o m p e t i t o r .  Further-  i  more, u n l i k e 3-MC,  BP c o m p e t i t i o n .produced a-Scatchard  p l o t f o r 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 a f f i n i t y binding component i n male and female rats. Where indicated adult male and female rats received either 3-methylcholanthrene (3-MC) (20 mg/kg/day i.p. i n corn o i l ) for 2 days or spironolactone (Spir ) (100 mg/kg/day l.p. in corn o i l ) 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 A f f i n i t y Component  (Ammonium Sulfate Fraction)  (Whole Cytosol Fraction)  "7( x l 0 M) l u  Capacity (fraol/mg protein)  (xlO M) 8  Capacity (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)  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)  0  0  Spir. Female  (E2)  9.1  232  0  0  Spir. Female  (3-MC)  5.5  38  0  0  13.9  No Scatchard  I  TABLE 27 The e f f e c t o f 4 days treatment w i t h p h e n o b a r b i t a l on t h e apparent Kd and c a p a c i t y o f the h e p a t i c c y t o s o l i c e s t r o g e n r e c e p t o r and moderate a f f i n i t y b i n d i n g component i n male and female r a t s . Where i n d i c a t e d a d u l t male and female r a t s r e c e i v e d p h e n o b a r b i t a l (Phenobarb) (80 mg/kg/day i . p . i n s a l i n e ) f o r 4 days p r i o r t o 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 t h e l a s t i n j e c t i o n . B i n d i n g parameters were determined u s i n g e i t h e r a 100 f o l d excess o f u n l a b e l e d e s t r a d i o l (E2) or a 1000 f o l d excess of b e n z o ( a ) p y r e n e (BP) as competitors f o r [ H ] - e s t r a d i o l b i n d i n g . A. t i s s u e p o o l from 4 animals was used f o r eacb/fg 3  Model: Competitor  Estrogen  Receptor  Moderate A f f i n i t y  (Ammonium S u l f a t e F r a c t i o n ) q (xlO K  M)  .  Component  (Whole C y t o s o l F r a c t i o n )  Capacity (fmol/mg p r o t e i n ) —  (xl0 M) — —  K  d  y  Capacity (pmol/mg p r o t e i n ) _  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  0.73  91  0  0  (BP)  ^VD  97  e s t r o g e n r e c e p t o r i n the female.  Competition  of  b i n d i n g t o the e s t r o g e n r e c e p t o r by BP r e s u l t e d  [ H]-estradiol i n lower  capacities  than those o b t a i n e d by e s t r a d i o l c o m p e t i t i o n i n c o n t r o l males females.  Although  apparent  treatment  and  w i t h p h e n o b a r b i t a l d i d reduce t h e  Kd of t h e estrogen r e c e p t o r by e s t r a d i o l c o m p e t i t i o n ,  r e d u c t i o n was  not  seen u s i n g BP as a  S i m i l a r t o 3-MC,  this  competitor.  p h e n o b a r b i t a l i n c r e a s e d the c a p a c i t y of  male moderate a f f i n i t y component.  Benzo(a)pyrene was  an  the  effective  3 competitor  for  [ H ] - e s t r a d i o l b i n d i n g to the moderate  component as w e l l . from BP  However, u n l i k e 3-MC,  c o m p e t i t i o n was  approximately  e s t r a d i o l as a c o m p e t i t o r . apparent  the c a p a c i t y t h a t r e s u l t e d  one-half t h a t o b t a i n e d  using  As w i t h the estrogen r e c e p t o r , the  Kd of the moderate a f f i n i t y  c o n t r o l male when BP was  affinity  component was  reduced  i n the  used i n s t e a d o f e s t r a d i o l t o compete f o r  3 I H]-estradiol binding. d i f f e r e n c e was were s m a l l , and  lost  However, as w i t h the e s t r o g e n r e c e p t o r , t h i s  f o l l o w i n g p h e n o b a r b i t a l treatment.  These changes  their 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,  A d m i n i s t r a t i o n of p h e n o b a r b i t a l d i d not produce any  detectable  moderate a f f i n i t y component i n the female (Table 27). To e v a l u a t e the d u r a t i o n of i n d u c t i o n , 3-MC were a d m i n i s t e r e d  and  to male and female r a t s f o r t e n days.  the e f f e c t of a s y n t h e t i c e s t r o g e n , m e s t r a n o l , was The 3-MC  e f f e c t of p h e n o b a r b i t a l  b i n d i n g are p r e s e n t e d  (80 mg/kg/day, i n t r a p e r i t o n e a l l y )  i n Table 28.  N e i t h e r 3-MC  nor  and  and  estrogen  phenobarbital  f o r t e n days s i g n i f i c a n t l y a l t e r e d the b i n d i n g  of the e s t r o g e n r e c e p t o r .  In a d d i t i o n ,  a l s o examined.  (20 mg/kg/day, i n t r a p e r i t o n e a l l y ) on h e p a t i c AHH  treatment  phenobarbital  characteristics  Furthermore, a d m i n i s t r a t i o n of p h e n o b a r b i t a l  98  for  this  time p e r i o d d i d n o t a f f e c t  the apparent Kd or c a p a c i t y of  the  moderate a f f i n i t y component i n the male.  ten  days of 3-MC,  However, f o l l o w i n g  t h e c a p a c i t y of the moderate a f f i n i t y component  i n the male decreased amount 20 f o l d , w i t h a near 50% r e d u c t i o n i n apparent Kd  (Table 28).  No moderate a f f i n i t y component was d e t e c t e d  i n the female f o l l o w i n g l o n g term a d m i n i s t r a t i o n of i n d u c e r s . The e f f e c t  of these agents on AHH  No change, or o n l y s l i g h t AHH  inhibition,  and s e v e r a l f o l d  As expected, 3-MC  i n males and females t o comparable  levels  induced AHH  activity  Inhibition  d e c r e a s e i n AHH  activity  at r e v e r s i n g  E s t r o g e n s a r e a l s o known t o reduce  (Gurtoo and P a r k e r , 1979).  estrogens on AHH  effective  a c t i v i t y and moderate a f f i n i t y b i n d i n g i n the  male f o l l o w i n g gonadectomy. AHH  i n d u c t i o n of  (Table 28).  As p r e v i o u s l y shown t e s t o s t e r o n e was the  as expected.  a c t i v i t y were seen f o l l o w i n g p h e n o b a r b i t a l treatment of male and  female r a t s r e s p e c t i v e l y .  b)  a c t i v i t y was  activity  i n the male was  The i n h i b i t o r y demonstrated  effect  of  following  a d m i n i s t r a t i o n o f m e s t r a n o l (1.0 mg/kg/day subcutaneously f o r ten days) as shown i n T a b l e 28. on AHH  a c t i v i t y i n the female.  specific the  M e s t r a n o l d i d not produce any  effect  Furthermore, m e s t r a n o l a b o l i s h e d  b i n d i n g t o the e s t r o g e n r e c e p t o r i n males and females as  moderate a f f i n i t y component i n males.  TABLE 28 The e f f e c t o f 10 days treatment w i t h mestranol, p h e n o b a r b i t a l and 3-methylcholanthrene on h e p a t i c c y t o s o l i c estrogen b i n d i n g c h a r a c t e r i s t i c s and h e p a t i c AHH a c t i v i t y i n male and female r a t s . A d u l t male and female r a t s r e c e i v e d e i t h e r m e s t r a n o l (1.0 mg/kg/day s.c. i n corn o i l ) f o r 14 days, p h e n o b a r b i t a l (Phenobarb) (80 mg/kg/day i . p . i n s a l i n e ) f o r 10 days o r 3-methylcholanthrene (3-MC) (20 mg/kg/day i . p . i n corn o i l ) f o r 10 days p r i o r t o 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 o r phenobarb and 48 hours a f t e r t h e l a s t i n j e c t i o n f o r m e s t r a n o l and 3-MC. T i s s u e from 4 animals was pooled f o r each group. D i f f e r e n c e s between groups were c o n s i d e r e d s i g n i f i c a n t a t p < 0.05 u s i n g t h e Newman-Keuls m u l t i p l e range t e s t . Significant d i f f e r e n c e s were indicated- by common l e t t e r s .  Model  Estrogen Receptor (Ammonium S u l f a t e F r a c t i o n ) Kd (xlO  C o n t r o l Male Mestranol  Male  1 0  M)  Capacity (fmol/mg p r o t e i n )  Moderate A f f i n i t y Component (Whole C y t o s o l F r a c t i o n ) Kd (xlO  8  M)  AHH A c t i v i t y (pmol/min/mg p r o t e i n ) ± S.E.M., (n=4)  Capacity (pmol/mg p r o t e i n )  5.1  199  14.2  19.8  0  0  0  0  12.5  19.4  801 ±  acei  132  ± 16 aceh 542 ± ace 1201 ±  Phenobarb Male  5.4  177  3-MC Male  9.4  153  7.4  0.9  C o n t r o l Female  5.3  145  0  0  Female  0  0  0  0  124  Phenobarb Female  7.5  217  0  0  405  3-MC Female  7.8  140  0  0  1290  Mestranol  2 5  e f  2 5  6 7  99 ± l l  a  b  ± 13 aceg ± bdfghi ± c d  3 ?  g 3  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 t h e p h y s i o l o g i c a l c o r r e l a t i o n o f p o l y c y c l i c  3 aromatic  hydrocarbon  (PAH) c o m p e t i t i o n  for  [ H]-estradiol binding,  t e s t o s t e r o n e replacement i n gonadectomized r a t s was i n v e s t i g a t e d . As  shown i n T a b l e 29, a d m i n i s t r a t i o n o f t e s t o s t e r o n e  increased  t h e b i n d i n g o f t h e moderate a f f i n i t y component as determined by e i t h e r 3-MC or e s t r a d i o l c o m p e t i t i o n .  Administration of t e s t o -  s t e r o n e t o t h e gonadectomized female, shown not t o 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 b i n d i n g , d i d n o t r e s u l t i n 3-MC competed b i n d i n g e i t h e r .  As i n d i c a t e d p r e v i o u s l y , 3-MC  3 competed f o r approximately to  the estrogen  one-half  the [ H]-estradiol binding  r e c e p t o r as d i d e s t r a d i o l .  With t h e e x c e p t i o n  of t h e gonadectomized female r e c e i v i n g t e s t o s t e r o n e , 3-MC and e s t r a d i o l competition  f o r binding to the estrogen receptor r e s u l t e d  i n s i m i l a r apparent Kd v a l u e s d)  R e l a t i o n s h i p of estrogen  (Table 2 9 ) . b i n d i n g components t o Ah-receptor  3 To probe any a s s o c i a t i o n t o t h e A h - r e c e p t o r , b i n d i n g was competed w i t h a 100 f o l d dioxin  excess o f t e t r a c h l o r o d i b e n z o - p -  (TCDD), 2 , 7 - d i c h l o r o d i b e n z o - p - d i o x i n  octachlorodibenzo-p-dioxin  (OCDD).  [H]-estradiol  (2,7-DCDD), or  A l l t h r e e d i o x i n congeners  3 competed f o r [ H ] - e s t r a d i o l b i n d i n g t o t h e e s t r o g e n males and females.  receptor i n  However, o n l y OCDD and 2,7-DCDD were e f f e c t i v e  3 competitors  f o r [ H ] - e s t r a d i o l , b i n d i n g t o the moderate a f f i n i t y  TABLE 29 The e f f e c t of gonadectomy and t e s t o s t e r o n e replacement on l i g a n d s p e c i f i c i t y of t h e h e p a t i c c y t o s o l i c e s t r o g e n r e c e p t o r and moderate a f f i n i t y b i n d i n g component i n male and female rats. A d u l t male and female r a t s were gonadectomized (Gx) 11 days p r i o r to s a c r i f i c e . Where i n d i c a t e d animals r e c e i v e d t e s t o s t e r o n e enanthate (Test ) (1.0 mg/kg/day s.c. i n c o r n o i l ) f o r 10 days. Animals were s a c r i f i c e d 24 hours a f t e r t h e l a s t i n j e c t i o n . Binding parameters were determined u s i n g a 100 f o l d excess of u n l a b e l e d e s t r a d i o l (E2) or a 1000 f o l d excess of u n l a b e l e d 3-methylcholanthrene (3-MC) as c o m p e t i t o r s f o r [ H ] - e s t r a d i o l . S t u d i e s were done on t i s s u e pooled from 4 animals. 3  Model: Competitor  Estrogen  Receptor  Moderate A f f i n i t y  (Ammonium S u l f a t e F r a c t i o n ) K  d  (xlO  9  M)  Capacity (fmol/mg p r o t e i n )  Component  (Whole C y t d s d l F r a c t i o n ) K d  (xlO  8  M)  Capacity (pmol/mg p r o t e i n )  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  (E2)  1.54  496  0  0  (3-MC)  1.12  201  0  0  Gx  Female  Gx Female Female +  Test  (E2)  1.01  345  0  0  Gx Female +  Test  (3-MC)  0.59  161  0  0  Gx  102  component  i n t h e male (Table 30).  p l o t s c o u l d o n l y be generated  Furthermore,  a c c e p t a b l e Scatchard  f o r t h e female e s t r o g e n r e c e p t o r  f o l l o w i n g c o m p e t i t i o n w i t h t h e s e d i o x i n congeners.  The apparent  Kd and c a p a c i t y f o r a l l d i o x i n congeners were s i m i l a r but lower than t h a t o b t a i n e d by e s t r a d i o l c o m p e t i t i o n 3-MC  (Table 31).  o r BP, s p e c i f i c b i n d i n g t h a t r e s u l t e d i n good S c a t c h a r d  a n a l y s i s c o u l d not be produced  w i t h these d i o x i n congeners f o r  t h e male e s t r o g e n r e c e p t o r o r moderate a f f i n i t y though d i s p l a c e a b l e b i n d i n g was observed of  Unlike  c o n t r o l apparent  component, even  ( T a b l e 30).  Kd v a l u e s and c a p a c i t i e s f o r the a d u l t  and male e s t r o g e n r e c e p t o r and male moderate a f f i n i t y are presented  A summary  i n T a b l e 32.  female  component  103  TABLE 30 Ligand s p e c i f i c y of t h e h e p a t i c c y t o s o l i c estrogen r e c e p t o r and the moderate a f f i n i t y s i t e f o r v a r i o u s c o m p e t i t o r s o f [ H ] - e s t r a d i o l b i n d i n g i n t h e male and female r a t . Unless otherwise noted c o m p e t i t o r s were added i n a 100 f o l d excess t o 0.5 nM [ H ] - e s t r a d i o l i n t h e ammonium s u l f a t e f r a c t i o n f o r d e t e r m i n a t i o n o f c o m p e t i t i o n f o r b i n d i n g t o t h e e s t r o g e n r e c e p t o r , o r t o 50 nM p H ] - e s t r a d i o l i n t h e whole c y t o s o l f r a c t i o n f o r d e t e r m i n a t i o n o f c o m p e t i t i o n f o r b i n d i n g t o t h e moderate a f f i n i t y component. I n c u b a t i o n s were c a r r i e d out f o r 90 minutes at 4 °C. T i s s u e pooled from 4 animals were used f o r each group. 3  3  Percent o f T o t a l E s t r o g e n Receptor Unlabeled Competitor Control Estradiol TCDD OCDD 2,7-DCDD  * 3-MC BP*  Female  [H]-estradiol  Moderate A f f i n i t y  Male  Male  100  100  100  47  54  66  59  75+ 72+ 70+  57 66  T  62 .57-  * 1000 f o l d excess u n l a b e l e d competitor t l i g a n d c o n c e n t r a t i o n = 0 . 1 nM TCDD Tetrachlorodibenzo-p-dioxin OCDD Octachlorodibenzo-p-dioxin 2,7-DCDD 2 , 7 - D i c h l o r o d i b e n z o-p-d i o x i n 3-MC 3-Methylcholanthrene BP Benzo(a)pyrene  100 58 67  40 52  Bound Site  104  TABLE 31 The apparent Kd and c a p a c i t y o f t h e e s t r o g e n r e c e p t o r i n a d u l t female r a t s as determined w i t h d i f f e r e n t d i o x i n eongenefs. A l l c o m p e t i t o r s were added i n a 100 f o l d excess. S t u d i e s were conducted on t i s s u e pooled from 4 animals.  Competitor  Apparent Kd ( x l O M) 9  Capacity (fmol/mg p r o t e i n )  Estradiol  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 o f c o n t r o l v a l u e s f o r apparent Kd and c a p a c i t y o f t h e e s t r o g e n r e c e p t o r and moderate a f f i n i t y component.  Female E s t r o g e n Recept or Kd(M) Capacity (fmol/mg p r o t e i n )  Male E s t r o g en Receptor Kd (M) Capacity (fmol/mg p r o t e i n )  Male Moderate A f f i n i t y Kd(M.) Capacity (pmol/mg p r o t e i n '  1.1 x I O "  9  368  1.1 x 1 0 "  9  261  14.3 x I O "  8  6.6  2.5 x 1 0 "  9  506  2.2 x 1 0 "  9  516  14.0 x I O "  8  31.4  3.7 x 1 0 "  9  641  2.8 x 1 0 ~  9  620  6.4 x I O "  8  11.0  555  9.0 x I O "  8  14.9  260  8.8 x I O "  8  19.3  405  17.5 x I O "  8  274  5.3 x I O "  8  10.3  0.82 x 1 0 "  9  188  1.05 x 1 0 "  9  1.52 x 1 0 "  9  477  1.11 x 10"  9  1 0  487  1.9 x 1 0 "  489  7.5 x 1 0 "  1 0  ,330'  7.8 x 1 0 "  1 0  263  14.3 x I O "  8  17.9 11.5  8.1 x 1 0 "  2.07 x 1 0 "  9  9  15.0  8.2 x 1 0 "  1 0  8.7 x 1 0 "  1 0  448  6.6 x 1 0 "  1 0  234  6.0 x I O "  8  9.6 x 1 0 "  1 0  337  8.4 x 1 0 "  1 0  251  8.9 x I O "  8  19.6  145  5.1 x 1 0 "  1 0  199  14.2 x I O "  8  19.8  437  9.1 x I O "  1 0  352  6.25x 10  496  2.25x 1 0 "  549  6.99x 1 0 "  5.3 x 10 7.8 x 1 0 "  1 0  2.17 x 1 0 "  9  1 .43 ± 0 . 2 5 x l 0 ~  9  411.5 ± 37.7  (3 .7 - 0.53xl0- )  (145 - 641)  9  9  1.29 ± 0 . 2 0 x l 0 "  341.5 ± 38.1  9  (2.8 - 0.5] x ,10" ) (199 - (520) 9  9.47  8  6.2  8  10.15 ± 1.14x10"!  