UBC Theses and Dissertations

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UBC Theses and Dissertations

Characterization studies of the wistar rat hepatic cytosolic methlytrienolone (R1881) binding protein Sunahara, Geoffrey I. 1984

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I CHARACTERIZATION STUDIES OF THE WISTAR RAT HEPATIC CYTOSOLIC METHYLTRIENOLONE (R1881) BINDING PROTEIN By G e o f f r e y I . S u n a h a r a B.Sc. U n i v e r s i t y o f T o r o n t o , 1976 M.Sc. U n i v e r s i t y o f T o r o n t o , 1979 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES FACULTY OF PHARMACEUTICAL SCIENCES UNIVERSITY OF BRITISH COLUMBIA We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA J u l y , 1984 Cc) G e o f f r e y I . S u n a h a r a , 1984 I n 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 o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e 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 c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e head o f my d e p a r t m e n t o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g 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 g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . G e o f f r e y I. Sunahara 208pQg§S&ax^ o f Pharmaceutical Sciences. The U n i v e r s i t y o f B r i t i s h C o l u m b i a 1956 Main M a l l V a n c o u v e r , Canada V6T 1Y3 Date August 29, 1984 ABSTRACT Age and sex d i f f e r e n c e s i n a d u l t r a t h e p a t i c m i c r o s o m a l d r u g m e t a b o l i s m a r e w e l l - k n o w n . A l t h o u g h t h e e x a c t mechanism i s u n c l e a r , t h e r o l e o f t e s t o s t e r o n e has been c l e a r l y i m p l i c a t e d . S i n c e a n d r o g e n s e n s i t i v e sex e n d - o r g a n t i s s u e s a r e known t o c o n t a i n s p e c i f i c a n d r o g e n r e c e p t o r s w h i c h m e d i a t e t h e a c t i o n o f t e s t o s t e r o n e i n t h e s e t i s s u e s , a s i m i l a r mechanism may be i n v o l v e d i n t e s t o s t e r o n e s e n s i t i v e h e p a t i c m i c r o s o m a l d r u g m e t a b o l i s m . To t e s t t h i s h y p o t h e s i s , t e s t o s t e r o n e s e n s i t i v e l i v e r m i c r o s o m a l b e n z o ( a ) p y r e n e h y d r o x y l a s e a c t i v i t i e s and c y t o s o l i c a n d r o g e n b i n d i n g p r o t e i n l e v e l s were measured i n v i t r o , u s i n g d i f f e r e n t a n i m a l m o d e l s . M e t h y l t r i e n o l o n e (R1881) was us e d as t h e l i g a n d f o r t h e c y t o s o l i c a n d r o g e n b i n d i n g a s s a y , s i n c e t e s t o s t e r o n e was s i g n i f i c a n t l y m e t a b o l i z e d i n v i t r o . S c a t c h a r d a n a l y s i s o f a d u l t male r a t l i v e r whole c y t o s o l i n d i c a t e d two R1881 b i n d i n g i s o t h e r m s : a h i g h a f f i n i t y - l o w c a p a c i t y b i n d i n g s i t e , and a l o w e r a f f i n i t y - h i g h e r c a p a c i t y component. A d d i t i o n o f e x c e s s t r i a m c i n o l o n e o r C o r t i s o l t o t h e a s s a y m i x t u r e e l i m i n a t e d the l o w e r a f f i n i t y b i n d i n g component. The h i g h a f f i n i t y component was n o t p r e s e n t i n t h e immature f e m a l e o r t h e i n t a c t o r the 10-day o v a r i e c t o m i z e d a d u l t f e m a l e r a t ; i t was p r e s e n t i n low g u a n t i t i e s i n t h e immature m a l e . I n h i b i t i o n s t u d i e s o f t h e h i g h a f f i n i t y component u s i n g d i f f e r e n t c o m p e t i t o r s i n d i c a t e d a h i g h s p e c i f i c i t y t o a n d r o g e n s , i n c l u d i n g t e s t o s t e r o n e , d i h y d r o t e s t o s t e r o n e , a n d r o s t e n e d i o n e , R1881 and m i b o l e r o n e , a moderate s p e c i f i c i t y t o c y p r o t e r o n e a c e t a t e , e s t r a d i o l and f l u t a m i d e h y d r o x i d e , and no s p e c i f i c i t y t o p r o g e s t e r o n e , t r i a m c i n o l o n e , d i e t h y l s t i l b e s t r o l and C o r t i s o l . S a t u r a t i o n a n a l y s i s o f t h e h i g h a f f i n i t y component i n d i c a t e d t h a t t e s t o s t e r o n e , d i h y d r o t e s t o s t e r o n e , a n d r o s t e n e d i o n e , m i b o l e r o n e , c y p r o t e r o n e a c e t a t e and e s t r a d i o l p r o d u c e d s i m i l a r b i n d i n g k i n e t i c s , i n d i c a t i n g t h a t t h e s e s t e r o i d s were b i n d i n g t o t h e same o r a s i m i l a r h i g h a f f i n i t y s i t e . F l u t a m i d e h y d r o x i d e y i e l d e d d i s p l a c e a b l e b u t n o n - s a t u r a b l e b i n d i n g . C a s t r a t i o n o f a d u l t male r a t s f o r 18 h r s , 4 o r 10 d a y s , o r hypophysectomy f o r 10-17 days d i d n o t have a s i g n i f i c a n t e f f e c t on t h e h i g h a f f i n i t y b i n d i n g p r o t e i n k i n e t i c s compared t o c o n t r o l s . Nor d i d t e s t o s t e r o n e a d m i n i s t r a t i o n t o t h e s e a n i m a l s a l t e r t h i s p r o t e i n ' s b i n d i n g k i n e t i c s . F l u t a m i d e o r f l u t a m i d e h y d r o x i d e t r e a t m e n t t o t e s t o s t e r o n e - t r e a t e d a d u l t male r a t s s i g n i f i c a n t l y r e d u c e d b o t h l i v e r m i c r o s o m a l b e n z o ( a ) p y r e n e h y d r o x y l a s e a c t i v i t i e s and h i g h a f f i n i t y b i n d i n g p r o t e i n l e v e l s compared t o t h e f l u t a m i d e u n t r e a t e d c o n t r o l s . S t r e p t o z o t o c i n t r e a t m e n t t o a d u l t m a l e s c a u s e d a marked d e c r e a s e i n t h e h i g h a f f i n i t y b i n d i n g p r o t e i n l e v e l s . T h e s e r e s u l t s i n d i c a t e t h a t t h e p r e s e n c e o f t h e h i g h a f f i n i t y component i n t h e a d u l t male r a t i s n o t d e p e n d e n t upon g o n a d a l o r p i t u i t a r y hormones i n v i v o , b u t i t i s a l t e r e d f o l l o w i n g f l u t a m i d e , f l u t a m i d e h y d r o x i d e o r s t r e p t o z o t o c i n a d m i n i s t r a t i o n . T h e s e s t u d i e s do n o t n e g a t e t h e h y p o t h e s i s t h a t t h e p r e s e n c e as w e l l asf t h e a g o n i s t o c c u p a n c y o f t h e h i g h a f f i n i t y - l o w c a p a c i t y a n d r o g e n b i n d i n g p r o t e i n a r e p r e r e q u i s i t e s f o r t e s t o s t e r o n e - s e n s i t i v e e f f e c t s i n t h e l i v e r . G a i l B. B e l l w a r d , Ph. D. S u p e r v i s o r V TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS v LIST OF TABLES x i i L IST OF FIGURES x v L I S T OF ABBREVIATIONS x v i i ACKNOWLEDGEMENTS x x i INTRODUCTION 1 1. Age and s e x - d e p e n d e n t h e p a t i c m i c r o s o m a l d r u g and s t e r o i d m e t a b o l i s m 1 2 . The r o l e o f t e s t o s t e r o n e i n h e p a t i c m i c r o s o m a l d r u g and s t e r o i d m e t a b o l i s m 4 3 . The r o l e o f an androgen r e c e p t o r i n h e p a t i c m i c r o s o m a l d r u g and s t e r o i d m e t a b o l i s m . . . 7 a . I n d i r e c t e f f e c t s o f t e s t o s t e r o n e on h e p a t i c m i c r o s o m a l d r u g and s t e r o i d m e t a b o l i s m 8 b . D i r e c t e f f e c t s o f t e s t o s t e r o n e on h e p a t i c m i c r o s o m a l d r u g and s t e r o i d m e t a b o l i s m 13 i . I_n v i v o e v i d e n c e 13 i i . In . v i t r o e v i d e n c e 16 4 . Summary 30 5 . O b j e c t i v e s o f t h i s s t u d y 32 METHODS AND MATERIALS 1. Chemica l s 2. Care and surgery o f an imal s 3 . Treatment of animals 4. S u b c e l l u l a r f r a c t i o n a t i o n a . P r e p a r a t i o n o f microsomes t o r enzyme s t u d i e s b . P r e p a r a t i o n of c y t o s o l and microsomes t o r r a d i o - l i g a n d b i n d i n g s t u d i e s 5. H e p a t i c microsomal enzyme assays a . H e p a t i c microsomal benzo(a )pyrene h y d r o x y l a s e assay b . H e p a t i c microsomal t e s t o s t e r o n e r e d u c t a s e assay 6 . Other assays 7. T e s t o s t e r o n e metabol i sm s t u d i e s a . P r e p a r a t i o n o f s t andard s t e r o i d s o l u t i o n s and c y t o s o l e x t r a c t s f o r chromatography . . b . S e p a r a t i o n o f t e s t o s t e r o n e from r e l a t e d m e t a b o l i t e s u s i n g t h i n - l a y e r chromatography (TLC) c . V i s u a l i z a t i o n o f t e s t o s t e r o n e and r e l a t e d m e t a b o l i t e s u s i n g UV l i g h t and c o l o u r reagent s v i i Page d. I d e n t i f i c a t i o n of testosterone and 5g-androstan-3a,17$-dioi using Gas Chromatography-Mass Spectrometry (GCMS) . 46 e. Analysis of bound trit i a t e d - t e s t o s t e r o n e and metabolites using TLC 48 8. Radio-ligand exchange binding studies 48 a. Incubation of cytosol and microsomes 4 ^ b. Separation of bound and tree steroid tractions 49 c. Sephadex G-25M mini-column chromatography 50 9. Analysis of r a d i o a c t i v i t y 51 10. S t a t i s t i c a l analysis 52 RESULTS 5 3 1. E f f e c t of testosterone enanthate and flutamide administration on serum testosterone l e v e l s , tissue:body weight r a t i o s and hepatic microsomal enzyme a c t i v i t i e s 53 a. Serum testosterone levels 53 b. Tissue:body weight (BW) r a t i o s 5 7 c. Hepatic microsomal drug and steroid metabolizing enzyme a c t i v i t i e s ^4 Development of the hepatic c y t o s o l i c testosterone binding assay a. Separation of bound vs. free steroids b. Optimization of the c y t o s o l i c testosterone binding protein assay i . E f f e c t of varying the protein concentrations on testosterone binding in adult male hepatic cytosol i i . E f f e c t of varying incubation time and temperature on testosterone binding i n adult male rat l i v e r cytosol and microsomes i i i . Displacement of t r i t i a t e d - t e s t o s t e r o n e binding by various competitors i n adult rat l i v e r whole cytosol and microsomes Testosterone metabolism studies a. Separation and detection of testosterone and related steroid standards using t h i n -layer chromatography (TLC) b. Separation and detection of testosterone and i t s metabolites from cytosol extract using TLC I X 92 1 0 0 Page c. I d e n t i f i c a t i o n of testosterone and related d i o l - d e r i v a t i v e s using gas chromatography-mass spectrometry (GCMS).... d. Formation of radiolabelled testosterone metabolites i n l i v e r whole cytosol i n v i t r o  Hepatic c y t o s o l i c methyltrienolone (R1881) binding assay 104 a. Ettect of protein d i l u t i o n and time course on incubation of R1881 binding i n adult 1 no male rat l i v e r whole cytosol u o b. Computer analysis of binding data c. E f f e c t of triamcinolone acetonide alone and i n combination with C o r t i s o l on R1881 binding i n rat l i v e r whole cytosol 119 d. Displacement of tritiated-R1881 trom c y t o s o l i c R1881 binding s i t e s using various s t e r o i d a l and non-steroidal competitors e. Separation of c y t o s o l i c protein bound and free steroid fractions f. Optimization of the hepatic c y t o s o l i c androgen binding assay 1 3 4 Characterization of the hepatic c y t o s o l i c high a t t i n i t y - l o w capacity (HALC) androgen binding protein 127 1 3 3 1 3 5 Page In v i t r o experiments i . Displacement of HALC R1881 binding i n male rat l i v e r whole cytosol •'•35 i i . R1881 saturation study using d i f f e r e n t competitors 1 1 1 . Tritiated-Mibolerone binding i n adult male rat cytosol 1^2 Different physiological and pharmacological animal models -L45 i . Age and sex differences i n R1881 binding i n male and female rat l i v e r cytosol i i . E f f e c t of gonadectomy on adult male and female rat l i v e r cytosol HALC R1881 binding protein 1 5 1 i i i . E f f e c t of hypophysectomy and testosterone enanthate administration on adult male rat l i v e r cytosol HALC R1881 binding protein 1 5 3 i v . E f f e c t of flutamide and flutamide hydroxide treatment on testosterone enanthate-treated castrated adult male rat l i v e r c y t o s o l i c HALC R1881 binding protein 1 5 4 x i Page v . E f f e c t o f s t r e p t o z o t o c i n (STZ) and p r o t a m i n e z i n c i n s u l i n (PZI) t r e a t m e n t on l i v e r c y t o s o l i c HALC R1881 b i n d i n g i n t h e a d u l t male r a t 156 DISCUSSION -L59 1. Age and sex d i f f e r e n c e s i n h e p a t i c m i c r o s o m a l d r u g and s t e r o i d m e t a b o l i s m 159 2 . T e s t o s t e r o n e m e t a b o l i s m i n v i t r o 164 3. C h a r a c t e r i z a t i o n o f t h e 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 165 FUTURE EXPERIMENTS 1 7 4 SUMMARY 1 7 9 BIBLIOGRAPHY 1 8 3 x i i L I S T OF TABLES Page 1. E r r e c t o f f l u t a m i d e on serum t e s t o s t e r o n e l e v e l s 56 2. E l f e c t o f f l u t a m i d e and t e s t o s t e r o n e t r e a t m e n t on l i v e r :body w e i g h t r a t i o s 6° 3. E t f e c t o f t e s t o s t e r o n e t r e a t m e n t on h e p a t i c m i c r o s o m a l b e n z o ( a ) p y r e n e h y d r o x y l a s e and t e s t o s t e r o n e r e d u c t a s e a c t i v i t i e s 63 4. S e p a r a t i o n t e c h n i q u e s f o r p r o t e i n bound and unbound s t e r o i d s 72 5. E f f e c t o f v a r y i n g p r o t e i n c o n t e n t on a d u l t male r a t l i v e r c y t o s o l i c t e s t o s t e r o n e b i n d i n g 75> 6. E f f e c t o f v a r y i n g i n c u b a t i o n p e r i o d on t e s t o s t e r o n e and DHT b i n d i n g i n l i v e r c e l l u l a r f r a c t i o n s 7 7 7. E t t e c t o f v a r y i n g i n c u b a t i o n t e m p e r a t u r e on a d u l t male r a t l i v e r c y t o s o l i c and m i c r o s o m a l t e s t o s t e r o n e b i n d i n g 7 ^ 8. D i s p l a c e m e n t o f t r l t l a t e d - t e s t o s t e r o n e t r o m l i v e r c y t o s o l i c and m i c r o s o m a l f r a c t i o n s by v a r i o u s c o m p e t i t o r s 81 (A-D). Single point displacement studies of t r i t i a t e d - t e s t o s t e r o n e by various competitors i n l i v e r c y t o s o l i c and microsomal fractions Separation of testosterone and related metabolites using thin-layer chromatography Separation of ethereal extracts taken from l i v e r cytosol incubated tor d i f f e r e n t time int e r v a l s and at various temperatures with testosterone and 53-DHT Comparison of linear and non-linear regression analysis of binding data Eff e c t of varying excess molar ra t i o s of triamcinolone acetonide or C o r t i s o l on R1881 binding i n adult male rat l i v e r whole cytosol... Single point displacement studies of 8 nM tritiated-R1881 by various competitors in l i v e r c y t o s o l i c fractions Single point displacement studies of 1 nM tritiated-R1881 by various competitors in l i v e r c y t o s o l i c fractions Different methods to separate bound and tree steroid fractions I n h i b i t i o n study of l i v e r c y t o s o l i c R1881 binding by various competitors i n adult male rat X I V Page 18. B i n d i n g k i n e t i c s o f l i v e r c y t o s o l i c R1881 b i n d i n g p r o t e i n by v a r i o u s c o m p e t i t o r s 19. Age d i f f e r e n c e s between male and female r a t l i v e r h i g h a f f i n i t y - l o w c a p a c i t y R1881 b i n d i n g p r o t e i n 148 20. E f f e c t o f c a s t r a t i o n and t e s t o s t e r o n e rep lacement on l i v e r c y t o s o l i c h i g h a f f i n i t y - l o w c a p a c i t y R1881 b i n d i n g p r o t e i n 150 21. E f f e c t o f hypophysectomy and t e s t o s t e r o n e rep lacement on l i v e r c y t o s o l i c h i g h a f t m i t y -low c a p a c i t y R1881 b i n d i n g p r o t e i n 152 22. E t f e c t o f f l u t a m i d e and f l u t a m i d e h y d r o x i d e a d m i n i s t r a t i o n on l i v e r c y t o s o l i c h i g h a t t i n i t y - l o w c a p a c i t y R1881 b i n d i n g p r o t e i n 155 23. E f f e c t o f s t r e p t o z o t o c i n and protamine z i n c i n s u l i n rep lacement on l i v e r c y t o s o l i c h i g h a f f i n i t y - l o w c a p a c i t y R1881 b i n d i n g p r o t e i n 157 XV L I S T OF FIGURES Page 1. E f f e c t o f t e s t o s t e r o n e e n a n t h a t e on serum t e s t o s t e r o n e l e v e l s i n sham-opera ted and c a s t r a t e d a d u l t male r a t s 55 2 . E f f e c t o f t l u t a m i d e on p r o s t a t e t o body w e i g h t r a t i o s i n a d u l t male r a t 59 3. E f f e c t o f f l u t a m i d e on h e p a t i c b e n z o ( a ) p y r e n e h y d r o x y l a s e i n t e s t o s t e r o n e - t r e a t e d c a s t r a t e d a d u l t male and i n t a c t a d u l t f emale r a t s 62 4 . E t t e c t o f t l u t a m i d e on h e p a t i c m i c r o s o m a l t e s t o s t e r o n e r e d u c t a s e i n t e s t o s t e r o n e - t r e a t e d c a s t r a t e d a d u l t male and i n t a c t a d u l t f e m a l e r a t s 67 5 . E t t e c t o f t l u t a m i d e and f l u t a m i d e h y d r o x i d e on h e p a t i c m i c r o s o m a l b e n z o ( a ) p y r e n e h y d r o x y l a s e and t e s t o s t e r o n e r e d u c t a s e i n t h e i n t a c t a d u l t f e m a l e r a t 7 0 6. ( A - C ) . Gas chromatography-mas s s p e c t r o m e t r y (GCMS) a n a l y s i s o f t e s t o s t e r o n e and t e s t o s t e r o n e m e t a b o l i t e s t a k e n f rom l i v e r c y t o s o l e x t r a c t 7 . ( A & B ) . T h i n - l a y e r r a d i o c h r o m a t o g r a m o f t e s t o s t e r o n e m e t a b o l i s m i n v i t r o i n a d u l t male r a t l i v e r w h o l e c y t o s o l 8 . (A&B) . T h i n - l a y e r c h r o m a t o g r a p h i c a n a l y s i s o f c y t o s o l i c t e s t o s t e r o n e m e t a b o l i s m i n v i t r o 106 97 102 x v i Page 9 . E t f e c t o f v a r y i n g t h e p r o t e i n c o n c e n t r a t i o n on c y t o s o l i c R1881 b i n d i n g i n t h e a d u l t male r a t l i v e r H O 1 0 . ( A - C ) . E f f e c t o f v a r y i n g p r o t e i n c o n c e n t r a t i o n s and i n c u b a t i o n t i m e i n t e r v a l s on l i v e r c y t o s o l i c R1881 b i n d i n g p r o t e i n ' . H 2 1 1 . (A&B) . Computer a n a l y s i s o f R1881 b i n d i n g d a t a 117 1 2 . (A&B) . E f f e c t o f 10 mM p o t a s s i u m c h l o r i d e on l i v e r c y t o s o l i c R1881 b i n d i n g i n a d u l t male r a t 124 1 3 . (A&B) . S p e c i f i c i t y s t u d i e s f o r a d u l t male r a t l i v e r c y t o s o l i c h i g h a f f i n i t y - l o w c a p a c i t y R1881 b i n d i n g p r o t e i n 137 14 . S c a t c h a r d p l o t f o r a d u l t male r a t h e p a t i c c y t o s o l i c t r i t i a t e d - m i b o l e r o n e b i n d i n g p r o t e i n 144 1 5 . Sex d i f f e r e n c e s i n a d u l t r a t h e p a t i c c y t o s o l i c R1881 b i n d i n g p r o t e i n 147 LIST OF ABBREVIATIONS ACTH A l d o s t e r o n e a l p h a 5 a l p h a / b e t a - A - 3 a l p h a / b e t a , 17 b e t a - d i o l Andros tened ione be ta bGH Bmax BP BPH C o r t i s o l C y p r o t e r o n e a c e t a t e DES DCC DHT EDTA a d r e n o c o r t i c o t r o p i c hormone 3 , 2 0 - d i k e t o - l l B , 1 8 - o x i d o -4 - p r e g n e n e - 1 8 , 2 1 - d i o i Greek l e t t e r (a) 5 a / g - a n d r o s t a n - 3 a / B , 1 7 B - d i o l A - 4 - a n d r o s t e n - 3 , 1 7 - d i o n e Greek l e t t e r (e) bov ine growth hormone maximum b i n d i n g c a p a c i t y b e n z o ( a ) p y r e n e ; 3 ,4-benzpyrene benzo(a )pyrene h y d r o x y l a s e h y d r o c o r t i s o n e ; 11B,17B, 2 1 - t r i h y d r o x y - 4 - p r e g n e n e - 3 , 20-dione 17 ot-acety l o x y - 6 - c h l o r o - l , 2-d i h y d r o - ( l B , 2 6 ) - 3 ' H -c y c l o p r o p a - ( 1 , 2 ) p r e g n a - 1 , 4 , 6 -t r i e n e - 3 , 2 0 - d i o n e d i e t h y l s t i l b e s t r o l d e x t r a n coated c h a r c o a l d i h y d r o t e s t o s t e r o n e ; 5a /B- a n d r o s t - 2 - e n , 17B-o l 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 E s t r a d i o l F FSH Flutamide Flutamide hydroxide fmol/mg Gx HALC Hypox IP Kd Ki LAHC LH Methy1trienolone Mibolerone MSTFA 3-MC MOX x v i i i l,3,5-estratriene-3,17 B-diol free steroid concentration f o l l i c l e stimulating hormone a,a,a,-trifluoro-2 -methyl-4'-nitro-m-propiono-tol u i d i d e a, a, a , - t r i f luoro-2-methyl-2-hydroxy-4 1-nitro-m-propionotoluidide femtomoles per milligram castrated high a f f i n i t y - l o w capacity hypophysectomized in t r a p e r i t o n e a l l y equi librium .^dissociation constant equilibrium i n h i b i t i o n constant low a f f i n i t y - h i g h capacity l u t e i n i z i n g hormone 17 B-hydroxy-17-methylestra-4,9,ll-trien-3-one 7a,17a-dimethyl-19-nortestosterone N-methyl-N-trimethylsilyl trifluoroacetamide 3-methylcholanthrene methoxyamine reagent NADPH r e d u c e d n i c o t i n a m i d e a d e n i n e . d i n u c l e o t i d e p h o s p h a t e NAD n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e Ovx o v a r i e c t o m i z e d P r o g e s t e r o n e A-4 - p r e g n e n e - 3 , 2 0 - d i o n e Ps p s e u d o h e r m a p h r o d i t e male PZI p r o t a m i n e z i n c i n s u l i n rGH r a t g r o w t h hormone R1881 m e t h y l t r i e n o l o n e RU23908 5 , 5 - d i m e t h y l - 3 - ( 4 - n i t r o - 3 - t r i f l u o r o - m e t h y l ) p h e n y l ) -i m i d a z o l i d i n e - 2 , 4 - d i o n e RU2956 1 7 6 - h y d r o x y - 2 , 2 , 1 7 a - t r i m e t h y l - e s t r a - 4 , 9 , 1 1 - t r i e n -3-one SB s p e c i t i c b i n d i n g sc s u b c u t a n e o u s l y S p i r o n o l a c t o n e 1 7 - h y d r o x y - 7 - m e r c a p t o - 3 - o x o -1 7 a - p r e g n-4 - e n e -2 1 - c a r b o x y l i c a c i d gamma l a c t o n e , 7 - a c e t a t e STZ s t r e p t o z o t o c i n S t r e p t o z o t o c i n 2 - d e o x y - 2 - ( ( m e t h y - n i t r o s o a m i n o ) c a r b o n y l ) - a m i n o ( - D - g l u c o p y r a n o s e ) TCDD TE TLC TR T e s t o s t e r o n e T r i a m c i n o l o n e a c e t o n i d e 2 , 3 , 7 , 8 - t e t r a c h l o r o d i b e n z o -p - d i o x i n t e s t o s t e r o n e e n a n t h a t e t h i n - l a y e r c h r o m a t o g r a p h y t e s t o s t e r o n e r e d u c t a s e 4 - a n d r o s t e n - 3 - o n e - 1 7 B - o l 9 a - f l u o r o - H B , 16a , 1 7 , 2 1 - t e t r a - o l - p r e g n a - l , 4 - d i e n e - 3 , 2 0 - d i o n e c y c l i c 17 a c e t a l w i t h a c e t o n e ACKNOWLEDGEMENTS I w i s h t o e x p r e s s my s i n c e r e a p p r e c i a t i o n t o my s u p e r v i s o r , P r o f e s s o r G a i l D. B e l l w a r d f o r h e r s t a l w a r d p a t i e n c e , i n s i g h t and g u i l d a n c e t h r o u g h o u t t h e c o u r s e of t h i s t h e s i s . I wo u l d a l s o w i s h t o thank t h e members o f c o m m i t t e e , t h e p r e v i o u s members o f o u r l a b o r a t o r y ( D r s . R.C.K. Pak, L.S. G o n t o v n i c k , B.L. Warren and M.J.P. F i n l a y s o n ) and Mr. A.B. F a w z i , f o r t h e i r s u p p o r t and v a l u a b l e s u g g e s t i o n s . 1 INTRODUCTION 1. Age and s e x - d e p e n d e n t h e p a t i c m i c r o s o m a l d r u g and s t e r o i d m e t a b o l i s m I t has been w e l l - d o c u m e n t e d t h a t i n c e r t a i n s p e c i e s , age and sex d i f f e r e n c e s a r e two f a c t o r s w h i c h i n f l u e n c e t h e r a t e of m e t a b o l i s m o f c e r t a i n d r u g s , and t h e r e f o r e a l t e r t h e > l a t t e r ' s d u r a t i o n and i n t e n s i t y o f a c t i o n . H o i c k e t a l . (1937) were one of t h e e a r l i e s t g r o u p s t o r e p o r t sex d i f f e r e n c e s i n t h e a n a e s t h e t i c a c t i o n o f b a r b i t u r a t e s on t h e r a t ; t h e y o b s e r v e d t h a t t h e d u r a t i o n of b a r b i t u r a t e a n a e s t h e s i a ( h e x o b a r b i t a l , a m o b a r b i t a l o r p h e n o b a r b i t a l ) i n the a d u l t f e m a l e r a t was l o n g e r t h a n t h a t o f t h e a d u l t m a l e . Endogenous s e c r e t i o n o f t e s t o s t e r o n e i n t h e a d u l t male was t h o u g h t t o be r e s p o n s i b l e f o r t h e sex d i f f e r e n c e s i n c e t h e s l e e p i n g t i m e s o f t h e c a s t r a t e d a d u l t male were s i m i l a r t o t h a t o f t h e i n t a c t a d u l t f e m a l e . F u r t h e r , t e s t o s t e r o n e r e p l a c e m e n t t o t h e c a s t r a t e r e - e s t a b l i s h e d t h e sex d i f f e r e n c e . F o l l o w i n g t h i s e a r l y r e p o r t , numerous w o r k e r s have c o n f i r m e d t h a t sex d i f f e r e n c e s e x i s t i n t h e r a t and o t h e r mammalian h e p a t i c d r u g m e t a b o l i z i n g s y s t e m s . F o r example, o b s e r v a t i o n s have been made u s i n g r a t s ( Q u i n n e t a l . 1 9 58), i n c e r t a i n s t r a i n s o f m ice (Conney 1967; Brown e t a l . 1978 ), and i n t h e t u p a i a o r t h e t r e e shrew ( L i t t e r s t e t a l . 1 9 7 6 ) . A d u l t male r a t s a r e known t o r e g u i r e h i g h e r d o s e s o f c e r t a i n d r u g s s u c h as h e x o b a r b i t a l , i n o r d e r t o show e q u i p o t e n t a n a e s t h e t i c e f f e c t s compared t o f e m a l e s of 2 e q u a l age ( Q u i n n e t a l . 1 9 5 8 ) . However, C a s t r a and G i l l e t t e ( 1 9 6 7 ) , L i t t e r s t e t a l . (1976) and o t h e r w o r k e r s have f o u n d t h a t many l a b o r a t o r y a n i m a l s p e c i e s , s u c h as t h e Rhesus and t h e s q u i r r e l monkey, t h e g u i n e a p i g and t h e New Z e a l a n d r a b b i t e x h i b i t no sex d i f f e r e n c e s i n d r u g m e t a b o l i s m . I t has been s u g g e s t e d by G u i d i c e l l i and T i l l e m e n t (1977) t h a t s e x d i f f e r e n c e s i n d r u g m e t a b o l i s m and a c t i o n may o c c u r i n man. Sex d e p e n d e n c y does not o c c u r i n a l l r a t h e p a t i c c y t o c h r o m e P-450 d e p e n d e n t m i c r o s o m a l d r u g m e t a b o l i z i n g enzyme s y s t e m s . A c t i v i t i e s o f a n i l i n e , z o x a z o l a m i n e and b i p h e n y l h y d r o x y l a s e i n t h e r a t and o t h e r s p e c i e s ( e x c l u d i n g t h e t u p a i a ) a r e s e x - i n d e p e n d e n t ( E l Defrawy E l Masry e t a l . 1974; L i t t e r s t e t a_l. 1976), w h i l e e t h y l m o r p h i n e , a m i n o p y r i n e N - d e m e t h y l a s e , and b e n z o ( a ) p y r e n e h y d r o x y l a s e (BPH) have been o b s e r v e d t o be s e x - d e p e n d e n t enzymes ( L i t t e r s t e t a l . 1976; G u r t o o and P a r k e r 1977; G o n t o v n i c k and B e l l w a r d 1 9 8 0 ) . A l t h o u g h sex d e p endency i n d r u g m e t a b o l i s m does n o t o c c u r i n a l l mammalian s p e c i e s , i t s measurement may depend on t h e s e n s i t i v i t y o f t h e a s s a y , t h e a p p r o p r i a t e a n i m a l model and t h e enzyme s y s t e m . E l Defrawy E l Masry e t a l . (1974) showed t h a t some h e p a t i c m i c r o s o m a l d r u g m e t a b o l i z i n g enzyme a c t i v i t i e s a r e a l s o d e p e n d e n t upon t h e a n i m a l ' s age, as would be p r e d i c t e d by c h a n g e s i n sex hormones. Immature male and f e m a l e r a t s had v e r y s i m i l a r r a t e s o f c y t o c h r o m e P-450 d e p e n d e n t h e p a t i c m i c r o s o m a l e t h y l m o r p h i n e d r u g m e t a b o l i s m , b u t as t h e s e 3 a n i m a l s aged, t h e a c t i v i t y o f t h i s enzyme ( e t h y l m o r p h i n e N - d e m e t h y l a s e ) i n t h e male r a t i n c r e a s e d r e l a t i v e t o t h e immature male and f e m a l e o f t h e same age, o r t h e immature f e m a l e . T h e s e r e s u l t s were c o n f i r m e d by G o n t o v n i c k and B e l l w a r d ( 1 9 8 0 ) , who measured a d u l t and immature male r a t l i v e r m i c r o s o m a l BPH enzyme a c t i v i t i e s . Age d i f f e r e n c e s i n enzyme a c t i v i t i e s have a l s o been d e m o n s t r a t e d by the i n h i b i t i o n of t h e c o n s t i t u t i v e c y t o c h r o m e P-450 d e p e n d e n t BPH enzyme a c t i v i t i e s i n v i t r o ( W i e b e l and G e l b o i n 1975). P o s t - n a t a l (6 d a ys a f t e r b i r t h ) f e m a l e and male BPH a c t i v i t i e s were s t i m u l a t e d i n v i t r o by 7 , 8 - b e n z o f l a v o n e , b u t were d i m i n i s h e d as t h e a n i m a l s grew o l d e r , and by day 20 t h e f l a v o n e a c t u a l l y i n h i b i t e d t h i s enzyme's a c t i v i t y . A f t e r day 20 however, c o i n c i d e n t w i t h t h e o n s e t o f r a t p u b e r t y , o n l y t h e enzyme f r o m t h e f e m a l e r a t c o n t i n u e d t o be i n h i b i t e d by t h e f l a v o n e , w h i l e t h a t f r o m t h e male r a t became r e s i s t a n t t o i n h i b i t i o n . T h e s e d a t a s u g g e s t t h a t t h i s enzyme a c t i v i t y i s a l t e r e d d u r i n g d e v e l o p m e n t , c o i n c i d e n t w i t h h o r m o n a l c h a n g e s . P e l k o n e n e t a l . (1973) showed t h a t human f e t a l (9-22 weeks o l d ) l i v e r m i c r o s o m e s m e t a b o l i z e d s u c h d r u g s as t e s t o s t e r o n e , a n i l i n e , e t h y l m o r p h i n e and a m i n o p y r i n e a t r a t e s a p p r o x i m a t i n g 30-35% of a d u l t l e v e l s ( p e r gram l i v e r w e i g h t ) . A ge-dependency of c e r t a i n enzyme a c t i v i t i e s i s t h e r e f o r e a phenomenon common t o b o t h man and o t h e r a n i m a l s . T h i s does n o t p r e c l u d e t h a t a common mechanism i s i m p l i c a t e d i n a l l s p e c i e s . D i f f e r e n c e s i n h o r m o n a l s t a t u s would make c o m p a r i s o n s and 4 i n t e r p r e t a t i o n s o f human and a n i m a l d a t a most d i f f i c u l t . N e v e r t h e l e s s , age and sex d i f f e r e n c e s t y p i f y i n g i n d u c t i o n o f h e p a t i c m i c r o s o m a l enzymes by s t e r o i d hormones and by d r u g s a r e i m p o r t a n t b o t h p h a r m a c o l o g i c a l l y and p h y s i o l o g i c a l l y . 2. The r o l e o f t e s t o s t e r o n e i n h e p a t i c m i c r o s o m a l d r u g and s t e r o i d m e t a b o l i s m I t i s not t h e p u r p o s e o f t h i s r e v i e w t o e x t e n s i v e l y c o v e r t h e p h y s i o l o g i c a l r o l e o f t e s t o s t e r o n e i n p r i m a r y and s e c o n d a r y s e x u a l d e v e l o p m e n t ; t h i s i n f o r m a t i o n c a n be f o u n d e l s e w h e r e (Lee and L a y c o c k 1978; T r i b e and E r a u t 1 9 7 9 ) . Of i t s v a r i o u s e f f e c t s , t e s t o s t e r o n e may be i n v o l v e d i n t h e r e g u l a t i o n o f s e x - d e p e n d e n t h e p a t i c m i c r o s o m a l c y t o c h r o m e P-450 d e p e n d e n t enzyme a c t i v i t i e s i n t h e r a t . C a s t r a t i o n of t h e a d u l t male r a t i m p a i r e d h e p a t i c m i c r o s o m a l a m i n o p y r i n e m e t a b o l i s m ( K a t o and G i l l e t t e 1965), and i n c r e a s e d t h e Km ( M i c h a e l i s - M e n t o n c o n s t a n t ) o f male r a t l i v e r m i c r o s o m a l e t h y l m o r p h i n e N - d e m e t h y l a s e t o n e a r f e m a l e l e v e l s ( E l Defrawy E l Masry and M a n n e r i n g 1 9 7 4 ) . T e s t o s t e r o n e r e p l a c e m e n t t o t h e s e a n i m a l s r e v e r s e d t h e e f f e c t s o f c a s t r a t i o n on t h e s e x - d e p e n d e n t h e p a t i c m i c r o s o m a l enzymes ( K a t o and G i l l e t t e 1965; E l Defrawy E l Masry and M a n n e r i n g 1 9 7 4 ) . On t h e o t h e r hand, o v a r i e c t o m y o f a d u l t f e m a l e r a t w i t h o r w i t h o u t e s t r o g e n r e p l a c e m e n t had l i t t l e e f f e c t on h e p a t i c m i c r o s o m a l enzyme a c t i v i t i e s . F u r t h e r m o r 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 a t a dose c a p a b l e o f m a i n t a i n i n g n o r m a l p r o s t a t e : b o d y w e i g h t r a t i o s and h e p a t i c 5 m i c r o s o m a l BPH and a m i n o p y r i n e N - d e m e t h y l a s e a c t i v i t i e s i n the a d u l t c a s t r a t e d male r a t , f a i l e d t o i n f l u e n c e t h e a c t i v i t i e s o f t h e s e enzymes i n t h e a d u l t o v a r i e c t o m i z e d f e m a l e ( B e l l w a r d e t a l . 1982). I n j e c t i o n o f t h e same dose o f t e s t o s t e r o n e t o t h e i n t a c t a d u l t male f a i l e d t o i n f l u e n c e t h e s e d r u g - m e t a b o l i z i n g enzyme a c t i v i t i e s . T h i s l a c k of e f f e c t may be due t o t h e a l r e a d y h i g h l e v e l s of plasma t e s t o s t e r o n e ( F o r e s t 1 9 7 9 ) . S i n c e t e s t o s t e r o n e may have a l r e a d y e x e r t e d i t s maximal e f f e c t s i n t h e male r a t , exogenous t e s t o s t e r o n e a d m i n i s t r a t i o n would n o t f u r t h e r a l t e r t h i s t e s t o s t e r o n e - d e p e n d e n t enzyme a c t i v i t y . D u r i n g t h e c o u r s e o f n o r m a l d e v e l o p m e n t , t h e g r o w i n g r a t i s e x p o s e d t o t h r e e p h a s e s of t e s t o s t e r o n e s e c r e t i o n ( F o r e s t 1 9 79): s h o r t s u r g e s p r e - and p o s t - n a t a l l y , and a f i n a l p r o l o n g e d s e c r e t i o n d u r i n g and a f t e r p u b e r t y . The e f f e c t o f n e o n a t a l a n d r o g e n s on t h e r e s p o n s i v i t y o f t h e a d u l t male r a t t o exogenous t e s t o s t e r o n e was f i r s t r e p o r t e d by DeMoor and Denef ( 1 9 6 8 ) . T h e s e w o r k e r s showed t h a t c a s t r a t i o n o f n e o n a t a l m a l e s d r a m a t i c a l l y a l t e r e d t h e a d u l t p a t t e r n o f C o r t i s o l m e t a b o l i s m i n v i t r o . T hese o b s e r v a t i o n s were l a t e r c o n f i r m e d by o t h e r w o r k e r s ( E i n a r s s o n e t a l . 1973; Chung e t a l . 1975), who measured t e s t o s t e r o n e and e t h y l m o r p h i n e m e t a b o l i s m i n v i t r o . The work by DeMoor and Denef (1968) i n d i c a t e d t h a t c a s t r a t i o n o f n e o n a t a l m a l e s r e s u l t e d i n t h e m a i n t e n a n c e o f t h e immature o r a d u l t f e m a l e - t y p e o f C o r t i s o l m e t a b o l i s m r a t h e r t h a n t h e c h a r a c t e r i s t i c male m e t a b o l i c p a t t e r n . The same w o r k e r s 6 (Denef and DeMoor 1972) showed t h a t r e s p o n s i v i t y o f t h e a d u l t r a t t o a n d r o g e n was d e p e n d e n t upon n e o n a t a l e x p o s u r e t o t h e same hormone. N e o n a t a l a n d r o g e n i m p r i n t i n g i n male o r f e m a l e was b l o c k e d by t h e a d m i n i s t r a t i o n of c y p r o t e r o n e a c e t a t e , an a n d r o g e n r e c e p t o r a n t a g o n i s t (Neumann e t a l . 1 9 7 0 ) . T h a t i s , a d d i t i o n of t h e a n d r o g e n a n t a g o n i s t m i m i c k e d t h e e f f e c t o f n e o n a t a l c a s t r a t i o n , s i n c e i t b l o c k e d n e o n a t a l i m p r i n t i n g . N e o n a t a l i m p r i n t i n g a l s o o c c u r s i n r a t h e p a t i c m i c r o s o m a l d r u g m e t a b o l i s m . C a s t r a t i o n a t b i r t h r e s u l t e d i n a f e m a l e p a t t e r n of e t h y l m o r p h i n e m e t a b o l i s m , b u t a s i n g l e t e s t o s t e r o n e r e p l a c e m e n t dose d u r i n g l e s s t h a n 10 days o f a g e , and a g a i n l a t e r i n p u b e r t y (day 2 0 ) , r e s u l t e d i n enzyme l e v e l s s i m i l a r t o t h o s e o f t h e n o n - c a s t r a t e d male (Chung e t a l . 1 9 7 5). T h e s e r e s u l t s a g r e e w i t h t h e p r e v i o u s r e p o r t s by DeMoor and Denef (1968) and E i n a r s s o n e t a l . (1973) t h a t t e s t o s t e r o n e i s i n v o l v e d i n t h e s e x u a l d i f f e r e n t i a t i o n o f r a t h e p a t i c s t e r o i d h y d r o x y l a s e s and r e d u c t a s e s . The r e q u i r e m e n t f o r t e s t o s t e r o n e a t " c r i t i c a l " p e r i o d s o f r a t p o s t - n a t a l d e v e l o p m e n t s u g g e s t s t h a t i r r e v e r s i b l e " i m p r i n t i n g " o r "programming" o f l i v e r enzymes t o t h e male t y p e may be o c c u r r i n g ; p u b e r t a l t e s t o s t e r o n e s e c r e t i o n a l l o w s t h i s e f f e c t t o be e x p r e s s e d . T h i s would e x p l a i n t h e l a c k o f t e s t o s t e r o n e r e s p o n s i v e n e s s i n h e p a t i c d r u g m e t a b o l i z i n g enzyme a c t i v i t e s i n t h e f e m a l e ( C o l b y 1980; B e l l w a r d e t a l . 1 9 8 2 ) . A l t h o u g h th e mechanism o f a g e - d e p e n d e n t enzyme i n d u c t i o n i s n o t c l e a r l y u n d e r s t o o d , t e s t o s t e r o n e - m e d i a t e d n e o n a t a l i m p r i n t i n g would e x p l a i n t h e age and sex d i f f e r e n c e s i n h e p a t i c m i c r o s o m a l enzyme a c t i v i t i e s i n t h e r a t . F u r t h e r m o r e , p u b e r t a l t e s t o s t e r o n e s e c r e t i o n would be t h e c a u s a t i v e a g e n t of h e p a t i c microsoma enzyme i n d u c t i o n i n t h e p u b e r t a l male and t h e l a c k o f t e s t o s t e r o n e i n t h e f e m a l e d u r i n g o r a f t e r p u b e r t y would e x p l a i n t h e s e x and age d i f f e r e n c e s i n r a t h e p a t i c d r u g m e t a b o l i s m as s u g g e s t e d by Ka t o ( 1 9 7 4 ) . Though t h e e x a c t . mechanism u n d e r l y i n g t h e phenomenon of n e o n a t a l i m p r i n t i n g and h e p a t i c m i c r o s o m a l enzyme i n d u c t i o n i s n o t r e a l l y c l e a r i t i s t h o u g h t t o i n v o l v e a c y t o s o l i c a n d r o g e n r e c e p t o r . 3. The r o l e o f an a n d r o g e n r e c e p t o r i n h e p a t i c m i c r o s o m a l d r u g and s t e r o i d m e t a b o l i s m A l t h o u g h t e s t o s t e r o n e s e c r e t i o n i s c l e a r l y i m p l i c a t e d i n age and sex d i f f e r e n c e s i n r a t h e p a t i c d r u g and s t e r o i d m e t a b o l i s m , t h e e x a c t mechanism i s n o t known. T h e r e i s s t r o n g e v i d e n c e t o s u g g e s t t h a t t e s t o s t e r o n e may be a c t i n g a t an e x t r a h e p a t i c l o c u s , s i n c e t e s t o s t e r o n e and d i h y d r o t e s t o s t e r o n e f a i l e d t o s t i m u l a t e h e p a t i c m i c r o s o m a l d r u g m e t a b o l i s m i n t h e h y p o p h y s e c t o m i z e d a d u l t male r a t (Kramer e t a l . 1 9 7 9 ) . T h e s e r e s u l t s , however, s u g g e s t t h a t t h e a c t i o n o f t e s t o s t e r o n e r e q u i r e s t h e p r e s e n c e o f an i n t a c t p i t u i t a r y ; t e s t o s t e r o n e c o u l d t h e r e f o r e a c t e i t h e r a t h e l e v e l o f t h e l i v e r ( d i r e c t a c t i o n ) , o r a t l o c i l o c a t e d a l o n g t h e h y p o t h a l a m i c - p i t u i t a r y - l i v e r a x i s ( i n d i r e c t a c t i o n ) , o r b o t h . 8 (a) I n d i r e c t e f f e c t s of t e s t o s t e r o n e on h e p a t i c m i c r o s o m a l d r u g and s t e r o i d m e t a b o l i s m The r o l e of t h e p i t u i t a r y has been i m p l i c a t e d i n sex d i f f e r e n c e s i n d r u g and s t e r o i d m e t a b o l i s m . E x t e n s i v e r e v i e w s o f t h i s l i t e r a t u r e c an be f o u n d e l s e w h e r e ( S k e t t and G u s t a f s s o n 1978; C o l b y 1980; F i n l a y s o n 1 9 83). The f o l l o w i n g s e c t i o n w i l l b r i e f l y c o v e r t h e e v i d e n c e s u p p o r t i n g t h e i n d i r e c t a c t i o n o f a n d r o g e n s on h e p a t i c m i c r o s o m a l enzyme d r u g and s t e r o i d m e t a b o l i s m by m o d u l a t i o n o f p i t u i t a r y s e c r e t i o n s . Denef (1974) was one o f t h e f i r s t w o r k e r s t o n o t e t h a t t h e r e m o v a l o f t h e p i t u i t a r y f r o m f e m a l e r a t s r e s u l t e d i n a s h i f t t o t h e m a l e - t y p e o f t e s t o s t e r o n e m e t a b o l i s m i n v i t r o , w h i l e t h e same manoeuver i n t h e male had no e f f e c t , s u g g e s t i n g t h a t t h e p i t u i t a r y i s f u n d a m e n t a l t o sex 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 m e t a b o l i s m . T h e s e e f f e c t s were not i n f l u e n c e d by t e s t o s t e r o n e a d m i n i s t r a t i o n t o e i t h e r male o r f e m a l e h y p o p h y s e c t o m i z e d r a t s (DeMoor e t a l . 