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

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I  CHARACTERIZATION HEPATIC  S T U D I E S OF T H E WISTAR  RAT  CYTOSOLIC METHYLTRIENOLONE  (R1881)  BINDING PROTEIN  By Geoffrey  A  I. Sunahara  B.Sc.  University  of Toronto,  1976  M.Sc.  University  o f Toronto,  1979  THESIS  SUBMITTED  THE  IN P A R T I A L  REQUIREMENTS DOCTOR OF  FOR  F U L F I L M E N T OF  THE DEGREE  OF  PHILOSOPHY in  THE  F A C U L T Y OF GRADUATE  FACULTY  OF  PHARMACEUTICAL  UNIVERSITY  We  accept to  THE  OF B R I T I S H  this  thesis  the required  UNIVERSITY  Geoffrey  SCIENCES  COLUMBIA  as  conforming  standard  OF B R I T I S H July,  Cc)  STUDIES  COLUMBIA  1984  I . Sunahara,  1984  In presenting requirements  this  thesis i n partial  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  of B r i t i s h Columbia, I agree that it  freely  available  agree t h a t  the Library s h a l l  f o r r e f e r e n c e and study.  permission  for scholarly  fulfilment of the  for  further  f o r extensive copying of t h i s  that  copying or p u b l i c a t i o n of t h i s  f i n a n c i a l gain  It  is  thesis  s h a l l n o t b e a l l o w e d w i t h o u t my w r i t t e n  permission.  Geoffrey  208pQg§S&ax^ o f  thesis  p u r p o s e s may b e g r a n t e d b y t h e h e a d o f my  department 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 . understood  I  make  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 D a t e A u g u s t 2 9 , 1984  I . Sunahara  ABSTRACT  Age drug  and sex d i f f e r e n c e s  metabolism  mechanism  are well-known.  i s unclear,  clearly  implicated.  tissues  a r e known  which  mediate  hepatic  microsomal  as  animal  capacity  affinity-higher triamcinolone lower  component intact  was  binding  site,  i n low g u a n t i t i e s  studies  of the high  microsomal  indicated a high  binding  isotherms: and a  in vitro.  excess  eliminated affinity  female female male.  using  specificity  high  of  The h i g h  immature  component  whole  lower  mixture  adult  used  assay,  a  Addition  i n t h e immature  affinity  ( R 1 8 8 1 ) was  rat liver  component.  androgen  using  metabolized  to the assay  i n the  sensitive  and c y t o s o l i c  male  ovariectomized  present  tissues, a  this  in vitro,  component.  binding  not present  test  androgen  binding  been  receptors  i n these  Methyltrienolone  or Cortisol  o r t h e 10-day  competitors  measured  significantly  capacity  affinity  To  activities  two R 1 8 8 1  has  androgen  sensitive liver  analysis of adult  indicated  affinity-low  was  exact  in testosterone  metabolism.  models.  microsomal  s e n s i t i v e sex end-organ  specific  involved  were  the  of testosterone  f o r the c y t o s o l i c  Scatchard  the  levels  testosterone  cytosol  be  drug  Although  androgen  hydroxylase  protein  the ligand  since  may  r a t hepatic  of testosterone  testosterone  benzo(a)pyrene  different  Since  the a c t i o n  mechanism  binding  the role  to contain  similar  hypothesis,  in adult  or the rat;  i t was  Inhibition  different  to androgens,  including  testosterone,  androstenedione, specificity hydroxide,  and  of  no  mibolerone,  were  affinity  to  t o t h e same  hydroxide  and  and  flutamide  Cortisol.  Saturation  indicated  that  androstenedione,  and  estradiol  indicating  that  or a s i m i l a r  yielded  moderate  estradiol  component  acetate  kinetics,  a  progesterone,  dihydrotestosterone,  cyproterone  binding  acetate,  diethylstilbestrol  binding  Flutamide  mibolerone,  specificity  the high  testosterone,  similar  and  to cyproterone  triamcinolone, analysis  R1881  dihydrotestosterone,  these  high  displaceable  produced steroids  affinity  but  site.  non-saturable  binding. Castration or  of  hypophysectomy  effect  on  adult  male  f o r 10-17  the high  affinity  compared  to c o n t r o l s .  Nor  to  animals  this  these  Flutamide  or  alter  flutamide  testosterone-treated both and  liver high  adult  protein  presence rat  caused  of  but  male  rats  These  affinity upon  administration  to  i t i s altered following  to the  treatment  i n the high  component  reduced activities  compared  Streptozotocin  gonadal  kinetics.  hydroxylase  results  days,  significant  significantly  levels  decrease  a  10  kinetics  binding  treatment  protein  a marked  the high  protein  protein's  controls.  i s not dependent  vivo,  binding  did testosterone  adult  levels.  hrs, 4 or  d i d n o t have  hydroxide  binding  untreated  males  binding  days  f o r 18  microsomal benzo(a)pyrene  affinity  flutamide  rats  indicate  affinity that  i n the a d u l t  or p i t u i t a r y flutamide,  to  the male  hormones flutamide  in  hydroxide do  not  the  or  streptozotocin  negate  agonist  androgen  the  hypothesis  occupancy  binding  protein  testosterone-sensitive  Gail  B.  Bellward,  Supervisor  of  Ph.  the  administration. that  the p r e s e n c e as  high  affinity-low  are p r e r e q u i s i t e s f o r  effects  D.  These  i n the  liver.  studies well  capacity  asf  V  TABLE OF CONTENTS Page ABSTRACT  i i  TABLE OF CONTENTS  v  L I S T OF TABLES  xii  L I S T OF FIGURES  xv  L I S T OF ABBREVIATIONS  xvii  ACKNOWLEDGEMENTS  xxi  INTRODUCTION  1  1.  Age and s e x - 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  2.  The r o l e o f  metabolism  testosterone i n  m i c r o s o m a l d r u g and s t e r o i d 3.  The r o l e o f an a n d r o g e n hepatic a.  1 hepatic metabolism  receptor  Indirect effects  steroid  on h e p a t i c  7  and  metabolism of  metabolism...  testosterone  microsomal drug  Direct effects  steroid  of  4  in  m i c r o s o m a l d r u g and s t e r o i d  on h e p a t i c  b.  microsomal  8 testosterone  microsomal drug  and  metabolism  13  i.  I_n v i v o e v i d e n c e  13  ii.  In. v i t r o  16  4.  Summary  5.  Objectives  evidence  30 of  this  study  32  METHODS AND MATERIALS 1.  Chemicals  2.  Care  3.  Treatment  4.  Subcellular a.  and s u r g e r y  5.  Hepatic a.  fractionation  enzyme  Hepatic  Hepatic  6.  Other  7.  Testosterone  b.  of  microsomal  and  binding  enzyme  microsomes  studies  assays  benzo(a)pyrene  assay  microsomal  testosterone  assay  assays metabolism  studies  Preparation  of  and c y t o s o l  extracts  Separation related  of  standard  light  for  using  solutions  chromatography.. from thin-layer  (TLC)  V i s u a l i z a t i o n of related  steroid  testosterone  metabolites  chromatography c.  cytosol  microsomal  reductase  a.  microsomes  radio-ligand  hydroxylase b.  of  studies  Preparation tor  animals  animals  Preparation tor  b.  of  of  testosterone  metabolites  and c o l o u r  u s i n g UV  reagents  and  vii Page 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 5g-androstan-3a,17$-dioi u s i n g Gas Chromatography-Mass Spectrometry  e.  (GCMS)  A n a l y s i s of bound  .  tritiated-testosterone  and m e t a b o l i t e s u s i n g TLC 8.  48  R a d i o - l i g a n d exchange b i n d i n g s t u d i e s  48  a.  Incubation of c y t o s o l and microsomes  b.  S e p a r a t i o n of bound and t r e e steroid  c.  tractions  Sephadex G-25M  46  4  ^  49  mini-column  chromatography  50  9.  A n a l y s i s of r a d i o a c t i v i t y  51  10.  Statistical  52  analysis  RESULTS 1.  5  3  E f f e c t of t e s t o s t e r o n e enanthate 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  levels,  t i s s u e : b o d y weight r a t i o s and h e p a t i c microsomal  enzyme a c t i v i t i e s  53  a.  Serum t e s t o s t e r o n e l e v e l s  53  b.  Tissue:body weight  (BW) r a t i o s  5  c.  Hepatic microsomal  drug 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  7  ^4  Development of the h e p a t i c  cytosolic  t e s t o s t e r o n e b i n d i n g assay a.  Separation  of bound v s . f r e e s t e r o i d s  b.  O p t i m i z a t i o n of the  cytosolic  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 assay i.  E f f e c t of v a r y i n g the p r o t e i n concentrations  on  testosterone  b i n d i n g i n a d u l t male hepatic cytosol ii.  E f f e c t of v a r y i n g i n c u b a t i o n time temperature on t e s t o s t e r o n e  and  binding  i n a d u l t male r a t l i v e r c y t o s o l and microsomes iii.  Displacement 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 competitors i n a d u l t r a t l i v e r whole c y t o s o l and microsomes  Testosterone a.  metabolism s t u d i e s  Separation and  and  d e t e c t i o n of  r e l a t e d s t e r o i d standards using  l a y e r chromatography b.  Separation and  testosterone  and  u s i n g TLC  (TLC)  d e t e c t i o n of  i t s metabolites  thin-  testosterone  from c y t o s o l e x t r a c t  I X  Page c.  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 r e l a t e d d i o l - d e r i v a t i v e s using gas chromatography-mass spectrometry  d.  (GCMS)....  Formation of 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 metabolites  i n l i v e r whole 100  cytosol i n v i t r o Hepatic  c y t o s o l i c methyltrienolone  (R1881)  b i n d i n g assay a.  92  104  E t t e c t of p r o t e i n d i l u t i o n and time  course  on i n c u b a t i o n of R1881 b i n d i n g i n a d u l t 1 no  male r a t l i v e r whole c y t o s o l  u o  b.  Computer a n a l y s i s of b i n d i n g data  c.  E f f e c t of t r i a m c i n o l o n e acetonide and  alone  i n combination with 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 d.  119  Displacement of t r i t i a t e d - R 1 8 8 1 trom c y t o s o l i c R1881 b i n d i n g  sites  using 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  127  competitors e.  Separation  of c y t o s o l i c p r o t e i n bound and  133  free steroid fractions f.  O p t i m i z a t i o n of the h e p a t i c  cytosolic  androgen b i n d i n g assay C h a r a c t e r i z a t i o n of the h e p a t i c c y t o s o l i c attinity-low capacity protein  1  3  4  high  (HALC) androgen b i n d i n g  135  Page In v i t r o experiments i.  Displacement of HALC R1881 binding  i n male r a t l i v e r  whole c y t o s o l ii.  •'•35  R1881 s a t u r a t i o n study  using  d i f f e r e n t competitors 111.  Tritiated-Mibolerone  binding i n  a d u l t male r a t c y t o s o l  1^2  D i f f e r e n t p h y s i o l o g i c a l and pharmacological animal models i.  Age and sex d i f f e r e n c e s i n R1881 binding rat  ii.  -L45  i n male and female  liver cytosol  E f f e c t of gonadectomy on a d u l t male and  female r a t l i v e r  HALC R1881 b i n d i n g iii.  cytosol  protein  5  1  1  5  3  1  5  4  E f f e c t of hypophysectomy and testosterone  enanthate  on a d u l t male r a t l i v e r HALC R1881 b i n d i n g iv.  1  E f f e c t of flutamide  administration cytosol  protein and  hydroxide treatment on  flutamide testosterone  enanthate-treated castrated  adult  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  protein  xi  Page v.  Effect  of  streptozotocin  protamine  zinc insulin  treatment  on l i v e r  (STZ)  and  (PZI)  cytosolic  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 1.  Age and s e x  -L59 differences  i n hepatic  microsomal  d r u g and s t e r o i d m e t a b o l i s m  159  2.  Testosterone metabolism 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 of  the h e p a t i c  androgen b i n d i n g p r o t e i n  cytosolic 165  FUTURE EXPERIMENTS  1 7 4  SUMMARY  1  7  9  BIBLIOGRAPHY  1  8  3  xii LIST  OF  TABLES Page  1.  Errect  of flutamide  testosterone 2.  Elfect  Etfect  serum  levels  56  of flutamide  treatment 3.  on  on  and  l i v e r :body w e i g h t  of testosterone  hepatic  microsomal  hydroxylase  testosterone  and  ratios  treatment  on  benzo(a)pyrene  testosterone  reductase  activities 4.  Separation and  5.  63 techniques  unbound  Effect adult  for protein  bound  steroids  of varying male  72  protein  rat liver  content  cytosolic  on  testosterone  binding 6.  Effect  75> of varying  testosterone cellular 7.  adult  male  by  DHT  binding  period in  on  liver  liver  various  7  incubation  rat liver  testosterone  Displacement trom  incubation  fractions  microsomal 8.  and  E t t e c t of varying on  6°  7  temperature  cytosolic  and  binding  7  ^  oft r l t l a t e d - t e s t o s t e r o n e  cytosolic  and  competitors  microsomal  fractions 81  (A-D).  S i n g l e p o i n t displacement s t u d i e s 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 f r a c t i o n s Separation  of t e s t o s t e r o n e  metabolites Separation  using  and  related  t h i n - l a y e r chromatography  of e t h e r e a l e x t r a c t s taken from  l i v e r c y t o s o l incubated i n t e r v a l s and testosterone  at v a r i o u s  t o r d i f f e r e n t time temperatures with  53-DHT  and  Comparison of l i n e a r and  non-linear  r e g r e s s i o n a n a l y s i s of b i n d i n g data E f f e c t of v a r y i n g triamcinolone binding  excess molar r a t i o s of  acetonide  or C o r t i s o l  on  R1881  i n a d u l t male r a t l i v e r whole c y t o s o l . . .  S i n g l e p o i n t displacement s t u d i e s of 8 nM tritiated-R1881 in  by v a r i o u s  competitors  liver cytosolic fractions  S i n g l e p o i n t displacement s t u d i e s of 1 nM tritiated-R1881 in  by v a r i o u s  competitors  liver cytosolic fractions  D i f f e r e n t methods to separate bound  and  tree s t e r o i d f r a c t i o n s 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 b i n d i n g by v a r i o u s a d u l t male r a t  competitors i n  R1881  X I V  Page 18.  19.  Binding  kinetics  binding  p r o t e i n by v a r i o u s  Age d i f f e r e n c e s liver  liver  cytosolic  Effect  between male and f e m a l e  Effect  of  castration  and  on l i v e r  testosterone  cytosolic  of  of  on l i v e r  flutamide  attinity-low Effect zinc  of  cytosolic  cytosolic  high  and f l u t a m i d e  on l i v e r  capacity  aftmity-  replacement  high  cytosolic  and on  affinity-low  R1881 b i n d i n g p r o t e i n  152 hydroxide high  R1881 b i n d i n g p r o t e i n  streptozotocin  insulin  testosterone  R1881 b i n d i n g p r o t e i n  administration  23.  150  h y p o p h y s e c t o m y and  low c a p a c i t y Etfect  R1881  protein  replacement  22.  R1881 148  high a f f i n i t y - l o w capacity  21.  rat  protein  replacement  binding  R1881  competitors  high a f f i n i t y - l o w capacity  binding 20.  of  155  protamine liver capacity 157  XV L I S T OF FIGURES Page 1.  E f f e c t of testosterone testosterone  2.  l e v e l s i n sham-operated  castrated  a d u l t male r a t s  E f f e c t of  t l u t a m i d e on p r o s t a t e  weight r a t i o s 3.  e n a n t h a t e on serum and 55 t o body  i n a d u l t male r a t  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  59 benzo(a)pyrene  hydroxylase i n testosterone-treated  castrated  a d u l t m a l e and i n t a c t a d u l t f e m a l e r a t s 4.  E t t e c t of  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  testosterone castrated  62  reductase  in  testosterone-treated  a d u l t male and i n t a c t  adult  female r a t s 5.  67  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 testosterone  reductase  i n the i n t a c t a d u l t  female r a t 6.  (A-C).  7  Gas c h r o m a t o g r a p h y - m a s s  (GCMS) a n a l y s i s testosterone liver 7.  (A&B). of  of testosterone  metabolites taken  and from 97  T h i n - l a y e r radiochromatogram  testosterone  (A&B).  spectrometry  cytosol extract  metabolism i n v i t r o  i n a d u l t male r a t 8.  0  Thin-layer  cytosolic  102  l i v e r whole c y t o s o l chromatographic a n a l y s i s  testosterone  metabolism i n v i t r o  of 106  xvi Page 9.  Etfect  o f v a r y i n g the p r o t e i n c o n c e n t r a t i o n on  cytosolic rat 10.  R1881 b i n d i n g i n t h e a d u l t  liver  (A-C).  HO  Effect  concentrations on l i v e r 11.  male  (A&B).  of v a r y i n g p r o t e i n and i n c u b a t i o n t i m e  cytosolic  intervals  R1881 b i n d i n g p r o t e i n ' .  Computer a n a l y s i s  of  R1881  binding data 12.  (A&B).  Effect  on l i v e r adult 13.  rat  of  cytosolic  10 mM p o t a s s i u m  chloride  R1881 b i n d i n g i n 124  Specificity studies for  liver  capacity 14.  117  male r a t  (A&B).  adult  male  cytosolic high a f f i n i t y - l o w R1881 b i n d i n g p r o t e i n  Scatchard  plot  f o r a d u l t male r a t  cytosolic  tritiated-mibolerone  137 hepatic  binding protein 15.  