8  (14.3 •- 5 . 3 x l 0 ) _ £S  14.8 ± 1.9 (6.2 - 31.4)  106  DISCUSSION  1)  E s t r o g e n b i n d i n g s t u d i e s : c h a r a c t e r i z a t i o n and  comparison  of h e p a t i c c y t o s o l i c e s t r o g e n b i n d i n g components w i t h previously published results  I n i t i a l c h a r a c t e r i z a t i o n of the h e p a t i c c y t o s o l i c b i n d i n g p r o t e i n s p r o v i d e d d a t a s i m i l a r t o t h a t r e p o r t e d by other in t h i s area.  The developmental  investigators  s t u d i e s e s t a b l i s h e d an o p t i m a l  i n c u b a t i o n time of n i n e t y minutes a t 4°C f o r both the e s t r o g e n r e c e p t o r and the moderate a f f i n i t y  component.  T h i s time was  long  enough to a l l o w f o r completion of l i g a n d complex f o r m a t i o n yet not so long as to observe exchange or d e g r a d a t i o n .  These b i n d i n g data  were p r o v i d e d by Dr. B. Warren and are p r e s e n t e d i n Appendix F i g u r e s A l , A2.  1,  A p a r a l l e l study p r e s e n t e d i n T a b l e 3 examined  t h e time c o u r s e of b i n d i n g a t 25°C.  Comparison of t h e s e  time s t u d i e s i n d i c a t e s that completion o f ligand-complex o c c u r r e d e a r l i e r a t 25°C, than at 4°C, as expected. i n c u b a t i o n time was  used  f o r subsequent  t o times employed by o t h e r i n v e s t i g a t o r s Jones e t ^ _ a l . , 1980,  1981;  temperatureformation  The 90 minute  s t u d i e s and was  comparable  (Eagon et a l . , 1980;  and Sloop et a l . , 1983).  the p r e v i o u s l y c i t e d s t u d i e s , molybdate was  Powell-  However, u n l i k e  i n c l u d e d i n the t r i s m a -  E D T A - d i t h i o t h r e i t o l b u f f e r system.  Sodium molybdate has been shown  t o prevent r e c e p t o r a g g r e g a t i o n and  i n c r e a s e s t a b i l i t y to heat  activa-  t i o n thus making the measurement o f s p e c i f i c b i n d i n g more p r o b a b l e (Moncharmont et a l . , 1982;  Noma et a l . , 1980).  In a d d i t i o n ,  107  c o n t r a r y t o the methodology of Powell-Jones and Sloop et a l . , (1983), exposure  t o DCC  et a l . , (1980,  was  reduced  1981)  from  thirty  to f i v e minutes t o a v o i d s t r i p p i n g o f f bound s t e r o i d as d e s c r i b e d by Peck and  C l a r k (1977).  S t u d i e s p e r t a i n i n g t o l i g a n d s p e c i f i c i t y o f the e s t r o g e n r e c e p t o r and the moderate a f f i n i t y p r o t e i n were examined f o r comparison  w i t h p u b l i s h e d c h a r a c t e r i s t i c s t o c o n f i r m the  of these b i n d i n g components.  identity  These competitor s t u d i e s i n d i c a t e d 3  t h a t e s t r o g e n s were e f f e c t i v e i n h i b i t o r s of in v i t r o  i n male ( T a b l e 6) and female  [ H]-estradiol binding  (Table A2, Appendix 1)  However, as r e p o r t e d by Eagon et a l . , (1980), Powell-Jones (1980, 1981), and  Sloop et a l . ,  b i n d i n g t o t h e moderate a f f i n i t y r e p o r t e d i n T a b l e 5.  (1983) DES  et a l . ,  does not compete f o r  component.  Furthermore,  rats.  Similar r e s u l t s are  androgens, which do not compete  f o r b i n d i n g t o the e s t r o g e n r e c e p t o r ( T a b l e 6; T a b l e A2, Appendix 3 a r e e f f e c t i v e i n h i b i t o r s o f [ H ] - e s t r a d i o l b i n d i n g i n v i t r o to the moderate a f f i n i t y component i n male whole c y t o s o l (Table 6).  1)  3 Other  s t e r o i d hormones a r e not e f f e c t i v e c o m p e t i t o r s f o r [ H ] - e s t r a d i o l  b i n d i n g t o t h e e s t r o g e n r e c e p t o r or the moderate a f f i n i t y  component.  A l l o f these r e s u l t s a r e i n agreement w i t h the p r e v i o u s l y c i t e d investigators.  I t was  c o n c l u d e d , t h e r e f o r e , t h a t t h e 50% ammonium  s u l f a t e f r a c t i o n i n both male and female r a t s c o n t a i n e d the e s t r o g e n r e c e p t o r and the male whole c y t o s o l f r a c t i o n c o n t a i n e d the moderate a f f i n i t y component as d e f i n e d by p r e v i o u s r e p o r t s (Aten et D i c k s o n et a l . , 1978; 1981;  Eagon et a l . ,  Sloop et a l . , 1983).  1980;  Powell-Jones  al.,1978;  et a l . , 1980,  108  F u r t h e r c o n f i r m a t i o n of the i d e n t i t y of the h e p a t i c c y t o s o l i c b i n d i n g components i n v o l v e d examination of e s t r a d i o l b i n d i n g i n different  p h y s i o l o g i c a l models.  In so d o i n g , p h y s i o l o g i c a l  r e l e v a n c e as w e l l as the v a l i d i t y of the b i n d i n g a s s a y s were e s t a b l i s h e d . P r e v i o u s s t u d i e s have demonstrated moderate a f f i n i t y 1980;  s i t e was  Powell-Jones e t a l . ,  Powell-Jones et a l . , can be demonstrated  age and 1980,  t h a t the presence o f the  sex-dependent  1981;  (Eagon et a l . ,  Sloop et a l . ,  1983).  Furthermore,  (1981) have r e p o r t e d t h a t no o n t o g e n i c d i f f e r e n c e f o r the e s t r o g e n r e c e p t o r i n r a t l i v e r  cytosol.  The sex-dependency o f the moderate a f f i n i t y component as w e l l as t h e non-sex-dependency of the e s t r o g e n r e c e p t o r has strated  i n the course of our assay development.  c e r t a i n minor  been demon-  There a r e , however,  d i s c r e p a n c i e s between p r e v i o u s r e p o r t s and the r e s u l t s  presented here.  Powell-Jones et a l . ,  (1980, 1981)  d e s c r i b e the  presence o f s m a l l amounts of the 4S p r o t e i n i n a d u l t female, and pre-pubescent male and female r a t s .  However, i t i s i n c o r r e c t t o  assume t h a t the 4S peak o f the immature animal or a d u l t female i s the moderate a f f i n i t y component as these a u t h o r s suggest without further supporting evidence.  Eagon et a l . ,  (1980) have  demonstrated  t h a t a sex d i f f e r e n c e i s p r e s e n t f o r not o n l y the c a p a c i t y 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.  Powell-Jones et a l . ,  (1980,1981) Eagon's group have conducted  competitive i n h i b i t i o n female c y t o s o l .  s t u d i e s on the 4S p r o t e i n i n b o t h male and  They have demonstrated  p r o t e i n can be p a r t i a l l y male. DES  Unlike  t h a t b i n d i n g t o the 4S  i n h i b i t e d by e s t r a d i o l but not DES  i n the  However,, i n the female whole c y t o s o l f r a c t i o n , both e s t r a d i o l and  inhibited  3 - [ H ] - e s t r a d i o l b i n d i n g t o the 4S peak.  Therefore,  109  the 4S peak i n the female was s p e c i e s of the e s t r o g e n component. here.  probably  r e p r e s e n t a t i v e o f the  r e c e p t o r and not  4S  the moderate a f f i n i t y  These r e s u l t s a r e c o n s i s t e n t w i t h o b s e r v a t i o n s  presented  That i s , no moderate a f f i n i t y b i n d i n g component c o u l d  detected  be  i n the a d u l t female or i n immature male or female r a t  h e p a t i c c y t o s o l (Tables 7-9,  11,  12,  23,  25-28).  Thus, i n g e n e r a l , the t r e n d s observed f o r the sucrose s t u d i e s which have c h a r a c t e r i z e d these e s t r o g e n were r e p r o d u c i b l e by Scatchard  analysis.  binding  Moreover, the  components d e s c r i b e d by p r e v i o u s workers possessed c h a r a c t e r i s t i c s to those we  gradient  proteins binding  similar  have demonstrated.  Eagon et a l . , (1980) r e p o r t e d a sex d i f f e r e n c e i n the q u a n t i t y of the e s t r o g e n  receptor present  i n the a d u l t .  Following  3 the i n c u b a t i o n of 2 nM s e p a r a t i o n r e v e a l e d 70%  [ H ] - e s t r a d i o l , sucrose d e n s i t y  of t o t a l r a d i o a c t i v i t y bound to the  r e g i o n i n the female, but male.  o n l y 15% bound t o the 8-9S  but  r e g i o n was  s a t u r a t e d at 4 nM  that concentrations  concentrations t o the 8-9S  r e g i o n was  Therefore,  [ H ] - e s t r a d i o l were  p r o t e i n i n the male.  g r e a t e r than 16 nM,  no  At l i g a n d  sex d i f f e r e n c e i n b i n d i n g  observed.  both Eagon et a l . , (1980) and 3  (1980) r e p o r t e d the sex d i f f e r e n c e i n t o t a l t o the 8-9S  sex  region. They found t h a t the 3 [ H ] - e s t r a d i o l i n the female, 3  i n excess of 16 nM  r e q u i r e d t o s a t u r a t e the 8-9S  8-9S  peak i n the  Powell-Jones et a l . , (1980) have a l s o demonstrated a  d i f f e r e n c e i n b i n d i n g t o the 8-9S 8-9S  gradient  Powell-Jones et a l . ,  [ H ] - e s t r a d i o l bound  r e g i o n at lower l i g a n d c o n c e n t r a t i o n s .  the ontogeny of the e s t r o g e n  r e c e p t o r was  However, when  i n v e s t i g a t e d , Powell-Jones  110  etal.,  (1981) found no sex difference i n s p e c i f i c binding of 4.0  nM  3 [ H]-estradiol between male and female rats using a 100 f o l d 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 t o t a l  3 binding of lower concentrations region was  of [ H]-estradiol to the  8-9S  probably a result of ligand binding to the moderate  a f f i n i t y component i n the male. The r e s u l t s 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. increase occurred between days 30 and 40.  The most rapid  After day 42 l e v e l s  were reported to plateau, with no consistent sex differences being evident.  However, examination of the r e s u l t s presented by  Powell-Jones et a l . , (1981)showed that the concentration of estrogen receptor varied at least two  f o l d a f t e r 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 i s not uncommon. The observation  of Powell-Jones et a l . , (1981) that there  i s no sex difference in the ontogeny of the estrogen receptor consistent with our findings.  Scatchard  was  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). although the capacity of the adult male and female  However,  was s i m i l a r ,  i t was noted that the capacity of the male was usually s l i g h t l y 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 m a n i p u l a t i o n  on h e p a t i c  estrogen  binding  a)  Gonadectomy  e f f e c t s on h e p a t i c estrogen  P h y s i o l o g i c a l manipulation the relevance  binding  o f t h i s system has f u r t h e r confirmed  o f t h e moderate a f f i n i t y  component.  We have shown  t h a t gonadectomy of the male reduces the c a p a c i t y of the moderate affinity  component w i t h a s l i g h t concomitant i n c r e a s e i n the c a p a c i t y  of t h e e s t r o g e n  receptor  (Tables 8, 9,24).  i n c a p a c i t y of t h e e s t r o g e n  r e c e p t o r was not always seen, and may  be w i t h i n normal v a r i a b i l i t y . et  However, the i n c r e a s e  I t i s of i n t e r e s t t h a t Powell-Jones 3  a l . , (1980,1981) observed an i n c r e a s e i n [ H ] - e s t r a d i o l b i n d i n g  t o t h e 8-9S r e g i o n f o l l o w i n g gonadectomy which was c o n s i s t e n t our r e s u l t s .  These data a r e i n agreement w i t h  sucrose  with  density  g r a d i e n t a n a l y s i s of male c y t o s o l by Eagon et a l . , (1980), who demonstrated a t e s t o s t e r o n e r e v e r s i b l e i n c r e a s e i n D E S - s e n s i t i v e 3  [ H ] - e s t r a d i o l binding  to t h e 8-9S r e g i o n f o l l o w i n g gonadectomy.  As w i t h the i n c r e a s e i n the c a p a c i t y of the e s t r o g e n did  n o t c o n s i s t e n t l y observe a t e s t o s t e r o n e  dectomy induced  receptor,  we  r e v e r s a l of a gona-  increase i n capacity.  Powell-Jones et a l . , (1980,1981) d i d not d e t e c t a decrease 3  in  [ H ] - e s t r a d i o l binding  of t h e male.  to the 4S r e g i o n f o l l o w i n g gonadectomy  T h i s i s i n marked c o n t r a s t t o t h e c o n s i s t e n t  t e s t o s t e r o n e r e v e r s i b l e decrease we have noted f o r the moderate affinity  component  i n the gonadectomized male (Tables 8, 9, 24).  I n t e r e s t i n g l y , Eagon e t a l . , (1980) have shown t h a t w h i l e dectomy does not a f f e c t  3  gona-  [ H ] - e s t r a d i o l b i n d i n g t o the 4S r e g i o n  112  per  se, i t does change t h e l i g a n d s p e c i f i c i t y of t h e 4S p r o t e i n .  R e c a l l i n g t h a t t h e c o n t r o l male 4S p r o t e i n 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  t o t h e 4S p r o t e i n  of unlabeled. competitive (estrogen  i n the p r e s e n c e o f a 100 f o l d  DES f o l l o w i n g gonadectomy. f o r [ H ] - e s t r a d i o l binding  receptor)  excess  Normally, DES i s o n l y  t o t h e 8-9S  protein  i n males and females and the 4S p r o t e i n i n  the female (Aten et a l . , 1978; Dickson e t a l . , 1978; Eagon e t a l . , 1980; E i s e n f e l d et a l . , 1977b; Powell-Jones e t a l . , 1980, 1981). However, Eagon et a l binding  to.the  (;1980) c l e a r l y •demonstrated .a 'DESv-sensitive . v  4S p r o t e i n i n t h e gonadectomized.male.  In a d d i t i o n ,  f o l l o w i n g g e l f i l t r a t i o n , Eagon and co-workers (1980) have shown the D E S - i n s e n s i t i v e , reduced 16 f o l d .  male s p e c i f i c 4S b i n d i n g  p r o t e i n t o have been  Furthermore, t h e e f f e c t s o f gonadectomy on DES-  s e n s i t i v e and - i n s e n s i t i v e b i n d i n g  i n the 4S r e g i o n of male  hepatic  c y t o s o l were shown t o be DHT r e v e r s i b l e (Eagon et a l . , 1980). observations  support our f i n d i n g s t h a t gonadectomy of t h e male  r e s u l t s i n decreased c a p a c i t y which i s r e v e r s e d  by  of t h e moderate a f f i n i t y  report  component,  testosterone.  The p r e c e e d i n g o b s e r v a t i o n s the recent  These  a r e o f i n t e r e s t i n l i g h t of  by Sloop and co-workers (1983) who were not 3  a b l e t o demonstrate DES c o m p e t i t i o n or n e o n a t a l l y results  (Table  f o r [ H ] - e s t r a d i o l i n female  gonadectomized male whole c y t o s o l f r a c t i o n s .  Our  1) and those o f Aten e t a l . , (1978), Eagon e t al..,"  (1980) and E i s e n f e l d e t 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 b i n d i n g fractions.  i n female whole c y t o s o l  However, t h e study by Sloop et a l . , (1983) employed  113  a c o n c e n t r a t i o n of 30 nM  [ H ] - e s t r a d i o l whereas the p r e v i o u s l y 3  cited  i n v e s t i g a t o r s used between 2 and 5 nM  the h i g h e r  [ H]-estradiol.  At  concentration of r a d i o - l a b e l e d _ t r a c e r 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 e s t r o g e n  r e c e p t o r would be  masked because a l l s i t e s would be s a t u r a t e d . —6 have shown t h i s t o be the case.  At 1 x 10  Dickson 3  et a l . , (1978)  M [ H]-estradiol in  female h e p a t i c c y t o s o l , no r e d u c t i o n i n b i n d i n g was observed u s i n g a 1000 f o l d excess of unlabeled  e s t r a d i o l as a c o m p e t i t o r .  male c y t o s o l an 80% decrease i n b i n d i n g was r e p o r t e d under conditions.  T h i s i s i n p a r t i a l agreement w i t h data  in Table  S i m i l a r t o Dickson  1.  t o generate a S c a t c h a r d unlabeled  plot  In these  presented  e t a l . , (1978) we were unable  i n female whole c y t o s o l u s i n g  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]-estradiol.  