1975). S i m i l a r s t u d i e s were done m e a s u r i n g t h e e f f e c t of hypophysectomy on h e p a t i c m i c r o s o m a l d r u g m e t a b o l i s m . Chung (1977) f o u n d t h a t hypophysectomy of t h e a d u l t male r a t s i g n i f i c a n t l y a l t e r e d t h e a p p a r e n t Km o f e t h y l m o r p h i n e N - d e m e t h y l a s e f r o m t h e m a l e - t y p e t o t h a t c h a r a c t e r i s t i c o f t h e f e m a l e r a t . Hypophysectomy had no e f f e c t on t h e f e m a l e r a t . F u r t h e r m o r e , s i m i l a r t o t h e l a c k of t e s t o s t e r o n e e f f e c t s on s t e r o i d m e t a b o l i s m m e n t i o n e d above, t e s t o s t e r o n e o r d i h y d r o t e s t o s t e r o n e a d m i n i s t r a t i o n d i d n o t i n f l u e n c e 9 h y p o p h y s e c t o m i z e d male r a t l i v e r d r u g m e t a b o l i s m , i n d i c a t i n g t h a t t h e e f f e c t o f a n d r o g e n s r e q u i r e d an i n t a c t h y p o t h a l a m i c - p i t u i t a r y s t a l k . T h i s o b s e r v a t i o n s u p p o r t e d t h o s e o f Denef ( 1 9 7 4 ) , G u s t a f s s o n and S t e n b e r g ( 1 9 7 4 ) , and Lax e t a l . ( 1 9 7 4 ) , who measured h e p a t i c m i c r o s o m a l t e s t o s t e r o n e m e t a b o l i s m , and was c o n f i r m e d by Kramer e t a l . (1979) who s t u d i e d h e p a t i c m i c r o s o m a l e t h y l m o r p h i n e N - d e m e t h y l a t i o n . The l o s s o f sex d i m o r p h i s m i n a d u l t r a t h e p a t i c d r u g and s t e r o i d m e t a b o l i s m f o l l o w i n g hypophysectomy, i e . s h i f t o f male t y p e d r u g m e t a b o l i z i n g enzyme a c t i v i t i t e s t o w a r d s t h o s e o f t h e f e m a l e , o r t h e s h i f t of f e m a l e - t y p e s t e r o i d m e t a b o l i z i n g enzyme a c t i v i t i e s t o w a r d s t h o s e o f t h e m a l e , l e d many r e s e a r c h e r s t o i n v e s t i g a t e w h i c h p i t u i t a r y f a c t o r s were u n d e r l y i n g t h i s phenomenon. S k e t t e t a_l. (1982) showed t h a t i m p l a n t a t i o n of an e c t o p i c p i t u i t a r y g l a n d u n d e r t h e r e n a l c a p s u l e of a d u l t r a t s c a u s e d " f e m i n i z a t i o n " o f s t e r o i d m e t a b o l i z i n g enzymes ( i e . s h i f t f r o m male t o f e m a l e - t y p e s t e r o i d m e t a b o l i s m ) , b u t t h e s e e f f e c t s d i d n o t c o r r e l a t e w i t h t h e h o s t serum g o n a d o t r o p h i n ( l u t e i n i z i n g hormone ( L H ) , f o l l i c l e s t i m u l a t i n g hormone (FSH) o r p r o l a c t i n ) l e v e l s , hence t h e i d e n t i f i c a t i o n o f t h e " f e m i n i z i n g " s u b s t a n c e was n o t known. They a l s o showed t h a t n o r m a l p i t u i t a r y e x t r a c t and t h a t s e c r e t e d f r o m p i t u i t a r y tumour c a u s e d " f e m i n i z a t i o n " o f t h e s t e r o i d m e t a b o l i z i n g c a p a c i t y i n v i t r o u s i n g c u l t u r e d hepatoma and i s o l a t e d h e p a t o c y t e s . The two t y p e s o f p i t u i t a r y e x t r a c t s w h i c h c o n t a i n e d " f e m i n i z i n g " a c t i v i t y were f o u n d i n t h e g r a n u l a r f r a c t i o n o f a s u c r o s e d e n s i t y g r a d i e n t . S c r e e n i n g o f v a r i o u s known r a t hormones s u c h as v a s o p r e s s i n , o x y t o c i n , c o r t i c o t r o p i n , s o m a t o t r o p i n , FSH, LH, p r o l a c t i n , a l o n e and i n c o m b i n a t i o n f a i l e d t o s t i m u l a t e h e p a t i c s t e r o i d m e t a b o l i z i n g enzyme a c t i v i t y i n v i t r o , and t h e i d e n t i t y o f t h e " f e m i n i z i n g " f a c t o r r e m a i n e d unknown. A l t h o u g h t h e t r u e n a t u r e o f t h e " f e m i n i z i n g " f a c t o r o r as S k e t t and G u s t a f s s o n (1979) had c o i n e d " f e m i n o t r o p i n " was n o t i d e n t i f i e d , t h e above s t u d i e s would s u p p o r t i t s p r e s e n c e i n t h e p i t u i t a r y . G u s t a f s s o n e t a l . (1978) showed t h a t d i s c r e t e h y p o t h a l a m i c l e s i o n s i n t h e a d u l t male r a t l e d t o a f e m a l e - t y p e o f h e p a t i c m i c r o s o m a l s t e r o i d m e t a b o l i s m , and p r o p o s e d t h a t a " f e m i n i z i n g " f a c t o r i s s e c r e t e d f r o m t h e p i t u i t a r y i n b o t h a d u l t male and f e m a l e r a t s , b u t t h e r e l e a s e o f t h i s f a c t o r i n t h e a d u l t male i s under t o n i c i n h i b i t i o n f r o m h i g h e r c e n t r e s , ( i e . h y p o t h a l a m i c n u c l e i and a d j a c e n t a r e a s ) by t h e a c t i o n of f e m i n o t r o p i n i n h i b i t i n g f a c t o r ( f e m i n o s t a t i n ) . S k e t t and G u s t a f f s o n (1978) f u r t h e r s u g g e s t e d t h a t s e c r e t i o n o f t h e l a t t e r was m o d u l a t e d by serum t e s t o s t e r o n e l e v e l s i n t h e a d u l t m a l e . The n e t e f f e c t would be t h e m a i n t e n a n c e o f a t o n i c i n h i b i t i o n o f " f e m i n i z i n g " f a c t o r on a d u l t male r a t h e p a t i c s t e r o i d m e t a b o l i s m . T h i s w o u l d s u g g e s t t h a t t e s t o s t e r o n e o r i t s m e t a b o l i t e may a c t a t a c e n t r a l l o c u s t o m o d u l a t e p i t u i t a r y s e c r e t i o n s a l o n g t h e h y p o t h a l a m i c - p i t u i t a r y - 1 i v e r a x i s . T h i s would r e q u i r e a n d r o g e n b i n d i n g s i t e s i n t h e b r a i n . S p e c i f i c a n d r o g e n r e c e p t o r s have been c h a r a c t e r i z e d i n r a t h y p o t h a l a m u s , c o r t e x and p i t u i t a r y g l a n d (Naess e t a l . 1975; G u s t a f s s o n e t a l . 1 9 7 6 ) . S t i m u l a t i o n o f t h e s e c e n t r a l n e r v o u s s y s t e m s t e r o i d r e c e p t o r s may l e a d i n p a r t t o t h e s e x u a l d i m o r p h i s m o f h e p a t i c s t e r o i d and d r u g m e t a b o l i s m . R i g o r o u s s t u d i e s t o t e s t t h i s h y p o t h e s i s have n o t been done. To f u r t h e r c o m p l e t e t h e d e s c r i p t i o n o f i n d i r e c t a n d r o g e n e f f e c t s on h e p a t i c enzyme m e t a b o l i s m , t h e s e a r c h f o r t h e " f e m i n i z i n g f a c t o r " l e d o t h e r w o r k e r s t o a t t r i b u t e sex and age d e p e n d e n t h e p a t i c d r u g and s t e r o i d m e t a b o l i s m t o th e s e c r e t i o n o f g r o w t h hormone (GH). The s e c r e t i o n of t h i s p i t u i t a r y hormone i s known t o a l t e r t h e m a l e - t y p e l i v e r d r u g and s t e r o i d m e t a b o l i s m t o w a r d s f e m a l e - t y p e l e v e l s . Rumbaugh and C o l b y (1980) r e p o r t e d t h a t b o v i n e GH, l i k e e s t r a d i o l a d m i n i s t r a t i o n f o r 10 d a y s , r e d u c e d h e p a t i c m i c r o s o m a l e t h y l m o r p h i n e d e m e t h y l a s e , a n i l i n e h y d r o x y l a s e and BPH, b u t i n c r e a s e d t e s t o s t e r o n e r e d u c t a s e (TR) a c t i v i t e s i n t h e c a s t r a t e d a d u l t m a l e . In a d d i t i o n , b o v i n e GH d e c r e a s e d e t h y l m o r p h i n e d e m e t h y l a s e and BPH, and had no e f f e c t on a n i l i n e h y d r o x y l a s e o r TR a c t i v i t i e s i n t h e h y p o p h y s e c t o m i z e d a d u l t m a l e . E s t r a d i o l a d m i n i s t r a t i o n d i d not s i g n i f i c a n t l y i n f l u e n c e t h e s e h e p a t i c m i c r o s o m a l enzyme a c t i v i t i e s . Hormone r e p l a c e m e n t s t u d i e s were done i n o r d e r t o r e c o v e r t h e s e enzyme a c t i v i t i e s w h i c h were l o s t f o l l o w i n g h y p o p hysectomy. T h e s e a u t h o r s f o u n d t h a t a d m i n i s t r a t i o n o f GH c a u s e d an e s t r o g e n - t y p e e f f e c t on h e p a t i c enzyme a c t i v i t i e s i n h y p o p h y s e c t o m i z e d m a l e s w h i c h were t r e a t e d w i t h th e c o m b i n a t i o n of t h y r o x i n e and ACTH; the a d m i n i s t r a t i o n of t h e l a t t e r hormones a l o n e had no e f f e c t . F u r t h e r m o r e , s i n c e e s t r o g e n d i d n o t i n f l u e n c e t h e s e a c t i v i t i e s i n t h e t h y r o x i n e p l u s ACTH t r e a t e d h y p o p h y s e c t o m i z e d m a l e s , i t was t h e n c o n c l u d e d t h a t t h e a c t i o n o f e s t r a d i o l may be m e d i a t e d by GH. G u s t a f s s o n ' s g r o u p a l s o examined t h e e f f e c t s o f GH on sex d i f f e r e n c e s i n h e p a t i c m i c r o s o m a l enzyme m e t a b o l i s m . Mode e t a l . (1981) r e p o r t e d a c o m p l e t e f e m i n i z a t i o n o f h e p a t i c m i c r o s o m a l 4 - a n d r o s t e n e - 3 , 1 7 , d i o n e m e t a b o l i s m i n a d u l t male r a t s w h i c h were t r e a t e d c o n t i n u o u s y w i t h human GH u s i n g o s m o t i c minipumps. T h i s e f f e c t was n o t i n f l u e n c e d by h ypophysectomy and c a s t r a t i o n i n t h e human GH t r e a t e d a d u l t m a l e s , o r by a d r e n a l e c t o m y and t h y r o i d e c t o m y i n t h e same a n i m a l s . S i m i l a r f e m i n i z i n g e f f e c t s on h e p a t i c s t e r o i d m e t a b o l i s m were f o u n d u s i n g r a t GH t r e a t m e n t . F u r t h e r , the l o s s o f t h e f e m a l e - t y p e o f h e p a t i c s t e r o i d m e t a b o l i s m i n t h e h y p o p h y s e c t o m i z e d - o v a r i e c t o m i z e d a d u l t f e m a l e was r e g a i n e d f o l l o w i n g c o n t i n u o u s human GH t r e a t m e n t . T h e s e r e s u l t s s u p p o r t t h e h y p o t h e s i s t h a t GH o r a r e l a t e d compound i s i n v o l v e d i n t h e s e x u a l d i f f e r e n t i a t i o n of h e p a t i c s t e r o i d m e t a b o l i s m . D i f f e r e n c e s i n t h e s e x u a l d i m o r p h i s m o f h e p a t i c s t e r o i d m e t a b o l i s m have been a t t r i b u t e d t o t h e p a t t e r n of s e c r e t i o n o f GH i n t h e r a t . On t h e b a s i s of t h e e a r l i e r s t u d i e s (Tannenbaum e t a l . 1976; S a u n d e r s e t a l . 1979; Eden e t a l . 1979) t h a t t h e a d u l t male r a t had an e p i s o d i c GH s e c r e t i o n p a t t e r n (3-4 h r s i n t e r v a l ) compared t o t h e more r e g u l a r p a t t e r n p r e s e n t i n t h e a d u l t f e m a l e r a t , Mode e t a_l (1982) compared d i f f e r e n t a d m i n i s t r a t i o n f r e q u e n c i e s o f human GH on h e p a t i c s t e r o i d m e t a b o l i s m i n t h e h y p o p h y s e c t o m i z e d a d u l t f e m a l e r a t . C o n t i n u o u s i n f u s i o n of human GH, o r a d m i n i s t r a t i o n o f t h i s hormone e v e r y 3-6 h o u r s l e d t o a r e f e m i n i z a t i o n o f t h e h y p o p h y s e c t o m i z e d a d u l t f e m a l e , w h i l e t r e a t m e n t e v e r y 12 h o u r s f a i l e d t o have t h i s e f f e c t . Measurement of p l a s m a GH l e v e l s i n t h e c a s t r a t e d a d u l t male r a t t r e a t e d w i t h o r w i t h o u t t e s t o s t e r o n e c o r r e l a t e d w e l l w i t h t h e c h a n g e s i n h e p a t i c s t e r o i d m e t a b o l i s m . T h e s e r e s u l t s s u g g e s t t h a t t h e p a t t e r n o f p l a s m a GH s e c r e t i o n s may be i n v o l v e d i n t h e r e g u l a t i o n o f sex d i f f e r e n c e s i n r a t h e p a t i c m i c r o s o m a l s t e r o i d m e t a b o l i s m . (b) D i r e c t e f f e c t s o f t e s t o s t e r o n e on h e p a t i c m i c r o s o m a l d r u g and s t e r o i d m e t a b o l i s m ( i ) In v i v o e v i d e n c e : The r o l e of t h e a n d r o g e n r e c e p t o r on h e p a t i c d r u g m e t a b o l i z i n g s y s t e m s has been i m p l i c a t e d u s i n g t h e S t a n l e y - G u m b r e c k p s e u d o h e r m a p h r o d i t i c (Ps) male r a t . The P male a n i m a l i s g e n o t y p i c a l l y male (XY) b u t i t i s p h e n o t y p i c a l l y e x p r e s s e d as a p s e u d o h e r m a p h r o d i t e ( S t a n l e y e t a l . 1 9 7 3 ) . I t i s t h o u g h t t h a t t h i s a n i m a l l a c k s a f u n c t i o n a l a n d r o g e n r e c e p t o r s i n c e i t does n o t r e s p o n d t o p h a r m a c o l o g i c a l d o s e s o f t e s t o s t e r o n e ( B a r d i n e t a l . 1973), e v e n t h o u g h s i m i l a r d o s e s of t h e same hormone r e d u c e d h e x o b a r b i t a l s l e e p i n g t i m e s and i n c r e a s e d e t h y l m o r p h i n e N - d e m e t h y l a s e ( B u l l o c k e t a l . 1971) and s t e r o i d m e t a b o l i z i n g enzyme a c t i v i t i e s ( E i n a r s s o n e t a l . 1972) i n b o t h i n t a c t f e m a l e s and c a s t r a t e d male r a t s . O t h e r w o r k e r s ( M i l a n and Roy 1973; D e c k e r and L e v i n s o n 1983) have f a i l e d t o show an a n d r o g e n r e c e p t o r i n t h e Ps male r a t compared t o t h e i n t a c t a d u l t m a l e . The l a c k o f a f u n c t i o n a l a n d r o g e n r e c e p t o r may not be t h e o n l y r e a s o n f o r t e s t o s t e r o n e i n s e n s i t i v i t y . One may p o s t u l a t e t h a t t h e p s e u d o h e r m a p h r o d i t i c male r a t may have a d e f e c t a t an i n t r a c e l l u l a r l o c u s o t h e r t h a n an a n d r o g e n r e c e p t o r , w h i c h c a n r e s u l t i n a f a u l t y enzyme i n d u c t i o n s y s t e m . Sonawane e t a l . (1979) have r e c e n t l y r e p o r t e d t h a t p h e n o b a r b i t a l c a n s t i m u l a t e a m i n o p y r i n e N - d e m e t h y l a s e and a n i l i n e h y d r o x y l a s e a c t i v i t i e s i n m a l e , f e m a l e and Ps r a t s . T h e s e r e s u l t s have been c o n f i r m e d by B e l l w a r d e t a l . (1982) m e a s u r i n g p h e n o b a r b i t a l ( P B ) - and 3 - m e t h y l c h o l a n t h r e n e (3-MC) i n d u c t i o n o f h e p a t i c m i c r o s o m a l BPH and TR i n t h e a d u l t m a l e , f e m a l e and Ps male r a t . Normal sex d i f f e r e n c e s between c o n t r o l male and f e m a l e BPH and TR a c t i v i t i e s were o b s e r v e d ; however none were s e e n when c o m p a r i n g t h e c o n t r o l Ps male t o f e m a l e r a t s . D e s p i t e h a v i n g above n o r m a l a d u l t male serum t e s t o s t e r o n e l e v e l s , t h e Ps male r a t d e m o n s t r a t e d BPH and TR a c t i v i t i e s t y p i c a l o f a f e m a l e - t y p e o f d r u g and s t e r o i d m e t a b o l i s m . In c o n t r a s t t o i t s n o n - r e s p o n s i t i v i t y t o t e s t o s t e r o n e compared t o t h e c a s t r a t e d a d u l t m a l e , t h e Ps male r a t was e q u a l l y as r e s p o n s i v e t o PB o r 3-MC as t h e f e m a l e r a t . T h e s e r e s u l t s s u g g e s t t h a t t h e d r u g i n d u c t i o n mechanisms were i n t a c t , and t h a t t e s t o s t e r o n e - t y p e enzyme i n d u c t i o n i s m e d i a t e d by a s e p a r a t e pathway. Though t h e e f f e c t s o f t e s t o s t e r o n e may be s i m i l a r t o t h e c l a s s i c a l enzyme i n d u c e r c l a s s t y p i f i e d by p h e n o b a r b i t a l , i t d i f f e r s i n some ways; one o f w h i c h p r e s u m a b l y i n v o l v e s t h e a n d r o g e n r e c e p t o r . R e c e p t o r - m e d i a t e d r e s p o n s e s seem t o be i n v o l v e d i n t h e i n h i b i t i o n o f t h e a n d r o g e n - s t i m u l a t e d c h a n g e s i n h e p a t i c m i c r o s o m a l d r u g and s t e r o i d m e t a b o l i z i n g enzyme a c t i v i t y c a u s e d by a d m i n i s t r a t i o n o f a n d r o g e n a n t a g o n i s t s . Brown e t a l . (1976) showed t h a t f l u t a m i d e , a s p e c i f i c n o n - s t e r o i d a l a n t i - a n d r o g e n , b l o c k e d t e s t o s t e r o n e - i n d u c e d h e p a t i c m i c r o s o m a l e t h y l m o r p h i n e N - d e m e t h y l a s e a c t i v i t y i n c a s t r a t e d BALB/cJ m i c e , and i n c a s t r a t e d and i n t a c t p r e - p u b e r t a l male F i s h e r r a t s . T h e s e e f f e c t s seemed t o be q u i t e d i s t i n c t f r o m f l u t a m i d e ' s n o n - s p e c i f i c a c t i o n on l i v e r w e i g h t and m i c r o s o m a l p r o t e i n c o n t e n t , e f f e c t s whose mechanism seemed t o be i n d e p e n d e n t o f t h e a n d r o g e n r e c e p t o r . More r e c e n t l y , Lax and S c h r i e f e r s (1981) have shown t h a t f l u t a m i d e g r e a t l y r e d u c e d d i h y d r o t e s t o s t e r o n e ( D H T ) - s t i m u l a t e d t e s t o s t e r o n e m e t a b o l i s m i n t h e f e m a l e r a t , b u t had no e f f e c t on t e s t o s t e r o n e m e t a b o l i s m i n t h e i n t a c t o r c a s t r a t e d male r a t . I t i s d i f f i c u l t t o draw c o n c l u s i o n s r e g a r d i n g t h e mechanism o f a c t i o n o f t e s t o s t e r o n e o r f l u t a m i d e on h e p a t i c d r u g and s t e r o i d m e t a b o l i s m f r o m s t u d i e s u s i n g v a r i o u s d o s e s and t y p e s o f a n d r o g e n s , g i v e n t o d i f f e r e n t a n i m a l m o d e l s , and m e a s u r i n g d i f f e r e n t enzyme s y s t e m s . F l u t a m i d e may be a c t i n g e i t h e r a t an h e p a t i c a n d r o g e n r e c e p t o r o r e l s e w h e r e , t o a l t e r d r u g and s t e r o i d m e t a b o l i s m ; e v i d e n c e t o s u g g e s t a d i r e c t a c t i o n a t t h e h e p a t i c a n d r o g e n r e c e p t o r has been l e s s t h a n c o n v i n c i n g , due t o t e c h n i c a l d i f f i c u l t i e s . ( i i ) In v i t r o e v i d e n c e : The r o l e o f a c y t o s o l i c r e c e p t o r (Ah r e c e p t o r ) i n h e p a t i c m i c r o s o m a l enzyme i n d u c t i o n has been p r e v i o u s l y p r o p o s e d by P o l a n d and c o - w o r k e r s (1974, 1976). T h e s e w o r k e r s ( P o l a n d e t a l . 1976) have shown t h a t 2 , 3 , 7 , 8 - t e t r a c h l o r o d i b e n z e n o - p - d i o x i n (TCDD), a p o t e n t i n d u c e r of h e p a t i c a r y l h y d r o c a r b o n h y d r o x y l a s e (AHH) i n most s p e c i e s ( P o l a n d and G l o v e r 1974) c a n b i n d t o h i g h a f f i n i t y c y t o s o l i c r e c e p t o r s l o c a t e d i n t h e l i v e r c y t o s o l o f C57BL/6J mice ( b i n d i n g a f f i n i t y (Kd)= 0.27 nM; maximum b i n d i n g c a p a c i t y (Bmax)= 84 fmol/mg c y t o s o l i c p r o t e i n ) , and i n t h a t o f S p r a g u e - D a w l e y r a t s (Kd= 0.2 - 0.3 nM; Bmax was s l i g h t l y l e s s t h a n 84 f m o l / m g ) . I t was a l s o shown t h a t t h e a f f i n i t i e s o f c e r t a i n TCDD i n d u c e r s were c o r r e l a t e d by r a n k - o r d e r t o t h e i r i n d u c i b i l i t y o f AHH, w h i l e d r u g s o f d i s s i m i l a r s t r u c t u r e s u c h as p h e n o b a r b i t a l and s t e r o i d hormones i n c l u d i n g t e s t o s t e r o n e , d i d n o t compete f o r t h i s c y t o s o l i c r e c e p t o r . In c o n t r a s t t o t h e l a t t e r d r u g s , AHH i n d u c t i o n by t h e TCDD-type d r u g s a p p e a r s t o i n v o l v e t h e TCDD c y t o s o l i c r e c e p t o r , and would be c o n s i s t e n t w i t h t h e o b s e r v a t i o n t h a t TCDD-type d r u g s i n d u c e a d i f f e r e n t s p e c t r u m of m i c r o s o m a l enzymes compared t o t h a t of t h e s t e r o i d - t y p e i n d u c e r s (Conney 1967; Kuntzman e t a l . 1 9 64). I t s h o u l d be n o t e d t h a t t h e b a s i c mechanism j u s t d e s c r i b e d i s i d e n t i c a l i n c o n c e p t t o t h a t o f t h e e f f e c t s o f sex hormones i n c l a s s i c e n d - o r g a n t i s s u e s (Grody e t a_l. 1 9 82). I f t e s t o s t e r o n e i s a c t i n g as an endogenous h e p a t i c m i c r o s o m a l d r u g m e t a b o l i z i n g enzyme i n d u c e r , t h e n i t may i n v o l v e a mechanism s i m i l a r t o i t s e f f e c t s i n o t h e r t e s t o s t e r o n e r e s p o n s i v e sex e n d - o r g a n t i s s u e s , and t o t h e i n d u c t i v e e f f e c t o f TCDD i n t h e l i v e r . T e s t o s t e r o n e w o u l d e n t e r t h e c e l l , b i n d t o a " r e c e p t o r " p r o t e i n l o c a t e d i n t h e c y t o s o l , t r a n s l o c a t e i n t o t h e n u c l e u s and s t i m u l a t e DNA-RNA t r a n s c r i p t i o n w h i c h would u l t i m a t e l y l e a d t o i n c r e a s e d a n d r o g e n - d e p e n d e n t p r o t e i n s y n t h e s i s . I t s h o u l d be n o t e d t h a t a l t h o u g h t e s t o s t e r o n e i s a c t i n g as an h e p a t i c enzyme i n d u c e r , i t i s t a c i t l y assumed t h a t t h i s e f f e c t i s a r e c e p t o r m e d i a t e d phenomenon. T h e r e i s s u b s t a n t i a l e v i d e n c e t o s u g g e s t t h a t c y t o s o l i c a n d r o g e n b i n d i n g p r o t e i n s a r e l o c a t e d i n many t i s s u e s ( H i i p a k k a e t a l . 1 9 78). Of t h e s e t i s s u e s , t h e a n d r o g e n r e c e p t o r i n p r o s t a t i c c y t o s o l has been w e l l - c h a r a c t e r i z e d . S t e r o i d a l a n t i - a n d r o g e n s s u c h as c y p r o t e r o n e a c e t a t e ( L i a o and Fang 1969; J u n g and B a u l i e u 1971), B.O.M.T. (Mangan and M a i n w a r i n g 1 9 7 2 ) , SKF 7690 o r 17 a l p h a - m e t h y 1 -n o r t e s t o s t e r o n e ( T v e t e r 1 971), RU 2956 and s p i r o n o l a c t o n e (Bonne and Raynaud 1974) have been u s e d s u c c e s s f u l l y i n c o m p e t i t i v e i n h i b i t i o n s t u d i e s o f r a d i o a c t i v e l y l a b e l l e d DHT o r l a b e l l e d m e t h y l t r i e n o l o n e , f r o m c y t o p l a s m i c a n d r o g e n b i n d i n g s i t e s i n v i t r o . Raynaud e t a l . (1979) d e s c r i b e d t h e f o l l o w i n g b i n d i n g p r o f i l e f o r t h e i n h i b i t i o n o f t r i t i a t e d - t e s t o s t e r o n e (5 nM) b i n d i n g t o t h e c y t o p l a s m i c a n d r o g e n r e c e p t o r o f r a t p r o s t a t e i n v i t r o ; t e s t o s t e r o n e > s t e r o i d a n t a g o n i s t s c y p r o t e r o n e a c e t a t e and RU 2956 > n o n - s t e r o i d a l a n t a g o n i s t f l u t a m i d e . T h e s e d a t a c o r r e l a t e d w e l l w i t h t h e o r d e r o f p o t e n c y f o r t h e i n h i b i t i o n o f t h e a n d r o g e n - i n d u c e d p r o s t a t e w e i g h t i n v i v o . A t t e m p t s have been made t o i d e n t i f y and c h a r a c t e r i z e t h e h e p a t i c c y t o s o l i c a n d r o g e n r e c e p t o r i n v i t r o , however, t h e r e i s l i t t l e a greement between t h e s e v a r i o u s s t u d i e s . E a r l i e r w o r k e r s s t u d i e d t h e h e p a t i c c y t o s o l i c a n d r o g e n r e c e p t o r by u s i n g t h e n a t u r a l a n d r o g e n s DHT, t e s t o s t e r o n e o r a n d r o s t e n e d i o n e , as l i g a n d s f o r t h e i r r a d i o - l i g a n d exchange e x p e r i m e n t s . However, more r e c e n t l y , w o r k e r s have u s e d the s y n t h e t i c a n d r o g e n m e t h y l t r i e n o l o n e (R1881), s i n c e t h e l a t t e r b i n d s s p e c i f i c a l l y t o t h e c y t o s o l i c a n d r o g e n r e c e p t o r w i t h o u t b i n d i n g t o serum sex b i n d i n g p r o t e i n s , and i t i s not m e t a b o l i z e d i n v i t r o (Bonne and Raynaud 1975, 1 9 76). G u s t a f s s o n e t a l . (1975) r e p o r t e d an u p t a k e of p u l s e - l a b e l l e d t r i t i a t e d - t e s t o s t e r o n e and i t s m e t a b o l i t e s i n t o c y t o s o l i c and t r i t i a t e d - a n d r o s t e n e d i o n e i n t o n u c l e a r f r a c t i o n s o f l i v e r s t a k e n f r o m male and f e m a l e r a t s . I n t e r e s t i n g l y , DHT was not d e t e c t e d i n t h e male r a t l i v e r n u c l e i . Column c h r o m a t o g r a p h y u s i n g Sephadex G25 o f h e p a t i c c y t o s o l i c f r a c t i o n s t a k e n f r o m c a s t r a t e d male r a t s w h i c h were i n j e c t e d w i t h t r i t i a t e d - t e s t o s t e r o n e i n d i c a t e d t h r e e t y p e s o f r a d i o a c t i v e l y l a b e l l e d f r a c t i o n s : a h i g h a f f i n i t y , low c a p a c i t y t r i t i a t e d - a n d r o s t e n e d i o n e b i n d i n g p r o t e i n ("complex A") w h i c h had a 10 S s e d i m e n t a t i o n c o - e f f i c i e n t , a f r a c t i o n c o n t a i n i n g p r o t e i n - b o u n d t r i t i a t e d - t e s t o s t e r o n e and m o n o - s u l f a t e d m e t a b o l i t e s (peak B) and a n o t h e r f r a c t i o n w i t h unbound m e t a b o l i t e s (peak C ) . T h e s e a u t h o r s r e p o r t e d e x t e n s i v e i n v i t r o m e t a b o l i s m o f t e s t o s t e r o n e and a n d r o s t e n e d i o n e i n male l i v e r c y t o s o l . They overcame t h i s c o m p l i c a t i o n by p r e - f r a c t i o n a t i n g t h e c y t o s o l u s i n g Sephadex G100 column c h r o m a t o g r a p h y p r i o r t o t h e i r b i n d i n g s t u d i e s . T h e i r r e s u l t s i n d i c a t e d t h a t s a t u r a t i o n o f t h e l i v e r c y t o s o l i c a n d r o g e n b i n d i n g p r o t e i n o c c u r r e d a t a n d r o s t e n e d i o n e c o n c e n t r a t i o n s g r e a t e r t h a n 100 nM, w i t h a b i n d i n g c a p a c i t y of 0.8 fmol/mg. T h e s e a u t h o r s s u g g e s t e d t h a t t e s t o s t e r o n e was r a p i d l y t a k e n up i n t o t h e l i v e r and was m e t a b o l i z e d i n t h e c y t o s o l t o a number of p r o d u c t s , i n c l u d i n g a n d r o s t e n e d i o n e . The l a t t e r was s p e c i f i c a l l y bound t o a c y t o s o l i c a n d r o s t e n e d i o n e b i n d i n g p r o t e i n , a f t e r w h i c h i t was t r a n s l o c a t e d t o t h e n u c l e u s . U n f o r t u n a t e l y , f u r t h e r c h a r a c t e r i z a t i o n s t u d i e s ( i n h i b i t i o n , age and s e x - d e p e n d e n c y , and t e s t o s t e r o n e r e s p o n s i v i t y ) were n o t c a r r i e d o u t , and one knows l i t t l e a b o u t t h i s p r o t e i n ' s s p e c i f i c i t y o r i t s p h y s i o l o g i c a l r o l e . O t h e r w o r k e r s have a l s o s u g g e s t e d t h a t a c y t o s o l i c a n d r o g e n r e c e p t o r may be l o c a t e d i n h e p a t i c c y t o s o l . Roy e t a l . (1974) i d e n t i f i e d and p a r t i a l l y c h a r a c t e r i z e d a 3.5 S a n d r o g e n - e s t r o g e n b i n d i n g p r o t e i n w h i c h was p r e s e n t i n a d u l t male r a t l i v e r c y t o s o l , and wh i c h c o r r e l a t e d w e l l w i t h t h e s e c r e t i o n o f h e p a t i c a l p h a - 2 u g l o b u l i n . S i n g l e p o i n t d i s p l a c e m e n t s t u d i e s of 400 nM t r i t i a t e d - D H T f r o m t h e 3.5 S p r o t e i n f r a c t i o n were done u s i n g d i f f e r e n t c o m p e t i t o r s , and r e v e a l e d r e l a t i v e s p e c i f i c i t y t o DHT (93% d i s p l a c e a b l e b i n d i n g ) , and t e s t o s t e r o n e ( 5 3 % ) , b u t l i t t l e s p e c i f i c i t y t o 5 a l p h a - a n d r o s t a n - 3 b e t a , 17 b e t a - d i o l ( 8 % ) , 17 a l p h a -m e t h y l t e s t o s t e r o n e o r c o r t i c o s t e r o n e ( b o t h 0 % ) . M e a s u r i n g t r i t i a t e d - D H T and t r i t i a t e d - e s t r a d i o l d i s p l a c e m e n t f r o m t h e 3.5S f r a c t i o n by t h e same u n l a b e l l e d s t e r o i d s , t h e y f o u n d t h a t b o t h u n l a b e l l e d s t e r o i d s d i s p l a c e d t r i t i a t e d - D H T t o the same e x t e n t (50% d i s p l a c e a b l e b i n d i n g ) , w h i l e DHT d i s p l a c e d a p p r o x i m a t e l y 40%, and e s t r a d i o l d i s p l a c e d a p p r o x i m a t e l y 70% o f t o t a l t r i t i a t e d - e s t r a d i o l b i n d i n g . F u r t h e r m o r e , t h e y n o t e d a d i f f e r e n t i a l l o s s o f t r i t i a t e d - D H T b i n d i n g f o l l o w i n g c y t o s o l i c d i l u t i o n , w h i l e t r i t i a t e d - e s t r a d i o l b i n d i n g r e m a i n e d s t a b l e . T h e s e a u t h o r s s u g g e s t e d t h a t DHT and e s t r a d i o l b i n d i n g may o c c u r a t two d i s t i n c t and s p e c i f i c s i t e s on t h e same r e c e p t o r m o l e c u l e . In c o n t r a s t t o G u s t a f s s o n ' s work, Roy's g r o u p d i d s a t u r a t i o n s t u d i e s u s i n g s u c r o s e d e n s i t y g r a d i e n t a n a l y s i s and measured t o t a l bound t r i t i a t e d - D H T o r t r i t i a t e d - e s t r a d i o l i n t h e 3.5 S p r o t e i n f r a c t i o n . T h e i r r e s u l t s i n d i c a t e d two t r i t i a t e d - D H T b i n d i n g s i t e s ( f o r b i n d i n g s i t e I , Kd= 450 nM and Bmax= 4.2 fmol/mg; and f o r b i n d i n g s i t e I I , Kd= 2000 nM and Bmax= 1.26 pmol/mg) and one b i n d i n g s i t e f o r t r i t i a t e d - e s t r a d i o l (Kd= 350 nM and Bmax= 42 pmoles/mg). Based on t h e b i n d i n g c a p a c i t y o f t h e DHT b i n d i n g s i t e I I , and t h a t o f e s t r a d i o l , t h e s e w o r k e r s n o t e d a 3:1 m o l a r r a t i o o f DHT t o e s t r a d i o l b i n d i n g , and s u g g e s t e d t h a t e s t r a d i o l m i g h t be a s t r o n g a l l o s t e r i c i n h i b i t o r o f DHT b i n d i n g t o t h e a n d r o g e n s p e c i f i c b i n d i n g s i t e , w h i l e DHT may be a weak i n h i b i t o r o f e s t r a d i o l b i n d i n g a t a d i f f e r e n t b i n d i n g s i t e . F u r t h e r , t h e y p o s t u l a t e d 4 p o s s i b l e b i n d i n g s i t e s on t h i s p r o t e i n : 3 r e c e p t o r s u b u n i t s f o r DHT and 1 r e g u l a t o r y s u b u n i t f o r e s t r a d i o l b i n d i n g . In t h e same p a p e r , Roy's gr o u p r e p o r t e d t h e a b s e n c e o f t h i s c y t o s o l i c p r o t e i n i n immature and aged m a l e s , and i n the immature and a d u l t f e m a l e r a t l i v e r s . C a s t r a t i o n o f t h e a d u l t male l e d t o a g r a d u a l d e c r e a s e , w h i l e e s t r a d i o l a d m i n i s t r a t i o n c o m p l e t e l y e l i m i n a t e d t h i s r e c e p t o r ' s a c t i v i t y . In a l l o f t h e a n i m a l models t e s t e d , i n c l u d i n g t h e Ps male r a t , Roy's g r o u p c l a i m e d a good c o r r e l a t i o n between t h e p r e s e n c e o f an h e p a t i c c y t o s o l i c a n d r o g e n b i n d i n g p r o t e i n and t e s t o s t e r o n e - d e p e n d e n t h e p a t i c s y n t h e s i s o f a l p h a - 2 u - g l o b u l i n , and t h e y s u g g e s t e d t h a t t h e f o r m e r was i n v o l v e d i n t h e r e g u l a t i o n of t h e l a t t e r . C o m p a r i s o n o f t h e r e s u l t s f r o m G u s t a f s s o n ' s and Roy's g r o u p s y i e l d a number of d i s c r e p a n c i e s , w h i c h may be e x p l a i n e d by t h e b i n d i n g o f d i f f e r e n t l i g a n d s (DHT and a n d r o s t e n e d i o n e ) t o d i f f e r e n t s p e c i f i c h e p a t i c c y t o s o l i c a n d r o g e n b i n d i n g p r o t e i n c o m p l e x e s , c o n s i s t e n t w i t h t h e m u l t i p l i c i t y o f a n d r o g e n r e c e p t o r s i n t h e p r o s t a t e c y t o s o l (Lea e t a l . 1 9 7 9 ) . However, o t h e r c o n s i d e r a t i o n s s u c h as d i f f e r e n t i n c u b a t i o n c o n d i t i o n s and s e p a r a t o r y t e c h n i q u e s c a n n o t be r u l e d o u t . B o t h g r o u p s used s i m i l a r t y p e s o f c r u d e c y t o s o l p r e p a r a t i o n s w h i c h r e g u i r e d h i g h p r o t e i n c o n c e n t r a t i o n s , g r e a t e r t h a n 40 mg/ml. G u s t a f s s o n ' s g r o u p n o t e d s i g n i f i c a n t m e t a b o l i s m o f t h e r a d i o l a b e l l e d l i g a n d i n v i t r o , and t h e y m i n i m i z e d t h i s p r o b l e m by p r e - f r a c t i o n a t i o n o f t h e c y t o s o l . In c o n t r a s t , Roy's gr o u p n e g l e c t e d t o measure DHT m e t a b o l i s m i n v i t r o , and p r e c a u t i o n s were n o t t a k e n a g a i n s t l i g a n d d e g r a d a t i o n . T h i s o v e r s i g h t may e x p l a i n t h e Roy g r o u p ' s i n c o n s i s t e n c y i n d i s p l a c e a b l e DHT b i n d i n g ( 4 0 - 9 3 % ) . S i n c e t h e c o n c e n t r a t i o n o f f r e e l i g a n d was n o t c o r r e c t e d f o r m e t a b o l i s m , t h e n t h e r e s u l t s o f the b i n d i n g a n a l y s i s were i n a c c u r a t e . Roy's s t o i c h i o m e t i c c o n c l u s i o n s r e g a r d i n g r e c e p t o r o c c u p a n c y were b a s e d on i n c o r r e c t measurements o f t r i t i a t e d - D H T c o n c e n t r a t i o n s , and woul d t h e r e f o r e be q u e s t i o n a b l e . F u r t h e r m o r e , d i f f e r e n c e s i n b i n d i n g k i n e t i c s i n t h e s a t u r a t i o n s t u d i e s may be p a r t i a l l y due t o t h e Roy g r o u p ' s measurement of " t o t a l " t r i t i a t e d - D H T bound i n t h e c y t o s o l , compared t o t h e measurement o f " d i s p l a c e a b l e " t r i t i a t e d - a n d r o s t e n e d i o n e b i n d i n g w h i c h was done by G u s t a f s s o n ' s g r o u p . T h i s , however, may n o t f u l l y a c c o u n t f o r t h e v a r i a t i o n i n b i n d i n g c a p a c i t i e s . O t h e r c o n s i d e r a t i o n s s h o u l d be made, i n c l u d i n g d i f f e r e n c e s i n t h e methods o f s e p a r a t i n g bound and f r e e s t e r o i d i n t h e c y t o s o l . G u s t a f s s o n ' s g r o u p u s e d t h e d e x t r a n - c o a t e d c h a r c o a l t e c h n i q u e w h i l e Roy's g r o u p u s e d s u c r o s e d e n s i t y g r a d i e n t a n a l y s i s . P e r h a p s t h e f o r m e r t e c h n i q u e " s t r i p p e d " more l i g a n d f r o m t h e moderate a f f i n i t y (Kd= 100-400 nM) a n d r o g e n b i n d i n g p r o t e i n complex, l e a d i n g t o r e l a t i v e l y l o w e r b i n d i n g c a p a c i t i e s compared t o t h a t o f th e l a t t e r t e c h n i q u e . T h u s , due t o i n a d e q u a t e s p e c i f i c i t y s t u d i e s done by G u s t a f s s o n ' s g r o u p and t h e l a c k o f c o n s i d e r a t i o n of i n v i t r o l i g a n d m e t a b o l i s m and " d i s p l a c e a b l e " DHT b i n d i n g by Roy's g r o u p , n e i t h e r g r o u p has p r o v i d e d c o n v i n c i n g e v i d e n c e t h a t an a n d r o g e n b i n d i n g p r o t e i n i s p r e s e n t i n male r a t h e p a t i c c y t o s o l . More r e c e n t l y , J a p a n e s e w o r k e r s l e d by S a t o e t a l . (1980) and Ota e t a l . (1980) r e - a t t e m p t e d t o c h a r a c t e r i z e t h e h e p a t i c c y t o s o l i c a n d r o g e n r e c e p t o r . T hese w o r k e r s u s e d c a s t r a t e d a d u l t male r a t h e p a t i c c r u d e c y t o s o l w h i c h was i n c u b a t e d w i t h t r i t i a t e d - t e s t o s t e r o n e o r t r i t i a t e d - a n d r o s t e n e d i o n e , i n t h e p r e s e n c e o r a b s e n c e o f e x c e s s c o m p e t i t o r . They r e p o r t e d a s a t u r a b l e and d i s p l a c e a b l e 10 S a n d r o g e n b i n d i n g s i t e w i t h t h e f o l l o w i n g b i n d i n g k i n e t i c s : Kd= 137-575 nM and Bmax= 170-313 fmol/mg u s i n g t r i t i a t e d - t e s t o s t e r o n e and ; Kd= 1130 nM and Bmax= 158 fmol/mg u s i n g t r i t i a t e d - a n d r o s t e n e d i o n e as l i g a n d s . T h i s p r o t e i n had a mo d e r a t e a f f i n i t y t o t e s t o s t e r o n e , DHT and a n d r o s t e n e d i o n e , b u t d i d not b i n d e s t r a d i o l . In l a t e r s t u d i e s , t h i s g r o u p u s e d ammonium s u l p h a t e p r e c i p i t a t e d l i v e r c y t o s o l . The l a t t e r was s u g g e s t e d by t h e s e a u t h o r s t o c o n t a i n l e s s a n d r o g e n m e t a b o l i z i n g components t h a n c r u d e c y t o s o l . S a t o e t a l . (1981) r e p o r t e d t h a t c a s t r a t i o n of t h e a d u l t male r a t i n c r e a s e d h e p a t i c c y t o s o l i c a n d r o g e n b i n d i n g , and d e c r e a s e d n u c l e a r a n d r o g e n b i n d i n g . B o t h of t h e s e e f f e c t s were r e v e r s e d f o l l o w i n g t e s t o s t e r o n e r e p l a c e m e n 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 Roy e t a l . ( 1 9 7 4 ) , t h e J a p a n e s e g r o u p f o u n d a s i g n i f i c a n t r e d u c t i o n i n h e p a t i c c y t o s o l i c t e s t o s t e r o n e b i n d i n g i n t h e a d u l t male r a t i n j e c t e d w i t h e s t r a d i o l . U n f o r t u n a t e l y , s a t u r a t i o n s t u d i e s were n o t done on t h e p a r t i a l l y p u r i f i e d c y t o s o l p r e p a r a t i o n s , and one does n o t know i f t h e a n d r o g e n b i n d i n g p r o t e i n was s t a b l e d u r i n g t h e p u r i f i c a t i o n p r o c e s s , and t h e r e f o r e , i t i s n o t known i f t h e s o - c a l l e d h e p a t i c a n d r o g e n b i n d i n g p r o t e i n i n t h e 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 c o u l d be r e l a t e d t o t h a t p r e s e n t i n t h e c r u d e c y t o s o l o f t h e a d u l t male o r t h e o t h e r a n i m a l models t e s t e d . P u b l i s h e d a r t i c l e s s u g g e s t i n g t h e p r e s e n c e o f an h e p a t i c a n d r o g e n r e c e p t o r r e m a i n q u i t e s p a r s e , and t h e c u r r e n t l i t e r a t u r e i s l i m i t e d t o a h a n d f u l of a b s t r a c t s . Rumbaugh and L u c i e r (1982) a t t e m p t e d t o c o r r e l a t e t h e p r e s e n c e of an h e p a t i c D H T - b i n d i n g p r o t e i n t o a n d r o g e n - i n d u c e d e t h y l m o r p h i n e N - d e m e t h y l a s e a c t i v i t y , u s i n g d i f f e r e n t a n i m a l m o d e l s . C o n s i s t e n t w i t h Roy et_ a l . ( 1 9 7 4 ) , t h e y r e p o r t e d t h a t h e p a t i c c y t o s o l i c DHT b i n d i n g p r o t e i n was p r e s e n t i n t h e male b u t n o t i n t h e immature male o r a d u l t f e m a l e r a t , and t h a t e s t r a d i o l a d m i n i s t r a t i o n i n h i b i t e d t h e p r e s e n c e o f t h i s b i n d i n g p r o t e i n i n t h e a d u l t m a l e . Hypophysectomy o f e i t h e r sex i n h i b i t e d and i n d u c e d t h i s b i n d i n g p r o t e i n i n t h e male and f e m a l e r a t s r e s p e c t i v e l y , an e f f e c t w h i c h was not i n f l u e n c e d by t e s t o s t e r o n e r e p l a c e m e n t . S i m i l a r t o Roy's g r o u p , and i n c o n t r a s t t o G u s t a f s s o n ' s and S a t o ' s g r o u p s , t h e s e a u t h o r s n e g l e c t e d t o c o n s i d e r l i g a n d m e t a b o l i s m i n v i t r o . F u r t h e r m o r e , i t was not e v i d e n t f r o m t h e a b s t r a c t i f t h e s e w o r k e r s were m e a s u r i n g a s p e c i f i c and s a t u r a b l e a n d r o g e n b i n d i n g p r o t e i n , n o r were p h y s i c o - c h e m i c a l b i n d i n g c h a r a c t e r i s t i c s r e p o r t e d . A l t h o u g h t h e r e seemed t o be a good c o r r e l a t i o n between t h e p r e s e n c e o f t h i s b i n d i n g p r o t e i n and h e p a t i c d r u g m e t a b o l i s m i n t h e s e d i f f e r e n t p h y s i o l o g i c a l and p h a r m a c o l o g i c a l a n i m a l m o d e l s , i t i s not known i f t h i s was a c a u s e - e f f e c t , o r m e r e l y a c o i n c i d e n t a l r e l a t i o n s h i p . The l a t t e r w o u l d seem p l a u s i b l e i f t h e s e w o r k e r s were m e a s u r i n g a n o n s p e c i f i c b i n d i n g p r o t e i n . The c o m p l i c a t i o n of l i g a n d m e t a b o l i s m has been overcome by t h e use o f a r e l a t i v e l y u n m e t a b o l i z a b l e l i g a n d , m e t h y l t r i e n o l o n e (R1881), a p o t e n t s y n t h e t i c a n d r o g e n w i t h p r o g e s t i n p r o p e r t i e s . R1881 has been shown t o b i n d t o a n d r o g e n r e c e p t o r s i n p r o s t a t e c y t o s o l ( B o e s e l and S h a i n 1974; P o u s e t t e e t a l . 1 979), and t h a t t a k e n f r o m r a t and human l i v e r c y t o s o l . Eagon e t a_l. (1983) r e p o r t e d t h e p r e s e n c e o f a s a t u r a b l e R1881 b i n d i n g p r o t e i n (Kd= 1.5 nM; c a p a c i t y = 10-15 fmol/mg) i n b o t h male and f e m a l e r a t l i v e r u n f r a c t i o n a t e d c y t o s o l c o n t a i n i n g t r i a m c i n o l o n e . T h i s b i n d i n g s i t e was d i s p l a c e d by r a d i o a c t i v e l y u n l a b e l l e d R1881 o r DHT. Gonadectomy o f e i t h e r s.ex l e d t o a t i m e - d e p e n d e n t d e c r e a s e i n R1881 b i n d i n g . D e c k e r and L e v i h s o n (1983) r e p o r t e d a R1881 h e p a t i c c y t o s o l i c b i n d e r w i t h a s i m i l a r a f f i n i t y (Kd= 1.4 nM) and h i g h e r c a p a c i t y (132 fmol/mg) compared t o Eagon's g r o u p ; however, i n c o n t r a s t t h e y u s e d male r a t u n f r a c t i o n a t e d l i v e r c y t o s o l i n c u b a t e d w i t h o u t t r i a m c i n o l o n e . T h i s b i n d i n g was d i s p l a c e d by t e s t o s t e r o n e and e s t r a d i o l b u t not by DES, s u g g e s t i n g t h a t R1881 was n o t b i n d i n g t o 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 . However, i n c o n t r a s t t o Eagon's work, t h e R1881 b i n d i n g p r o t e i n was n o t p r e s e n t i n t h e f e m a l e o r i n t h e Ps male r a t l i v e r c y t o s o l . T h e s e two s t u d i e s i n d i c a t e t h a t an a n d r o g e n b i n d i n g p r o t e i n i s p r e s e n t i n a d u l t male r a t h e p a t i c c y t o s o l . I n c o n s i s t e n c i e s i n t h e b i n d i n g k i n e t i c s o f t h i s p r o t e i n i n t h e d i f f e r e n t a n i m a l s t e s t e d by Eagon's, and D e c k e r and L e v i n s o n ' s g r o u p s may be due t o t h e d i f f e r e n c e s i n a s s a y c o n d i t i o n s , i e . t h e f o r m e r ' s g r o u p ' s use o f t r i a m c i n o l o n e i n t h e i r a s s a y m i x t u r e . O t h e r t e c h n i c a l f a c t o r s c a n n o t be a s s e s s e d s i n c e we a r e l i m i t e d by t h e l a c k o f d e s c r i p t i o n of t h e s e s t u d i e s . E i s e n f e l d e t a l . (1983) r e p o r t e d a R1881 b i n d i n g p r o t e i n i n a d u l t f e m a l e r a b b i t ammonium s u l p h a t e f r a c t i o n a t e d l i v e r c y t o s o l c o n t a i n i n g t r i a m c i n o l o n e and C o r t i s o l (Kd= 0.3 nM; c a p a c i t y = 79 fmo l / m g ) , w i t h s p e c i f i c i t y t o R1881, t e s t o s t e r o n e , and DHT, bu t n o t p r o g e s t e r o n e o r DES. A t e n e t a l . (1983) r e p o r t e d d i s p l a c e a b l e and s a t u r a b l e R1881 b i n d i n g i n human l i v e r c y t o s o l i n c u b a t e d w i t h t r i a m c i n o l o n e and C o r t i s o l (Kd= 1-2 nM; c a p a c i t y = 30-50 f m o l / m g ) . T h i s b i n d i n g was d i s p l a c e d by t e s t o s t e r o n e , DHT and s e v e r a l s y n t h e t i c p r o g e s t i n s . R e c e n t l y , a male s p e c i f i c 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 p r o t e i n has been d e s c r i b e d . I n a d d i t i o n t o d i s p l a c i n g r a d i o a c t i v e l y l a b e l l e d a n d r o g e n s f r o m t h e h e p a t i c c y t o s o l i c a n d r o g e n b i n d i n g p r o t e i n (Roy e t a l . 1974), u n l a b e l l e d a n d r o g e n s s u c h as DHT and t e s t o s t e r o n e d i s p l a c e d t r i t i a t e d - e s t r a d i o l f r o m a m o d e r a t e a f f i n i t y , m o d erate c a p a c i t y 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 p r o t e i n ; t h e l a t t e r i s p r e s e n t i n t h e a d u l t male b u t a b s e n t i n t h e f e m a l e ( D i c k s o n e t a l . 1978; P o w e l l - J o n e s e t a l . 