H2  Sex d i f f e r e n c e s  144 i n adult  R1881 b i n d i n g p r o t e i n  rat  hepatic  cytosolic 147  L I S T OF ABBREVIATIONS  ACTH  adrenocorticotropic  hormone  Aldosterone  3,20-diketo-llB,18-oxido4-pregnene-18,21-dioi  alpha  Greek  5 alpha/beta-Aalpha/beta,  17  3  letter  (a)  5a/g-androstan-3a/B,17B-diol  beta-diol  Androstenedione  A-4-androsten-3,17-dione  beta  Greek  bGH  b o v i n e g r o w t h hormone  Bmax  maximum b i n d i n g  capacity  BP  benzo(a)pyrene;  3,4-benzpyrene  BPH  benzo(a)pyrene  Cortisol  hydrocortisone;  letter  (e)  hydroxylase 11B,17B,  21-trihydroxy-4-pregnene-3, 20-dione Cyproterone  acetate  17 ot-acety l o x y - 6 - c h l o r o - l , 2dihydro-(lB,26)-3'Hcyclopropa-(1,2)pregna-1,4,6triene-3,20-dione  DES  diethylstilbestrol  DCC  dextran  DHT  dihydrotestosterone; 5a/B-  EDTA  coated  androst-2-en,  ethylenediamine acid  charcoal  17B-ol  tetraacetic  xviii Estradiol  l,3,5-estratriene-3,17  F  free steroid  FSH  follicle  Flutamide  a,a,a,-trifluoro-2  B-diol  concentration  s t i m u l a t i n g hormone  -methyl-4'-nitro-m-propionotoluidide Flutamide hydroxide  a, a, a , - t r i f l u o r o - 2 methyl-2-hydroxy-4 -nitro-m1  propionotoluidide fmol/mg  femtomoles per m i l l i g r a m  Gx  castrated  HALC  high a f f i n i t y - l o w  Hypox  hypophysectomized  IP  intraperitoneally  Kd  equi l i b r i u m . ^ d i s s o c i a t i o n  capacity  constant Ki  e q u i l i b r i u m i n h i b i t i o n constant  LAHC  low a f f i n i t y - h i g h  LH  l u t e i n i z i n g hormone  Methy1trienolone  17 B-hydroxy-17-methylestra-  capacity  4,9,ll-trien-3-one Mibolerone  7a,17a-dimethyl-19nortestosterone  MSTFA  N-methyl-N-trimethylsilyl trifluoroacetamide  3-MC  3-methylcholanthrene  MOX  methoxyamine reagent  NADPH  reduced  nicotinamide  dinucleotide NAD  nicotinamide  adenine.  phosphate adenine  dinucleotide Ovx  ovariectomized  Progesterone  A-4-pregnene-3,20-dione  Ps  pseudohermaphrodite  PZI  protamine  rGH  rat  R1881  methyltrienolone  RU23908  5,5-dimethyl-3-(4-nitro-3-tri  zinc  growth  male  insulin  hormone  fluoro-methyl)phenyl)imidazolidine-2,4-dione RU2956  176-hydroxy-2,2,17a-tri methyl-estra-4,9,11-trien3-one  SB  specitic  binding  sc  subcutaneously  Spironolactone  17-hydroxy-7-mercapto-3-oxo17a-pregn-4-ene21-carboxylic lactone,  STZ Streptozotocin  a c i d gamma  7-acetate  streptozotocin 2-deoxy-2-((methy-nitroso amino)carbonyl)-amino (-D-glucopyranose)  2,3,7,8-tetrachlorodibenzo-  TCDD  p-dioxin TE  testosterone  TLC  thin-layer  TR  testosterone  Testosterone  4-androsten-  Triamcinolone  acetonide  enanthate chromatography reductase 3-one-17B-ol  9 a - f l u o r o - H B , 16a , 17,21-tetra-ol-pregna-l, 4-diene-3,20-dione 17  acetal  with  cyclic  acetone  ACKNOWLEDGEMENTS  I  wish  to express  supervisor, patience, this  Professor  insight  thesis.  committee,  my Gail  sincere  D. B e l l w a r d  and g u i l d a n c e  I would  also  the previous  wish  members  R.C.K. P a k , L . S . G o n t o v n i c k , Finlayson) valuable  t o my  f o rher  throughout t o thank  stalward  the course of  t h e members o f  of our laboratory (Drs.  B.L. Warren  and Mr. A.B. F a w z i , suggestions.  appreciation  f o rtheir  a n d M.J.P. support  and  1  INTRODUCTION  1.  Age  steroid  age  and  sex-dependent  has  been  and  sex  differences  of  duration  (1937) were  one  differences  in  they  of  of  adult  that  rat  secretion  thought  be  that  of  to  the  replacement  to  observations  1958),  in  al.  1978  and  al.  1976). of  hepatic  ),  certain in  Adult  certain  equipotent  the  of of  than  the  adult  female.  drug  have  of  tupaia  or  male  rats  are  drugs  such  as  the  on  adult  adult  difference male  the  were  to  male. was  since  the  similar  to  testosterone the  in  sex  the  workers  rat  and  systems. rats  (Conney  tree  in  male  numerous  using  known  al.  sex  the  the  exist  mice  the  phenobarbital)  report,  made  s t r a i n s of  anaesthetic  or  metabolizing  been  et  barbiturates  re-established  differences  the  alter  Hoick  Further,  early  influence  barbiturate  in  sex  species,  report  that  testosterone  this  sex  to  amobarbital  castrate  that  action  castrated  Following  al.  doses  and  therefore  action.  duration  for  which  and  groups  longer  of  the  the  mammalian  example,  show  of  confirmed  other  was  intact adult  difference. have  the  responsible  times  drugs,  anaesthetic  Endogenous  sleeping  drug  in certain  factors  earliest  (hexobarbital, female  two  that  i n t e n s i t y of  the  the  are  certain  and  observed  anaesthesia the  well-documented  metabolism  latter's  rat;  microsomal  metabolism  It  rate  hepatic  (Quinn  1967;  shrew  e f f e c t s compared  Brown  et et  higher  in order to  et  (Litterst  reguire  hexobarbital,  For  to  females  of  >  2  equal  age  (1967), that the  (Quinn  et  a l . 1958).  Litterst  et  a l . (1976)  many  laboratory  squirrel  rabbit  animal  monkey,  exhibit  has  been  sex  differences  no  the  sex  suggested  However, and  Guidicelli  i n drug  workers  such  p i g and  differences  by  other  species,  guinea  C a s t r a and  as  the  i n drug and  metabolism  have  the New  found  Rhesus  metabolism.  action  and  Zealand  Tillement  and  Gillette  It  (1977) may  that  occur  in  man. Sex  dependency  cytochrome enzyme  hydroxylase  Litterst  aminopyrine (BPH)  have  (Litterst and  et  metabolism  a l . 1976; 1980).  does  not  measurement  may  appropriate  animal  El hepatic also by had  very  i n sex  similar  microsomal  species  benzo(a)pyrene  enzymes  dependency  drug  and et  sensitivity the  enzyme  a l . (1974)  animal's  hormones. of  ethylmorphine  age,  Immature  cytochrome drug  Gontovnick  in  drug  species, of  the  its  assay,  the  system. showed  m e t a b o l i z i n g enzyme  the  rates  1977;  i n a l l mammalian the  et a l .  hydroxylase  sex-dependent  sex  on  (excluding  ethy lmorphine,  Although occur  and  E l Masry  Parker  E l Masry  upon  be  other  and  model  microsomal  changes  to  zoxazolamine  ( E l Defrawy  while  and  drug m e t a b o l i z i n g  Gurtoo  depend  Defrawy  dependent  r a t and  a_l. 1976),  observed  in a l l rat hepatic  aniline,  sex-independent  been  Bellward  of  i n the  N-demethylase,  et  occur  microsomal  Activities  tupaia) are  1974;  not  dependent  systems.  biphenyl the  P-450  does  as  P-450  metabolism,  some  activities  would  male  that  and  be  predicted  female  dependent but  as  are  rats hepatic  these  3  animals  aged,  the  N-demethylase)  activity  i n the  male  male  female.  These  Bellward  (1980),  who  measured  microsomal  BPH  enzyme  enzyme  female  results  activities  inhibition  of  BPH  activities  enzyme  the  Post-natal  (6  activities  were  were  flavone day  20  only  the  enzyme  inhibited became this  by  coincident showed  that  metabolized  of  adult  to  man  a  common  P-450  male  flavone, while  drugs  as  activities other  of  from  rat  the  data  20  the  After  puberty, be rat  suggest  that  development,  Pelkonen  weeks  at  liver  old)  rates  et  liver  a l .  (1973)  microsomes  approximating  weight).  is therefore a  animals.  This  is implicated  i n hormonal  activity.  but  testosterone, aniline,  aminopyrine  ( p e r gram  day  male  changes. (9-22  by  that  during  fetal  and  to  is altered  hormonal  BPH  rat continued  These  1975).  7,8-benzoflavone,  onset  inhibition.  in  dependent  and  female  rat  the  female  the  to  by  Gelboin  grew  and  male  and  with  the  immature  (Wiebel  birth)  the  differences  demonstrated  coincident  mechanism  Differences  Age  cytochrome  animals  to  Gontovnick  activities. been  the  immature  enzyme's  and  and  and  this  such  enzyme  adult  by  inhibited  human  certain both  the  activity  levels  confirmed  or  older,  the  ethylmorphine  age,  as  from  with  same  by  resistant  enzyme  the  in vitro  after  (ethylmorphine  stimulated in vitro  actually however,  also  enzyme  increased relative  constitutive  days  diminished  of  were  have  this  rat  immature  liver  and  of  status  does  30-35%  Age-dependency phenomenon not  common  preclude  that  in a l l species.  would  make  of  comparisons  and  4  interpretations Nevertheless, hepatic are  of  age  microsomal  The  steroid It cover  role  of  i s not  the  (Lee  various  regulation  adult  male  castration  and  induction and  by  of  drugs  physiologically.  microsomal  drug  and  Tribe  activities  may  and  these the  On  the  with  without  Mannering animals  1965;  of  normal  enzyme  rat liver  near  female  the  the  Castration aminopyrine the  Km  microsomal  levels  (El  effects  of  had  and  enzymes  Mannering  adult  female  rat  little  effect  on  Furthermore,  dose  prostate:body weight  of  microsomal  E l Masry  activities. a  in  Of  Testosterone  hepatic  at  found  1979).  increased  male  1974).  be  and  cytochrome  rat.  and  ovariectomy  testosterone  Eraut  microsomal  estrogen replacement  microsomal  can  involved  i n the  E l Defrawy  o t h e r hand,  and be  reversed  sex-dependent  in primary  microsomal  1965),  to  extensively  information  hepatic  N-demethylase  1974).  maintaining  1978;  Gillette  to  testosterone  this  constant) of  Gillette  administration  of  review  rat impaired hepatic  to on  role  this  testosterone  enzyme  E l Masry  replacement  hepatic  difficult.  hormones  i n hepatic  of  Laycock  ( K a t o and  ethylmorphine  and  purpose  sex-dependent  (Michaelis-Menton  or  most  typifying  steroid  pharmacologically  effects, of  metabolism  (Kato  by  development;  and  dependent  Defrawy  data  differences  enzymes  physiological  elsewhere  the  sex  animal  testosterone  the  sexual  P-450  and  metabolism  secondary  its  and  important both  2.  human  capable ratios  of  and  hepatic  of  5  microsomal the  adult  BPH  of  of  (Bellward  may  be  testosterone already  exogenous alter  this  final  of  to  short  Since  during  r a t to exogenous and  Denef  pattern  Cortisol  of  later Chung  other  et a l . 1975),  ethylmorphine (1968)  by  metabolism indicated  who  i n the maintenance  female-type  of  characteristic  Cortisol male  after  the  growing  secretion  was  showed  The  reported that  work  rather  by  et a l .  than  The  and  DeMoor  neonatal or  adult  observations  testosterone  immature  pattern.  the  a l t e r e d the These  a  The  first  (Einarsson  measured  the  further  puberty.  workers  workers  metabolism  metabolic  not  r e s p o n s i v i t y of  c a s t r a t i o n of of  have  activity.  in vitro.  in vitro.  that  would  dramatically  metabolism  plasma may  of  p o s t - n a t a l l y , and  and  These  males  of  testosterone  the  influence  lack  levels  testosterone  (1968).  neonatal  confirmed  on  to  dose  This  development,  of  same  i n the male r a t ,  s u r g e s p r e - and  androgens  failed  enzyme  normal  the  the  testosterone  effects  phases  secretion  neonatal  male  resulted  high  in  ovariectomized  male  administration  three  of  Denef  1979).  of  influence  activities.  already  i t s maximal  castration  1973;  adult  enzyme  the  the course  1979):  DeMoor  were  intact  activities  I n j e c t i o n of  testosterone-dependent  prolonged  effect  to  (Forest  i s exposed  (Forest  by  the  testosterone  During  adult  to  to  i n the a d u l t  e t a l . 1982).  due  exerted  rat, failed  enzymes  drug-metabolizing  effect  rat  male  these  testosterone  these  aminopyrine N-demethylase  castrated  activities female  and  and  males  adult the  same  workers  6  (Denef adult  and  DeMoor  rat  to  the  or  female  to  same  acetate, 1970).  1972)  androgen  an  blocked  the  neonatal  imprinting.  effect  Neonatal  a  female  and  levels al.  DeMoor  Denef is  for  may  allows lack  be this  of  or  Bellward  (1968)  the  enzyme  et  pubertal  but 10  male  previous  a  in  single  days in  of enzyme  (Chung  et  reports  a l . (1973)  by  that  differentiation  reductases.  of  The  periods  of  rat  irreversible  liver  enzymes  testosterone  expressed.  This  would  in hepatic  the  female  Although  the  mechanism  i s not  to  the  male  secretion  in  induction  blocked  resulted  resulted  that  of  i t  20),  "critical"  responsiveness  a l . 1982). enzyme  and  at  activites  et a l .  than  sexual  suggests  be  birth  non-castrated  hydroxylases  to  cyproterone  less  Einarsson  "programming"  at  the  male  hepatic  metabolism,  with  in  antagonist  in rat  (day  agree  in  of  castration, since  during  the  testosterone  effect  age-dependent  dose  exposure  (Neumann  androgen  occurs  the  imprinting  Castration  and  involved  testosterone  et  also  results  occurring;  metabolizing  the  in puberty  development  "imprinting"  of  of  neonatal  antagonist  neonatal  those  upon  androgen  ethylmorphine  steroid  requirement  type  to  These  hepatic  post-natal  of  later  similar  testosterone rat  of  r e s p o n s i v i t y of  administration  receptor  replacement  again  and  the  metabolism.  pattern  1975).  by  imprinting  drug  testosterone age,  dependent  i s , addition  mimicked  that  Neonatal  androgen  That  microsomal  was  hormone.  was  showed  explain drug  (Colby  clearly  the  1980;  of understood,  testosterone-mediated age  and sex d i f f e r e n c e s  activities secretion enzyme  would  induction  explain  mechanism  as suggested underlying  hepatic  role  drug  and s t e r o i d Although  the exact  metabolism  (Kramer  locus,  intact  of  (1974).  Though  androgen  receptor  secretion  drug  the exact . imprinting clear  receptor.  i n hepatic  is clearly  i n r a t hepatic  microsomal  drug  i s n o t known.  both.  implicated and  testosterone  may  since  testosterone  and  to stimulate  results,  requires  testosterone (direct  could  steroid  There i s  that  These  testosterone  of the l i v e r  or  would  i s not r e a l l y  hepatic adult  however,  therefore  be  acting  microsomal male r a t suggest  the presence  that  of an  act either a  action), or at l o c i  the 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 axis  action),  of  puberty  of neonatal  i n the hypophysectomized  pituitary;  level  along  failed  e t a l . 1979).  action  the lack  i n r a t hepatic  induction  mechanism  to suggest  an e x t r a h e p a t i c  drug  microsoma  metabolism  testosterone  evidence  and  the  testosterone  of hepatic  or after  a cytosolic  o f an a n d r o g e n  dihydrotestosterone  the  enzyme  age and sex d i f f e r e n c e s  metabolism,  the  by Kato  to involve  The  at  during  t h e phenomenon  microsomal  i s thought  strong  male  explain  enzyme  pubertal  agent  i n the pubertal  i n the female  3.  in  Furthermore,  the causative  would  microsomal  t h e s e x and age d i f f e r e n c e s  metabolism  and  be  imprinting  i n hepatic  i n the r a t .  testosterone  it  neonatal  located  (indirect  8  (a)  Indirect  microsomal The  drug  role  differences reviews  of  section  and  of  steroid  the  i n drug this  Gustafsson  e f f e c t s of  will  and  Colby  briefly  hepatic  has  been  implicated  s t e r o i d metabolism. can  1980;  cover  be  action  drug  s t e r o i d metabolism  found  Finlayson  the  indirect and  of  on  metabolism  pituitary  literature  1978;  testosterone  androgens  sex  Extensive  elsewhere 1983).  (Skett  The  supporting  the  hepatic  microsomal  enzyme  modulation  of  and  following  evidence  on by  in  pituitary  secretions. Denef the  (1974)  removal  shift  to  while  the  of  the  that  male  or  the  Similar  of  found  by  were on  that  from  rat.  on  the  similar  no  of  apparent  resulted  in a  in  had  the  to  sex  These  Chung  rat  c h a r a c t e r i s t i c of  of  mentioned  either  a l . 1975).  male  e f f e c t on  lack  to  ethylmorphine  that  administration  et  effects  metabolism.  adult  of  no  vitro,  e f f e c t of  drug  Km  to  the  that  effect,  (DeMoor  the  microsomal  to  note  administration  measuring  s t e r o i d metabolism  dihydrotestosterone  had  rats  the male-type  to  metabolism  metabolism.  testosterone  done  rats  i s fundamental  Hypophysectomy  Furthermore,  effects or  rat.  female  i n the male  hypophysectomy  N-demethylase  workers  testosterone  hepatic  altered  female  first  from  testosterone  significantly  the  the  hypophysectomized  studies  hypophysectomy (1977)  of  pituitary  influenced female  of  pituitary  same m a n o e u v e r  differentiation not  one  the male-type  suggesting  were  was  the  female  testosterone  above,  d i d not  testosterone influence  9  hypophysectomized that  the  effect  male  of  rat liver  androgens  drug  required  hypothalamic-pituitary  stalk.  