Furthermore, a Scatchard  was generated under s i m i l a r c o n d i t i o n s i n the male. u n l i k e Sloop  plot  However,  et a l . , (1983), we observed d i s p l a c e a b l e b i n d i n g  and were a b l e t o produce a Scatchard  p l o t u s i n g a 100 f o l d  excess  3  of DES i n female whole c y t o s o l between 25 and 1000 nM Therefore  [ H]-estradiol.  t h e decrease i n moderate a f f i n i t y b i n d i n g  we  observed i n t h e male f o l l o w i n g gonadectomy, n o t r e p o r t e d by o t h e r 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 c o n c e n t r a t i o n o f [ H ] - e s t r a d i o l used i n t h e i r sucrose  gradient a n a l y s i s studies.  The h i g h e r  concentration  3  of  [ H ] - e s t r a d i o l may have s a t u r a t e d t h e D E S - s e n s i t i v e  masked the decrease i n the D E S - i n s e n s i t i v e s i t e .  s i t e and  T h i s would r e s u l t  i n no n o t i c e a b l e change i n t o t a l b i n d i n g t o the 4S peak as t h e D E S - s e n s i t i v e p r o t e i n would b i n d e s t r a d i o l as w e l l as t h e male  114  s p e c i f i c 4S  DES-insensitive protein.  T h i s must be the  case  3  r e g a r d l e s s of the c o n c e n t r a t i o n of  [ H ] - e s t r a d i o l used as n e i t h e r  Eagon et a l . , (1980) nor Powell-Jones et a l . , (1980,1981) r e p o r t e d 3  gross changes i n t o t a l b i n d i n g of  [ H ] - e s t r a d i o l to the 4S  of sucrose g r a d i e n t s f o l l o w i n g gonadectomy.  The  peak  gonadectomized  male resembles the female w i t h r e s p e c t t o b i n d i n g parameters more so than the i n t a c t male. s e n s i t i v e 4S peak was  Therefore,  s a t u r a t e d and  i t i s p o s s i b l e t h a t the  DES-  c o u l d not be demonstrated a t  3  30 nM  [ H ] - e s t r a d i o l j u s t as the e s t r o g e n  r e c e p t o r c o u l d not  be  demonstrated a t e l e v a t e d t r a c e r l e v e l s d e s c r i b e d above.  This  p o s s i b i l i t y becomes probable  apparent  Kd and be  when one  c o n s i d e r s t h a t the  c a p a c i t y of t h e D E S - s e n s i t i v e 4S p r o t e i n were r e p o r t e d  0.36  nM  1980).  and  0.16  Therefore  fmol/mg p r o t e i n r e s p e c t i v e l y (Eagon et a l . ,  the d i s c r e p a n c y  between our o b s e r v a t i o n  gonadectomy reduces the c a p a c i t y of the moderate a f f i n i t y and  to  that component  those o f Powell-Jones et a l . , (1980,1981) a r e p o s s i b l y the  r e s u l t of measuring o n l y t o t a l b i n d i n g i n sucrose analysis.  However, t h i s too was  density gradient  c o n t r a r y t o our r e s u l t s as  c o n s i s t e n t l y observed a decrease i n t o t a l as w e l l as  we  specific  3  b i n d i n g of  [ H ] - e s t r a d i o l to the moderate a f f i n i t y  component  f o l l o w i n g gonadectomy of the a d u l t male. i) Imprinting, of the ^moderate a f f i n i t y ' component 3  Powell-Jones and co-workers (1981) have r e p o r t e d t h a t e s t r a d i o l b i n d i n g t o the 4S p r o t e i n c o u l d be a b o l i s h e d o n l y neonatal  gonadectomy of the male.  by Sloop  et a l . , (1983).  T h i s o b s e r v a t i o n was  They f u r t h e r demonstrated t h a t  [ H]by.  confirmed the  115  decrease i n [ H ] - e s t r a d i o l binding  t o the HCLA s i t e  ( t h e i r term f o r  moderate a f f i n i t y component) i n a d u l t male whole c y t o s o l n e o n a t a l gonadectomy c o u l d be r e v e r s e d  following administration  a l a r g e dose of t e s t o s t e r o n e ( 5 7 . 5 mg/kg) on days 2, life.  In a d d i t i o n , the l i g a n d s p e c i f i c i t y of the  r e s t o r e d HCLA s i t e was  following  6,  9 and  of  13  of  testosterone  i d e n t i c a l to the i n t a c t a d u l t male.  As  a  r e s u l t of these s t u d i e s Sloop et a l . , (1983) have suggested t h a t the m o d e r a t e ' a f f i n i t y  component or HCLA s i t e i s n e o n a t a l l y  imprinted  by t e s t i c u l a r androgens i n the male r a t . Sloop and  co-workers (1983) have shown a f i v e f o l d  increase  3 in  total  [ H ] - e s t r a d i o l binding  in adult  gonadectomized female  whole c y t o s o l f o l l o w i n g the a d m i n i s t r a t i o n mg/kg) on days 2,  6,  9 and  13 of l i f e .  when i n t a c t females were t r e a t e d et  of testosterone  (57.5  However, t h i s was  similarly.  a l . , (1978) have demonstrated a four f o l d  not  seen  C o n v e r s e l y , Dickson increase  in  specific  3 [ H ] - e s t r a d i o l b i n d i n g to i n t a c t female whole c y t o s o l f o l l o w i n g the a d m i n i s t r a t i o n of DHT (0.3 yg/g/day) f o r seven days. However, 3  t h i s increase  i n [ H ] - e s t r a d i o l binding  so i t does not more, a 30%  revealed  DHT  represent  was  shown to be  DES-sensitive  a moderate a f f i n i t y or HCLA s i t e .  Further-  ammonium s u l f a t e f r a c t i o n a t i o n of the whole c y t o s o l 3 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 w i t h those of Sloop et a l . , (1983) , show not  o n l y the m i s i n t e r p r e t a t i o n  t h a t do not  t h a t can a r i s e from  investigate s p e c i f i c binding  the d i s c r e p a n c y between l i g a n d b i n d i n g study d i d not  and  studies  ligand s p e c i f i c i t y ,  i n crude v e r s e s  but  partially  purified cytosol.  Our  demonstrate changes of  the  magnitude r e p o r t e d  by Dickson et a l . , (1978) or Sloop et a l . , (1983)  116  f o r t o t a l or s p e c i f i c b i n d i n g of  [ H ] - e s t r a d i o l t o the ammonium  s u l f a t e or whole c y t o s o l f r a c t i o n s f o l l o w i n g a d m i n i s t r a t i o n  of  t e s t o s t e r o n e t o sham-operated or• gonadectomized females> (Tables-9_, 23, The  2  d i f f e r e n c e s i n t h e methodology employed by v a r i o u s i n v e s t i g a t o r s  make comparisons between s t u d i e s d i f f i c u l t .  One  may  conclude,  however, t h a t i m p r i n t i n g of the moderate a f f i n i t y component i s h i g h l y suspect.  This i s e s p e c i a l l y true i n regard  to  the  d i f f e r e n c e s i n l i g a n d s p e c i f i c i t y of the 4S p r o t e i n t h a t following p h y s i o l o g i c a l manipulation (1980).  Before  competition  as shown by Eagon et a l . ,  the HCLA s i t e i s c o n s i d e r e d  s t u d i e s should  occurred  to be  be conducted a t lower  imprinted, concentrations  3  of  [ H ] - e s t r a d i o l to determine i f t h i s b i n d i n g  same b i n d i n g  ii)  s i t e r e t a i n s the  characteristics.  Sucrose d e n s i t y g r a d i e n t v e r s u s  competitive  binding  A p o t e n t i a l confounding f a c t o r e x i s t s w i t h r e g a r d comparison of r e s u l t s from sucrose competitive  binding studies.  the  d e n s i t y g r a d i e n t a n a l y s i s and  T h i s i n v o l v e s the p r a c t i c e of l a b e l i n g  the c y t o s o l p r i o r t o g r a d i e n t gradient  to  studies  separation.  A l l the sucrose  s t u d i e s d e s c r i b e d were conducted on p r e - l a b e l e d  While t h i s t e c h n i q u e i s a c c e p t a b l e  density  gradients.  f o r measurement of the  estrogen  receptor,  i t poses a problem i n c o n s i d e r a t i o n of moderate a f f i n i t y  binding.  T h i s has been demonstrated f o r a s i m i l a r lower  higher  c a p a c i t y 4S  from r a t u t e r u s  estrogen  b i n d i n g p r o t e i n (type I I s i t e )  ( C l a r k et a l . , 1978)  (MXT-3590) (Watson and  affinity  C l a r k , 1980).  isolated  and mouse mammary tumor These workers have demonstrated  1 1 7  that  s i g n i f i c a n t u n d e r - e s t i m a t i o n of the type I I s i t e o c c u r s i f 3  gradients  are pre-labeled with  [ H]-estradiol.  The u n d e r - e s t i m a t i o n  i s a r e s u l t of t h e d i s s o c i a t i o n t h a t o c c u r s d u r i n g u l t r a c e n t r i f u g a t i o n . The a u t h o r s p o i n t out t h a t as the c e n t r i f u g a t i o n i s u s u a l l y 16-18 hours, the h a l f - l i f e o f the l i g a n d r e c e p t o r a lower a f f i n i t y b i n d i n g  complex, e s p e c i a l l y  s i t e , would be accommodated.  As such,  the a u t h o r s suggest p o s t - l a b e l i n g g r a d i e n t s and have demonstrated an i n c r e a s e 8-9S  i n estrogen receptor  assay s e n s i t i v i t y f o r b o t h t h e  and i n p a r t i c u l a r t h e 4-5S r e g i o n .  Thus the q u e s t i o n  persists  as t o what t h e l i g a n d s p e c i f i c i t y of t h e p r o t e i n sedimenting i n the 4S  r e g i o n might-be. •'In addition., .one may. s p e c u l a t e  that the l a c k of  d i f f e r e n c e i n 4 S . b i n d i n g f o l l o w i n g gonadectomy .reported - by Powell-Jone& et a l . ,- (1980) , was a f u n c t i o n of d i s s o c i a t i o n p r i o r t o measurement.  b)  E f f e c t s of hypophysectomy on h e p a t i c  estrogen  The androgen requirement f o r m a i n t a i n i n g moderate a f f i n i t y component Following  binding  t h e c a p a c i t y of the  i n males was c o n f i r m e d ( T a b l e s  t h i s , the degree of p i t u i t a r y  involvement was  9, 24, 29).  investigated.  We have found t h a t hypophysectomy reduced t h e c a p a c i t y of t h e . e s t r o g e n receptor (Tables  i n male and female r a t s i n a time-dependent f a s h i o n 10, 12, 13, 25).  a f f i n i t y component (Tables  Furthermore, t h e c a p a c i t y o f the moderate  i n the male was a l s o decreased post-hypophysectomy  11, 12, 14, 25; F i g u r e 3 ) .  s i t e , which was n o r m a l l y not present f o r the f i r s t hypophysectomy  time by Scatchard (Table 11).  However, the moderate a f f i n i t y i n t h e female, was  detected  a n a l y s i s two weeks f o l l o w i n g  At t h i s time the c a p a c i t y of the  female moderate a f f i n i t y s i t e was o n e - t e n t h t h a t o f the hypo-  118  physectomized male. of t h i s s i t e was  At f o u r weeks post-hypophysectomy  the c a p a c i t  a p p r o x i m a t e l y one h a l f , and by s i x weeks e q u a l t o ,  t h a t o f the hypophysectomized  male ( T a b l e s 11, 12, 14, 25; F i g u r e  These r e s u l t s agree w i t h those r e p o r t e d by Powell-Jones et a l . , (1980) . T h i s r e p o r t was  the f i r s t  demonstration of hypophyseal-  hypothalamic i n f l u e n c e on the b i n d i n g of e s t r a d i o l t o the h e p a t i c c y t o s o l i c e s t r o g e n r e c e p t o r and the moderate a f f i n i t y  site.  p r e - l a b e l e d s u c r o s e g r a d i e n t s , Powell-Jones and co-workers  Using  (1980)  3 have shown t h a t f o l l o w i n g hypophysectomy to the 8-9S  r e g i o n was  abolished  i n both males and  Furthermore, b i n d i n g t o the 4S r e g i o n was and  [ H]-estradiol binding females.  reduced i n the male  i n c r e a s e d i n the female t o a p p r o x i m a t e l y one h a l f the hypo-  physectomized male l e v e l a t t h r e e weeks p o s t - s u r g e r y . Powell-Jones et a l . , ,4S  However,  (1980) r e p o r t e d t h a t a t f i v e weeks the  b i n d i n g p r o f i l e s of the hypophysectomized  male and  were the same as t h a t observed at t h r e e weeks. a f f i n i t y component i n the hypophysectomized  female  The moderate  male and female  equivalent on t h e b a s i s of t h e i r b i n d i n g c h a r a c t e r i s t i c s . the b i n d i n g c h a r a c t e r i s t i c s of t h i s p r o t e i n may  (1980) demonstrated  p r o t e i n f o l l o w i n g gonadectomy.  T h e r e f o r e , i t would be o f  protein  However,  change f o l l o w i n g  hypophysectomy as Eagon et a l . ,  t o a d d r e s s the l i g a n d  appear  f o r the 4S interest  s p e c i f i c i t y o f the post-hypophysectomy  4S  (moderate a f f i n i t y b i n d i n g component) at 2-5 nM and 30  nM  3  [ H]-estradiol.  T h i s i s of importance as the apparent Kd of b o t h  male and female moderate a f f i n i t y p r o t e i n appears t o decrease by s i x t o e i g h t weeks a f t e r  hypophysectomy.  119  3)  Comparison binding  a)  of h e p a t i c enzyme a c t i v i t y t o h e p a t i c e s t r o g e n  components  G e n e r a l c o n c l u s i o n s from p u b l i s h e d o b s e r v a t i o n s  We  have observed a c o r r e l a t i o n w i t h p h y s i o l o g i c a l  m a n i p u l a t i o n between- AHH' a c t i v i t y , and the b i n d i n g of e s t r o g e n s t o c y t o s o l i c p r o t e i n s i n the l i v e r .  A l t h o u g h changes  i n the c a p a c i t y o f the e s t r o g e n r e c e p t o r were observed, the b i n d i n g of the moderate a f f i n i t y component was a p o i n t o f m o d u l a t i o n o f AHH To r e i t e r a t e ,  particularly interesting  activity.  .a s e x u a l dimorphism  e x i s t s with respect to  s e v e r a l h e p a t i c mixed f u n c t i o n o x i d a s e a c t i v i t i e s (Kato and G i l l e t t e , et a l . ,  (1974a,  1965", Quinn et a l . ,  1974b) have demonstrated  P-450 dependent N-demethylase a c t i v i t y r e l a t i v e to females.  i n the r a t ,  1958). E l Defrawy E l Masry that r a t hepatic  i n c r e a s e d w i t h age  cytochrome i n males  Furthermore, the d i f f e r e n c e s i n t h e s e  a c t i v i t i e s between a d u l t male and female r a t s were reduced gonadectomy o r e s t r o g e n treatment o f the male. not a f f e c t t h e s e a c t i v i t i e s  i n the female.  of gonadectomy of the male was T h i s demonstrated  as  by  Gonadectomy d i d  Furthermore, t h e e f f e c t  shown t o be t e s t o s t e r o n e r e v e r s i b l e .  the involvement of androgens  i n the e x p r e s s i o n  of sex d i f f e r e n c e s f o r c e r t a i n h e p a t i c mixed f u n c t i o n o x i d a s e activities. Rat h e p a t i c AHH age and  activity  i s known t o respond  sex dependency has been d e s c r i b e d  similarly.  (Oesch et a l . ,  An  1976)  as have requirements f o r t e s t i c u l a r androgens  (Gontovnick et a l . ,  1979;  1975).  Gurtoo and P a r k e r , 1977;  Kramer et a l . ,  Moreover,  120  the t e s t o s t e r o n e r e v e r s i b i l i t y o f t h e decrease f o l l o w i n g gonadectomy and the non-responsiveness  i n AHH  o f the a d u l t  female t o gonadectomy have been p r e v i o u s l y r e p o r t e d 1979;. Gurtoo and P a r k e r ,  1977).  activity  We have confirmed  (Kramer et a l . , these  observations  3  and have noted  t h a t t h e b i n d i n g o f [ H ] - e s t r a d i o l t o 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 f o l l o w i n g p h y s i o l o g i c a l manipulation  b)  (Table 3 3 ) .  Age and sex dependency  We have shown t h a t t h e l e v e l s of AHH a c t i v i t y were g r e a t e r i n the a d u l t c o n t r o l male than female ( T a b l e s 15-19, 25, 28). The c a p a c i t y of the e s t r o g e n r e c e p t o r was not d i f f e r e n t the a d u l t male and female ( T a b l e s 7-10, 12, 25-28). moderate a f f i n i t y component c o u l d o n l y be d e t e c t e d c o n t r o l male.  I t was not found  between  However, the  i n the adult  i n t h e c o n t r o l female ( T a b l e s 7-9,  23, 25-28). The AHH a c t i v i t y o f t h e immature male o r female was shown to be s i m i l a r t o t h a t o f t h e a d u l t female (Bellward e t a l . , 1982). The c a p a c i t y o f t h e e s t r o g e n r e c e p t o r , w h i l e reduced animal,  d i d not e x h i b i t any sex d i f f e r e n c e .  