1980; Warren 1982; F i n l a y s o n 1 9 83). D i c k s o n e t a l . (1978) r e p o r t e d t h a t 1 0 - f o l d m o l a r r a t i o e x c e s s r a d i o i n e r t DHT b u t not t e s t o s t e r o n e , d i s p l a c e d 1000 nM r a d i o a c t i v e l y l a b e l l e d e s t r a d i o l f r o m a d u l t male r a t l i v e r whole c y t o s o l by 40%, compared t o 70-80% d i s p l a c e d by r a d i o i n e r t e s t r a d i o l a t t h e same m o l a r r a t i o . T h i s s o l u b l e p r o t e i n had a b i n d i n g a f f i n i t y of 480 nM and c a p a c i t y of 480 fmol/mg p r o t e i n . P o w e l l - J o n e s e t a l . (1980) m e a s u r i n g a s i m i l a r p r o t e i n , c o n f i r m e d t h e l a t t e r g r o u p ' s r e s u l t s u s i n g a l o w e r e s t r a d i o l c o n c e n t r a t i o n w h i c h was c l o s e r t o t h e b i n d i n g a f f i n i t y (30 nM t r i t i a t e d - e s t r a d i o l i n t h e p r e s e n c e o r a b s e n c e o f , 1 0 0 - f o l d m o l a r r a t i o e x c e s s c o m p e t i t o r ) . They f o u n d t h e f o l l o w i n g amount of d i s p l a c e m e n t ( e x p r e s s e d as a % of t o t a l b i n d i n g ) : e s t r a d i o l ( 8 0 % ) ; DHT ( 6 3 % ) ; t e s t o s t e r o n e ( 3 7 % ) ; 5 a l p h a - a n d r o s t a n e - 3 a l p h a , 1 7 b e t a - d i o l ( 5 8 % ) ; a n d r o s t e n e d i o n e (0%) and DES ( 0 % ) . B o t h o f t h e s e s t u d i e s r e p o r t e d a r e l a t i v e s e l e c t i v i t y o f t h e m o d e r a t e a f f i n i t y , m o d e rate c a p a c i t y e s t r o g e n b i n d i n g p r o t e i n t o n a t u r a l e s t r o g e n s and some a n d r o g e n s , b u t n o t t o DES o r a n d r o s t e n e d i o n e , s u g g e s t i n g t h a t t h i s b i n d i n g p r o t e i n r e g u i r e d a d e g r e e o f s t r u c t u r a l s p e c i f i c i t y b u t was n o t t h e 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 r e c e p t o r , s i n c e DES b i n d s s p e c i f i c a l l y t o t h e l a t t e r . U n f o r t u n a t e l y , s a t u r a t i o n s t u d i e s u s i n g v a r i o u s c o n c e n t r a t i o n s o f l a b e l l e d e s t r a d i o l d i s p l a c e d by u n l a b e l l e d a n d r o g e n were n o t done, and t h e r e f o r e one does n o t know i f t h e s e s i n g l e p o i n t d i s p l a c e m e n t s t u d i e s u s i n g a n d r o g e n s c o u l d be r e l a t e d t o a s a t u r a b l e p r o t e i n s u c h as t h e mo d e r a t e a f f i n i t y - m o d e r a t e c a p a c i t y e s t r o g e n o r a n d r o g e n b i n d i n g p r o t e i n , o r p e r h a p s a d i s p l a c e a b l e , n o n s a t u r a b l e s t e r o i d b i n d i n g p r o t e i n . Our l a b o r a t o r y a t t e m p t e d t o answer some o f t h e s e q u e s t i o n s . Warren (1982) and F i n l a y s o n (1983) measured a mo d e r a t e a f f i n i t y , m o d e r a t e c a p a c i t y e s t r o g e n - a n d r o g e n b i n d i n g p r o t e i n . They r e p o r t e d a 15 t o 50% d i s p l a c e m e n t o f 30 nM t r i t i a t e d - e s t r a d i o l u s i n g t h e f o l l o w i n g r a d i o i n e r t c o m p e t i t o r s a t v a r i o u s m o l a r r a t i o s (10 t o 1000 f o l d e x c e s s ) : DHT, t e s t o s t e r o n e and a n d r o s t e n e d i o n e . M a x i m a l d i s p l a c e m e n t of t o t a l e s t r a d i o l b i n d i n g by u n l a b e l l e d e s t r a d i o l o c c u r r e d a t m o l a r r a t i o s g r e a t e r t h a n 1 0 0 - f o l d , w h i l e t h e a n d r o g e n s r e q u i r e d a t l e a s t 1 0 0 0 - f o l d m o l a r r a t i o s . S a t u r a t i o n s t u d i e s u s i n g a d u l t male whole c y t o s o l i n c u b a t e d w i t h v a r i o u s c o n c e n t r a t i o n s o f t r i t i a t e d - e s t r a d i o l , i n t h e a b s e n c e o r p r e s e n c e of 1 0 0 0 - f o l d e x c e s s r a d i o i n e r t e s t r a d i o l o r DHT gave t h e f o l l o w i n g b i n d i n g k i n e t i c s (n=5): Kd= 144 +/- 50 nM and Bmax= 17 +/- 4 pmol/mg u s i n g u n l a b e l l e d e s t r a d i o l ; Kd= 104 +/- 38 nM and Bmax= 14 +/- 3 pmol/mg u s i n g u n l a b e l l e d DHT. The s i m i l a r i t y i n b i n d i n g k i n e t i c s between t h e s e two d i f f e r e n t c o m p e t i t o r s w o u l d i n d i c a t e d i s p l a c e m e n t o f t h e r a d i o l a b e l l e d e s t r a d i o l f r o m t h e same b i n d i n g s i t e . T h e s e r e s u l t s c o n f i r m t h e r e s u l t s o f t h e e a r l i e r w o r k e r s . B ased on t h e i n h i b i t i o n s t u d i e s , t h i s p r o t e i n has a h i g h e r a f f i n i t y f o r e s t r a d i o l t h a n a n d r o g e n s . The e x a c t p h y s i o l o g i c a l r o l e of t h i s p r o t e i n i s n o t c l e a r . I t has been shown t o be age and sex d e p e n d e n t , i e . p r e s e n t i n t h e a d u l t male but a b s e n t o r i n low q u a n t i t i e s i n th e a d u l t f e m a l e , immature male and f e m a l e ( P o w e l l - J o n e s e t a l . 1980; F i n l a y s o n 1 9 8 3 ) . F i n l a y s o n (1983) r e p o r t e d a r e d u c t i o n i n b i n d i n g o f t h i s p r o t e i n f o l l o w i n g c a s t r a t i o n , an e f f e c t r e v e r s i b l e a f t e r t e s t o s t e r o n e a d m i n i s t r a t i o n ; i n c o n t r a s t , P o w e l l - J o n e s e t a l . (1980) f a i l e d t o see t h e s e e f f e c t s . L i k e t h e h e p a t i c m i c r o s o m a l d r u g and s t e r o i d m e t a b o l i z i n g enzymes, t h e p r e s e n c e of t h i s p r o t e i n was d e p e n d e n t on p i t u i t a r y f a c t o r s , s i n c e hypophysectomy o f t h e a d u l t male r e d u c e d b i n d i n g ( P o w e l l - J o n e s e t a l . 1980; F i n l a y s o n 1 9 8 3 ) . A l t h o u g h i t i s n o t t r a n s l o c a t e d i n t o t h e n u c l e u s ( P o w e l l - J o n e s e t a l . 1 980), t h i s p r o t e i n i s t h o u g h t t o be i n v o l v e d i n t h e r e g u l a t i o n of 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 s o f a n d r o g e n s and e s t r o g e n s , o r t h e i r m e t a b o l i t e s ( D i c k s o n e t al. 1978 ). In summary, i t i s n o t known i f t h e i n c o n s i s t e n c i e s 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 f t h e h e p a t i c a n d r o g e n b i n d i n g p r o t e i n a r e due t o d i f f e r e n c e s between s p e c i e s - o r s e x - d e p e n d e n t a n d r o g e n b i n d i n g p r o t e i n s , o r due t o t e c h n i c a l d i f f e r e n c e s i n l i g a n d u s e d (DHT, t e s t o s t e r o n e , a n d r o s t e n e d i o n e o r R1881), c y t o s o l p r e p a r a t i o n ( f r a c t i o n e d w i t h o r w i t h o u t ammonium, s u l p h a t e ) , o r i n c u b a t i o n c o n d i t i o n s ( w i t h o r w i t h o u t t r i a m c i n o l o n e and C o r t i s o l ) . D e s p i t e t h e s e d i s c r e p a n c i e s , an h e p a t i c c y t o s o l i c a n d r o g e n b i n d i n g p r o t e i n has been d e t e c t e d i n s e v e r a l mammalian s p e c i e s . I n o r d e r t o a c t as an a n d r o g e n r e c e p t o r , t h i s b i n d i n g p r o t e i n w o u l d have t o s e r v e a p h y s i o l o g i c a l r o l e . The i n t r a c e l l u l a r r o l e of t h e h e p a t i c c y t o s o l i c a n d r o g e n r e c e p t o r as a means of n u c l e a r t r a n s l o c a t i o n of a n d r o g e n l e a d i n g t o t h e s t i m u l a t i o n o f a n d r o g e n s e n s i t i v e p r o t e i n s y n t h e s i s , i s b a s e d on h e p a t i c n u c l e a r u p t a k e s t u d i e s i n v i v o ( E i s e n f e l d e t a l . 1983) and i n v i t r o ( D e c k e r and L e v i n s o n 1 9 8 3 ) . T h e s e s t u d i e s a r e p r e l i m i n a r y and more r e s e a r c h i s r e q u i r e d i n o r d e r t o b e t t e r u n d e r s t a n d t h i s p r o t e i n ' s p h y s i o l o g i c a l f u n c t i o n . 4. Summary Age- and s e x - d i f f e r e n c e s i n r a t h e p a t i c c y t o c h r o m e P-450 d e p e n d e n t d r u g m e t a b o l i z i n g enzymes a c t i v i t i e s have been a t t r i b u t e d t o i n c r e a s e d serum t e s t o s t e r o n e c o n c e n t r a t i o n s i n t h e male f o l l o w i n g p u b e r t y ( K a t o and G i l l e t t e 1965; E l Defrawy E l Masry e t a l . 1974); w h e t h e r t h i s phenomenon i s r e g u l a t e d by a r e c e p t o r s y s t e m i s n o t y e t c l e a r . T h i s i s i n c o n t r a s t w i t h t h e c l a s s i c a l a n d r o g e n t a r g e t t i s s u e s s u c h as p r o s t a t e o r e p i d i d y m u s , w h i c h a r e r e s p o n s i v e t o t e s t o s t e r o n e and t e s t o s t e r o n e a n t a g o n i s t s , and where a r e c e p t o r i s c l e a r l y i m p l i c a t e d . L i a o and Fang (1969) and o t h e r s have r e p o r t e d t h a t a n d r o g e n a n t a g o n i s t s s u c h as c y p r o t e r o n e a c e t a t e , f l u t a m i d e and f l u t a m i d e h y d r o x i d e r e d u c e d t a r g e t o r g a n w e i g h t . T h e s e e f f e c t s c o u l d be c o r r e l a t e d w i t h t h e c o m p e t i t i v e d i s p l a c e m e n t o f r a d i o l a b e l l e d a n d r o g e n s f r o m c y t o s o l i c a n d r o g e n b i n d i n g s i t e s i n v i t r o (Raynaud e_t a l . 1979 ). I n d i r e c t e v i d e n c e s u g g e s t i n g a r e g u l a t o r y r o l e f o r t h e h e p a t i c a n d r o g e n r e c e p t o r i n d r u g m e t a b o l i s m has been p r o v i d e d by Brown e t a l . (1976) who showed f l u t a m i d e b l o c k e d t e s t o s t e r o n e i n d u c e d h e p a t i c m i c r o s o m a l e t h y l m o r p h i n e N - d e m e t h y l a s e a c t i v i t y i n c a s t r a t e d m ice and p r e p u b e r t a l r a t s . T e c h n i c a l i n a d e q u a c i e s have made i t d i f f i c u l t t o i d e n t i f y a c y t o s o l i c h e p a t i c a n d r o g e n b i n d i n g p r o t e i n i n r a t l i v e r c y t o s o l ( G u s t a f s s o n e t a l . 1975; Roy e t a l . 1976; S a t o e t a l . 1980; L u c i e r and Rumbaugh 1983). The j u d i c i o u s use of R1881 has c i r c u m v e n t e d some o f t h e s e p r o b l e m s and has made t h i s s y n t h e t i c a n d r o g e n - p r o g e s t i n a s u c c e s s f u l t o o l t o i d e n t i f y t h e h e p a t i c a n d r o g e n b i n d i n g p r o t e i n (Eagon e t a l . 1983; D e c k e r and L e v i n s o n 1983; E i s e n f e l d e t a l . 1 9 8 3 ) . The above s t u d i e s p r o v i d e some e v i d e n c e s u g g e s t i n g t h a t an h e p a t i c a n d r o g e n r e c e p t o r e x i s t s i n l i v e r c y t o s o l . The l a c k of a p p r o p r i a t e c h a r a c t e r i z a t i o n s t u d i e s has made i t d i f f i c u l t t o a s s e s s t h i s p r o t e i n ' s p h y s i o l o g i c a l f u n c t i o n o r p h a r m a c o l o g i c a l r e l e v a n c e t o d r u g m e t a b o l i s m ; a s y s t e m a t i c a p p r o a c h c o r r e l a t i n g t h e p r e s e n c e of an h e p a t i c c y t o s o l i c a n d r o g e n r e c e p t o r t o t e s t o s t e r o n e d e p e n d e n t h e p a t i c m i c r o s o m a l enzyme i n d u c t i o n has not been p u r s u e d . A l t h o u g h t h e r e i s e v i d e n c e t o s u g g e s t a r e g u l a t o r y r o l e f o r c y t o s o l i c a n d r o g e n r e c e p t o r s i n e x t r a h e p a t i c t a r g e t t i s s u e s , d r u g m e t a b o l i s m r e s e a r c h has been l i m i t e d by t h e f a i l u r e t o d e m o n s t r a t e t h e e x i s t e n c e o f a s p e c i f i c h e p a t i c a n d r o g e n r e c e p t o r i_n v i v o and i n v i t r o . 5. O b j e c t i v e s of t h i s s t u d y Sex- and age d i f f e r e n c e s i n h e p a t i c m i c r o s o m a l BPH a c t i v i t i e s a r e w e l l - k n o w n ; however, t h e mechanism u n d e r l y i n g t h e s e phenomena i s u n c l e a r . T e s t o s t e r o n e s e c r e t i o n i n t h e a d u l t male has been i m p l i c a t e d . S i n c e t e s t o s t e r o n e s e n s i t i v e sex e n d - o r g a n t i s s u e s a r e known t o c o n t a i n c y t o s o l i c a n d r o g e n r e c e p t o r s w h i c h may be r e s p o n s i b l e f o r t h e a c t i o n o f a n d r o g e n s i n t h e s e t i s s u e s , a s i m i l a r r e c e p t o r mechanism may be m e d i a t i n g t e s t o s t e r o n e d e p e n d e n t l i v e r m i c r o s o m a l BPH i n d u c t i o n . To d e t e r m i n e i f h e p a t i c t e s t o s t e r o n e - i n d u c e d enzyme i n d u c t i o n i s a r e c e p t o r - m e d i a t e d phenomenon, enzyme m e t a b o l i s m and a n d r o g e n b i n d i n g p r o t e i n s t u d i e s were done, u s i n g d i f f e r e n t a n i m a l m o d els i n v a r i o u s e n d o c r i n o l o g i c a l and p h a r m a c o l o g i c a l s t a t e s . C o r r e l a t i o n s between t h e s e s t u d i e s w o u l d i m p l y a r e g u l a t o r y r o l e f o r t h e a n d r o g e n r e c e p t o r i n t e s t o s t e r o n e d e p e n d e n t h e p a t i c d r u g m e t a b o l i s m . The W i s t a r r a t was u s e d s i n c e i t i s a c o n v e n i e n t l a b o r a t o r y a n i m a l model t o s t u d y ; i t i s v e r y w e l l - c h a r a c t e r i z e d , and b e c a u s e i t has c h a r a c t e r i s t i c sex and age d e p e n d e n t h e p a t i c d r u g and s t e r o i d m e t a b o l i s m . The g o a l s o f t h i s s t u d y were: (a) t o s t u d y t h e e f f e c t s of t e s t o s t e r o n e on immature and a d u l t male and f e m a l e h e p a t i c m i c r o s o m a l d r u g m e t a b o l i z i n g enzymes, by e x a m i n i n g t h e r e s p o n s i v i t y o f t h e s e a n i m a l s t o t h e male hormone. (b) t o s t u d y t h e e f f e c t o f a n d r o g e n a n t a g o n i s t s on t e s t o s t e r o n e - i n d u c e d enzyme a c t i v a t i o n , u s i n g t h e a d u l t c a s t r a t e d male r a t . R e s u l t s o f t h e s e s t u d i e s may i n d i c a t e t h a t t e s t o s t e r o n e - i n d u c e d h e p a t i c enzyme a c t i v a t i o n i s m e d i a t e d by a s p e c i f i c a n d r o g e n r e c e p t o r mechanism. ( c ) as a f i r s t a p p r o a c h t o i d e n t i f y and c h a r a c t e r i z e t h e h e p a t i c a n d r o g e n r e c e p t o r i n v i t r o , a n d r o g e n b i n d i n g was s t u d i e d u s i n g t h e c y t o s o l i c f r a c t i o n o f l i v e r homogenate s i n c e i t has been p u r p o r t e d t o c o n t a i n t h e a n d r o g e n r e c e p t o r (Roy e t a l . 1974; S a t o e t a l . 1 9 8 0 ) . Changes i n t h e b i n d i n g k i n e t i c s o f t h i s h e p a t i c c y t o s o l i c a n d r o g e n b i n d i n g p r o t e i n may be c o r r e l a t e d t o a l t e r a t i o n s i n h e p a t i c m i c r o s o m a l enzyme a c t i v i t i e s , u s i n g d i f f e r e n t p h y s i o l o g i c a l and p h a r m a c o l o g i c a l a n i m a l m o d e l s . MATERIALS AND METHODS 1. C h e m i c a l s The f o l l o w i n g c h e m i c a l s were p u r c h a s e d f r o m Sigma C h e m i c a l Co. ( S t . L o u i s , MO): NAD, NADH, NADP, NADPH, g l u c o s e - 6 - p h o s p h a t e , g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e , b o v i n e serum a l b u m i n ( B S A ) , t e s t o s t e r o n e e n a n t h a t e , s t r e p t o z o t o c i n ( S T Z ) , b e n z o ( a ) p y r e n e , t e s t o s t e r o n e , a n d r o s t e n e d i o n e , 5 a l p h a - d i h y d r o t e s t o s t e r o n e (5 a l p h a - D H T ) , 5 b e t a - d i h y d r o t e s t o s t e r o n e (5 beta- D H T ) , t r i a m c i n o l o n e a c e t o n i d e , 5 b e t a - a n d r o s t a n - 3 b e t a , 1 7 b e t a - d i o l (5 b e t a - A - 3 b e t a , 17 b e t a - d i o l ) , e s t r a d i o l , s p i r o n o l a c t o n e , h y d r o c o r t i s o n e , d i e t h y l s t i l b e s t r o l ( D E S ) , p r o g e s t e r o n e , t r i s ( h y d r o x y m e t h y 1 ) a m i n o m e t h a n e ( T r i z m a - b a s e ) , 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 (EDTA), D L - d i t h i o t h r e i t o l ( C l e l a n d ' s R e a g e n t , D T T ) , H i d e Powder A z u r e , sodium m o l y b d a t e , a c t i v a t e d c h a r c o a l and p r o t a m i n e s u l p h a t e . P r o t a m i n e z i n c i n s u l i n (100 u n i t s / m l ) was o b t a i n e d f r o m Connaught L a b o r a t o r i e s ( W i l l o w d a l e , O n t . ) . F l u t a m i d e and f l u t a m i d e h y d r o x i d e ( S c h l 6 4 2 3 ) were g i f t s f r o m S c h e r i n g Co. ( B l o o m f i e l d , N J ) , w h i l e c y p r o t e r o n e a c e t a t e was d o n a t e d by S c h e r i n g AG ( B e r l i n , Germany). RU23908 and RU2956 were g i f t s f r o m R o u s s e l U c l a f ( R o u m a i n v i l l e , F r a n c e ) . S i l i c a g e l w i t h f l u o r e s c e n t i n d i c a t o r t h i n l a y e r c h r o m a t o g r a p h y s h e e t s (20x20 cm), g l y c e r o l and d e x t r a n T70 were o b t a i n e d f r o m Eastman Kodak Co. ( R o c h e s t e r , NY), B r i t i s h Drug House C h e m i c a l s ( V a n c o u v e r , B C ) , and P h a r m a c i a F i n e C h e m i c a l s ( U p p s a l a , Sweden), r e s p e c t i v e l y . N-methyl-N-t r i m e t h y l s i l y l t r i f l u o r o a c e t a m i d e (MSTFA) and methoxyamine r e a g e n t (MOX; 2% methoxyamine h y d r o c h l o r i d e i n p y r i d i n e ) were s u p p l i e d by P i e r c e C h e m i c a l Co. ( R o c k f o r d , I L ) . 2 , 4 - d i n i t r o p h e n y l h y d r a z i n e was o b t a i n e d f r o m B r i t i s h Drug House C h e m i c a l s ( P o o l e , E n g l a n d ) . B e n z o ( a ) p y r e n e was f u r t h e r . p u r i f i e d by s o l u b i l i z a t i o n i n b e n z e n e , f i l t r a t i o n and f i n a l l y r e c r y s t a l l i z a t i o n i n c o l d m e t h a n o l . T h i s p r o c e d u r e y i e l d e d f i n e and n e e d l e - l i k e y e l l o w c r y s t a l s . R a d i o a c t i v e l y l a b e l l e d (17 a l p h a - m e t h y 1 -t r i t i a t e d ) - m e t h y l t r i e n o l o n e (R1881, s p e c i f i c a c t i v i t y : 87 Ci/mmol) and r a d i o i n e r t R1881, 1 a l p h a , 2 a l p h a - t r i t i a t e d ( N ) - t e s t o s t e r o n e ( s p e c i f i c a c t i v i t y : 50-70 Ci/mmol) and 1 , 2 - t r i t i a t e d ( N ) - d i h y d r o t e s t o s t e r o n e (DHT, s p e c i f i c a c t i v i t y : 52 Ci/mmol) were o b t a i n e d f r o m New E n g l a n d N u c l e a r (NEN)- Canada L t d . , ( M o n t r e a l , PQ) u n d e r l i c e n c e d agreement w i t h 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 ) . L a b e l l e d (17 a l p h a - m e t h y 1 - t r i t i a t e d ) - and r a d i o i n e r t m i b o l e r o n e (7 a l p h a , 1 7 a l p h a - d i m e t h y l - 1 9 - n o r t e s t o s t e r o n e ) , were p u r c h a s e d f r o m Amersham Co. ( O a k v i l l e , Ont) under l i c e n c e d a greement f r o m U p j o h n Co. (Kalamazoo, M I ) . The s p e c i f i c a c t i v i t y o f t h i s r a d i o - l i g a n d was 72 Ci/mmol. The f o l l o w i n g s t e r o i d s were g r a c i o u s l y d o n a t e d by Dr. P. R e n n i e ( C a n c e r C o n t r o l A g e n cy o f B C ) : 5 a l p h a - a n d r o s t a n - 3 b e t a , 1 7 b e t a - d i o l (5 a l p h a - A - 3 b e t a , 17 b e t a - d i o l ) , 5 b e t a - a n d r o s t a n - 3 a l p h a , 1 7 b e t a - d i o l (5 b e t a - A - 3 a l p h a , 1 7 b e t a - d i o l ) and 5 a l p h a - a n d r o s t a n - 3 a l p h a , 17 b e t a - d i o l (5 36 a l p h a - A - 3 a l p h a , 1 7 b e t a - d i o l ) , w h i l e 1,2 n a p h t h o q u i n o n e - 4 - s u l p h o n i c a c i d - s o d i u m s a l t was p r o v i d e d by Mr. M. Coombs ( U n i v e r s i t y o f T o r o n t o ) . A l l s o l v e n t s and o t h e r r e a g e n t s were o f t h e r e a g e n t g r a d e . 2. C a r e and s u r g e r y o f a n i m a l s A d u l t (60-85 days o l d ) male (275-350 g) and f e m a l e (180-200 g ) , and immature male and f e m a l e (50 g, 20-24 days o l d , 8 d a y s p o s t - w e a n i n g ) W i s t a r r a t s were p u r c h a s e d l o c a l l y (UBC A n i m a l C a r e U n i t ) , o r f r o m C a n a d i a n B r e e d i n g L a b o r a t o r i e s (La P r a i r e , P.Q.). A l l a n i m a l s were housed i n g r o u p s of f o u r , i n p l a s t i c c a g e s c o n t a i n i n g c o r n c o b b e d d i n g (Lobund g r a d e , P a x t o n P r o c e s s i n g L t d . , P a x t o n , I L ) , u n d e r s t a n d a r d c o n d i t i o n s (22 d e g r e e s C, l i g h t s ON 06:00-20:00 h o u r s ) and m a i n t a i n e d on P u r i n a L a b o r a t o r y Chow and w a t e r ad  l i b i t u m . F o r some e x p e r i m e n t s , a d u l t male r a t s were h y p o p h y s e c t o m i z e d by C h a r l e s R i v e r L a b o r a t o r i e s ( B o s t o n , MA) and were s h i p p e d s e v e n d a y s p o s t s u r g e r y . C o m p l e t e n e s s of hypophysectomy was a s s e s s e d by v i s u a l i n s p e c t i o n and by t h e d e c r e a s e i n body w e i g h t compared t o t h e s h a m - o p e r a t e d c o n t r o l . In o t h e r e x p e r i m e n t s , c a s t r a t i o n and o v a r i e c t o m y o f r a t s were done u s i n g t h e s c r o t a l and d o r s a l r o u t e s , r e s p e c t i v e l y . 3. T r e a t m e n t o f A n i m a l s 0 In o r d e r t o d e t e r m i n e a s u i t a b l e dose o f t e s t o s t e r o n e e n a n t h a t e w h i c h i n d u c e s maximum a n d r o g e n i c r e s p o n s e s , d i f f e r e n t d a i l y s u b c u t a n e o u s ( s c ) d o s e s of t e s t o s t e r o n e e n a n t h a t e (0, 1.25, 2.5, o r 5 m i c r o m o l e s / k g i n c o r n o i l v e h i c l e ) were i n j e c t e d i n t o 10-day c a s t r a t e d a d u l t male r a t s f o r 10 d a y s . R e s u l t s f r o m p r e l i m i n a r y e x p e r i m e n t s showed c o n s i d e r a b l e d i f f e r e n c e s i n r e s p o n s e t o low d o s e s of t e s t o s t e r o n e e n a n t h a t e , t h e r e f o r e a dose of 5 m i c r o m o l e s / k g was c h o s e n as a s u i t a b l e dose t o m a i n t a i n c o n s i s t e n t p h y s i o l o g i c a l l e v e l s of p r o s t a t e w e i g h t and s e x - d e p e n d e n t h e p a t i c m i c r o s o m a l d r u g m e t a b o l i z i n g enzyme a c t i v i t i e s i n t h e a d u l t c a s t r a t e d male r a t , d e s p i t e b i o l o g i c a l v a r i a t i o n . The f l u t a m i d e dose r e s p o n s e c u r v e was d e t e r m i n e d u s i n g t e s t o s t e r o n e e n a n t h a t e (5 m i c r o m o l e s / k g , s c . , 10 d a y s ) t r e a t e d c a s t r a t e d a d u l t male r a t s , i n j e c t e d w i t h c o r n o i l v e h i c l e o r d i f f e r e n t d o s e s o f f l u t a m i d e (5 t o 50 m i c r o m o l e s / k g , s c . , 10 d a y s ) . A d u l t f e m a l e r a t s were i n j e c t e d s c . d a i l y w i t h 25 m i c r o m o l e s / k g f l u t a m i d e o r f l u t a m i d e h y d r o x i d e f o r 10 d a y s . In o t h e r s t u d i e s , a d u l t m ales were i n t r a v e n o u s l y ( i v ) i n j e c t e d w i t h s t r e p t o z o t o c i n (STZ; 60 mg/kg) d i s s o l v e d i n 0.1 M c i t r a t e b u f f e r (pH 4.5) f o u r d a y s p r i o r t o t h e e x p e r i m e n t . C o n t r o l r a t s were t r e a t e d w i t h e q u a l volumes o f t h e c i t r a t e b u f f e r . A s e p a r a t e g r o u p o f S T Z - t r e a t e d a n i m a l s were i n j e c t e d s c . d a i l y f o r 4 d a y s p o s t STZ i n j e c t i o n , w i t h 10 u n i t s / k g p r o t a m i n e z i n c i n s u l i n ( P Z I ) , i n o r d e r t o r e v e r s e t h e e f f e c t s o f d i a b e t e s ( u s i n g g l u c o s u r i a as an i n d e x ) . The i n s u l i n r e p l a c e m e n t e x p e r i m e n t s were done 24 h o u r s a f t e r t h e l a s t PZI i n j e c t i o n . A l l o f t h e d i a b e t i c r a t s ( e x c l u d i n g t h e P Z I - t r e a t e d a n i m a l s ) t e s t e d had u r i n e g l u c o s e l e v e l s >0.5%, u s i n g th e T e s - T a p e ( E l i L i l l y and Co. L t d . , T o r o n t o , O n t a r i o ) method. 4. S u b c e l l u l a r f r a c t i o n a t i o n : (a) P r e p a r a t i o n o f m i c r o s o m e s f o r enzyme s t u d i e s E a ch a n i m a l was d e c a p i t a t e d , t h e l i v e r was e x s a n g u i n a t e d by p e r f u s i o n w i t h i c e c o l d aqueous KCI (1.15% w/v). The l i v e r was q u i c k l y d i s s e c t e d and was immersed i n i c e c o l d i s o t o n i c KCI s o l u t i o n . F o l l o w i n g t h i s , t h e l i v e r was b l o t t e d and t h e wet w e i g h t was d e t e r m i n e d by d i f f e r e n c e . A p p r o x i m a t e l y f i v e grams of e a c h a n i m a l ' s l i v e r t i s s u e , i n c l u d i n g t h e l a r g e s t l i v e r l o b e , were p l a c e d i n t o a 50 ml s i z e A P o t t e r - E l v j h e i m t i s s u e h o m o g e n i z e r c o n t a i n i n g 20 ml o f c o l d i s o t o n i c KCI s o l u t i o n . The a n i m a l ' s t i s s u e was h o mogenized s e p a r a t e l y f o r 75 s e c o n d s o r a p p r o x i m a t e l y 11-20 p a s s e s , was p o u r e d i n t o p l a s t i c c e n t r i f u g e t u b e s , and was c e n t r i f u g e d a t 10,000 x g f o r 10 m i n u t e s a t 4 d e g r e e s C, by means of an IEC Model B-20 c e n t r i f u g e . F o l l o w i n g t h i s , the l i p i d l a y e r c o v e r i n g t h e s u p e r n a t a n t was c a r e f u l l y removed and 8 ml v olumes o f t h e l a t t e r were p i p e t t e d i n t o s e p a r a t e 10 ml p o l y c a r b o n a t e u l t r a c e n t r i f u g e t u b e s ; the r e m a i n i n g p e l l e t was d i s c a r d e d . The s u p e r n a t a n t was c e n t r i f u g e d f o r a n o t h e r 65 m i n u t e s a t 105,000 x g a t 4 d e g r e e s C, by means of a Beckman Model L5-50 u l t r a c e n t r i f u g e . A f t e r c e n t r i f u g a t i o n , t h e s o l u b l e f r a c t i o n was d i s c a r d e d and t h e p e l l e t s ( m i c r o s o m a l p r e c i p i t a t e ) were c a r e f u l l y washed w i t h t h r e e 4 ml volumes of i c e - c o l d aqueous 0 .1 M phosphate b u f f e r (pH 1.2), and r e - s u s p e n d e d i n 4 ml of the same. (b) P r e p a r a t i o n of c y t o s o l and microsomes f o r r a d i o - l i g a n d b i n d i n g s t u d i e s T h i s p r o t o c o l f o l l o w e d a s i m i l a r methodo logy as a b o v e . I n b r i e f , each a n i m a l was s t u n n e d by a blow t o the h e a d , was d e c a p i t a t e d and was e x s a n g u i n a t e d . The l i v e r was q u i c k l y d i s s e c t e d and p e r f u s e d w i t h i c e - c o l d TED b u f f e r (pH 7.4) c o n t a i n i n g : 0 .05 M T r i s - b a s e ; 1.5 mM E D T A - d i s o d i u m ; 0 .5 mM DTT; 10% g l y c e r o l ; w i t h o r w i t h o u t 20 mM sodium m o l y b d a t e ; w i t h o r w i t h o u t 10 mM K C I . I n d i v i d u a l l i v e r s were we ighed and were homogenized s e p a r a t e l y i n c o l d b u f f e r u s i n g a p r e - c o o l e d P o t t e r - E l v e j h e i m homogenizer and t e f l o n p e s t l e . Homogenates were t h e n c e n t r i f u g e d a t 10 ,000 x g f o r 10 min a t 4 degrees C , and the s u p e r n a t a n t was r e - c e n t r i f u g e d a t 105,000 x g f o r 65 m i n a t 4 degree s C . The r e s u l t i n g s u p e r n a t a n t s (or c y t o s o l i c f r a c t i o n ) and re su spended p e l l e t s ( c o n t a i n i n g the m i c r o s o m a l f r a c t i o n ) were d i l u t e d w i t h i c e - c o l d b u f f e r t o the d e s i r e d p r o t e i n c o n c e n t r a t i o n . I n o t h e r e x p e r i m e n t s , r a t p r o s t a t e c y t o s o l was p r e p a r e d . O n l y t h e s t r a w - c o l o u r e d g l a n d u l a r p r o s t a t i c t i s s u e was used f o r the se e x p e r i m e n t s . The p r o s t a t i c c y t o s o l was p r e p a r e d as d e s c r i b e d a b o v e . 40 5. H e p a t i c m i c r o s o m a l enzyme a s s a y s (a) H e p a t i c m i c r o s o m a l b e n z o ( a ) p y r e n e h y d r o x y l a s e a s s a y The BPH a s s a y ( N e b e r t and G e l b o i n 1968) was c a r r i e d o u t by t h e a d d i t i o n o f m i c r o s o m a l p r o t e i n (7.5 mg wet l i v e r w e i g h t ) t o a 50 mM T r i s b u f f e r (pH 7.5) s o l u t i o n c o n t a i n i n g : 80 mM BP; 0.44 mM NADPH; 0.47 mM NADH; 3 mM magnesium c h l o r i d e and 0.6% b o v i n e serum a l b u m i n (BSA). The i n c u b a t i o n o f samples and b l a n k s ( i n d u p l i c a t e ) was c a r r i e d o u t f o r 5 m i n u t e s a t 37 d e g r e e s C, and was s t o p p e d by a d d i t i o n o f c o l d .acetone and hexane. The major m e t a b o l i t e o f BP, 3-OH BP was e x t r a c t e d f r o m t h e hexane p h a s e , by a d d i t i o n o f t h e l a t t e r t o 1 N NaOH. The o r g a n i c phase was s e p a r a t e d f r o m t h e aqueous f r a c t i o n by d i f f e r e n t i a l f r e e z i n g u s i n g a d r y i c e - a c e t o n e b a t h . A f t e r t h e o r g a n i c p o r t i o n o f the m i x t u r e was d i s c a r d e d , t h e aqueous NaOH phase was m e l t e d t o room t e m p e r a t u r e . The 3-OH BP was measured by f l u o r o m e t r i c a n a l y s i s a t a c t i v a t i o n and e m i s s i o n (nm) w a v e l e n g t h s of 396 and 522 r e s p e c t i v e l y , u s i n g a q u i n i n e s u l p h a t e p r e - c a l i b r a t e d T u r n e r S p e c t r o f l u o r o m e t e r Model 430. (b) H e p a t i c m i c r o s o m a l t e s t o s t e r o n e r e d u c t a s e a s s a y M i c r o s o m a l TR was measured f o l l o w i n g t h e a s s a y d e s c r i b e d by M c G u i r e and Tomkins ( 1 9 5 9 ) . T h i s s p e c t r o p h o t o m e t r i c a s s a y i n v o l v e s measurement o f t h e e n z y m a t i c r e d u c t i o n o f t h e t e s t o s t e r o n e C4-C5 c o n j u g a t e d d o u b l e bond. In t h i s a s s a y , m i c r o s o m e s (6 mg p r o t e i n ) were added t o an aqueous 0.1 M p h o s p h a t e b u f f e r (pH 7.2) s o l u t i o n c o n t a i n i n g : 0.15 mM t e s t o s t e r o n e ; 6.3 mM magnesium c h l o r i d e ; .0.5 mM NADP; 10 mM g l u c o s e - 6 - p h o s p h a t e and 1 u n i t g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e , and were i n c u b a t e d f o r 10 m i n u t e s a t 37 d e g r e e s C. The r e a c t i o n was t e r m i n a t e d u s i n g d i c h l o r o m e t h a n e . The l a t t e r was s haken and t h e aqueous phase and i n t e r p h a s e were a s p i r a t e d . The r e m a i n i n g o r g a n i c p h a s e was u s e d f o r s p e c t r o p h o t o m e t r i c s c a n n i n g (210-290 nm), u s i n g a P e r k i n - E l m e r Model 124 d o u b l e beam s p e c t r o p h o t o m e t e r . The amount of t e s t o s t e r o n e r e d u c e d was c a l c u l a t e d by t h e d i f f e r e n c e i n t h e a b s o r b a n c e i n t h e c o n c e n t r a t i o n o f t e s t o s t e r o n e p r e s e n t b e f o r e and a f t e r i n c u b a t i o n . Samples and b l a n k s were i n c u b a t e d i n d u p l i c a t e . 6. O t h e r a s s a y s Serum t e s t o s t e r o n e c o n c e n t r a t i o n s were d e t e r m i n e d u s i n g t h e f o l l o w i n g p r o t o c o l . A n i m a l s were k i l l e d and b l o o d s a m p l e s were c o l l e c t e d i n h e p a r i n i z e d t e s t t u b e s . B l o o d was a l l o w e d t o c l o t f o r 30 m i n u t e s and t h e serum was p r e p a r e d by c e n t r i f u g a t i o n . Serum t e s t o s t e r o n e c o n c e n t r a t i o n s were d e t e r m i n e d u s i n g t h e p r o t o c o l o u t l i n e d i n t h e RSL N o s o l v e x ( I o d i n e - 1 2 5 ) - t e s t o s t e r o n e r a d i o i m m u n o a s s a y k i t ( R a d i o a s s a y System L a b o r a t o r i e s I n c . , C a r s o n , CA 9 0 7 4 6 ) . The a s s a y was s p e c i f i c ( c r o s s - r e a c t i v i t y t o 5 a l p h a - d i h y d r o t e s t o s t e r o n e was 10.3% and t o o t h e r s t e r o i d s <0.01%) and was s e n s i t i v e t o t e s t o s t e r o n e f r o m 0.05 t o 25 ng/ml. R a d i o a c t i v i t y was measured u s i n g a N u c l e a r C h i c a g o S e r i e s 1185 A u t o m a t i c Gamma C o u n t i n g System. C y t o s o l i c p r o t e o l y t i c a c t i v i t y was d e t e r m i n e d u s i n g t h e s p e c t r o p h o t o m e t r i c method d e s c r i b e d by R i n d e r k n e c h t e t a l . ( 1 9 6 8 ) . In b r i e f , t h e a s s a y measures t h e p r o t e o l y t i c a c t i v i t y by t h e t o t a l s o l u b i l i z a t i o n of R e m a z o l b r i l l i a n t B l u e - H i d e powder (RBB-Hide; H i d e Powder A z u r e ) , an i n s o l u b l e d y e - p r o t e i n complex. L i v e r whole c y t o s o l ( c o n t a i n i n g 40 mg/ml p r o t e i n i n TED b u f f e r ) was i n c u b a t e d i n T r i s b u f f e r (pH 7.2) c o n t a i n i n g 2 mM c a l c i u m c h l o r i d e , f o r 3 o r 18 h o u r s a t 37 d e g r e e s C. The o p t i c a l a b s o r b a n c e was measured a t 595 nm, u s i n g a P e r k i n - E l m e r Model 124 d o u b l e beam s p e c t r o p h o t o m e t e r . T h i s a s s a y gave a l i n e a r s t a n d a r d c u r v e f o r t r y p s i n w i t h a s e n s i t i v i t y f r o m 1 t o 2 ng t r y p s i n p e r ml a s s a y m i x t u r e ( R i n d e r k n e c h t e t a_l. 1968 ). M i c r o s o m a l and c y t o s o l i c p r o t e i n c o n t e n t s were d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y u s i n g t h e Coumassie B r i l l a n t B l u e G method as d e s c r i b e d by B r a d f o r d ( 1 9 7 6 ) . D i l u t i o n s o f a BSA s t o c k s o l u t i o n (0.05-0.50 mg/ml) p r o d u c e d a l i n e a r s t a n d a r d c u r v e , u s i n g a P e r k i n - E l m e r Model 124 d o u b l e beam s p e c t r o p h o t o m e t e r . 7. T e s t o s t e r o n e M e t a b o l i s m S t u d i e s (a) P r e p a r a t i o n o f s t a n d a r d s t e r o i d s o l u t i o n s and c y t o s o l e x t r a c t s f o r c h r o m a t o g r a p h y S t a n d a r d s o l u t i o n s o f r a d i o a c t i v e l y u n l a b e l l e d t e s t o s t e r o n e and r e l a t e d m e t a b o l i t e s were p r e p a r e d (1 mg/ml) i n a b s o l u t e e t h a n o l o r d r y d i e t h y l e t h e r , and were s t o r e d i n g l a s s t u b e s f i t t e d w i t h f o i l - w r a p p e d c o r k s , a t 4 d e g r e e s C. L i v e r whole c y t o s o l (40 mg/ml) was p r e p a r e d f o l l o w i n g h o m o g e n i z a t i o n and d i f f e r e n t i a l c e n t r i f u g a t i o n s t e p s . An a l i q u o t (5 ml) o f c y t o s o l was i n c u b a t e d w i t h 2 m i c r o g r a m s t e s t o s t e r o n e f o r 0 o r 3 h o u r s , a t 4 o r 37 d e g r e e s C. I n c u b a t i o n was t e r m i n a t e d by a d d i t i o n o f 10 volumes of d i e t h y l e t h e r and t h e s t e r o i d s were e x t r a c t e d u s i n g t h e same o r g a n i c s o l v e n t . In a s e p a r a t e e x p e r i m e n t , 5 beta-DHT was s u b s t i t u t e d f o r t e s t o s t e r o n e . F o r t h e t r i t i a t e d - t e s t o s t e r o n e t h i n - l a y e r c h r o m a t o g r a p h y e x p e r i m e n t s , c y t o s o l was i n c u b a t e d i n d u p l i c a t e , w i t h v a r y i n g c o n c e n t r a t i o n s of t r i t i a t e d - t e s t o s t e r o n e (2-40 nM) i n t h e p r e s e n c e o r a b s e n c e o f 1 0 0 - f o l d e x c e s s m o l a r r a t i o r a d i o a c t i v e l y u n l a b e l l e d t e s t o s t e r o n e f o r 3 h o u r s a t 4 d e g r e e s C. F o l l o w i n g i n c u b a t i o n , bound and f r e e s t e r o i d f r a c t i o n s were s e p a r a t e d u s i n g the 2.5% d e x t r a n - c o a t e d c h a r c o a l t e c h n i q u e . An a l i q u o t o f t h e s u p e r n a t a n t ( c o n t a i n i n g t h e bound f r a c t i o n ) was added t o 10 volumes o f d i e t h y l e t h e r f o r s t e r o i d e x t r a c t i o n . L a b e l l e d and u n l a b e l l e d s t e r o i d s were e x t r a c t e d f r o m c y t o s o l u s i n g t h r e e washes o f d i e t h y l e t h e r ( 1 0 : 1 ) . E t h e r e a l e x t r a c t s were c o l l e c t e d and were d r i e d under a s t r e a m o f d r y n i t r o g e n , o r by u s i n g a r o t a r y vacuum e v a p o r a t o r . T h i s e x t r a c t i o n method was 97% e f f i c i e n t . (b) S e p a r a t i o n o f t e s t o s t e r o n e f r o m r e l a t e d m e t a b o l i t e s u s i n g t h i n - l a y e r c h r o m a t o g r a p h y (TLC) A l l s o l v e n t s were p r e - d r i e d f o r a t l e a s t 18 h o u r s p r i o r t o e x p e r i m e n t , u s i n g c a l c i u m c h l o r i d e o r p o t a s s i u m c a r b o n a t e . The s o l v e n t s y s t e m s were p r e p a r e d a c c o r d i n g t o L i s b o a ( 1 9 6 2 ) : s y s t e m I - e t h y l a c e t a t e : c y c l o h e x a n e (50:50, v : v ) , s y s t e m I I - b e n z e n e : a b s o l u t e e t h a n o l (95:5, v : v ) . TLC s h e e t s were p r e - d r i e d a t 90 d e g r e e s C f o r 10-20 mins p r i o r t o s t e r o i d a p p l i c a t i o n . Each d r i e d e t h e r e a l e x t r a c t was r e d i s s o l v e d i n a s m a l l volume of d r y d i e t h y l e t h e r ; t h i s o r a volume of s t a n d a r d s t e r o i d s o l u t i o n ( c o n t a i n i n g 1 t o 10 m i c r o g r a m s s t e r o i d ) was c a r e f u l l y a p p l i e d and was d r i e d as s e p a r a t e s p o t s ( d i a m e t e r <1 cm), o n t o t h e TLC s h e e t s (2 cm f r o m b o t t o m e d g e ) , u s i n g a m o d i f i e d 10 m i c r o l i t e r H a m i l t o n s y r i n g e and a b l o w - d r y e r . The g l a s s c h r o m a t o g r a p h y chamber c o n t a i n i n g 100 ml s o l v e n t and f l u t e d f i l t e r p a p e r was p r e - e q u i l i b r a t e d a t room t e m p e r a t u r e f o r a t l e a s t 2 h o u r s p r i o r t o c h r o m a t o g r a p h y . Each TLC s h e e t was p l a c e d i n t o a s e p a r a t e chamber, and t h e s o l v e n t f r o n t was a l l o w e d t o r u n f o r 15-17 cm above t h e o r i g i n . The a v e r a g e r u n n i n g t i m e was 65 mins b e f o r e t h e s o l v e n t f r o n t was marked and e a c h TLC s h e e t was a l l o w e d t o d r y a t room t e m p e r a t u r e b e f o r e s t e r o i d v i s u a l i z a t i o n and i d e n t i f i c a t i o n . (c) V i s u a l i z a t i o n of t e s t o s t e r o n e and r e l a t e d s m e t a b o l i t e s u s i n g UV l i g h t and c o l o u r r e a g e n t s The p o l y a c r y l i c b i n d e r c o n t a i n e d i n t h e s i l i c a g e l s h e e t s p r e c l u d e d c h a r r i n g t e c h n i q u e s u s i n g s t r o n g a c i d s . F o r t h i s r e a s o n , t h e f o l l o w i n g a l t e r n a t i v e t e c h n i q u e s were u s e d : ( i ) UV v i s u a l i z a t i o n o f s t e r o i d s c o n t a i n i n g t h e d e l t a - 4 u n s a t u r a t e d d o u b l e bond ( e g . t e s t o s t e r o n e o r d e l t a - 4 A - d i o n e ) was done u s i n g a Chromato-Vue c a b i n e t s e t a t 265 nm ( U l t r a - v i o l e t P r o d . I n c . , San G a b r i e l , C A ) . T h i s e a s y and c o n v e n i e n t t e c h n i q u e was r o u t i n e l y done p r i o r t o a d d i t i o n o f d i f f e r e n t c o l o u r r e a g e n t s . ( i i ) D i n i t r o p h e n y l h y d r a z i n e r e a g e n t was p r e p a r e d a c c o r d i n g t o V o g e l ( 1 9 5 7 ) . In b r i e f , 2 g o f 2 , 4 - d i n i t r o p h e n y l h y d r a z i n e r e a g e n t was s u s p e n d e d i n 100 ml d r y m e t h a n o l . F o u r m i l l i l i t r e s o f c o n c e n t r a t e d s u l p h u r i c a c i d were c a u t i o u s l y added and t h e s o l u t e was c a r e f u l l y d i s s o l v e d . The r e m a i n i n g u n d i s s o l v e d p a r t i c l e s were removed u s i n g vacuum f i l t r a t i o n . The r e a g e n t was l i g h t l y s p r a y e d and t h o s e s t e r o i d s c o n t a i n i n g 3- o r 17- k e t o g r o u p s c o u l d be q u i c k l y and e a s i l y v i s u a l i z e d as o r a n g e s p o t s on a y e l l o w b a c k g r o u n d , w i t h a s e n s i t i v i t y of 1 m i c r o g r a m . ( i i i ) N a p h t h o q u i n o n e P e r c h l o r i c a c i d r e a g e n t was p r e p a r e d a c c o r d i n g t o R i c h t e r ( 1 9 6 5 ) . A 0.1% (w/v) s o l u t i o n of 1,2 n a p h t h o q u i n o n e 4 - s u l p h o n i c a c i d sodium s a l t was p r e p a r e d i n a m i x t u r e o f a b s o l u t e e t h a n o l : 60% p e r c h l o r i c a c i d : 40% f o r m a l d e h y d e : w a t e r (2: 1: 0.1: 0 . 9 ) . The f i n a l s o l u t i o n was f i l t e r e d t o remove u n d i s s o l v e d p a r t i c l e s . The r e a g e n t was s p r a y e d u n i f o r m l y and a l l o w e d t o d r y f o r 10 min i n an oven ( s e t a t 80-100 d e g r e e s C ) . T h i s t e c h n i q u e a l l o w e d v i s u a l i z a t i o n o f o x o - s t e r o i d s ( b l u e on w h i t e b a c k g r o u n d ) as w e l l as h y d r o x y - s t e r o i d s (brown on w h i t e b a c k g r o u n d ) , a t a s e n s i t i v i t y o f 1-2 m i c r o g r a m s . F o l l o w i n g t h e s e t e c h n i q u e s , e a c h s p o t was marked i n p e n c i l , and t h e Rf v a l u e s were c a l c u l a t e d . (d) I d e n t i f i c a t i o n of t e s t o s t e r o n e and 5 b e t a - a n d r o s t a n -3 a l p h a , 17 b e t a - d i o l u s i n g Gas Chromatography-Mass s p e c t r o m e t r y (GCMS) C o n f i r m a t i o n of t e s t o s t e r o n e and 5 b e t a - A - 3 a l p h a , 17 b e t a - d i o l i n t h e c y t o s o l was done u s i n g GCMS. A H e w l e t t - P a c k a r d HP 5987A GCMS s y s t e m f i t t e d w i t h a f u s e d s i l i c a c r o s s l i n k e d d i m e t h y l s i l i c o n e c a p i l l a r y column (12.5 m x 0.2 mm i d ; HP p a r t number 19091-60312) was u s e d . The e l e c t r o n i m p a c t s p e c t r a were r e c o r d e d a t 80 eV, i o n s o u r c e p r e s s u r e 8.0 m i c r o T o r r . TIC p l o t s were b a s e d on m/z 40 t o 500 amu. The i n j e c t i o n volume was 1 m i c r o l i t r e , u s i n g p y r i d i n e as v e h i c l e . I n j e c t i o n s were done u s i n g a n o n - s p l i t mode. The r u n n i n g c o n d i t i o n s were as f o l l o w s : C h r o m a t o g r a p h i c t e m p e r a t u r e s -50 d e g r e e s f o r 1 min, t h e n i n c r e a s e d a t 20 d e g r e e s / m i n t o 280 d e g r e e s and h e l d f o r 10 mins E m i s s i o n - 300 m i c r o a m p e r e s Scan c y c l e - 3.9 s e c o n d s E t h e r e a l e x t r a c t s were c h r o m a t o g r a p h e d on s e p a r a t e p r e p a r a t i v e TLC s h e e t s u s i n g s o l v e n t s y s t e m I I . Zones w i t h Rf v a l u e s c o r r e s p o n d i n g t o t e s t o s t e r o n e o r 5 b e t a - A - 3 a l p h a , 17 b e t a - d i o l were c u t - o u t f r o m TLC s h e e t s and were e x t r a c t e d u s i n g d i e t h y l e t h e r . F o r c y t o s o l w h i c h was i n c u b a t e d f o r 0 h o u r s a t 4 d e g r e e s C, zone Rf=0.43 was u s e d ; f o r c y t o s o l i n c u b a t e d f o r 3 h o u r s a t 4 o r 37 d e g r e e s C, zone Rf=0.26 was u s e d . The e t h e r e a l e x t r a c t s were t h e n e v a p o r a t e d t o d r y n e s s f o r GCMS a n a l y s i s . F u l l y s i l y l a t e d t r i m e t h y l e t h e r s and O-methyloxime d e r i v a t i v e s were p r e p a r e d u s i n g MSTFA and MOX r e a g e n t s r e s p e c t i v e l y , u s i n g t h e method o f G a r d i n e r and H o r n i n g ( 1 9 6 6 ) . In b r i e f , t h e f o r m e r r e a g e n t r e a c t s w i t h h y d r o x y - s t e r o i d s , w h i l e t h e l a t t e r r e a c t s w i t h k e t o - s t e r o i d s . T h i s t e c h n i q u e y i e l d e d s h a r p and s e p a r a t e p e a k s f o r 5 b e t a -A- 3 a l p h a , 17 b e t a - d i o l and t e s t o s t e r o n e . The r e t e n t i o n t i m e s were were 12 and 13 mins r e s p e c t i v e l y , d e p e n d i n g on d a i l y c h r o m a t o g r a p h y c o n d i t i o n s . E x t r a c t e d and n o n - e x t r a c t e d s t a n d a r d s gave t h e same r e t e n t i o n t i m e s . The m/z v a l u e s f o r t h e M+ i o n s f o r 5 b e t a - A - 3 a l p h a , 17 b e t a - d i o l and t e s t o s t e r o n e d e r i v a t i v e s were 436 and 389 r e s p e c t i v e l y . (e) A n a l y s i s o f bound t r i t i a t e d - t e s t o s t e r o n e and m e t a b o l i t e s u s i n g TLC Each TLC s h e e t was c u t i n t o l o n g i t u d i n a l t r a c k s c o n t a i n i n g t h e e l u e n t s o f e a c h c y t o s o l i c e x t r a c t . E a c h t r a c k was f u r t h e r c u t i n t o 1-2 cm s t r i p s w h i c h were t h e n p l a c e d i n t o s e p a r a t e s c i n t i l l a t i o n v i a l s f o r r a d i o a c t i v i t y a n a l y s i s . The r a d i o a c t i v i t y o f e a c h s t r i p was p l o t t e d as a f u n c t i o n o f i t s d i s t a n c e f r o m t h e o r i g i n ; t h i s d i s t a n c e was e x p r e s s e d as a f r a c t i o n o f t h e t o t a l d i s t a n c e e l u t e d by t h e s o l v e n t f r o n t . T h i s p l o t gave a chromatogram o f t h e e l u t i o n o f r a d i o l a b e l l e d t e s t o s t e r o n e and i t s m e t a b o l i t e s . 