those  Gustafsson  Lax  of  et  Denef  (1974),  a l . (1974),  testosterone (1979)  who  who  studied  This  measured  metabolism,  metabolism,  and  an  intact  observation supported  and  Stenberg  hepatic  was  indicating  and  microsomal  confirmed  hepatic microsomal  (1974),  by  Kramer  et a l .  ethylmorphine  N-demethylation. The and of  loss  steroid male  those  drug  the  many  were  caused  (ie.  shift  effects  stimulating  secreted steroid hepatoma  male  hormone of  showed from  under  female-type those  which  to  of  shift  towards steroid  the  male,  pituitary  factors  that  hormone  normal  the  (LH),  adult  caused  metabolism),  host  serum  levels,  hence  was  extract  not  and  The  using two  cultured types  the known.  that  "feminization"  in vitro  but  follicle  substance  pituitary  hepatocytes.  of  an  m e t a b o l i z i n g enzymes  prolactin)  tumour  capsule  steroid  with  "feminizing"  pituitary  isolated  steroid  correlate  or  i m p l a n t a t i o n of  renal  female-type  (FSH)  the  that  the  of  metabolizing capacity and  ie.  activitites  towards  showed  (luteinizing  identification also  gland  d i d not  gonadotrophin  of  drug  phenomenon.  "feminization" from  shift  investigate  a_l. (1982)  pituitary  rats  the  activities  underlying this  ectopic  They  or  rat hepatic  hypophysectomy,  m e t a b o l i z i n g enzyme  enzyme  et  in adult  following  researchers to  Skett  these  dimorphism  female,  metabolizing led  sex  metabolism  type  of  of  of  of  the  pituitary were  extracts  found  in  gradient.  the  prolactin,  the  steroid  identity  Although Skett not in  the  true  various  enzyme  nature  a  failed  to  activity  "feminizing"  (1979)  had  coined  FSH,  in vitro,  and  unknown.  f a c t o r or  "feminotropin"  would  LH,  stimulate  remained  the  as  support  as was  i t s presence  pituitary.  Gustafsson hypothalamic female-type proposed  release  et  a l . (1978)  lesions of  that  pituitary  in  hepatic a  the  adult  microsomal  "feminizing"  i n both  showed  adult  male  and  rat  rats,  in  inhibition  from  higher  centres,  by  the  action  of  feminotropin  (feminostatin).  Skett  and  Gustaffson  suggested  that  secretion  serum  testosterone  would  be  the  levels  maintenance  "feminizing"  f a c t o r on  metabolism.  This  metabolite  may  secretions  along  at  the  a  the  in of  adult  would  act  of  from  but  i s under  ( i e . hypothalamic  the a  male  latter adult  tonic male  was  suggest  that  central  locus  the tonic nuclei  (1978)  male.  The  and  further  net  by effect  of steroid  testosterone to  the  modulated  hepatic  and  inhibiting  inhibition  rat  a  metabolism,  factor  areas)  to  is secreted  female  adult  led  this  adjacent  the  discrete  steroid  factor  male  that  of  factor  such  somatotropin,  factor  studies  density  hormones  of  above  activity  sucrose  rat  corticotropin,  "feminizing"  the  of  known  in combination  Gustafsson  identified, the  and  "feminizing"  fraction  metabolizing  of  the  and  of  oxytocin,  alone  contained  granular  Screening  vasopressin,  hepatic  which  modulate  hypothalamic-pituitary-1iver  or  its  pituitary axis.  This  would  Specific  require  androgen  hypothalamus, Gustafsson nervous sexual  steroid  To  studies further  androgen  l e d other  sex  and age dependent  the  secretion  hepatic  of growth  s t e r o i d metabolism  and  Colby  ethylmorphine  f o r 10  to a l t e r  towards  reported  (GH).  that  days,  demethylase,  reduced  reductase  castrated  adult  In a d d i t i o n ,  aniline  demethylase  hydroxylase  o r TR  adult  not  influence  activities. to  recover  Hormone these  hypophysectomy. GH  caused  male.  These  these  an e s t r o g e n - t y p e  GH,  to attribute  secretion  like  of  to  this  liver  levels.  bovine  drug  Rumbaugh  estradiol  microsomal a n d BPH,  GH  but  i n the  decreased  a n d h a d no e f f e c t o n i n the  hepatic studies  activities authors  search  Estradiol administration  replacement  enzyme  The  hepatic  activities  hypophysectomized significantly  the  (TR) a c t i v i t e s  a n d BPH,  done.  indirect  a n i l i n e hydroxylase  testosterone  ethylmorphine  not been  s t e r o i d metabolism  female-type  increased  male.  have  the male-type  bovine  to the  metabolism.  workers  and  e t a l . 1975;  in part  of  inrat  central  metabolism,  drug  hormone  i s known  and  administration  the d e s c r i p t i o n  factor"  the "feminizing  of these  hypothesis  enzyme  for  (Naess  lead  hepatic  brain.  characterized  gland  may  i n the  s t e r o i d and drug  this  complete  (1980)  been  receptors  to test  hormone  sites  Stimulation  of hepatic  e f f e c t s on  pituitary  have  and p i t u i t a r y  e t a l . 1976).  dimorphism  binding  receptors  cortex  system  Rigorous  androgen  which  found  e f f e c t on  that  microsomal  did  enzyme  were done  in  were  following  lost  order  administration  hepatic  enzyme  of  activities with  the  in  hypophysectomized  combination  administration Furthermore, activities  of  the  hypophysectomized action  of  differences  Mode  et  al.  hepatic  males,  group  in  rats  using  osmotic  or  by  animals. metabolism of  the  then  support  and  were  treated This  castration  found  using  female-type  of  in  involved  in  metabolism. steroid  studies et  of  GH  Differences have  in  the  that  et  the  in been  rat.  the  On  male  with  in  human  influenced  GH  treated in  the  hepatic  sexual  of  the  was  same  dimorphism  hepatic  pattern  the  Saunders  et  a l . 1979;  an  is  of  of  had  the  steroid  basis  rat  in  results  compound  the  the  regained  hepatic  to  by  steroid  These  related  GH  adult  Further,  female  attributed  a l . 1976; adult  a  on  of  metabolism  not  treatment.  differentiation  sexual  GH  s t e r o i d metabolism  the  GH  feminization  human  adult  or  the  metabolism.  treatment.  hepatic  GH  that  e f f e c t s of  enzyme  e f f e c t s on  GH  these  continuousy  the  effect.  GH. the  that  (Tannenbaum  a l . 1979)  by  thyroidectomy  rat  no  concluded  hypothesis  metabolism  secretion  human  had  treated  treated  e f f e c t was  and  feminizing  continuous  the  the  complete  hypophysectomized-ovariectomized following  ACTH;  4-androstene-3,17,dione  adrenalectomy  were  ACTH  examined  a  were  alone  microsomal  reported  which  influence  mediated  hepatic  which  Similar  plus  also  minipumps.  hypophysectomy  not  i t was be  and  hormones did  thyroxine  microsomal  male  loss  latter  estrogen  (1981)  adult  males,  thyroxine  e s t r a d i o l may  Gustafsson's sex  the  since  in  of  males  of  earlier  episodic  Eden GH  secretion regular (1982)  pattern  pattern  on  led  GH,  hepatic  to a  female,  female  GH  of  treatment  well  with  These  differences  the  female  with  or  in  hours  GH  r a t , Mode  levels  e t a_l of  the infusion  every  failed  without  more  frequencies  hormone  3-6  of  hours  adult  to  i n the  have  this  castrated  testosterone  in hepatic that  involved  in rat hepatic  the  hypophysectomized  suggest  be  to  Continuous  this  changes  results may  rat.  12  plasma  the  secretions  of  every  of  rat treated  metabolism.  sex  adult  Measurement  correlated  adult  s t e r o i d metabolism  administration  while  male  plasma  i n the  refeminization  effect. adult  or  i n t e r v a l ) compared  different administration  hypophysectomized human  hrs  present  compared  human GH  (3-4  the  i n the  microsomal  steroid pattern  of  regulation  of  steroid  metabolism.  (b)  Direct  microsomal  e f f e c t s of  drug  (i)  In  The  role  and  vivo  metabolizing  animal  steroid  of  the  systems  receptor  has  implicated  been  pseudohermaphroditic  phenotypically  expressed  et  It  functional  as  i s thought  androgen  pharmacological  hepatic  metabolism  androgen  is genotypically  a l . 1973).  on  evidence:  Stanley-Gumbreck male  testosterone  receptor  doses  of  male  (XY)  on  hepatic using  the  (Ps) male but  drug  rat.  since  this  animal  i t does  testosterone  P  i t is  a pseudohermaphrodite that  The  not  (Bardin  (Stanley  lacks  a  respond et  to  a l . 1973),  even  though  s i m i l a r doses  hexobarbital  sleeping  N-demethylase enzyme females Roy  and  1973;  androgen adult  The  not  be  may  postulate  have  a  and  receptor  male.  only  defect  androgen  reported  reason  that an  rats.  Bellward  et  and  TR  Normal  sex  and  activities  TR  comparing  These  the  above  the  male  of  a  contrast to  the  adult  to  were  control normal  in a  stimulate  r e s u l t s have  of  induction  male,  female  between  the  Ps  adult  male  drug  and  adult  male,  and  however  serum  BPH  rat  than  TR  the  Ps  aminopyrine in  (PB)-  hepatic Ps  male,  confirmed  by  and  microsomal  male r a t . and  none  female  were  rats.  seen  when  levels,  activities  r a t was  BPH  Despite  typical In  testosterone  male  an  recently  s t e r o i d metabolism. to  may  have  testosterone  and  may One  enzyme  male  to female  male  an  intact  faulty  been  of  control  observed;  show  receptor  phenobarbital  i t s non-responsitivity  castrated  to  other  a l . (1979)  can  r a t demonstrated  female-type  to  and  insensitivity.  locus  result et  (3-MC)  differences  having Ps  can  a l . (1982) m e a s u r i n g  i n the  failed  androgen  intact  (Milan  a n i l i n e hydroxylase a c t i v i t i e s  3-methylcholanthrene BPH  i n both  r a t compared  intracellular  phenobarbital  female  metabolizing  workers  have  functional  Sonawane  and  Ps  male  ethylmorphine  p s e u d o h e r m a p h r o d i t i c male  which  N-demethylase and  a  Other  1983)  reduced  steroid  for testosterone  the  system. that  of  and  a l . 1972)  rats.  Ps  hormone  increased  et  Levinson  i n the  receptor,  induction  and  same  e t a l . 1971)  male  lack  at  the  (Einarsson  castrated  Decker  the  times  (Bullock  activities  of  compared  equally  as  responsive suggest that  to  that  PB  or  the  3-MC  drug  similar  pathway. to  the  presumably  Though  involves  inhibition  of  microsomal  drug  al.  by  the  the  anti-androgen, microsomal BALB/cJ Fisher  rats.  flutamide's microsomal to Lax  be  reduced  protein  testosterone  the  It  is difficult  of  action  types  of  of  female  draw  androgens,  from  be  in  to  on  but  given  be  to  may  be  by  enzyme  activity Brown  in  castrated  pre-pubertal quite weight  More  intact  or  castrated  different  recently, greatly  on  the  hepatic  animal  seemed  testosterone  effect  various  from  and  flutamide  regarding  male  distinct  no  using  et  hepatic  had  on  the  non-steroidal  receptor. that  in  hepatic  whose m e c h a n i s m  flutamide  studies  in  activity  liver  conclusions or  a  which  antagonists.  shown  the  by  involved  (DHT)-stimulated  rat,  testosterone  metabolism  of  specific  effects  have  and  typified  changes  intact  seemed  androgen  (1981)  to  a  and  action  the  metabolism  one  to  N-demethylase  content, of  class  testosterone-induced  dihydrotestosterone in  seem  androgen  effects  intact,  receptor.  flutamide,  non-specific  Schriefers  metabolism  steroid  of  results  testosterone  metabolizing  in castrated  These  independent  and  that  blocked  and  of  some w a y s ;  responses  steroid  These  i s mediated  inducer  androgen  ethylmorphine  mice,  effects  androgen-stimulated  and  showed  in  rat.  mechanisms were  enzyme  administration  (1976)  female  induction  the  i t differs  Receptor-mediated  caused  enzyme  classical  phenobarbital,  the  induction  testosterone-type  separate  as  male  rat.  mechanism drug  doses  and  and  models,  and  measuring either alter  enzyme  a t an h e p a t i c drug  direct than  different  The hepatic  role  workers  of a c y t o s o l i c  by P o l a n d (Poland  most  enzyme  receptor  induction  and co-workers  e t a l . 1976) have  of hepatic  species  aryl  (Poland  affinity  cytosolic  C57BL/6J  mice  and G l o v e r  receptors  (binding  in  of Sprague-Dawley less  (Bmax)=  than  rank-order  to their  dissimilar  structure  receptor.  induction  by t h e TCDD-type  cytosolic  receptor, that  (TCDD),  drugs  and would  a  potent (AHH) i n to  high  i n the l i v e r nM;  cytosolic  o f AHH,  maximum p r o t e i n ) , and nM;  while and  to the l a t t e r  induce  Bmax  shown  was  that the  drugs of steroid  d i d n o t compete  appears  cytosol of  c o r r e l a t e d by  f o r this  drugs,  to involve  be c o n s i s t e n t  drugs  These  hydroxylase  were  in  that  as p h e n o b a r b i t a l  In c o n t r a s t  TCDD-type  1976).  I t was a l s o  inducers  testosterone,  cytosolic  observation  less  previously  (Kd= 0.2 - 0.3  inducibility such  has been  ( K d ) = 0.27  84 f m o l / m g ) .  o f c e r t a i n TCDD  including  located  rats  (Ah r e c e p t o r )  shown  84 f m o l / m g  affinities  hormones  a  has been  1974) c a n b i n d  affinity  capacity  slightly  receptor  (1974,  hydrocarbon  binding that  to suggest  difficulties.  2,3,7,8-tetrachlorodibenzeno-p-dioxin inducer  be a c t i n g  evidence:  microsomal  proposed  may  or elsewhere, to  evidence  androgen  due t o t e c h n i c a l  In v i t r o  Flutamide  receptor  metabolism;  at the hepatic  convincing,  (ii)  androgen  and s t e r o i d  action  systems.  with  AHH  t h e TCDD  the  a different  spectrum  of  microsomal  inducers noted in  (Conney  that  end-organ  to that tissues  involve  drug  inductive enter  metabolizing  effect  similar  There  protein  assumed  that  proteins  Of  anti-androgens 1969; Jung 1972),  nortestosterone  to  located  DNA-RNA  be  a s an h e p a t i c this  i n the  increased  I t should  effect  such  SKF 7690  noted enzyme  isa  1971),  studies  that  i n many  cytosolic  tissues  t i s s u e s , the androgen  has been  well-characterized.  as cyproterone 1971),  o r 17  1974) have  inhibition  to suggest  these  and B a u l i e u  (Tveter  and Raynaud  competitive  would  and s t i m u l a t e  are located  in prostatic cytosol  Mainwaring  i n other  phenomenon.  e t a l . 1978).  Steroidal  i t may  Testosterone  lead  i s acting  hepatic  t i s s u e s , and t o t h e  synthesis.  testosterone  binding  in classic  then  protein  ultimately  i s s u b s t a n t i a l evidence  (Hiipakka  (Bonne  to i t s effects  i n the l i v e r .  would  mediated  Fang  inducer,  the nucleus  receptor  and  enzyme  into  i t i s tacitly  receptor  as an endogenous  translocate  inducer,  be  i s identical  o f s e x hormones  "receptor"  which  I t should  described  to a  although  androgen  just  sex end-organ  o f TCDD  androgen-dependent that  e t a l . 1964).  bind  transcription  of the s t e r o i d - t y p e  e t a_l. 1982).  i s acting  responsive  the c e l l ,  cytosol,  mechanism  (Grody  a mechanism  testosterone  to that  of the effects  testosterone  microsomal  compared  1967; Kuntzman  the basic  concept  If  enzymes  acetate  B.O.M.T.  (Liao  (Mangan and  alpha-methy1-  RU been  2956 used  and  spironolactone  successfully in  of r a d i o a c t i v e l y l a b e l l e d  DHT  or  labelled  binding  methyltrienolone,  sites  following  in vitro.  binding  androgen steroid  Raynaud  profile  tritiated-testosterone receptor  of  antagonists  (5  rat  antagonist  well  order  the  androgen-induced Attempts the  hepatic  there  receptor  by  androstenedione, experiments. synthetic latter without  as  binds  metabolized  to  serum  in vitro  Gustafsson  et  a l .  and  these  sex  and  (1975)  DHT,  cytosolic  Interestingly, nuclei. cytosolic  Column  DHT  was  not  from  reported  an  male  from  using  and in  not  of  into  nuclear  rats.  male  male  i t is  metabolites  female  castrated  receptor  1976).  its  Sephadex  the  the  and  uptake  the  used  since  1975,  or  exchange  have  proteins,  Raynaud  detected  chromatography  f r a c t i o n s taken  androgen  androgen  tritiated-androstenedione taken  studies.  testosterone  (R1881),  and  livers  the  however,  radio-ligand  into  of  of  characterize  cytosolic  their  binding  >  correlated  various  t r i t i a t e d - t e s t o s t e r o n e and  fractions  data  in vitro,  androgens  the  2956  >  vivo.  pulse-labelled cytosolic  RU  r e c e n t l y , workers  to  (Bonne  cytoplasmic  inhibition  receptor  methyltrienolone  the  testosterone  and  identify  for  more  specifically  binding  the  hepatic  ligands  the  These  in  between  the  to  described  of  in vitro;  for  to  natural  However,  inhibition  acetate  androgen  the  androgen  binding  made  studied  using  nM)  weight  agreement  workers  the  potency  androgen  a l . (1979)  flutamide.  