component  c o u l d be d e t e c t e d  i n t h e immature  No_ moderate a f f i n i t y  i n t h e immature r a t (Table 8 ) .  T h e r e f o r e t h e i n c r e a s e i n AHH a c t i v i t y w i t h age i n t h e male i s c o n s i s t e n t w i t h the d e t e c t i o n of the moderate a f f i n i t y component  i n the male and i n c o n s i s t e n t w i t h t h e i n c r e a s e i n  e s t r o g e n r e c e p t o r i n both males- arid  females,  1 2 1  TABLE 33 Summary o f r e l a t i v e changes i n c a p a c i t y of t h e e s t r o g e n r e c e p t o r , moderate a f f i n i t y b i n d i n g component and AHH a c t i v i t y r e l a t i v e t o the a d u l t male ( u n l e s s otherwise n o t e d ) . Binding to Moderate Affinity Component  B i n d i n g to Estrogen Receptor  Model  H e p a t i c AHH Activity  4-  Female  +  0  Pseudo  f  0  Immature male  4-  0  Immature female  4-  0  4-  + 4~>  4-  I  4->  +'  t*  ->  0  4-  Gx male Gx male +  Testosterone  Gx female  ->-  0  0  0  Male + pimozide  +  ->  Gx male + pimozide  4  4-  Female + p e r g o i i d e  -y  0  Gx female + p e r g o i i d e  4-  0  Hx male  0  4-  Hx male + rGH  0  0  Hx  0  t  Hx Female + rGH  0  0  Male +  3-MC  4-  Male +  Phenobarbital  4-  +  Gx female + Male +  Testosterone  Mestranol  Female  Female +  3-MC  4-  0  Female +  Phenobarbital  4-  0  *• R e l a t i v e ** R e l a t i v e t Relative tt Relative  to to to to  4-  tt  Gx male Gx female sham-operated female Hx female  Changes i n s p e c i f i c b i n d i n g were i n d i c a t e d as f o l l o w s : 4- = decrease; -»- = no change; 0 = none d e t e c t e d .  t  = increase;  122  c)  Androgen dependency  We  have shown t h a t androgens p l a y a r o l e i n m a i n t a i n i n g  l e v e l s of AHH  activity  reduces h e p a t i c AHH  i n the male.  a c t i v i t y and  Gonadectomy of the a d u l t male  the c a p a c i t y of the moderate  a f f i n i t y component i n a t e s t o s t e r o n e 9,  24,  29).  r e v e r s i b l e manner ( T a b l e s  appeared to be r e v e r s e d  i n the male which a t times  by t e s t o s t e r o n e .  However, these  changes i n c a p a c i t y of the e s t r o g e n r e c e p t o r physiologically relevant.  Furthermore, we  dectomy does not  activity  nor were any  d)  8,  Gonadectomy d i d produce s l i g h t f l u c t u a t i o n s i n the  c a p a c i t y of the e s t r o g e n r e c e p t o r  (Tables  the  a l t e r AHH  small  were p r o b a b l y not  have shown t h a t gona-  i n the female ( T a b l e s 8,  9,  22),  changes seen i n e s t r o g e n b i n d i n g c h a r a c t e r i s t i c s ,  20-22).  E f f e c t of  I t has  estrogens  been demonstrated t h a t the a d m i n i s t r a t i o n of e s t r o g e n s  to males w i l l produce e f f e c t s s i m i l a r t o gonadectomy w i t h t o sex dependent mixed f u n c t i o n oxidase a c t i v i t y E l Masry and  Mannering, 1974;  AHH  a c t i v i t y was  was  not  ( E l Defrawy  Sladek et a l . , 1974).  demonstrated t h a t f o l l o w i n g the a d m i n i s t r a t i o n  respect  We  have  of m e s t r a n o l , male  reduced t o female l e v e l s , whereas female AHH  a f f e c t e d (Table 28).  The  administration  activity  of m e s t r a n o l  3  abolished  [ H ] - e s t r a d i o l b i n d i n g to the moderate a f f i n i t y component  i n the male whole c y t o s o l f r a c t i o n .  Mestranol a l s o  abolished  3  [ H ] - e s t r a d i o l b i n d i n g t o the h e p a t i c and  female r a t s .  T h i s was  not  estrogen receptor  c o n s i s t e n t w i t h the  i n male  increase  in  123  s p e c i f i c b i n d i n g t o the 8-9S  r e g i o n r e p o r t e d by Eagon et a l . , (1980)  f o l l o w i n g a d m i n i s t r a t i o n of DES However, our o b s e r v a t i o n s  or e s t r a d i o l to i n t a c t a d u l t male r a t s .  c o u l d be the r e s u l t  of t h e f o l l o w i n g :  (1) n u c l e a r 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 e s t r o g e n (2) down r e g u l a t i o n o f the c y t o s o l i c r e c e p t o r , a l l unoccupied c y t o s o l i c e s t r o g e n or  receptor,  (3) o c c u p a t i o n  of  r e c e p t o r b i n d i n g s i t e s by m e s t r a n o l  (4) a combination of these p o s s i b i l i t i e s .  Whereas most treatments  were d i s c o n t i n u e d 24 hours p r i o r t o s a c r i f i c e , m e s t r a n o l  was  d i s c o n t i n u e d 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 t o circumvent these  effects. T h i s o b s e r v a t i o n p o i n t s out an  e q u i l i b r i u m r e c e p t o r assay system. f o r the determination  i n h e r e n t problem w i t h the  The methodology o n l y  of unoccupied r e c e p t o r  sites.  To  non-  allows fully  understand the mechanism(s) i n v o l v e d i n r e c e p t o r mediated pathways t o t a l b i n d i n g s i t e s should a l s o be determined -- which would the determination  e)  of  permit  occupied.receptors.  Pseudohermaphroditic r a t model  Assays were c a r r i e d out  i n pseudohermaphroditic r a t s as a  f u r t h e r c h a r a c t e r i z a t i o n of androgen involvement i n t h e modulation of sex dependent h e p a t i c mixed f u n c t i o n o x i d a s e s estrogen  as w e l l as  hepatic  binding.  The  pseudohermaphroditic r a t i s g e n o t y p i c a l l y a male but  d e s p i t e h i g h serum t e s t o s t e r o n e l e v e l s i s p h e n o t y p i c a l l y a pseudohermaphrodite, ( S t a n l e y et a l . , 1973). to be due  T h i s i s thought  t o a d e f i c i e n c y i n the androgen r e c e p t o r pathway  124  ( B a r d i n et a l . , 1973).  The  pseudohermaphrodite i s an  interesting  model s i n c e h e p a t i c mixed f u n c t i o n oxidase a c t i v i t i e s have been shown t o resemble the female i n both c o n t r o l and (Sonawane et a l . , 197 9).  Our  induced  states  r e s u l t s confirm t h i s r e p o r t .  F o l l o w i n g the a d m i n i s t r a t i o n of v a r i o u s i n d u c e r s of drug metabolism ( p h e n o b a r b i t a l , 3-MC  and  s p i r o n o l a c t o n e ) the AHH  activity  of  t h e pseudohermaphroditic r a t p a r a l l e l e d t h a t of t h e female, not of the male, i n a l l cases  ( T a b l e s 15,  A l l t h r e e drugs i n c r e a s e d AHH  16).  a c t i v i t y i n the female  pseudohermaphrodite t o comparable l e v e l s .  In the male, as  expected,  The  i n AHH  o n l y 3-MC  induced AHH  and  activity.  slight  and  decrease  a c t i v i t y produced by p h e n o b a r b i t a l arid -^h:±bit;ion,' of'-AHH.  a c t i v i t y by s p i r o n o l a c t o n e i n the' -male 'eon.fi^med-'Cpreyious ••<repo-r'fcs.':;  (Gontovnick  e t a l . ,"1981-;.. S t r i p p et - a l ; , 1973); ... "  •  I n t e r e s t i n g l y , as w i t h the female, no moderate a f f i n i t y b i n d i n g was  detected  i n the pseudohermaphrodite ( T a b l e 7 ) .  more, w h i l e the c a p a c i t y of the e s t r o g e n r e c e p t o r was  Further-  reduced  in  the l i t t e r m a t e c o n t r o l male, t h e r e were e s s e n t i a l l y no d i f f e r e n c e s between the female and  pseudohermaphrodite.  Thus, the pseudo-  hermaphrodite not o n l y resembled the female w i t h r e s p e c t t o e s t r o g e n b i n d i n g but  i n response to i n d u c e r s of drug metabolism  as w e l l .  f)  Hypophysectomy model  The  e f f e c t s of hypophysectomy on h e p a t i c mixed f u n c t i o n  125  oxidase  a c t i v i t y have been w e l l documented  (Colby et a l . , 1973;  Kramer et a l . , 1975, 1979; Rumbaugh and Colby, androgens and estrogens h e p a t i c drug steroid  Furthermore,  have been found t o produce no e f f e c t  and Stenberg, 1974, 1976; Lax et a l . , 1974)  activities.  T h i s was  i n agreement w i t h our r e s u l t s .  Hypophysectomy reduced t h e 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 the a d u l t male and female r a t . activity  on  (Colby et a l . , 1973; Kramer et a l . , 1975, 1979) or  (Gustafsson  metabolizing  1980).  between  We observed a decrease i n AHH  i n t h e male and a s m a l l but c o n s i s t e n t l y s i g n i f i c a n t  i n c r e a s e i n t h e female (Tables 17-19, 25). The e f f e c t o f hypophysectomy on e s t r o g e n b i n d i n g i n h e p a t i c c y t o s o l was of i n t e r e s t .  As i l l u s t r a t e d  of hypophysectomy on t h e e s t r o g e n  i n Table 25, the e f f e c t  r e c e p t o r d i d not p a r a l l e l changes  i n AHH a c t i v i t y of male and female r a t s . 3 The b i n d i n g o f [ H ] - e s t r a d i o l t o the e s t r o g e n  receptor  was  a b o l i s h e d i n b o t h males and females f o l l o w i n g hypophysectomy (Tables 13, 25). a f f i n i t y component  We a l s o observed the c a p a c i t y of the moderate i n t h e male t o be reduced by t h i s procedure  (Tables 11, 12, 14, 25).  Most s t r i k i n g was t h e appearance o f the  moderate a f f i n i t y component ( T a b l e s 11, 12, 14, 25).  i n t h e hypophysectomized female  T h i s was c o n s i s t e n t w i t h t h e i n c r e a s e  i n AHH a c t i v i t y and i n c o n s i s t e n t w i t h the decrease i n e s t r o g e n receptor.  Furthermore, u n l i k e the gonadectomy model, a d m i n i s t r a t i o n  of t e s t o s t e r o n e d i d not have any e f f e c t on AHH 3 In a d d i t i o n , t e s t o s t e r o n e d i d not a f f e c t of e i t h e r the e s t r o g e n  activity  (Table 18).  [ H]-estradiol binding  r e c e p t o r or t h e moderate a f f i n i t y  component  i n t h e hypophysectomized r a t s of e i t h e r sex, (Tables 13, 14).  126  These r e s u l t s 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 r e s u l t s of the hypophysectomy study were consistent with the hypothesis that the p i t u i t a r y 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 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  We had i n i t i a l l y hypothesized  that PRL may  be the p i t u i t a r y  hormone responsible for the observed levels of hepatic enzyme a c t i v i t y i n the female for the following reasons: PRL regulates i t s own 1975)  (Bohnet et a l . , 1976)  as well as estrogen  (Chamness et a l . ,  receptor levels i n the l i v e r ; castration r e s u l t s i n a  testosterone r e v e r s i b l e increase in hepatic PRL et a l . , 1976); and estrogens  induce  receptors(Aragona  hepatic PRL receptors and  hypophysectomy reduces hepatic PRL binding i n 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 i s the p i t u i t a r y 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 e a r l i e r i t was not clear whether these e f f e c t s were due to the somato- or l a c t o - t r o p i c 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 o f p i t u i t a r y hormone m a n i p u l a t i o n a d u l t male sham-operated and gonadectomized r a t s were t r e a t e d w i t h the dopamine a n t a g o n i s t pimozide.  The dose of pimozide used  r e p o r t e d l y produces an approximate 6 f o l d PRL l e v e l s  (Ojeda et a l . ,  1974).  In a d d i t i o n , f o l l o w i n g a s i n g l e  i n j e c t i o n of pimozide, PRL s y n t h e s i s was days and GH s y n t h e s i s s l i g h t l y G o r s k i , 1977).  i n c r e a s e i n plasma  induced f o r f o u r  i n h i b i t e d f o r two days (Maurer and  These workers f u r t h e r demonstrated t h a t a d m i n i s -  t r a t i o n o f e s t r a d i o l t o male and female r a t s i n c r e a s e d s y n t h e s i s w h i l e GH s y n t h e s i s was u n a f f e c t e d .  PRL  In a d d i t i o n ,  pimozide has been r e p o r t e d t o reduce d i u r n a l f l u c t u a t i o n s and s t r e s s - i n d u c e d r e l e a s e of GH  (Schwinn et a l . ,  1976).  We observed t h a t pimozide, w h i l e not a f f e c t i n g AHH d i d i n c r e a s e A4 r e d u c t a s e a c t i v i t y o f t h e sham-operated  activity,  male.  In a d d i t i o n , pimozide i n h i b i t e d the a c t i o n of t e s t o s t e r o n e on A4 r e d u c t a s e i n t h e gonadectomized male  (Table 22).  Subsequent  b i n d i n g s t u d i e s were conducted i n c o n j u n c t i o n w i t h AHH and s i m i l a r e f f e c t s were observed (Table 24). a l t e r AHH  activity  determinations,  Pimozide d i d not  i n any model but d i d a b o l i s h t h e d e t e c t i o n o f  the moderate a f f i n i t y component i n the gonadectomized male.  It  a l s o i n h i b i t e d the r e s t o r a t i o n of t h e moderate a f f i n i t y component by t e s t o s t e r o n e i n t h i s model". T h i s suggests that  i n t h e gonadectomized male the e l e v a t i o n  128  i n PRL,  and/or r e d u c t i o n of GH,  component. gonize 1982)  The  i s i n h i b i t o r y to the moderate  a c t i o n of t e s t o s t e r o n e i n t h i s case may  the e l e v a t i o n i n PRL  produced by pimozide (Giguere  d i s c u s s e d , the a c t i o n of pimozide was cally significant  changes i n AHH  as p r e v i o u s l y  not r e f l e c t e d by  activity.  I t i s of  however, t h a t pimozide d i d decrease the n u m e r i c a l  interest,  v a l u e of  the  females.  E f f e c t s of  The utilized  pergoiide  second approach to the i n v i v o m a n i p u l a t i o n  the dopamine a g o n i s t p e r g o i i d e to reduce PRL  r a t i o n a l e was  t h a t i f PRL  female may  of  sex  i n the  reduce the sex d i f f e r e n c e s i n b a s a l a c t i v i t y  enhance the a b i l i t y of t e s t o s t e r o n e to e l i c i t  PRL  levels.  were r e s p o n s i b l e f o r the  d i f f e r e n c e i n enzyme a c t i v i t y , t h e n r e d u c t i o n of PRL  we  statisti-  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 t h a t  found i n  The  et a l . ,  However, t h e reduced r e l e a s e o f GH must be c o n s i d e r e d . W h i l e  we d i d observe p a r a l l e l e f f e c t s w i t h A4 r e d u c t a s e  c)  anta-  thus i n h i b i t i n g the r e d u c t i o n of the moderate a f f i n i t y  component.  AHH  be to  affinity  or  a response.  However,  found the a c t i o n of p e r g o i i d e t o be c o n t r a r y t o our p r e d i c t i o n .  P e r g o i i d e i n h i b i t e d the e f f e c t o f t e s t o s t e r o n e on AHH a d u l t gonadectomized females (Table 20) gonadectomized p r e - p u b e s c e n t l y  i n adult  However, when h e p a t i c  a c t i v i t y were s t u d i e d c o n c u r r e n t l y  t e s t o s t e r o n e d i d not  females  (Table 21) r e c e i v i n g p e r g o i i d e  throughout t h e p u b e r t a l p e r i o d . b i n d i n g and AHH  and  activity in  i n c r e a s e AHH  activity.  (Table  Therefore,  of p e r g o i i d e t o i n h i b i t t e s t o s t e r o n e i n d u c t i o n was  estrogen  not  23)  the  effect  demonstrated.  1 2 9  Furthermore, no moderate a f f i n i t y b i n d i n g was t h e a d m i n i s t r a t i o n of t e s t o s t e r o n e . the c a p a c i t y of the e s t r o g e n  estrogen binding  (Norstedt  P e r g o l i d e d i d tend to reduce  receptor  which i s c o n s i s t e n t w i t h the d i r e c t  detected f o l l o w i n g  i n the gonadectomized male,  involvement of PRL  with  hepatic  et a l . , 1981b), U n l i k e pimozide, p e r g o l i d e  d i d not a f f e c t the. a c t i o n of t e s t o s t e r o n e on A4  reductase.  I f PRL were the hormone r e s p o n s i b l e f o r the observed  levels  of female enzyme a c t i v i t y , t h e n one would expect the a c t i o n s of t e s t o s t e r o n e to be p o t e n t i a t e d i n a h y p o p r o l a c t i n e m i c However, p e r g o l i d e i s a dopamine a g o n i s t . to be  s e l e c t i v e f o r PRL  expected. GH  Therefore  (and not PRL)  then we  female.  