8. R a d i o - l i g a n d e xchange b i n d i n g s t u d i e s (a) I n c u b a t i o n o f c y t o s o l and m i c r o s o m e s C y t o s o l and m i c r o s o m e s were i n c u b a t e d f o l l o w i n g c o n d i t i o n s o u t l i n e d by t h e f o l l o w i n g w o r k e r s : K i r c h o f f e t a l . (1979) and B o e s e l and S h a i n (1974) f o r p r o s t a t i c c y t o s o l , S a t o e t a l . (1980) f o r h e p a t i c c y t o s o l , and Yamada and M i y a g i (1982) f o r h e p a t i c m i c r o s o m e s . An a l i q u o t (0.5 ml) o f c y t o s o l o r m i c r o s o m e s was i n c u b a t e d i n TED b u f f e r o r T E D - s u p p l e m e n t e d w i t h 10 mM KCI b u f f e r c o n t a i n i n g v a r y i n g c o n c e n t r a t i o n s o f t r i t i a t e d - t e s t o s t e r o n e , t r i t i a t e d - 5 alpha-DHT o r t r i t i a t e d - R l 8 8 1 , i n t h e p r e s e n c e o r a b s e n c e o f 1 1 0 0 - f o l d ( u n l e s s o t h e r w i s e s t a t e d ) e x c e s s m o l a r r a t i o c o m p e t i t o r . F o r some a s s a y s , r a d i o i n e r t t r i a m c i n o l o n e a c e t o n i d e ( 1 0 0 - f o l d e x c e s s m o l a r r a t i o ; u n l e s s o t h e r w i s e s t a t e d ) was added t o t h e i n c u b a t i o n m i x t u r e . T h i s m i x t u r e was i n c u b a t e d f o r 3 h o u r s a t 4 d e g r e e s C ( u n l e s s o t h e r w i s e s t a t e d ) . P r o t e i n c o n c e n t r a t i o n s were d e t e r m i n e d u s i n g the p r o t e i n - d y e method d e s c r i b e d by B r a d f o r d ( 1 9 7 6 ) . (b) S e p a r a t i o n o f bound and f r e e s t e r o i d f r a c t i o n s F o l l o w i n g i n c u b a t i o n , f r e e s t e r o i d was removed u s i n g d e x t r a n - c o a t e d c h a r c o a l i n TED b u f f e r (DCC) w h i c h was p r e p a r e d as a 2.5% c h a r c o a l s u s p e n s i o n i n TED b u f f e r (pH 7.4) c o n t a i n i n g 0.25% d e x t r a n T70 ( u n l e s s o t h e r w i s e s t a t e d ) . An a l i q u o t (0.5 ml) o f 2.5% DCC s l u r r y was added t o a l l i n c u b a t i o n t u b e s . T h i s s u s p e n s i o n was mixed t h o r o u g h l y and c e n t r i f u g e d a t 1500 x g f o r 10 m i n u t e s a t 4 d e g r e e s C b e f o r e s a m p l i n g f o r r a d i o a c t i v i t y a n a l y s i s . T h i s t e c h n i q u e i n v o l v e s t h e r a p i d a d s o r p t i o n and p r e c i p i t a t i o n o f f r e e o r unbound s t e r o i d f r o m t h e c y t o s o l ( B e a t o and F e i g e l s o n 1972; C l a r k and Peck 1980) . In o t h e r e x p e r i m e n t s , 0.5 ml p r o t a m i n e s u l p h a t e s o l u t i o n (2 mg/ml i n TED b u f f e r w i t h o u t m o l y b d a t e ) was added t o t h e i n c u b a t i o n m i x t u r e , f o l l o w i n g t h e p r o t o c o l o f Menon e t a l . ( 1 9 7 8 ) . T h i s m i x t u r e was a l l o w e d t o s t a n d f o r 5 min b e f o r e c e n t r i f u g a t i o n a t 1500 x g f o r 10 m i n . The s u p e r n a t a n t f r a c t i o n was p o u r e d o n t o a pre-made 2.5% d e x t r a n c o a t e d c h a r c o a l p e l l e t , and was c e n t r i f u g e d and sampled (as d e s c r i b e d a b o v e ) , i n o r d e r t o d e t e r m i n e " p r o t a m i n e s u l p h a t e n o n - p r e c i p i t a b l e b i n d i n g " . The p r o t a m i n e s u l p h a t e p r e c i p i t a t e was washed and a s p i r a t e d t w i c e u s i n g TED b u f f e r . F o l l o w i n g t h e f i n a l a s p i r a t i o n , 0.5 ml e t h a n o l was added t o th e p r e c i p i t a t e and t h e t u b e s were s e t a t room t e m p e r a t u r e f o r 1 h o u r , b e f o r e s a m p l i n g f o r r a d i o a c t i v i t y a n a l y s i s . F o l l o w i n g c e n t r i f u g a t i o n , an a l i q u o t o f t h e DCC o r p r o t a m i n e s u l p h a t e s u p e r n a t a n t (0.5 ml) was p l a c e d i n t o s c i n t i l l a t i o n v i a l s c o n t a i n i n g B i o f l u o r (NEN-Canada). R a d i o a c t i v i t y was a n a l y s e d u s i n g t h e Mark I I I l i q u i d s c i n t i l l a t i o n s y s t e m Model 6880 ( S e a r l e A n a l y t i c I n c . , Des P l a i n e s , I L 60 0 1 8 ) . Q u e n c h i n g o f t r i t i u m a v e r a g e d 35-47%. R a d i o a c t i v i t y d a t a was r e c o r d e d o n t o a m i c r o c o m p u t e r d i s k e t t e u s i n g an A p p l e I I - p l u s d i s c - o p e r a t i n g s y s t e m (DOS; A p p l e Computer I n c . , C u p e r t i n o , CA 95014), w h i c h was c o n n e c t e d t o t h e s c i n t i l l a t i o n c o u n t e r u s i n g a s e r i a l c a r d i n t e r f a c e . (c) Sephadex G-25M m i n i - c o l u m n c h r o m a t o g r a p h y An a l i q u o t (0.5 ml) o f l i v e r whole c y t o s o l (40 mg/ml) i n c u b a t i o n m i x t u r e was c a r e f u l l y added t o p r e - e q u i l i b r a t e d p r e p a c k e d d i s p o s a b l e Sephadex G-25M m i n i - c o l u m n s (PD-10; P h a r m a c i a F i n e C h e m i c a l s , U p p s a l a , Sweden). The columns were t h e n e l u t e d a t 4 d e g r e e s C w i t h TED b u f f e r , and t h e f r a c t i o n s were c o l l e c t e d f o r r a d i o a c t i v i t y a n a l y s i s . T h i s t e c h n i q u e p e r m i t s p r o t e i n bound f r a c t i o n s , t o be s e p a r a t e d f r o m n o n - p r o t e i n bound o r f r e e f r a c t i o n s a c c o r d i n g t o m o l e c u l a r s i z e . 9. A n a l y s i s of r a d i o a c t i v i t y N o n - p r o t e i n bound ( f r e e ) s t e r o i d c o n c e n t r a t i o n ( F ; nM) was c a l c u l a t e d f r o m t h e amount o f r a d i o a c t i v i t y added t o t h e i n c u b a t i o n t u b e i n t h e a b s e n c e o f c y t o s o l . The d e g r e e o f c y t o s o l i c o r m i c r o s o m a l s t e r o i d t o t a l b i n d i n g ( i n c u b a t i o n i n th e a b s e n c e o f c o m p e t i t o r ) was d e t e r m i n e d f r o m t h e r a d i o a c t i v i t y measured i n t h e DCC o r p r o t a m i n e s u l p h a t e s u p e r n a t a n t o r t h e PD-10 e f f l u e n t . N o n - s p e c i f i c s t e r o i d b i n d i n g was d e t e r m i n e d i n a s i m i l a r f a s h i o n u s i n g c y t o s o l o r m i c r o s o m e s w h i c h were i n c u b a t e d i n t h e p r e s e n c e o f c o m p e t i t o r . S p e c i f i c bound (SB) s t e r o i d was c a l c u l a t e d a s th e a r i t h e m a t i c d i f f e r e n c e between t o t a l and n o n - s p e c i f i c bound f r a c t i o n . B i n d i n g was e x p r e s s e d as fmol/mg p r o t e i n ( u n l e s s o t h e r w i s e s t a t e d ) , and was a n a l y s e d u s i n g n o n - l i n e a r s a t u r a t i o n (SB x F) and l i n e a r S c a t c h a r d - t y p e (SB/F x SB; S c a t c h a r d 1949) p l o t s . The r a t i o o f SB/F was e x p r e s s e d as l i t r e s p e r mg p r o t e i n . The b i n d i n g k i n e t i c s were e x p r e s s e d as t h e a p p a r e n t d i s s o c i a t i o n c o n s t a n t o f e q u i l i b r i u m (Kd; nM) and t h e a p p a r e n t maximum number o f b i n d i n g s i t e s o r b i n d i n g c a p a c i t y (Bmax; f m o l / m g ) . E x p e r i m e n t a l d a t a were a n a l y s e d u s i n g a n o n - l i n e a r r e g r e s s i o n c o mputer p r o g r a m ( p r o v i d e d by Dr. B.D. R o u f o g a l i s , UBC), w h i c h was r u n on an AMDAHL-470 computer (UBC Computing C e n t r e ) . T h i s i t e r a t i v e p r o g r a m i n v o l v e s t h e i n p u t o f t h e f o l l o w i n g d a t a : t h e s p e c i f i c bound (SB) v a l u e a t e a c h f r e e l i g a n d c o n c e n t r a t i o n (F) u s e d , f o r t h e c a l c u l a t i o n o f b e s t f i t . The a v e r a g e r u n n i n g t i m e f o r c a l c u l a t i o n was 0.8 s e c , d e p e n d i n g upon t h e a c c u r a c y o f t h e i n i t i a l e s t i m a t e s . A n o t h e r computer program w h i c h c a n a n a l y s e SB/F v s . SB d a t a , and c a n be r u n on an A p p l e I I - p l u s m i c r o c o m p u t e r was d e v e l o p e d by Mr. G l e n C o l l i n s ( D e p t . o f P h a r m a c o l o g y , UBC). In b r i e f , t h i s l i n e a r r e g r e s s i o n p r o g r a m d e t e r m i n e s e a c h l i n e a r p hase o f t h e t w o - p a r t i s o t h e r m by r e p e t i t i v e l y e s t i m a t i n g t h e two Kd and t h e two Bmax v a l u e s , s u c h t h a t t h e sum o f s q u a r e s ( t h e o r e t i c a l v s . o b s e r v e d ) i s m i n i m i z e d . The a v e r a g e r u n n i n g t i m e of c a l c u l a t i o n r a n g e d f r o m 4 t o 8 min, d e p e n d i n g upon t h e a c c u r a c y o f t h e i n i t i a l e s t i m a t e s . 10. S t a t i s t i c a l A n a l y s i s In v i v o and in v i t r o e x p e r i m e n t a l d a t a were a n a l y s e d u s i n g t h e t w o - t a i l e d S t u d e n t ' s t - t e s t f o r u n p a i r e d d a t a ; d i f f e r e n c e s were s t a t i s t i c a l l y s i g n i f i c a n t a t t h e p< 0.05 l e v e l . The Duncan m u l t i p l e r a n g e t e s t (p< 0.05) was used f o r s t a t i s t i c a l a n a l y s i s i f i n t e r a c t i o n between t r e a t m e n t g r o u p s was s u s p e c t e d . RESULTS 1. E f f e c t o f t e s t o s t e r o n e e n a n t h a t e and f l u t a m i d e a d m i n i s t r a t i o n on serum t e s t o s t e r o n e l e v e l s , t i s s u e i b o d y w e i g h t s r a t i o s and h e p a t i c m i c r o s o m a l enzyme a c t i v i t i e s i n t h e W i s t a r r a t (a) Serum t e s t o s t e r o n e l e v e l s In o r d e r t o d e t e r m i n e what dose o f t e s t o s t e r o n e e n a n t h a t e would m a i n t a i n n o r m a l p h y s i o l o g i c a l l e v e l s of p r o s t a t e / b o d y w e i g h t r a t i o s and h e p a t i c m i c r o s o m a l BPH and TR a c t i v i t i e s i n t h e c a s t r a t e d a d u l t male r a t , t h e f o l l o w i n g t e s t o s t e r o n e d o s e - r e s p o n s e e x p e r i m e n t s were c a r r i e d o u t . W i t h t h i s i n f o r m a t i o n , we would have more p r e c i s e c o n t r o l o v e r a n d r o g e n l e v e l s d e s p i t e t h e i n d i v i d u a l b i o l o g i c a l v a r i a t i o n . Serum t e s t o s t e r o n e c o n c e n t r a t i o n s i n t h e i n t a c t a d u l t male r a t v a r i e d f r o m 2.4 t o 7.6 ng/ml ( f i g u r e 1 ) , b u t t h e y were w i t h i n t h e p h y s i o l o g i c a l r a n g e ( F o r e s t 1979). D a i l y a d m i n i s t r a t i o n of 5 m i c r o m o l e s / k g s c . t e s t o s t e r o n e e n a n t h a t e f o r 10 days d i d n o t s i g n i f i c a n t l y a l t e r i n t a c t a d u l t male serum t e s t o s t e r o n e l e v e l s compared t o c o r n o i l i n j e c t e d c o n t r o l a n i m a l s . C a s t r a t i o n f o r 10 days s i g n i f i c a n t l y l o w e r e d t h e s e l e v e l s compared t o i n t a c t a d u l t c o n t r o l m a l e s . Serum t e s t o s t e r o n e c o n c e n t r a t i o n s were s i g n i f i c a n t l y i n c r e a s e d i n the 10 days p o s t - c a s t r a t e d a d u l t male r a t s t r e a t e d w i t h v a r y i n g d o s e s (1.3 - 5 m i c r o m o l e s / k g s c . , 10 d a y s ) o f t e s t o s t e r o n e e n a n t h a t e ( l a s t i n j e c t i o n was 24 h o u r s p r i o r t o e x p e r i m e n t ) compared t o t h e c o r n o i l 4 Figure 1. EFFECT OF TESTOSTERONE ENANTHATE ON SERUM TESTOSTERONE LEVELS IN SHAM-OPERATED AND CASTRATED ADULT MALE RATS Serum testosterone levels (expressed i n ng/ml) were determined i n sham-operated (Sh) or 10 day post-castrated (Gx) adult male rats (280-300 g) treated with corn o i l vehicle (C) or varying doses of testosterone enanthate (TE; 1.25-5.0 micromoles/kg sc., 10 days). Experiments were done 24 hours following l a s t i n j e c t i o n . Data were expressed as average +/- S.E.M.; numbers in brackets denote the numbers of animals used. * : s i g n i f i c a n t difference (p< 0.05) compared to sham-operated adult male of the same treatment group. **: s i g n i f i c a n t difference (p< 0.05) compared to corn o i l treated castrated control group. F i g u r e 1. 55 T a b l e 1 EFFECT OF FLUTAMIDE ON SERUM TESTOSTERONE LEVELS The e f f e c t o f f l u t a m i d e on serum t e s t o s t e r o n e l e v e l s was d e t e r m i n e d i n c a s t r a t e d a d u l t male r a t s s u p p l e m e n t e d w i t h t e s t o s t e r o n e e n a n t h a t e ( T E ) . C o n t r o l s h a m - o p e r a t e d a d u l t male r a t s (280-300 g) were i n j e c t e d w i t h c o r n o i l f o r 10 d a y s . Ten day c a s t r a t e d a d u l t males were i n j e c t e d w i t h c o r n o i l o r TE (5 m i c r o m o l e s / k g , 10 d a y s ) . Some o f t h e l a t t e r a n i m a l s were a l s o i n j e c t e d w i t h v a r y i n g d o s e s o f f l u t a m i d e (5-50 m i c r o m o l e s / k g s c . , 10 d a y s ) . E x p e r i m e n t s were done 24 h o u r s a f t e r l a s t i n j e c t i o n . Serum t e s t o s t e r o n e l e v e l s were e x p r e s s e d i n ng t e s t o s t e r o n e / m l serum. Data were e x p r e s s e d as a v e r a g e +/- S.E.M. Numbers i n b r a c k e t s d e n o t e numbers o f a n i m a l s t e s t e d . p.moles= m i c r o m o l e s A n i m a l models u s e d : Serum T e s t o s t e r o n e L e v e l (a) Sham-operated a d u l t male 4.6 +/- 1-7 (6) (b) C a s t r a t e d a d u l t male i n j e c t e d w i t h : ( i ) C o r n o i l ( i i ) TE (5 p m o l e s / k g ) o n l y ( i i i ) TE (5 /jmoles/kg) p l u s f l u t a m i d e : 5 j m o l e s / k g 25 p m o l e s / k g 50 pjmoles/kg 0.3 +/- 0.2 (8) * 1.2 +/- 0.2 (4) ** 1.4 +/" 0.2 (4) ** 1.0 +/- 0.1 (3) ** 0.9 +/- 0.1 (4) ** * d e n o t e s s i g n i f i c a n t d i f f e r e n c e compared t o s h a m - o p e r a t e d c o n t r o l (p< 0. 0 5 ) . ** d e n o t e s s i g n i f i c a n t d i f f e r e n c e compared t o c a s t r a t e d c o n t r o l (p< 0. 0 5 ) . 57 i n j e c t e d c a s t r a t e d c o n t r o l m a l e s . Normal p h y s i o l o g i c a l l e v e l s were a c h i e v e d a t 2.5 and 5 m i c r o m o l e s / k g d o s e s . The e f f e c t o f f l u t a m i d e on serum t e s t o s t e r o n e l e v e l s was d e t e r m i n e d t o e l i m i n a t e t h e p o s s i b l i t y t h a t f l u t a m i d e may be h a v i n g a d i r e c t e f f e c t on serum t e s t o s t e r o n e c o n c e n t r a t i o n s ( t a b l e 1 ) . C o n s i s t e n t w i t h t h e r e s u l t s m e n t i o n e d above, c a s t r a t i o n f o r 10 d a y s s i g n i f i c a n t l y l o w e r e d and t e s t o s t e r o n e e n a n t h a t e r e p l a c e m e n t s i g n i f i c a n t l y i n c r e a s e d , t e s t o s t e r o n e l e v e l s i n t h e i n t a c t and c a s t r a t e d male r e s p e c t i v e l y , compared t o c o n t r o l a n i m a l s . In t h e t e s t o s t e r o n e - t r e a t e d c a s t r a t e d m a l e s , f l u t a m i d e , a t v a r i o u s d a i l y d o s e s (5, 25 and 50 m i c r o m o l e s / k g s c . , 10 d ays) had no s i g n i f i c a n t e f f e c t on serum t e s t o s t e r o n e l e v e l s compared t o t h e f l u t a m i d e u n t r e a t e d c o n t r o l a n i m a l s . (b) T i s s u e : b o d y w e i g h t (BW) r a t i o s I n o r d e r t o e l i m i n a t e t h e p o s s i b i l i t y o f n o n - s p e c i f i c e f f e c t s due t o a n i m a l s u r g e r y o r d r u g t r e a t m e n t , t i s s u e and body w e i g h t s were a l s o m e a s u r e d . T h e r e were no s i g n i f i c a n t e f f e c t s on l i v e r : B W r a t i o s between a n i m a l s due t o d i f f e r e n c e s i n s u r g i c a l p r o c e d u r e , o r d r u g ( t e s t o s t e r o n e e n a n t h a t e o r f l u t a m i d e ) t r e a t m e n t ( t a b l e 2 ) . P r o s t a t e : B W r a t i o s were n o t s i g n i f i c a n t l y a l t e r e d i n s h a m - o p e r a t e d a d u l t male r a t s t r e a t e d w i t h 25 m i c r o m o l e s / k g f l u t a m i d e compared t o s h a m - o p e r a t e d c o r n o i l t r e a t e d c o n t r o l m a l e s ( f i g u r e 2 ) . The 10 day p o s t - c a s t r a t e d a d u l t male r a t p r o s t a t e : B W r a t i o s were r e d u c e d s i g n i f i c a n t l y , an e f f e c t w h i c h was f u l l y F i g u r e 2. EFFECT OF FLUTAMIDE ON PROSTATE TO BODY WEIGHT RATIOS IN ADULT MALE RAT P r o s t a t e wet weight to body weight r a t i o s (expressed as mg/100 g) were determined i n sham-operated (Sh) and 10 day p o s t - c a s t r a t e d (Gx) a d u l t male r a t s (280-300 g) which were t r e a t e d w i t h c o r n o i l (C) or t e s t o s t e r o n e enanthate (TE; 5 m i c r o m o l e s / k g s c . , 10 days) a lone or i n combina t ion w i t h v a r y i n g doses of l i u t a m i d e (F; 5-50 mic romole s /kg s c . , 10 d a y s ) . Exper iments were done 24 hours a f t e r l a s t i n j e c t i o n . R a t i o s were expres sed as average +/- S . E . M . Numbers i n b r a c k e t s denote the numbers o f an imals t e s t e d . * : s i g n i f i c a n t d i f f e r e n c e (p< 0.05) compared t o sham-operated a d u l t male o f the same t reatment g roup . * * : s i g n i f i c a n t d i f f e r e n c e (p< 0.05) compared to c o r n o i l - t r e a t e d c a s t r a t e d a d u l t male . Figure 2. 59 lflfln LD O 5 80-g 68-2) ec m 43-20-(4) (4) (4) (4) (4) ( 8 ) ( 8 ) i ( 8 ) ( 8 ) F25 C F25 T E 5 TE+FgTE+F^TE+F^TE+F, ShcT GxcT 5° T a b l e 2 EFFECT OF FLUTAMIDE AND TESTOSTERONE TREATMENT ON LIVER:BODY WEIGHT RATIOS The e f f e c t o f t l u t a m i d e and t e s t o s t e r o n e e n a n t h a t e (TE) t r e a t m e n t was d e t e r m i n e d on l i v e r : b o d y w e i g h t r a t i o s i n s h a m - o p e r a t e d and 10 day c a s t r a t e d a d u l t male r a t s (280-300 g ) . A n i m a l s were i n j e c t e d w i t h c o r n o i l , o r v a r i o u s d o s e s o f TE (1.3 t o 5 m i c r o m o l e s / k g s c . , 10 days) a l o n e , o r i n c o m b i n a t i o n w i t h v a r i o u s d o s e s o f f l u t a m i d e (5 t o 50 m i c r o m o l e s / k g s c . , 10 d a y s ) . The e x p e r i m e n t was done 24 h o u r s a f t e r l a s t i n j e c t i o n . R a t i o s were e x p r e s s e d as gram wet t i s s u e p e r 100 g body w e i g h t . D a t a were e x p r e s s e d as a v e r a g e +/- S.E.M. Numbers i n b r a c k e t s d e n o t e numbers o f a n i m a l s t e s t e d . umoles= m i c r o m o l e s A n i m a l models t e s t e d L i v e r : B o d y w e i g h t r a t i o s (a) S h a m - o p e r a t e d a d u l t male t r e a t e d w i t h : C o r n o i l 4. .16 + /- 0. ,14 (4) TE (5 umoles/kg) o n l y 4. .17 + /- 0. .13 (4) F l u t a m i d e (25 pmoles/kg) o n l y 4. .20 + /- 0, .03 (4) (b) C a s t r a t e d a d u l t males t r e a t e d w i t h : C o r n o i l TE o n l y : 1.3 p m o l e s / k g 2.5 p m o l e s / k g 5.0 p m o l e s / k g F l u t a m i d e (25 umoles/kg) o n l y 4. 40 + /- 0. 30 (4) 4. 50 + /- 0. 12 (4) 4. 72 + /- 0. 07 (4) 4. 42 + /- 0. 09 (4) 3. 67 + /- 0. 23 (3) TE (5 umoles/kg) 5 jumoles/kg 15 p m o l e s / k g 25 p m o l e s / k g bO p m o l e s / k g p l u s F l u t a m i d e 3 .91 + /- 0. 15 (8) 4 .02 + /- 0. 05 (8) 4 .12 + /- 0. 13 (8) 4 .37 + /- 0. 09 (4) F i g u r e 3 . EFFECT OF FLUTAMIDE ON HEPATIC BENZO(A)PYRENE HYDROXYLASE IN TESTOSTERONE-TREATED CASTRATED ADULT MALE AND INTACT ADULT FEMALE RATS H e p a t i c m i c r o s o m a l b e n z o ( a ) p y r e n e (BP) h y d r o x y l a s e a c t i v i t i e s were measured i n t h e s h a m - o p e r a t e d (Sh) a d u l t male (280-300 g) o r f ema le (180-200 g) and t h e 10 day c a s t r a t e d (Gx) a d u l t male r a t . A n i m a l s were t r e a t e d w i t h c o r n o i l v e h i c l e ( C ) , o r t e s t o s t e r o n e e n a n t h a t e (T; 5 r n i c r o m o l e s / k g s c . , 10 days) a l o n e , o r i n c o m b i n a t i o n w i t h v a r y i n g doses o f f l u t a m i d e (F ; 5-50 m i c r o m o l e s / k g s c . , 10 d a y s ) . E x p e r i m e n t s were done 24 h o u r s f o l l o w i n g l a s t i n j e c t i o n . Enzyme a c t i v i t i e s were e x p r e s s e d as pmoles 3-OH BP t o r m e d / m i n / m g p r o t e i n +/- S . E . M . Numbers i n b a r s denote t h e numbers o f a n i m a l s u s e d . * : s i g n i f i c a n t d i f f e r e n c e (p< 0 .05) compared t o s h a m - o p e r a t e d a d u l t male o f t h e same t r e a t m e n t g r o u p . * * : s i g n i f i c a n t d i f f e r e n c e (p< 0 .05) compared t o a p p r o p r i a t e c a s t r a t e d a d u l t male c o n t r o l g r o u p . For c o m p a r i s o n s between t h e t e s t o s t e r o n e p l u s f l u t a m i d e - t r e a t e d g roups and t h e f l u t a m i d e u n t r e a t e d c o n t r o l g r o u p , t h e Duncan m u l t i p l e r ange t e s t was u s e d . 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 . 0 5 ; s i g n i f i c a n t d i f f e r e n c e s between g roups were i n d i c a t e d by common l e t t e r s . F i g u r e 3. 62 C C F25 9 <* Sh F25 T5 T + F 5 T + % T + F 2 5 ' T + F 5 0 GxCC T a b l e 3 EFFECT OF TESTOSTERONE TREATMENT ON HEPATIC MICROSOMAL BENZO(A)PYRENE HYDROXYLASE AND TESTOSTERONE REDUCTASE A C T I V I T I E S E f f e c t o f t e s t o s t e r o n e e n a n t h a t e (TE) a d m i n i s t r a t i o n (1 .3 t o 5 m i c r o m o l e s / k g s c . , 10 days) on h e p a t i c m i c r o s o m a l b e n z o ( a ) p y r e n e h y d r o x y l a s e (BPH) and t e s t o s t e r o n e r e d u c t a s e (TR) a c t i v i t i e s was measured i n sham-opera ted and 10 d a y - c a s t r a t e d a d u l t male r a t s (280-300 g ) . C o n t r o l a n i m a l s were i n j e c t e d w i t h t h e c o r n o i l v e h i c l e . BPH a c t i v i t i e s were e x p r e s s e d as nmoles 3-OH b e n z o ( a ) p y r e n e fo rmed/min /mg p r o t e i n +/- S . E . M . TR a c t i v i t i e s were e x p r e s s e d as nmoles t e s t o s t e r o n e r e d u c e d / m i n / m g p r o t e i n +/- S . E . M . The numbers i n b r a c k e t s denote t h e numbers o f a n i m a l s t e s t e d . umoles= m i c r o m o l e s . A n i m a l model u s e d : M i c r o s o m a l Enzyme A c t i v i t i e s BPH TR Sham-opera ted a d u l t m a l e : C o n t r o l 3 .2 +/- 0 .1 3 .6 +/- 1.2 (7) TE (5 u m o l e s / k g ) 3 .1 +/- 0 . 3 2 .9 +/- 0 .8 (3) C a s t r a t e d a d u l t m a l e : C o n t r o l 2 .2 +/- 0 . 1 * 8 .3 +/- 0 . 8 * (4) TE t r e a t e d w i t h : 1.3 u m o l e s / k g 2 .7 +/- 0 . 2 * * 8 .0 +/- 0 .5 (4) 2 .5 u m o l e s / k g 3 .0 +/- 0 . 1 * * 8 .3 +/- 0 .6 (4) 5 . 0 u m o l e s / k g 3 .8 +/- 0 . 4 * * 6 .2 +/- 0 . 9 * * (4) * d e n o t e s s i g n i f i c a n t d i f f e r e n c e compared t o s h a m - o p e r a t e d male o f t h e same t r e a t m e n t g r o u p (p< 0 . 0 5 ) . * * d e n o t e s s i g n i f i c a n t d i f f e r e n c e compared t o c o n t r o l c o r n o i l t r e a t e d c a s t r a t e d a n i m a l s (p< 0 . 0 5 ) . r e v e r s e d by t e s t o s t e r o n e e n a n t h a t e r e p l a c e m e n t (5 m i c r o m o l e s / k g s c . , 10 d a y s ) . W h i l e f l u t a m i d e (25 m i c r o m o l e s / k g ) had no s i g n i f i c a n t e f f e c t on p r o s t a t e r B W r a t i o s i n t h e c o r n o i l t r e a t e d c a s t r a t e d a d u l t male, i t s i g n i f i c a n t l y r e d u c e d t h e s e r a t i o s , i n a d o s e - d e p e n d e n t manner ( f r o m 5 t o 50 m i c r o m o l e s / k g ) , i n t h e t e s t o s t e r o n e e n a n t h a t e t r e a t e d (5 m i c r o m o l e s / k g s c . , 10 d a y s ) c a s t r a t e d m a l e s compared t o t h e f l u t a m i d e u n t r e a t e d c o n t r o l a n i m a l s . ( c ) H e p a t i c m i c r o s o m a l d r u g and s t e r o i d m e t a b o l i z i n g enzyme a c t i v i t i e s In o r d e r t o d e t e r m i n e t h e a p p r o p r i a t e dose o f t e s t o s t e r o n e e n a n t h a t e w h i c h would r e v e r s e t h e e f f e c t s o f c a s t r a t i o n on a d u l t male h e p a t i c m i c r o s o m a l BPH and TR a c t i v i t i e s b u t m a i n t a i n p h y s i o l o g i c a l l e v e l s o f serum t e s t o s t e r o n e and p r o s t a t e : b o d y w e i g h t r a t i o s , dose r e s p o n s e s t u d i e s were done. T h e s e two enzyme s y s t e m s were c h o s e n s i n c e b o t h a r e known t o be s e x - d e p e n d e n t and s e n s i t i v e t o serum t e s t o s t e r o n e l e v e l s i n t h e a d u l t m a l e . In a d d i t i o n , BPH i s h i g h e r i n t h e m a l e , w h i l e TR i s h i g h e r i n t h e f e m a l e . C o n t r o l s h a m - o p e r a t e d a d u l t male r a t l i v e r m i c r o s o m a l BPH a c t i v i t i e s were c o n s i s t e n t w i t h G o n t o v n i c k and B e l l w a r d (1980; f i g u r e 3 and t a b l e 3 ) . C a s t r a t i o n o f t h e a d u l t male f o r 10 days s i g n i f i c a n t l y r e d u c e d BPH v a l u e s by 31% compared t o t h e s h a m - o p e r a t e d c o n t r o l ( t a b l e 3 ) . I n c r e a s i n g d a i l y s c . d o s e s o f t e s t o s t e r o n e e n a n t h a t e (1.3 t o 5 m i c r o m o l e s / k g , 10 d a y s ) s i g n i f i c a n t l y r e v e r s e d t h e c a s t r a t i o n e f f e c t t o c o n t r o l l e v e l s . T e s t o s t e r o n e e n a n t h a t e (5 m i c r o m o l e s / k g ) a d m i n i s t r a t i o n t o t h e c o r n - o i l t r e a t e d s h a m - o p e r a t e d c o n t r o l m a l e s had no s i g n i f i c a n t e f f e c t on BPH a c t i v i t i e s . In t h e same a n i m a l s as above, h e p a t i c m i c r o s o m a l TR a c t i v i t e s were measured, i n o r d e r t o e l i m i n a t e t h e p o s s i b i l i t y o f a n o n - s p e c i f i c a c t i o n o f c a s t r a t i o n o r a n d r o g e n r e p l a c e m e n t on h e p a t i c m i c r o s o m a l enzyme a c t i v i t y . C a s t r a t i o n l e d t o a s i g n i f i c a n t s t i m u l a t i o n o f TR a c t i v i t i e s (by 131%; t a b l e 3 ) , compared t o t h e s h a m - o p e r a t e d c o n t r o l , an e f f e c t w h i c h was s i g n i f i c a n t l y , b u t p a r t i a l l y r e v e r s e d by t e s t o s t e r o n e e n a n t h a t e t r e a t m e n t (1.3 t o 5 m i c r o m o l e s / k g ) . S i m i l a r t o the BPH a c t i v i t i e s , TR a c t i v i t i e s were n o t s i g n i f i c a n t l y i n f l u e n c e d by t e s t o s t e r o n e e n a n t h a t e t r e a t m e n t t o t h e s h a m - o p e r a t e d a d u l t m a l e . T h i s s t u d y s u g g e s t s t h a t a s c . d o s e o f 5 m i c r o m o l e s / k g t e s t o s t e r o n e e n a n t h a t e f o r 10 d a y s , s i g n i f i c a n t l y r e v e r s e d t h e e f f e c t o f c a s t r a t i o n i n t h e a d u l t male h e p a t i c m i c r o s o m a l BPH and TR a c t i v i t i e s , as w e l l as m a i n t a i n i n g n o r m a l p h y s i o l o g i c a l serum t e s t o s t e r o n e l e v e l s and p r o s t a t e : b o d y w e i g h t r a t i o s . H e p a t i c m i c r o s o m a l BPH i s a s e x - d e p e n d e n t enzyme i n t h e a d u l t W i s t a r r a t ( G o n t o v n i c k and B e l l w a r d 1 9 8 0 ) . T h i s enzyme a c t i v i t y was s i g n i f i c a n t l y h i g h e r i n t h e a d u l t male t h a n i n t h e a d u l t f e m a l e ( f i g u r e 3 ) . In c o n t r a s t t o t h e p r e v i o u s s t u d y , t e s t o s t e r o n e e n a n t h a t e a d m i n i s t r a t i o n d i d n o t c o n s i s t e n t l y f u l l y r e v e r s e t h e s e c a s t r a t i o n e f f e c t s t o w a r d s i n t a c t male l e v e l s ( f i g u r e 3 ) , b u t t h e l e v e l s were s i g n i f i c a n t l y g r e a t e r t h a n t h e c a s t r a t e d c o n t r o l s . F i g u r e 4. EFFECT OF FLUTAMIDE ON HEPATIC MICROSOMAL TESTOSTERONE REDUCTASE IN TESTOSTERONE-TREATED CASTRATED ADULT MALE AND INTACT ADULT FEMALE RATS H e p a t i c m i c r o s o m a l t e s t o s t e r o n e r e d u c t a s e a c t i v i t i e s were measured i n s h a m - o p e r a t e d a d u l t male (280-300 g) o r f e m a l e (180-200 g ) , and t h e 10 d a y - c a s t r a t e d (Gx) a d u l t male r a t . A n i m a l s were t r e a t e d w i t h c o r n o i l v e h i c l e ( C ) , o r t e s t o s t e r o n e e n a n t h a t e (T; 5 m i c r o m o l e s / k g s c . , 10 days) a i o n e , o r i n c o m b i n a t i o n w i t h v a r y i n g d o s e s o f f l u t a m i d e (F 5-50 m i c r o m o l e s / k g , s c . , 10 d a y s ) . E x p e r i m e n t s were done 2 h o u r s a l t e r t h e l a s t i n j e c t i o n . Enzyme a c t i v i t i e s were e x p r e s s e d as pmoles t e s t o s t e r o n e reduced/min/mg p r o t e i n +/-S.E.M. Numbers i n b r a c k e t s d e n o t e t h e numbers o f a n i m a l s t e s t e d . * : s i g n i f i c a n t d i f f e r e n c e (p< 0.05) compared t o s h a m - o p e r a t e d a d u l t male o f t h e same t r e a t m e n t g r o u p . ** : s i g n i f i c a n t d i f f e r e n c e (p< 0.05) compared t o t h e a p p r o p r i a t e c a s t r a t e d a d u l t male c o n t r o l g r o u p . F o r c o m p a r i s o n s between t h e t e s t o s t e r o n e p l u s t l u t a m i d e - t r e a t e d g r o u p s , and t h e t l u t a m i d e - u n t r e a t e d c o n t r o l g r o u p , t h e Duncan m u l t i p l e r a n g e t e s t was u s e d . D i l l e r e n c e s were c o n s i d e r e d s i g n i t i c a n t a t p< 0.05; s i g n i f i c a n t d i f f e r e n c e s between g r o u p s were i n d i c a t e d by common l e t t e r s . F i g u r e 4. 68 F l u t a m i d e (25 m i c r o m o l e s / k g ) i n j e c t e d d a i l y had no s i g n i f i c a n t e f f e c t on BPH a c t i v i t y i n t h e i n t a c t male, b u t s i g n i f i c a n t l y r e d u c e d t h e enzyme's a c t i v i t y i n t h e c a s t r a t e d male t o w a r d s t h a t o f t h e f e m a l e r a t ( f i g u r e 3 ) . F u r t h e r m o r e , f l u t a m i d e ( f r o m 15 t o 50 m i c r o m o l e s / k g ) s i g n i f i c a n t l y i n h i b i t e d t h e t e s t o s t e r o n e e f f e c t on BPH a c t i v i t y i n t h e 10 d a y - c a s t r a t e d a d u l t male r a t s (Duncan's m u l t i p l e r a n g e t e s t ; p< 0.05). The maximal BPH i n h i b i t i o n o c c u r r e d a t f l u t a m i d e d o s e s g r e a t e r t h a n o r e q u a l t o 15 m i c r o m o l e s / k g . L i k e BPH, h e p a t i c m i c r o s o m a l TR i s a s e x - d e p e n d e n t enzyme i n t h e a d u l t r a t (Kramer e t a l . 1 9 7 9 ) . However, u n l i k e BPH, TR a c t i v i t y was h i g h e r i n t h e a d u l t f e m a l e r a t t h a n i n t h e a d u l t male ( f i g u r e 4 ) . I n t h e a d u l t m a l e , c a s t r a t i o n f o r 10 days s i g n i f i c a n t l y i n c r e a s e d TR a c t i v i t i e s t o w a r d s t h o s e o f t h e f e m a l e . D a i l y a d m i n i s t r a t i o n of f l u t a m i d e (25 m i c r o m o l e s / k g , 10 d a y s ) had no s i g n i f i c a n t e f f e c t on TR a c t i v i t i e s i n t h e i n t a c t male compared t o t h e c o n t r o l . However, i n c r e a s i n g d o s e s of t h i s d r u g (5 t o 50 m i c r o m o l e s / k g ) s i g n i f i c a n t l y s t i m u l a t e d t h i s enzyme's a c t i v i t y i n t h e t e s t o s t e r o n e - t r e a t e d c a s t r a t e d male t o w a r d s f e m a l e l e v e l s (Duncan's m u l t i p l e r a n g e t e s t ) , w i t h maximal e f f e c t s o c c u r r i n g a t d o s e s e q u a l t o o r g r e a t e r t h a n 25 m i c r o m o l e s / k g . In o r d e r t o d e t e r m i n e sex s p e c i f i c i t y o f f l u t a m i d e and f l u t a m i d e h y d r o x i d e , t h e e f f e c t o f f l u t a m i d e and i t s m e t a b o l i t e f l u t a m i d e h y d r o x i d e were measured on a d u l t f e m a l e i F i g u r e 5 . EFFECT OF FLUTAMIDE AND FLUTAMIDE HYDROXIDE ON HEPATIC MICROSOMAL BENZO(A)PYRENE HYDROXYLASE AND TESTOSTERONE REDUCTASE IN THE INTACT ADULT FEMALE RAT H e p a t i c m i c r o s o m a l b e n z o ( a ) p y r e n e h y d r o x y l a s e (BPH; l e f t p a n e l ) and t e s t o s t e r o n e r e d u c t a s e (TR; r i g h t p a n e l ) a c t i v i t i e s were measured i n t h e i n t a c t a d u l t f e m a l e r a t (180-200 g ) . A n i m a l s were t r e a t e d w i t h c o r n o i l v e h i c l e ( C ) , t l u t a m i d e (F ; 25 m i c r o m o l e s / k g s c . , 10 days) o r f l u t a m i d e h y d r o x i d e ( F L - O H ; 25 m i c r o m o l e s / k g s c . , 10 days) Enzyme a c t i v i t i e s t o r BPH and TR were e x p r e s s e d as pmoles 3-OH b e n z o ( a ) p y r e n e fo rmed/min/mg p r o t e i n +/- S . E . M . , and nmoles t e s t o s t e r o n e r e d u c e d / m i n / m g p r o t e i n +/- S . E . M . , r e s p e c t i v e l y . Numbers i n t h e b a r s denote t h e numbers o f a n i m a l s u s e d . * : s i g n i f i c a n t d i f f e r e n c e (p< 0 .05) compared t o c o r n o i l - t r e a t e d a d u l t f e m a l e . PMOLES/MIN/MS H-c ro NMOLES/MIN/MS 71 h e p a t i c m i c r o s o m a l BPH and TR a c t i v i t i e s . C o n s i s t e n t w i t h i t s e f f e c t s on t h e i n t a c t m a l e , f l u t a m i d e a d m i n i s t r a t i o n (25 m i c r o m o l e s / k g , 10 d a y s ) had no s i g n i f i c a n t e f f e c t on h e p a t i c m i c r o s o m a l BPH o r TR a c t i v i t i e s i n t h e i n t a c t a d u l t f e m a l e . On t h e o t h e r hand, t h e same dose o f f l u t a m i d e h y d r o x i d e i n t h e i n t a c t f e m a l e s i g n i f i c a n t l y d e p r e s s e d BPH a c t i v i t i e s below, and s t i m u l a t e d TR a c t i v i t i e s above t h e c o r n o i l t r e a t e d c o n t r o l f e m a l e l e v e l s , r e s p e c t i v e l y ( f i g u r e 5 ) . 2. Development o f t h e h e p a t i c c y t o s o l i c t e s t o s t e r o n e b i n d i n g a s s a y (a) S e p a r a t i o n o f bound v s f r e e s t e r o i d s As a f i r s t a p p r o x i m a t i o n i n d e v e l o p i n g an a s s a y o f h e p a t i c a n d r o g e n r e c e p t o r , t h e t e c h n i q u e s and c o n d i t i o n s w e l l - k n o w n i n t h e measurement o f p r o s t a t i c a n d r o g e n r e c e p t o r were u s e d . P r o s t a t i c t i s s u e was us e d s i n c e i t p r o v i d e d an abun d a n t s o u r c e o f c y t o s o l i c a n d r o g e n r e c e p t o r . F u r t h e r m o r e , t e c h n i q u e s t o s e p a r a t e t h i s p r o t e i n f r o m unbound s t e r o i d have been w e l l - d o c u m e n t e d ( C l a r k and Peck 1 9 8 0 ) . Two d i f f e r e n t methods u s i n g Sephadex G-25M (PD-10) column c h r o m a t o g r a p h y and d e x t r a n - c o a t e d c h a r c o a l (DCC) p r e c i p i t a t i o n f o r s e p a r a t i n g p r o t e i n bound and f r e e s t e r o i d s were compared. S i n c e some w o r k e r s i n t h e f i e l d have o b t a i n e d b e t t e r r e s u l t s u s i n g t h e PD-10 m i n i c o l u m n s t h a n t h e DCC t e c h n i q u e ( D r . P. R e n n i e , p e r s o n n a l c o m m u n i c a t i o n ) , t h e f o l l o w i n g e x p e r i m e n t s were done. T a b i a 4. SEPARATION TECHNIQUES FOR PROTEIN BOUND AND UNBOUND STEROIDS For prostate experiments, adult male rats (280-300 g) were castrated 18-20 hrs p r i o r to experiment. Prostate cytoso l (1-3 mg/ml) was prepared as described i n Methods sec t ion , and was incubated w i t h 3H-dihydrotestosterone (DHT), i n the presence or absence of 1000-fold excess molar r a t i o rad io iner t DHT or R1881, for 18 hrs at 4 ° C. Intact adult male r a t l i v e r whole cy toso l (40 mg/ml) was prepared as described i n Methods sect ion, and was incubated wi th %-testosterone, i n the presence or absence of 100- or 1000-fold excess molar r a t i o rad io iner t testosterone. Cytosol preparations were then treated w i t h 0.5% DCC for prostate or 2.5% DCC for l i v e r c y t o s o l . In a separate set of tubes, cy toso l preparations were eluted through Sephadex G-25M (PD-10) ininicolumns. Rad ioac t iv i ty was analysed and Scatchard analysis was used to determine the apparent binding k i n e t i c s (Kd denotes binding a f f i n i t y i n nM; Bmax denotes binding capacity i n fmol/mg). Data was expressed as average + S .E .M. Numbers i n brackets denote the numbers of animals tested. Tissue cytoso l Ligand Competitor DCC Apparent Binding K i n e t i c s Kd (nM) Bmax (fmol/mg) PD-10 Kd (nM) Bmax (fmol/mg) Prostate DHT DHT 0.5 166 (2) 0.7 1 3 4 ( 1 ) R1881 0.6 137 (2) 0.7 175 (1) L i v e r DHT DHT no detectable b inding* no detectable b inding* Te (HA) Te (MA) Te Te 2o6 + 0.8 1.9 + 0.5 (3) 39 + 14 44 + 14 (5) 2.7 + 0.9 6.8 + 1.5 (3) * : no detectable displaceable and saturable binding Te : testosterone l igand range (HA denotes 0.05 to 2.0 nM; MA denotes 2.0 t o 60 nM) A d u l t male r a t p r o s t a t e and l i v e r whole ( u n f r a c t i o n a t e d ) c y t o s o l were p r e p a r e d as d e s c r i b e d i n t h e Methods s e c t i o n . B o t h DCC and PD-10 column methods f o r s e p a r a t i o n o f bound f r o m f r e e s t e r o i d s y i e l d e d s i m i l a r r e s u l t s f o r a n d r o g e n b i n d i n g p r o t e i n l e v e l s ( t a b l e 4 ) . P r o s t a t e c y t o s o l w h i c h was i n c u b a t e d i n v a r y i n g c o n c e n t r a t i o n s o f t r i t i a t e d - D H T o r R1881 (0.05 - 2 nM), w i t h o r w i t h o u t 1 0 0 0 - f o l d e x c e s s m o l a r r a t i o r a d i o i n e r t DHT y i e l d e d d e t e c t a b l e s a t u r a b l e and d i s p l a c e a b l e b i n d i n g w i t h t h e f o l l o w i n g b i n d i n g k i n e t i c s ( t a b l e 4 ) : a p p a r e n t (app.) Kd= 0.5 nM and app. Bmax= 166 fmol/mg u s i n g t h e DCC t e c h n i q u e , s i m i l a r t o t h o s e r e p o r t e d by B o e s l and S h a i n (1974) and K i r c h o f f e t a l . ( 1 9 8 0 ) , w h i l e the PD-10 column s e p a r a t i o n t e c h n i q u e o f f e r e d c o m p a r a b l e r e s u l t s (app. Kd= 0.7 nM; app. Bmax= 134 f m o l / m g ) . I n c u b a t i o n o f l i v e r c y t o s o l w i t h t h e same c o n c e n t r a t i o n s o f t r i t i a t e d - D H T and c o m p e t i t o r , f a i l e d t o show d e t e c t a b l e d i s p l a c e a b l e b i n d i n g u s i n g e i t h e r s e p a r a t i o n t e c h n i q u e ( t a b l e 4 ) . L i v e r whole c y t o s o l (40 mg/ml) t a k e n f r o m i n t a c t a d u l t male r a t s was i n c u b a t e d i n t r i t i a t e d - t e s t o s t e r o n e (0.05 t o 2 nM; o r 2 t o 60 nM) w i t h o r w i t h o u t 100- o r 1 0 0 0 - f o l d e x c e s s m o l a r r a t i o r a d i o i n e r t t e s t o s t e r o n e f o r 3 h o u r s a t 4 d e g r e e s C. F o l l o w i n g i n c u b a t i o n , t h e c y t o s o l was t h e n t r e a t e d w i t h 2.5% DCC s e p a r a t i o n t e c h n i q u e . T h e s e two r a n g e s o f t e s t o s t e r o n e c o n c e n t r a t i o n s y i e l d e d two d i f f e r e n t d i s p l a c e a b l e and s a t u r a b l e b i n d i n g i s o t h e r m s w i t h t h e f o l l o w i n g b i n d i n g k i n e t i c s ( t a b l e 4 ) : one b i n d i n g i s o t h e r m , app. Kd= 2.6 +/- 0.8 nM and app. Bmax= 1.9 +/- 0.5 fmol/mg u s i n g 0.05 - 2.0 nM; and a n o t h e r i s o t h e r m app. Kd= 39 +/- 14 nM and app. Bmax= 44 +/- 14 fmol/mg u s i n g 2.0 - 60 nM. S i m i l a r b i n d i n g k i n e t i c s were a l s o f o u n d u s i n g 0.05 - 2 nM t r i t i a t e d - t e s t o s t e r o n e and t h e PD-10 column s e p a r a t i o n t e c h n i q u e : app. Kd= 2.7 nM +/- 0.9 (n=3), app. Bmax= 6.8 fmol/mg +/- 1.5 (n=3). S i n c e b o t h s e p a r a t i o n t e c h n i q u e s y i e l d e d s i m i l a r r e s u l t s , t h e DCC method was us e d r o u t i n e l y , as i t was more c o n v e n i e n t and e c o n o m i c a l . The b i n d i n g k i n e t i c s o f t h e s e c o n d c y t o s o l i c b i n d i n g i s o t h e r m were i n a s i m i l a r r a n g e as t h a t d e s c r i b e d by o t h e r w o r k e r s ( S a t o e t a l . 1980; Ota e t a l . 1 9 8 0 ) . However, u n d e r o u r e x p e r i m e n t a l c o n d i t i o n s , t h e a s s a y was f o u n d t o y i e l d i n c o n s i s t e n t d i s p l a c e a b l e h e p a t i c c y t o s o l i c t e s t o s t e r o n e b i n d i n g v a l u e s . As a r e s u l t , p r o t e i n c o n c e n t r a t i o n d e p e n d e n c y , i n c u b a t i o n t i m e and t e m p e r a t u r e s t u d i e s were p u r s u e d i n o r d e r t o o p t i m i z e t h i s a s s a y . F u r t h e r , a r e p o r t by Yamada and M i y a j i (1982) d e s c r i b e d an a n d r o g e n b i n d i n g p r o t e i n i n t h e h e p a t i c m i c r o s o m a l f r a c t i o n . S i n c e t h e l a t t e r i s a l s o known t o c o n t a i n p r o g e s t e r o n e and g l u c o c o r t i c o i d r e c e p t o r s (Drangova and F e u e r 1980; A m b e l l a n e t a l . 