been  cytosolic  et  prostate  prostate  have  is little  Earlier  of  cytoplasmic  for  cyproterone  non-steroidal with  from  rat  G25 rats  liver  of  hepatic  which  were  injected  types low  of  with  radioactively labelled fractions: a  capacity  ("complex fraction  tritiated-androstenedione  A")  which  had  containing  mono-sulfated unbound  tritiated-testosterone indicated  in  10  metabolites  vitro  S  (peak  (peak  C).  B)  of  i n male  complication  pre-fractionating  G100  by  column  Their  chromatography  results  cytosolic  indicated  androgen  androstenedione binding that was  metabolized  to  which  i t was  a  translocated  and  and or  to  The  to  a  studies  testosterone  one  knows  workers  have  little  may  identified  also  be and  up  Sephadex studies.  liver  100  nM,  authors into  the of  with  liver  and  products,  binding  protein,  nucleus.  a  suggested  specifically after  Unfortunately,  (inhibition, responsivity) this  age  and  were  not  protein's  role.  suggested  located  this  was  about  with  at  than  number  latter  the  its physiological  receptor  (1974)  These  using  the  occurred  taken  overcame  binding  of  and  and  cytosol  greater  cytosol  fraction  a  reported  They  saturation  rapidly  the  another  their  c y t o s o l i c androstenedione  out,  Other  to  fmol/mg.  characterization  specificity  al.  was in  sex-dependency,  androgen  0.8  co-efficient,  authors  the  protein  androstenedione.  bound  carried  binding  of  testosterone  further  prior  that  and  cytosol.  concentrations  capacity  including  liver  protein  testosterone  androstenedione  affinity,  tritiated-testosterone  These  metabolism  binding  sedimentation  protein-bound  metabolites  extensive  a  high  three  in  partially  that  hepatic  a cytosolic cytosol.  characterized  a  Roy 3.5  et S  androgen-estrogen male  rat liver  secretion  binding),  done  t o DHT  and t r i t i a t e d - e s t r a d i o l  3.5S  fraction  b y t h e same  that  both  same  extent  total  noted  sites  stable.  0%).  Measuring  displacement steroids,  from the  they  tritiated-DHT  authors  receptor  gradient  binding.  found  Their  results  sites  binding  site  approximately  binding  group  indicated I , Kd= Kd=  following  specific  In c o n t r a s t  to  studies  and measured  total  i n t h e 3.5 S  and  f o r binding  site  II,  nM  a n d Bmax=  and  one b i n d i n g  site  fortritiated-estradiol  using bound  protein  two t r i t i a t e d - D H T  4 5 0 nM a n d Bmax=  2000  they  DHT a n d  and  d i d saturation  analysis  70%  binding  that  a t two d i s t i n c t molecule.  to the  displaced  Furthermore,  suggested  or tritiated-estradiol  fraction.  DHT  tritiated-estradiol  occur  Roy's  displaced  of tritiated-DHT  while  may  work,  density  loss  These  binding  Gustafsson's  (for  alpha-  while  o n t h e same  sucrose  (both  17  binding),  dilution,  estradiol  (8%),  (50% d i s p l a c e a b l e  40%, and e s t r a d i o l  and  specificity to  tritiated-DHT  a differential  remained  competitors,  displaced  tritiated-estradiol  cytosolic  t h e 3.5 S  steroids  approximately of  unlabelled  point  (93% displaceable  17 b e t a - d i o l  tritiated-DHT  i n adult  with the  from  but l i t t l e  or corticosterone  unlabelled  well  Single  different  (53%),  3 beta,  methyltestosterone  correlated  tritiated-DHT  using  and t e s t o s t e r o n e  was p r e s e n t  globulin.  specificity  alpha-androstan-  which  and which  o f 400 nM  were  relative  protein  alpha-2u  studies  fraction  revealed  5  cytosol,  of hepatic  displacement protein  binding  4.2  binding fmol/mg;  1.26 p m o l / m g )  (Kd= 350 nM a n d  Bmax= DHT  42  pmoles/mg).  binding  noted  a  site  3:1  that  inhibitor  of  site, at  a  while  DHT  DHT  the  same p a p e r ,  cytosolic immature  male  male  the  Roy's in  adult  led  activity.  In  to  a  protein  and  an  in  the  yield  explained  by  a  and  of  the  number  the  binding  androgen  protein  binding  et  of  aged  a  a l . 1979).  of  the  specific  other  in  the  of  the  receptor's including  the  between  binding  synthesis the  of  former  was  and  Roy's  Gustafsson's  different  complexes,  However,  this  correlation  that  In  latter.  from  receptors  of  and  tested,  discrepancies, of  subunits  estradiol  androgen  suggested  4  binding.  Castration while  binding  postulated  absence  hepatic  and  binding  estradiol  males,  good  ,  receptor  this  cytosolic  different  androgen  the  models  claimed  binding  estradiol  eliminated animal  3  the  workers  specific  they  livers.  results  to  (Lea  for  these  of  allosteric  of  protein:  decrease,  they  of  strong  androgen  and  rat  hepatic  androstenedione)  multiplicity  a  reported  gradual  group  be  subunit  group  the  estradiol  Further,  this  regulation  Comparison groups  on  estradiol,  inhibitor  site.  capacity  to  the  weak  binding  testosterone-dependent  alpha-2u-globulin, involved  DHT  to  completely  of  of  immature  a l l of  the  might  female  r a t , Roy's  presence  a  regulatory  administration  Ps  sites  protein and  be  binding  for  adult  may  binding 1  of  binding  DHT  and  ratio  on  that  estradiol  different  possible  I I , and  molar  suggested  Based  which  ligands hepatic  consistent in  the  may  be  (DHT  and  cytosolic with  prostate  considerations  the cytosol  such  as  different can  incubation  n o t be r u l e d  crude  cytosol  and they  taken  DHT  against  ligand  (40-93%).  binding  analysis  conclusions incorrect would in  however,  may  differences  was  done  Other  Roy's  occupancy  density  charcoal gradient  of free  DHT  ligand  stoichiometic were  based  on  concentrations,  Furthermore, studies  measurement  differences may  of  compared  and  be  "total"  to the  tritiated-androstenedione group.  This,  f o r the v a r i a t i o n i n binding  considerations  i n the cytosol.  may  the r e s u l t s of the  by G u s t a f s s o n ' s account  to  were n o t  oversight  of tritiated-DHT  i n t h e methods  group  in displaceable  then  i n the c y t o s o l ,  not f u l l y  dextran-coated sucrose  receptor  of "displaceable"  which  capacities.  steroid  This  the concentration  due t o t h e Roy g r o u p ' s  measurement  neglected  and p r e c a u t i o n s  be q u e s t i o n a b l e .  bound  protein  by p r e - f r a c t i o n a t i o n  group  k i n e t i c s i n the saturation  tritiated-DHT  binding  Roy's  inaccurate.  measurements  binding  partially  were  high  Gustafsson's  problem  f o r metabolism,  regarding  therefore  this  inconsistency  Since  s i m i l a r types of  reguired  degradation.  binding  techniques  of the r a d i o l a b e l l e d ligand i n  in vitro,  t h e Roy g r o u p ' s  not corrected  used  40 m g / m l .  In c o n t r a s t ,  explain  was  which  metabolism  metabolism  and s e p a r a t o r y  groups  than  minimized  the c y t o s o l .  measure  Both  greater  significant  vitro, of  out.  preparations  concentrations, noted  conditions  should  of separating  Gustafsson's technique analysis.  be m a d e , bound  group  while  and f r e e  used the  Roy's  Perhaps  including  group  used  the former  technique (Kd= to the  "stripped"  100-400  studies  lower  "displaceable"  i s present  More (1980) the  recently,  hepatic  (1980)  castrated  adult  incubated  with  male  binding using  They  k i n e t i c s : Kd=  using  protein  had a moderate  androstenedione,  contain  less  cytosol. adult  receptor. crude  male  et a l .  to characterize These  cytosol  i n the presence  workers which  used  was  site  with  nM a n d Bmax=  affinity  et a l .  fmol/mg  a n d Bmax=  as l i g a n d s .  sulphate  In  This  DHT a n d later  by t h e s e  that  158  precipitated  components  reported  hepatic  170-313  estradiol.  metabolizing  r a t increased  the following  1130 nM  was s u g g e s t e d  (1981)  and  to testosterone,  ammonium  The l a t t e r  o r absence of  a saturable  binding  137-575  used  androgen  Sato  binding  l e d by S a t o  re-attempted  but d i d not bind  group  cytosol.  group has  cytosol.  tritiated-androstenedione  this  neither  an a n d r o g e n  reported  10 S a n d r o g e n  fmol/mg  liver  group,  t r i t i a t e d - t e s t o s t e r o n e a n d ; Kd=  studies,  specificity  tritiated-testosterone or  competitor.  displaceable  to that of  and t h e l a c k o f  workers  r a t hepatic  tritiated-androstenedione, excess  leading  metabolism and  that  c y t o s o l i c androgen  affinity  complex,  compared  r a t hepatic  Japanese  and Ota e t a l .  group  by Roy's  evidence i n male  protein  due t o i n a d e q u a t e  ligand  DHT b i n d i n g  convincing  protein  Thus,  of i n v i t r o  the moderate  capacities  by G u s t a f s s o n ' s  consideration  from  binding  binding  technique.  done  provided  ligand  nM) a n d r o g e n  relatively latter  more  authors to  than  crude  castration  c y t o s o l i c androgen  of the  binding,  and  decreased  effects  were  Consistent Japanese  injected  with  the r e s u l t s found  with  therefore,  o f Roy e t a l .  binding  and one does during  purified  n o t know  protein  i n the p a r t i a l l y  related  to that  present  or the other Published  articles  hepatic  androgen  current  literature  models  and L u c i e r  presence  of an h e p a t i c  (1982)  different  models.  reported  present female  that  i n the male r a t , and t h a t  presence  of t h i s  Hypophysectomy binding effect  of e i t h e r  protein which  was  with DHT  o f an and t h e  activity,  R o y et_ a l . binding male  administration i n the adult  sex i n h i b i t e d and female  not i n f l u e n c e d  be  to c o r r e l a t e the  N-demethylase  protein  i n the male  could  protein to  cytosolic  estradiol  androgen  of a b s t r a c t s .  b u t n o t i n t h e immature  binding  and  of the adult  sparse,  to a handful  Consistent  hepatic  fraction  cytosol  quite  DHT-binding  ethylmorphine  they  process,  the presence  attempted  androgen-induced animal  binding  tested.  remain  i s limited  Rumbaugh  cytosol  purified  suggesting  receptor  studies  i f the androgen  i n the crude  animal  male r a t  i f the s o - c a l l e d hepatic  binding  male  i n hepatic  saturation  the p u r i f i c a t i o n  i t i s n o t known  these  replacement.  reduction  Unfortunately,  of  (1974), the  i n the adult  on t h e p a r t i a l l y  stable  Both  testosterone  a significant  estradiol.  preparations, was  binding.  following  testosterone  n o t done  protein  androgen  reversed  group  cytosolic  were  nuclear  (1974),  protein or  was  adult  inhibited  the  male.  and induced rats  using  this  r e s p e c t i v e l y , an  by t e s t o s t e r o n e  replacement.  Similar Sato's  to  Roy's  groups,  metabolism the  these  physico-chemical there of  seemed  this  be  binding  binding  a  neglected  workers  binding  to  in contrast  Furthermore,  i f these  androgen  and  authors  in vitro.  abstract  saturable  group,  to  consider not  nor  correlation hepatic  a  reported.  between  drug  presence  metabolism animal  it  known  a  or  coincidental if  these  relationship.  workers  were  cause-effect,  The  latter  measuring  a  and  Although  the  pharmacological  was  from  were  p h y s i o l o g i c a l and if this  ligand  specific  different i s not  and  evident  were m e a s u r i n g  protein,  and  Gustafsson's  i t was  characteristics  good  protein  to  would  in  these  models,  merely  a  seem p l a u s i b l e  nonspecific  binding  protein. The by  the  complication  use  of  a  androgen  receptors  Pousette  human  liver  presence  et  a  10-15  unfractionated  or  DHT.  site  reported  a  R1881  et  been  that  i n both  male  containing  of  either  binding. hepatic  by  shown  taken  to  bind and  from  (Kd=  female  triamcinolone.  with  to Shain  rat  reported  protein and  overcome  androgen  (Boesel  a_l. (1983) binding  been  ligand,  synthetic  cytosol  and  R1881  displaced  Gonadectomy i n R1881  has  Eagon  cytosol  decrease  potent  a l . 1979),  fmol/mg)  was  a  in prostate  saturable  has  unmetabolizable  R1881  cytosol.  of  capacity=  binding  (R1881),  properties.  1974;  ligand metabolism  relatively  methyltrienolone progestin  of  and  the 1.5  rat  nM; liver  This  radioactively unlabelled s.ex  Decker  cytosolic  led  to  and  Levihson  binder  a  R1881  time-dependent  with  (1983) a  similar  affinity  (Kd= 1.4  compared  t o Eagon's  male  estradiol  binding  protein liver  This  Decker  These  i n the d i f f e r e n t and L e v i n s o n ' s  triamcinolone factors  groups  assay  be a s s e s s e d  d e s c r i p t i o n of these a R1881 b i n d i n g  ammonium  sulphate  fmol/mg), but  cytosol nM;  specificity  and s a t u r a b l e  incubated  capacity=  testosterone, Recently,  with  30-50 DHT  we  a male  group's Other  i n adult  this  use of technical by t h e l a c k  Aten  This  female  cytosol nM;  (1983)  rabbit  containing  capacity=  79  testosterone,  et a l .  triamcinolone  specific  of  by Eagon's, and  R1881 b i n d i n g  and s e v e r a l  kinetics  Eisenfeld et a l .  t o R1881,  fmol/mg).  androgen  r a t hepatic  are limited  (Kd= 0.3  o r DES.  an  be d u e t o t h e d i f f e r e n c e s  fractionated liver  not progesterone  displaceable  protein  and C o r t i s o l  with  tested  studies.  reported  triamcinolone  male  mixture.  since  binding  o r i n t h e Ps male r a t  i e .the former's  in their  cannot  may  R 1 8 8 1 was n o t  receptor.  i n the binding animals  testosterone  indicate that  i n adult  used  without  t h e R1881  i n the female  i s present  conditions,  work,  by  that  estrogen  two s t u d i e s  they  incubated  was d i s p l a c e d  t o Eagon's  Inconsistencies  assay  cytosol  cytosolic  not present  protein  protein  liver  (132 fmol/mg)  in contrast  b u t n o t b y DES, s u g g e s t i n g  cytosol.  cytosol.  capacity  however,  binding  in contrast was  binding  of  group;  to the hepatic  However,  in  and h i g h e r  rat unfractionated  triamcinolone. and  nM)  (1983)  reported  i n human  and C o r t i s o l binding  synthetic hepatic  was  a n d DHT,  liver (Kd= 1-2  displaced  progestins. cytosolic  estrogen  by  binding  protein  displacing cytosolic  has  been  androgen  binding  androgens  tritiated-estradiol  latter  hepatic  et  Finlayson 10-fold  to  same m o l a r affinity  Powell-Jones the  concentration nM  male  and  al.  which  molar  following  amount  binding):  estradiol  androstenedione  of  a  moderate  capacity  estrogens  and  some  by  in  excess  and  DES  of  480 a  the  had  the  the  female  Warren  1982;  that  not  a  by  40%,  protein.  similar  protein,  a  (63%);  of  absence They  but  ,  found  as  of  a  %  these  moderate  protein not  of  (30  of  the total  (37%);  (58%);  Both the  estradiol  affinity  testosterone  beta-diol (0%).  lower  binding or  the  binding  fmol/mg  (expressed  binding  androgens,  in  cytosol  using  presence  selectivity  estrogen  but  competitor).  alpha,17  protein;  reported  protein  to  DHT  moderate  a l . 1980;  whole  results  the  displaced  r a d i o i n e r t e s t r a d i o l at  capacity  (80%);  3  relative  et  liver  closer  1974),  absent  (1978)  displacement  (0%)  reported  but  hepatic  radioactively labelled  soluble  was  ratio  alpha-androstane-  rat  group's  tritiated-estradiol  100-fold  binding  r a d i o i n e r t DHT nM  al.  the  testosterone  (1980) m e a s u r i n g  latter  et  to  from  affinity,  male  al.  1000  This  nM  et  et  displaced  ratio. 480  and  moderate  excess  adult  70-80%  of  confirmed  addition  (Roy  Powell-Jones  displaced  from  DHT  adult  Dickson  ratio  testosterone,  as a  the  a l . 1978;  molar  compared  from  in  1983).  estradiol  such  protein  c y t o s o l i c estrogen  i s present  (Dickson  In  r a d i o a c t i v e l y l a b e l l e d androgens  unlabelled  capacity  described.  to  to  DES  studies  affinity,  natural or  5  androstenedione, reguired high  a degree  affinity,  binds  suggesting  of s t r u c t u r a l  low c a p a c i t y  specifically  studies  using  that  to the l a t t e r .  