Although i t i s p u r p o r t e d  d e p l e t i o n , e l e v a t i o n s i n GH would  i f , as s e v e r a l p r e v i o u s l y c i t e d  i s r e s p o n s i b l e f o r female l e v e l s of  would expect p e r g o l i d e - i n d u c e d  the a c t i o n of t e s t o s t e r o n e .  As  studies  d)  to  this  was  experimentally.  P i t u i t a r y hormone replacement: E f f e c t s on h e p a t i c AHH and  inhibit  would not expect to d e t e c t  a moderate a f f i n i t y b i n d i n g component i n t h i s model and confirmed  indicate,  activity,  e l e v a t i o n s of GH  such, we  be  c y t o s o l i c estrogen  I t was  activities  binding  c l e a r t h a t the use  not produce c o n c l u s i v e r e s u l t s .  of p e r g o l i d e and Therefore,  of p i t u i t a r y hormones on enzyme and v a r i o u s hormones were a d m i n i s t e r e d  estrogen  pimozide d i d  to determine the  b i n d i n g parameters,  to hypophysectomized  While t h i s hormone replacement model was  role  animals.  more i n v a s i v e , and  produced changes i n l a r g e r numbers of hormones, i t d i d  provide  130  more c o n c l u s i v e r e s u l t s . The has  e f f e c t of bGH  on h e p a t i c mixed f u n c t i o n o x i d a s e  been w e l l c h a r a c t e r i z e d .  (1970,1973) have shown GH i n t a c t and  Kramer and  activities  Colby (1976), arid W i l s o n  t o reduce sex dependent a c t i v i t i e s  gonadectomized male r a t .  effect.  Rumbaugh and  activity  i n c a s t r a t e d males, yet  However, GH has  Colby (1980) r e p o r t e d increased  t h a t bGH AHH  a  "paradoxical"  reduced  activity  i n the  AHH  i n hypo-  physectomized males. To was  administered  (1980).  c h a r a c t e r i z e the e f f e c t s of t h i s hormone,  according  t o the p r o t o c o l of Rumbaugh and  T h i s regimen s p e c i f i e d i n j e c t i o n of bGH  achieving we  initially  4.0  I.U./kg/day.  Following  twice  t h i s treatment  bGH  Colby  daily  schedule  observed a pronounced decrease i n the c a p a c i t y of the moderate  a f f i n i t y component  (Table 14).  I f GH  i s the p i t u i t a r y f a c t o r  r e s p o n s i b l e f o r female l e v e l s of a c t i v i t y , and  we  hypothesize  t h a t 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 , f o l l o w i n g bGH not  then the decrease i n the moderate a f f i n i t y i s c o n s i s t e n t w i t h the h y p o t h e s i s .  c o n s i s t e n t w i t h the  Rumbaugh and  Colby  increase  i n AHH  component  However, t h i s i s  a c t i v i t y reported  by  (1980).  Subsequent s t u d i e s by Mode et a l . , (1981) c o n c l u s i v e l y demonstrated f e m i n i z a t i o n of h e p a t i c  s t e r o i d metabolism f o l l o w i n g  continuous i n f u s i o n of GH  i n adult  and  , Interestingly,  gonadectomized r a t s .  there  i s a sex d i f f e r e n c e i n the  i n the r a t .  i n t a c t , hypophysectomized,  s e c r e t o r y p a t t e r n of GH  .  Males e x h i b i t a p u l s a t i l e s e c r e t o r y p a t t e r n w i t h a  131  h i g h peak t o trough r a t i o a t r e g u l a r t h r e e t o f o u r hour  intervals.  Females have an i r r e g u l a r p u l s a t i l e r e l e a s e w i t h h i g h e r l e v e l s between peaks than males ( Eden, 1979;  T e r r y , et al., 1977)  T h e r e f o r e , i t i s l o g i c a l t o suggest t h a t d i f f e r e n c e s between t h e o b s e r v a t i o n s o f Rumbaugh and Colby (1980) and those of Mode et a l . , (1981) were due t o t h e regimen mimicking the i n v i v o r e l e a s e  pattern  of t h e male and female r e s p e c t i v e l y . Because o f t h i s , we c a r r i e d o u t subsequent  replacement  s t u d i e s a c c o r d i n g t o t h e p r o t o c o l e s t a b l i s h e d by Mode et a l . , (1981), u s i n g A l z e t ® osmotic minipumps.  The v a l i d i t y of t h i s  t e c h n i q u e has been confirmed by Norstedt  et a l . ,  (1981a,  1981b,  1983).  i)  Hormone replacement  i n male r a t s  We found t h a t bGH replacement by c o n t i n u o u s i n f u s i o n (0.01 I.U./hour) reduced AHH a c t i v i t y male r a t s . ( T a b l e 2 5 ) .  i n sham-operated  T h i s o b s e r v a t i o n was c o n s i s t e n t w i t h t h e  f e m i n i z a t i o n o f h e p a t i c s t e r o i d metabolism et a l . ,  and-hypophysectomized  (1981) and i n disagreement  by Rumbaugh and Colby (1980).  r e p o r t e d by Mode  with the r e s u l t s  described  As p r e v i o u s l y mentioned  this i s  p r o b a b l y due t o t h e method o f hormone d e l i v e r y : I n t e r e s t i n g l y , a l t h o u g h c o n t i n u o u s i n f u s i o n o f bGH reduced AHH a c t i v i t y hypophysectomized  i n the sham-operated  and  male, no obvious changes i n c a p a c i t y o f the  moderate a f f i n i t y component were d e t e c t e d as they had been f o l l o w i n g i n j e c t i o n o f hypophysectomized  males w i t h bGH.  t h e bGH a d m i n i s t e r e d possessed 62% l a c t o g e n i c a c t i v i t y .  However, Furthermore,  132  when oPRL was  a d m i n i s t e r e d t o the sham-operated  male u s i n g osmotic  minipumps, we observed an even g r e a t e r r e d u c t i o n of AHH without any change i n the c a p a c i t y of t h e moderate component.  activity  affinity  T h e r e f o r e , the l a c t o t r o p i c p r o p e r t i e s of t h e s e  hormones may  produce t h e r e d u c t i o n i n AHH  activity.  The i n t e r p l a y between s o m a t o t r o p i c and l a c t o t r o p h i c was more pronounced  i n the hypophysectomized  activity  r a t (Table 25).  Continuous i n f u s i o n o f GH o r PRL d i d not r e s t o r e b i n d i n g t o the e s t r o g e n r e c e p t o r i n t h e hypophysectomized  rat.  rGH,  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 o n l y reduced AHH  which activity  to c o n t r o l female l e v e l s , but a b o l i s h e d t h e d e t e c t i o n of the moderate  a f f i n i t y component on b o t h male and female r a t s .  T h e r e f o r e , 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 i n f u s i o n of "rPRL d i d not reduce AHH c a p a c i t y of the moderate  a f f i n i t y component.  a c t i v i t y or the Furthermore, the  rPRL as w e l l as oPRL used had n e g l i g i b l e s o m a t o t r o p i c a c t i v i t y . T h e r e f o r e , one must c o n c l u d e t h a t t h e l a c t o t r o p i c a c t i o n i s not m o d u l a t i n g AHH component. (1981) who  a c t i v i t y or the c a p a c i t y o f t h e moderate  T h i s i s i n agreement  affinity  w i t h the r e s u l t s of Mode et a l . ,  demonstrated that c o n t i n u o u s i n f u s i o n of rPRL d i d not  a f f e c t h e p a t i c s t e r o i d metabolism i n hypophysectomized male r a t s . The e f f e c t  of oPRL i n the sham-operated  animal might  be  due t o i n c r e a s e d PRL m o d u l a t i o n o f o t h e r p i t u i t a r y f a c t o r s , or an i n h e r e n t d i f f e r e n c e between t h e two l a c t o t r o p h s . the e f f e c t s o f oPRL i n t h e hypophysectomized  To examine  a n i m a l , the c o n c u r r e n t  a d m i n i s t r a t i o n o f t e s t o s t e r o n e must be taken i n t o a c c o u n t .  133  Testosterone  was a d m i n i s t e r e d  c o n c u r r e n t l y w i t h bGH and  oPRL t o determine i f i n t h e presence of these p i t u i t a r y AHH a c t i v i t y c o u l d be i n c r e a s e d .  However, t h e presence of t e s t o -  s t e r o n e d i d not a l t e r t h e e f f e c t o f bGH on AHH a c t i v i t y hypophysectomized male. by i t s e l f estrogen  hormones  i n the  T h i s might be expected s i n c e t e s t o s t e r o n e  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 h e p a t i c  b i n d i n g i n the hypophysectomized animal.  T h i s does not  mean that GH and PRL a r e not the p i t u i t a r y hormones i n v o l v e d i n t h e a c t i o n of t e s t o s t e r o n e . l e v e l s administered,  Rather, i t i n d i c a t e d t h a t at the  bGH and oPRL d i d not modify t h e b i n d i n g  components s u f f i c i e n t l y t o permit  a t e s t o s t e r o n e response.  It i s  obvious t h a t t h e l e v e l s o f GH and PRL a r e low as demonstrated by the i n a b i l i t y to d e t e c t any unoccupied estrogen  receptor  sites  i n the hypophysectomized r a t r e c e i v i n g hormone replacement. i n a b i l i t y to detect estrogen receptor the non-equilibrium  The  s i t e s may be the r e s u l t of  assay, o r the requirement o f an a d r e n a l f a c t o r  as suggested by L u c i e r et a l . , (1981), and Norstedt  et a l . , (1981b).  However, these hormones d i d not a l t e r t h e c a p a c i t y of the e s t r o g e n r e c e p t o r i n t h e sham-operated male e i t h e r ( T a b l e 25).  ii)  The  Hormone replacement i n female r a t s  e f f e c t s o f hormone replacement i n t h e female were  s e p a r a t e l y (Table 25).  I n i t i a l grouping  analyzed  of t h e female and male  models from T a b l e 25 f o r s t a t i s t i c a l a n a l y s i s i n d i c a t e d t h a t hormone replacement, and even hypophysectomy, d i d not r e s u l t i n significant  changes i n female AHH a c t i v i t y a c c o r d i n g t o the Duncan  134  m u l t i p l e range t e s t . hypophysectomy  However, we have c o n s i s t e n t l y demonstrated  i n c r e a s e s AHH  t h e hypophysectomized  a c t i v i t y i n the female.  Therefore  female model was a n a l y z e d s e p a r a t e l y .  a r e s u l t we have demonstrated, as expected, hypophysectomy an i n c r e a s e i n AHH  As produces  a c t i v i t y concomitant w i t h the appearance of a  moderate a f f i n i t y b i n d i n g component.  Continuous i n f u s i o n of rGH  reduced AHH  female l e v e l s and a b o l i s h e d  a c t i v i t y t o sham-operated  moderate  a f f i n i t y component  T h i s was  t h e same e f f e c t observed i n t h e hypophysectomized male  f o l l o w i n g rGH i n f u s i o n . clear.  that  the  i n the hypophysectomized female.  However, the e f f e c t of rPRL was not as  U n l i k e t h e male model, rPRL reduced AHH  hypophysectomized female t o sham-operated s p e c i f i c b i n d i n g t o the moderate  activity  i n the  female v a l u e s . Furthermore,  a f f i n i t y component was  evident,  and a l t h o u g h a n e g a t i v e r e l a t i o n s h i p between bound/-free v e r s u s bound was  p r e s e n t , l i n e a r r e g r e s s i o n d i d n o t p r o v i d e an a c c e p t a b l e  correlation  coefficient.  The c o n c u r r e n t a d m i n i s t r a t i o n of t e s t o s t e r o n e w i t h e i t h e r bGH  or oPRL d i d not induce AHH  female.  On the c o n t r a r y AHH  a c t i v i t y i n t h e hypophysectomized  a c t i v i t y was reduced i n both models.  However,as. t h e a p p r o p r i a t e c o n t r o l s were not a v a i l a b l e i t i s d i f f i c u l t t o resolve t h e e f f e c t s of the combined  treatment.  135  5)  The e f f e c t s o f x e n o b i o t i c s on h e p a t i c AHH a c t i v i t y and c y t o s o l i c e s t r o g e n b i n d i n g parameters  a)  In v i t r o  i)  effects  Ah r e c e p t o r  In a d d i t i o n t o sex s t e r o i d s and p i t u i t a r y hormones, a v a r i e t y of x e n o b i o t i c s a r e known t o induce activities  (Conney, 1967).  h e p a t i c mixed f u n c t i o n  The p o l y c y c l i c aromatic  (PAH)  " a r e predominently m e t a b o l i z e d  PAH  such as 3-MC w i l l  oxidase  hydrocarbons  by AHH, and a d m i n i s t r a t i o n of  induce AHH a c t i v i t y .  I n d u c t i o n of AHH  a c t i v i t y i s c o n s i d e r e d t o be mediated by t h e Ah ( a r y l hydrocarbon) receptor Glover,  (Nebert 1976).  e t a l . , 1982; Okey e t a l . , 197 9; Poland and This receptor  i s o f t e n r e f e r r e d t o as t h e TCDD  r e c e p t o r because i t s most potent dioxin  (TCDD).  agonist  i s tetrachlorodibenzo-p-  C h a r a c t e r i z a t i o n o f t h e Ah r e c e p t o r has r e v e a l e d  an 8-9S s p e c i e s on hypotonic  sucrose  density gradient a n a l y s i s .  However, b i n d i n g o f BP and;TCDD i n t h e 4§ r e g i o n has'been observed  -  (Okey.:et a l . , 1.979.).  To a s s e s s t h e r e l a t i o n s h i p between the Ah r e c e p t o r , t h e estrogen  r e c e p t o r , and t h e moderate a f f i n i t y b i n d i n g s i t e ,  d i o x i n congeners of d i f f e r i n g Ah b i n d i n g c h a r a c t e r i s t i c s  three  (Poland  3 and  Glover,  1976) were used as c o m p e t i t o r s  f o r [ H]-estradiol  binding. We have demonstrated t h a t a l l t h r e e d i o x i n congeners were 3  e f f e c t i v e competitors  of [ H ] - e s t r a d i o l b i n d i n g t o the e s t r o g e n  r e c e p t o r i n male and female r a t s ( T a b l e 30). The Scatchard  plots  136  of t h e female estrogen  r e c e p t o r produced an apparent Kd and c a p a c i t y  that were lower than t h a t o b t a i n e d Scatchard  by u n l a b e l e d  estradiol  p l o t s y i e l d i n g l i n e a r regression of acceptable  (Table 3 1 ) . correlation  c o u l d not be generated f o r t h e male w i t h any o f t h e d i o x i n congeners. This occurred  d e s p i t e t h e f a c t t h a t a l l congeners competed f o r  [ H . ] - e s t r a d i o l b i n d i n g to t h e estrogen  receptor.  A s i m i l a r r e s u l t was noted f o r t h e moderate a f f i n i t y U n l i k e t h e estrogen for  r e c e p t o r , o n l y 2,7-DCDD and OCDD competed  ["^H]-estradiol b i n d i n g to t h e moderate a f f i n i t y  i n t h e male. acceptable  However, l i k e t h e estrogen  Scatchard  congeners.  component.  receptor  component  i n the male,  p l o t s c o u l d not be generated w i t h  Furthermore, as w i t h a l l o t h e r c o m p e t i t o r s ,  these none o f  the d i o x i n congeners l e d t o d e t e c t i o n o f moderate a f f i n i t y i n t h e female. male estrogen  The i n a b i l i t y  to generate Scatchard  p l o t s f o r the  r e c e p t o r w i t h d i o x i n was not due to d i f f i c u l t i e s  a s s o c i a t e d w i t h a dextran-coated as evidenced  binding  c h a r c o a l assay (Okey et a l . , 1979)  by t h e r e s u l t s f o r t h e female estrogen  do not f e e l t h e b i n d i n g i n t h e female r e p r e s e n t s i t would have been d e t e c t e d  i n t h e male as w e l l .  receptor.  We  t h e Ah r e c e p t o r as Poland  and G l o v e r ,  (1976) have shown 1 7 B - e s t r a d i o l does not compete f o r TCDD b i n d i n g to the Ah r e c e p t o r .  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 o r t h e b i n d i n g s i t e r a t h e r than l i g a n d s p e c i f i c i t y . any  case,  t h e r e s u l t s of the d i o x i n c o m p e t i t i o n  In  for [ H]-estradiol 3  b i n d i n g i n male c y t o s o l i c f r a c t i o n s f u r t h e r demonstrate t h e problems w i t h r e l y i n g on s i n g l e p o i n t c o m p e t i t i o n binding  components.  a n a l y s i s to detect  specific  137  ii)  Hepatic  c y t o s o l i c BP./S^MC• b i n d i n g ;prot,e,ih.  In a d d i t i o n to the Ah r e c e p t o r , a 4S BP b i n d i n g component has been i d e n t i f i e d In a d d i t i o n , 3-MC  i n h e p a t i c c y t o s o l (Weaver et a l . , 1980).  