1982) w h i c h may be i n v o l v e d i n t h e r e g u l a t i o n o f i n t r a c e l l u l a r s t e r o i d c o n c e n t r a t i o n s , o r may p l a y a d i r e c t r o l e i n p r o t e i n s y n t h e s i s a t t h e t r a n s l a t i o n a l l e v e l , i t was d e c i d e d t o i n v e s t i g a t e t h e p r e s e n c e o f t h e a n d r o g e n b i n d i n g p r o t e i n i n t h i s f r a c t i o n as w e l l . T a b l e 5 EFFECT OF VARYING PROTEIN CONTENT ON ADULT MALE RAT LIVER P o o l e d a d u l t male r a t (280-300 g; n=10 a n i m a l s ) l i v e r w hole c y t o s o l was p r e p a r e d i n TED b u t t e r (pH 7.4) s u p p l e m e n t e d w i t h 10 mM p o t a s s i u m c h l o r i d e , and v a r y i n g p r o t e x n c o n c e n t r a t i o n s (11-74 mg/ml), u s i n g t r i t i a t e d - t e s t o s t e r o n e (2.0-60 nM) i n t h e p r e s e n c e o r a b s e n c e o f 100 f o l d e x c e s s m o l a r r a t i o r a d i o i n e r t t e s t o s t e r o n e , t o r 3 h o u r s a t 4 d e g r e e s C. C y t o s o l was t r e a t e d w i t h 2.5% DCC. R a d i o a c t i v i t y was a n a l y s e d and b i n d i n g k i n e t i c s (app. Kd i n nM; app. Bmax i n fmol/mg) were c a l c u l a t e d u s i n g S c a t c h a r d a n a l y s i s and l e a s t s q u a r e s method t o r l i n e a r r e g r e s s i o n . P r o t e i n C o n t e n t A p p a r e n t B i n d i n g K i n e t i c s CYTOSOLIC TESTOSTERONE BINDING (mg/ml) Kd (nM) Bmax (fmol/mg) 11 no d e t e c t a b l e b i n d i n g 20 no d e t e c t a b l e b i n d i n g * 32 286 60 65 132 30 74 182 36 * d e n o t e s no d e t e c t a b l e d i s p l a c e a b l e b i n d i n g . (b) O p t i m i z a t i o n o f t h e c y t o s o l i c t e s t o s t e r o n e b i n d i n g p r o t e i n a s s a y ( i ) E f f e c t o f v a r y i n g t h e p r o t e i n c o n c e n t r a t i o n on t e s t o s t e r o n e b i n d i n g i n a d u l t male h e p a t i c c y t o s o l A d u l t male r a t l i v e r c y t o s o l o f v a r y i n g p r o t e i n c o n c e n t r a t i o n s (11 t o 74 mg/ml) was i n c u b a t e d f o r 3 h o u r s a t 4 d e g r e e s C, i n t r i t i a t e d - t e s t o s t e r o n e (2 t o 60 nM), w i t h o r w i t h o u t 1 0 0 - f o l d e x c e s s m o l a r r a t i o r a d i o i n e r t t e s t o s t e r o n e . P r o t e i n c o n c e n t r a t i o n s g r e a t e r t h a n 32 mg/ml y i e l d e d d i s p l a c e a b l e t e s t o s t e r o n e b i n d i n g (app. Kd= 132-286 nM; app. Bmax= 30-60 fmol/mg), w h i l e no d e t e c t a b l e b i n d i n g was f o u n d a t l o w e r p r o t e i n c o n c e n t r a t i o n s ( t a b l e 5 ) . S i n c e a h e p a t i c c y t o s o l i c t e s t o s t e r o n e b i n d i n g p r o t e i n c o u l d o n l y be f o u n d a t r e l a t i v e l y h i g h p r o t e i n c o n c e n t r a t i o n s , i e . >30 mg/ml, the p o s s i b i l i t y o f m i c r o s o m a l c o n t a m i n a t i o n was s t u d i e d . D i f f e r e n c e s i n t h e b i n d i n g k i n e t i c s between t h e s e two f r a c t i o n s , u n d e r v a r i o u s i n c u b a t i o n c o n d i t i o n s would make th e p o s s i b i l i t y o f m i c r o s o m a l c o n t a m i n a t i o n u n l i k e l y . ( i i ) E f f e c t of v a r y i n g i n c u b a t i o n t i m e and t e m p e r a t u r e on t e s t o s t e r o n e b i n d i n g i n a d u l t male r a t l i v e r c y t o s o l and m i c r o s o m e s L i v e r c y t o s o l i c (40 mg/ml) and m i c r o s o m a l f r a c t i o n s (1 mg/ml) were p r e p a r e d as d e s c r i b e d i n t h e Methods s e c t i o n and were s e p a r a t e l y i n c u b a t e d i n t r i t i a t e d - t e s t o s t e r o n e (2 t o 60 nM) w i t h o r w i t h o u t 1 0 0 - f o l d e x c e s s r a d i o i n e r t t e s t o s t e r o n e , a t 4 d e g r e e s f o r v a r y i n g p e r i o d s o f t i m e (3, 12 and 18 T a b l e 6 EFFECT OF VARYING INCUBATION PERIOD ON TESTOSTERONE AND DHT BINDING IN LIVER CELLULAR FRACTIONS P o o l e d a d u l t male r a t (280-300 g; n= 16 a n i m a l s ) l i v e r c e l l t r a c t i o n s were p r e p a r e d as d e s c r i b e d i n Methods s e c t i o n , and were i n c u b a t e d i n t r i t i a t e d - t e s t o s t e r o n e (2-60 nM) f o r c y t o s o l (40 mg/ml) and mi c r o s o m e s (1 mg/ml), o r t r i t i a t e d -DHT (2-60 nM) t o r microso m e s f o r 3, 12 o r 18 h o u r s a t 4 d e g r e e s C, i n t h e a b s e n c e o r p r e s e n c e o f 1 0 0 - f o l d e x c e s s m o l a r r a t i o r a d i o i n e r t t e s t o s t e r o n e o r DHT. The 2.5% DCC s e p a r a t i o n t e c h n i q u e was u s e d . B i n d i n g k i n e t i c s were d e t e r m i n e d u s i n g S c a t c h a r d a n a l y s i s and t h e l e a s t s q u a r e s method t o r l i n e a r r e g r e s s i o n . C e l l f r a c t i o n I n c u b a t i o n A p p a r e n t B i n d i n g K i n e t i c s u s e d p e r i o d T e s t o s t e r o n e DHT Kd Bmax Kd Bmax (nM) (fmol/mg) (nM) (fmol/mg) C y t o s o l 3 h o u r s 37 131 12 h o u r s 54 40 18 h o u r s 21 15 M i c r o s o m a l 3 h o u r s 101 173 5.7 205 12 h o u r s 73 205 8.5 233 18 h o u r s 48 106 7.5 162 h o u r s ) . I n a p a r a l l e l s e t of t e s t t u b e s , m i c r o s o m e s were i n c u b a t e d i n t r i t i a t e d - D H T (2-60 nM) w i t h o r w i t h o u t 1 0 0 - f o l d e x c e s s m o l a r r a t i o DHT, u s i n g t h e same i n c u b a t i o n c o n d i t i o n s as a b o v e . I n c u b a t i o n of t h e c y t o s o l i c t e s t o s t e r o n e b i n d i n g p r o t e i n d e c r e a s e d t h e app. Bmax f r o m 131 t o 15 fmol/mg a f t e r 3 t o 18 h o u r s i n c u b a t i o n r e s p e c t i v e l y a t 4 d e g r e e s C ( t a b l e 6 ) . In c o n t r a s t , i n c u b a t i o n o f t r i t i a t e d - t e s t o s t e r o n e o r t r i t i a t e d - D H T b i n d i n g p r o t e i n i n t h e m i c r o s o m a l f r a c t i o n f o r 3 t o 18 h o u r s ( t a b l e 6 ) , y i e l d e d s i m i l a r b i n d i n g c a p a c i t i e s ( r a n g e f o r t e s t o s t e r o n e b i n d i n g was 106-205 fmol/mg and f o r DHT b i n d i n g was 162-233 fmol/mg). The c y t o s o l i c and m i c r o s o m a l t e s t o s t e r o n e b i n d i n g p r o t e i n a p p a r e n t Kd v a l u e s a p p e a r e d t o d e c r e a s e somewhat, f r o m 37 nM ( a f t e r 3 h o u r s i n c u b a t i o n ) t o 21 nM ( a f t e r 18 h o u r s i n c u b a t i o n ) f o r c y t o s o l , and f r o m 101 nM ( a f t e r 3 h o u r s i n c u b a t i o n ) t o 48 nM ( a f t e r 18 h o u r s i n c u b a t i o n ) f o r m i c r o s o m e s . I n c o n t r a s t , m i c r o s o m a l DHT b i n d i n g p r o t e i n a p p a r e n t b i n d i n g a f f i n i t y r e m a i n e d r e l a t i v e l y unchanged ( r a n g e 5.7 t o 7.5 nM). The e f f e c t o f v a r y i n g i n c u b a t i o n t e m p e r a t u r e was s t u d i e d u s i n g a d u l t male r a t l i v e r c y t o s o l w h i c h was i n c u b a t e d f o r 3 h o u r s i n t r i t i a t e d - t e s t o s t e r o n e , w i t h o r w i t h o u t 1 0 0 - f o l d e x c e s s m o l a r r a t i o u n l a b e l l e d t e s t o s t e r o n e c o m p e t i t o r ( t a b l e 7 ) . I n c r e a s i n g t h e i n c u b a t i o n t e m p e r a t u r e f r o m 4 t o 22 d e g r e e s C e l i m i n a t e d d i s p l a c e a b l e t e s t o s t e r o n e b i n d i n g f r o m t h e c y t o s o l i c f r a c t i o n . On t h e o t h e r hand, u s i n g t h e same i n c u b a t i o n c o n d i t i o n s , b o t h t h e app. Kd and Table 7 EFFECT OF VARYING INCUBATION TEMPERATURE ON ADULT MALE RAT LIVER CYTOSOLIC AND MICROSOMAL TESTOSTERONE BINDING Pooled a d u l t male r a t (280-300 g ; n= 8 an imal s ) c y t o s o l i c (40 mg/ml) and microsomal (1 mg/ml) c e l l t r a c t i o n s were prepared as d e s c r i b e d i n Methods s e c t i o n , and were i n c u b a t e d i n t r i t i a t e d - t e s t o s t e r o n e (2-60 nM) w i t h or w i t h o u t 1 0 0 - f o l d excess molar r a t i o r a d i o i n e r t t e s t o s t e r o n e , f o r 3 hours a t 4 or 22 degrees C . B i n d i n g k i n e t i c s were de termined u s i n g S c a t c h a r d a n a l y s i s . C e l l f r a c t i o n I n c u b a t i o n temperature Apparent used T e s t o s t e r o n e B i n d i n g K i n e t i c s Kd (nM) Bmax (fmol/mg) C y t o s o l 4 degrees C 26 36 22 degrees C no b i n d i n g * Microsomes 4 degrees C 27 268 22 degrees C 331 657 * denotes no d e t e c t a b l e d i s p l a c e a b l e s a t u r a b l e b i n d i n g app. Bmax o f t h e m i c r o s o m a l t e s t o s t e r o n e b i n d i n g p r o t e i n were i n c r e a s e d f r o m 27 nM ( a t 4 d e g r e e s C) t o 331 nM ( a t 22 d e g r e e s C ) , and f r o m 268 fmol/mg ( a t 4 d e g r e e s C) t o 657 fmol/mg ( a t 22 d e g r e e s C ) f r e s p e c t i v e l y . T h e r e seemed t o be l i t t l e l i k e l i h o o d o f i n t e r f r a c t i o n a l c o n t a m i n a t i o n b a s e d on: th e l a c k o f DHT b i n d i n g t o c y t o s o l ( t a b l e 4) compared t o mi c r o s o m e s ( t a b l e 6) a f t e r 3 h r s i n c u b a t i o n ; t h e t i m e d e p e n d e n t d e c r e a s e i n t e s t o s t e r o n e b i n d i n g i n c y t o s o l compared t o r e l a t i v e l y no change i n m i c r o s o m e s ; and t h e l a c k o f t e s t o s t e r o n e b i n d i n g i n c y t o s o l compared t o m i c r o s o m e s , a t 22 d e g r e e s C i n c u b a t i o n ( t a b l e 7 ) . To f u r t h e r s u p p o r t t h i s h y p o t h e s i s , s a t u r a t i o n s t u d i e s and s p e c i f i c i t y s t u d i e s were done, u s i n g t h e s e f r a c t i o n s and c o m p a r i n g t e s t o s t e r o n e d i s p l a c e m e n t by d i f f e r e n t c o m p e t i t o r s . ( i i i ) D i s p l a c e m e n t of t r i t i a t e d - t e s t o s t e r o n e b i n d i n g by v a r i o u s c o m p e t i t o r s i n a d u l t male r a t l i v e r whole c y t o s o l and m i c r o s o m e s T e s t o s t e r o n e b i n d i n g was compared i n c y t o s o l and m i c r o s o m a l f r a c t i o n s , u s i n g a v a r i e t y o f c o m p e t i t o r s ( t a b l e 8 ) . In c y t o s o l , d i s p l a c e m e n t o f t r i t i a t e d - t e s t o s t e r o n e by 1 0 0 - f o l d e x c e s s m o l a r r a t i o r a d i o i n e r t t e s t o s t e r o n e , DHT, p r o g e s t e r o n e , e s t r a d i o l o r R1881 i n d i c a t e d s i m i l a r a p p a r e n t Kd v a l u e s (50, 37, 38, 78 and 25 nM, r e s p e c t i v e l y ) and a p p a r e n t Bmax v a l u e s (139, 144, 99, 105 and 101 fmol/mg, r e s p e c t i v e l y ) . I n t e r e s t i n g l y , t r i a m c i n o l o n e a c e t o n i d e d i d no t d e t e c t a b l y d i s p l a c e l i g a n d f r o m l i v e r c y t o s o l . T a b l e 8 DISPLACEMENT OF TRITIATED-TESTOSTERONE FROM LIVER CYTOSOLIC AND MICROSOMAL FRACTIONS BY VARIOUS COMPETITORS P o o l e d a d u l t male r a t (280-300g ; n= 12 a n i m a l s ) l i v e r c y t o s o l (40 mg/ml) and microsomes ( lmg/ml) were p r e p a r e d as d e s c r i b e d i n Methods s e c t i o n , and were i n c u b a t e d i n t r i t i a t e d - t e s t o s t e r o n e (2-60 nM) w i t h o r w i t h o u t 1 0 0 - f o l d e x c e s s m o l a r r a t i o c o m p e t i t o r . F r a c t i o n s were i n c u b a t e d t o r 3 h o u r s a t 4 d e g r e e s C b e f o r e 2.5% DCC t r e a t m e n t and r a d i o a c t i v i t y a n a l y s i s . A p p a r e n t b i n d i n g k i n e t i c s were d e t e r m i n e d u s i n g S c a t c h a r d a n a l y s i s . A p p a r e n t T e s t o s t e r o n e B i n d i n g K i n e t i c s C y t o s o l Micro somes C o m p e t i t o r s Kd Bmax Kd Bmax (nM) (fmol/mg) (nM) (fmol/mg) T e s t o s t e r o n e 50 139 25 572 DHT 37 144 41 856 P r o g e s t e r o n e 38 99 31 730 E s t r a d i o l 78 105 61 612 R1881 ( m e t h y l t r i e n o l o n e ) 25 101 108 1351 T r i a m c i n o l o n e a c e t o n i d e no b i n d i n g * 119 1626 * d e n o t e s no d e t e c t a b l e d i s p l a c e a b l e and s a t u r a b l e b i n d i n g . 8 2 The m i c r o s o m a l t e s t o s t e r o n e b i n d i n g p r o t e i n i n c u b a t e d u n d e r t h e same c o n d i t i o n s as d e s c r i b e d a bove, was d i s p l a c e d e q u a l l y by 100 f o l d e x c e s s m o l a r r a t i o r a d i o i n e r t t e s t o s t e r o n e , DHT, p r o g e s t e r o n e o r e s t r a d i o l (app. Kd= 25, 41, 31 o r 61 nM r e s p e c t i v e l y , and app. Bmax= 572, 856, 730 o r 612 fmol/mg r e s p e c t i v e l y ) . R1881 d i s p l a c e d t r i t i a t e d - t e s t o s t e r o n e b i n d i n g i n m i c r o s o m e s w i t h an app. Kd= 108 nM and app. Bmax= 1351 fmol/mg. I n a d d i t i o n , t r i a m c i n o l o n e d i s p l a c e d m i c r o s o m a l t e s t o s t e r o n e b i n d i n g (app. Kd= 119 nM; app. Bmax= 1626 fmol/mg). In a n o t h e r e x p e r i m e n t , c y t o s o l and m i c r o s o m e s were i n c u b a t e d i n f i x e d c o n c e n t r a t i o n s o f t r i t i a t e d - t e s t o s t e r o n e (5 o r 40 nM f o r c y t o s o l ; 1 o r 40 nM f o r m i c r o s o m e s ) , w i t h o r w i t h o u t i n c r e a s i n g m o l a r r a t i o s (10 t o 1 0 0 0 - f o l d e x c e s s ) o f v a r i o u s c o m p e t i t o r s ( t a b l e s 9 A - D ) . T h e s e l i g a n d c o n c e n t r a t i o n s were c h o s e n s i n c e t h e y were w i t h i n t h e r a n g e o f t h e two c y t o s o l i c t e s t o s t e r o n e b i n d i n g s i t e s ( t a b l e 4 ) . D i s p l a c e m e n t o f 5 nM t r i t i a t e d - t e s t o s t e r o n e ( w i t h i n t h e rang e o f t h e h i g h a f f i n i t y - l o w c a p a c i t y t e s t o s t e r o n e b i n d i n g p r o t e i n , i e . Kd= 2.6 nM) by a v a r i e t y o f s t e r o i d a l and n o n - s t e r o i d a l c o m p e t i t o r s i n d i c a t e d some r e l a t i v e s p e c i f i c i t y f o r t e s t o s t e r o n e and p r o g e s t e r o n e o n l y a t t h e l a r g e m o l a r r a t i o of 1 0 0 0 - f o l d ( t a b l e 9 A ) . On t h e o t h e r hand, i n c u b a t i o n a t 40 nM y i e l d e d a n o n - s e l e c t i v e b i n d i n g p r o f i l e , i n c l u d i n g t e s t o s t e r o n e , DHT, p r o g e s t e r o n e and e s t r a d i o l a t a l l m o l a r r a t i o s t e s t e d ( t a b l e 9 B ) . DES, f l u t a m i d e o r f l u t a m i d e h y d r o x i d e d i d n o t p r o d u c e a p p r e c i a b l e 83 T a b l e 9A SINGLE POINT DISPLACEMENT STUDIES OF TRITIATED-TESTOSTERONE BY VARIOUS COMPETITORS IN LIVER CYTOSOLIC AND MICROSOMAL FRACTIONS P o o l e d a d u l t male r a t (280-300 g; n=8 a n i m a l s ) l i v e r c y t o s o l (40 mg/ml) and m i c r o s o m e s (1 mg/ml) were p r e p a r e d as d e s c r i b e d i n Methods, and were i n c u b a t e d w i t h 1, 5 o r 40 nM t r i t i a t e d - t e s t o s t e r o n e w i t h o u t o r w i t h v a r y i n g e x c e s s m o l a r r a t i o s ( c o m p e t i t o r : 1 i g a n d ) o f c o m p e t i t o r . C y t o s o l and m i c r o s o m e s were t r e a t e d w i t h 2.5% DCC t o s e p a r a t e bound and f r e e s t e r o i d f r a c t i o n s . Data were e x p r e s s e d as % bound r e l a t i v e t o c o n t r o l . C o n t r o l c y t o s o l d i d n o t c o n t a i n c o m p e t i t o r ( i e . 100%= no d i s p l a c e m e n t ) . (A) C y t o s o l i c f r a c t i o n : T r i t i a t e d - t e s t o s t e r o n e (5 nM) d i s p l a c e d by v a r y i n g m o l a r r a t i o s o f r a d i o i n e r t c o m p e t i t o r s . C o m p e t i t o r s M o l a r R a t i o ( c o m p e t i t o r : l i g a n d ) lOx lOOx lOOOx C o n t r o l 100 100 100 T e s t o s t e r o n e 88 92 91 D i h y d r o t e s t o s t e r o n e 86 91 93 P r o g e s t e r o n e 93 91 94 E s t r a d i o l 89 90 100 DES 93 94 93 A l d o s t e r o n e 100 100 R1881 ( m e t h y l t r i e n o l o n e ) 100 99 100 F l u t a m i d e 100 100 100 T r i a m c i n o l o n e a c e t o n i d e 100 100 100 T a b l e 9B (B) C y t o s o l i c f r a c t i o n : T r i t i a t e d - t e s t o s t e r o n e (40 nM) d i s p l a c e d by v a r y i n g m o l a r r a t i o s o f c o m p e t i t o r . Data r e p r e s e n t r e s u l t s o f two e x p e r i m e n t s . C o m p e t i t o r s M o l a r R a t i o ( c o m p e t i t o r : 1 i g a n d ) lOx lOOx lOOOx C o n t r o l 100 100 100 T e s t o s t e r o n e 72-68 69-88 62-78 D i h y d r o t e s t o s t e r o n e 75-89 70-88 62-78 P r o g e s t e r o n e 79-85 68-83 68-83 E s t r a d i o l 63 70 75 DES 92 93 T r i a m c i n o l o n e a c e t o n i d e 93 91 F l u t a m i d e h y d r o x i d e 95 92 T a b l e 9C (C) M i c r o s o m a l f r a c t i o n : T r i t i a t e d - t e s t o s t e r o n e (1 nM) d i s p l a c e d by v a r y i n g m o l a r r a t i o s o f d i f f e r e n t c o m p e t i t o r . C o m p e t i t o r s M o l a r R a t i o ( c o m p e t i t o r : l i g a n d ) lOx lOOx lOOOx C o n t r o l 100 100 100 T e s t o s t e r o n e 89 81 81 D i h y d r o t e s t o s t e r o n e 89 83 85 P r o g e s t e r o n e 79 75 87 E s t r a d i o l 94 92 91 DES 87 90 95 A l d o s t e r o n e 93 85 89 R1881 ( m e t h y l t r i e n o l o n e ) 85 81 82 F l u t a m i d e 87 73 79 F l u t a m i d e h y d r o x i d e 87 81 85 C y p r o t e r o n e a c e t a t e 83 83 85 T r i a m c i n o l o n e a c e t o n i d e 89 87 83 A n d r o s t e n e d i o n e 86 81 80 S p i r o n o l a c t o n e 84 82 77 T a b l e 9D (D) M i c r o s o m a l f r a c t i o n : T r i t i a t e d - t e s t o s t e r o n e (40 nM) d i s p l a c e d by v a r y i n g m o l a r r a t i o s o f d i f f e r e n t c o m p e t i t o r . Data r e p r e s e n t r e s u l t s f r o m two e x p e r i m e n t s . C o m p e t i t o r s M o l a r R a t i o ( c o m p e t i t o r : 1 i g a n d ) lOx lOOx lOOOx C o n t r o l 100 100 100 T e s t o s t e r o n e 46 -59 41--54 50' -69 D i h y d r o t e s t o s t e r o n e 43 -57 42 -53 46 -62 P r o g e s t e r o n e 40 -52 40--48 4 5 . -59 E s t r a d i o l 48 -65 52 -61 49 -57 DES 46 48 45 R1881 ( m e t h y l t r i e n o l o n e ) 49 -61 52 -57 50 -55 F l u t a m i d e 51 -78 51 -73 50 -69 F l u t a m i d e h y d r o x i d e 51 51 48 C y p r o t e r o n e a c e t a t e 50 49 53 T r i a m c i n o l o n e a c e t o n i d e 52 -63 48 -60 50 A n d r o s t e n e d i o n e 60 -71 60 -65 67 -76 87 d i s p l a c e m e n t , a l t h o u g h t h e maximum m o l a r r a t i o used was o n l y 1 0 0 - f o l d e x c e s s . In m i c r o s o m e s , t h e r e was l i t t l e i n d i c a t i o n o f s e l e c t i v e b i n d i n g , s i n c e a l l c o m p e t i t o r s seemed t o e q u a l l y and w e a k l y d i s p l a c e t h e t e s t o s t e r o n e l i g a n d a t 1 nM (between 5-27% o f t o t a l bound was d i s p l a c e d ) , w h i l e a t 40 nM t r i t i a t e d - t e s t o s t e r o n e between 40-60% o f t o t a l bound was d i s p l a c e d by a l l c o m p e t i t o r s t e s t e d ( t a b l e s 9C and 9D r e s p e c t i v e l y ) . D i f f e r e n c e s i n t h e b i n d i n g p r o f i l e and t h e r e l a t i v e s t a b i l i t y o f t h e m i c r o s o m a l compared t o t h e c y t o s o l i c t e s t o s t e r o n e b i n d i n g p r o t e i n , i e . d e c r e a s e d c y t o s o l i c t e s t o s t e r o n e b i n d i n g as a f u n c t i o n o f t i m e o r t e m p e r a t u r e , i n d i c a t e s t h e u n l i k e l i n e s s of i n t e r f r a c t i o n a l c o n t a m i n a t i o n . However, t h e p r o t e i n d e p e n d e n t d e c r e a s e i n c y t o s o l i c t e s t o s t e r o n e b i n d i n g may be e x p l a i n e d by e i t h e r l i g a n d o r p r o t e i n m e t a b o l i s m i n v i t r o , o r b o t h . The e f f e c t of 1 mM PMSF, a known s e r i n e p r o t e a s e i n h i b i t o r , on t h e t e s t o s t e r o n e b i n d i n g p r o t e i n was s t u d i e d u s i n g a d u l t male r a t l i v e r whole c y t o s o l w i t h v a r y i n g c o n c e n t r a t i o n s o f t r i t i a t e d - t e s t o s t e r o n e , i n t h e p r e s e n c e o r a b s e n c e o f 1 0 0 - f o l d e x c e s s r a d i o i n e r t t e s t o s t e r o n e . A d d i t i o n o f t h i s p r o t e a s e i n h i b i t o r t o t h e l i v e r homogenate d u r i n g p r e p a r a t i o n d i d n o t improve t h e a s s a y ' s c o n s i s t e n c y . ^ I n a d d i t i o n , i n c u b a t i o n o f male r a t l i v e r whole c y t o s o l w i t h H i d e-powder f o r 3 o r 18 h o u r s a t 37 d e g r e e s C y i e l d e d no d e t e c t a b l e p r o t e a s e a c t i v i t y , s u g g e s t i n g t h a t p r o t e o l y s i s was n o t a f a c t o r and p e r h a p s l i g a n d m e t a b o l i s m s h o u l d be T a b l e 10 SEPARATION OF TESTOSTERONE AND RELATED METABOLITES USING THIN-LAYER CHROMATOGRAPHY S t a n d a r d s t e r o i d s o l u t i o n s (1 mg/ml) were p r e p a r e d u s i n g d r y d i e t h y l e t h e r o r a b s o l u t e e t h a n o l . The s o l v e n t s y s t e m s u s e d were: s y s t e m I - e t h y l a c e t a t e : c y c l o h e x a n e (50:50) , and s y s t e m I I - b e n z e n e : a b s o l u t e e t h a n o l ( 9 5 : 5 ) . The v i s u a l i z a t i o n t e c h n i q u e s u s e d were: UV d e n o t e s u l t r a v i o l e t l i g h t s e t a t 456 nm, DPH d e n o t e s d i n i t r o p h e n y l h y d r a z i n e r e a g e n t (see Methods f o r p r e p a r a t i o n ) , and NPA d e n o t e s n a p h t h o q u i n o n e p e r c h l o r i c a c i d r e a g e n t (see Methods t o r p r e p a r a t i o n ) . E a c h R f - v a l u e was c a l c u l a t e d as t h e r a t i o o f t h e d i s t a n c e s (cm) e l u t e d f r o m o r i g i n by t h e s t e r o i d compared t o t h a t by t h e s o l v e n t f r o n t . R t - v a l u e s V i s u a l i z a t i o n S o l v e n t s y s t e m s T e c h n i q u e s u s e d S t a n d a r d s u s e d I I I UV DPH NPA T e s t o s t e r o n e 0.35 0.43 x x x 5 a - D i h y d r o t e s t o s t e r o n e 0.39 0.28 x x 5 B - D i h y d r o t e s t o s t e r o n e 0.39 0.47 x x 5 a - A n d r o s t a n - 3 a , 1 7 6 - d i o l 0.33 0.35 x 5 B - A n d r o s t a n - 3 a , 1 7 6 - d i o l 0.20 0.26 x 5 a - A n d r o s t a n - 3 B , 1 7 B - d i o l 0.35 0.32 x 5 B - A n d r o s t a n - 3 B , 1 7 6 - d i o l 0.38 x A n d r o s t e n e d i o n e 0.51 0.50 x x x i n v e s t i g a t e d . 3. T e s t o s t e r o n e m e t a b o l i s m s t u d i e s B e c a u s e t h e p r e v i o u s r e s u l t s had i n d i c a t e d t h a t t h e l a c k o f c o n s i s t e n c y i n t h e t e s t o s t e r o n e b i n d i n g a s s a y may be due t o l i g a n d m e t a b o l i s m , t h e f o l l o w i n g e x p e r i m e n t s were c a r r i e d o u t . (a) S e p a r a t i o n and d e t e c t i o n o f t e s t o s t e r o n e and r e l a t e d s t e r o i d s t a n d a r d s u s i n g t h i n - l a y e r c h r o m a t a g r a p h y (TLC) T h i n l a y e r c h r o m a t o g r a p h y u s i n g s o l v e n t s y s t e m I and I I , a d e q u a t e l y s e p a r a t e d a n d r o s t e n e d i o n e , t e s t o s t e r o n e , 5 a l p h a - and 5 beta-DHT, 5 a l p h a - and 5 b e t a - A - 3 a l p h a , 1 7 b e t a - d i o l and 5 a l p h a - and 5 b e t a - A- 3 b e t a , 17 b e t a - d i o l ( t a b l e 1 0 ) . O n l y t e s t o s t e r o n e and a n d r o s t e n e d i o n e were v i s u a l i z e d u s i n g UV l i g h t , c o n f i r m i n g t h e method's s e n s i t i v i t y t o t h e c o n j u g a t e d C4-C5 d o u b l e bond, w h i l e t e s t o s t e r o n e , a n d r o s t e n e d i o n e , and 5 a l p h a - and 5 beta-DHT were d e t e c t e d u s i n g t h e d i n i t r o p h e n y l h y d r a z i n e c o l o u r r e a g e n t c o n f i r m i n g t h e l a t t e r ' s s e n s i t i v i t y t o t h e o x o - g r o u p s a t C3. A l l o f t h e s t e r o i d s t e s t e d were d e t e c t e d u s i n g t h e n a p h t h o q u i n o n e s u l p h o n i c a c i d c o l o u r r e a g e n t s i n c e a l l c o n t a i n e d e i t h e r o xo- and h y d r o x y - g r o u p s a t C3 o r C17 p o s i t i o n s . A l t h o u g h 5 a l p h a - and 5 beta-DHT had s i m i l a r Rf v a l u e s (Rf=0.39) u s i n g s o l v e n t s y s t e m I , b o t h had d i s t i n c t Rf v a l u e s u s i n g s o l v e n t s y s t e m I I (Rf=0.28 and Rf=0.47, r e s p e c t i v e l y ) . I n a d d i t i o n t o UV a n a l y s i s , t h e d i f f e r e n c e 90 i n Rf v a l u e s between t e s t o s t e r o n e and 5 a l p h a - A - 3 a l p h a , 17 b e t a - d i o l was l a r g e r u s i n g s y s t e m I I (Rf=0.43 and Rf=0.35 r e s p e c t i v e l y ) t h a n i n s y s t e m I (Rf=0.33 and Rf=0.35 r e s p e c t i v e l y ) . F u r t h e r m o r e , i t was a d v a n t a g e o u s t o use b o t h s o l v e n t s y s t e m s when i d e n t i f y i n g 5 a l p h a -A- 3 b e t a , 17 b e t a - d i o l ; t h i s s t e r o i d had a Rf v a l u e u s i n g s y s t e m I o f 0.35 ( s i m i l a r t o t e s t o s t e r o n e ) , but a Rf v a l u e o f 0.32 u s i n g s y s t e m I I ( d i s s i m i l a r t o t e s t o s t e r o n e ) . 5 b e t a - A - 3 b e t a , 17 b e t a - d i o l had an Rf v a l u e o f 0.38 u s i n g s y s t e m I I ; t h i s v a l u e was q u i t e d i f f e r e n t f r o m b o t h 5 b e t a - A - 3 a l p h a , 17 b e t a - d i o l o r t e s t o s t e r o n e . (b) S e p a r a t i o n and d e t e c t i o n o f t e s t o s t e r o n e and i t s m e t a b o l i t e s f r o m c y t o s o l e x t r a c t s u s i n g TLC E t h e r e a l e x t r a c t s o f a d u l t male r a t l i v e r c y t o s o l (40 mg/ml) i n c u b a t e d w i t h r a d i o i n e r t t e s t o s t e r o n e o r 5 beta-DHT f o r 0 t o 3 h r s a t 4 o r 37 d e g r e e s C, were d r i e d on TLC s i l i c a g e l s h e e t s f o r c h r o m a t o g r a p h y . The e l u t i o n o f t h e s e e x t r a c t s y i e l d e d a number of s p o t s ( t a b l e 1 1 ) , i n c l u d i n g t e s t o s t e r o n e (Rf v a l u e s = 0.37 u s i n g s y s t e m I and 0.45 u s i n g s y s t e m I I ) , 5 b e t a - A - 3 a l p h a , 17 b e t a - d i o l (Rf v a l u e = 0.23 u s i n g s y s t e m I and 0.27 u s i n g s y s t e m I I ) , and o t h e r endogenous m a t e r i a l (Rf v a l u e s 0-0.12 and 0.58-0.90). The e l u t i o n o f t h e endogenous m a t e r i a l d i d n o t seem t o i n f l u e n c e t h e m o b i l i t i e s o f e i t h e r t e s t o s t e r o n e and 5 b e t a - A - 3 a l p h a , 17 b e t a - d i o l . T e s t o s t e r o n e was d e t e c t e d i n e x t r a c t s o f • c y t o s o l w h i c h were i n c u b a t e d f o r 0 h r s a t 4 d e g r e e s , w h i l e 5 T a b l e 11 SEPARATION OF ETHEREAL EXTRACTS TAKEN FROM LIVER CYTOSOL INCUBATED FOR DIFFERENT TIME INTERVALS AND AT VARIOUS TEMPERATURES WITH TESTOSTERONE AND 5B-DHT A d u l t male r a t l i v e r whole c y t o s o l (40 mg/ml) was p r e p a r e d as d e s c r i b e d i n Methods s e c t i o n , and was i n c u b a t e d a t d i f f e r e n t t i m e p e r i o d s and t e m p e r a t u r e s u s i n g 2 m i c r o g r a m s r a d i o i n e r t t e s t o s t e r o n e (T) o r 5 B - d i h y d r o t e s t o s t e r o n e (DHT) as s u b s t r a t e . S t e r o i d s were e x t r a c t e d and were s e p a r a t e l y a p p l i e d o n t o TLC s h e e t s , a l o n e w i t h s t a n d a r d s t e r o i d s o l u t i o n s ( t e s t o s t e r o n e and 5 B - a n d r o s t a n - 3 a , 1 7 B - d i o i ; t h e l a t t e r was d e n o t e d by t h e l e t t e r A ) . S t e r o i d s were s e p a r a t e d u s i n g t h e a p p r o p r i a t e s o l v e n t sy s tem and were i d e n t i f i e d u s i n g t h e a p p r o p r i a t e d e t e c t i o n method . The R l - v a l u e o f each e l u e n t was e x p r e s s e d as t h e r a t i o o f d i s t a n c e m i g r a t e d (cm) by s t e r o i d : t o t a l d i s t a n c e t r a v e l l e d by s o l v e n t t r o n t . S o l v e n t sys tems used w e r e : s y s t e m I -e t h y l a c e t a t e : c y c l o h e x a n e ( 5 0 : 5 0 ) , and sys tem I I - b e n z e n e : a b s o l u t e e t n a n o l ( 9 5 : 5 ) . pg= micrograms C y t o s o l i c E x t r a c t I n c u b a t i o n c o n d i t i o n s R f - v a l u e s u s i n g S t a n d a r d s o l v e n t sytems R f - v a l u e s S u b s t r a t e Time Temp. I I I I I I (hr s ) (deg .C) ug T 0 4 0 - 0 . 1 0 - 0 . 1 9 0 .39 0 .43 0 .37 0 .45 (T) 0 . 5 6 - 0 . 7 0 0 . 5 5 - 0 . 8 0 2 ug T 3 4 0 - 0 . 1 0 - 0 . 1 9 0 .24 0 .27 0 .23 0 .27 (A) 0 . 5 7 - 0 . 7 6 0 . 5 7 - 0 . 7 8 pg 37 0 - 0 . 2 0 0 - 0 . 2 7 0 .23 0 .30 0 .23 0 .27 (A) O . b O - 0 . 7 8 0 . 5 7 - 0 . 8 4 2 p g DHT 3 37 0 - 0 . 1 7 0 - 0 . 2 3 0 .20 0 .27 0 .23 0 .27 (A) 0 . 4 3 - 0 . 4 8 0 . 4 5 - 0 . 9 0 no 3 4 0 - 0 . 1 2 exogenous 0 . 5 8 - 0 . 9 0 s t e r o i d s added b e t a - A - 3 a l p h a - 17 b e t a - d i o l c o u l d o n l y be d e t e c t e d i n c y t o s o l w h i c h was i n c u b a t e d a f t e r 3 h o u r s a t 4 o r 37 d e g r e e s C. In a d d i t i o n , 5 b e t a - A - 3 a l p h a , 17 b e t a - d i o l was d e t e c t e d f r o m c y t o s o l w h i c h was i n c u b a t e d w i t h 5 beta-DHT a t 37 d e g r e e s f o r 3 h r s . T h e s e r e s u l t s s u g g e s t t h a t t e s t o s t e r o n e i s m e t a b o l i z e d i n v i t r o ( v i a 5 beta-DHT r e d u c t i o n ) t o 5 b e t a - A - 3 a l p h a , 1 7 b e t a - d i o l , p e r h a p s by c y t o s o l i c 3 a l p h a - s t e r o i d d e h y d r o g e n a s e and 5 b e t a - r e d u c t a s e (Tomkins e t a l . 1 9 57). C o n f i r m a t i o n of t h e TLC e x p e r i m e n t s was c a r r i e d o u t by gas c h r o m a t o g r a p h y - m a s s s p e c t r o m e t r y . ( c ) I d e n t i f i c a t i o n o f t e s t o s t e r o n e - and r e l a t e d d i o l -d e r i v a t i v e s u s i n g gas c h r o m a t o g r a p h y - m a s s s p e c t r o m e t r y (GCMS). The gas c h r o m a t o g r a p h - m a s s s p e c t r o m e t e r has become a p o w e r f u l t o o l i n t h e s e p a r a t i o n and i d e n t i f i c a t i o n of compounds w i t h s i m i l a r s t r u c t u r e s , s u c h as t e s t o s t e r o n e and i t s v a r i o u s a n d r o s t a n e - d i o l m e t a b o l i t e s . T h i s has been f u r t h e r a i d e d by t h e use o f MSTFA and MOX d e r i v a t i z a t i o n r e a g e n t s ( B o u r n o t e t a l . 1 9 7 4 ) . F o l l o w i n g t h e t e c h n i q u e o f B o u r n o t e t a_l. ( 1 9 7 4 ) , s e p a r a t e s t a n d a r d s t o c k s o l u t i o n s o f t e s t o s t e r o n e and a n d r o s t a n e - d i o l s were p r e p a r e d , and l a t e r d e r i v a t i z e d t o O-methyloxime (MO), t r i m e t h y l s i l o x y (TMS) t e s t o s t e r o n e and T M S - a n d r o s t a n e d i o l i s o m e r s f o r GCMS a n a l y s i s . D i f f e r e n c e s i n t h e r a t e of a d s o r p t i o n by t h e v a r i o u s e l u e n t s t o t h e l i q u i d p hase on t h e c a p i l l a r y column a r e r e f l e c t e d by t h e i r d i f f e r e n t r e t e n t i o n t i m e s . T h e s e compounds were f u r t h e r c h a r a c t e r i z e d by t h e i r mass s p e c t r a o f m/z v a l u e s , w h i c h were d e t e r m i n e d by the r e l a t i v e d e f l e c t i o n o f t h e s e compounds i n an e l e c t r o m a g n e t i c f i e l d . C h r o m a t o g r a p h y r e t e n t i o n t i m e s (Rt) o f t h e d e r i v a t i z e d a n d r o g e n s t e s t e d v a r i e d on a d a i l y b a s i s ; c o m p a r i s o n o f Rt v a l u e s were b e s t done u n d e r t h e same r u n n i n g c o n d i t i o n s o f t h a t d a y . The Rt v a l u e f o r t e s t o s t e r o n e (MO-TMS) was 13.5 min, w h i l e t h a t f o r 5 b e t a - A - 3 a l p h a , 17 b e t a - d i o l (TMS) was 12.4 m i n . D e s p i t e d a i l y d i f f e r e n c e s i n Rt v a l u e s , t e s t o s t e r o n e (MO-TMS) c o n s i s t e n t l y e l u t e d a t a s l o w e r r a t e t h a n 5 b e t a - A - 3 a l p h a , 17 b e t a - d i o l (TMS). The f o l l o w i n g d a t a i n d i c a t e d t h a t t h e Rt v a l u e s f o r 5 b e t a - A - 3 a l p h a , 17 b e t a d i o i (TMS; Rt= 9.3 min) d i d not r e l i a b l y d i f f e r w i t h t h o s e of t h e o t h e r T M S - a n d r o s t a n e d i o l s t e s t e d (Rt v a l u e s r a n g e f r o m 9.0 t o 9.8 m i n s ) , and t h a t due t o p o o r s e p a r a t i o n , d i f f e r e n c e s i n t h e Rt v a l u e s c o u l d n o t be us e d t o c h a r a c t e r i z e t h e a n d r o s t a n e - d i o l s t e r e o i s o m e r s . The l a t t e r was a c c o m p l i s h e d by e x a m i n i n g t h e i r mass s p e c t r a . 5 b e t a - A - 3 a l p h a , 17 b e t a - d i o l (TMS) Rt= 9. 3 min 5 a l p h a - A - 3 a l p h a , 17 b e t a - d i o l (TMS) Rt= 9. 0 min 5 a l p h a - A - 3 b e t a , 17 b e t a - d i o l (TMS) Rt= 9. 8 min 5 b e t a - A - 3 b e t a , 17 b e t a - d i o l (TMS) Rt= 9. 3 min S e p a r a t e i n j e c t i o n s o f t e s t o s t e r o n e and t h e a n d r o s t a n e - d i o l d e r i v a t i v e s were a n a l y z e d u s i n g GCMS, and y i e l d e d t h e f o l l o w i n g mass s p e c t r a v a l u e s : (a) t e s t o s t e r o n e ( m o l e c u l a r w e i g h t 288) m/z v a l u e s 389 (m+ 5 8 % ) , 153 ( 1 0 0 ) , 358 (12) (b) 5 b e t a - A - 3 a l p h a , 17 b e t a - d i o l ( m o l e c u l a r w e i g h t 292) m/z v a l u e s 436 (m+, 8 % ) , 129 ( 1 0 0 ) , 256 ( 7 2 ) , 346 ( 2 2 ) , 241 (50),421 (m-15, 4) (c) 5 a l p h a - A - 3 a l p h a , 17 b e t a - d i o l ( m o l e c u l a r w e i g h t 292) m/z v a l u e s 436 (m+, 8 5 ) , 166 ( 1 0 0 ) , 129 ( 6 0 ) , 241 ( 2 0 ) , 256 ( 1 0 ) , 331 ( 8 ) , 346 ( 8 ) , 256 (8) (d) 5 a l p h a - A - 3 b e t a , 17 b e t a - d i o l ( m o l e c u l a r w e i g h t 292) m/z v a l u e s 436 (m+, 1 6 % ) , 129 ( 1 0 0 ) , 346 ( 2 0 ) , 214 ( 2 0 ) , 421 (m-15, 18) (e) 5 b e t a - A - 3 b e t a , 17 b e t a - d i o l ( m o l e c u l a r w e i g h t 292) m/z v a l u e s 436 (m+, <4%), 129 ( 1 0 0 ) , 256 ( 2 2 ) , 346 ( 1 8 ) , 241 (12) The t e s t o s t e r o n e m o l e c u l e d i f f e r s f r o m t h e a n d r o s t a n e - d i o l s t e s t e d i n a t l e a s t two ways: t h e c o n j u g a t e d d o u b l e bond a t t h e C4-C5 p o s i t i o n and t h e e n o l g r o u p a t t h e C3 p o s i t i o n , w h i l e t h e l a t t e r i s i n t h e r e d u c e d f o r m and c o n t a i n s a h y d r o x y - g r o u p a t t h e C3 p o s i t i o n . T h e s e d i f f e r e n c e s a r e r e f l e c t e d by t h e i r m o l e c u l a r w e i g h t s , and a r e f u r t h e r a c c e n t u a t e d f o l l o w i n g d e r i v a t i z a t i o n . F o r i n s t a n c e , ( T M S , M O ) - t e s t o s t e r o n e had a m o l e c u l a r i o n o f m/z= 389, w h i c h was d i s t i n g u i s h e d f r o m t h e T M S - a n d r o s t a n e d i o l i s o m e r s w h i c h had m o l e c u l a r i o n s o f m/z= 436. Of t h e s t e r e o i s o m e r s o f 5 a l p h a / b e t a A- 3 a l p h a / b e t a , 17 b e t a - d i o l s d e r i v a t i v e s t e s t e d , a l l c o n t a i n e d v a r y i n g q u a n t i t i e s o f t h e m o l e c u l a r i o n (m/z= 4 3 6 ) , r a n g i n g f r o m 4 t o 16% o f t h e i r r e l a t i v e b ase p e a k s . D i f f e r e n c e s between t h e s e compounds can be d e t e r m i n e d on t h e b a s i s o f t h e i r mass s p e c t r a . 5 a l p h a - A - 3 a l p h a , 17 b e t a - d i o l was c h a r a c t e r i z e d by t h e p r e s e n c e o f t h e m/z 166 i o n o r t h e base peak, compared t o t h e o t h e r a n d r o s t a n e d i o l s t e r e o i s o m e r s . 5 a l p h a - A - 3 b e t a , 17 b e t a - d i o l was d i s t i n g u i s h e d f r o m 5 b e t a - A - 3 a l p h a , 1 7 b e t a - d i o l by t h e l a c k o f t h e i o n m/z= 256, as a m a j o r p r o d u c t . 5 b e t a - A - 3 b e t a , 17 b e t a - d i o l d i f f e r e d w i t h b o t h 5 a l p h a - A - 3 b e t a , 17 b e t a - d i o l and 5 b e t a - A - 3 a l p h a , 17 b e t a - d i o l s i n c e i t l a c k e d t h e m/z= 421 i o n (m-15), w h i c h may r e p r e s e n t t h e d e m e t h y l a t e d f o r m o f t h e m o l e c u l a r i o n , as a m a j o r f r a g m e n t a t i o n p r o d u c t . GCMS a n a l y s i s was done on t h e e t h e r e a l e x t r a c t o f s i l i c a g e l u s i n g zone Rf= 0.43 ( s o l v e n t s y s t e m I I ) w h i c h was t a k e n f r o m t h e TLC s e p a r a t i o n o f a d u l t male r a t l i v e r whole c y t o s o l (40 mg/ml) i n c u b a t e d f o r 0 h r a t 4 d e g r e e s C ( f i g u r e 6 A ) . T h i s a n a l y s i s y i e l d e d a m a j o r peak a t Rt= 13.5, w h i c h was i d e n t i f i e d as t e s t o s t e r o n e (m/z= 3 8 9 ) . O t h e r p e a k s F i g u r e s 6 (A-C) GAS CHROMATOGRAPHY-MASS SPECTROMETRY (GCMS) ANALYSIS OF TESTOSTERONE AND TESTOSTERONE METABOLITES TAKEN FROM L I V E R CYTOSOL EXTRACT A d u l t male r a t (280-300 g) w h o l e l i v e r c y t o s o l (40 mg/ml) was p r e p a r e d as d e s c r i b e d i n Methods s e c t i o n , and was i n c u b a t e d a t d i f f e r e n t t i m e i n t e r v a l s and t e m p e r a t u r e s w i t h r a d i o i n e r t t e s t o s t e r o n e . S t e r o i d s were e x t r a c t e d u s i n g d i e t h y l e t h e r and s e p a r a t e d u s i n g t h i n - l a y e r c h r o m a t o g r a p h y ( T L C ) . TLC zones c o r r e s p o n d i n g t o t e s t o s t e r o n e (T) and 56 - a n d r o s t a n - 3 a , 1 7 6 - d i o l (A) were e l u t e d and t h e r e s u l t i n g e x t r a c t was d e r i v a t i z e d p r i o r t o GCMS a n a l y s i s . Da ta were compared t o d e r i v a t i z e d s t a n d a r d s . Upper p a n e l : gas c h r o m a t o g r a p h . Lower p a n e l : mass s p e c t r u m . I n c u b a t i o n C o n d i t i o n s : (A) 0 h o u r s a t 4 d e g r e e s C , (B) 3 h o u r s a t 4 degree s C , and (C) 3 h o u r s =at 37 degree s C . figure 30080-1 Etwid8tH . 00000H hi 4°C,0h -A ">1 i 1 1 1 1 r- • ' 1 i 13 .fl 14 . 1 & . <j le.tf i P .0 I 8 . 8 1 9 . tf 2 figure i 98 -J a • i i • J • ' > \ l t i t > I. 30M09H ->>1M -•88888* c S 8 8 W H » 9 8 8 0 » - 'l| > 4°C,3h .i4 9 . W • | I « I • | • I • I | • < • 8 13.8 14.8 1 &.8 1 & . 8 F i l e J; Boh Mi I c kn 195 .?? t i n . fyurc t C W . S - b N . I M i l . 4 8 S8 — . I • i i 1 I I I I I i- L 1 i 1 * - J L - i - !• 168 288 240 238 10000 'I 37°C,3h •8.8 : B . 5 . 1 . 8 i i .8 T-Dk ?t. T.S.S68 1138-, -Sc ari 1'. I i 30H .4 1: 346 j II I I I ,• fliilliiii 43* !• i JL f.rr---- --...1 . 1 . . . 100 f a i l e d t o show r e c o g n i z a b l e mass s p e c t r a . C y t o s o l w h i c h was i n c u b a t e d f o r 3 h o u r s a t 4 d e g r e e s C y i e l d e d 5 b e t a - A - 3 a l p h a , 17 b e t a - d i o l as a m a j o r peak a t Rt= 12.8 min i n TLC zone Rf= 0.26 ( s o l v e n t s y s t e m I I . F i g u r e 6B shows t h e mass s p e c t r u m o f t h i s p r o d u c t , m/z v a l u e s = 436(m+,28), 256 ( 1 0 0 ) , 241 ( 7 0 ) , 346 ( 7 0 ) . A n a l y s i s o f o t h e r p e a k s i n t h i s s p e c t r u m f a i l e d t o show r e c o g n i z a b l e mass s p e c t r a . In a d i f f e r e n t e x p e r i m e n t , a n a l y s i s o f t h e same zone (Rf= 0.26; s o l v e n t s y s t e m I I ) t a k e n f r o m c y t o s o l w h i c h was i n c u b a t e d f o r 3 h r s a t 37 d e g r e e s C, y i e l d e d 5 b e t a - A - 3 a l p h a , 17 b e t a - d i o l (Rt= 9.9 min) as a m a j o r p r o d u c t ( f i g u r e 6C) m/z v a l u e s = 436 (m+,10%), 129 ( 1 0 0 ) , 256 ( 9 0 ) , 241 ( 5 0 ) , 346 ( 3 0 ) , 421 ( 8 ) . T e s t o s t e r o n e o r o t h e r compounds were n o t d e t e c t e d i n t h e s e z o n e s s i n c e t h e y were p r e - f r a c t i o n a t e d u s i n g T L C . T h e s e d a t a c o n f i r m s t h e e a r l i e r TLC r e s u l t s t h a t t e s t o s t e r o n e i s m e t a b o l i z e d t o 5 b e t a - A - 3 a l p h a , 17 b e t a - d i o l i n v i t r o , u n d e r t h e s e e x p e r i m e n t a l c o n d i t i o n s . (d) F o r m a t i o n o f r a d i o l a b e l l e d t e s t o s t e r o n e m e t a b o l i t e s i n l i v e r whole c y t o s o l i_n v i t r o In o r d e r t o e s t i m a t e t h e q u a n t i t a t i v e i m p o r t a n c e o f t h e m e t a b o l i s m o f t e s t o s t e r o n e t o 5 b e t a - A - 3 a l p h a , 17 b e t a - d i o l , f u r t h e r e x p e r i m e n t s were u n d e r t a k e n u s i n g c y t o s o l w h i c h was i n c u b a t e d w i t h t r i t i a t e d - t e s t o s t e r o n e and t r e a t e d u n d e r t h e same c o n d i t i o n s as t h o s e u s e d f o r o u r p r e v i o u s t e s t o s t e r o n e b i n d i n g s t u d i e s . A n a l y s i s o f t r i t i a t e d - t e s t o s t e r o n e TLC o f e t h e r e a l e x t r a c t t a k e n f r o m Figures 7 (A&B) THIN-LAYER RADIOCHROMATOGRAM OF TESTOSTERONE METABOLISM IN VITRO IN ADULT MALE RAT LIVER WHOLE CYTOSOL Cytosol (40 mg/ml) was prepared as described i n Methods section, and was incubated with 40 nM tri t i a t e d - t e s t o s t e r o n e , in the absence ( f i g . 7A; broken line) or i n the presence ( f i g 7B; dotted line) of 100-fold excess molar r a t i o radioinert testosterone. Each radiochromatogram was sectioned into 1-2 cm s t r i p s and analysed for r a d i o a c t i v i t y . The r a d i o a c t i v i t y (ordinate) was plotted as a function ot the Rt-value for each thin-layer radiochromatogram s t r i p (abscissa). The major peaks were i d e n t i f i e d using radioinert standard steroids (T denotes testosterone; A denotes 53-androstan-3a,176-dioi). 102 F i g u r e 7A CYTOSOLIC TESTOSTERONE METABOLISM IN_ VITRO 2 5 0 3 2 0 0 D W 1 5 0 H 0 -P X 0 E LT) • \ 0_ u i o o H 5 0 H o ( O) no competitor- added T \ / i I 1 — 0 10 20 30 40 Rf values/100 5 0 60 F i g u r e 7B C Y T O S O L I C T E S T O S T E R O N E M E T A B O L I S M I N V I T R O 2 5 0 -] ( • ) w i t h competitor- added o • 0 (/> 0 +> X u E in • \ a. u 2 0 0 1 5 0 1 0 0 H 5 0 H o • a n • R • P • • • • n - n T r-10 2 0 3 0 4 0 5 0 R f v a l u e s / 1 0 0 6 0 the p r o t e i n bound f r a c t i o n o f a d u l t male r a t l i v e r whole c y t o s o l w h i c h was i n c u b a t e d w i t h v a r y i n g c o n c e n t r a t i o n s o f t r i t i a t e d - t e s t o s t e r o n e i n t h e p r e s e n c e o r a b s e n c e of 100 f o l d e x c e s s m o l a r r a t i o r a d i o i n e r t t e s t o s t e r o n e , f o r 3 h r s a t 4 d e g r e e s C, i n d i c a t e d two m a j o r p e a k s (Rf= 0.21 and Rf= 0 . 3 8 ) . T h e s e p e a k s c o r r e s p o n d e d t o 5 b e t a - A - 3 a l p h a , 17 b e t a - d i o l 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 ( f i g u r e 7 A&B). The f o r m a t i o n of t r i t i a t e d - 5 b e t a - A - 3 a l p h a , 17 b e t a - d i o l was d e p e n d e n t on t h e amount o f t r i t i a t e d - t e s t o s t e r o n e added t o t h e i n c u b a t i o n m i x t u r e , r a n g i n g f r o m 22-32% of t h e t r i t i a t e d - t e s t o s t e r o n e p r e s e n t i n t h e bound f r a c t i o n ( f i g u r e 8 A ) . F o r m a t i o n of t r i t i a t e d - 5 b e t a - A - 3 a l p h a , 17 b e t a - d i o l was g r e a t l y enhanced f o l l o w i n g a d d i t i o n of 1 0 0 - f o l d e x c e s s r a d i o i n e r t t e s t o s t e r o n e ( f i g u r e 8 B ) . T h e s e d a t a f u r t h e r s t r e n g t h e n t h e c o n c l u s i o n t h a t a m a j o r f r a c t i o n o f t e s t o s t e r o n e i s b e i n g m e t a b o l i z e d i_n v i t r o t o 5 b e t a - A - 3 a l p h a , 17 b e t a - d i o l . B e c a u s e o f t h i s , t h e c a l c u l a t i o n o f f r e e l i g a n d c o n c e n t r a t i o n must be c o r r e c t e d f o r m e t a b o l i s m i n o r d e r t o p r o p e r l y a n a l y s e t h e b i n d i n g d a t a . In o r d e r t o a v o i d t h i s d i f f i c u l t y , a r e l a t i v e l y n o n - m e t a b o l i z a b l e l i g a n d was c h o s e n f o r f u r t h e r s t u d i e s . 