various  therefore  one does  saturable capacity  studies  protein  displaceable,  moderate binding 30  nM  Warren  protein.  excess):  displacement estradiol while  They  occurred  Saturation  incubated  with  radioinert  binding  kinetics  pmol/mg  using  Kd=  these  (1983) measured  at least  144 +/-  unlabelled estradiol;  of  radioinert  than  1000-fold  adult  o r DHT  a  fold Maximal  by u n l a b e l l e d  greater  i n the absence  (n=5):  some o f  (10 t o 1000  binding  ratios  a  protein.  the following  using  to a  estrogen-androgen  concentrations  estradiol  be r e l a t e d  and a n d r o s t e n e d i o n e .  required studies  point  15 t o 5 0 % d i s p l a c e m e n t  ratios  estradiol  various  tritiated-estradiol, excess  molar  a t molar  the androgens  ratios.  using  estradiol  p r o t e i n , or perhaps  binding  capacity a  saturation  affinity-moderate  t o answer  reported  testosterone of t o t a l  could  and F i n l a y s o n  moderate  at various  DHT,  steroid  attempted  tritiated-estradiol  competitors  single  binding  DES  n o t done, and  as the moderate  (1982)  affinity,  were  not the  since  of l a b e l l e d  androgens  nonsaturable  b u t was  Unfortunately,  i f these  or androgen  laboratory  questions.  using  such  estrogen  Our  n o t know  protein  receptor,  concentrations  by u n l a b e l l e d androgen  binding  specificity  estrogen  displaced  displacement  this  male  100-fold, molar  whole  cytosol  of  or presence gave  1000-fold  the following  50 nM Kd=  of  a n d Bmax=  104 +/-  38  17 +/nM a n d  4  Bmax= in  14 +/-  binding  would from  3 pmol/mg  kinetics  indicate t h e same  results  than  between  binding  unlabelled these  displacement  this protein  The  two d i f f e r e n t  similarity  competitors  These  workers.  results  Based  has a h i g h e r  on  confirm the  affinity  the  inhibition  for estradiol  androgens. The  exact  physiological  clear.  I t has been  present  i n the adult  the  adult  al.  1980; F i n l a y s o n  female,  shown male  role  of t h i s  but absent  immature 1983).  male  Finlayson  of t h i s p r o t e i n  an  reversible  after  contrast,  Powell-Jones  effects.  Like  metabolizing dependent adult  male  Finlayson nucleus be  on p i t u i t a r y f a c t o r s , reduced 1983).  Although  (Powell-Jones  involved  concentrations metabolites In  binding  summary,  e t al.  t o see  of  in  these  and s t e r o i d was  hypophysectomy  of the  e t a l . 1980;  i t i s not translocated  i n the regulation  (Dickson  castration,  of t h i s p r o t e i n  (Powell-Jones  e t a l . 1980),  of androgens  drug  since  a  administration;  failed  microsomal  the presence  reported  following  (1980)  in  (Powell-Jones e t  (1983)  testosterone  et a l .  the hepatic  enzymes,  i s not  o r i n low q u a n t i t i e s  and female  i n binding  effect  protein  t o be a g e a n d s e x d e p e n d e n t , i e .  reduction  to  DHT.  of the r a d i o l a b e l l e d e s t r a d i o l  site.  of the e a r l i e r  studies,  using  into the  this protein  i s thought  intracellular  and e s t r o g e n s ,  or their  1978 ) .  i t i s n o t known  i f the inconsistencies  binding  c h a r a c t e r i s t i c s of the hepatic  androgen  protein  a r e due  species-  to differences  between  binding or  in  sex-dependent differences  androgen  in  ligand  binding used  proteins,  (DHT,  or  with  ammonium, s u l p h a t e ) ,  (with  without  or  without  discrepancies, has  been  act  as  to  hepatic  detected  an  serve The  in  androgen a  cytosol preparation  triamcinolone  an  and  this  role  androgen  receptor  means o f  androgen  leading  to  protein  synthesis,  studies  in vivo  and  Levinson  research  P-450  These  androgen  conditions  Despite binding  species.  binding  the  on  these  protein  In  order  p r o t e i n would  hepatic  nuclear of  to  have  t r a n s l o c a t i o n of  nuclear  a l . 1983)  to  cytosolic  androgen  hepatic  studies  in order  and  are  sensitive uptake  in vitro  preliminary  better  understand  (Decker  and  more  this  physiological function.  and  sex-differences  dependent  drug  a t t r i b u t e d to  increased  in  the  Gillette  El  Defrawy  1965;  phenomenon  clear. target  This  is  tissues  male  rat  E l Masry  in contrast as  serum  by with  prostate  hepatic  enzymes  a  activities  et  puberty  the  (Kato  a l . 1974);  receptor  or  cytochrome have  testosterone  following  is regulated  such  in  metabolizing  concentrations  this  Cortisol).  stimulation  ( E i s e n f e l d et  1983).  (fractioned  Summary Age-  been  of  i s based  is required  protein's  4.  the  technical  role.  intracellular a  to  incubation  mammalian  receptor,  as  or  cytosolic  several  physiological  due  testosterone,  androstenedione or  R1881),  or  system  classical  epididymus,  and  whether i s not  androgen which  are  yet  responsive where  a receptor  (1969) such  have  as c y p r o t e r o n e reduced  sites  suggesting receptor al.  showed  mice  have  i tdifficult  al.  and p r e p u b e r t a l  binding  1983).  of these  in rat liver  problems  a n d h a s made  a successful protein  evidence  exists  (Eagon  induced  activity  in  inadequacies hepatic  (Gustafsson  et  suggesting  studies  h a s made  to identify  that  the presence  the hepatic  The above an h e p a t i c  The l a c k  of  i tdifficult  physiological function metabolism;  synthetic  e t a l . 1983; D e c k e r and  cytosol.  t o drug  correlating  by Brown e t  testosterone  this  tool  in liver  characterization  relevance  androgen  e t a l . 1980; L u c i e r and  1983; E i s e n f e l d e t a l . 1983).  protein's  evidence  cytosol  Levinson  this  Indirect  a cytosolic  binding  receptor  binding  Technical  androgen  some  of  androgen  provided  rats.  could  T h e j u d i c i o u s u s e o f R1881 h a s c i r c u m v e n t e d  androgen-progestin  provide  effects  N-demethylase  to identify  protein  antagonists  These  blocked  1975; Roy e t a l . 1976; S a t o  Rumbaugh some  flutamide  and Fang  displacement  has been  and  flutamide  f o r the hepatic  ethylmorphine  castrated  androgen  weight.  cytosolic  role  Liao  and  e_t a l . 1979 ) .  metabolism  microsomal  made  organ  antagonists,  androgen  flutamide  from  a regulatory  i n drug  that  the competitive  (Raynaud  ( 1 9 7 6 ) who  hepatic  acetate,  androgens  in vitro  implicated.  reported  target  c o r r e l a t e d with  radiolabelled  and t e s t o s t e r o n e  i s clearly  and o t h e r s  hydroxide be  to testosterone  or  a systematic  o f an h e p a t i c  studies androgen  appropriate to  assess  pharmacological approach  cytosolic  androgen  receptor  to  induction  testosterone  has  not  Although for  there  cytosolic  tissues, failure androgen  5.  and  activities  age  are  phenomena  adult  male  has  cytosolic  androgen  mechanism microsomal To  BPH  and  in  Wistar  animal  in  a  target  limited  by  specific  the  hepatic  the  microsomal mechanism  Testosterone Since  which  these  BPH  underlying  secretion  in  the  testosterone  known may  to  be  contain  responsible  tissues,  testosterone  i f hepatic  androgen  a  similar  dependent  testosterone-induced  animal  imply  binding models  states. a  r a t was to  used  study;  phenomenon, protein  for  receptor liver  dependent since  i t i s very  for  hepatic  i t is a  were  done,  endocrinological  Correlations role  enzyme  enzyme  studies  in various  regulatory  testosterone  model  of  in hepatic  however,  receptors  pharmacological  receptor The  and  would  been  role  vitro.  receptor-mediated  different  studies  regulatory  induction.  is a  metabolism  has  t i s s u e s are  mediating  determine  induction  using  be  in  implicated.  androgens  may  a  enzyme  study  end-organ  of  and  is unclear.  sex  action  this  been  suggest  existence  differences  sensitive  the  the  well-known;  these  microsomal  in extrahepatic  research  i_n v i v o  of  to  receptors  demonstrate  Objectives Sex-  i s evidence  metabolism  receptor  hepatic  pursued.  androgen  drug to  been  dependent  between the  these  androgen  drug  convenient  metabolism. laboratory  well-characterized,  and  because drug  and  i t has  characteristic  and  steroid  The  goals  (a)  to  adult  metabolizing animals  to  (b)  of  this  to  the  and  female  male  study  study  by  the  effect  Results  rat.  testosterone-induced  mediated  by  a  specific  (c)  as  a  first  hepatic  studied since (Roy  i t has et  may  be  enzyme  of  on  microsomal  drug  the  activation, of  these  hepatic  androgen to  receptor  activities,  pharmacological  to  activation is  in vitro, of  contain  a l . 1980). cytosolic  animal  different  models.  characterize  androgen liver the  androgen  binding  was  homogenate  androgen  Changes  a l t e r a t i o n s in hepatic  using  indicate  and  to  hepatic  may  identify  purported et  studies  on  adult  mechanism.  been  correlated  these  the  receptor  fraction  Sato  antagonists using  enzyme  cytosolic  this  immature  r e s p o n s i v i t y of  androgen  the  a l . 1974;  kinetics  of  approach  androgen  using  hepatic  hormone.  castrated  the  dependent  testosterone  examining  enzyme  that  of  hepatic  testosterone-induced male  age  were:  effects  enzymes,  the  and  metabolism.  study  male  sex  in  the  binding  receptor binding protein  microsomal  physiological  and  MATERIALS  1.  AND  METHODS  Chemicals The  following  Chemical  c h e m i c a l s were  Co. ( S t . L o u i s ,  MO):  purchased  NAD,  NADH,  glucose-6-phosphate,  glucose-6-phosphate  bovine  (BSA),  serum  albumin  streptozotocin  androstenedione, 5  beta,  testosterone,  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  5 beta-androstan-3  17 b e t a - d i o l ) ,  hydrocortisone,  beta,17  estradiol,  diethylstilbestrol  ethylenediamine-tetraacetic (Cleland's  Reagent,  molybdate,  activated  DTT), Hide  Protamine  zinc  Connaught  Laboratories  flutamide  hydroxide  Schering gifts with  AG  from  insulin  fluorescent  (20x20 Eastman  Kodak  Chemicals  (EDTA),  DL-dithiothreitol  Azure,  and protamine  were  gifts  sulphate. from  F l u t a m i d e and from  Germany).  RU23908  S c h e r i n g Co.  a n d RU2956  (Roumainville, France). thin  layer  and d e x t r a n T70 were  Silica gel  obtained  B C ) , and P h a r m a c i a  Drug  Fine  by  were  chromatography  Co. ( R o c h e s t e r , NY), B r i t i s h  (Vancouver,  sodium  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  indicator  3  progesterone,  (Willowdale, Ont.).  Uclaf  cm), g l y c e r o l  (5 b e t a - A -  ( 1 0 0 u n i t s / m l ) was o b t a i n e d  NJ), while  Roussel  beta-diol  (DES),  Powder  (Schl6423)  (Berlin,  triamcinolone  (Trizma-base),  acid  charcoal  alpha-DHT),  spironolactone,  tris(hydroxymethy1)aminomethane  (Bloomfield,  NADPH,  enanthate,  b e t a - d i h y d r o t e s t o s t e r o n e (5 b e t a - D H T ) ,  acetonide,  NADP,  Sigma  dehydrogenase,  testosterone  (STZ), benzo(a)pyrene,  from  sheets from  House  Chemicals  (Uppsala,  Sweden),  respectively.  N-methyl-N-  trimethylsilyltrifluoroacetamide reagent were  (MOX;  2%  methoxyamine  s u p p l i e d by  Pierce  Chemicals  (Poole,  further.purified and  finally  procedure  by  was  and  1,2-tritiated activity: (NEN)with  52  Canada  Ltd.,  Roussel-Uclaf  were  Amersham  from  Upjohn  this  r a d i o - l i g a n d was The  alpha,2  obtained PQ)  Co.  (Oakville,  (Kalamazoo, 72  (Cancer  beta-diol  Ont) MI).  Ci/mmol)  (DHT,  from  beta-androstanbeta-diol)  and  3 5  under  87  and  specific  New  under  The  England  licenced  Nuclear  agreement  Labelled  mibolerone  (7  were  licenced  specific  (17  purchased agreement  activity  of  Ci/mmol. g r a c i o u s l y donated  C o n t r o l Agency (5  crystals.  alpha-tritiated  50-70  radioinert  f o l l o w i n g s t e r o i d s were  Rennie  beta,17  Co.  This  activity:  alpha-dimethyl-19-nortestosterone),  from  P.  specific  activity:  and  was  filtration  methanol.  (Romainville, France).  alpha-methy1-tritiated)alpha,17  1  (Montreal,  Drug  alpha-methy1-  (N)-dihydrotestosterone Ci/mmol)  British  i n benzene,  (R1881,  R1881,  (specific  from  IL).  Benzo(a)pyrene  in cold  (17  tritiated)-methyltrienolone  (N)-testosterone  in pyridine)  needle-like yellow  labelled  radioinert  methoxyamine  (Rockford,  obtained  solubilization  yielded fine  and  Co.  England).  recrystallization  Radioactively  Ci/mmol)  hydrochloride  Chemical  2,4-dinitrophenylhydrazine House  (MSTFA) and  alpha-A-  alpha,17  3  of  BC):  beta,  beta-diol  alpha-androstan-  3  17 (5  5  Dr.  alpha-androstan-3  beta-diol), beta-A-  alpha,  by  17  3  5  alpha,17  beta-diol  (5  36  alpha-A-  3 alpha,17  beta-diol),  naphthoquinone-4-sulphonic Mr.  M.  other  2.  Coombs  (University  r e a g e n t s were  Care  (60-85  days  g ) , and immature post-weaning)  (UBC  Animal  Care  Laboratories  hours)  Unit),  grade,  Paxton  F o r some  were  shipped  hypophysectomy decrease  of  rats  cages  Processing  on P u r i n a  experiments, by C h a r l e s  seven  was  i n body  control.  P.Q.).  (22 d e g r e e s  and m a i n t a i n e d  hypophysectomized and  was  A l l  provided  solvents  by  and  grade.  days  using  g) and  post  compared  female  ( 5 0 g , 20-24  were  Canadian  purchased  containing  locally  lights  housed  corncob  L t d . , Paxton, C,  days  Breeding  A l l a n i m a l s were  ON  IL),  in  bedding under  06:00-20:00  L a b o r a t o r y Chow  and water  adult  were  male  surgery.  (Boston,  Completeness  inspection  to the  ad  MA)  of  and by t h e  sham-operated  castration  the s c r o t a l  rats  Laboratories  a s s e s s e d by v i s u a l  weight  done  rats  River  In other experiments, were  (275-350  and female  Wistar  in plastic  conditions  libitum.  male  o r from  (La P r a i r e ,  of four,  standard  salt  of Toronto).  o l d ) male  8 days  (Lobund  acid-sodium  of the reagent  old,  groups  1,2  and s u r g e r y of a n i m a l s  Adult (180-200  while  and o v a r i e c t o m y  and d o r s a l  routes,  respect ively.  3.  Treatment In  order  enanthate  of Animals to determine  which  0  a suitable  i n d u c e s maximum  dose  androgenic  of  testosterone  responses,  different  daily  enanthate  (0,  vehicle) for  10  subcutaneous  1.25,  were  2.5,  injected  days.  Results  from  differences  testosterone  enanthate,  chosen  as  a  physiological hepatic the  levels  microsomal  adult The  flutamide  testosterone  castrated  vehicle  or  flutamide In injected 0.1  M  with  citrate  experiment. the  citrate  were 10  units/kg  reverse index). hours  male  doses  s c . , 10  the The  after  of  studies,  25  (STZ;  60  A  protamine effects insulin the  last  4.5)  of  four  were  for 4  zinc  variation.  determined  (5  days)  with to  using  corn o i l  50  rats  were  flutamide  or  days  insulin  replacement  intravenously  days  treated  diabetes  PZI  in  prior with  of  post  to  in  the  equal  volumes  of  STZ-treated animals STZ  (PZI),  (using  injection,  in order  glucosuria  experiments  injection.  (iv)  mg/kg) d i s s o l v e d  s e p a r a t e group  sc. daily  activities  days.  streptozotocin  rats  micromoles/kg  s c . , 10  micromoles/kg  showed  sex-dependent  female  were  buffer.  and  flutamide  rats  consistent  was  males  (pH  5  male  of  biological  adult  buffer  of  injected  Adult  adult  doses  enzyme  curve  rats,  days).  with  Control  injected  weight  rat, despite  h y d r o x i d e f o r 10  other  low  (5 m i c r o m o l e s / k g ,  adult  sc. daily  to  a dose  metabolizing  response  in corn o i l  experiments  to maintain  prostate  dose  different  micromoles/kg,  dose  male  enanthate  treated  injected  of  testosterone  castrated  preliminary  therefore  drug  castrated  10-day  i n response  suitable  of  5 micromoles/kg  into  considerable  was  or  (sc) doses  A l l of  were the  with  to as  an  done  24  diabetic  rats  (excluding  glucose Ltd.,  4.  the  levels  >0.5%,  Toronto,  animal  exsanguinated w/v).  The cold  was  blotted  by  size of  A  the  KCI  the  KCI  separately  passes,  poured  centrifuged  at  means  of  IEC  lipid  layer  and  ml  10  8 ml  another a  Model  covering  volumes  of  was 65  Beckman  Model  centrifugation, pellets  the  (microsomal  for  of  x  g  75  for  B-20  the  cold  had  urine and  Co.  x  g  was  precipitate)  were  tissue, 50  ml  20  ml  was  into  the  11-20  and  was  degrees  C,  by  this,  the  removed separate  remaining  centrifuged  degrees  was  a  tissue  carefully  tubes;  fraction  liver  liver  Following  4  in  difference.  