has been shown t o i n t e r a c t n o n - c o v a l e n t l y  a 4S b i n d i n g p r o t e i n i n h e p a t i c c y t o s o l as w e l l 1980) .  C o l l i n s and M a r i e t t a  with  ( T i e r n e y et a l . ,  (1983) have demonstrated t h e  cytosolic  4S BP b i n d i n g p r o t e i n i s not r e l a t e d to the Ah r e c e p t o r or S - t r a n s f e r a s e B. (Banderia 1977).  et a l . , 1982;  The  i s capable  However, 3-MC  and  BP do b i n d to these  D i x i t e t a l . , 1982,  1983;  s i t e s as w e l l  Guenther et a l . ,  4S h e p a t i c c y t o s o l i c p r o t e i n t h a t b i n d s 3-MC of n u c l e a r  et a l . , 1980,  1981;  glutathione  and  t r a n s l o c a t i o n (Holder et a l . , 1981;  Weaver et a l . , 1980).  d i d not r e q u i r e an a c t i v a t i o n step  Tierney  This translocation  ( T i e r n e y et a l . , 1980;  et a l . , 1980), as i s seen w i t h the e s t r o g e n  BP  receptor  Weaver  (Jensen  and  DeSombre, 1973) . The v a l u e s r e p o r t e d f o r the apparent Kd and 3-MC and  b i n d i n g protein,2.8 nM,  770  t h e BP b i n d i n g p r o t e i n , 2.5  c a p a c i t y of  the  fmol/mg ( T i e r n e y et a l . , 1980) nM,  530  fmol/mg (Holder et a l . .  1981) , a r e below those v a l u e s observed f o r the moderate a f f i n i t y component. T i e r n e y et a l . . (1980) have demonstrated t h a t i n a d d i t i o n to an a r r a y of PAH's, 1 7 g - e s t r a d i o l i s an e f f e c t i v e competitor 3 [ H]3-MC b i n d i n g t o the 4S p r o t e i n .  A 100  f o l d excess of  for unlabeled  3  17 3 - e s t r a d i o l competed f o r 55% of hepatic cytosol. BP  The  [ H]3-MC b i n d i n g i n male r a t  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  b i n d i n g p r o t e i n to t h e moderate a f f i n i t y  and  s i t e i s presently unclear.  138  The e s t r o g e n r e c e p t o r has been shown t o b i n d a v a r i e t y of x e n o b i o t i c s : 3-diethylaminoethyl-2,2-diphenylpentanoate  (SKF 525-A)  (Bulger and K u p f e r , 1981), o,p'-DDT (Kupfer and B u l g e r , 1976), Kepone (Bulger et a l . , 1979) Coogan, 1983).  S i n c e we  and  2-acetylaminofluorene (Farley  a r e i n t e r e s t e d i n the modulation  of  and  AHH  a c t i v i t y by PAH's as w e l l as sex s t e r o i d s , t h e r e l a t i o n s h i p of t h e 3-MC/BP b i n d i n g s i t e and the moderate a f f i n i t y component investigated.  u n l a b e l e d 3-MC  was  and BP were used as c o m p e t i t o r s f o r  3 [ H ] - e s t r a d i o l b i n d i n g i n h e p a t i c c y t o s o l f r a c t i o n s i n male and female  rats. 3 We  have shown t h a t both 3-MC  and BP competed f o r [ H ] - e s t r a d i o l  b i n d i n g t o t h e e s t r o g e n r e c e p t o r i n male and female and  f o r the  moderate a f f i n i t y component i n the male r a t . ( T a b l e 30). the c o m p e t i t i o n data produced the S c a t c h a r d t e c h n i q u e .  good l i n e a r p l o t s when a n a l y z e d by  T h i s was  t r u e i n both the males and  u n l i k e t h e d a t a from the d i o x i n study. c o m p e t i t i o n was the apparent was  no  Furthermore,  The apparent Kd f o r  s i m i l a r i n both male and female, and  females, 3-MC  similar, to  Kd o b t a i n e d by e s t r a d i o l c o m p e t i t i o n ( T a b l e 26).  sex d i f f e r e n c e i n the c a p a c i t y , which was  t h a t d e t e c t e d by e s t r a d i o l c o m p e t i t i o n .  There  approximately  50%  With r e s p e c t to BP c o m p e t i t i o n ,  (Table 27) b o t h the apparent Kd and c a p a c i t y were s i m i l a r but  reduced  i n both male and female r e l a t i v e t o e s t r a d i o l c o m p e t i t i o n . F o l l o w i n g i n d u c t i o n w i t h 3-MC  or p h e n o b a r b i t a l we  observed  a decrease i n the c a p a c i t y o f the e s t r o g e n r e c e p t o r when e s t r a d i o l was  used as a c o m p e t i t o r .  But t h i s decrease was not  f o l l o w i n g i n d u c t i o n u s i n g e i t h e r 3-MC i t appears  observed  o r BP as c o m p e t i t o r s .  t h a t the PAH's a r e competing  Therefore,  f o r e s t r a d i o l b i n d i n g to a  139  subclass of receptor s i t e s .  Competition  for binding to the moderate  a f f i n i t y component by 3-MC and BP resulted i n similar s h i f t s of apparent Kd and capacity r e l a t i v e to e s t r a d i o l competition as were reported for the estrogen receptor. It may be argued that 3-MC and BP are displacing [ H.]-estradiol 3  bound to s i t e s other than the moderate a f f i n i t y component.  However,  following gonadectomy (Table 29) or induction (Table 26) we observed p a r a l l e l s h i f t s i n displacement e s t r a d i o l or 3-MC as competitors  of labeled estradiol using unlabeled 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 i n capacities may be due to a difference i n ligand a v i d i t y for the moderate a f f i n i t y binding s i t e . We have demonstrated an interaction between PAH's, the estrogen receptor, and the moderate a f f i n i t y component.  I t would be of interest  to examine the ontogenetic p r o f i l e of the BP/3-MC binding protein i n r e l a t i o n to the moderate a f f i n i t y s i t e .  This has yet to be addressed.  In f a c t , investigation of the 4S BP/3-MC binding protein has so far been conducted i n adult males only. On the basis of the s i m i l a r i t y of ligand s p e c i f i c i t y that we have shown, and the s i m i l a r i t y 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 i d e n t i t y .  A l t e r n a t i v e l y , there  may be a family of heterogeneous proteins with p a r t i a l l y overlapping binding a f f i n i t i e s .  Heterogeneity  of hepatic estrogen binding s i t e s  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  c o r r e l a t i o n between the l e v e l s of AHH the moderate a f f i n i t y component. described  discrepancies with  a c t i v i t y and  the presence of  These i n c o n s i s t e n c i e s were  i n the hormone models, and  the  first  have been observed f o l l o w i n g  treatment w i t h x e n o b i o t i c s as w e l l .  b)  In v i v o e f f e c t s of  xenobiotics  D u v i v i e r et a l . , (1981) have demonstrated a c o r r e l a t i o n between 3-MC  i n d u c t i o n and  the c a p a c i t y of t h e e s t r o g e n  the a d m i n i s t r a t i o n of 3-MC to the e s t r o g e n  receptor  i n ovariectomized  i n i n t a c t male and  that p h e n o b a r b i t a l Our  data  When AHH  female r a t s .  female r a t s .  produced a s l i g h t  induced  increase i n estrogen  w i t h 3-MC  phenobarbital  which was  estrogen  shown not  reported  binding.  produced a receptor.  i n the male, an  i n the c a p a c i t y of the moderate a f f i n i t y component was However, p h e n o b a r b i t a l ,  have  These a u t h o r s a l s o  change i n the c a p a c i t y of the estrogen  a c t i v i t y was  We  change i n c a p a c i t y of the  i n d i c a t e s t h a t s i m i l a r t o 3-MC,  decrease or no  Following  t h i s group observed a decrease i n b i n d i n g  observed e i t h e r a decrease or no receptor  receptor.  increase  a l s o noted.  to induce AHH  activity  i n the a d u l t male, a l s o i n c r e a s e d the c a p a c i t y of the moderate a f f i n i t y component.  Furthermore, f o l l o w i n g the a d m i n i s t r a t i o n of  spironolactone activity  or p h e n o b a r b i t a l ,  (which were shown to induce  i n the f e m a l e ) , no moderate a f f i n i t y b i n d i n g  c o u l d be d e t e c t e d phenobarbital  3-MC,  i n the female.  Administration  component  of 3-MC  and  f o r t e n days ( i n s t e a d of the customary 2 and  respectively) s t i l l  d i d not r e s u l t  4 days  i n d e t e c t i o n of a moderate  component i n t h e female.even:though AHH  a c t i v i t y was  AHH  affinity  induced. I n t e r e s t i n g  141  the prolonged a d m i n i s t r a t i o n  o f 3-MC t o males n e a r l y a b o l i s h e d t h e  moderate a f f i n i t y component, a l t h o u g h AHH a c t i v i t y remained The  induced.  reason f o r t h e decrease i n c a p a c i t y of t h e moderate a f f i n i t y  component i n t h i s  i n s t a n c e may be much t h e same as t h a t  following administration  of mestranol.  s i t e s and, a s was d i s c u s s e d  observed  We were measuring unoccupied  e a r l i e r , t h e 3-MC b i n d i n g t o t h e s e  sites  may have masked d e t e c t i o n . Thus, i n t h e female, the o n l y procedure t h a t produced an increase  i n AHH a c t i v i t y and moderate a f f i n i t y b i n d i n g was  hypophysectomy. increased  Furthermore, we have n o t e d procedures t h a t  t h e c a p a c i t y of t h e moderate a f f i n i t y component i n t h e  male without a f f e c t i n g reduced AHH a c t i v i t y  AHH a c t i v i t y  (spironolactone).  decreases i n AHH a c t i v i t y  o r which  We have a l s o observed  i n t h e male t h a t have not .produced decreases  the c a p a c i t y o f t h i s b i n d i n g the c o r r e l a t i o n  (phenobarbital)  component  (oPRL, bGH).  Therefore,  o f AHH a c t i v i t y w i t h the c a p a c i t y o f t h e moderate  a f f i n i t y component i s n o t c o n s i s t e n t .  We have suggested t h a t  t h i s may be due t o d i f f e r e n c e s i n the form o f t h e moderate a f f i n i t y component f o l l o w i n g p h y s i o l o g i c a l m a n i p u l a t i o n . c h a r a c t e r i z a t i o n d i d not d e t e c t the p o s s i b i l i t y  t h i s but o t h e r s  (Eagon et a l . , 1980).  Our have  limited reported  A l t e r n a t i v e l y , these  d i s c r e p a n c i e s may be due t o t h e m u l t i p l i c i t y  o f cytochromes P-450.  142  6)  M u l t i p l i c i t y o f Cytochromes P-450  Cytochromes P-450 comprise a group o f c o n s t i t u t i v e and i n d u c i b l e isozymes e x h i b i t i n g o p t i m a l  and o v e r l a p p i n g  substrate  s p e c i f i c i t i e s , as w e l l as r e 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 o n l y a f i n i t e  number o f P-450 isozymes have been p u r i f i e d t o 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 f o r t h e P-450 system  has l e a d Nebert et a l . , (1982) t o compare i t t o t h e immune system. They suggest t h a t be s y n t h e s i z e d  i n response t o x e n o b i o t i c s , P-450 isozymes may  t h a t a r e s p e c i f i c f o r metabolism o f that  i . e . a s i m i l a r process to antigen production  xenobiotic,  stimulation of s p e c i f i c antibody  occurs.  Recently phenobarbital  a m i c r o h e t e r o g e n e i t y has been r e p o r t e d  f o r the  i n d u c i b l e form o f P-450 i n t h e r a t (Waxman and  Walsh et a l . , 1982) t h a t v a r i e s between s p e c i e s and s t r a i n s o r colonies  (Vlasuk  subclasses  et a l . , 1982).  Moreover, c a t a l y t i c a l l y  distinct  o f r a b b i t P-450 3b a c t i v e towards h y d r o x y l a t i o n o f  p r o g e s t e r o n e have been c h a r a c t e r i z e d  (Johnson e t a l . , 1983).  These d i f f e r e n c e s may be due t o p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n such as a l l o s t e r i c r e g u l a t i o n genomic v a r i a t i o n .  (Johnson et a l . , 1983) r a t h e r than  Or, p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n may occur  s i m i l a r t o t h a t demonstrated f o r s y n t h e s i s c h i c k blastoderm.  of f e t a l hemoglobin by  Mainwaring and I r v i n g (1980) have r e p o r t e d  53- reduced s t e r o i d s enhance g l o b i n s y n t h e s i s cytoplasmic  p r o t e i n s not found i n t h e y o l k .  that  following binding to This binding  t r a n s l a t i o n o f s p e c i f i c mRNA coded f o r g l o b i n s y n t h e s i s .  increased The  143  cytoplasmic  proteins are recognized  t o be cap b i n d i n g  proteins  which i n c r e a s e t r a n s l a t i o n of capped but not uncapped mRNA (Sonenberg et a l . j 1980).  T h i s means that t h e amount o f enzyme  present  t o m o d i f i c a t i o n at several p o i n t s ,  i n vivo  i s subject  which g r e a t l y enhances b o t h the c o m p l e x i t y and f i n e c o n t r o l o f t h i s system. It  i s o f i n t e r e s t t h a t t h e forms o f P-450 present  i n female  r a t s have been shown t o be d i f f e r e n t from those i n males w i t h respect  to substrate a c t i v i t y  (Kamataki et a l . , 1981, 1982).  In  a d d i t i o n t o a sex d i f f e r e n c e , t h e presence o f t h e P-450 isozyme r e s p o n s i b l e f o r the 1 6 a - h y d r o x y l a t i o n  of testosterone  i n the  a d u l t male was shown t o be a f u n c t i o n of n e o n a t a l i m p r i n t i n g by androgens (Chao and Chung, 1982; Chung and Chao, 1980).  These  i n v e s t i g a t o r s a l s o noted t h a t no q u a l i t a t i v e d i f f e r e n c e s were evident  i n t h e column e l u t i o n p r o f i l e s o f P-450 from  imprinted  or n o n - i m p r i n t e d r a t s .  differences i n substrate  neonatally  I t was suggested that  these  s p e c i f i c i t y between s i m i l a r forms of  P-450 i n male and female r a t s a r e r e s p o n s i b l e f o r sex d i f f e r e n c e s i n mixed f u n c t i o n o x i d a s e a c t i v i t i e s Based on t h i s type o f data,  i n the a d u l t .  one may s p e c u l a t e  t h a t t h e moderate  affinity  component may be a s s o c i a t e d w i t h a P-450 isozyme t h a t i s  imprinted  and s p e c i f i c t o t h e male, and a l s o t h a t BP i s i n c l u d e d  in i t s substrate p r o f i l e .  This i s of i n t e r e s t considering the reports  of Sloop e t a l . , (1983) t h a t t h e HCLA s i t e i s n e o n a t a l l y  imprinted.  In t h i s case, we would n o t expect t o f i n d t h e moderate a f f i n i t y component i n t h e d i f f e r e n t i a t e d female. to i n d u c i n g  S i m i l a r l y t h e response  agents may be d i f f e r e n t w i t h r e s p e c t  t o P-450 isozyme  144  substrate  specificity  i n male and female r a t s .  i n d u c t i o n of AHH a c t i v i t y male i m p r i n t e d  Therefore,  i n t h e female may not be r e l a t e d t o the  form of P-450.  The p l e i o t r o p i c response of the  female would n o t be expected to i n c l u d e p r o d u c t i o n moderate a f f i n i t y component. to observe an i n c r e a s e  an  of t h e  As a r e s u l t , we would expect  i n AHH a c t i v i t y  without a concomitant  d e t e c t i o n of the moderate a f f i n i t y component, i n c e r t a i n c i r c u m s t a n c e s .  7)  Proposed experiments  We have h y p o t h e s i z e d t h a t t h e moderate a f f i n i t y p r o t e i n i s a s s o c i a t e d w i t h t h e l e v e l s of c e r t a i n h e p a t i c i n the a d u l t male.  Studies  p r o t e i n may not simply but  of the 4S p r o t e i n  following  T h i s suggests t h a t the moderate a f f i n i t y  be reduced i n c a p a c i t y f o l l o w i n g  that a conformational  specificity  activities  by Eagon et a l . , (1980) have demonstrated  a change i n the l i g a n d s p e c i f i c i t y gonadectomy of t h e male.  enzyme  change may  occur.  Therefore,  of t h e moderate a f f i n i t y component  should  gonadectomy the l i g a n d  be i n v e s t i g a t e d  f o l l o w i n g procedures such as gonadectomy and hypophysectomy to determine i f t h e b i n d i n g component  present  site  i s r e p r e s e n t a t i v e o f the same  i n t h e sham-operated a n i m a l .  (1980) r e p o r t t h e moderate a f f i n i t y component  I f as Eagon et a l . , becomes  DES-sensitive,  then i t would n e c e s s i t a t e r e - e v a l u a t i o n o f t h e b i o c h e m i c a l  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 o f the p r o t e i n . I t would a l s o be of i n t e r e s t t o determine i f a n e o n a t a l l y gonadectomized a d u l t male r e t a i n s t h e t e s t o s t e r o n e  r e v e r s a l of  the r e d u c t i o n  component  i n c a p a c i t y of the moderate a f f i n i t y  145  observed f o l l o w i n g gonadectomy of the a d u l t .  T h i s would examine  the p o s s i b i l i t y of i m p r i n t i n g suggested by Sloop et a l . , (1983). I f n e o n a t a l gonadectomy does i r r e v e r s i b l y a b o l i s h the male moderate a f f i n i t y component, then a d m i n i s t r a t i o n  of t e s t o s t e r o n e  neonatally  the f i r s t  should  gonadectomized female d u r i n g  be attempted.  production  week of  T h i s would be done i n an e f f o r t t o  life induce  of the moderate a f f i n i t y component i n the a d u l t  following testosterone  challenge.  t h i s f a s h i o n then i t would c o n f i r m The  to a  female  I f the female d i d respond i n the  imprinting  e f f e c t s of g l u c o c o r t i c o i d s and  process.  thyroxine  should  s t u d i e d to f u r t h e r c l a r i f y the hormonal m i l i e u r e q u i r e d the maintenance of the e s t r o g e n r e c e p t o r  and  be  for  t h e moderate a f f i n i t y  component f o l l o w i n g hypophysectomy.; These..hormones., i n . / a d d i t i o n to GH,  have been r e p o r t e d  t o be n e c e s s a r y f o r f e m i n i z a t i o n of  metabolism and maintenance of the e s t r o g e n r e c e p t o r . the a d d i t i o n of DHT,  t h e s e hormones a r e - r e q u i r e d  maintenance of the DHT  binding protein.  for  With.  Roy  et a l . , (1973) r e p o r t e d  r e s t o r e d the DHT for  c o u l d be due  This i s inconsistent with  to the hormone regimen.  binding protein.  increase  testosterone We of sex  i n AHH  discussed, In any  case  that t h i s combination of hormones If this effect  the moderate a f f i n i t y component then i t may  observe an  . '  the  a f e m i n i z a t i o n of metabolism, a l t h o u g h , as p r e v i o u s l y t h i s discrepancy  hepatic  c o u l d be  repeated  be p o s s i b l e to  a c t i v i t y following administration  of  to the hypophysectomized a n i m a l .  have a l s o p r e s e n t e d p r e l i m i n a r y r e s u l t s on the i n t e r a c t i o n  s t e r o i d b i n d i n g p r o t e i n s w i t h d i o x i n congeners.  d i o x i n congeners, we  Using  have noted d i f f e r e n c e s between the male  female i n v i t r o b i n d i n g  characteristics.  and  146  T h i s suggests t h a t t h e moderate a f f i n i t y component may be i n t e r f e r i n g with d i o x i n binding to t h e estrogen male.  i n the  Examination o f i n v i t r o b i n d i n g o f d i o x i n s i n t h e gona-  dectomized male should r e s o l v e t h i s q u e s t i o n . behind  receptor  t h i s proposal  i s t h a t gonadectomy reduces t h e c a p a c i t y  of t h e moderate a f f i n i t y estrogen  receptor  The r a t i o n a l e  component  but does n o t a f f e c t t h e  .torancappreclable.-degree.  TCDD i s a v e r y potent  i n d u c e r o f t h e Ah r e c e p t o r and AHH  activity.  We have e s t a b l i s h e d t h a t TCDD binds  receptor.  I t would be of i n t e r e s t  t o induce  to the estrogen  b o t h male and female  r a t s w i t h TCDD and measure t h e h e p a t i c c y t o s o l i c e s t r o g e n parameters as TCDD may w e l l induce  the-estrogen  receptor.  We have suggested t h a t t h e moderate a f f i n i t y r e l a t e d t o t h e c y t o s o l i c 3-MC/BP b i n d i n g p r o t e i n . of i n t e r e s t  binding  component i s I t would be  t o conduct i n c u b a t i o n s u s i n g 3-MC and e s t r a d i o l 3  ( s e p a r a t e l y ) as c o m p e t i t o r s incubations  t o [ H]-3—MC b i n d i n g .  Parallel  be conducted u s i n g 3-MC and e s t r a d i o l  should  (separately)  3  as c o m p e t i t o r s  to [ H]-estradiol binding.  By examining t h e changes  i n t h e b i n d i n g parameters, f o l l o w i n g p h y s i o l o g i c a l m a n i p u l a t i o n of the animals,  one c o u l d determine t h e r e l a t i o n s h i p o f t h e s e  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 a c i d  of t h e v a r i o u s b i n d i n g p r o t e i n s must be c a r r i e d o u t . monoclonal a n t i b o d y b i o l o g i c a l studies.  techniques  sequencing  Specific  would prove i n v a l u a b l e f o r f u r t h e r  147  8.  P h y s i o l o g i c a l relevance  We have suggested t h a t t h e moderate a f f i n i t y  component i s a  male s p e c i f i c sex s t e r o i d b i n d i n g p r o t e i n t h a t i s r e g u l a t e d by b o t h hypothalamic-hypophyseal and gonadal f a c t o r s . affinity  While the moderate  component may possess " f u n c t i o n a l i d e n t i t y w i t h  cytosolic  3-MC/BP b i n d i n g p r o t e i n s , i t does appear t o r e q u i r e t h e presence o f other  f a c t o r ( s ) t o e x h i b i t a response t o t e s t o s t e r o n e .  As demonstrated  i n t h e hypophysectomy model which r e s u l t e d i n t h e l o s s o f t h e estrogen  r e c e p t o r , t e s t o s t e r o n e was unable t o produce a response  even though the moderate a f f i n i t y a high a f f i n i t y ,  supported  a response, and, l i k e t h e e s t r o g e n  f o l l o w i n g hypophysectomy.  by r e c e n t  a high a f f i n i t y ,  Therefore,  low c a p a c i t y r e c e p t o r may be r e q u i r e d f o r t e s t o -  sterone to e l i c i t i t may be l o s t  component was p r e s e n t .  receptor,  This p o s s i b i l i t y i s  r e p o r t s that have i n d i c a t e d t h e presence o f  low c a p a c i t y androgen r e c e p t o r i n t h e l i v e r  (Eagon e t a l . , 1983; E i s e n f e l d e t a l . , 1983; Decker e t a l . , 1983). Therefore,  the h e p a t i c moderate a f f i n i t y  component may be s i m i l a r i n  f u n c t i o n t o a b i n d i n g p r o t e i n found i n t h e u t e r u s .  C l a r k et a i . , . (19.78) ,  and Watson and C l a r k (1980) have c h a r a c t e r i z e d a 4S e s t r o g e n  binding  component i n r a t u t e r u s and mouse mammary tumor, r e s p e c t i v e l y . T h i s b i n d i n g p r o t e i n (type II) was present  i n both c y t o s o l i c and  n u c l e a r f r a c t i o n s and was shown t o be o f h i g h e r affinity was  than t h e t y p e I-estrogen  n o t capable  of nuclear  (1979) r e p o r t e d that these  receptor  site.  c a p a c i t y and lower The type I I s i t e  t r a n s l o c a t i o n . Markaverich  and C l a r k  sites 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 t r a n s l o c a t i o n or nuclear interaction.  Furthermore, t h e type I I s i t e was c o r r e l a t e d w i t h t h e  148  delayed  and  prolonged a c t i v a t i o n of RNA  as u t e r i n e hypertrophy and authors reported  hyperplasia  polymerase I and  (Hardin et a l . , 1976).  type I (estrogen receptor)  s u s t a i n e d e l e v a t i o n of the type I I s i t e . i n c r e a s e d u t e r i n e wet s i m i l a r l y , d i d not  weight.  produce any  s i t e and  well These  t h a t a s i n g l e i n j e c t i o n of e s t r a d i o l produced  e l e v a t i o n o f the n u c l e a r  it  I I as  an  a  This c o r r e l a t e d with  However, e s t r i o l  administered  e l e v a t i o n i n t y p e I I b i n d i n g nor  did  s t i m u l a t e u t e r i n e growth or produce a prolonged e l e v a t i o n i n  RNA  polymerase I and  II.  Thus i t was  concluded t h a t u t e r i n e growth  was  associated with  sustained elevations in type I I binding.  T h i s i s of i n t e r e s t when c o n s i d e r i n g the a c t i o n s of e s t r o g e n s on the hypothalamus and have r e p o r t e d  Kelner  and  Peck J r . (1981) ,  t h a t n e i t h e r the hypothalmus or p i t u i t a r y  the type I I s i t e . not  pituitary.  contain  Furthermore, a d m i n i s t r a t i o n of e s t r a d i o l d i d  produce an a c t i v a t i o n of RNA  polymerase I .  RNA  polymerase I I  e x h i b i t e d a t r a n s i e n t a c t i v a t i o n w i t h a time course t h a t c o r r e l a t e d with nuclear The  accumulation o f e s t r a d i o l  authors speculate  (Kelner et a l . , 1980).  t h a t the type I I s i t e a c t s to p r o l o n g  nuclear  r e t e n t i o n of e s t r a d i o l thus i n c r e a s i n g g e n e t i c i n t e r a c t i o n . T h i s would a l l o w f o r p r o l o n g e d a c t i v a t i o n of RNA and  IT r e s u l t i n g i n the end  s y n t h e s i s , hypertrophy and  polymerase I  p l e i o t r o p i c responses: increased hyperplasia.  Furthermore, the type I I  s i t e s i n r a t u t e r i n e c y t o s o l ( E r i k s s o n et a l . , 1978) a s i m i l a r f a s h i o n " t h a t i s , to p r o l o n g c r e a t i n g an estrogen by the e s t r o g e n  protein  may  elevated estrogen  act i n levels,  r i c h environment f o r n u c l e a r t r a n s l o c a t i o n  receptor.  149  That the f u n c t i o n of the h e p a t i c moderate a f f i n i t y may  be  s i m i l a r to t h a t suggested f o r the uterine, type.11 srt;e i s supported  by the  striking  similarity  (both p h y s i o l o g i c a l l y and  between the moderate a f f i n i t y component and and  component  e s t r a d i o l binding  the male h e p a t i c  protein previously described.  binding protein i s correlated with synthesis  biochemically)  of the  This  DHT  DHT  urinary  protein c ^ - g l o b u l i n . M i l i n and  Roy(1973)and Roy  et a l . , ( l 9 7 4 ) r e p o r t e d t h i s  p r o t e i n to be absent i n immature male and pseudohermaphrodite r a t s .  female, a d u l t  T h i s p r o t e i n was  manner.  reversible  b i n d i n g as w e l l as u r i n a r y output of c ^ - g l o b u l i n .  et a l . , (1976) have c o r r e l a t e d the a d m i n i s t r a t i o n of DHT  a r i s e i n u r i n a r y o ^ ^ g l o b u l i n and l a t a b l e mRNA f o r o ^ - g l o b u l i n .  an  increase  in hepatic  Furthermore, Roy  which c o u l d o n l y be r e s t o r e d f o l l o w i n g simultaneous thyroxine,  C o r t i s o l and  In c o n c l u s i o n , apparent Kd,  there  they a r e the t o be  trans-  synthesis  administration  DHT.  i s a s i m i l a r i t y between l i g a n d  specificity,  s e d i m e n t a t i o n c o e f f i c i e n t , hormonal r e g u l a t i o n ,  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 the DHT  to  et a l . , (1973)  have observed t h a t hypophysectomy reduced o ^ ^ g l o b u l i n  and  o^-globulin.  of e s t r a d i o l t o i n t a c t a d u l t male r a t s r e s u l t e d i n  a d e c r e a s e i n DHT  of GH,  and  androgen  P a r a l l e l changes were seen f o r u r i n a r y output of  Administration  Roy  female  shown t o be  dependent, reduced by gonadectomy i n a t e s t o s t e r o n e  binding  binding protein. same b i n d i n g  T h i s s i t e has  i n v o l v e d i n the t r a n s l a t i o n p r o c e s s and,  p r o v i d e a f u n c t i o n i n the l i v e r  component  T h i s l e a d s .one t o ; s p e c u l a t e  site.  that  been shown by therefore,  s i m i l a r to that described  and  others  may for  the  150  u t e r i n e s i t e I I component. a f f i n i t y component may and  A l t e r n a t i v e l y , b i n d i n g to the moderate  r e t a r d the m e t a b o l i c  s t e r o i d - l i k e compounds.  T h i s may  degradation  of  steroid  a l l o w them to a c t i n the  c y t o s o l 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 m o d i f i c a t i o n of p r o t e i n s y n t h e s i s as suggested by Mainwaring and  I r v i n g (1980) f o r  5a-reduced s t e r o i d s i n c h i c k b l a s t o d e r m , i . e . , to  increase  t r a n s l a t i o n o f s p e c i f i c mRNA coded f o r s p e c i f i c p r o t e i n s . We  cannot p r e c i s e l y d e f i n e t h e r o l e of t h e h e p a t i c moderate  a f f i n i t y component-in the male. can be  However, a r e a s o n a b l e  suggested. The  h e p a t i c androgen r e c e p t o r r e c e n t l y d e s c r i b e d  et a l . , (1983), E i s e n f e l d et a l . , (1983), and was  detected  i n both male and  gonadectomy.  shown  a l t e r e d by  These a r e unexpected c h a r a c t e r i s t i c s f o r a r e c e p t o r However, we  the h e p a t i c moderate a f f i n i t y component to be  responsive  by Eagon  Decker e t a l . , (1983)  female r a t s and not  i n v o l v e d i n maintenance of a sex d i f f e r e n c e .  and  possibility  to gonadectomy.  As  sex  have dependent  such, the androgen r e c e p t o r does  not have to e x h i b i t 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 p r o v i d e a s t a b l e p o o l of androgen a v a i l a b l e f o r b i n d i n g to a h i g h e r a f f i n i t y ,  lower c a p a c i t y androgen r e c e p t o r  f o r subsequent n u c l e a r t r a n s l o c a t i o n . t h i s s i t u a t i o n may thereby  reducing  The  a c t i o n of e s t r a d i o l i n  simply be to compete f o r b i n d i n g to t h i s p r o t e i n  i n t r a c e l l u l a r c o n c e n t r a t i o n of androgen a v a i l a b l e  f o r n u c l e a r t r a n s l o c a t i o n by the androgen r e c e p t o r . to t h i s component prevents m e t a b o l i c b r i n g the s t e r o i d  degradation  Whether b i n d i n g  of the s t e r o i d  i n t o the p r o x i m i t y of a n u c l e a r t r a n s l o c a t i o n  to  151  p r o t e i n , or f o r p o s t - t r a n s c r i p t i o n a l m o d i f i c a t i o n , u n c l e a r and- warrents  further  study.  or both, i s  SUMMARY  Despite  i n c o n s i s t e n c i e s w i t h the female we have observed a  c o r r e l a t i o n w i t h the l e v e l s of AHH  activity  i n the male r a t and the  c a p a c i t y of the moderate a f f i n i t y component. on Scatchard  These r e s u l t s were based  a n a l y s i s , and not sucrose d e n s i t y g r a d i e n t  as were many of the s t u d i e s we have c i t e d .  determinations  These i n c l u d e the f o l l o w i n g  points: 1)  an age dependent sex d i f f e r e n c e i n the c a p a c i t y of the moderate a f f i n i t y  2)  f o r AHH  i s known to be age r e l a t e d , Bellward  gonadectomy produced a t e s t o s t e r o n e i n b o t h AHH affinity  4)  observed.  