4. H e p a t i c c y t o s o l i c m e t h y l t r i e n o l o n e (R1881) b i n d i n g a s s a y M e t h y l t r i e n o l o n e (R1881) i s p u r p o r t e d t o be a n o n - m e t a b o l i z a b l e and s t a b l e a n d r o g e n (Bonne and Raynaud 1975, 1 9 7 6 ) . To d e t e r m i n e i t s p r o p e r t i e s i n o u r s y s t e m , we F i g u r e s 8 (A&B) THIN-LAYER CHROMATOGRAPHIC ANALYSIS OF CYTOSOLIC TESTOSTERONE METABOLISM IN VITRO A d u l t male r a t (280-300 g) l i v e r w hole c y t o s o l (40 mg/ml) was p r e p a r e d as d e s c r i b e d i n Methods s e c t i o n , and was i n c u b a t e d w i t h v a r y i n g c o n c e n t r a t i o n s o f t r i t i a t e d -t e s t o s t e r o n e , w i t h o u t ( f i g 8A; " T o t a l Bound" f r a c t i o n ) o r w i t h ( f i g 8B; N o n - s p e c i f i c Bound o r "NSB" f r a c t i o n ) 1 0 0 - f o l d e x c e s s m o l a r r a t i o r a d i o i n e r t t e s t o s t e r o n e . C y t o s o l was t r e a t e d w i t h 2.5% DCC t o s e p a r a t e bound f r o m unbound s t e r o i d f r a c t i o n s . The bound f r a c t i o n c o n t a i n i n g t r i t i a t e d - t e s t o s t e r o n e and m e t a b o l i t e s was e x t r a c t e d u s i n g d i e t h y l e t h e r and was a n a l y s e d u s i n g t h i n - l a y e r r a d i o c h r o m a t o g r a p h y ( T L C ) . R a d i o a c t i v i t y ( o r d i n a t e ) c o n t a i n e d on TLC zones Rf= 0.38 (or t e s t o s t e r o n e ) and Rf= 0.21 ( 5 B - a n d r o s t a n - 3 a , na - d i o i ) u s i n g s o l v e n t s y s t e m I I , was p l o t t e d as a f u n c t i o n o f t h e c o n c e n t r a t i o n o f t r i t i a t e d - t e s t o s t e r o n e added t o t h e i n c u b a t i o n m i x t u r e ( a b s c i s s a ) . F i g 8A shows t h e p l o t o f c y t o s o l i n c u b a t e d i n t h e a b s e n c e o f r a d i o i n e r t t e s t o s t e r o n e ; a t e a c h t e s t o s t e r o n e c o n c e n t r a t i o n , c i r c l e s d e n o t e r a d i o a c t i v i t y on zone Rf= 0.38, w h i l e s q u a r e s d e n o t e r a d i o a c t i v i t y on zone Rf= 0.21. I n f i g 8B, c y t o s o l i n c u b a t e d i n t h e p r e s e n c e o f r a d i o i n e r t t e s t o s t e r o n e i s p l o t t e d ; a t e a c h t r i t i a t e d - t e s t o s t e r o n e c o n c e n t r a t i o n , s q u a r e s d e n o t e r a d i o a c t i v i t y on zone Rf= 0.21, w h i l e c i r c l e s d e n o t e r a d i o a c t i v i t y on zone Rf= 0.38. 106 F i g u r e 8A. Rod i o — chromatograph of" " T o t a l Bound" f r a c t i o n 15Q-. < CO "T e s t o s t e r o n e " remaining C • > "DIDL" formed 120 -• 10 2 0 3 0 4D 5 0 Free Te added <nNO 1 0 7 F i g u r e 8B. Rodio—chromatograph of "NSB" f r a c t i o n 150 - i (CO " T e s t o s t e r o n e " remaining C •> "•IOL" formed 120 -a o o E D_ "D T3 C 0 0 CD 10 2D 3D 40 Free Te added (nM) c o n d u c t e d t h e f o l l o w i n g e x p e r i m e n t s . (a) E f f e c t o f p r o t e i n d i l u t i o n and t i m e c o u r s e on i n c u b a t i o n o f R1881 b i n d i n g i n a d u l t male r a t l i v e r whole c y t o s o l C y t o s o l was p r e p a r e d as d e s c r i b e d i n Methods s e c t i o n , and was i n c u b a t e d a t v a r i o u s p r o t e i n c o n c e n t r a t i o n s (0.87-3.4 mg/ml) f o r d i f f e r e n t t i m e p e r i o d s (1.5, 3 o r 18 h r s ) a t 4 d e g r e e s C. A n a l y s i s o f t h e h i g h a f f i n i t y - l o w c a p a c i t y R1881 component of c y t o s o l i n c u b a t e d f o r 18 h o u r s a t 4 d e g r e e s , i n d i c a t e d p r o t e i n c o n c e n t r a t i o n dependency ( f i g u r e 9; r= 1 . 0 ) . The e f f e c t of t i m e of i n c u b a t i o n on c y t o s o l R1881 b i n d i n g i s shown i n f i g u r e s 10 A-C. A t a l l p r o t e i n c o n c e n t r a t i o n s , t h e r e was a t i m e d e p e n d e n t l o s s of t h e l o w e r a f f i n i t y - h i g h e r c a p a c i t y component, f r o m 1.5 h o u r s ( f i g 10A) t o 18 h o u r s ( f i g 10C), where i t was c o m p l e t e l y a b s e n t . A l t h o u g h t h e r e i s some e x p e r i m e n t a l v a r i a t i o n between t h e 18 hour S c a t c h a r d p l o t s , t h e b i n d i n g c a p a c i t y was d e p e n d e n t on t h e p r o t e i n c o n c e n t r a t i o n of t h e c y t o s o l ( f i g u r e s 9 and 10C). I t became a p p a r e n t t h a t d i r e c t g r a p h i c a l a n a l y s i s of S c a t c h a r d p l o t s i n v o l v i n g m u l t i p l e b i n d i n g s i t e s m i g h t l e a d t o i n a c c u r a c i e s i n t h e d e t e r m i n a t i o n of b i n d i n g k i n e t i c s . I n o r d e r t o overcome t h i s c o m p l i c a t i o n , two a p p r o a c h e s were p u r s u e d : ( i ) computer a n a l y s i s u s i n g two d i f f e r e n t methods f o r e s t i m a t i n g m u l t i p l e b i n d i n g k i n e t i c s , and ( i i ) c h e m i c a l l y b i n d i n g ( o r b l o c k i n g ) t h e l o w e r a f f i n i t y - h i g h e r c a p a c i t y d i s p l a c e a b l e n o n - s p e c i f i c b i n d i n g s i t e s , u s i n g F i g u r e 9. EFFECT OF VARYING THE PROTEIN CONCENTRATION ON CYTOSOLIC R1881 BINDING IN THE ADULT MALE RAT LIVER C y t o s o l c o n t a i n i n g d i f f e r e n t p r o t e i n c o n c e n t r a t i o n s (1-4 mg/ml) was p r e p a r e d as d e s c r i b e d i n Methods s e c t i o n , and was i n c u b a t e d w i t h v a r y i n g c o n c e n t r a t i o n s o f t r i t i a t e d - R 1 8 8 1 (0.05-2.0 nM) i n t h e a b s e n c e o r p r e s e n c e o f 1 0 0 - f o l d e x c e s s m o l a r r a t i o r a d i o i n e r t R1881, f o r 18 h o u r s a t 4 d e g r e e s C. A p p a r e n t number o f b i n d i n g s i t e s ( e x p r e s s e d i n f m o l e s ) were e s t i m a t e d u s i n g S c a t c h a r d a n a l y s i s , and were e x p r e s s e d p e r 0.5 ml c y t o s o l . T h e s e v a l u e s were d i r e c t l y p r o p o r t i o n a l (r= 1.0) t o t h e c y t o s o l i c p r o t e i n c o n t e n t (mg/ml) added t o t h e i n c u b a t i o n m i x t u r e , u s i n g l e a s t s q u a r e s method f o r l i n e a r r e g r e s s i o n . 1 1 0 F i g u r e 9. F i g u r e s 10 ( A - C ) . EFFECT OF VARYING PROTEIN CONCENTRATIONS AND INCUBATION TIME INTERVALS ON LIVER CYTOSOLIC R1881 BINDING PROTEIN A d u l t male r a t (280-300 g) l i v e r c y t o s o l was p r e p a r e d i n v a r y i n g 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 , and was i n c u b a t e d f o r d i f f e r e n t t i m e p e r i o d s a t 4 d e g r e e s C, u s i n g t r i t i a t e d - R 1 8 8 1 (0.05-2.0 nM) i n t h e a b s e n c e o r p r e s e n c e o f 1 0 0 - f o l d e x c e s s m o l a r r a t i o r a d i o i n e r t R1881. P r o t e i n c o n c e n t r a t i o n s u s e d were : ( c i r c l e s ) 0.87 mg/ml; ( s q u a r e s ) 1.64 mg/ml; ( t r i a n g l e s ) 3.41 mg/ml. I n c u b a t i o n t i m e p e r i o d s were 1.5 h o u r s ( f i g 10A), 3 h o u r s ( f i g 10B) and 18 h o u r s ( f i g 1 0 C ) . A p p a r e n t b i n d i n g a f f i n i t y , Kd was e x p r e s s e d i n nM, and was d e t e r m i n e d u s i n g l e a s t s q u a r e s method f o r l i n e a r r e g r e s s i o n . F i g u r e 10A. TIME COURSE C1.5 hi~s> & PRDTEIN DILUTION • H 1 1 1 1 1 1 D 10 20 3D 40 50 60 S p e c i f i c Bound (fmol/mg) 113 F i g u r e 10B. TIME CDURSE C3 hrs) & PROTEIN DILUTION o H 1 1 — i 1 1 1 O 10 20 30 40 50 60 S p e c i f i c Bound (fmol/mg) F i g u r e IOC. TIME COURSE C18 hrs) & PROTEIN DILUTION C O > D. B7 m g / m l C D ) 1. 6 4 m g / m l C A ) 3 . 4 1 m g / m l KcfcO.15-025 1 1 1 1 1 1 0 10 2D 30 40 50 60 S p e c i f i c B i n d i n g (fmol/mg) T a b l e 12 COMPARISON OF LINEAR AND NON-LINEAR REGRESSION ANALYSIS OF BINDING DATA A d u l t male r a t l i v e r whole c y t o s o l (1-3 mg/ml) was i n c u b a t e d w i t h v a r y i n g c o n c e n t r a t i o n s o f t r i t i a t e d - R 1 8 8 1 (0.05-2.0 nM), w i t h o u t o r w i t h 1 0 0 - f o l d e x c e s s m o l a r r a t i o r a d i o i n e r t R1881, f o r 3 h o u r s a t 4 d e g r e e s C. C y t o s o l was t r e a t e d w i t h 2.5% DCC b e f o r e r a d i o a c t i v i t y a n a l y s i s . A p p a r e n t b i n d i n g k i n e t i c s were d e t e r m i n e d u s i n g l i n e a r r e g r e s s i o n f o r S c a t c h a r d a n a l y s i s , o r n o n - l i n e a r r e g r e s s i o n f o r s a t u r a t i o n ( f r e e v s . bound) a n a l y s i s . The AMDAHL computer (UBC Computing C e n t r e ) and t h e A p p l e I I p l u s m i c r o c o m p u t e r were us e d f o r t h e s e p u r p o s e s . Kd i s t h e e q u i l i b r i u m d i s s o c i a t i o n c o n s t a n t ( e x p r e s s e d i n nM), and Bmax i s t h e b i n d i n g c a p a c i t y ( e x p r e s s e d i n f m o l / m g ) . HALC d e n o t e s h i g h a f f i n i t y - l o w c a p a c i t y and LAHC d e n o t e s l o w e r a f f i n i t y - h i g h e r c a p a c i t y b i n d i n g p r o t e i n s . E s t i m a t e s f o r B i n d i n g HALC LAHC Method o f a n a l y s i s Kd Bmax Kd Bmax N o n - 1 i n e a r 0. 22 5.6 12.2 138 R e g r e s s i o n A n a l y s i s u s i n g AMDAHL L i n e a r R e g r e s s i o n A n a l y s i s u s i n g A p p l e I I p l u s 0.25 5.3 10.0 135 F i g u r e s 11 (A&B). COMPUTER ANALYSIS OF R1881 BINDING DATA A d u l t male r a t (280-300 g) l i v e r w hole c y t o s o l (1-3 mg/ml) was p r e p a r e d as d e s c r i b e d i n Methods, and was i n c u b a t e d w i t h v a r y i n g c o n c e n t r a t i o n s o f t r i t i a t e d - R 1 8 8 1 (0.05-2.0 nM) w i t h o r w i t h o u t 1 0 0 - f o l d e x c e s s m o l a r r a t i o r a d i o i n e r t R1881, f o r 3 h o u r s a t 4 d e g r e e s C. C y t o s o l was t r e a t e d w i t h 2.5% DCC and r a d i o a c t i v i t y was a n a l y s e d . A p p a r e n t b i n d i n g k i n e t i c s were d e t e r m i n e d by u s i n g n o n - l i n e a r r e g r e s s i o n o f t h e s a t u r a t i o n c u r v e ( f i g 11A; F r e e R1881 added v s . S p e c i f i c Bound) by AMDAHL c o m p u t e r , o r by u s i n g l i n e a r r e g r e s s i o n o f t h e S c a t c h a r d p l o t ( f i g 11B; S p e c i f i c B o u n d / ( F r e e R1881) v s . S p e c i f i c Bound) by A p p l e I I - p l u s m i c r o c o m p u t e r . C i r c l e s d e n o t e v a l u e s f o r o b s e r v e d d a t a , and s q u a r e s d e n o t e v a l u e s f o r c o m p u t e r - f i t t e d d a t a . F i g u r e 11A. 1 1 7 N O N - L I N E A R R E G R E S S I O N A N A L Y S I S 70 -i 0) 60 -E \ r-l 0 50 -E <+ 40 -TJ C D 0 OQ 30 -U •ri U- 20 -u ai •_ 10 -0 -C O ) O b s e r v e d d a t a C D ) C o m p u t e t — f i t t e d Kd:122 nM -T 1 1 1 1 1 2 3 4 5 6 7 F r e e R1881 CnM) F i g u r e 1 1 B . LINEAR REGRESSION ANALYSIS 40 - i CO) Observed data CD) M i c r o c o m p u t e i — f i t t e d 20 -: l o -• Kd 025 nM Kd 10 nM 1 1 1 0 20 40 60 S p e c i f i c Bound Cfmol 80 /mg) 100 e x c e s s t r i a m c i n o l o n e a c e t o n i d e as d e s c r i b e d by Eagon e t a l . ( 1 9 8 3 ) . L i v e r whole c y t o s o l was a d j u s t e d t o 1-3 mg/ml and i n c u b a t e d f o r 3 h r s a t 4 d e g r e e s C, b e c a u s e t h e s e c o n d i t i o n s had p r o v i d e d r e l i a b l e d a t a and a c o n v e n i e n t t i m e p e r i o d . (b) Computer a n a l y s i s of b i n d i n g d a t a Two d i f f e r e n t methods o f computer a n a l y s i s , l i n e a r and n o n - l i n e a r r e g r e s s i o n of two s a t u r a b l e b i n d i n g s i t e s were compared u s i n g t h e same s e t o f d a t a ( t a b l e 12, f i g u r e 11 A&B). B o t h methods gave s i m i l a r r e s u l t s : a h i g h a f f i n i t y - l o w c a p a c i t y s i t e (app. Kd= 0.22-0.25 nM; app. Bmax= 5.3-5.6 fmol/mg), and a l o w e r a f f i n i t y - h i g h e r c a p a c i t y s i t e ( a pp. Kd= 10-12 nM; app. Bmax= 135-138 f m o l / m g ) . I t s h o u l d be n o t e d t h a t t h e t i m e r e q u i r e d f o r a n a l y s i s was much l e s s when u s i n g t h e n o n - l i n e a r r e g r e s s i o n p r o g r a m compared t o t h e l i n e a r r e g r e s s i o n program; t h i s may be a t t r i b u t e d t o t h e d i f f e r e n c e i n c a p a c i t i e s between t h e two c o m p u t e r s . F o r t h e sake of c o n v e n i e n c e and t i m e , d a t a u t i l i z i n g t h e s e e x p e r i m e n t a l c o n d i t i o n s were a n a l y s e d u s i n g t h e AMDAHL computer i e . t h e n o n - l i n e a r r e g r e s s i o n method. ( c ) E f f e c t o f t r i a m c i n o l o n e a c e t o n i d e a l o n e and i n c o m b i n a t i o n w i t h C o r t i s o l on R1881 b i n d i n g i n r a t l i v e r whole c y t o s o l M e t h y l t r i e n o l o n e was shown t o b i n d w i t h h i g h a f f i n i t y t o c y t o s o l i c a n d r o g e n and p r o g e s t e r o n e b i n d i n g p r o t e i n s i n t h e human p r o s t a t e and b r e a s t t i s s u e s (Zava e t a l . 1 9 7 9 ) . In o r d e r t o s e p a r a t e t h e s e e f f e c t s , Zava e t a l . (1979) have added t h e s y n t h e t i c p r o g e s t i n t r i a m c i n o l o n e a c e t o n i d e t o t h e p r o s t a t e c y t o s o l m i x t u r e , a t e c h n i q u e w h i c h i s t h o u g h t t o block t h e l o w e r a f f i n i t y R1881 b i n d i n g component, w i t h o u t a f f e c t i n g 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 . A s i m i l a r a p p r o a c h was u s e d i n t h e measurement of t h e h e p a t i c a n d r o g e n r e c e p t o r by Eagon e t a l . (1983) and o t h e r s . In a d d i t i o n t o t r i a m c i n o l o n e , E i s e n f e l d e t a l . (1983) and A t e n e t a l . (1983) added C o r t i s o l t o t h e i r h e p a t i c c y t o s o l m i x t u r e . I n th e f o l l o w i n g s t u d i e s , we d e c i d e d t o t e s t t h e e f f e c t s of t r i a m c i n o l o n e a c e t o n i d e and C o r t i s o l on R1881 b i n d i n g f o l l o w i n g t h e same i n c u b a t i o n c o n d i t i o n s as t h a t u s e d f o r th e t e s t o s t e r o n e b i n d i n g s t u d i e s . C y t o s o l was i n c u b a t e d w i t h v a r i o u s c o n c e n t r a t i o n s o f t r i t i a t e d - R 1 8 8 1 , i n t h e p r e s e n c e o r a b s e n c e of 1 0 0 - f o l d e x c e s s m o l a r r a t i o r a d i o i n e r t R1881, f o r 3 h r s a t 4 d e g r e e s C. In a d i f f e r e n t s e t o f t u b e s , d i f f e r e n t m o l a r r a t i o s (100, 500 o r 1 0 0 0 - f o l d ) o f e x c e s s t r i a m c i n o l o n e a c e t o n i d e o r C o r t i s o l were a l s o added t o e a c h i n c u b a t i o n m i x t u r e . M u l t i p l e b i n d i n g a n a l y s i s of c y t o s o l i n c u b a t e d w i t h o u t t r i a m c i n o l o n e o r C o r t i s o l showed two components ( t a b l e 1 3 ) , a h i g h a f f i n i t y - l o w c a p a c i t y component (app. Kd= 0.22 nM; app. Bmax= 5.6 fmol/mg) and a low a f f i n i t y - h i g h c a p a c i t y component ( app. Kd= 12.2 nM; app. Bmax= 138 fm o l / m g ) . S i n g l e component l i n e a r r e g r e s s i o n a n a l y s i s o f S c a t c h a r d - t y p e p l o t s o f t h e same c y t o s o l i c p r e p a r a t i o n i n d i c a t e d t h a t a d d i t i o n o f t r i a m c i n o l o n e a c e t o n i d e (100-1000 EFFECT OF VARYING EXCESS MOLAR RATIOS OF TRIAMCINOLONE ACETONIDE OR CORTISOL ON R1881 BINDING IN ADULT MALE RAT LIVER WHOLE CYTOSOL A d u l t male r a t (280-300 g) l i v e r w h o l e c y t o s o l (40 mg/ml) was p r e p a r e d as d e s c r i b e d i n Methods s e c t i o n , and was i n c u b a t e d w i t h v a r y i n g c o n c e n t r a t i o n s o f t r i t i a t e d - R 1 8 8 1 ( 0 . 0 5 - 2 n M ) , i n t h e absence o r p r e s e n c e o f e x c e s s r a d i o i n e r t t r i a m c i n o l o n e a c e t o n i d e (100-1000 t o l d ) o r Cortisol (100-500 t o l d ) , w i t h o u t o r w i t h r a d i o i n e r t R1881 , f o r 3 h o u r s a t 4 d e g r e e s . C y t o s o l was t r e a t e d w i t h 2.5% DCC and r a d i o a c t i v i t y was a n a l y s e d . B i n d i n g k i n e t i c s were d e t e r m i n e d u s i n g l i n e a r o r n o n - l i n e a r r e g r e s s i o n methods . HALC d e n o t e s h i g h a t t i n i t y - l o w c a p a c i t y component ; LAHC d e n o t e s l o w e r a f f i n i t y - h i g h e r c a p a c i t y component . E x c e s s m o l a r r a t i o A p p a r e n t B i n d i n g K i n e t i c s T r i a m c i n o l o n e C o r t i s o l Kd (nM) Bmax (fmol/mg) a c e t o n i d e 0 0 0 .22 5 .6 (HALC) 12 .20 13 .8 (LAHC) 0 500 0 .33 6 .6 100 0 0 .22 6 .9 5U0 0 0 .29 6 .5 1000 0 0 .28 6 .8 100 100 0 .28 6 .5 100 500 0 .30 5 .6 f o l d e x c e s s ) a l o n e o r i n c o m b i n a t i o n w i t h C o r t i s o l (100 o r 500 f o l d e x c e s s ) y i e l d e d one b i n d i n g i s o t h e r m w i t h t h e f o l l o w i n g a p p a r e n t b i n d i n g k i n e t i c s ( t a b l e 1 3 ) : app. Kd= 0.27 +/- 0.01 nM; app. Bmax= 6.5 +/- 0.2 fmol/mg; t h e s e v a l u e s were s i m i l a r t o t h e b i n d i n g k i n e t i c s o f t h e h i g h a f f i n i t y - l o w c a p a c i t y R1881 b i n d i n g component c o n t a i n e d i n t h e c y t o s o l i n c u b a t e d i n t h e a b s e n c e o f t r i a m c i n o l o n e a c e t o n i d e o r C o r t i s o l . F u r t h e r m o r e , t h e a d d i t i o n o f C o r t i s o l (100 o r 500 e x c e s s ) t o c y t o s o l c o n t a i n i n g t r i a m c i n o l o n e a c e t o n i d e (100 o r 500 f o l d e x c e s s ) d i d n o t s i g n i f i c a n t l y change R1881 b i n d i n g . F o r t h i s r e a s o n , t h e f o r m e r was o m i t t e d f r o m t h e i n c u b a t i o n m i x t u r e . I t s h o u l d be n o t e d t h a t i n c u b a t i o n o f c y t o s o l i c m i x t u r e s w i t h e x c e s s t r i a m c i n o l o n e a c e t o n i d e f o r 3 h o u r s y i e l d e d a s i n g l e b i n d i n g i s o t h e r m w i t h b i n d i n g k i n e t i c s s i m i l a r t o t h o s e of t h e same m i x t u r e i n c u b a t e d w i t h o u t t r i a m c i n o l o n e f o r 18 h o u r s . In o u r p r e v i o u s t e s t o s t e r o n e b i n d i n g s t u d i e s , we u s e d t h e same i n c u b a t i o n c o n d i t i o n s as t h a t d e s c r i b e d by S a t o e t a l . (1980) and Ota e t a l . ( 1 9 8 0 ) , i e . t h e TED b u f f e r was s u p p l e m e n t e d w i t h 10 mM p o t a s s i u m c h l o r i d e . To t e s t t h e e f f e c t of t h i s c a t i o n on R1881 b i n d i n g i n h e p a t i c c y t o s o l , t h e f o l l o w i n g e x p e r i m e n t s were c o n d u c t e d . Whole c y t o s o l (1-3 mg/ml) was p r e p a r e d f r o m a d u l t male r a t l i v e r and was i n c u b a t e d i n t h e u s u a l manner u s i n g e i t h e r TED b u f f e r o r T E D - s u p p l e m e n t e d w i t h 10 mM p o t a s s i u m . A s e p a r a t e a l i q u o t o f t h e TED p r e p a r e d c y t o s o l was i n c u b a t e d i n t h e p r e s e n c e o f 10 mM p o t a s s i u m . A l l c y t o s o l f r a c t i o n s were i n c u b a t e d w i t h F i g u r e s 12 (A&B). EFFECT OF 10 mM POTASSIUM CHLORIDE ON LIVER CYTOSOLIC R1881 BINDING IN ADULT,MALE RAT C y t o s o l (1-3 mg/ml) was p r e p a r e d as d e s c r i b e d i n Methods, and was i n c u b a t e d w i t h t r i t i a t e d - R 1 8 8 1 (0.05-2.0 nM) w i t h o r w i t h o u t 1 0 0 - f o l d e x c e s s m o l a r r a t i o r a d i o i n e r t R1881. In a d d i t i o n t o t h e a b o v e , c y t o s o l was i n c u b a t e d i n t h e a b s e n c e ( f i g 12A) o r p r e s e n c e ( f i g 12B) o f 1 0 0 - f o l d e x c e s s m o l a r r a t i o r a d i o i n e r t t r i a m c i n o l o n e a c e t o n i d e . TED b u f f e r m i x t u r e i n c l u d e d : ( c i r c l e s ) 10 mM p o t a s s i u m c h l o r i d e added d u r i n g p r e p a r a t i o n o f c y t o s o l and m a i n t a i n e d t h r o u g h o u t i n c u b a t i o n ; ( s q u a r e s ) no p o t a s s i u m added; ( t r i a n g l e s ) 10 mM p o t a s s i u m c h l o r i d e added p r i o r t o i n c u b a t i o n . A p p a r e n t b i n d i n g a f f i n i t y (Kd) was e x p r e s s e d i n nM. B i n d i n g k i n e t i c s were c a l c u l a t e d u s i n g n o n - l i n e a r r e g r e s s i o n ( f o r f i g 12A) o r l e a s t s q u a r e s method f o r l i n e a r r e g r e s s i o n ( f i g 1 2 B ) . F i g u r e 1 2 A . EFFECT OF KCI ON CYTOSOLIC R1881 BINDING 120 -i m GO 100 80 H 60 40 H 20 H 0 . 2 3 - 0 . 2 9 1 O 20 40 60 80 100 S p e c i f i c Bound (fmol/mg) F i g u r e 12B. E F F E C T O F K C I O N C Y T O S O L I C H A L C R 1 8 8 1 B I N D I N G 120 -| CD OD 1D0 cr CD cu L LL S C D • T) C D 0 CO u •ri u-•ri u ai o. LO 80 H 60 H 40 H 20 H Kd:0.14-0.24 T T 1 1 20 40 60 80 S p e c i f i c Bound Cfmol/mg) 1 0 0 t r i t i a t e d - R 1 8 8 1 , w i t h o r w i t h o u t 1 0 0 - f o l d e x c e s s m o l a r r a t i o r a d i o i n e r t R1881. A p a r a l l e l s e t of i n c u b a t i o n t u b e s c o n t a i n e d 1 0 0 - f o l d e x c e s s t r i a m c i n o l o n e a c e t o n i d e . A l l m i x t u r e s were t r e a t e d w i t h 2.5% DCC i n t h e a p p r o p r i a t e b u f f e r and were c e n t r i f u g e d b e f o r e r a d i o a c t i v i t y a n a l y s i s . R i g o r o u s a n a l y s i s o f d a t a u s i n g c y t o s o l i n c u b a t e d i n t h e a b s e n c e o f t r i a m c i n o l o n e a c e t o n i d e was l i m i t e d by t h e number of f r e e l i g a n d c o n c e n t r a t i o n s u s e d t o d e t e r m i n e t h e h i g h a f f i n i t y - l o w c a p a c i t y and l o w e r a f f i n i t y - h i g h e r c a p a c i t y b i n d i n g p r o t e i n s . D e s p i t e t h i s , S c a t c h a r d a n a l y s i s f o r two s a t u r a b l e b i n d i n g s i t e s i n d i c a t e d t h a t a d d i t i o n of 10 mM p o t a s s i u m t o TED b u f f e r d i d n o t a f f e c t R1881 b i n d i n g ( f i g u r e s 12 A&B). The h i g h a f f i n i t y - l o w c a p a c i t y R1881 b i n d i n g p r o t e i n was not a l t e r e d by t h e p r e s e n c e o r a b s e n c e of 10 mM KCI i n t h e i n c u b a t i o n m i x t u r e c o n t a i n i n g 1 0 0 - f o l d e x c e s s m o l a r r a t i o t r i a m c i n o l o n e a c e t o n i d e (app. Kd= 0.14 +/- 0.24 nM; app. Bmax= 17-22 f m o l / m g ) . T h e s e s t u d i e s s u g g e s t t h a t 10 mM p o t a s s i u m does n o t i n f l u e n c e R1881 b i n d i n g i n h e p a t i c c y t o s o l i n v i t r o , and t h e r e f o r e c o u l d be o m i t t e d f r o m t h e a s s a y . In summary, on t h e b a s i s o f s a t u r a t i o n s t u d i e s , a d u l t male r a t whole l i v e r c y t o s o l c o n t a i n s two R1881 b i n d i n g s i t e s : a h i g h a f f i n i t y - l o w c a p a c i t y s i t e ( a p p. Kd= 0.22 +/-0.02 nM; app. Bmax= 5.61 +/*- 0.97 fmol/mg, n=8) and a l o w e r a f f i n i t y - h i g h e r c a p a c i t y s i t e ( app. Kd= 10.5 +/- 1.2 nM; app. Bmax= 131 +/- 29 fmol/mg, n=8). The a d d i t i o n o f 1 0 0 - f o l d m o l a r r a t i o e x c e s s t r i a m c i n o l o n e a c e t o n i d e t o a l l i n c u b a t i o n m i x t u r e s e l i m i n a t e d t h e l a t t e r s i t e , and p r o v i d e d a c o n v e n i e n t means o f a s s a y i n g t h e f o r m e r s i t e i n v i t r o . (d) D i s p l a c e m e n t o f t r i t i a t e d - R 1 8 8 1 f r o m c y t o s o l i c R1881 b i n d i n g s i t e s u s i n g v a r i o u s s t e r o i d a l and n o n - s t e r o i d a l c o m p e t i t o r s To d e t e r m i n e t h e s p e c i f i c i t y o f t h e R1881 b i n d i n g s i t e , s i n g l e p o i n t s c r e e n i n g e x p e r i m e n t s were done u s i n g c y t o s o l i n c u b a t e d w i t h o r w i t h o u t v a r y i n g m o l a r r a t i o s (50-1000 f o l d e x c e s s ) o f s t e r o i d o r n o n - s t e r o i d c o m p e t i t o r s . D i s p l a c e m e n t o f t r i t i a t e d - R 1 8 8 1 (1 nM) f r o m t h e h i g h a f f i n i t y - l o w c a p a c i t y s i t e was done by u s i n g c y t o s o l c o n t a i n i n g 1 0 0 - f o l d e x c e s s t r i a m c i n o l o n e a c e t o n i d e . S i m i l a r s t u d i e s were done by u s i n g c y t o s o l w h i c h was i n c u b a t e d w i t h t r i t i a t e d - R l 8 8 1 (8 nM) i n t h e a b s e n c e o f t r i a m c i n o l o n e a c e t o n i d e , i n o r d e r t o measure t h e l o w e r a f f i n i t y - h i g h e r c a p a c i t y component. T h e s e l i g a n d c o n c e n t r a t i o n s were c h o s e n b e c a u s e t h e y a p p r o x i m a t e d the Kd v a l u e s o f t h e r e s p e c t i v e b i n d i n g s i t e s , and t h e y p r o d u c e d d e t e c t a b l e d e g r e e s of t r i t i a t e d - R 1 8 8 1 d i s p l a c e m e n t by t h e v a r i o u s c o m p e t i t o r s f r o m t h e b i n d i n g s i t e s . D i s p l a c e m e n t of 8 nM t r i t i a t e d - R l 8 8 1 f r o m l i v e r c y t o s o l by v a r i o u s c o m p e t i t o r s i n d i c a t e d t h a t t h e l o w e r a f f i n i t y - h i g h e r c a p a c i t y b i n d i n g s i t e was n o n - s e l e c t i v e ( t a b l e 1 4 ) . F u r t h e r m o r e , o f t h e 17 c o m p e t i t o r s t e s t e d ( i n c l u d i n g p r o g e s t i n s , g l u c o c o r t i c o i d s , e s t r o g e n s , a n d r o g e n s and a n t i - a n d r o g e n s ) u s i n g m o l a r r a t i o s f r o m 50 t o 1 0 0 0 - f o l d 128 T a b l e 14 SINGLE POINT DISPLACEMENT STUDIES OF 8 nM TRITIATED-R1881 BY VARIOUS COMPETITORS IN LIVER CYTOSOLIC FRACTIONS A d u l t male r a t (280-300 g) l i v e r w h o l e c y t o s o l (1-3 mg/ml) was p r e p a r e d as d e s c r i b e d i n M e t h o d s , and was i n c u b a t e d w i t h 8 nM t n t i a t e d - R l b 8 1 w i t h o u t o r w i t h v a r y i n g e x c e s s m o l a r r a t i o s ( c o m p e t i t o r : l i g a n d ) o f c o m p e t i t o r . C y t o s o l was t r e a t e d w i t h 2.b% DCC t o s e p a r a t e bound and t r e e s t e r o i d f r a c t i o n s . Data were e x p r e s s e d as % bound r e l a t i v e t o c o n t r o l . C o n t r o l c y t o s o l was i n c u b a t e d i n t h e absence o f c o m p e t i t o r ( i e . 100%= no d i s p l a c e m e n t ) . C o m p e t i t o r s M o l a r R a t i o ( c o m p e t i t o r : l i g a n d ) 50x lOOx 500x 1000X C o n t r o l 100 100 100 100 R1881 ( m e t h y l t r i e n o l o n e ) 80 79 81 73 P r o g e s t e r o n e 96 88 73 69 T r i a m c i n o l o n e a c e t o n i d e 80 78 83 86 C o r t i s o l 77 78 78 75 E s t r a d i o l 88 82 75 71 DES 91 82 77 73 F l u t a m i d e 100 100 100 95 F l u t a m i d e h y d r o x i d e 99 100 94 95 C y p r o t e r o n e a c e t a t e 70 65 70 66 T e s t o s t e r o n e 84 78 68 65 5 a - D i h y d r o t e s t o s t e r o n e 100 96 90 82 5 3 - D i h y d r o t e s t o s t e r o n e 100 92 96 86 A n d r o s t e n e d i o n e 94 92 81 72 5 a - A n d r o s t a n - 3 a , 1 7 8 - d i o i 97 92 87 85 5 6 - A n d r o s t a n - 3 a , 1 7 8 - d i o l 100 99 100 95 5 a - A n d r o s t a n - 3 B , 1 7 8 - d i o l 100 95 97 87 5 B - A n d r o s t a n - 3 3 , 1 7 8 - d i o l 100 100 95 85 T a b l e 15 SINGLE POINT DISPLACEMENT STUDIES OF InM TRITIATED-Rl881 BY VARIOUS COMPETITORS IN LIVER CYTOSOLIC FRACTIONS A d u l t male r a t (280-300 g) l i v e r whole c y t o s o l (1-3 mg/ml) was p r e p a r e d as d e s c r i b e d i n Methods, and was i n c u b a t e d w i t h 1 nM t r i t i a t e d - R 1 8 8 1 and 100 nM t r i a m c i n o l o n e a c e t o n i d e , w i t h o u t o r w i t h v a r y i n g e x c e s s m o l a r r a t i o s ( c o m p e t i t o r : 1 i g a n d ) o f c o m p e t i t o r . C y t o s o l was t r e a t e d w i t h 2.5% DCC t o s e p a r a t e bound and f r e e s t e r o i d f r a c t i o n s . Data were e x p r e s s e d as % bound r e l a t i v e t o c o n t r o l . C o n t r o l c y t o s o l was i n c u b a t e d i n t h e a b s e n c e o f c o m p e t i t o r ( i e . 100%= no d i s p l a c e m e n t ) . C o m p e t i t o r s M o l a r R a t i o ( c o m p e t i t o r : l i g a n d ) 50x 100 500 1000 500( C o n t r o l 100 100 100 100 100 R1881 ( m e t h y l t r i e n o l o n e ) 79 80 78 81 83 P r o g e s t e r o n e 99 100 93 83 T r i a m c i n o l o n e a c e t o n i d e 100 100 99 100 C o r t i s o l 100 100 100 100 E s t r a d i o l 100 96 78 76 73 DES 100 100 98 99 F l u t a m i d e 100 100 99 98 F l u t a m i d e h y d r o x i d e 100 91 ' 81 77 77 C y p r o t e r o n e a c e t a t e 88 83 77 76 79 T e s t o s t e r o n e 84 75 76 76 5 a - D i h y d r o t e s t o s t e r o n e 81 73 73 78 5 3 - D i h y d r o t e s t o s t e r o n e 100 100 91 87 A n d r o s t e n e d i o n e 89 84 78 81 5 a - A n d r o s t a n - 3 a , 17 3 - d i o l 87 76 75 73 5 3 - A n d r o s t a n - 3 a , 1 7 3 - d i o l 98 100 91 87 80 5 a - A n d r o s t a n - 3 3,17 3 - d i o l 95 94 76 78 80 5 3 - A n d r o s t a n - 3 6 , 1 7 3 - d i o l 100 100 85 85 83 RU23908 99 100 97 97 RU2956 97 98 96 94 S p i r o n o l a c t o n e 99 94 92 92 e x c e s s , 5 b e t a - A - 3 a l p h a , 17 b e t a - d i o l , f l u t a m i d e o r f l u t a m i d e h y d r o x i d e d i s p l a c e d o n l y up t o 5-6% o f t h e t o t a l bound R1881; i n o t h e r words, 94-95% o f t o t a l bound r e m a i n e d i n t h e bound f r a c t i o n . T h i s i s i n c o n t r a s t t o t h e r e s t o f t h e c o m p e t i t o r s w h i c h y i e l d e d up t o 35% d i s p l a c e a b l e b i n d i n g ( o r 65% t o t a l bound r e m a i n i n g ) a t m o l a r r a t i o s v a r y i n g f r o m 50-1000 f o l d . D i s p l a c e m e n t o f 1 nM t r i t i a t e d - R 1 8 8 1 f r o m t h e h i g h a f f i n i t y - l o w c a p a c i t y s i t e y i e l d e d a d i f f e r e n t s p e c t r u m o f r e s u l t s ( t a b l e 1 5 ) . A t f i f t y - f o l d e x c e s s m o l a r r a t i o , t r i t i a t e d - R 1 8 8 1 was d i s p l a c e d by r a d i o i n e r t R1881, w h i l e m o l a r r a t i o s g r e a t e r t h a n o r e q u a l t o 5 0 0 - f o l d e x c e s s r a d i o i n e r t t e s t o s t e r o n e , 5 alpha-DHT, a n d r o s t e n e d i o n e , 5 a l p h a - A - 3 a l p h a , 17 b e t a - d i o l , 5 a l p h a - A - 3 b e t a , 17 b e t a - d i o l , e s t r a d i o l , c y p r o t e r o n e a c e t a t e and f l u t a m i d e h y d r o x i d e were needed t o m a x i m a l l y d i s p l a c e t h i s l i g a n d . The maximum r a n g e o f d i s p l a c e m e n t was more t h a n 20-25% o f t o t a l bound R1881 ( o r 75-80% t o t a l bound r e m a i n i n g i n t h e f r a c t i o n ) . A h i g h e r m o l a r r a t i o ( 1 0 0 0 - f o l d e x c e s s ) was r e q u i r e d f o r 5 beta-DHT and 5 b e t a - A - 3 a l p h a , 17 b e t a - d i o l . O t h e r c o m p e t i t o r s s u c h DES, f l u t a m i d e , RU23908 (a h i g h l y p o t e n t f l u t a m i d e a n a l o g u e ) , RU2956 (a R1881 a n a l o g u e w i t h a m t i - a n d r o g e n p r o p e r t i e s ) , and s p i r o n o l a c t o n e were p o o r d i s p l a c e r s o f t h e l i g a n d . Even a t t h e s e m o l a r r a t i o s , t h e l a t t e r g r o u p o f c o m p e t i t o r s i n s i g n i f i c a n t l y d i s p l a c e d o f t o t a l bound R1881 f r o m t h i s s i t e . T r i a m c i n o l o n e a c e t o n i d e and C o r t i s o l a t 1 0 0 0 - f o l d d i d n o t d i s p l a c e t h e l i g a n d f r o m t h e h i g h a f f i n i t y s i t e . On t h e b a s i s of t h e s e p r e l i m i n a r y s c r e e n i n g s t u d i e s , t h e h i g h a f f i n i t y - l o w c a p a c i t y (HALC) s i t e d e m o n s t r a t e d c o n s i d e r a b l y more s p e c i f i c i t y t h a n d i d t h e l o w e r a f f i n i t y - h i g h e r c a p a c i t y (LAHC) s i t e . F u r t h e r m o r e , f l u t a m i d e h y d r o x i d e ( t h e a c t i v e m e t a b o l i t e o f f l u t a m i d e ) and n o t f l u t a m i d e d i s p l a c e d R1881 f r o m th e HALC s i t e . More r i g o r o u s d i s p l a c e m e n t s t u d i e s a r e d e s c r i b e d l a t e r . T h e s e b i n d i n g s t u d i e s j u s t d e s c r i b e d l e n d s u p p o r t t o o u r d e c i s i o n t o f o c u s on t h e h i g h a f f i n i t y - l o w c a p a c i t y s i t e . To r e c o n f i r m t h a t DCC s u i t a b l y s e p a r a t e s bound and unbound f r a c t i o n s i n o u r p r e p a r a t i o n , two s e p a r a t i o n t e c h n i q u e s (DCC and p r o t a m i n e s u l p h a t e ) e m p l o y i n g d i f f e r e n t p r i n c i p l e s ( a d s o r p t i o n and p r e c i p i t a t i o n , r e s p e c t i v e l y ) were compared. T h e s e t e c h n i q u e s have t h e a d v a n t a g e of r a p i d i t y and e a s e of a p p l i c a t i o n , compared t o o t h e r t e c h n i q u e s e g . column f r a c t i o n a t i o n , s u c r o s e d e n s i t y g r a d i e n t , e t c . A c o n c e n t r a t i o n of 2.5% DCC was u s e d , f o l l o w i n g t h e same c o n d i t i o n s as t h e t e s t o s t e r o n e b i n d i n g s t u d i e s . However, s i n c e l o w e r 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 a r e r e q u i r e d f o r t h e R1881 b i n d i n g a s s a y compared t o t h a t o f t h e t e s t o s t e r o n e b i n d i n g a s s a y , t h e p o s s i b i l i t y of u s i n g a l o w e r DCC c o n c e n t r a t i o n (0.5%) was t e s t e d . T a b l e 16 DIFFERENT METHODS TO SEPARATE BOUND AND FREE STEROID FRACTIONS C y t o s o l (1-3 mg/ml) was p r e p a r e d f r o m a d u l t male r a t (280-300 g) whole l i v e r , and was i n c u b a t e d w i t h v a r y i n g c o n c e n t r a t i o n s of t r i t i a t e d - R l 8 8 1 (0.25-2.0 nM) and 1 0 0 - f o l d e x c e s s m o l a r r a t i o r a d i o i n e r t t r i a m c i n o l o n e a c e t o n i d e , w i t h o r w i t h o u t 1 0 0 - f o l d e x c e s s m o l a r r a t i o r a d i o i n e r t R1881, f o r 3 h o u r s a t 4 d e g r e e s C. The bound and f r e e f r a c t i o n s were s e p a r a t e d u s i n g 0.5% o r 2.5% DCC o r p r o t a m i n e s u l p h a t e , f o l l o w e d by c e n t r i f u g a t i o n . The p r o t a m i n e s u l p h a t e n o n - p r e c i p i t a t a b l e f r a c t i o n was f u r t h e r a n a l y s e d u s i n g 2.5% DCC a d s o r p t i o n . B i n d i n g k i n e t i c s were c a l c u l a t e d u s i n g S c a t c h a r d a n a l y s i s and l e a s t s q u a r e s method f o r l i n e a r r e g r e s s i o n . S e p a r a t i o n T e c h n i q u e s A p p a r e n t R1881 B i n d i n g K i n e t i c s Kd (nM) Bmax (fmol/mg) A. D e x t r a n c o a t e d c h a r c o a l 2.5% 0.23 2.94 0.5% no d e t e c t a b l e b i n d i n g * B. P r o t a m i n e s u l p h a t e f r a c t i o n p r e c i p i t a b l e 0.10 2.90 n o n - p r e c i p i t a b l e no d e t e c t a b l e b i n d i n g * * d e n o t e s no d i s p l a c e a b l e o r s a t u r a b l e b i n d i n g (e) S e p a r a t i o n of c y t o s o l i c p r o t e i n bound and f r e e s t e r o i d f r a c t i o n s Whole c y t o s o l (1-3 mg/ml) was p r e p a r e d i n t h e u s u a l manner and i n c u b a t e d w i t h v a r y i n g c o n c e n t r a t i o n s of t r i t i a t e d - R l 8 8 1 and 1 0 0 - f o l d e x c e s s m o l a r r a t i o t r i a m c i n o l o n e a c e t o n i d e , i n t h e p r e s e n c e o r a b s e n c e o f 1 0 0 - f o l d e x c e s s m o l a r r a t i o r a d i o i n e r t R1881. F o l l o w i n g i n c u b a t i o n , s e p a r a t i o n o f bound f r o m f r e e s t e r o i d s was done u s i n g t h r e e d i f f e r e n t c o n d i t i o n s : p r e c i p i t a t i o n by u s i n g 0.5% o r 2.5% DCC, o r by u s i n g p r o t a m i n e s u l p h a t e . A d e q u a t e s e p a r a t i o n o f bound v s . f r e e s t e r o i d was f o u n d u s i n g 2.5% DCC, b u t not 0.5% DCC. A n a l y s i s o f c y t o s o l t r e a t e d w i t h 2.5% DCC i n d i c a t e d a d i s p l a c e a b l e s p e c i f i c b i n d i n g component ( app. Kd= 0.23 nM; app. Bmax= 2.9 fmol/mg; t a b l e 1 6 ) . T h i s i s i n c o n t r a s t t o t h e 0.5% DCC t r e a t e d c y t o s o l w h i c h r e s u l t e d i n no d i s p l a c e a b l e b i n d i n g . The 2.5% DCC p r e c i p i t a t i o n t e c h n i q u e was r e l a t i v e l y r a p i d and r e q u i r e d a p p r o x i m a t e l y 15 min p e r b i n d i n g a s s a y ( i n c l u d i n g 10 min and 3-5 min s a m p l i n g and d e c a n t i n g t i m e ) . P r o t a m i n e s u l p h a t e p r e c i p i t a t i o n r e q u i r e d a much l o n g e r a s s a y t i m e ( a p p r o x i m a t e l y 90 m i n ) , and y i e l d e d s i m i l a r r e s u l t s t o the 2.5% DCC t e c h n i q u e , w i t h a s l i g h t l y h i g h e r a f f i n i t y ( a p p . Kd= 0.1 nM) and a s i m i l a r b i n d i n g c a p a c i t y (app. Bmax= 2.9 f m o l / m g ) . The c y t o s o l i c f r a c t i o n w h i c h was n o t p r e c i p i t a t e d by p r o t a m i n e s u l p h a t e p r o d u c e d no d i s p l a c e a b l e b i n d i n g . B o t h methods, 2.5% DCC o r p r o t a m i n e s u l p h a t e p r e c i p i t a t i o n , c o u l d be u s e d f o r t h e a n a l y s i s o f t h e h i g h - a f f i n i t y low c a p a c i t y R1881 b i n d i n g p r o t e i n ; however, f o r t h e p u r p o s e o f o u r s t u d i e s , t h e f o r m e r was c h o s e n due t o the c o n v e n i e n c e o f d e c r e a s e d t o t a l s a m p l i n g t i m e . ( f ) O p t i m i z a t i o n of t h e h e p a t i c c y t o s o l i c a n d r o g e n b i n d i n g a s s a y On t h e b a s i s o f t h e p r e v i o u s l y d e s c r i b e d p r e l i m i n a r y e x p e r i m e n t s , t h e f o l l o w i n g p r o t o c o l was a d o p t e d f o r t h e measurement o f t h e h e p a t i c c y t o s o l i c a n d r o g e n b i n d i n g p r o t e i n . C y t o s o l was p r e p a r e d f o l l o w i n g h o m o g e n i z a t i o n and d i f f e r e n t i a l c e n t r i f u g a t i o n s t e p s . The p r o t e i n c o n c e n t r a t i o n was a d j u s t e d (1-3 mg/ml), u s i n g i c e c o l d TED b u f f e r (pH 7 . 4 ) . The d i l u t e d c y t o s o l was i n c u b a t e d w i t h v a r y i n g c o n c e n t r a t i o n s o f t r i t i a t e d - R 1 8 8 1 (0.05 t o 5 nM) and 100 f o l d e x c e s s m o l a r r a t i o t r i a m c i n o l o n e a c e t o n i d e , w i t h o r w i t h o u t c o m p e t i t o r , f o r 3 h o u r s a t 4 d e g r e e s C. Bound and f r e e s t e r o i d f r a c t i o n s were s e p a r a t e d by a d d i t i o n o f 2.5% DCC, f o l l o w e d by c e n t r i f u g a t i o n . a t 1500 x g f o r 10 mins a t 4 d e g r e e s C. An a l i g u o t (0.5 ml) of s u p e r n a t a n t was d e c a n t e d and p l a c e d i n t o s c i n t i l l a t i o n v i a l s f o r r a d i o a c t i v i t y a n a l y s i s . A p p a r e n t b i n d i n g k i n e t i c s were e s t i m a t e d u s i n g t h e l e a s t s q u a r e s method f o r l i n e a r r e g r e s s i o n . 5. C h a r a c t e r i z a t i o n o f t h e h e p a t i c c y t o s o l i c h i g h a f f i n i t y - l o w c a p a c i t y (HALC) a n d r o g e n b i n d i n g p r o t e i n (a) In v i t r o e x p e r i m e n t s : ( i ) D i s p l a c e m e n t o f HALC R1881 b i n d i n g i n male r a t l i v e r whole c y t o s o l P r e l i m i n a r y s t u d i e s i n d i c a t e d t h a t r a d i o i n e r t t e s t o s t e r o n e , a n d r o s t e n e d i o n e , DHT, e s t r a d i o l and f l u t a m i d e h y d r o x i d e ( a t 5 0 0 - f o l d m o l a r r a t i o ) d i s p l a c e d s i m i l a r amounts o f t r i t i a t e d - R 1 8 8 1 as t h a t by 1 0 0 - f o l d e x c e s s m o l a r r a t i o r a d i o i n e r t R1881. In o r d e r t o g a i n a more q u a n t i t a t i v e a p p r e c i a t i o n of t h e s p e c i f i c i t y o f t h e s e c o m p e t i t o r s f o r t h e HALC R1881 b i n d i n g p r o t e i n , more r i g o r o u s i n h i b i t i o n s t u d i e s were c o n d u c t e d . F o r t h i s p u r p o s e , m o l a r r a t i o s w h i c h c a u s e d maximal and sub-maximal t r i t i a t e d - R 1 8 8 1 d i s p l a c e m e n t were u s e d . C y t o s o l was p r e p a r e d as d e s c r i b e d e a r l i e r , and i n c u b a t e d w i t h 1 nM t r i t i a t e d - R 1 8 8 1 and 100 nM t r i a m c i n o l o n e a c e t o n i d e , i n t h e p r e s e n c e o r a b s e n c e o f v a r y i n g c o n c e n t r a t i o n s (0.25 t o 1000 nM) o f c o m p e t i t o r , f o r 3 h r s a t 4 d e g r e e s C. The i n h i b i t i o n c u r v e (% SB r e m a i n i n g ) was p l o t t e d as a f u n c t i o n of f r e e (F) c o m p e t i t o r a d d e d . The IC50 v a l u e o f e a c h c o m p e t i t o r was c a l c u l a t e d by u s i n g a l o g i t (%SB r e m a i n i n g ) v s . l o g (F) p l o t , and by u s i n g t h e l e a s t - s g u a r e s method f o r l i n e a r r e g r e s s i o n . The K i ( i n h i b i t o r e q u i l i b r i u m c o n s t a n t ) was c a l c u l a t e d u s i n g t h e e q u a t i o n : app. K i = (IC50) / (1 + L/app. K d ) , a c c o r d i n g t o W e i l a n d and M o l i n o f f ( 1 9 8 1 ) , where IC50 F i g u r e s 13 (A&B) S P E C I F I C I T Y STUDIES FOR ADULT MALE RAT LIVER CYTOSOLIC HIGH AFFINITY-LOW CAPACITY R1881 BINDING PROTEIN A d u l t male r a t (280-300 g) whole l i v e r c y t o s o l (1-3 mg/ml) was i n c u b a t e d w i t h 1 nM t r i t i a t e d - R 1 8 8 1 and 1 0 0 - f o l d e x c e s s m o l a r r a t i o r a d i o i n e r t t r i a m c i n o l o n e a c e t o n i d e , i n t h e p r e s e n c e o r a b s e n c e o f 1 0 0 - f o l d e x c e s s m o l a r r a t i o r a d i o i n e r t R1881, f o r 3 h o u r s a t 4 d e g r e e s C. P e r c e n t a g e (%) s p e c i f i c b i n d i n g ( o r d i n a t e ) was c a l c u l a t e d as t h e f r a c t i o n o f R1881 d i s p l a c e d r e l a t i v e t o t o t a l R1881 bound, m u l t i p l i e d by 100. T h i s v a l u e was p l o t t e d as a f u n c t i o n o f t h e l o g a r i t h m o f t h e f r e e c o m p e t i t o r c o n c e n t r a t i o n added t o t h e i n c u b a t i o n m i x t u r e ( a b s c i s s a ) . F i g 13A shows t r i t i a t e d - R 1 8 8 1 i n h i b i t i o n c u r v e s f o r r a d i o i n e r t R1881 ( m e t h y l t r i e n o l o n e ) , t e s t o s t e r o n e ( T ) , a n d r o s t e n e d i o n e ( A - d i o n e ) and 5 a - d i h y d r o t e s t o s t e r o n e (DHT). In f i g 13B i s shown i n h i b i t i o n c u r v e s f o r d i s p l a c e m e n t o f t r i t i a t e d - R 1 8 8 1 by r a d i o i n e r t c y p r o t e r o n e a c e t a t e ( C A ) , e s t r a d i o l (E) and f l u t a m i d e h y d r o x i d e ( F l u t - O H ) ; R1881 i n h i b i t i o n c u r v e was r e - p l o t t e d f o r r e f e r e n c e . 