into  4  pipetted  at  by  tubes,  at  was  supernatant  the  containing  minutes  were  (1.15%  approximately  ultracentrifuge.  soluble  this,  centrifuge  supernatant  105,000  L5-50  or  KCI  immersed  placed  centrifuge.  The  was  animal's  seconds  ultracentrifuge  at  and  animal's  were  The  latter  was  determined each  10  studies  aqueous  homogenizer  plastic  the  discarded. minutes  was  lobe,  tissue  into  10,000  polycarbonate  pellet  of  an  grams  liver  liver  Following  solution.  homogenized was  ice  enzyme  dissected  weight  five  for the  solution.  wet  largest  isotonic  with  quickly  Potter-Elvjheim  cold  (Eli Lilly  decapitated,  was  Approximately including  Tes-Tape  microsomes  perfusion  isotonic and  tested  method.  of  was  liver  ice  the  animals)  fractionation:  Preparation  Each  using  Ontario)  Subcellular (a)  PZI-treated  C,  by  for means  After discarded  carefully  and  washed  the with  three  4 ml v o l u m e s  buffer  of  i c e - c o l d aqueous 0.1  (pH 1.2), and r e - s u s p e n d e d  (b)  P r e p a r a t i o n of  radio-ligand binding  M phosphate  i n 4 ml o f  the  c y t o s o l and m i c r o s o m e s  same.  for  studies  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 methodology In b r i e f ,  dissected  and was e x s a n g u i n a t e d .  and p e r f u s e d  containing:  0.05  with  M Tris-base;  1.5  pre-cooled  I n d i v i d u a l l i v e r s were  separately  in cold buffer  were t h e n c e n t r i f u g e d  at  4 d e g r e e s C , and t h e s u p e r n a t a n t  105,000 x g f o r supernatants (containing  65 m i n a t  weighed  using a  (or c y t o s o l i c  fraction)  the m i c r o s o m a l f r a c t i o n )  buffer  to the d e s i r e d rat  the s t r a w - c o l o u r e d  glandular  these experiments. above.  pestle.  10,000 x g f o r  10 m i n  was r e - c e n t r i f u g e d  4 degrees C.  other experiments,  described  molybdate;  P o t t e r - E l v e j h e i m h o m o g e n i z e r and t e f l o n  Homogenates  ice-cold  (pH 7.4)  mM E D T A - d i s o d i u m ; 0 . 5 mM  10% g l y c e r o l ; w i t h o r w i t h o u t 20 mM s o d i u m  and were h o m o g e n i z e d  The  at  resulting  and r e s u s p e n d e d were d i l u t e d  pellets  with  protein concentration.  p r o s t a t e c y t o s o l was p r e p a r e d . prostatic  The p r o s t a t i c  was  The l i v e r was q u i c k l y  i c e - c o l d TED b u f f e r  w i t h o r w i t h o u t 10 mM K C I .  at  above.  e a c h a n i m a l was s t u n n e d by a b l o w t o t h e h e a d ,  decapitated  DTT;  as  t i s s u e was u s e d  c y t o s o l was p r e p a r e d  In Only for as  40  5.  Hepatic  by  (a)  Hepatic  The  BPH a s s a y  the addition  weight) 80  mM  of samples  of cold  B P , 3-OH  addition  the  protein  0.47 mM serum  the aqueous  room  fluorometric wavelengths sulphate  (b)  analysis  microsomal  Microsomal  by McGuire  double  In t h i s  by d i f f e r e n t i a l  was  freezing  the organic portion of NaOH  phase  was  melted  by  and e m i s s i o n using  (nm)  a quinine  testosterone reductase following  involves  assay,  by  The o r g a n i c phase  (1959).  This  measurement  microsomes  assay  the assay  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  bond.  metabolite  S p e c t r o f l u o r o m e t e r Model 430.  and Tomkins  assay  carried by  phase,  B P was m e a s u r e d  TR was m e a s u r e d  spectrophotometric enzymatic  After  Turner  The  The major  t h e hexane  at activation  pre-calibrated  containing:  C, a n d was s t o p p e d  fraction  T h e 3-OH  liver  magnesium  (BSA).  o f 396 a n d 522 r e s p e c t i v e l y ,  Hepatic  described  3 mM  was d i s c a r d e d , t h e a q u e o u s  temperature.  wet  ( i n d u p l i c a t e ) was  t o 1 N NaOH.  a dry ice-acetone bath.  mixture  NADH;  BP was e x t r a c t e d f r o m  from  ( 7 . 5 mg  and hexane.  assay  1 9 6 8 ) was c a r r i e d o u t  albumin  a t 37 d e g r e e s .acetone  hydroxylase  (pH 7.5) s o l u t i o n  and blanks  of the l a t t e r  separated  and G e l b o i n  buffer  NADPH;  f o r 5 minutes  using  Tris  a n d 0.6% b o v i n e  addition of  (Nebert  assays  benzo(a)pyrene  of microsomal  B P ; 0.44 mM  incubation out  enzyme  microsomal  t o a 50 mM  chloride  to  microsomal  (6 mg  of the conjugated protein)  were  added  to  an  containing: .0.5  mM  aqueous  0.1  0.15  testosterone;  NADP;  mM  10  mM  at  37  and  interphase  phase  was  used  using  a  for  spectrophotometer. the  incubation.  6.  the  of  were  The  Serum  testosterone  allowed  to  collected  clot  for  30  centrifugation.  Serum  determined  the  using  chloride;  of  were  test  the  in  Carson,  was  10.3%  (cross-reactivity and  testosterone measured  to  other  from  0.05  using  a  to  5  steroids to  Nuclear  25  the after  in duplicate.  and  blood  tubes. was  the  Blood  90746).  was  prepared  RSL  kit  using  by  were Nosolvex  (Radioassay The  assay  was  alpha-dihydrotestosterone <0.01%) and  ng/ml.  Chicago  was  determined  serum  System  specific  in  concentrations  outlined  CA  nm),  reduced  and  killed  radioimmunoassay  Inc.,  organic  (210-290  incubated  in heparinized  testosterone  using  aqueous  remaining  before  were  and  10  beam  present  minutes  the  absorbance  were  for  terminated  testosterone  the  Animals  unit incubated  and  (Iodine-125)-testosterone Laboratories  1  scanning  concentrations  protocol  magnesium  The  double  blanks  protocol.  solution  were  shaken  in  testosterone  assays  were  was  amount  and  7.2)  r e a c t i o n was  124  difference  (pH  and  and  aspirated.  Model  Other  samples  The  latter  Samples  following  mM  spectrophotometric  Perkin-Elmer  concentration  C.  The  phase  by  buffer  6.3  dehydrogenase,  degrees  dichloromethane.  calculated  phosphate  glucose-6-phosphate  glucose-6-phosphate minutes  M  was  sensitive  Radioactivity  Series  1185  to  was  Automatic  Gamma  Counting  System.  Cytosolic  proteolytic  activity  spectrophotometric  method  (1968).  In b r i e f ,  the assay  activity  by the t o t a l  Blue-Hide  powder  dye-protein mg/ml (pH at nm,  (RBB-Hide;  using  C.  with  mixture  determined  Dilutions a  linear  double  7.  an  insoluble  was  incubated  in Tris  calcium  chloride,  f o r 3 o r 18  absorbance  ( c o n t a i n i n g 40 buffer  was m e a s u r e d  assay  gave  a linear  a sensitivity  from  1 t o 2 ng t r y p s i n  This  a t 595  beam standard  curve p e r ml  e t a _ l . 1968 ) . protein  contents  were  the Coumassie  as d e s c r i b e d by B r a d f o r d  curve,  hours  solution using  (0.05-0.50  a Perkin-Elmer  (1976).  mg/ml) Model  produced 124  spectrophotometer.  Metabolism  Preparation  Studies  of standard  steroid  s o l u t i o n s and  e x t r a c t s f o r chromatography  Standard testosterone in  Azure),  124 d o u b l e  G method  Testosterone  cytosol  Powder  Model  o f a BSA s t o c k  beam  (a)  of R e m a z o l b r i l l i a n t  cytosol  and c y t o s o l i c  standard  et a l .  the proteolytic  spectrophotometrically using  Blue  using the  whole  (Rinderknecht  Microsomal  Brillant  2 mM  a Perkin-Elmer  trypsin  assay  Liver  Hide  The o p t i c a l  spectrophotometer. for  by R i n d e r k n e c h t  measures  i n TED b u f f e r )  7.2) c o n t a i n i n g 37 d e g r e e s  described  solubilization  complex.  protein  was d e t e r m i n e d  absolute  solutions and r e l a t e d  ethanol  of r a d i o a c t i v e l y metabolites  or dry diethyl  unlabelled  were  prepared  (1 mg/ml)  ether,  and were  stored i n  glass  tubes  fitted  Liver  whole  cytosol  homogenization  and  aliquot  of  (5  ml)  testosterone Incubation  for  was  ether  organic  solvent.  For  differential  0  or  3  the In  a  testosterone  using  the  aliquot  varying  was  for  to  and  dry  free  supernatant  10  volumes  extracts  evaporator.  of  2  10  C.  volumes  extracted  of  using  5  An  micrograms  degrees  experiment,  nM)  at  using  cytosol  (2-40  hours  from  of  37  the  beta-DHT  same was  4  nitrogen, This  of  three  in  the  degrees  steroid  C.  or  absence  washes  collected  of  e x t r a c t i o n method  separated  technique. the  ether  bound  for  An fraction)  steroid  s t e r o i d s were  diethyl  and  using  Following  f r a c t i o n s were  charcoal  diethyl  by  presence  radioactively unlabelled  unlabelled  or  in  of  (containing  and  were  incubated  concentrations  ratio  Labelled  stream  or  C.  following  with  c y t o s o l was  molar  extraction.  Ethereal  4  addition  dextran-coated  the  added  3  bound  2.5%  of  at  separate  experiments,  excess  incubation,  degrees  testosterone.  with  100-fold  by  4  centrifugation steps.  incubated  hours,  at  prepared  s t e r o i d s were  tritiated-testosterone of  mg/ml) was  corks,  tritiated-testosteronethin-layer  chromatography duplicate,  (40  c y t o s o l was  and  for  the  foil-wrapped  terminated  diethyl  substituted  with  were a  ether dried  rotary  was  97%  extracted (10:1).  under  vacuum efficient.  a  (b) using  thin-layer All  to  Separation  carbonate. Lisboa v:v),  (1962): system  sheets to  were  steroid  were  using  The  testosterone  chromatography  solvents  experiment,  of  solvent system  ethyl  pre-dried  at  90  application.  a  standard steroid  micrograms separate from  steroid)  spots  bottom  syringe  and  The solvent  small  using  glass  temperature  f o r at  least  sheet front  origin.  The  solvent  front  dry  at  room  C  cm),  prior  potassium  (95:5,  f o r 10-20  ethereal  onto 10  v:v). mins  ether;  (containing  applied the  to  (50:50,  extract  diethyl  solution  chromatography filter  solvent  hours  TLC  prior was this  1  to  and  was  TLC  sheets  10  dried  microliter  or  as  (2  cm  Hamilton  blow-dryer.  fluted  TLC  18  prepared according  dry  a modified  and  Each  of  carefully  ( d i a m e t e r <1  edge), a  was  or  ethanol  dried  volume  least  acetate:cyclohexane  degrees  Each  in a  of  were  I I - benzene:absolute  redissolved volume  f o r at  chloride  systems  I-  related metabolites  (TLC)  pre-dried  calcium  from  was was  was  allowed  into to  running  marked  temperature  identification.  paper  was  2 hours  placed  average  chamber  and  a  run  100  ml  pre-equilibrated  at  prior  containing  to  chromatography.  s e p a r a t e chamber, f o r 15-17  time  was  65  each  TLC  sheet  before steroid  room  cm  mins  and  above before  was  the  the the  allowed  visualization  and  to  (c)  Visualization  metabolites The sheets For  using  UV  this  testosterone  light  polyacrylic  precluded  of  binder  charring  reason,  and  the  and  colour  s  reagents  contained  techniques  related  i n the using  silica  strong  following alternative  gel  acids.  techniques  were  used:  (i)  UV  visualization  unsaturated A-dione)  double  was  done  (Ultra-violet convenient different (ii)  bond using  Prod.  a  was  Chromato-Vue San  to  were  (1957).  using and  The  vacuum  those  quickly  background,  according  filtration.  mixture  of  brief,  reagent  and  set  This  prior  to  at  265  easy  nm  and  addition  of  of  suspended  concentrated  the  solute  was  reagent 3-  or  as  orange  of  17-  1  A  acid  ethanol:  0.1%  were  on  removed  sprayed  groups  spots  reagent  (w/v)  sodium 60%  ml  carefully  lightly  keto  100  a  could  be  yellow  microgram.  Perchloric acid  (1965).  in  sulphuric  was  sensitivity  4-sulphonic  of  The  with  absolute  was  g  prepared  particles  visualized  Richter  2  was  undissolved  easily  naphthoquinone a  remaining  a  reagent  millilitres  Naphthoquinone to  delta-4  delta-4  G a b r i e l , CA).  In  steroids containing  and  (iii)  Four  c a u t i o u s l y added  dissolved.  the  reagents.  Vogel  methanol.  or  cabinet  r o u t i n e l y done  2,4-dinitrophenylhydrazine  acid  testosterone  Dinitrophenylhydrazine  according  dry  steroids containing  (eg.  Inc.,  technique colour  of  salt  was  solution was  prepared of  prepared  perchloric acid:  40%  1,2 in  formaldehyde: was  filtered  was  sprayed  oven  water  t o remove uniformly  ( s e t a t 80-100  visualization well  and a l l o w e d  degrees  pencil,  o f 1-2  these  pressure  8.0 m i c r o  mode.  background) as  background),  at a  spot  was m a r k e d i n  and 5  beta-androstan  Gas Chromatography-Mass  of testosterone  i d ; HP p a r t  impact  pyridine  each  of testosterone  HP 5 9 8 7 A  electron  amu.  on white  allowed  were c a l c u l a t e d .  GCMS  crosslinked dimethyl  m x 0.2 mm  technique  on w h i t e  i n t h e c y t o s o l was d o n e  Hewlett-Packard silica  reagent  (GCMS)  Confirmation beta-diol  The  t o d r y f o r 10 m i n i n a n This  techniques,  17 b e t a - d i o l u s i n g  spectrometry  solution  micrograms.  Identification  alpha,  particles.  (blue  (brown  and t h e Rf v a l u e s  (d)  C).  of oxo-steroids  Following  500  undissolved  as h y d r o x y - s t e r o i d s  sensitivity  -3  ( 2 : 1: 0 . 1 : 0 . 9 ) . T h e f i n a l  Torr.  The i n j e c t i o n as v e h i c l e .  were  using  system  GCMS.  fitted  silicone  number  spectra  and 5 beta-A-  3 alpha, A  with  capillary  a  fused  column (12.5  1 9 0 9 1 - 6 0 3 1 2 ) was u s e d . recorded  TIC plots volume  The  a t 80 e V , i o n s o u r c e  were  based  o n m/z  was 1 m i c r o l i t r e ,  I n j e c t i o n s were  17  done  using  40 t o  using a non-split  The  running  c o n d i t i o n s were  Chromatographic 50  degrees  20  degrees/min for  e x t r a c t s were  TLC  sheets  17  beta-diol  diethyl  incubated  for  hours  cytosol  Rf=0.26  evaporated trimethyl  used. to  dryness  ethers  and  For  degrees 3  hours  TLC  5  C, at  zone 4  or  O-methyloxime  MOX  reagents  Gardiner  and  Horning  beta-Aand  Rf=0.43  was  degrees  C,  zone  silylated were  using the  hydroxy-steroids, while  used;  then  Fully  brief,  were  was  derivatives  In  with  3  which  37  respectively,  (1966).  Zones  sheets  cytosol  f o r GCMS a n a l y s i s .  and  reacts with  from  separate  II.  e t h e r e a l e x t r a c t s were  MSTFA  reagent  4  at  and  on  system  t e s t o s t e r o n e or  ether.  for  The  solvent  cut-out  at  incubated  was  to  were  using 0  chromatographed  using  corresponding  280  increased  mins  seconds  extracted  prepared  the  method  former  the  latter  with keto-steroids.  This beta  degrees  3.9  values  reacts  to  Scan  alpha,  of  then  microamperes  preparative  using  10  1 min,  300  Ethereal  for  for  Emissioncycle-  follows:  temperatures-  held  Rf  as  -A-  technique 3  alpha,  retention  times  depending  on  non-extracted  yielded  17  were  daily  sharp  beta-diol were  12  and  and  chromatography  standards  gave  and  the  separate  peaks  testosterone.  13  mins  5  The  respectively,  conditions. same  for  retention  Extracted times.  and The  m/z  values  beta-diol  f o r t h e M+ and  ions  f o r 5 beta-A- 3 alpha,  testosterone  derivatives  were  436  17  and  389  respectively.  (e)  Analysis  metabolites Each  track  analysis.  of  8.  cut  and  and  ml)  of  concentrations or  of  strips  cytosol  and  and  Shain  10 mM  strip  then  plotted  as  this  distance  a  was  eluted  by  of  elution  the  the  studies microsomes incubated  following  workers: Kirchoff  (1974)  cytosol,  incubated buffer  et  for prostatic  An  and  i n TED  (0.5  buffer  or  varying  tritiated-  i n the presence  Yamada  aliquot  containing  tritiated-testosterone,  tritiated-Rl881,  were  i t s metabolites.  microsomes.  KCI  Each  was  distance  f o r hepatic  was  tracks  for radioactivity  a chromatogram  were  f o r hepatic  with  each  the f o l l o w i n g and  which  vials  binding  or microsomes  TED-supplemented  alpha-DHT  by  Boesel  (1982)  and  extract.  the o r i g i n ;  gave  e t a l . (1980)  cytosol  cm  the t o t a l  microsomes  al.  Miyagi  of  plot  of  outlined  and  from  exchange  conditions  Sato  1-2  testosterone  Incubation  longitudinal  scintillation  This  Radio-ligand  Cytosol  into  each c y t o s o l i c  into  a fraction  radiolabelled  cytosol,  of  i t s distance  as  (1979)  cut  r a d i o a c t i v i t y of  front.  (a)  was  separate  of  expressed solvent  sheet  The  tritiated-testosterone  TLC  further  into  function  bound  the e l u e n t s  was  placed  using  TLC  containing  of  5  or absence of  1  100-fold  (unless  competitor. acetonide stated) was  F o r some  (100-fold  was  added  incubated  stated).  