a sex d i f f e r e n c e was a l s o r e p o r t e d (and  3)  component was  activity  component  et a l . , 1982)  r e v e r s i b l e decrease  and the c a p a c i t y of the moderate  i n the male  hypophysectomy a b o l i s h e d b i n d i n g receptor,  activity  and reduced both AHH  t o the e s t r o g e n  activity  and the  c a p a c i t y of the moderate a f f i n i t y component  i n the  male 5)  hypophysectomy a b o l i s h e d receptor,  binding  and i n c r e a s e d both AHH  t o the e s t r o g e n activity  and the  c a p a c i t y of the moderate a f f i n i t y component  i n the  female 6)  the a d m i n i s t r a t i o n o f rGH to both hypophysectomized male or female r a t s reduced AHH moderate a f f i n i t y b i n d i n g ; reduced AHH a c t i v i t y  activity  and  abolished  i n a d d i t i o n , bGH and oPRL  i n the sham-operated male without  153  altering  hepatic estrogen  b i n d i n g parameters  the a d m i n i s t r a t i o n of m e s t r a n o l to the a d u l t male reduced AHH both the  activity  estrogen  and  intact  abolished binding  r e c e p t o r and  to  the moderate a f f i n i t y  component the a d m i n i s t r a t i o n of p h e n o b a r b i t a l , and  3-MC  spironolactone  to the a d u l t male r a t i n c r e a s e d the c a p a c i t y  of the moderate a f f i n i t y component, a l t h o u g h 3-MC  produced an  i n c r e a s e i n AHH  activity  f o l l o w i n g a d m i n i s t r a t i o n of p h e n o b a r b i t a l , s p i r o n o l a c t o n e , the changes i n AHH  only  3-MC  or  of  the  activity  pseudohermaphrodite p a r a l l e l e d those of the t r e a t e d female and not  similarly  the male  no moderate a f f i n i t y component was  detected  i n the  p seu dohermaphr od i t i c r a t 3  pimozide a b o l i s h e d  [ H ] - e s t r a d i o l b i n d i n g to  the  moderate a f f i n i t y component i n the gonadectomized male no procedure,  other than hypophysectomy,  resulted  i n d e t e c t i o n of t h e moderate a f f i n i t y component i n the  female  both 3-MC  BP were shown to compete f o r  specific  e s t r a d i o l b i n d i n g to the e s t r o g e n r e c e p t o r  i n males  and  and  females and  the male r a t  the moderate a f f i n i t y component i n  154  d i o x i n congeners d i d not compete f o r s p e c i f i c e s t r a d i o l b i n d i n g to the male e s t r o g e n  receptor  or moderate a f f i n i t y component as d i d 3-MC  and BP  a l l d i o x i n congeners demonstrated s p e c i f i c i t y f o r the h e p a t i c e s t r o g e n  receptor  i n the female  155  BIBLIOGRAPHY  A l v a r e s , A.P., S c h i l l i n g , G., Garbut, A., and Kuntzman, R., S t u d i e s on the h y d r o x y l a t i o n of 3,4-benzpyrene by h e p a t i c microsomes, Biochem. 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T i e r n e y , B., and B r e s n i c k , E., D i f f e r e n c e s i n the b i n d i n g o f 3-methylcholanthrene and p h e n o b a r b i t a l t o r a t l i v e r c y t o s o l i c and n u c l e a r p r o t e i n f r a c t i o n s , A r c h . Biochem. Biophys. 210: 729-739, 1981. Thompson, C , Powell-Jones, W., and L u c i e r , G.,W., Sex d i f f e r e n c e s i n h e p a t i c o e s t r o g e n - b i n d i n g p r o t e i n s , Biochem. J . 194: 1-8, 1981.  167  V l a s u k , G.P., Ghrayeb, J . , Ryan, D.E., Reik, L., Thomas, P.E., L e v i n , W., and Walz J r . , F., M u l t i p l i c i t y , s t r a i n d i f f e r e n c e s , and t o p o l o g y of p h e n o b a r b i t a l - i n d u c e d cytochromes P-450 i n r a t l i v e r microsomes, B i o c h e m i s t r y 21: 789-798, 1982. Watson, C.S., and C l a r k , J.H., H e t e r o g e n e i t y of e s t r o g e n b i n d i n g s i t e s i n mouse mammary cancer, J . Receptor Research 1: 91-111, 1980. Waxman, D.J., and Walsh, C., P h e n o b a r b i t a l - i n d u c e d r a t l i v e r cytochrome P-450 p u r i f i c a t i o n and c h a r a c t e r i z a t i o n of two c l o s e l y r e l a t e d i s o z y m i c forms, J . B i o l . Chem. 257: 10446-10457, 1982. Weaver, D., Amos, A., T i e r n e y , B., and B r e s n i c k , E., I n t e r a c t i o n of DNA w i t h c y t o s o l i c 3-methylcholanthrene b i n d i n g p r o t e i n s from e i t h e r r a t or mouse l i v e r , C a r i n o g e n e s i s 1: 481-486, 1980. W i l s o n , J.T., I d e n t i f i c a t i o n of somatotropin as the hormone i n a m i x t u r e o f somatotropin a d r e n o c o r t i c o t r o p i c hormone and p r o l a c t i n which decreased l i v e r drug metabolism i n the r a t , Biochem. Pharmacol. 18: 2029-2031, 1969. W i l s o n , J.T., A l t e r a t i o n o f normal development of drug metabolism by i n j e c t i o n of growth hormone, Nature 225: 861-863, 1970. W i l s o n , J.T., L i v e r drug metabolism i n -seraa to tropin, (STH) t r e a t e d r a t s : E f f e c t o f e n d o c r i n e n u t r i t i o n and s u b s t r a t e parameters, Fed.. Proc. 3 0 : 3 8 1 , 1 9 7 1 .  168  APPENDIX I  3 1)  A d d i t i o n a l s t u d i e s of  [ H ] - e s t r a d i o l b i n d i n g to the  r e c e p t o r and moderate a f f i n i t y  estrogen  b i n d i n g component i n r a t h e p a t i c  cytosol. *Assays were performed by Dr. B. Warren, F a c u l t y of S c i e n c e s , U n i v e r s i t y of B r i t i s h  The  data c o n t a i n e d  time c o u r s e and Results section.  Columbia.  i n t h i s s e c t i o n were p a r a l l e l  l i g a n d s p e c i f i c i t y s t u d i e s to those  a)  An  temperature  presented  These s t u d i e s were i n c l u d e d because of  importance i n d e f i n i n g o p t i m a l assay c o n d i t i o n s and binding  Pharmaceutical  estradiol  characteristics.  Determination  of o p t i m a l  i n c u b a t i o n time course was  i n c u b a t i o n time at  4°C  conducted at 4°C t o determine  [ H ] - e s t r a d i o l b i n d i n g to the h e p a t i c c y t o s o l i c  receptor  i n male and  female r a t s , and  componenf.:in:.the:;male. the e s t r o g e n  As  r e c e p t o r was  estrogen  the moderate a f f i n i t y  binding  shown i n F i g u r e A l , maximum b i n d i n g t o reached by two  n i n e t y minutes i n the male.  hours i n the female,  Binding to the.estrogen  s t a b l e f o r a t l e a s t f o u r hours i n both male and  receptor  found to be maximal by one  hour ( F i g u r e A2).  convenience a l l subsequent assays were incubated  and was  female r a t s .  S p e c i f i c e s t r a d i o l b i n d i n g to the male moderate a f f i n i t y  minutes.  their  hepatic  specific  was  i n the  component  Therefore, at 4°C  for  for ninety  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 b i n d i n g t o the e s t r o g e n  r e c e p t o r a t 4°C i n male and female r a t l i v e r , sulfate fraction.  50% ammonium  The b i n d i n g i n each sex was determined on  3 a s i n g l e t i s s u e p o o l a t 2 nM and  100-fold  [ H ] - e s t r a d i o l ligand concentration  excess e s t r a d i o l c o m p e t i t o r .  P o i n t s j o i n e d by a  dashed l i n e were determined i n more than 1 t i s s u e p o o l .  170  MCUBATON "nME (hours)  171  Figure  A2.  Time c o u r s e o f s p e c i f i c  3 [ H ] - e s t r a d i o l binding  t o t h e moderate  a f f i n i t y component a t 4°C i n male r a t l i v e r , whole c y t o s o l fraction.  The b i n d i n g  was determined on a s i n g l e t i s s u e p o o l  3 a t 200 nM  [ H ] - e s t r a d i o l ligand concentration  excess e s t r a d i o l c o m p e t i t o r .  Points  were determined i n more than 1 t i s s u e  and 1 0 0 - f o l d  j o i n e d by a dashed l i n e pool.  172  173  b)  Determination  of l i g a n d s p e c i f i c i t y  i n female r a t h e p a t i c  cytosol  The  a b i l i t y of v a r i o u s u n l a b e l e d  l i g a n d s to compete f o r  3 [ H ] - e s t r a d i o l b i n d i n g to the h e p a t i c c y t o s o l i c estrogen i n the ammonium s u l f a t e f r a c t i o n and  receptor  the moderate a f f i n i t y  component  i n the whole c y t o s o l f r a c t i o n were i n v e s t i g a t e d i n the female r a t . As  shown i n T a b l e 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 for  [ H ] - e s t r a d i o l b i n d i n g to t h e e s t r o g e n  sulfate fraction.  Moxestrol  e f f e c t i v e l y than e s t r a d i o l .  and  DES  receptor  i n the ammonium  competed s i m i l a r l y though l e s s  Androgens, p r o g e s t i n s and g l u c o c o r t i c o i d s  3 did not  compete f o r [ H ] - e s t r a d i o l b i n d i n g t o t h e h e p a t i c  estrogen  receptor  i n the female r a t (Table A l ) .  cytosolic  Competition  for  3 specific  [ H ] - e s t r a d i o l b i n d i n g i n the moderate a f f i n i t y range i s  shown i n T a b l e A2. unlabeled  W i t h the  exception  of e s t r a d i o l , none of  the  s t e r o i d s competed f o r r a d i o - l a b e l e d e s t r a d i o l b i n d i n g to  female r a t whole c y t o s o l . 2)  Determination  *Work was  of serum PRL  c a r r i e d out by Mr.  U n i v e r s i t y of B r i t i s h  Serum PRL and  l e v e l s by  bioassay  P. Gout, Department of  Biochemistry,  Columbia.  l e v e l s were determined by b i o a s s a y  in five control  f i v e p e r g o l i d (0.1 mg/kg/day, i . p . f o r eleven days) t r e a t  rats according  to the method of Tanaka et a l . , (1980)."''  female  T h i s assay  Tanaka, T., S h i u , R.P., Gout, P.W., Beer, C.T., Noble, R.L., and F r i e s e n , H.G., A new s e n s i t i v e and s p e c i f i c b i o a s s a y f o r l a c t o g e n i c hormones: measurement of p r o l a c t i n and growth hormone i n human serum, J . C l i n . E n d o c r i n o l . 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 b i n d i n g 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 a t 4°C w i t h [ 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 l i g a n d . 3  % of c o n t r o l binding"'" i n the presence of Competitor  e  10-fold  x  c  e  s  s  competitor  100-fold  1000-fold  control estradiol diethylstilbestrol moxestrol  100 63 61 79  100 36 61 64  100 24 63 64  testosterone dihydrotestosterone androstenedione methyltrienelone  100 100 100 100  100 100 100 100  100 100 100 100  progesterone promegestone  100 98  99 100  96 100  triamcinolone dexamethasone  100 100  100 100  100 100  The  b i n d i n g of  [ H ] - e s t r a d i o l i n the presence of competitor  as % of c o n t r o l v a l u e s competitor).  ( c o n t r o l = b i n d i n g i n the absence of  i s given  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 b i n d i n g s i t e s i n the whole c y t o s o l f r a c t i o n of l i v e r from female r a t s . Samples of whole c y t o s o l were incubated f o r 90 minutes at 4°C w i t h [ H ] - e s t r a d i o l (50 nM) alone or i n the presence of 10, 100, or 1 0 0 0 - f o l d excess of competing l i g a n d . 3  % of c o n t r o l b i n d i n g i n t h e presence o f excess competitor 1  Competitor 10-fold  100-fold  1000-fold  control estradiol diethylstilbestrol moxestrol  100 79 80 90  100 76 81 89  100 100 100 92  testosterone dihydrotestosterone an dr o s t en e d i on e methyltrienelone  100 100 100 99  100 100 100 97  100 100 100 100  progesterone promegestone  95 91  99 98  100 100  triamcinolone dexamethasone  99 98  98 100  100 100  The b i n d i n g of  [ H ] - e s t r a d i o l i n t h e presence of competitor  g i v e n as % of c o n t r o l v a l u e s of  competitor).  is  ( c o n t r o l = b i n d i n g i n the absence  176  a s s a y u t i l i z e s c e l l s from a t r a n s p l a n t a b l e lymphoma produced i n lymph nodes of e s t r o g e n i z e d Node c e l l s ) . the Nb  The  male Noble r a t s (and  b a s i s of t h i s b i o a s s a y  so d e s i g n a t e d  i s t h a t the growth of  2 Node lymphoma c e l l c u l t u r e i s s p e c i f i c a l l y  l a c t o g e n i c hormones, (Gout et a l . , 1980). Serum was  obtained  as d e s c r i b e d  stimulated  i n the methods s e c t i o n .  were as f o l l o w s .  in bioassay,  2 Node lymphoma c e l l s were t r a n s f e r r e d to  medium which was  supplemented w i t h f e t a l c a l f  (50 pg/ml) and  Details  Approximately 24 hours b e f o r e  serum (10%), 2-mercaptoethanol (.0.1 streptomycin  by  2  of the b i o a s s a y Nb  Nb2  mM),  incubated  Fischers  serum (1%) , horse  penicillin  (50 U/ml) ,  f o r 24 hours a t 37°C.  were then c o l l e c t e d b y . c e n t r i f u g a t i o n and  use  resuspended to 1-2  Cells x  10^  c e l l s / m l i n F i s c h e r s medium supplemented as above w i t h the e x c e p t i o n the f e t a l c a l f serum. dishes  Aliquots  f o r determination  (2 ml)  were t r a n s f e r r e d to c u l t u r e  of growth promoting a c t i v i t y .  assayed f o r growth promoting a c t i v i t y were added to the d i s h i n 50 p i p h o s p h a t e - b u f f e r e d s a l i n e t h a t contained albumin ( 0 . 1 % ) . 72 hours a t 37°C. counter  C u l t u r e s were incubated  (from 23.5  2  FL.).  zero f o l l o w i n g 72 hour incubat i o n .  ± 2.7  Sera to  t o 1.6  than 90% r e d u c t i o n  ± 0.3  ng/ml PRL)  be  culture bovine serum  incubator  C e l l numbers were determined u s i n g a  assay i n d i c a t e d a g r e a t e r  of  i n a CO2  (Counter E l e c t r o n i c s , Inc., H i a l e a h ,  c u l t u r e was  of  for  Coulter  Growth i n c o n t r o l  Results  from  i n serum PRL  f o l l o w i n g the  this  levels  administration  pergoiide.  Gout, P.W., Beer, C.T., and Noble, R.L., P r o l a c t i n - s t i m u l a t e d growth of c e l l c u l t u r e s e s t a b l i s h e d from malignant Nb r a t lymphomas, Cancer Res., 40: 2433-2436, 1980.  17-7  TABLE A3  S p e c i f i c a t i o n s o f p i t u i t a r y hormones used i n the c o n t i n u o u s i n f u s i o n s t u d i e s .  PERCENT CONTAMINATION (by weight)  HORMONE  **  Bovine Growth Hormone (bGH) NIH-bGH-B18 (.81 I.U./mg)  TSH LH PRL  6 .1% 3 .0%** 62 .0%**  Rat Growth Hormone (rGH) NIAMDD-rGH-B-7 (1.6 I.U./mg)  TSH LH FSH PRL  0 .1%* 0 .5%* 0 .5%* 0 .1%*  Rat P r o l a c t i n (rPRL) NIADDK-rPRL-B-4 (20.0 I.U./mg)  TSH LH FSH GH  0 .1%* 0 .5%* 0.5%* 1 .5%  Ovine P r o l a c t i n NIAMDD-oPRL-15  TSH LH FSH GH  0 .17* 0 .57* 0 .5%* 0 .5%*  (oPRL) (30.5 I.U./mg)  * Determined by radioimmunoassay **Determined by b i o a s s a y  Papers M.J. F i n l a y s o n * and G.D. B e l l w a r d , Introductory Remarks: Cytochrome P-450 Mixed Function Oxidases, Proc. West. Pharmacol. Soc. 23, 1-2 (1980). G.D. G.I. Drug NRC,  B e l l w a r d , R.C.K. Pak, L.S. Gontovnick, B.L. Warren, M.J.P. F i n l a y s o n , and Sunahara, Endogenous Mechanisms f o r the Regulation of Hepatic Microsomal and S t e r o i d Mixed Function Oxidases, i n "The Combined E f f e c t of X e n o b i o t i c s , pubs." pg 97-126 (1981).  M.J. Finlayson and G.D. B e l l w a r d , E f f e c t s of P e r i n a t a l Exposure o f Wistar Rats to l-tx-Acetylmethadol-HCl on Hepatic Microsomal Mixed Function Oxidase Enzyme A c t i v i t y i n the A d u l t , Drug Metab. Dispos., 10, 74-76 (1982). Betty L. Warren, Raphael Pak, Malcolm Finlayson, Larry Contovnick, Geoffrey Sunahara and G a l l D. Bellward, Different E f f e c t s 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 l n Hepatic Cytosol with Changes in Hepatic Monooxygenase A c t i v i t y i n 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 E f f e c t s of 1-oc-Acetylmethadol (LAAM) on Neonatal Androgen Induced Imprinting i n the Rat, Pharmacologist 22, 243 (1980). B.L. Warren, M. Finlayson, L. Gontovnick, R. Pak, G. Sunahara and G.D. Bellward, Microsomal Monooxygenase (MO) A c t i v i t i e s i n Genetically (BB) Diabetic Wistar Rats, Pharmacologist 23, 165 (1981). Raphael Pak, Malcolm Finlayson, Larry Gontovnick, Geoffery Sunahara, Betty Warren and G a i l Bellward, Effects of Hypophysectomy on Hepatic Microsomal Drug and Steroid Metabolizing Enzymes i n 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 A c t i v i t i e s i n Various Physiological Models, Can. Fed. B i o l . S c i . (Proc.) 25, 391 (1982). B.L. Warren, M.J. Finlayson* and G.D. Bellward, Characterization of High and Moderate A f f i n i t y Binding of Sex Hormones i n Rat Hepatic Cytosol, Can. Fed. B i o l . S c i . (Proc.) 25, 392 (1982). M.J. Finlayson* and G.D. Bellward, Hormonal Modulation of Rat Hepatic Monooxygenase A c t i v i t y : Possible Control Mechanisms, Fed. Am. Soc. Exp. B i o l . ( P r o c ) , Chicago, A p r i l (1983). * Presented by M.J. Finlayson  

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