137 F i g u r e 13A I N H I B I T I O N CURVE FQR C Y T O S O L I C HALC R1B81 BINDING C CO R1881 CN-0 j QQ _ C •) Testosterone <N~) ( A ) Androstenedione CN") C V) Dihydrotestosterone CN") • \ • 8 0 -CO C •i-i V c 6 0 -m u •rt t+-0 4 0 0) 0 _ tn 2 0 -dione — i — i II i u i | 1—rr - 1 • L o g ( C o m p e t i t o r a d d e d ; n M ) R1881 if—i " rrrfp" r e n^ frrrn 1 2 3 138 F i g u r e 13B I N H I B I T I O N C U R V E F O R C Y T O S O L I C H A L C R I B B 1 B I N D I N G Log ( C o m p e t i t o r added;nM) T a b l e 17 INHIBITION STUDY OF LIVER CYTOSOLIC R1881 BINDING BY VARIOUS COMPETITORS IN ADULT MALE RAT A d u l t male r a t (280-300 g) l i v e r w h o l e c y t o s o l (1-3 mg/ml) was p r e p a r e d as d e s c r i b e d i n Methods s e c t i o n , and was i n c u b a t e d w i t h 1 nM t r i t i a t e d - R 1 8 8 1 and 100 nM t r i a m c i n o l o n e a c e t o n i d e , i n t h e p r e s e n c e o r absence o f d i f f e r e n t m o l a r r a t i o s o f r a d i o i n e r t c o m p e t i t o r s , f o r 3 h o u r s a t 4 degree s C . C y t o s o l was t h e n t r e a t e d w i t h 2.5% DCC and r a d i o a c t i v i t y was a n a l y s e d . IC50 v a l u e s were d e t e r m i n e d u s i n g a l o g i t - l o g p l o t and t h e l e a s t s q u a r e s method o f l i n e a r r e g r e s s i o n . U s i n g t h e same c y t o s o l , a s a t u r a t i o n s t u d y was done t o e s t i m a t e t h e a p p . Kd o f t h e R1881 b i n d i n g s i t e . The e q u i l i b r i u m i n h i b i t i o n c o n s t a n t , a p p . K i was c a l c u l a t e d u s i n g t h e e q u a t i o n d e s c r i b e d i n t e x t . Da ta were e x p r e s s e d i n nM. I n some c a s e s , e x p e r i m e n t s were r e p e a t e d and t h e d a t a were e x p r e s s e d as average +/- S . E . M . Numbers i n b r a c k e t s d e n o t e numbers o f a n i m a l s t e s t e d . C o m p e t i t o r s R1881 ( m e t h y l t r i e n o l o n e ) T e s t o s t e r o n e A n d r o s t e n e d i o n e 5 o i - D i h y d r o t e s t o s t e r o n e E s t r a d i o l F l u t a m i d e h y d r o x i d e C y p r o t e r o n e a c e t a t e A p p a r e n t K i (nM) 0 .06 +/- 0 .02 (n=3) 0 .12 0 .12 0 .07 0 .63 7 7 . 0 +/- 6 4 . 0 (n=3) 0 .38 140 f o r e a c h c o m p e t i t o r i s e s t i m a t e d f r o m t h e i n h i b i t i o n c u r v e , L i s t h e f r e e l i g a n d c o n c e n t r a t i o n , and t h e app. Kd i s t h e a p p a r e n t e q u i l i b r i u m d i s s o c i a t i o n c o n s t a n t . The app. Kd v a l u e was e s t i m a t e d s e p a r a t e l y by d o i n g a s a t u r a t i o n s t u d y f o r e a c h c y t o s o l p r e p a r a t i o n . A n a l y s i s o f t h e d i s p l a c e m e n t c u r v e s ( f i g u r e 13 A&B) and a p p a r e n t K i v a l u e s ( t a b l e 17) i n d i c a t e d t h e f o l l o w i n g d i s p l a c e m e n t p r o f i l e o f c o m p e t i t o r s ( t h e app. K i , i n nM): the a f f i n i t y of R1881 (0.06 +/- 0.02) t o t h e HALC R1881 b i n d i n g p r o t e i n i s s i m i l a r t o a n d r o s t e n e d i o n e , t e s t o s t e r o n e and 5 alpha-DHT (0.07 t o 0.12). T h e s e c o m p e t i t o r s have a h i g h e r a f f i n i t y t o t h i s p r o t e i n t h a n do c y p r o t e r o n e a c e t a t e ( 0 . 3 8 ) , e s t r a d i o l (0.63) o r f l u t a m i d e h y d r o x i d e (77 +/- 6 6 ) . In c o n t r a s t t o t h e r e s t o f t h e c o m p e t i t o r s t e s t e d , f l u t a m i d e h y d r o x i d e was r e l a t i v e l y i n e f f e c t i v e i n d i s p l a c i n g R1881 f r o m t h e HALC b i n d i n g s i t e , and p r o d u c e d h i g h l y v a r i a b l e r e s u l t s . ( i i ) R1881 s a t u r a t i o n s t u d y u s i n g d i f f e r e n t c o m p e t i t o r s The p r e v i o u s l y d e s c r i b e d i n h i b i t i o n s t u d i e s i n d i c a t e d r e l a t i v e s p e c i f i c i t y o f t h e c o m p e t i t o r s t e s t e d t o t h e R1881 b i n d i n g s i t e . To d e t e r m i n e i f t h e s e c o m p e t i t o r s were b i n d i n g t o t h e same s i t e , t h e f o l l o w i n g e x p e r i m e n t s were c o n d u c t e d . C y t o s o l (1-3 mg/ml) was p r e p a r e d as u s u a l , and i n c u b a t e d w i t h v a r y i n g c o n c e n t r a t i o n s o f t r i t i a t e d - R 1 8 8 1 and 1 0 0 - f o l d e x c e s s t r i a m c i n o l o n e a c e t o n i d e , i n t h e p r e s e n c e o r a b s e n c e o f 5 0 0 - f o l d e x c e s s m o l a r r a t i o c o m p e t i t o r , e x c e p t T a b l e 18 BINDING KINETICS OF LIVER CYTOSOLIC R1881 BINDING PROTEIN BY VARIOUS COMPETITORS A d u l t male r a t (280-230 g) l i v e r whole c y t o s o l (1-3 mg/ml) was p r e p a r e d as d e s c r i b e d i n Methods s e c t i o n , and was i n c u b a t e d w i t h t r i t i a t e d - R 1 8 8 1 (0.05-2.0 nM) and 1 0 0 - f o l d e x c e s s m o l a r r a t i o t r i a m c i n o l o n e a c e t o n i d e , w i t h o u t o r w i t h 5 0 0 - f o l d e x c e s s m o l a r r a t i o c o m p e t i t o r , f o f 3 h o u r s a t 4 d e g r e e s C. I n c o n t r a s t t o t h e o t h e r c o m p e t i t o r s , 1 0 0 - l o l d R1881 and 100- o r ' 5 0 0 - f o l d e x c e s s m o l a r r a t i o m i b o l e r o n e were u s e d . C y t o s o l was t r e a t e d w i t h 2.5% DCC; r a d i o a c t i v i t y was a n a l y s e d . B i n d i n g k i n e t i c s were c a l c u l a t e d u s i n g S c a t c h a r d a n a l y s i s , and t h e l e a s t s q u a r e s method t o r l i n e a r r e g r e s s i o n . D a t a were e x p r e s s e d as t h e a v e r a g e +/- S.E.M. The numbers i n b r a c k e t s d e n o t e numbers o f a n i m a l s u s e d . A p p a r e n t B i n d i n g K i n e t i c s C o m p e t i t o r s u s e d Kd (nM) Bmax (fmol/mg) R l b 8 1 ( m e t h y l t r i e n o l o n e ) 0 .26 +/- 0 .08 5 .42 +/- 0 .65 (3) M i b o l e r o n e 0 .12 +/- 0 .04 4 .45 +/- 1.77 (3) ns T e s t o s t e r o n e 0 .29 3 .8 5 a - D i h y d r o t e s t o s t e r o n e 0 .32 3 .41 A n d r o s t e n e d i o n e 0 .17 2 .9 E s t r a d i o l 0 .82 +/- 0 .41 ; 8 .19 +/- 3 .61 (3) ns C y p r o t e r o n e a c e t a t e 0 .35 2 .00 F l u t a m i d e h y d r o x i d e 3 h r s . i n c u b a t i o n 18 h r s . i n c u b a t i o n no d e t e c t a b l e s p e c i f i c b i n d i n g * ns d e n o t e s no s i g n i f i c a n t d i f f e r e n c e compared t o R1881 as c o m p e t i t o r (p> 0 .05). * d e n o t e s no d e t e c t a b l e d i s p l a c e a b l e o r s a t u r a b l e b i n d i n g f o r r a d i o i n e r t R1881 o r m i b o l e r o n e , i n w h i c h a 1 0 0 - f o l d m o l a r r a t i o was u s e d . M i b o l e r o n e i s a p o t e n t s y n t h e t i c a n d r o g e n w h i c h i s m i n i m a l l y m e t a b o l i z e d i n p r o s t a t e ( L i a o e t al. 1 9 7 3 ) . A n a l y s i s o f S c a t c h a r d - t y p e p l o t s ( t a b l e 18) i n d i c a t e d t h a t R1881, m i b o l e r o n e , t e s t o s t e r o n e , 5 alpha-DHT, a n d r o s t e n e d i o n e , c y p r o t e r o n e a c e t a t e and e s t r a d i o l had s i m i l a r b i n d i n g a f f i n i t i e s ( a pp. Kd= 0.17 t o 0.35) and c a p a c i t i e s (app. Bmax= 2.9 t o 8.2 fmol/mg), s u g g e s t i n g t h a t t h e l a t t e r compounds were b i n d i n g t o t h e same b i n d i n g s i t e . D e s p i t e h a v i n g d i s p l a c e a b l e t r i t i a t e d - R l 8 8 1 b i n d i n g (6-20% o f t o t a l b o u n d ) , f l u t a m i d e h y d r o x i d e ( a t 500 f o l d e x c e s s ) w h i c h was i n c u b a t e d f o r 3 o r 18 h r s f a i l e d t o d e m o n s t r a t e s a t u r a b l e b i n d i n g ( i e . no S c a t c h a r d p l o t ) . ( i i i ) T r i t i a t e d - M i b o l e r o n e b i n d i n g i n a d u l t male r a t c y t o s o l The p r e v i o u s s t u d y i n d i c a t e d t h a t r a d i o i n e r t m i b o l e r o n e d i s p l a c e d t r i t i a t e d - R 1 8 8 1 f r o m t h e HALC R1881 b i n d i n g s i t e , and i t was b i n d i n g t o t h e HALC a n d r o g e n b i n d i n g s i t e . U n f r a c t i o n a t e d c y t o s o l (1-3 mg/ml) was p r e p a r e d as u s u a l , and was i n c u b a t e d w i t h t r i t i a t e d - m i b o l e r o n e ( f r o m 0.05 t o 10 nM) i n t h e p r e s e n c e o r a b s e n c e of 5 0 0 - f o l d e x c e s s m o l a r r a t i o r a d i o i n e r t m i b o l e r o n e . A s e p a r a t e s e t of t u b e s c o n t a i n e d 1 0 0 - f o l d e x c e s s t r i a m c i n o l o n e a c e t o n i d e . F o l l o w i n g i n c u b a t i o n , c y t o s o l was t r e a t e d w i t h 2.5% DCC, b e f o r e r a d i o a c t i v i t y a n a l y s i s . S c a t c h a r d a n a l y s i s o f c y t o s o l ( f i g u r e 14) w h i c h was i n c u b a t e d i n t h e a b s e n c e o f F i g u r e 14 SCATCHARD PLOT FOR ADULT MALE RAT HEPATIC CYTOSOLIC TRITIATED-MIBOLERONE BINDING PROTEIN A d u l t male r a t (280-300 g) l i v e r w hole c y t o s o l (1-3 mg/ml) was i n c u b a t e d w i t h v a r y i n g c o n c e n t r a t i o n s o f t r i t i a t e d - m i b o l e r o n e (0.05-2.0 nM), i n t h e a b s e n c e o r p r e s e n c e o f 5 0 0 - f o l d e x c e s s m o l a r r a t i o r a d i o i n e r t m i b o l e r o n e . I n a d d i t i o n t o t h e above, c y t o s o l was i n c u b a t e d i n t h e a b s e n c e o f ( c i r c l e s ) o r p r e s e n c e o f ( s q u a r e s ) 1 0 0 - f o l d e x c e s s m o l a r r a t i o r a d i o i n e r t t r i a m c i n o l o n e a c e t o n i d e . D a t a were a n a l y s e d u s i n g l e a s t s q u a r e s f o r l i n e a r r e g r e s s i o n . A p p a r e n t Kd was e x p r e s s e d i n nM. 144 F i g u r e 14. RAT LIVER MIBOLERONE BINDING • _ | 1 1 1 1 1 1 O 2 4 6 B 10 12 S p e c i f i c B i n d i n g (fmol/mg) t r i a m c i n o l o n e a c e t o n i d e i n d i c a t e d a d i s p l a c e a b l e and s a t u r a b l e m i b o l e r o n e b i n d i n g s i t e , ( app. Kd= 1.9 nM; app. Bmax= 14.2 fmol/mg; l a s t 5 p o i n t s u s e d ) . M u l t i p l e b i n d i n g s i t e a n a l y s i s f a i l e d t o g i v e r e l i a b l e r e s u l t s . A n o t h e r s i t e may be p r e s e n t , b u t a c c u r a t e e s t i m a t i o n s would r e g u i r e more p o i n t s a t l o w e r l i g a n d c o n c e n t r a t i o n s . On t h e o t h e r hand, a d d i t i o n of 1 0 0 - f o l d e x c e s s m o l a r r a t i o t r i a m c i n o l o n e a c e t o n i d e t o t h e m i x t u r e y i e l d e d a s i n g l e HALC i s o t h e r m (app. Kd= 0.54 nM; app. Bmax= 3.5 fmo l / m g ) , c o n s i s t e n t w i t h t h e b i n d i n g k i n e t i c s o f t h e HALC R1881 b i n d i n g p r o t e i n . The r e s u l t s o f t h i s s t u d y s u p p o r t t h e e a r l i e r c o n c l u s i o n t h a t R1881 and m i b o l e r o n e a r e b i n d i n g t o t h e same s i t e , p r e s u m a b l y t h e HALC a n d r o g e n b i n d i n g p r o t e i n . (b) D i f f e r e n t p h y s i o l o g i c a l and p h a r m a c o l o g i c a l a n i m a l models In o u r p r e v i o u s enzyme s t u d i e s , we have shown t h a t t e s t o s t e r o n e - r e s p o n s i v e h e p a t i c m i c r o s o m a l d r u g and s t e r o i d m e t a b o l i z i n g enzymes a r e s e n s i t i v e t o t h e a n i m a l ' s hormonal s t a t u s . I f t h e h e p a t i c c y t o s o l i c a n d r o g e n r e c e p t o r 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 o f t h e s e a n d r o g e n d e p e n d e n t enzyme a c t i v i t i e s , t h e n i t s h o u l d s h a r e t h e same s e n s i t i v i t y t o t h e h o r m o n a l e n v i r o n m e n t as t h e h e p a t i c m i c r o s o m a l enzymes. I n o r d e r t o t e s t t h i s h y p o t h e s i s , t h e f o l l o w i n g e x p e r i m e n t s were done. F i g u r e 15. SEX DIFFERENCES IN ADULT RAT HEPATIC CYTOSOLIC R1881 BINDING PROTEIN L i v e r whole c y t o s o l (1-3 mg/ml) were p r e p a r e d f r o m age-matched a d u l t male and f e m a l e r a t s as d e s c r i b e d i n Methods s e c t i o n , and were i n c u b a t e d w i t h v a r y i n g c o n c e n t r a t i o n s o f t r i t i a t e d - R l 8 8 1 , i n t h e a b s e n c e o r p r e s e n c e of 1 0 0 - f o l d e x c e s s m o l a r r a t i o r a d i o i n e r t R1881. C i r c l e s d e n o t e a d u l t male and o p e n - u p r i g h t t r i a n g l e s d e n o t e a d u l t f e m a l e r a t l i v e r c y t o s o l i n c u b a t e d i n t h e a b s e n c e of t r i a m c i n o l o n e a c e t o n i d e ; s q u a r e s d e n o t e a d u l t male and t h e f i l l e d - i n v e r t e d t r i a n g l e d e n o t e a d u l t f e m a l e r a t l i v e r c y t o s o l i n c u b a t e d i n t h e p r e s e n c e of 1 0 0 - f o l d e x c e s s m o l a r r a t i o r a d i o i n e r t t r i a m c i n o l o n e a c e t o n i d e . Note : t h e r e was no d i s p l a c e a b l e R1881 b i n d i n g d e t e c t e d i n a d u l t f e m a l e r a t l i v e r c y t o s o l w h i c h was i n c u b a t e d i n t h e p r e s e n c e o f t r i a m c i n o l o n e a c e t o n i d e . 147 F i g u r e 15. SEX DIFFERENCES IN RAT LIVER CYTOSOLIC R 1 B B 1 BINDING 50 -i • 20 40 60 60 100 S p e c i f i c B i n d i n g (fmol/mg) T a b l e 19 AGE DIFFERENCES BETWEEN MALE AND FEMALE RAT LIVER HIGH AFFINTIY-LOW CAPACITY R1B81 BINDING PROTEIN C y t o s o l (1-3 mg/ml) was p r e p a r e d f rom a d u l t male (70 d a y s ; 280-300 g) and immature male and t e m a l e (24 days o l d ; 8 day p o s t - w e a n i n g ; 50-60 g) r a t l i v e r s as d e s c r i b e d i n Methods s e c t i o n , and was i n c u b a t e d w i t h t r i t i a t e d - R 1 8 8 1 ( 0 . 0 5 - 2 . 0 nM) and 1 0 0 - f o l d e x c e s s m o l a r r a t i o t r i a m c i n o l o n e a c e t o n i d e w i t h o u t o r w i t h 1 0 0 - l o l d e x c e s s m o l a r r a t i o R1881 , t o r 3 h o u r s a t 4 d e g r e e s C . C y t o s o l was t r e a t e d w i t h 2.5% DCC; r a d i o a c t i v i t y was a n a l y s e d . B i n d i n g k i n e t i c s were c a l c u l a t e d u s i n g S c a t c h a r d a n a l y s i s and t h e l e a s t s q u a r e s method t o r l i n e a r r e g r e s s i o n . Data were e x p r e s s e d as t h e a v e r a g e +/- S . E . M . The numbers i n b r a c k e t s denote numbers o f a n i m a l s u s e d . A p p a r e n t B i n d i n g K i n e t i c s A n i m a l model used Kd (nM) Bmax (fmol/mg) A d u l t male 0 .27 +/- 0 .10 8 .00 +/- 2 .28 (3) (3) Immature male 0 .96 +/- 0 .55 ns 1.96 +/- 1.07 (3) (4) Immature t e m a l e no d e t e c t a b l e S c a t c h a r d * * (4) * : s i g n i f i c a n t d i f f e r e n c e compared t o a d u l t male (p< 0 .05) ns : n o t s i g n i t i c a n t l y d i f f e r e n t compared t o a d u l t male (p>0.05) * * : no d e t e c t a b l e d i s p l a c e a b l e and s a t u r a b l e b i n d i n g ( i ) Age and sex d i f f e r e n c e s i n R1881 b i n d i n g i n male and f e m a l e r a t l i v e r c y t o s o l H e p a t i c c y t o s o l i c R1881 b i n d i n g p r o t e i n was measured i n th e a d u l t (70 days o l d ) o r immature (24 days o l d ; 8 d a y s p o s t w eaning) male and f e m a l e r a t s . B o t h a d u l t male and f e m a l e r a t l i v e r c y t o s o l d e m o n s t r a t e d d i s p l a c e a b l e R1881 b i n d i n g i n t h e a b s e n c e of t r i a m c i n o l o n e a c e t o n i d e ; n o t a b l y , l i v e r c y t o s o l t a k e n f r o m t h e a d u l t f e m a l e y i e l d e d o n l y a s i n g l e b i n d i n g i s o t h e r m (app. Kd= 13.9 nM; app. Bmax= 167 fmol/mg; f i g u r e 1 5 ) . When t r i a m c i n o l o n e a c e t o n i d e was added t o t h e c y t o s o l , i t became c l e a r t h a t an HALC component w h i c h was p r e s e n t i n t h e a d u l t male ( c o n s i s t e n t w i t h o u r p r e v i o u s s t u d i e s ) was n o t p r e s e n t i n t h e a d u l t and immature f e m a l e r a t l i v e r c y t o s o l ( t a b l e 1 9 ) . L i v e r c y t o s o l t a k e n f r o m t h e immature male r a t had l o w e r o r no d e t e c t a b l e HALC R1881 b i n d i n g compared t o a d u l t male r a t s . Of t h e 4 immature male r a t s t e s t e d , t h r e e had l o w e r b u t d e t e c t a b l e HALC b i n d i n g compared t o t h e a d u l t male; w h i l e one had no d e t e c t a b l e d i s p l a c e a b l e and s a t u r a b l e R1881 b i n d i n g ( i e . no S c a t c h a r d p l o t ) . A l t h o u g h t h e HALC component i n t h e immature male r a t s had a s i m i l a r b i n d i n g a f f i n i t y ( app. Kd= 0.96 +/- 0.55 nM), i t had a s i g n i f i c a n t l y l o w e r c a p a c i t y ( a p p. Bmax= 1.96 +/- 1.07 fmol/mg) compared t o t h e a d u l t m a l e . T h e s e r e s u l t s i n d i c a t e t h a t t h e HALC R1881 b i n d i n g p r o t e i n s h a r e some c h a r a c t e r i s t i c s s i m i l a r t o t h e h e p a t i c d r u g and s t e r o i d m e t a b o l i z i n g enzymes i n t h e r a t , i e . sex and age d e p e n d e n c y . O t h e r e x p e r i m e n t s were c o n d u c t e d t o f u r t h e r c h a r a c t e r i z e t h e T a b l e 20 EFFECT OF CASTRATION AND TESTOSTERONE REPLACEMENT ON LIVER CYTOSOLIC HIGH AFFINITY-LOW CAPACITY R1881 BINDING PROTEIN The e f f e c t o f c a s t r a t i o n and t e s t o s t e r o n e e n a n t h a t e r e p l a c e m e n t (5 m i c r o m o l e s / k g s c . , 10 d a y s ) on a d u l t male and f e m a l e r a t h i g h a f f i n i t y - l o w c a p a c i t y R1881 b i n d i n g component was d e t e r m i n e d u s i n g c y t o s o l (1-3 mg/ml) w h i c h was p r e p a r e d f r o m i n t a c t and 18 h r s t o 10 day g o n a d e c t o m i z e d a d u l t r a t s ( m a l e s , 280-300 g; f e m a l e s , 180-200 g) as d e s c r i b e d i n Methods s e c t i o n . E x p e r i m e n t was done 24 h o u r s a f t e r l a s t i n j e c t i o n . C y t o s o l was i n c u b a t e d w i t h t r i t i a t e d - R l 8 8 1 (0.05-2.0 nM) and 1 0 0 - f o l d e x c e s s m o l a r r a t i o t r i a m c i n o l o n e a c e t o n i d e , w i t h o u t o r w i t h 1 0 0 - f o l d e x c e s s m o l a r r a t i o R1881, f o r 3 h o u r s a t 4 d e g r e e s C. C y t o s o l was t r e a t e d w i t h 2.5% DCC; r a d i o a c t i v i t y was a n a l y s e d . B i n d i n g k i n e t i c s were c a l c u l a t e d u s i n g S c a t c h a r d a n a l y s i s and t h e l e a s t s q u a r e s method f o r l i n e a r r e g r e s s i o n . Data were e x p r e s s e d as t h e a v e r a g e +/- S.E.M. The numbers i n b r a c k e t s d e n o t e numbers o f a n i m a l s u s e d . A p p a r e n t B i n d i n g K i n e t i c s A n i m a l model u s e d Kd (nM) Bmax (fmol/mg) I n t a c t a d u l t male 0.17 +/- 0.03 7.50 +/- 1.11 (4) C a s t r a t e d (Gx) a d u l t male 18 h o u r s 4 days 10 days T e s t o s t e r o n e e n a n t h a t e -t r e a t e d (10 da y s ) Gx a d u l t male I n t a c t a d u l t f e m a l e 10 d a y - O v a r i e c t o m i z e d (Ovx) a d u l t f e m a l e T e s t o s t e r o n e e n a n t h a t e -t r e a t e d 10 d a y s -Ovx a d u l t f e m a l e 0. 24 +/- 0. 07 0. 18 +/- 0. 04 0. 25 +/- 0. 06 0. 16 +/- 0. 03 no d e t e c t a b l e no d e t e c t a b l e no d e t e c t a b l e 5.86 +/- 0.88 (4) 7.75 +/- 1.62 (4) 4.76 +/- 1.77 (4) 4.27 +/- 0.76 (4) d i s p l a c e m e n t (4) * d i s p l a c e m e n t (4) * d i s p l a c e m e n t (4) * * : no d e t e c t a b l e d i s p l a c e a b l e and s a t u r a b l e b i n d i n g HALC R1881 b i n d i n g component i n v i v o . ( i i ) E f f e c t of gonadectomy on a d u l t male and f e m a l e r a t l i v e r c y t o s o l HALC R1881 b i n d i n g p r o t e i n B e c a u s e t h e R1881 b i n d i n g p r o t e i n had shown s p e c i f i c i t y m a i n l y f o r a n d r o g e n s and was f o u n d o n l y i n m a l e s , t h e e f f e c t o f gonadectomy was o f i n t e r e s t . T h i s was measured u s i n g l i v e r c y t o s o l t a k e n f r o m 18 h o u r s , 4 o r 10 day c a s t r a t e d a d u l t male r a t s . Some 10 d a y - g o n a d e c t o m i z e d a d u l t male and f e m a l e r a t s were t r e a t e d w i t h t e s t o s t e r o n e e n a n t h a t e (5 m i c r o m o l e s / k g s c . , 10 d a y s ) . C a s t r a t i o n f o r 18 h o u r s , 4 o r 10 days had no s i g n i f i c a n t e f f e c t on t h e h e p a t i c c y t o s o l i c HALC R1881 b i n d i n g p r o t e i n i n a d u l t m a l e , compared t o c o n t r o l s ( t a b l e 2 0 ) . C o n s i s t e n t w i t h o u r e a r l i e r r e s u l t s , t h i s b i n d i n g p r o t e i n was a b s e n t i n t h e i n t a c t a d u l t f e m a l e r a t . F u r t h e r m o r e , t e s t o s t e r o n e e n a n t h a t e r e p l a c e m e n t f o r 10 d a y s p r o d u c e d no s i g n i f i c a n t e f f e c t s on HALC R1881 b i n d i n g compared t o t h e c o n t r o l s i n e i t h e r s e x . U n l i k e t h e h e p a t i c m i c r o s o m a l d r u g and s t e r o i d m e t a b o l i z i n g enzymes, t h e s e r e s u l t s s u g g e s t t h a t , u n d e r t h e s e e x p e r i m e n t a l c o n d i t i o n s , g o n a d a l f a c t o r s (endogenous t e s t o s t e r o n e o r e s t r a d i o l ) o r exogenous ( t e s t o s t e r o n e ) s t e r o i d s do n o t seem t o i n f l u e n c e t h e p r e s e n c e ( i n t h e male) o r t h e a b s e n c e ( i n t h e f e m a l e ) o f the l i v e r c y t o s o l i c HALC R1881 b i n d i n g p r o t e i n i n t h e a d u l t W i s t a r r a t . 152 T a b l e 21 EFFECT OF HYPOPHYSECTOMY AND TESTOSTERONE REPLACEMENT ON LIVER CYTOSOLIC HIGH AFFINITY-LOW CAPACITY The e l f e e t o f hyp o p h y s e c t o m y and t e s t o s t e r o n e e n a n t h a t e (5 m i c r o m o l e s / k g s c . , 10 days) a d m i n i s t r a t i o n on a d u l t male r a t l i v e r w h o l e c y t o s o l i c h i g h a f f i n i t y - l o w c a p a c i t y R1881 b i n d i n g p r o t e i n was d e t e r m i n e d u s i n g c y t o s o l (1-3 mg/ml) w h i c h was p r e p a r e d f r o m i n t a c t , 10 and 17-days h y p o p h y s e c t o m i z e d (Hypox) a d u l t male r a t s . Some o f t h e 17 day Hypox a n i m a l s were a l s o i n j e c t e d w i t h t e s t o s t e r o n e e n a n t h a t e (5 m i c r o m o l e s / k g s c . ) f o r 10 d a y s . E x p e r i m e n t s were done 24 h o u r s a l t e r t h e l a s t i n j e c t i o n . C y t o s o l was p r e p a r e d as d e s c r i b e d i n Methods s e c t i o n , and was i n c u b a t e d w i t h t r i t i a t e d - R 1 8 8 1 (0.05-2.0 nM) and 1 0 0 - t o l d e x c e s s m o l a r r a t i o r a d i o i n e r t t r i a m c i n o l o n e a c e t o n i d e , w i t h o u t o r w i t h 1 0 0 - t o l d e x c e s s m o l a r r a t i o r a d i o i n e r t R1881, t o r 3 h o u r s a t 4 d e g r e e s C. C y t o s o l was t r e a t e d w i t h 2.5% DCC; r a d i o a c t i v i t y was a n a l y s e d . B i n d i n g k i n e t i c s were c a l c u l a t e d u s i n g S c a t c h a r d a n a l y s i s and t h e l e a s t s q u a r e s method t o r l i n e a r r e g r e s s i o n . D a t a were e x p r e s s e d as t h e a v e r a g e +/- S.E.M. The numbers i n b r a c k e t s d e n o t e numbers o f a n i m a l s u s e d . R1881 BINDING PROTEIN A p p a r e n t B i n d i n g K i n e t i c s A n i m a l model u s e d Kd (nM) Bmax (fmol/mg) I n t a c t a d u l t male 0.28 +/- 0.07 4.50 +/- 0.07 (4) Hypox (10-17 days) a d u l t male 0.46 +/- 0.02 5.64 +/- 0.93 (6) T e s t o s t e r o n e e n a n a t h a t e - 0.18 +/- 0.03 t r e a t e d Hypox a d u l t male 5.84 +/- 0.35 (9) ( i i i ) E f f e c t o f hypophysectomy and t e s t o s t e r o n e e n a n t h a t e a d m i n i s t r a t i o n on a d u l t male r a t l i v e r c y t o s o l HALC R1881 b i n d i n g p r o t e i n I n a d d i t i o n t o d e l e t i n g p i t u i t a r y hormones and serum t e s t o s t e r o n e l e v e l s , hypophysectomy a l s o d e c r e a s e d sex d i f f e r e n c e s i n h e p a t i c m i c r o s o m a l enzyme m e t a b o l i s m i n t h e r a t , an e f f e c t w h i c h was not r e v e r s e d by t e s t o s t e r o n e ( B e l l w a r d e t a l . 1982) o r DHT (Kramer e t a l . 1979) r e p l a c e m e n t . To t e s t t h e s e n s i t i v i t y o f t h e HALC b i n d i n g p r o t e i n t o r e m o v a l o f c i r c u l a t i n g p i t u i t a r y hormones and r e d u c t i o n i n serum t e s t o s t e r o n e , t h e R1881 b i n d i n g p r o t e i n was measured i n t h e h y p o p h y s e c t o m i z e d male r a t i n j e c t e d w i t h t e s t o s t e r o n e e n a n t h a t e (5 m i c r o m o l e s / k g s c . , 10 d a y s ) . H i g h a f f i n i t y - l o w c a p a c i t y R1881 b i n d i n g was measured u s i n g l i v e r c y t o s o l t a k e n f r o m a d u l t male c o n t r o l o r h y p o p h y s e c t o m i z e d m a l e s t r e a t e d w i t h ( f o r 10 da y s ) o r w i t h o u t ( f o r 10 o r 17 d a y s ) t e s t o s t e r o n e e n a n t h a t e (5 m i c r o m o l e s / k g s c . , 10 d a y s ) . P o o l e d r e s u l t s o f two s u c h e x p e r i m e n t s a r e summarized i n t a b l e 21. Hypophysectomy f o r 10 o r 17 days had no s i g n i f i c a n t e f f e c t on HALC R1881 b i n d i n g k i n e t i c s i n a d u l t male l i v e r c y t o s o l , compared t o t h e i n t a c t s h a m - o p e r a t e d c o n t r o l a d u l t male r a t . F u r t h e r m o r e , t e s t o s t e r o n e e n a n t h a t e a d m i n i s t r a t i o n t o t h e h y p o p h y s e c t o m i z e d a n i m a l had no s i g n i f i c a n t e f f e c t on t h e HALC R1881 b i n d i n g s i t e compared t o t h e c o r n o i l v e h i c l e t r e a t e d h y p o p h y s e c t o m i z e d c o n t r o l . T h e s e r e s u l t s i n d i c a t e t h a t t h e HALC R1881 b i n d i n g p r o t e i n i s n o t s e n s i t i v e t o r e m o v a l o f p i t u i t a r y hormones, and c o n f i r m t h e e a r l i e r r e s u l t s t h a t t h e l a t t e r i s n o t d e p e n d e n t on serum t e s t o s t e r o n e l e v e l s . ( i v ) E f f e c t o f f l u t a m i d e and f l u t a m i d e h y d r o x i d e t r e a t m e n t on t e s t o s t e r o n e e n a n t h a t e - t r e a t e d c a s t r a t e d a d u l t male r a t l i v e r c y t o s o l i c HALC R1881 b i n d i n g p r o t e i n F l u t a m i d e a d m i n i s t r a t i o n was p r e v i o u s l y shown t o d e c r e a s e t e s t o s t e r o n e d e p e n d e n t h e p a t i c m i c r o s o m a l BPH a c t i v i t i e s i n t h e a d u l t male r a t , w i t h o u t a f f e c t i n g serum t e s t o s t e r o n e l e v e l s . F u r t h e r m o r e , f l u t a m i d e h y d r o x i d e ( t h e a c t i v e m e t a b o l i t e o f f l u t a m i d e ) d i s p l a c e d R1881 f r o m t h e h e p a t i c c y t o s o l i c HALC R1881 b i n d i n g p r o t e i n i n v i t r o . To d e t e r m i n e w h e t h e r t h e s e two phenomena a r e r e l a t e d , t h e e f f e c t o f f l u t a m i d e and f l u t a m i d e h y d r o x i d e was t e s t e d on HALC R1881 b i n d i n g u s i n g c a s t r a t e d a d u l t male r a t s t r e a t e d w i t h and w i t h o u t t e s t o s t e r o n e e n a n t h a t e (5 m i c r o m o l e / k g , s c . 10 d a y s ) . Some t e s t o s t e r o n e e n a n t h a t e - t r e a t e d and u n t r e a t e d a n i m a l s were a l s o i n j e c t e d w i t h a s c . dose o f e i t h e r f l u t a m i d e o r f l u t a m i d e h y d r o x i d e (25 m i c r o m o l e s / k g ) f o r 10 d a y s b e f o r e t h e e x p e r i m e n t . E x p e r i m e n t s were done 24 h r s a f t e r t h e l a s t i n j e c t i o n . C a s t r a t i o n f o r 10 days w i t h o r w i t h o u t t e s t o s t e r o n e t r e a t m e n t , had no s i g n i f i c a n t e f f e c t on l i v e r c y t o s o l i c HALC R1881 b i n d i n g compared t o t h e i n t a c t male c o n t r o l ( t a b l e 22) c o n f i r m i n g o u r e a r l i e r r e s u l t s . F l u t a m i d e a d m i n i s t r a t i o n t o c a s t r a t e s s i g n i f i c a n t l y l o w e r e d R1881 a p p a r e n t Kd v a l u e s w i t h o u t a f f e c t i n g t h e b i n d i n g c a p a c i t y ( a pp. Kd=0.81 +/- 0.20 nM; app. Bmax= 4.27 T a b l e 22 EFFECT OF FLUTAMIDE AND FLUTAMIDE HYDROXIDE ADMINISTRATION ON LIVER CYTOSOLIC HIGH AFFINITY-LOW CAPACITY R1881 BINDING PROTEIN The e f f e c t o f f l u t a m i d e and f l u t a m i d e h y d r o x i d e a d m i n i s t r a t i o n was d e t e r m i n e d on 10 day c a s t r a t e d (Gx) a d u l t male r a t l i v e r c y t o s o l i c h i g h a f f i n i t y - l o w c a p a c i t y R1881 b i n d i n g p r o t e i n . C o n t r o l a n i m a l s were i n j e c t e d w i t h c o r n o i l w h i l e some o f t h e c a s t r a t e d a n i m a l s were i n j e c t e d w i t h t e s t o s t e r o n e e n a n t h a t e (5 m i c r o m o l e s / k g s c . ) o r f l u t a m i d e o r f l u t a m i d e h y d r o x i d e (dose o f l a t t e r d r u g s , 25 m i c r o m o l e s / k g s c . , 10 d a y s ) . E x p e r i m e n t s were done 24 h o u r s a f t e r t h e l a s t i n j e c t i o n . C y t o s o l (1-3 mg/ml) was p r e p a r e d as d e s c r i b e d i n Methods s e c t i o n t rom i n t a c t , 10 day c a s t r a t e d a d u l t male r a t s (280-300 g ) , and was i n c u b a t e d w i t h t r i t i a t e d - R l « 8 1 ( 0 . 0 5 - 2 . 0 nM) and 1 0 0 - f o l d e x c e s s m o l a r r a t i o r a d i o i n e r t t r i a m c i n o l o n e a c e t o n i d e , w i t h o u t o r w i t h 1 0 0 - r o l d e x c e s s m o l a r r a t i o r a d i o i n e r t R1881 , f o r 3 h o u r s a t 4 degree s C . C y t o s o l was t r e a t e d w i t h 2.5% DCC; r a d i o a c t i v i t y was a n a l y s e d . B i n d i n g k i n e t i c s were c a l c u l a t e d u s i n g S c a t c h a r d a n a l y s i s and t h e l e a s t s q u a r e s method t o r l i n e a r r e g r e s s i o n . Da ta were e x p r e s s e d as t h e a v e r a g e +/- S . E . M . The numbers i n b r a c k e t s d e n o t e numbers ot a n i m a l s u s e d . 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 . S i g n i t i c a n t d i t t e r e n c e s between g r o u p s were i n d i c a t e d by common l e t t e r s . A p p a r e n t B i n d i n g K i n e t i c s A n i m a l model used Kd (nM) Bmax (fmol/mg) I n t a c t a d u l t male 0 . 20 + / - 0 (4) .04 4 . 50 + / - 0 . (4) 07 Gx a d u l t male 0 . 24 + / - 0 (4 ; a) .03 3 . 90 + / - 1. (4) 26 T e s t o s t e r o n e e n a n a t h a t e -t r e a t e d Gx a d u l t male 0 . 29 + / - 0 (4) .06 3 . 24 + / - 0 . (4 ;a ) 40 F l u t a m i d e - t r e a t e d Gx a d u l t male 0 . 81 + / - 0 (4 ;a ) .20 4 . 27 + / - 0 . (4 ;b) 92 T e s t o s t e r o n e e n a n t h a t e p l u s f l u t a m i d e - t r e a t e d 0 . 53 + / - 0 (3) .11 0 . 89 + / - 0 . (8 ; ab) 52 Gx a d u l t male T e s t o s t e r o n e e n a n t h a t e 0 .34 p l u s t l u t a m i d e h y d r o x i d e -t r e a t e d Gx a d u l t male +/- 0 .17 1.52 (3) +/- 0 .57 (4) +/- 0.92 fmol/mg), compared t o t h e c a s t r a t e d c o n t r o l (app. Kd= 0.24 +/- 0.03 nM; Bmax= 3.90 +/- 1*26 fmol/mg, u s i n g Duncan's m u l t i p l e r a n g e t e s t ) . T h i s e f f e c t was r e v e r s e d by t e s t o s t e r o n e e n a n t h a t e r e p l a c e m e n t (5 m i c r o m o l e s / k g s c . , 10 d a y s ) . F l u t a m i d e o r f l u t a m i d e h y d r o x i d e ( b o t h 25 m i c r o m o l e s / k g s c . , 10 d a y s ) a d m i n i s t r a t i o n t o t e s t o s t e r o n e e n a n t h a t e t r e a t e d c a s t r a t e d a d u l t male r a t s d i d n o t s i g n i f i c a n t l y a l t e r t h e HALC R1881 b i n d i n g a f f i n i t y compared t o t h e t e s t o s t e r o n e t r e a t e d c a s t r a t e d c o n t r o l s . F l u t a m i d e t r e a t m e n t s i g n i f i c a n t l y r e d u c e d o r e l i m i n a t e d t h e d e t e c t a b l e d i s p l a c e a b l e b i n d i n g c a p a c i t y (app. Bmax= 0.89 +/- 0.52 fmol/mg, n=8) compared t o t h e t e s t o s t e r o n e - t r e a t e d c a s t r a t e d c o n t r o l s (app. Bmax= 3.24 +/- 0.4 fmol/mg; p< 0.05 u s i n g Duncan's m u l t i p l e r a n g e t e s t ) . About 50% o f t h e f l u t a m i d e -and t e s t o s t e r o n e e n a n t h a t e t r e a t e d , c a s t r a t e d a n i m a l s d e m o n s t r a t e d no d i s p l a c e a b l e HALC R1881 b i n d i n g . F l u t a m i d e h y d r o x i d e a d m i n i s t r a t i o n a p p e a r e d t o r e d u c e HALC a n d r o g e n b i n d i n g i n t h e f l u t a m i d e h y d r o x i d e and t e s t o s t e r o n e t r e a t e d c a s t r a t e d a d u l t male; however, a n a l y s i s o f v a r i a n c e was n o t done s i n c e b i n d i n g k i n e t i c s were not d e t e r m i n e d on t h e f l u t a m i d e h y d r o x i d e t r e a t e d c a s t r a t e d a d u l t male r a t . (v) E f f e c t o f s t r e p t o z o t o c i n (STZ) and p r o t a m i n e z i n c i n s u l i n ( P Z I ) t r e a t m e n t on l i v e r c y t o s o l i c HALC R1881 b i n d i n g i n t h e a d u l t male r a t S t r e p t o z o t o c i n i s a d i a b e t o g e n , w h i c h i n a d d i t i o n t o d e c r e a s i n g serum t e s t o s t e r o n e and p r o s t a t e a n d r o g e n r e c e p t o r T a b l e 23 EFFECT OF STREPTOZOTOCIN AND PROTAMINE ZINC INSULIN REPLACEMENT ON LIVER CYTOSOLIC HIGH A F F I N I T Y -LOW CAPACITY R1881 BINDING PROTEIN The e f f e c t o f s t r e p t o z o t o c i n (STZ) and p r o t a m i n e z i n c i n s u l i n (PZI) r e p l a c e m e n t was d e t e r m i n e d on a d u l t male r a t (280-300 g) l i v e r c y t o s o l i c h i g h a f f i n i t y - l o w c a p a c i t y R1881 b i n d i n g p r o t e i n . C o n t r o l S T Z - u n t r e a t e d a n i m a l s were i n j e c t e d i v . w i t h 0 .1 N c i t r a t e b u f f e r (pH=4 .5 ) . The STZ dose was 60 mg/kg i v s i n g l e dose 4 days p r i o r t o e x p e r i m e n t , w h i l e t h e PZI dose was 10 u n i t s / k g s c . f o r 4 d a y s . E x p e r i m e n t was done 24 h o u r s a l t e r t h e l a s t i n j e c t i o n . C y t o s o l was p r e p a r e d as d e s c r i b e d i n Methods s e c t i o n , and was i n c u b a t e d w i t h t r i t i a t e d - R 1 8 8 1 ( 0 . 0 5 - 2 . 0 nM) and 1 0 0 - f o l d e x c e s s m o l a r r a t i o r a d i o i n e r t t r i a m c i n o l o n e a c e t o n i d e , w i t h o u t o r w i t h 1 0 0 - t o l d e x c e s s m o l a r r a t i o r a d i o i n e r t R1881 , f o r 3 h o u r s a t 4 degree s C . C y t o s o l was t r e a t e d w i t h 2.5% DCC; r a d i o a c t i v i t y was a n a l y s e d . B i n d i n g k i n e t i c s were c a l c u l a t e d u s i n g S c a t c h a r d a n a l y s i s and t h e l e a s t s q u a r e s method t o r l i n e a r r e g r e s s i o n . Da ta were e x p r e s s e d as t h e average +/- S . E . M . The numbers i n b r a c k e t s d e n o t e numbers o f a n i m a l s u s e d . A p p a r e n t B i n d i n g K i n e t i c s A n i m a l model used Kd (nM) Bmax (fmol/mg) C o n t r o l a d u l t male 0 .22 +/- 0 .02 5 .71 +/- 0 .97 (4) (4) S t r e p t o z o t o c i n (STZ) 0 .24 +/- 0 .08 2 .53 +/- 0 .47 (7) (8i * t r e a t e d a d u l t male S t r e p t o z o t o c i n p l u s 0 . 2 1 +/- 0 .04 2 .23 +/- 0 .11 (4) (4) ns P Z I - t r e a t e d a d u l t male * d e n o t e s s i g n i f i c a n t d i f f e r e n c e (p< 0 .05) compared t o c o n t r o l u n t r e a t e d a d u l t m a l e . ns d e n o t e s no s i g n i f i c a n t d i f f e r e n c e compared t o s t r e p t o z o t o c i n t r e a t e d a d u l t m a l e . 158 l e v e l s ( T e s o n e e t al. 1980), d e c r e a s e s h e p a t i c m i c r o s o m a l BPH a c t i v i t i e s (Warren 1982) i n t h e a d u l t male r a t . A s i n g l e i v dose o f s t r e p t o z o t o c i n (60 mg/kg) s i g n i f i c a n t l y r e d u c e d t h e b i n d i n g c a p a c i t y o f t h e HALC R1881 b i n d i n g p r o t e i n i n t h e a d u l t male l i v e r c y t o s o l (app. Kd= 0.24 +/- 0.08 nM; app. Bmax= 2.53 +/- 0.47 fmol/mg) compared t o t h e u n t r e a t e d c o n t r o l (app. Kd= 0.22 +/- 0.02 nM; app. Bmax= 5.71 +/- 0.97 fmol/mg; t a b l e 2 3 ) . F u r t h e r m o r e , one o f t h e 8 S T Z - t r e a t e d a n i m a l s t e s t e d had no d e t e c t a b l e d i s p l a c e a b l e HALC R1881 b i n d i n g . D a i l y sc i n j e c t i o n s o f p r o t a m i n e z i n c i n s u l i n (10 u n i t s / k g ) r e v e r s e d t h e g l u c o s u r i c e f f e c t s o f STZ, b u t showed no s i g n i f i c a n t e f f e c t on t h e HALC R1881 b i n d i n g p r o t e i n . DISCUSSION 1. Age and sex d i f f e r e n c e s i n h e p a t i c m i c r o s o m a l d r u g and s t e r o i d m e t a b o l i s m Age and sex d e p e n d e n t h e p a t i c m i c r o s o m a l d r u g and s t e r o i d m e t a b o l i s m was shown u s i n g d i f f e r e n t a n i m a l s m o d els w i t h v a r y i n g h ormonal s t a t u s ( f i g u r e s 3 and 4 ) . Sex d i f f e r e n c e s i n h e p a t i c m i c r o s o m a l TR and c y t o c h r o m e P-450 d e p e n d e n t BPH a c t i v i t i e s have been shown u s i n g t h e a d u l t r a t ; t h e s e d i f f e r e n c e s were s i g n i f i c a n t l y r e d u c e d 10 d a y s f o l l o w i n g c a s t r a t i o n o f t h e a d u l t m a l e , w i t h an i n c r e a s e i n TR, and a d e c r e a s e i n BPH a c t i v i t i e s t o w a r d s f e m a l e l v e l s . T e s t o s t e r o n e r e p l a c e m e n t u s i n g t e s t o s t e r o n e e n a n t h a t e (5 m i c r o m o l e s / k g , s c . , 10 d a y s ) p a r t i a l l y r e v e r s e d t h e s e e f f e c t s . T h e s e e f f e c t s a r e c o n s i s t e n t w i t h t h o s e o f o t h e r w o r k e r s ( G o n t o v n i c k and B e l l w a r d 1980; G u r t o o and P a r k e r 1977; Kramer e t a l . 1 979), and s u g g e s t t h a t sex and age d i f f e r e n c e s a r e r e l a t e d t o t e s t o s t e r o n e s e c r e t i o n . The a n t i - a n d r o g e n f l u t a m i d e , a t t h e d o s e s u s e d , c a u s e d a d e c r e a s e i n a n d r o g e n - d e p e n d e n t p r o s t a t e / B W r a t i o s i n 10 day p o s t - c a s t r a t e d a d u l t male r a t s t r e a t e d w i t h t e s t o s t e r o n e e n a n t h a t e (5 m i c r o m o l e s . k g s c . , 10 d a y s ; f i g u r e 2 ) . T h i s i s 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 P e e t s e t a_l. (1974) and o t h e r s , and a g r e e s w i t h t h e h y p o t h e s i s t h a t f l u t a m i d e o r i t s m e t a b o l i t e may a c t by t h e c o m p e t i t i v e d i s p l a c e m e n t o f a n d r o g e n s f r o m a c y t o s o l i c a n d r o g e n r e c e p t o r . The e f f e c t d oes n o t i n v o l v e t h e a l t e r a t i o n o f serum t e s t o s t e r o n e l e v e l s s i n c e t h e s e v a l u e s r e m a i n e d unchanged i n t h e c a s t r a t e d male t r e a t e d w i t h t e s t o s t e r o n e e n a n t h a t e and f l u t a m i d e ( t a b l e 1 ) . F l u t a m i d e ( a t d o s e s g r e a t e r t h a n 15 m i c r o m o l e s / k g , s c . , 10 d a y s ) i n h i b i t e d t e s t o s t e r o n e - s t i m u l a t e d h e p a t i c m i c r o s o m a l BPH a c t i v i t y i n 10 day p o s t - c a s t r a t e d a d u l t male r a t s ( f i g u r e 3 ) . T h e s e r e s u l t s a g r e e w i t h Brown e t a l . (1976) who showed a s i m i l a r i n h i b i t i o n o f h e p a t i c m i c r o s o m a l e t h y l m o r p h i n e N - d e m e t h y l a s e a c t i v i t y i n v i t r o i n p r e p u b e r t a l male r a t s , b u t d i f f e r w i t h o t h e r r e s u l t s by t h e same a u t h o r s , s i n c e f l u t a m i d e a t t h e d o s e s u s e d i n o u r s t u d y i f a i l e d t o have a s i g n i f i c a n t e f f e c t on BPH a c t i v i t i e s o r p r o s t a t e / B W r a t i o s i n t h e i n t a c t a d u l t r a t . The l a c k o f f l u t a m i d e e f f e c t i n t h e i n t a c t a d u l t male r a t may be due t o c o m p e n s a t o r y i n c r e a s e s i n serum t e s t o s t e r o n e l e v e l s as s u g g e s t e d by C l a r k e t a l . ( 1 9 8 1 ) . As w i t h h e p a t i c m i c r o s o m a l BPH a c t i v i t i e s , t h e same d o s e s o f f l u t a m i d e a n t a g o n i z e d t e s t o s t e r o n e - d e p e n d e n t i n h i b i t i o n o f TR a c t i v i t i e s i n t h e c a s t r a t e d male ( f i g u r e 4 ) . Lax and S c h r i e f e r s (1981) showed t h a t f l u t a m i d e d i d n o t have a s i g n i f i c a n t e f f e c t on TR a c t i v i t i e s i n t h e D H T - t r e a t e d c a s t r a t e d male r a t . T h i s d i s c r e p a n c y i s l i k e l y due t o d i f f e r e n c e s i n p u b e r t a l e x p o s u r e t o t e s t o s t e r o n e , s i n c e o u r s t u d y i n v o l v e d male r a t s c a s t r a t e d as a d u l t s , w h i l e t h e l a t t e r s t u d y i n v o l v e d a d u l t m a l e s w h i c h were c a s t r a t e d a t an e a r l i e r age (day 2 1 ) . The d i f f e r e n c e i s c o n s i s t e n t w i t h t h e h y p o t h e s i s t h a t p r e v i o u s e x p o s u r e o f t h e male r a t t o h i g h c o n c e n t r a t i o n s o f a n d r o g e n s d u r i n g p u b e r t y may i n d u c e a " c l a s s i c a l " r e c e p t o r w h i c h i s f l u t a m i d e - s e n s i t i v e (Lax and S c h r i e f e r s 1 9 81). In c o n t r a s t t o t h e r e s u l t s o f Lax and S c h r i e f e r s ( 1 9 8 1 ) , f l u t a m i d e d i d n o t have a s i g n i f i c a n t e f f e c t on h e p a t i c m i c r o s o m a l BPH o r TR a c t i v i t i e s i n t h e s h a m - o p e r a t e d a d u l t f e m a l e ( f i g u r e 5 ) . However f l u t a m i d e h y d r o x i d e , t h e a c t i v e m e t a b o l i t e o f f l u t a m i d e c a u s e d a s m a l l b u t s i g n i f i c a n t s t i m u l a t i o n o f a d u l t f e m a l e r a t TR a c t i v i t i e s above c o n t r o l f e m a l e l e v e l s . The l a c k o f f l u t a m i d e a c t i v i t y i n t h e f e m a l e r a t may be due t o e i t h e r f l u t a m i d e ' s low e f f i c a c y , o r due t o t h e l a c k o f a d e q u a t e f l u t a m i d e h y d r o x y l a t i o n , o r b o t h . The e f f e c t o f f l u t a m i d e h y d r o x i d e on t h e f e m a l e may be due t o i t s a c t i o n s i n d e p e n d e n t of t h e h e p a t i c a n d r o g e n r e c e p t o r , as p r o p o s e d by Brown e t a l . ( 1 9 7 6 ) . A d m i n i s t r a t i o n of f l u t a m i d e t o t h e c a s t r a t e d a d u l t male r e s u l t e d i n an i n h i b i t i o n o f BP m e t a b o l i s m and a s t i m u l a t i o n o f h e p a t i c m i c r o s o m a l t e s t o s t e r o n e m e t a b o l i s m ( f i g u r e s 3 and 4 ) . T h e s e r e s u l t s s u g g e s t t h a t f l u t a m i d e may be a c t i n g upon a d r e n a l l y s y n t h e s i z e d a n d r o g e n s t i l l p r e s e n t i n t h e c a s t r a t e d a d u l t male r a t . F u r t h e r e x p e r i m e n t s u s i n g a d r e n a l e c t o m i z e d and c a s t r a t e d a n i m a l s would have t o be done i n o r d e r t o t e s t t h i s h y p o t h e s i s . The a n t i - a n d r o g e n i c e f f e c t s o f f l u t a m i d e on t h e s e two t e s t o s t e r o n e s e n s i t i v e h e p a t i c m i c r o s o m a l enzyme s y s t e m s i n t h e male r a t a r e v e r y s i m i l a r t o i t s a n t i - a n d r o g e n i c a c t i o n on t h e p r o s t a t e , and a r e c o n s i s t e n t w i t h t h e h y p o t h e s i s t h a t f l u t a m i d e r e v e r s e s t h e e f f e c t s o f t e s t o s t e r o n e on r a t l i v e r m i c r o s o m a l d r u g and s t e r o i d m e t a b o l i s m by a n t a g o n i s m a t a c y t o s o l i c a n d r o g e n r e c e p t o r . To t e s t t h i s h y p o t h e s i s , a h e p a t i c a n d r o g e n b i n d i n g p r o t e i n a s s a y was d e v e l o p e d f o l l o w i n g t h e p r o t o c o l o f o t h e r s (Roy e t a l . 