excess  incubation centrifuged  incubation, charcoal  the rapid  and Peck other  adsorption  from  experiments,  was  fractions  removed  i n TED  using  was  buffer  otherwise  was  added  was m i x e d  This  (pH stated).  to a l l  thoroughly  a t 4 degrees  (Beato  the  (1976).  C  and before  technique  and p r e c i p i t a t i o n  0.5  of free  and F e i g e l s o n  et  a l . (1978).  This  centrifugation fraction  charcoal  ml p r o t a m i n e  i n TED b u f f e r w i t h o u t  the incubation mixture,  coated  using  or  1972;  1980) .  (2 mg/ml  supernatant  mixture  otherwise  (DCC) w h i c h  slurry  the c y t o s o l  to  before  (unless  (unless  analysis.  otherwise This  steroid  suspension  suspension  involves  solution  C  a t 1 5 0 0 x g f o r 10 m i n u t e s  steroid  triamcinolone unless  steroid  T70  ratio  determined  i n TED b u f f e r  for radioactivity  In  free  ( 0 . 5 m l ) o f 2 . 5 % DCC This  were  and f r e e  0.25% d e x t r a n  tubes.  molar  by B r a d f o r d  o f bound  sampling  Clark  ratio;  a t 4 degrees  a s a 2.5% c h a r c o a l  aliquot  unbound  radioinert  molar  described  Separation  7.4) c o n t a i n i n g  excess  to the incubation mixture.  method  dextran-coated  An  assays,  f o r 3 hours  Following  prepared  stated)  Protein concentrations  protein-dye  (b)  otherwise  sulphate  molybdate)  was  allowed  to stand  a t 1500 x g f o r 10 m i n .  pellet,  added  f o l l o w i n g t h e p r o t o c o l o f Menon  mixture  was  was  poured a n d was  onto  a pre-made  f o r 5 min  The 2.5%  dextran  c e n t r i f u g e d and sampled  (as  described  above),  non-precipitable precipitate Following  i n order binding".  was w a s h e d  the f i n a l  the  precipitate  for  1 hour,  protamine  and a s p i r a t e d  and t h e tubes sampling  Radioactivity  was a n a l y s e d  using  scintillation  system  6880  Radioactivity diskette Apple  containing  IL 60018). data  using  Computer  connected  Biofluor  Quenching was  Inc.,  temperature analysis.  o f t h e DCC o r into  (NEN-Canada).  t h e Mark (Searle  II-plus  Cupertino,  to the s c i n t i l l a t i o n  onto  a  III liquid  Analytic  Inc.,  Des  35-47%.  microcomputer  disc-operating CA 9 5 0 1 4 ) , counter  buffer.  was a d d e d t o  of t r i t i u m averaged  recorded  an A p p l e  TED  ( 0 . 5 m l ) was p l a c e d  vials  Plaines,  using  s e t a t room  an a l i q u o t  scintillation  Model  twice  forradioactivity  supernatant  sulphate  sulphate  0.5 m l e t h a n o l  were  centrifugation,  sulphate  "protamine  The protamine  aspiration,  before  Following  to determine  system  which  using  (DOS;  was  a serial  card  interface.  (c) An  Sephadex aliquot  G-25M  (0.5 ml) o f l i v e r  incubation  mixture  prepacked  disposable  Pharmacia  Fine  were  eluted  then  were  technique  permits  Sephadex  whole  protein bound  G-25M  Uppsala,  at 4 degrees  collected  non-protein  chromatography  was c a r e f u l l y a d d e d  Chemicals,  fractions  from  mini-column  cytosol to  pre-equilibrated  mini-columns Sweden).  C with  ( 4 0 mg/ml)  (PD-10;  The columns  TED b u f f e r ,  and the  f o rradioactivity analysis. bound  or free  fractions, fractions  t o be  This  separated  according  to  molecular  9.  size.  Analysis  of  Non-protein was  bound  c a l c u l a t e d from  incubation cytosolic the  tube  absence  supernatant binding  measured  which  litres  1949) p l o t s . protein.  capacity  Experimental regression Roufogalis,  data  computer  Computing  Centre).  of the following  fashion  steroid  using  cytosol or  was c a l c u l a t e d a s  total  and n o n - s p e c i f i c  as fmol/mg using  Scatchard-type  kinetics  constant number  protein non-linear  ( S B / F x SB;  o f S B / F was  The b i n d i n g  expressed were  as  expressed  o f e q u i l i b r i u m (Kd;  of binding  sites  or  fmol/mg). were  program  UBC), which  sulphate  i n the presence of  The r a t i o  maximum  (Bmax;  from the  a n d was a n a l y s e d  dissociation  to the  (incubation i n  Non-specific  (SB) s t e r o i d  (SB x F) a n d l i n e a r  added  or protamine  was e x p r e s s e d  stated),  and t h e apparent  input  bound  ( F ; nM)  The degree o f  binding  was d e t e r m i n e d  in a similar  Binding  the apparent  (UBC  total  d i f f e r e n c e between  otherwise  p e r mg  binding  steroid  incubated  Specific  saturation  nM)  were  fraction.  Scatchard  of c y t o s o l .  i n t h e DCC  was d e t e r m i n e d  concentration  of radioactivity  i n the absence  arithematic  (unless  t h e amount  of competitor)  competitor.  bound  steroid  o r t h e PD-10 e f f l u e n t .  microsomes  the  (free)  or microsomal  radioactivity  as  radioactivity  was  analysed (provided  a  non-linear  b y D r . B.D.  r u n o n a n AMDAHL-470  This data:  using  iterative  program  the s p e c i f i c  bound  computer involves the (SB) v a l u e  at  each  free  ligand  c o n c e n t r a t i o n (F)  used,  running  calculation  of  best  f i t .  calculation  was  0.8  sec,  The  depending  initial  estimates.  Another  analyse  SB/F  data,  vs.  SB  microcomputer  was  Pharmacology,  UBC).  program isotherm  by  observed)  ranged  accuracy  the  10.  In using  vivo  the  and  phase  estimating  the  from  sum  The 4  to  in  vitro  Student's  Duncan m u l t i p l e  was  an  the  can  Apple  II-plus  (Dept.  of  regression  of  the  the  two  two-part Kd  and  the  (theoretical  running  time  depending  experimental  level.  groups  for  two vs.  of  upon  the  Analysis  were  statistical  which  Collins  squares  8 min,  time  accuracy of  on  linear  average  the  estimates.  differences  for  of  run  Glen  linear  two-tailed  The  Mr.  each  initial  Statistical  by  the  program  be  this  that  calculation  can  brief,  i s minimized.  of  and  In  repetitively  Bmax v a l u e s , s u c h  upon  computer  developed  determines  average  for  statistically  analysis  suspected.  t-test  data  analysed  f o r unpaired  significant  range  were  test  i f interaction  (p<  at  the  0.05)  between  data; p<  0.05  was  used  treatment  RESULTS  1.  Effect  of  testosterone  administration  on  weights  and  the  ratios  Wistar (a) In  testosterone  hepatic  order  to determine maintain  what  ratios  TR  i n the  castrated  activities  testosterone  dose-response  With  this  information,  over  androgen  they Daily  were  Serum  injected  control  significantly rats  sc.,  10  hours  of  Serum  days) prior  of  2.4  male  in  more  with  concentrations  i n the  to  7.6  ng/ml  levels  testosterone  to experiment)  10  (figure  (Forest sc.  significantly  testosterone  varying  control  biological  levels  i n the  out.  individual  alter  f o r 10  compared  doses  compared  -  intact  days to  5  (last to  but  1979).  intact  the  adult  were  post-castrated  (1.3  enanthate  1),  to corn o i l  concentrations days  intact  testosterone  compared  Castration  these  and  following  carried  precise  of  BPH  r a t , the  were  5 micromoles/kg  d i d not  increased  treated  have  the  testosterone  lowered  males.  adult  microsomal  p h y s i o l o g i c a l range  c o n t r o l animals.  male  activities  testosterone  hepatic  would  from  days  serum  significantly  24  the  f o r 10  we  of  experiments  testosterone  administration  male  enzyme  physiological levels  and  despite  rat varied within  enanthate adult  levels  dose  normal  weight  male  tissueibody  levels  prostate/body  adult  flutamide  levels,  microsomal  testosterone  would  variation.  and  rat  Serum  enanthate  serum  enanthate  adult  micromoles/kg injection corn o i l  was  4  F i g u r e 1.  EFFECT OF TESTOSTERONE ENANTHATE ON SERUM TESTOSTERONE LEVELS IN SHAM-OPERATED AND CASTRATED ADULT MALE RATS  Serum t e s t o s t e r o n e l e v e l s determined i n sham-operated (Gx) a d u l t male r a t s vehicle  (expressed i n ng/ml) were (Sh) or 10 day p o s t - c a s t r a t e d  (280-300 g) t r e a t e d with corn o i l  (C) or v a r y i n g doses of t e s t o s t e r o n e enanthate (TE;  1.25-5.0 micromoles/kg s c . , 10 d a y s ) . 24 hours f o l l o w i n g l a s t i n j e c t i o n .  Experiments were done  Data were expressed as  average +/- S.E.M.; numbers i n b r a c k e t s denote the numbers of animals used.  * : significant difference sham-operated  (p< 0.05) compared to  a d u l t male of the same treatment group.  **: s i g n i f i c a n t d i f f e r e n c e treated castrated control  (p< 0.05) compared t o c o r n o i l group.  Figure  1.  55  Table EFFECT  OF F L U T A M I D E  ON  1  SERUM  TESTOSTERONE  LEVELS  T h e e f f e c t o f f l u t a m i d e o n s e r u m t e s t o s t e r o n e l e v e l s was determined i n c a s t r a t e d a d u l t male r a t s supplemented w i t h testosterone enanthate (TE). C o n t r o l sham-operated adult m a l e r a t s ( 2 8 0 - 3 0 0 g ) w e r e i n j e c t e d w i t h c o r n o i l f o r 10 days. T e n 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 T E ( 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 - 5 0 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 w e r e d o n e 24 hours 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 levels w e r e 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 s e r u m . Data were e x p r e s s e d a s 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  Animal  models  used:  (a)  Sham-operated  (b)  C a s t r a t e d a d u l t male injected with: (i) (ii) (iii)  adult  Serum  male  Corn o i l TE  (5 p m o l e s / k g )  only  4.6  Testosterone Level  +/-  1-7 ( 6 )  0.3 +/-  0.2  (8) *  1.2 +/-  0.2  ( 4 ) **  1.4 +/" 1.0 +/0.9 +/-  0.2 0.1 0.1  (4) ** ( 3 ) ** (4) **  T E (5 / j m o l e s / k g ) plus flutamide: 5 jmoles/kg 25 pmoles/kg 50 p j m o l e s / k g  *  denotes  significant difference c o n t r o l (p< 0 . 0 5 ) .  compared  to  sham-operated  **  denotes  significant difference c o n t r o l (p< 0 . 0 5 ) .  compared  to castrated  57  injected levels  castrated  were  The  achieved  effect  was  determined  may  be  having  lowered  a  and  at  eliminate  (table  testosterone testosterone  doses  (5,  significant  (b) In effects  due  on  enanthate  male to  or  were  10  day  with corn  reduced  the  intact  or  drug  treatment  sc.,  or  o i l treated  of  were  drug  (table  altered  adult  10  levels  animals  castrated In  the  at  various  days)  had  no  compared  to  non-specific  no  due  effect  significant  (testosterone  2).  Prostate:BW  flutamide  was  adult  compared  (figure  r a t prostate:BW which  and  to  c o n t r o l males  male  tissue  i n sham-operated  micromoles/kg  an  and  treatment,  There  between  significantly,  significantly  flutamide,  possibility  procedure,  post-castrated  significantly  ratios  measured.  25  results  animals.  (BW)  significantly  flutamide  c o n t r o l animals.  control  ratios  the  replacement  testosterone  surgery  also  with  days  i n the  to  that  levels  testosterone  micromoles/kg  weight  flutamide)  treated  serum  10  levels  50  surgical  not  on  doses.  testosterone  possiblity  enanthate  eliminate  were  in  the  Consistent  serum  animal  sham-operated  The  on  liver:BW  were  rats  to  to  differences  ratios  and  untreated  order  serum  c a s t r a t e d males,  Tissue:body  weights  effects  25  effect  flutamide  body  1).  Normal p h y s i o l o g i c a l  5 micromoles/kg  on  effect  r e s p e c t i v e l y , compared  daily  and  castration for  testosterone-treated  the  2.5  flutamide  direct  above,  increased, male  of to  concentrations mentioned  c o n t r o l males.  fully  2).  ratios  Figure  2.  EFFECT OF FLUTAMIDE ON PROSTATE TO BODY WEIGHT RATIOS IN ADULT MALE RAT  Prostate mg/100  wet w e i g h t  g)  with  (Gx)  corn  adult  oil  male r a t s  (C)  or t e s t o s t e r o n e  sc.,  varying  doses  of  days).  E x p e r i m e n t s were done 24 h o u r s  :  denote  significant  sham-operated  **  liutamide  were e x p r e s s e d  brackets  *  10 d a y s )  oil-treated  (F;  difference  or  5-50  average  t h e numbers o f  adult  : significant  as  alone  after  S.E.M.  animals  0.05)  last  were  (TE; 5  tested.  compared  difference  (p<  0.05)  to group.  compared t o  10  injection.  Numbers i n  same t r e a t m e n t  male.  which  day  micromoles/kg s c . ,  the  adult  and 10  enanthate  male o f  castrated  as  i n combination with  +/-  (p<  (Sh)  (280-300 g)  micromoles/kg  Ratios  (expressed  were d e t e r m i n e d i n s h a m - o p e r a t e d  post-castrated treated  t o body w e i g h t r a t i o s  corn  59  F i g u r e 2.  lflfln  LD O 5  80-  g  68-  (4)  (4)  (4)  43-  2)  ec m  (8)  20-  (4) F  25  ShcT  C  (4) F  25  (8)  i  (8)  (8)  T E 5 TE+FgTE+F^TE+F^TE+F,  GxcT  5°  Table EFFECT  OF  FLUTAMIDE ON  AND  2 TESTOSTERONE  L I V E R : B O D Y WEIGHT  TREATMENT  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 o n 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 a n d 10 d a y c a s t r a t e d a d u l t m a l e 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 T E ( 1 . 3 t o 5 m i c r o m o l e s / k g s c . , 10 d a y 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 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 d o n e 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 a s g r a m w e t t i s s u e p e r 100 g b o d y w e i g h t . Data w e r e e x p r e s s e d a s 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  Animal  (a)  models  tested  Liver:Body weight  Sham-operated adult treated with:  male  Corn o i l TE  (5 u m o l e s / k g )  Flutamide  (b)  ratios  only  (25 p m o l e s / k g )  only  4..16  + /- 0.,14  (4)  4..17  + /- 0..13  (4)  4..20  + /- 0,.03  (4)  4. 40  + /- 0. 30  (4)  4. 50 4. 72 4. 42  + /- 0. 12 + /- 0. 07 + /- 0. 09  (4) (4) (4)  3. 67  + /- 0. 23  (3)  + /+ /+ /+ /-  (8) (8) (8) (4)  Castrated adult males treated with: Corn o i l TE  only:  Flutamide  TE  1.3 2.5 5.0 (25  pmoles/kg pmoles/kg pmoles/kg umoles/kg)  (5 u m o l e s / k g ) p l u s 5 jumoles/kg 15 p m o l e s / k g 25 p m o l e s / k g bO pmoles/kg  only  Flutamide 3 4 4 4  .91 .02 .12 .37  0. 0. 0. 0.  15 05 13 09  Figure  3.  EFFECT OF FLUTAMIDE ON HEPATIC BENZO(A)PYRENE HYDROXYLASE IN TESTOSTERONE-TREATED CASTRATED ADULT MALE AND INTACT ADULT FEMALE RATS  Hepatic microsomal benzo(a)pyrene activities male  were measured  ( 2 8 0 - 3 0 0 g)  castrated  (Gx)  v a r y i n g doses of days).  ( 1 8 0 - 2 0 0 g)  a d u l t male r a t .  10 d a y s )  alone,  flutamide  (F;  t h e numbers o f a n i m a l s  a d u l t male o f  : significant difference  appropriate  castrated  For comparisons flutamide-treated group,  (T;  5  or i n combination w i t h  5-50 m i c r o m o l e s / k g s c . ,  S.E.M.  (p<  0.05)  as  10  last p m o l e s 3-OH  Numbers i n b a r s  denote  compared  group.  (p<  to  0.05)  compared  a d u l t male c o n t r o l  group.  between the t e s t o s t e r o n e  groups  to  t h e same t r e a t m e n t  plus  and t h e f l u t a m i d e u n t r e a t e d  t h e Duncan m u l t i p l e range  were c o n s i d e r e d s i g n i f i c a n t a t differences  with  used.  : significant difference  sham-operated **  enanthate  Enzyme a c t i v i t i e s were e x p r e s s e d  BP t o r m e d / m i n / m g p r o t e i n +/-  adult  and t h e 10 day  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  injection.  *  (Sh)  A n i m a l s were t r e a t e d  (C), or testosterone  rnicromoles/kg s c . ,  hydroxylase  i n the sham-operated  or female  corn o i l v e h i c l e  (BP)  between groups  t e s t was u s e d .  p< 0 . 0 5 ;  control  Differences  significant  were i n d i c a t e d by common  letters.  62 Figure  C  C  F  25  9 <* Sh  3.  F  25  T  5  T + F  5  T +  %  T + F  GxCC  2 ' 5  T + F  50  Table 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 d a y s ) 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 reductase (TR) a c t i v i t i e s was m e a s u r e d i n s h a m - o p e r a t e d and 10 d a y - c a s t r a t e d a d u l t male r a t s ( 2 8 0 - 3 0 0 g ) . Control animals w e r e 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 n m o l e s 3-OH b e n z o ( a ) p y r e n e f o r m e d / m i n / m g p r o t e i n +/- S . E . M . TR a c t i v i t i e s w e r e e x p r e s s e d as n m o l e s 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 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 . umoles= micromoles. 