1974; S a t o e t a l . 1980) u s i n g n a t u r a l a n d r o g e n s ( t e s t o s t e r o n e and DHT) as l i g a n d s and c o m p e t i t o r s . D i s p l a c e a b l e and s a t u r a b l e t e s t o s t e r o n e b i n d i n g was d e t e c t e d i n h e p a t i c c y t o s o l i c and m i c r o s o m a l f r a c t i o n s ( t a b l e s 6-8) w i t h b i n d i n g c h a r a c t e r i s t i c s c o n s i s t e n t w i t h o t h e r w o r k e r s ( S a t o e t a l . 1980; Yamada and M i y a j i 1 9 82). However, i n c o n t r a s t t o Roy e t a l . ( 1 9 7 4 ) , t h e r e was no d e t e c t a b l e d i s p l a c e a b l e DHT b i n d i n g i n t h e l i v e r c y t o s o l ( t a b l e 4 ) . The r e q u i r e m e n t o f v e r y h i g h p r o t e i n c o n c e n t r a t i o n s i n v i t r o (>30 mg/ml; t a b l e 5) s u g g e s t e d t h a t t h e c y t o s o l i c t e s t o s t e r o n e b i n d i n g p r o t e i n was e i t h e r low i n b i n d i n g c a p a c i t y and would r e q u i r e h i g h p r o t e i n c o n c e n t r a t i o n s f o r d e t e c t i o n , o r was s i m p l y c o n t a m i n a t i o n f r o m t h e m i c r o s o m a l f r a c t i o n . The l a t t e r seemed u n l i k e l y b a s e d on d i f f e r e n c e s i n DHT b i n d i n g ( t a b l e 4 ) , b i n d i n g d i f f e r e n c e s under v a r i o u s i n c u b a t i o n t e m p e r a t u r e s ( t a b l e 7 ) , and t h e o b s e r v e d b i n d i n g d i f f e r e n c e s a f t e r t h e a d d i t i o n o f t r i a m c i n o l o n e a c e t o n i d e t o th e c y t o s o l i c compared t o t h e m i c r o s o m a l f r a c t i o n ( t a b l e 8 ) . O t h e r h e p a t i c s t e r o i d r e c e p t o r s y s t e m s a r e known t o r e q u i r e much l o w e r c o n c e n t r a t i o n s o f p r o t e i n , e g . 1-3 mg/ml, ( P o w e l l - J o n e s e t a l . 1 9 8 0 ) . The p o o r d i s p l a c e m e n t p r o f i l e of t h e h e p a t i c c y t o s o l i c t e s t o s t e r o n e b i n d i n g p r o t e i n s y s t e m r e f l e c t e d n o n - s p e c i f i c i t y ( t a b l e s 9A and 9 B ) . T h e s e r e s u l t s a r e i n c o n t r a s t w i t h t h e h e p a t i c t e s t o s t e r o n e b i n d i n g p r o t e i n r e p o r t e d by S a t o e t a l . ( 1 9 8 0 ) . However, r e s u l t s f r o m t h e i r l a b o r a t o r y have p r o v i d e d n e i t h e r c o n c l u s i v e nor c o n s i s t e n t e v i d e n c e t o s u g g e s t s p e c i f i 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 t e s t o s t e r o n e r e c e p t o r . S a t o e t a l . (1980) r e p o r t e d t h a t e s t r a d i o l d i d n o t d i s p l a c e t e s t o s t e r o n e f r o m t h e a n d r o g e n b i n d i n g p r o t e i n , w h i l e f r o m t h e same l a b o r a t o r y , Ota e t a l . (1980) r e p o r t e d t h a t e s t r a d i o l d i s p l a c e d 25-30% o f t h a t d i s p l a c e d by t e s t o s t e r o n e . F u r t h e r m o r e , Ota e t a l . (1980) r e p o r t e d 50% l e s s t e s t o s t e r o n e b i n d i n g t h a n d i d S a t o e t a l . ( 1 9 8 0 ) , u n d e r s c o r i n g t h e v a r i a b i l i t y i n v o l v e d i n m e a s u r i n g t h i s b i n d i n g p r o t e i n . Our a t t e m p t s t o r e p r o d u c e t h e s e r e s u l t s were met w i t h o n l y p a r t i a l s u c c e s s . Two m a j o r p r o b l e m s were e n c o u n t e r e d : ( i ) t h e r e q u i r e m e n t o f v e r y h i g h 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 (40-60 mg/ml), c o n s i s t e n t w i t h t h e c o n d i t i o n s c i t e d by t h e s e w o r k e r s , and ( i i ) i n c o n s i s t e n t r e p r o d u c i b i l i t y o f d i s p l a c e a b l e and s a t u r a b l e t e s t o s t e r o n e b i n d i n g . A t t h e s e h i g h p r o t e i n c o n c e n t r a t i o n s , t e s t o s t e r o n e b i n d i n g was r e d u c e d i n a c o n c e n t r a t i o n d e p e n d e n t manner, s u g g e s t i n g e i t h e r p r o t e i n d e g r a d a t i o n by p r o t e o l y t i c a c t i v i t y , o r t h a t l i g a n d m e t a b o l i s m may be o c c u r r i n g i n  v i t r o . The f o r m e r p o s s i b i l i t y was l e s s l i k e l y s i n c e a d d i t i o n o f PMSF, a p r o t e a s e i n h i b i t o r , d i d n o t i m prove c o n s i s t e n c y ; i n a d d i t i o n , t h e R e m a z o l b r i l l i a n t B l u e method f o r d e t e c t i o n o f p r o t e o l y t i c a c t i v i t y i n d i c a t e d no d e t e c t a b l e p r o t e a s e a c t i v i t y i n v i t r o ( s e e R e s u l t s 2b s e c t i o n i i i ) . T h e s e r e s u l t s s u g g e s t t h a t p r o t e o l y s i s was n o t a f a c t o r and p e r h a p s t h a t l i g a n d m e t a b o l i s m was a p o s s i b i l i t y . 2. T e s t o s t e r o n e m e t a b o l i s m i n v i t r o TLC s t u d i e s i n d i c a t e d t h a t t e s t o s t e r o n e was m e t a b o l i z e d t o 5 b e t a - A - 3 a l p h a , 1 7 b e t a - d i o l i n v i t r o ( f i g u r e s 7 and 8 ) , u s i n g t h e same i n c u b a t i o n c o n d i t i o n s as t h o s e f o r t h e measurement of t h e h e p a t i c t e s t o s t e r o n e b i n d i n g p r o t e i n . T h e s e c o n d i t i o n s t e n d t o f a v o u r t e s t o s t e r o n e m e t a b o l i s m : ( i ) h i g h p r o t e i n c o n c e n t r a t i o n s , s o l u b l e t e s t o s t e r o n e m e t a b o l i z i n g enzymes, and t h e n e c e s s a r y c o f a c t o r s (NADPH), ( i i ) a d d i t i o n o f e x c e s s u n l a b e l l e d t e s t o s t e r o n e would f a v o u r e n z y m a t i c r e d u c t i o n and h y d r o g e n a t i o n r e a c t i o n s , f o l l o w i n g t h e law o f mass a c t i o n : t e s t o s t e r o n e > 5 beta-DHT ^ ^ > 5 b e t a - A n d r o s t a n - 3 a l p h a , 1 7 b e t a - d i o l In o t h e r words, t h e a d d i t i o n o f e x c e s s r a d i o i n e r t t e s t o s t e r o n e t o t h e c y t o s o l i c m i x t u r e w o u l d i n c r e a s e t h e t e s t o s t e r o n e c o n c e n t r a t i o n t o w a r d t h e Km v a l u e f o r t e s t o s t e r o n e m e t a b o l i s m i n v i t r o , t h e r e b y l e a d i n g t o i n c r e a s e d o v e r a l l ( l i g a n d and r a d i o i n e r t ) t e s t o s t e r o n e m e t a b o l i s m . T h e s e r e s u l t s l e a d one t o c o n c l u d e t h a t g r o s s i n a c c u r a c i e s w o u l d be made i n e s t i m a t i n g t h e f r e e t e s t o s t e r o n e l i g a n d c o n c e n t r a t i o n s u s e d f o r s a t u r a t i o n s t u d i e s , and t h u s w o u l d i n v a l i d a t e S c a t c h a r d a n a l y s i s . A more s u i t a b l e l i g a n d had t o be c h o s e n , w h i c h was n o t s i g n i f i c a n t l y m e t a b o l i z e d under t h e s e b i n d i n g c o n d i t i o n s . M e t h y l t r i e n o l o n e (R1881) was us e d f o r t h i s p u r p o s e , s i n c e i t i s a v e r y p o t e n t a n d r o g e n (Raynaud e t a l . 1979), and d e m o n s t r a t e d m i n i m a l m e t a b o l i s m i n v i t r o , u s i n g l i v e r ( D e c k e r and L e v i n s o n 1983) o r p r o s t a t i c c y t o s o l (Bonne and Raynaud 1976; P o u s e t t e e t a l . 1 9 79). 3. C h a r a c t e r i z a t i o n o f t h e h e p a t i c c y t o s o l i c a n d r o g e n b i n d i n g p r o t e i n I n c u b a t i o n o f R1881 i n a d u l t male l i v e r whole c y t o s o l i n d i c a t e d two b i n d i n g s i t e s ( f i g u r e 11B), as d e t e r m i n e d u s i n g S c a t c h a r d a n a l y s i s : a s p e c i f i c h i g h a f f i n i t y - l o w c a p a c i t y (Kd= 0.22 +/- 0.02 nM; Bmax= 5.61 +/- 0.97 fmol/mg, n=8) and a n o n s p e c i f i c l o w e r a f f i n i t y - h i g h e r c a p a c i t y (Kd= 10.4 +/- 1.2 nM; Bmax= 131 +/- 29 fmol/mg, n=8) b i n d i n g s i t e . Measurement o f t h e l a t t e r was b l o c k e d by a d d i t i o n o f e x c e s s t r i a m c i n o l o n e a c e t o n i d e ( t a b l e 13 and f i g u r e 1 2 B). Th e s e r e s u l t s a r e c o n s i s t e n t w i t h t h e r e c e n t a b s t r a c t s o f Eagon e t a l . ( 1 9 8 3 ) , D e c k e r and L e v i n s o n (1983) and E i s e n f e l d e t a_l. ( 1 9 8 3 ) , who us e d s i m i l a r c o n d i t i o n s i n t h e i r h e p a t i c a n d r o g e n " r e c e p t o r " b i n d i n g s t u d i e s . Most r e c e n t l y , a c o n t r o v e r s y has a r i s e n r e g a r d i n g t h e i n t r a c e l l u l a r l o c a t i o n o f t h e s t e r o i d r e g e p t o r . K i n g and G r e e n e (1984) and Welshons e t a l . (1984) u s i n g d i f f e r e n t h i s t o c y t o c h e m i c a l a p p r o a c h e s , have c h a l l e n g e d t h e h y p o t h e s i s 166 t h a t u n o c c u p i e d e s t r o g e n r e c e p t o r s r e s i d e p r i m a r i l y i n t h e c y t o p l a s m of e s t r o g e n - r e s p o n s i v e c e l l s when the s t e r o i d i s a b s e n t . K i n g and Greene (1984) f o u n d t h a t s p e c i f i c e s t r o g e n r e c e p t o r a n t i b o d y s t a i n i n g o f e s t r o g e n - r e s p o n s i v e t i s s u e s i n d i c a t e d t h e p r e s e n c e o f o n l y n u c l e a r e s t r o g e n b i n d i n g , b o t h i n t h e p r e s e n c e o r a b s e n c e o f exogenous s t e r o i d . W e l s h o n s e t a l . (1984) f o u n d t h a t e n u c l e a t i o n of p i t u i t a r y d e r i v e d GH3 c e l l s by c y t o c h a l a s i n B c a u s e d n e a r l y a c o m p l e t e l o s s o f e s t r o g e n r e c e p t o r s , c o n f i r m i n g t h e c o n c l u s i o n o f t h e f o r m e r g r o u p , t h a t t h e e s t r o g e n r e c e p t o r r e s i d e s p r i m a r i l y i n t h e n u c l e u s . B o t h g r o u p s s u g g e s t t h a t u n f i l l e d c y t o p l a s m i c e s t r o g e n r e c e p t o r measurement u s i n g c o n v e n t i o n a l r a d i o l i g a n d exchange t e c h n i g u e s , may be an a r t e f a c t o f c y t o s o l p r e p a r a t i o n , o c c u r r i n g d u r i n g t h e h o m o g e n i z a t i o n and c e n t r i f u g a t i o n s t e p s . T h e s e r e c e p t o r s may be l o o s e l y bound n u c l e a r r e c e p t o r s w h i c h become p a r t i t i o n e d i n t o t h e c y t o s o l d u r i n g c e l l d i s r u p t i o n . S c h r a d e r (1984) o f f e r e d an i n t e r e s t i n g r e b u t t a l t o t h e e a r l i e r c i t e d p a p e r s , n o t i n g t h a t t h e i r e v i d e n c e was b a s e d on l i g h t m i c r o s c o p y t e c h n i q u e s , and t h a t p e r i n u c l e a r s t a i n i n g does n o t d i s t i n g u i s h between u n f i l l e d e s t r o g e n r e c e p t o r s l o c a t e d i n t h e n u c l e u s o r on t h e n u c l e u s . The c o n t r o v e r s y c o n t i n u e s ; however t h i s does n o t d i s c o u n t t h e i m p o r t a n c e o f hormone r e c e p t o r s i n c e l l p h y s i o l o g y . T h u s , whether o u r e x p e r i m e n t s i n v o l v e a n u c l e a r c o n t a m i n a n t o r n o t , t h e c h a r a c t e r i z a t i o n s t u d i e s ( s p e c i f i c i t y , s a t u r a b i l i t y and i n v i v o p r e s e n c e o f t h e HALC component) w o u l d s t i l l be v a l i d , and t h e p r e s e n c e o f an HALC a n d r o g e n b i n d i n g p r o t e i n c o u l d s t i l l be r e l a t e d t o t e s t o s t e r o n e - d e p e n d e n t h e p a t i c enzyme i n d u c t i o n . Of c o u r s e , f u r t h e r e x p e r i m e n t s u t i l i z i n g t h e above h i s t o c y t o c h e m i c a l t e c h n i q u e s may h e l p t o answer t h e v e r y i m p o r t a n t q u e s t i o n r e g a r d i n g t h e i n t r a c e l l u l a r l o c a t i o n of t h e HALC a n d r o g e n b i n d i n g p r o t e i n . The h i g h a f f i n i t y - l o w c a p a c i t y (HALC) a n d r o g e n b i n d i n g p r o t e i n showed r e l a t i v e s p e c i f i c i t y f o r t h e s y n t h e t i c (R1881) and n a t u r a l ( t e s t o s t e r o n e and i t s r e l a t e d m e t a b o l i t e s ) a n d r o g e n s , and t h e s t e r o i d a l and n o n - s t e r o i d a l a n t i - a " n d r o g e n s , c y p r o t e r o n e a c e t a t e and f l u t a m i d e h y d r o x i d e , and i n t e r e s t i n g l y , f o r e s t r a d i o l ( t a b l e 1 5 ) . B o t h t h e a p p a r e n t d i s s o c i a t i o n and i n h i b i t o r y c o n s t a n t s were w i t h i n t h e s u b - n a n o m o l a r r a n g e ( t a b l e 1 7 ) , w e l l w i t h i n t h e known p h y s i o l o g i c a l c o n c e n t r a t i o n r a n g e of sex hormones i n t h e a d u l t male r a t ? serum t e s t o s t e r o n e c o n c e n t r a t i o n s were a p p r o x i m a t e l y 20 nM ( t a b l e 1 ) , w h i l e serum e s t r o g e n l e v e l s a r e a p p r o x i m a t e l y s u b n a n o m o l a r i n r a n g e ( P a t s c h e t a t . 1 9 8 0 ) . F l u t a m i d e h y d r o x i d e however, showed d i s p l a c e a b l e b u t n o n - s a t u r a b l e b i n d i n g ( t a b l e 1 8 ) . T h i s i s c o n s i s t e n t w i t h B o u t o n e t a l . (1981) and Raynaud e t a l . (1979) m e a s u r i n g p r o s t a t e a n d r o g e n r e c e p t o r , who c o n c l u d e d t h a t f l u t a m i d e h y d r o x i d e had a v e r y r a p i d o f f - r a t e c o n s t a n t . The h e p a t i c HALC a n d r o g e n b i n d i n g p r o t e i n was n o t p r e s e n t i n t h e i n t a c t o r o v a r i e c t o m i z e d a d u l t f e m a l e compared t o t h e a d u l t male ( t a b l e 2 0 ) , c o n s i s t e n t w i t h t h e o b s e r v a t i o n s by D e c k e r and L e v i n s o n ( 1 9 8 3 ) . F u r t h e r , i t was n o t p r e s e n t i n t h e immature f e m a l e r a t s compared t o t h e immature male; t h o u g h i n t h e l a t t e r , t h e HALC p r o t e i n had a l o w e r a f f i n i t y and l o w e r b i n d i n g c a p a c i t y compared t o t h e a d u l t male ( t a b l e 1 9 ) . The p r e s e n c e o f t h i s p r o t e i n i n t h e a d u l t male b u t n o t i n t h e f e m a l e , w o u l d s u g g e s t t h e i n v o l v e m e n t of t e s t o s t e r o n e s e c r e t i o n w h i c h may o c c u r b e f o r e and d u r i n g b i r t h ( i m p r i n t i n g p h a s e ) , as w e l l as d u r i n g p u b e r t y . D e c k e r and L e v i n s o n (1983) r e p o r t e d t h e a b s e n c e of t h i s p r o t e i n i n t h e l i v e r c y t o s o l o f t h e p s e u d o h e r m a p h r o d i t i c male r a t , w h i c h may be e x p l a i n e d by e i t h e r g e n e t i c d e f e c t s l e a d i n g t o t h e l a c k o f a d e q u a t e n e o n a t a l i m p r i n t i n g o r i m p r o p e r a n d r o g e n r e c e p t o r s y n t h e s i s . C a s t r a t i o n d i d n o t s i g n i f i c a n t l y a l t e r HALC a n d r o g e n b i n d i n g i n t h e a d u l t male compared t o t h e s h a m - o p e r a t e d male ( t a b l e 2 0 ) . T h i s l a c k o f change s u g g e s t s t h a t a l t e r a t i o n s i n g o n a d a l s e c r e t i o n s s u c h as t e s t o s t e r o n e d i d n o t i n f l u e n c e 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 i n v i v o . The m a i n t e n a n c e o f an a n d r o g e n r e c e p t o r may be due t o a t e s t o s t e r o n e i n d e p e n d e n t i n d u c t i o n s y s t e m ( S u l l i v a n and S t r o t t 1 9 7 3 ) . T h e s e w o r k e r s r e p o r t e d a marked d e c r e a s e (80%) i n DHT b i n d i n g i n a d u l t male r a t p r o s t a t e c y t o s o l a f t e r 1-4 days c a s t r a t i o n , f o l l o w e d by a s l o w i n c r e a s e and r e s t o r a t i o n t o above c o n t r o l l e v e l s by day 8-12 p o s t - c a s t r a t i o n . Hypophysectomy (10-17 d a y s ) o f t h e a d u l t male r a t d i d not i n f l u e n c e t h e h e p a t i c HALC a n d r o g e n p r o t e i n ( t a b l e 2 1 ) . T h i s would s u g g e s t t h a t n e i t h e r t h e serum t e s t o s t e r o n e , s e c o n d a r y t o LH l e v e l s n o r t h e p r e s e n c e o f o t h e r p i t u i t a r y f a c t o r s a r e r e s p o n s i b l e f o r t h e m a i n t e n a n c e o f c e l l u l a r l e v e l s o f t h i s p r o t e i n . T h e s e r e s u l t s were s i m i l a r t o S u l l i v a n and S t r o t t ( 1 9 7 3 ) , who showed t h a t hypophysectomy and a d r e n a l e c t o m y d i d n o t s i g n i f i c a n t l y a l t e r p r o s t a t i c a n d r o g e n b i n d i n g p r o t e i n , s u g g e s t i n g t h a t t h e d i s a p p e a r a n c e and l a t e r r e a p p e a r a n c e o f t h e a n d r o g e n r e c e p t o r f o l l o w i n g c a s t r a t i o n was not a s e c o n d a r y r e s p o n s e t o e i t h e r t h e i n c r e a s e i n p i t u i t a r y o u t f l o w o r a d r e n a l s e c r e t i o n s . T h e s e a u t h o r s s p e c u l a t e d t h a t t h e p r e s e n c e o f t h i s r e c e p t o r as a c o n s t i t u t i v e p r o t e i n w i t h a p r e - s e t t i s s u e c o n c e n t r a t i o n may be d e t e r m i n e d d u r i n g e m b r y o n i c d i f f e r e n t i a t i o n , o r m a i n t a i n e d by some unknown s t i m u l i f o r d e v e l o p m e n t . A d m i n i s t r a t i o n o f f l u t a m i d e o r f l u t a m i d e h y d r o x i d e , t h e a c t i v e m e t a b o l i t e , t o t e s t o s t e r o n e - t r e a t e d c a s t r a t e d a d u l t male r a t s d e c r e a s e d HALC a n d r o g e n b i n d i n g compared t o t e s t o s t e r o n e - t r e a t e d c o n t r o l s ( t a b l e 2 2 ) . T h i s e f f e c t was n o t l i k e l y due t o f l u t a m i d e h y d r o x i d e a c c u m u l a t i o n i n v i v o , t h e r e b y l e a d i n g t o d e c r e a s e d a p p a r e n t R1881 b i n d i n g i n v i t r o , s i n c e t h e serum h a l f - l i f e o f f l u t a m i d e h y d r o x i d e i n th e r a t was 5-6 h o u r s (Buxbaum, p e r s o n n e l c o m m u n i c a t i o n ) , and o u r e x p e r i m e n t s were done 24 h r s a f t e r t h e l a s t i n j e c t i o n . S i n c e t h e r e d u c t i o n o f serum t e s t o s t e r o n e had no e f f e c t on HALC b i n d i n g , t h e h y p o t h e s i s t h a t f l u t a m i d e h y d r o x i d e c o m p e t i t i v e l y i n h i b i t e d a n d r o g e n b i n d i n g , l e a d i n g t o l o w e r t e s t o s t e r o n e t r a n s l o c a t i o n , and a r e d u c t i o n i n HALC s y n t h e s i s , w o u l d be u n l i k e l y . M o r e o v e r , r e d u c e d b i n d i n g a f f i n i t i e s i n t h e f l u t a m i d e - t r e a t e d c a s t r a t e d male, and l a c k of e f f e c t i n t h e h y p o p h y s e c t o m i z e d male s u p p o r t s g o n a d a l -and p i t u i t a r y - i n d e p e n d e n t mechanisms f o r HALC a n d r o g e n r e c e p t o r s y n t h e s i s . S t r e p t o z o t o c i n (STZ) t r e a t m e n t r e d u c e d HALC a n d r o g e n b i n d i n g i n t h e a d u l t male r a t ( t a b l e 2 3 ) . S i m i l a r e f f e c t s were f o u n d u s i n g t h e same dose o f STZ and m e a s u r i n g a d u l t male r a t 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 l e v e l s (Rodway e t a l . 1 9 8 4 ) . Our r e s u l t s a r e c o n s i s t e n t w i t h t h o s e r e p o r t e d by T e s o n e e t a l . ( 1 9 8 0 ) , who r e p o r t e d an 86% d e c r e a s e i n p r o s t a t e a n d r o g e n b i n d i n g p r o t e i n i n S T Z - t r e a t e d d i a b e t i c male r a t s compared t o u n t r e a t e d c o n t r o l s . However, r e s u l t s f r o m t h e c a s t r a t i o n s t u d i e s d i s a g r e e w i t h t h e l a t t e r w o r k e r s ' c o n c l u s i o n t h a t STZ m e d i a t e d d e c r e a s e s i n serum t e s t o s t e r o n e a c c o u n t f o r r e d u c e d a n d r o g e n r e c e p t o r . I n s u l i n f a i l e d t o s i g n i f i c a n t l y r e v e r s e t h e i n h i b i t o r y e f f e c t on t h e HALC p r o t e i n i n t h e S T Z - t r e a t e d male ( t a b l e 2 3 ) . T h i s i s i n c o n t r a s t t o T e s o n e e t a l . (1980) who showed a p a r t i a l r e s t o r a t i o n (43% o f c o n t r o l ) t o c o n t r o l l e v e l s . T h i s may be due t o d i f f e r e n c e s i n t h e i n s u l i n r e g i m e n , i n s u l i n s e n s i t i v i t y , o r t i s s u e d i f f e r e n c e s i n t h e r e c e p t o r s y n t h e t i c pathway. The r o l e of t h e HALC a n d r o g e n b i n d i n g p r o t e i n i n t e s t o s t e r o n e - d e p e n d e n t d r u g m e t a b o l i s m c a n be d i s c u s s e d by c o r r e l a t i n g t h e enzyme d a t a w i t h t h e i n v i t r o and i n v i v o b i n d i n g d a t a . Sex and a g e - d i f f e r e n c e s i n HALC a n d r o g e n p r o t e i n c o r r e l a t e w e l l w i t h t h e same d i f f e r e n c e s i n BPH and TR a c t i v i t i e s . F u r t h e r m o r e , t h e l a c k o f t h e r e s p o n s e o f t h e s e h e p a t i c m i c r o s o m a l d r u g m e t a b o l i z i n g enzyme a c t i v i t i e s t o p h y s i o l o g i c a l d o s e s of t e s t o s t e r o n e i n t h e a d u l t f e m a l e r a t ( C o l b y 1980) c a n be e x p l a i n e d by t h e a b s e n c e o f t h e HALC p r o t e i n i n t h i s m o d e l . M a i n t e n a n c e o f c o n s t i t u t i v e l e v e l s o f h e p a t i c m i c r o s o m a l BPH i n t h e f e m a l e r a t i s a p p a r e n t l y due t o t h e m u l t i p l i c i t y o f c y t o c h r o m e P-450's (Lu and West 1 9 7 9 ) . T h a t i s , t h e HALC a n d r o g e n b i n d i n g p r o t e i n p r e s e n t i n t h e a d u l t male i s s t i m u l a t i n g a t e s t o s t e r o n e - d e p e n d e n t c y t o c h r o m e P-450 d i f f e r e n t f r o m t h e c o n s t i t u t i v e l e v e l s f o rms o f f e m a l e r a t s . P r e s e n c e o f t h e HALC a n d r o g e n b i n d i n g p r o t e i n and h e p a t i c m i c r o s o m a l enzyme t e s t o s t e r o n e - r e s p o n s i v i t y i n t h e a d u l t male may be d e t e r m i n e d n e o n a t a l l y ( i e . n e o n a t a l i m p r i n t i n g , Chung e t a l . 1975) . A g e - r e l a t e d c h a n g e s i n BPH and TR a c t i v i t i e s c a n be e x p l a i n e d by t h e p u b e r t a l s e c r e t i o n o f t e s t o s t e r o n e and t h e p r e s e n c e o f t h e a n d r o g e n r e c e p t o r i n t h e g r o w i n g young m a l e . C a s t r a t i o n l e d t o d e c r e a s e d BPH enzyme a c t i v i t y , b u t no change i n t h e HALC b i n d i n g k i n e t i c s . T h i s a p p a r e n t d i c h o t o m y c a n be e x p l a i n e d by d e c r e a s e d " a g o n i s t " o r serum t e s t o s t e r o n e l e v e l s l e a d i n g t o d e c r e a s e d o c c u p a n c y o f t h e a n d r o g e n r e c e p t o r p r e s e n t and d e c r e a s e d enzyme s y n t h e s i s . T h i s d e c r e a s e i n enzyme a c t i v i t y i s r e a d i l y r e v e r s i b l e f o l l o w i n g t e s t o s t e r o n e r e p l a c e m e n t , an e f f e c t w h i c h t e n d s t o s u p p o r t o u r h y p o t h e s i s . F l u t a m i d e a d m i n i s t r a t i o n r e d u c e d h e p a t i c m i c r o s o m a l BPH a c t i v i t i e s i n t h e t e s t o s t e r o n e - t r e a t e d c a s t r a t e d a d u l t male r a t , w h i l e f l u t a m i d e and f l u t a m i d e h y d r o x i d e ( a t t h e same d o s e s ) were f o u n d t o r e d u c e HALC a n d r o g e n b i n d i n g . The a c t i o n of f l u t a m i d e h y d r o x i d e c a n be e x p l a i n e d by a t l e a s t two d i f f e r e n t ways: ( i ) by c o m p e t i t i v e d i s p l a c e m e n t of a n d r o g e n s a t t h e a n d r o g e n r e c e p t o r l e v e l , t h e r e b y r e d u c i n g t h e a g o n i s t e f f e c t on t e s t o s t e r o n e - d e p e n d e n t enzyme s y n t h e s i s , o r ( i i ) by r e d u c e d HALC b i n d i n g p r o t e i n s y n t h e s i s l e a d i n g t o d e c r e a s e d amounts o f a g o n i s t t r a n s l o c a t e d t o t h e n u c l e u s , and t h e r e d u c t i o n o f t e s t o s t e r o n e - d e p e n d e n t enzyme s y n t h e s i s . D e s p i t e d i f f e r e n c e s i n t h e s e mechanisms, b o t h i n v o l v e a d e c r e a s e i n r e c e p t o r o c c u p a n c y . T h e r e a r e l i k e l y t o be o t h e r mechanisms u n d e r l y i n g h e p a t i c m i c r o s o m a l d r u g and s t e r o i d m e t a b o l i z i n g enzyme i n d u c t i o n w h i c h may n o t depend s o l e l y on t h e p r e s e n c e o r o c c u p a n c y o f t h e h e p a t i c a n d r o g e n r e c e p t o r . The h y p o p h y s e c t o m i z e d a d u l t male, w i t h o r w i t h o u t t e s t o s t e r o n e t r e a t m e n t , d e m o n s t r a t e d d e c r e a s e d h e p a t i c m i c r o s o m a l BPH a c t i v i t i e s t o w a r d s f e m a l e l e v e l s , y e t no change was f o u n d i n HALC a n d r o g e n b i n d i n g p r o t e i n . I t would seem t h a t o t h e r " p i t u i t a r y f a c t o r s " s u c h as g r o w t h hormone a r e i n v o l v e d i n t h e h ypophysectomy e f f e c t on BPH and TR a c t i v i t y (Kramer e t a l . 1 9 7 9 ) . However, t h e s e o b s e r v a t i o n s do n o t d i s c o u n t t h e i n v o l v e m e n t o f an h e p a t i c a n d r o g e n r e c e p t o r i n t e s t o s t e r o n e s e n s i t i v e h e p a t i c m i c r o s o m a l d r u g m e t a b o l i z i n g enzyme s y n t h e s i s . In c o n t r a s t , s t r e p t o z o t o c i n r e d u c e d h e p a t i c m i c r o s o m a l BPH a c t i v i t i e s i n a d u l t m a l e s (Warren 1982), as w e l l as r e d u c e d HALC a n d r o g e n b i n d i n g . T h i s may be e x p l a i n e d by r e d u c e d serum t e s t o s t e r o n e l e v e l s as w e l l as by d e c r e a s e d HALC a n d r o g e n b i n d i n g p r o t e i n c o n c e n t r a t i o n s , l e a d i n g t o r e d u c e d r e c e p t o r o c c u p a n c y , r e d u c e d t r a n s l o c a t i o n , and r e d u c e d BPH s y n t h e s i s . A l t h o u g h i n s u l i n i s known t o r e v e r s e t h e STZ e f f e c t on BPH a c t i v i t i e s (Warren 1982), i t d i d n o t s i g n i f i c a n t l y i n f l u e n c e t h e p r e s e n c e o f t h e HALC p r o t e i n . F u r t h e r e x p e r i m e n t s a r e r e q u i r e d t o g a i n a b e t t e r u n d e r s t a n d i n g o f t h e mechanism of STZ on h e p a t i c d r u g m e t a b o l i s m and t h e HALC a n d r o g e n b i n d i n g p r o t e i n . In c o n c l u s i o n , an h e p a t i c c y t o s o l i c a n d r o g e n r e c e p t o r may be i n v o l v e d i n t h e t e s t o s t e r o n e - d e p e n d e n t BPH i n d u c t i o n p r o c e s s , however, t h i s seems t o be o n l y one s t e p i n a v e r y complex s y n t h e t i c pathway. 174 FUTURE EXPERIMENTS The f o l l o w i n g e x p e r i m e n t s a r e i n t e n d e d t o f u r t h e r c h a r a c t e r i z e t h e h e p a t i c androgen b i n d i n g p r o t e i n iin v i v o and i n v i t r o . R e s u l t s f rom t h e s e e x p e r i m e n t s w o u l d s t r e n g t h e n our c o n c l u s i o n s as w e l l as p r o v i d e a g r e a t e r b a s i s t o r a p h y s i o l o g i c a l r o l e o f t h e HALC androgen b i n d i n g p r o t e i n . 1. Based on t h e s a t u r a t i o n s t u d i e s , we have r e p o r t e d t h a t b o t h c a s t r a t e d and h y p o p h y s e c t o m i z e d a d u l t male r a t l i v e r c y t o s o l i c HALC R1881 b i n d i n g k i n e t i c s r e m a i n e d r e l a t i v e l y unchanged compared t o i n t a c t c o n t r o l s , and s u g g e s t e d t h a t t h i s p r o t e i n was i n s e n s i t i v e t o t h e l o s s o f g o n a d a l and p i t u i t a r y hormones . These r e s u l t s , h o w e v e r , were based on s a t u r a t i o n s t u d i e s and i n o r d e r t o s t r e n g t h e n our c o n c l u s i o n s , o t h e r s t u d i e s s u c h as i n h i b i t i o n s t u d i e s s h o u l d be done u s i n g a d u l t male r a t l i v e r c y t o s o l i n c u b a t e d w i t h 1 nM t r i t i a t e d - R 1 8 8 1 and 100 f o l d e x c e s s ; molar r a t i o t r i a m c i n o l o n e a c e t o n i d e , i n t h e p r e s e n c e o r absence o f v a r y i n g c o n c e n t r a t i o n s o f d i f f e r e n t c o m p e t i t o r s . From t h i s d a t a , t h e a p p . K i v a l u e s c o u l d be c a l c u l a t e d and one c o u l d d e t e r m i n e t h e s p e c i f i c i t y o f t h e HALC p r o t e i n p r e s e n t i n t h e c a s t r a t e d and h y p o p h y s e c t o m i z e d a d u l t male r a t l i v e r c y t o s o l t r a c t i o n s . I f t h e h e p a t i c c y t o s o l i c HALC androgen b i n d i n g p r o t e i n i s i n s e n s i t i v e t o t h e a l t e r a t i o n o f i t s h o r m o n a l e n v i r o n m e n t , t h e n i t s s p e c i f i c i t y w o u l d r e m a i n r e l a t i v e l y unchanged compared to that present i n the sham-operated in t a c t adult male controls. Other characterization studies including the determination of the sedimentation c o - e t t i c i e n t s may be useful i n supporting our conclusions. Eagon et a l . (1980) reported a s h i f t i n the sedimentation c o - e t t i c i e n t of the estrogen binding protein from 10 S to 4 S, in the 30 day castrated adult male compared to the in t a c t c o n t r o l . If the s p e c i f i c i t y or the sedimentation c o - e f f i c i e n t of the HALC androgen binding protein are altered i n the castrated or hypophysectomized animal models compared to controls, then time course studies of these ettects should be done. In addition, these techniques would be useful in confirming other conclusions that ontogeny i s important to the presence of the HALC androgen binding protein. 2. Our studies indicate that the presence of the HALC androgen binding protein i s dependent on the animal's age, i e . absent or i n low quantities i n the immature male rat compared to the adult male. In order to study the role of gonadal factors (ie. testosterone) and p i t u i t a r y factors on the regulation of thi s age-dependent protein, the HALC protein could be measured i n the prepubertally castrated or hypophysectomized adult male r a t . Continued absence or presence of low levels of the HALC protein in these animals compared to sham-operated controls would suggest the importance of hormonal secretions during maturity on this androgen b i n d i n g p r o t e i n . I f t h e s e e f f e c t s a r e r e v e r s e d t o n o r m a l a d u l t l e v e l s by t e s t o s t e r o n e e n a n t h a t e t r e a t m e n t f o l l o w i n g p r e p u b e r t a l c a s t r a t i o n o r h y p o p h y s e c t o m y , t h e n i t w o u l d be l i k e l y t h a t t e s t o s t e r o n e s e c r e t i o n i s i n v o l v e d i n t h e o n t o g e n i c p r o c e s s . 3 . The p r e s e n c e o f t h e HALC androgen b i n d i n g p r o t e i n i n t h e t e s t o s t e r o n e e n a n t h a t e t r e a t e d c a s t r a t e d a d u l t male and i t s absence i n t h e s i m i l a r l y t r e a t e d o v a r i e c t o m i z e d a d u l t t e m a l e may be due t o t h e l a c k o f n e o n a t a l i m p r i n t i n g . A s i m i l a r mechanism i s i n v o l v e d i n sex d i f f e r e n c e s i n h e p a t i c m i c r o s o m a l d r u g and s t e r o i d m e t a b o l i s m ( S k e t t and G u s t a f s s o n 1979 ; C o l b y 1 9 8 0 ) . I f sex d i f f e r e n c e s i n HALC androgen b i n d i n g p r o t e i n a r e due t o n e o n a t a l i m p r i n t i n g , t h e n n e o n a t a l c a s t r a t i o n w o u l d l e a d t o t h e l o s s o f t h i s p r o t e i n i n t h e a d u l t m a l e . T h i s e x p e r i m e n t s h o u l d be c a r r i e d o u t . F u r t h e r m o r e , i f t h e h y p o t h e s i s i s t r u e , t h e n t h i s e f f e c t s h o u l d be r e v e r s e d by t e s t o s t e r o n e t r e a t m e n t f o l l o w i n g c a s t r a t i o n and t h r o u g h o u t m a t u r i t y . To f u r t h e r t e s t t h i s h y p o t h e s i s , t h e p r e s e n c e o f t h e HALC p r o t e i n c o u l d be measured i n t h e n e o n a t a l l y o v a r i e c t o m i z e d a d u l t f ema le w h i c h was t r e a t e d w i t h t e s t o s t e r o n e f o l l o w i n g gonadectomy and d u r i n g m a t u r i t y . I n a d d i t i o n t o t h e b i n d i n g s t u d i e s , h e p a t i c m i c r o s o m a l BPH a c t i v i t i e s c o u l d be measured u s i n g t h e s e a n i m a l m o d e l s , i n o r d e r t o c o r r e l a t e t h e p r e s e n c e o f t h e HALC p r o t e i n w i t h t e s t o s t e r o n e dependent h e p a t i c m i c r o s o m a l BPH i n d u c t i o n . 177 4 . F l u t a m i d e t r e a t m e n t r e d u c e d b o t h t h e p r e s e n c e o f t h e h e p a t i c c y t o s o l i c HALC androgen b i n d i n g p r o t e i n and t e s t o s t e r o n e dependent h e p a t i c m i c r o s o m a l BPH a c t i v i t i e s i n t h e t e s t o s t e r o n e e n a n t h a t e t r e a t e d c a s t r a t e d a d u l t male r a t . I f t h e r e d u c t i o n i n t h e l e v e l s o f t h e HALC p r o t e i n l e a d s t o d e c r e a s e d n u c l e a r t r a n s l o c a t i o n ot t h e a g o n i s t and d e c r e a s e d t e s t o s t e r o n e dependent h e p a t i c m i c r o s o m a l BPH i n d u c t i o n , t h e n r e v e r s a l o f t h e t l u t a m i d e e f f e c t s h o u l d l e a d t o i n c r e a s e d HALC p r o t e i n l e v e l s accompanied by s t i m u l a t i o n o f BPH a c t i v i t i e s . I n c r e a s e s i n HALC p r o t e i n l e v e l s w i t h o u t i n c r e a s e s i n BPH a c t i v i t y , o r i n c r e a s e s i n BPH a c t i v i t y w i t h o u t i n c r e a s e s i n HALC l e v e l s w o u l d t e n d t o d i s p u t e t h e h y p o t h e s i s . R e v e r s a l o f t h e f l u t a m i d e e f f e c t may be t e s t e d by a number o f w a y s : (1) a f l u t a m i d e d o s e - r e s p o n s e s t u d y on t h e p r e s e n c e o f t h e HALC p r o t e i n i n f l u t a m i d e - t r e a t e d c a s t r a t e d a d u l t m a l e s . E x p e r i m e n t s s h o u l d be done 24 h r s 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 t o a v o i d c a r r y - o v e r o f f l u t a m i d e o r t l u t a m i d e m e t a b o l i t e s i n v i t r o ; (2) a t e s t o s t e r o n e d o s e - r e s p o n s e s t u d y on t h e p r e s e n c e o f t h e HALC p r o t e i n i n t l u t a m i d e - t r e a t e d a d u l t m a l e s ; (3) a t i m e c o u r s e s t u d y o f t h e e f f e c t o f d i s c o n t i n u i n g f l u t a m i d e t r e a t m e n t on HALC p r o t e i n and BPH l e v e l s i n a d u l t male r a t s w h i c h a r e m a i n t a i n e d on t e s t o s t e r o n e t h e r a p y . A s i m i l a r a p p r o a c h may be used t o r e v e r s e t h e e f f e c t s o f STZ on HALC p r o t e i n and BPH l e v e l s i n t h e a d u l t m a l e , u s i n g d i f f e r e n t doses o r t i m e p e r i o d s o f i n s u l i n t r e a t m e n t . 5. P u r i f i c a t i o n s t u d i e s o f t h e h e p a t i c c y t o s o l i c a n d r o g e n b i n d i n g p r o t e i n w o u l d have t o be done. F o r t h i s p u r p o s e , d i f f e r e n t t e c h n i q u e s may be u s e d , s u c h as ammonium s u l p h a t e p r e c i p i t a t i o n , s u c r o s e d e n s i t y g r a d i e n t u l t r a c e n t r i f u g a t i o n , and column c h r o m a t o g r a p h y . C h a r a c t e r i z a t i o n s t u d i e s o f t h e p u r i f i e d p r o t e i n w o u l d e n a b l e c o m p a r i s o n t o t h o s e o f t h e whole c y t o s o l . SUMMARY The p r e s e n t s t u d i e s s u p p o r t t h e h y p o t h e s i s t h a t t e s t o s t e r o n e i n d u c e d h e p a t i c m i c r o s o m a l d r u g and s t e r o i d m e t a b o l i s m i s an a n d r o g e n r e c e p t o r m o d u l a t e d p r o c e s s . T h i s c o n c l u s i o n i s b a s e d on r e l a t i o n s h i p s between h e p a t i c m i c r o s o m a l c y t o c h r o m e P-450 de p e n d e n t b e n z o ( a ) p y r e n e h y d r o x y l a s e (BPH) a c t i v i t y and a n d r o g e n b i n d i n g k i n e t i c s , i n d i f f e r e n t a n i m a l models whose hormonal s t a t u s has been a l t e r e d f o l l o w i n g p h y s i o l o g i c a l and p h a r m a c o l o g i c a l m a n i p u l a t i o n s . 1. T e s t o s t e r o n e m e t a b o l i s m s t u d i e s i n d i c a t e d t h a t t h e h e p a t i c c y t o s o l i c t e s t o s t e r o n e b i n d i n g p r o t e i n a s s a y was c o m p l i c a t e d by l i g a n d m e t a b o l i s m i n v i t r o . C o n c l u s i o n s f r o m e a r l i e r s t u d i e s u s i n g t e s t o s t e r o n e as a l i g a n d a r e t h e r e f o r e q u e s t i o n a b l e . 2. M e t h y l t r i e n o l o n e (R1881) was a u s e f u l t o o l f o r t h e measurement o f t h e h e p a t i c c y t o s o l i c a n d r o g e n b i n d i n g p r o t e i n . 3. The a d u l t male r a t l i v e r c y t o s o l c o n t a i n e d two a n d r o g e n b i n d i n g components: a s p e c i f i c h i g h a f f i n i t y (Kd i n t h e s u b n a n o m o l a r r a n g e ) - l o w c a p a c i t y ( i n t h e fmol/mg r a n g e ) o r HALC a n d r o g e n b i n d i n g p r o t e i n , and a r e l a t i v e l y n o n - s p e c i f i c l o w e r a f f i n i t y - h i g h e r c a p a c i t y (LAHC) a n d r o g e n b i n d i n g component. 4. The HALC a n d r o g e n b i n d i n g p r o t e i n had a h i g h s p e c i f i c i t y t o n a t u r a l ( i n c l u d i n g t e s t o s t e r o n e , 5 alpha-DHT, and a n d r o s t e n e d i o n e ) , as w e l l as s y n t h e t i c a n d r o g e n s ( i n c l u d i n g R1881 and m i b o l e r o n e ) , b u t had a l o w e r s p e c i f i c i t y t o e s t r a d i o l , c y p r o t e r o n e a c e t a t e and f l u t a m i d e h y d r o x i d e ( t h e a c t i v e m e t a b o l i t e o f f l u t a m i d e ) . T h i s p r o t e i n d i d n o t b i n d s i g n i f i c a n t l y t o o t h e r s t e r o i d s s u c h as DES, p r o g e s t e r o n e , t r i a m c i n o l o n e a c e t o n i d e , C o r t i s o l o r f l u t a m i d e . 5. The LAHC b i n d i n g component was r e l a t i v e l y n o n - s p e c i f i c and bound t o many d i f f e r e n t s t e r o i d c l a s s e s i n c l u d i n g p r o g e s t e r o n e , C o r t i s o l , e s t r a d i o l , DES, t e s t o s t e r o n e , DHT and a n d r o s t e n e d i o n e , b u t n o t t o f l u t a m i d e o r f l u t a m i d e h y d r o x i d e . 6. A d d i t i o n of t r i a m c i n o l o n e a c e t o n i d e t o t h e l i v e r whole c y t o s o l e l i m i n a t e d t h e LAHC b i n d i n g component d u r i n g a n a l y s i s . T h i s p r o c e d u r e a l l o w e d f o r e a s y S c a t c h a r d a n a l y s i s o f t h e HALC a n d r o g e n b i n d i n g p r o t e i n . 7. S a t u r a t i o n s t u d i e s u s i n g t e s t o s t e r o n e , 5 alpha-DHT, a n d r o s t e n e d i o n e , c y p r o t e r o n e a c e t a t e and e s t r a d i o l p r o d u c e d s i m i l a r b i n d i n g k i n e t i c s s u g g e s t i n g t h a t t h e s e s t e r o i d s were b i n d i n g t o t h e same o r a s i m i l a r h i g h a f f i n i t y s i t e . F l u t a m i d e h y d r o x i d e , on t h e o t h e r hand, f a i l e d t o y i e l d c o n s i s t e n t s a t u r a b l e b i n d i n g ( i e . no S c a t c h a r d p l o t s ) . 8. T r i t i a t e d - m i b o l e r o n e c o u l d be s u b s t i t u t e d f o r t r i t i a t e d - R l 8 8 1 f o r t h e measurement o f t h e HALC a n d r o g e n b i n d i n g s i t e i n v i t r o . 9. The HALC a n d r o g e n b i n d i n g p r o t e i n was s e x - and a g e - d e p e n d e n t , b e i n g p r e s e n t i n t h e a d u l t male r a t l i v e r b u t not i n t h e a d u l t f e m a l e . F u r t h e r m o r e , i t was d e t e c t e d i n low q u a n t i t i e s i n t h e immature male and a b s e n t i n t h e immature f e m a l e r a t . These c h a r a c t e r i s t i c s c o r r e l a t e w e l l w i t h sex and a g e - d e p e n d e n t h e p a t i c m i c r o s o m a l BPH a c t i v i t i e s i n t h e r a t . 10. A l t h o u g h h e p a t i c m i c r o s o m a l BPH a c t i v i t i e s were s e n s i t i v e t o c a s t r a t i o n , an e f f e c t w h i c h was r e v e r s i b l e by t e s t o s t e r o n e r e p l a c e m e n t , t h e HALC a n d r o g e n b i n d i n g p r o t e i n r e m a i n e d r e l a t i v e l y unchanged f o l l o w i n g c a s t r a t i o n w i t h o r w i t h o u t t e s t o s t e r o n e t r e a t m e n t . 11. Hypophysectomy (10-17 d a y s ) , w i t h o r w i t h o u t t e s t o s t e r o n e a d m i n i s t r a t i o n , d i d not s i g n i f i c a n t l y a l t e r t h e b i n d i n g k i n e t i c s o f t h e a d u l t male r a t h e p a t i c c y t o s o l i c HALC a n d r o g e n b i n d i n g p r o t e i n . 12. A d m i n i s t r a t i o n o f f l u t a m i d e t o t h e t e s t o s t e r o n e - t r e a t e d and u n t r e a t e d c a s t r a t e d a d u l t male r a t r e d u c e d t e s t o s t e r o n e d e p e n d e n t h e p a t i c m i c r o s o m a l BPH a c t i v i t i e s . A d m i n i s t r a t i o n o f t h i s d r u g t o t h e a d u l t f e m a l e had no s i g n i f i c a n t e f f e c t on BPH a c t i v i t i e s , however t r e a t m e n t w i t h f l u t a m i d e h y d r o x i d e ( t h e a c t i v e m e t a b o l i t e of f l u t a m i d e ) c a u s e d a s m a l l b u t s i g n i f i c a n t d e c r e a s e i n t h i s enzyme's a c t i v i t y r e l a t i v e t o c o n t r o l f e m a l e l e v e l s . 13. C o n s i s t e n t w i t h t h e e f f e c t s o f f l u t a m i d e , a d m i n i s t r a t i o n o f f l u t a m i d e h y d r o x i d e t o t h e a d u l t male r a t a p p e a r e d t o r e d u c e HALC a n d r o g e n b i n d i n g . 14. A d m i n i s t r a t i o n o f s t r e p t o z o t o c i n ( a t t h e d o s e s used) d e c r e a s e d HALC a n d r o g e n b i n d i n g i n t h e a d u l t male r a t . 15. T h e s e s t u d i e s s u g g e s t t h a t b o t h p r e s e n c e and o c c u p a n c y o f t h e HALC a n d r o g e n b i n d i n g p r o t e i n a r e n e c e s s a r y i n o r d e r f o r t h e l a t t e r t o be a r e c e p t o r w h i c h m o d u l a t e s t e s t o s t e r o n e d e p e n d e n t h e p a t i c m i c r o s o m a l BPH a c t i v i t y i n t h e r a t . BIBLIOGRAPHY A m b e l l a n , E . , Swanson, and D a v i d s o n . ( 1 9 8 1 ) . G l u c o c o r t i c o i d b i n d i n g t o r a t l i v e r m i c r o s o m a l f r a c t i o n s i n . v i t r o . J . S t e r o i d B i o c h e m . 14_: 421-428. A t e n , R.F., E i s e n f e l d , A . J . and B e a t t i e , C.W. ( 1 9 8 3 ) . A n d r o g e n and e s t r o g e n r e c e p t o r s i n human l i v e r . The E n d o c r i n e S o c i e t y . 65_: 245. ( a b s t r a c t #660) B a r d i n , C.W., B u l l o c k , L.P., S h a r i n s , R . J . , Mowszowicz, I . and B l a c k b u r n , W.R. ( 1 9 7 3 ) . 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