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  Sham-operated  adult  TR  male:  Control  3.2  +/-  0.1  3.6  +/-  1.2  (7)  TE  3.1  +/-  0.3  2.9  +/-  0.8  (3)  2.2  +/-  0.1*  8.3  +/-  0.8*  (4)  Castrated  (5 u m o l e s / k g ) adult  male:  Control TE t r e a t e d  with:  1.3  umoles/kg  2.7  +/-  0.2**  8.0  +/-  0.5  (4)  2.5  umoles/kg  3.0  +/-  0.1**  8.3  +/-  0.6  (4)  5.0  umoles/kg  3.8  +/-  0.4**  6.2  +/-  0.9**  (4)  *  denotes  s i g n i f i c a n t d i f f e r e n c e compared t o sham-operated m a l e o f t h e same t r e a t m e n t g r o u p (p< 0 . 0 5 ) .  **  denotes  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 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 ) .  corn  reversed  by  testosterone  micromoles/kg  s c . , 10  micromoles/kg) ratios  i n the  (from  enanthate males  enzyme  5  to  50  treated  order  to  the  activities  but  adult  and  studies  done.  adult  s c . , 10  and  male,  i t  dose-dependent  untreated  drug  (25  prostaterBW  i n the  testosterone days)  castrated  control  steroid  were  both  are  serum  testosterone  i s higher  Control  figure  for  days  to  days)  of  would  two  be  levels  animals.  metabolizing  consistent  3 and  table  significantly  male  3).  significantly  systems  (table  enanthate  reversed  the  TR  response chosen  sensitive In  i s higher  BPH  of  serum  were  and  i n the  and of  female.  v a l u e s by  adult 31%  3).  Increasing  (1.3  to  castration  BPH  Bellward  the  5  to  addition,  microsomal  Gontovnick  reduced  and  dose  male.  Castration  effects  of  ratios,  rat liver  of  BPH  levels  adult  w h i l e TR  control  testosterone  enzyme  with  the  microsomal  i n the  adult  dose  reverse  sex-dependent  i n the male,  sham-operated  doses  appropriate  hepatic  These  known  were  (1980;  which  male  sham-operated  activities  the  prostate:body weight  since  10  on  in a  maintain physiological  testosterone  sc.  flutamide  enanthate  on  the  castrated  micromoles/kg),  to determine  castration  to  effect  (5 m i c r o m o l e s / k g  (5  flutamide  ratios,  Hepatic microsomal  testosterone  10  While  significant  these  replacement  activities  In  BPH  no  reduced  compared  (c)  days).  corn o i l treated  significantly manner  had  enanthate  male  compared daily  micromoles/kg, effect  to  control  levels.  administration males  had  no  as above,  i n order  non-specific hepatic  compared  action  activities, by  sham-operated of  hepatic  maintaining and  Wistar  enzyme than  activities  male.  reversed  a n d TR  weight  (figure  testosterone  i n t a c t male  and  was  Similar  to  significantly  enanthate  activities,  to the that  a sc.  f o r 10  days,  i n the  adult  as w e l l  testosterone  than  higher 3).  as  levels  these  (figure  enzyme  1980).  i n the adult  did  effects  3), but the l e v e l s  the c a s t r a t e d  male  to the  administration  castration  i n the  This  In c o n t r a s t  enanthate  reverse  levels  greater  not  Bellward  significantly  fully  3),  testosterone  i s a sex-dependent  r a t (Gontovnick  consistently  table  ratios. BPH  female  on  led to a  e f f e c t which  suggests  p h y s i o l o g i c a l serum  was  of a  the e f f e c t of c a s t r a t i o n BPH  were  replacement  treatment  study  In the  (by 131%;  an by  were  testosterone  microsomal  study,  significantly  reversed  This  i n the adult  towards  control,  adult  activity  previous not  TR  microsomal  Hepatic adult  activities  control  activites  Castration  enanthate  prostate:body  TR  or androgen  testosterone  normal  activities.  (1.3 t o 5 m i c r o m o l e s / k g ) .  5 micromoles/kg  significantly male  o f TR  sham-operated  the p o s s i b i l i t y  activity.  but p a r t i a l l y  treatment  influenced  dose  enzyme  BPH  (5 m i c r o m o l e s / k g )  microsomal  of c a s t r a t i o n  to the sham-operated  enanthate BPH  hepatic  stimulation  significantly,  the  e f f e c t on  to eliminate  microsomal  significant  enanthate  to the c o r n - o i l t r e a t e d  significant  same a n i m a l s measured,  Testosterone  controls.  were  Figure  4.  EFFECT  OF  REDUCTASE INTACT  FLUTAMIDE  HEPATIC  FEMALE  microsomal  testosterone  i n sham-operated  (180-200  g ) , and were  5-50  or  t h e 10  treated  testosterone aione,  expressed S.E.M.  adult  corn  the  as  pmoles  male  activities  (280-300 (Gx)  g)  or  adult  AND  (T; 5 m i c r o m o l e s / k g  s c . , 10  varying  days).  injection.  days)  were  activities  reduced/min/mg  denote  female  of flutamide  Experiments  Enzyme  testosterone  i n brackets  doses  were  male r a t .  (C), or  s c . , 10  last  Numbers  MALE  o i lvehicle  i n combination with  alter  reductase  day-castrated  with  enanthate  micromoles/kg,  hours  TESTOSTERONE  RATS  measured  Animals  MICROSOMAL  IN TESTOSTERONE-TREATED CASTRATED ADULT  ADULT  Hepatic  ON  done  of  : significant  sham-operated **  adult  : significant  appropriate For  male  comparisons  group,  (p<  adult  (p<  t h e Duncan  multiple  Dillerences  were  significant  d i f f e r e n c e s between  common  letters.  group.  compared  to the  control  the  to  treatment  group.  the testosterone  and  considered  compared  0.05)  male  between  groups,  0.05)  o f t h e same  difference  castrated  tlutamide-treated control  difference  plus  tlutamide-untreated range  signiticant groups  test a t p<  were  was  +/-  animals  tested. *  2  were  protein  t h e numbers  (F  used.  0.05;  indicated  by  Figure  4.  68  Flutamide  (25 m i c r o m o l e s / k g )  significant  effect  significantly male  towards  reduced that  Furthermore,  on  of  activity  i n the  the  enzyme's  activity  the  i n the  multiple  range  occurred  at  10  female  (from  inhibited  activity  the  rat  15  to  p<  flutamide  0.05).  doses  had  50  greater  male,  i n the  but  castrated  3).  micromoles/kg)  adult The  no  intact  (figure  testosterone  day-castrated  test;  daily  BPH  flutamide  significantly  injected  effect  male  on  rats  (Duncan's  maximal  BPH  than  equal  or  BPH  inhibition to  15  micromoles/kg. Like  BPH,  enzyme  i n the  unlike  BPH,  than  towards  adult  TR  i n the  castration  hepatic  microsomal  rat  (Kramer  activity  was  higher  (figure  f o r 10  days  significantly  the  female.  flutamide  (25 m i c r o m o l e s / k g ,  effect  TR  control.  activities  However,  micromoles/kg) activity female effects  i n the  male  of  levels  In  Daily 10  increasing  significantly  i n the  4 ) .  i n the  occurring  at  adult  the  had  intact doses  female  adult  no  male  of  stimulated  equal  However,  increased  days)  multiple  doses  sex-dependent  TR  activities of  significant  compared  this  rat  male,  administration  testosterone-treated  (Duncan's  is a  e t a l . 1979).  adult  those  on  TR  drug  this  (5  to  the  to  50  enzyme's  castrated  range  test),  to or  greater  male with  towards maximal  than  25  micromoles/kg. In  order  flutamide metabolite  to determine  hydroxide, the flutamide  sex  effect  specificity of  of  flutamide  h y d r o x i d e were measured  i  flutamide  and  and i t s on  adult  female  Figure  5.  EFFECT OF FLUTAMIDE AND FLUTAMIDE HYDROXIDE ON HEPATIC MICROSOMAL BENZO(A)PYRENE REDUCTASE  HYDROXYLASE AND TESTOSTERONE  IN THE INTACT ADULT FEMALE RAT  Hepatic microsomal benzo(a)pyrene  hydroxylase  panel)  (TR; r i g h t  panel)  i n the i n t a c t a d u l t  female  and t e s t o s t e r o n e r e d u c t a s e  activities  were measured  (180-200 g ) . (C),  A n i m a l s were t r e a t e d  tlutamide  flutamide  (F;  Enzyme a c t i v i t i e s  *  10 d a y s )  25 m i c r o m o l e s / k g s c . ,  f o r m e d / m i n / m g p r o t e i n +/-  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 +/-  respectively. animals  (FL-OH;  Numbers i n t h e b a r s d e n o t e  left  rat  vehicle  t o r BPH and TR were e x p r e s s e d as  3-OH b e n z o ( a ) p y r e n e nmoles  with corn o i l  25 m i c r o m o l e s / k g s c . ,  hydroxide  (BPH;  or 10 d a y s ) pmoles  S.E.M., S.E.M.,  t h e numbers  used.  : significant  oil-treated  adult  difference female.  (p<  0.05)  compared  and  to  corn  of  PMOLES/MIN/MS  H-  c  ro  NMOLES/MIN/MS  71 hepatic its  microsomal  effects  on  BPH  a n d TR a c t i v i t i e s .  the intact  micromoles/kg,  10 d a y s )  microsomal  o r TR  On  BPH  the other  the  intact  below,  female  control  Development  binding  As  androgen  well-known  above  hepatic female.  hydroxide  BPH  (25  in  activities  the corn o i l  respectively (figure 5).  source  approximation receptor,  cytosolic  testosterone  steroid  1980).  Two  have  different  chromatography  precipitation  Since  better  technique  been  used  since  androgen  were  i t provided  protein  and  G-25M  charcoal  bound  (PD-10) (DCC)  and f r e e  i n the f i e l d  Peck  steroids  have  t h e PD-10  minicolumns  personnal  communication),  done.  an  from  (Clark  Sephadex  protein  receptor  receptor.  this  using  some w o r k e r s  ( D r . P. R e n n i e ,  conditions  of p r o s t a t i c androgen  and d e x t r a n - c o a t e d  using  an a s s a y o f  and  well-documented  methods  results  experiments  steroids  i n developing  to separate  f o r separating  compared.  following  vs free  the techniques  of c y t o s o l i c  techniques  unbound  obtained  o f bound  P r o s t a t i c t i s s u e was  Furthermore,  DCC  levels,  i n t h e measurement  used.  abundant  were  activities  on  adult  of flutamide depressed  of the hepatic  Separation a first  hepatic  column  TR  effect  i n the intact  significantly  female  administration  h a d no s i g n i f i c a n t  t h e same d o s e  with  assay  (a)  were  flutamide  activities  and s t i m u l a t e d  treated  2.  hand,  male,  Consistent  than the the  T a b i a 4.  SEPARATION TECHNIQUES FOR PROTEIN BOUND AND UNBOUND STEROIDS  For p r o s t a t e experiments, a d u l t male r a t s (280-300 g) were c a s t r a t e d 18-20 h r s p r i o r t o experiment. Prostate c y t o s o l (1-3 mg/ml) was prepared as described i n Methods s e c t i o n , and was incubated w i t h 3 H-dihydrotestosterone (DHT), i n the presence o r absence of 1000-fold excess molar r a t i o r a d i o i n e r t DHT o r R1881, f o r 18 hrs a t 4 ° C. Intact 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 prepared as described i n Methods s e c t i o n , and was incubated w i t h %-testosterone, i n the presence o r absence o f 100- o r 1000-fold excess molar r a t i o r a d i o i n e r t testosterone. C y t o s o l preparations were then t r e a t e d w i t h 0.5% DCC f o r prostate or 2.5% DCC f o r l i v e r c y t o s o l . In a separate set of tubes, c y t o s o l preparations were e l u t e d through Sephadex G-25M (PD-10) ininicolumns. R a d i o a c t i v i t y was analysed and Scatchard a n a l y s i s was used t o determine the apparent b i n d i n g k i n e t i c s (Kd denotes b i n d i n g a f f i n i t y i n nM; Bmax denotes b i n d i n g capacity i n fmol/mg). Data was expressed as average + S . E . M . Numbers i n brackets denote the numbers of animals t e s t e d . Tissue c y t o s o l  Ligand  Prostate  DHT  Liver  Apparent B i n d i n g K i n e t i c s  Competitor Kd (nM)  DCC  Bmax (fmol/mg)  Kd (nM)  PD-10  Bmax (fmol/mg)  DHT  0.5  166  (2)  0.7  134  (1)  R1881  0.6  137  (2)  0.7  175  (1)  DHT  DHT  no detectable b i n d i n g *  Te (HA)  Te  Te (MA)  Te  2o6  + 0.8  39 + 14  1.9 + 0.5  (3)  44 + 14  (5)  no detectable b i n d i n g * 2.7 + 0.9  * : no detectable displaceable and saturable b i n d i n g Te : testosterone l i g a n d range (HA denotes 0.05 t o 2.0 nM; MA denotes 2.0 t o 60 nM)  6.8 + 1.5  (3)  Adult  male  r a tprostate  (unfractionated) Methods  results  Both  o f bound  without  yielded  detectable  was  following  Kd=  0.5 nM a n d a p p . Bmax= similar  and K i r c h o f f  separation 0.7  nM;  cytosol  a p p . Bmax= with  competitor, using  nM;  either  rats  2.5%  separation  whole was  ratio  cytosol  incubated  and  Shain  results of  column  ( a p p . Kd= liver  displaceable  binding  (table 4). from  intact  adult  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 o r without  100- o r 1 0 0 0 - f o l d  technique.  displaceable  and s a t u r a b l e kinetics  f o r 3 hours  t h e c y t o s o l was t h e n  concentrations  binding  t h e DCC  o f t r i t i a t e d - D H T and  detectable  testosterone  following  with  (app.)  t h e PD-10  ( 4 0 mg/ml) t a k e n  incubation,  separation  using  Incubation  technique  binding  4 ) : apparent  comparable  with  r a d i o i n e r t DHT  while  radioinert testosterone  Following DCC  (1980),  t o show  (table 4).  by B o e s l  concentrations  o r 2 t o 60 nM) w i t h  molar C.  t h e same  ratio  reported  offered  similar  i n varying  (table  134 f m o l / m g ) .  failed  Liver male  et a l .  technique  levels  166 f m o l / m g  to those  methods f o r  and d i s p l a c e a b l e  kinetics  i nthe  o r R1881 (0.05 - 2 nM),  molar  saturable  the  (1974)  protein  incubated  excess  binding  as d e s c r i b e d  steroids yielded  of tritiated-DHT  1000-fold  technique,  prepared  free  binding  c y t o s o l which  concentrations  whole  DCC a n d PD-10 c o l u m n  from  f o r androgen  Prostate  or  c y t o s o l were  section.  separation  and l i v e r  These  yielded binding (table  at 4  excess degrees  treated  with  two r a n g e s o f  two  different  isotherms  with the  4 ) : one b i n d i n g  isotherm,  app.  Kd=  using  2.6 +/-  0.8  nM a n d a p p . Bmax=  0.05 - 2.0 nM;  nM a n d a p p . Bmax= Similar  binding  and a n o t h e r  44 +/-  tritiated-testosterone technique: fmol/mg  +/-  Since results,  that al.  1.5  separation  cytosolic  cytosolic  binding  However,  were  workers  to yield  pursued  The b i n d i n g were  (Sato  6.8  binding  values.  incubation  i n order  b y Yamada  time  to optimize  the l a t t e r  i s also  known  a l . 1982) which  may  steroid  and M i y a j i  (Drangova  of the  protein  i n this  as w e l l .  range  as  hepatic protein  temperature  this  assay. described  microsomal  an  fraction.  p r o g e s t e r o n e and 1980; A m b e l l a n  i n the regulation of o r may  play  at the t r a n s l a t i o n a l  to i n v e s t i g a t e the presence fraction  and  and Feuer  concentrations,  decided  more  conditions, the  (1982)  to contain  be i n v o l v e d  synthesis  kinetics  As a r e s u l t ,  i n the hepatic  receptors  similar  inconsistent displaceable  protein  in protein  a p p . Bmax=  our experimental  binding  role  separation  e t a l . 1980; O t a e t  androgen  intracellular  14  0.05 - 2 nM  in a similar  a report  et  nM.  r o u t i n e l y , a s i t was  isotherm  dependency,  glucocorticoid  2.0 - 60  yielded  Further,  Since  39 +/-  using  (n=3),  techniques  under  testosterone  concentration  found  0.9  was u s e d  by o t h e r  was f o u n d  studies  2.7 nM +/-  and e c o n o m i c a l .  1980).  also  fmol/mg  (n=3).  both  described  assay  were  using  0.5  a p p . Kd=  a n d t h e PD-10 c o l u m n  t h e DCC m e t h o d  convenient second  a p p . Kd=  isotherm  14 f m o l / m g  kinetics  1.9 +/-  a  direct  level,  of the androgen  i t was  binding  Table EFFECT  OF  VARYING  PROTEIN  CYTOSOLIC  5  CONTENT ON  TESTOSTERONE  ADULT MALE  RAT  LIVER  BINDING  P o o l e d a d u l t m a l e r a t ( 2 8 0 - 3 0 0 g ; n=10 a n i m a l s ) l i v e r w h o l e c y t o s o l was p r e p a r e d i n T E D b u t t e r (pH 7.4) supplemented w i t h 10 mM p o t a s s i u m c h l o r i d e , a n d v a r y i n g protexn concentrations (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 - 6 0 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 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 , t o r 3 hours 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 a n d b i n d i n g k i n e t i c s ( a p p . Kd i n nM; a p p . Bmax i n f m o l / m g ) w e r e c a l c u l a t e d u s i n g Scatchard 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 .  Protein Content (mg/ml)  Apparent  Kd  *  denotes  (nM)  Binding  Bmax  Kinetics  (fmol/mg)  11  no  detectable  binding  20  no  detectable  binding  32  286  60  65  132  30  74  182  36  no  detectable  displaceable  binding.  *  (b) protein  Optimization  Effect  testosterone Adult  degrees  of varying  binding  male  concentrations C,  rat liver  concentrations  displaceable  lower  (ii) on  on  cytosol protein  incubated  f o r 3 hours a t  protein  various  testosterone  (table  Since only  between  was  contamination  male  was a  found  hepatic  be  found  time  studied.  these  conditions  incubation  nM; a p p .  i e . >30 mg/ml,  contamination  i n adult  yielded  binding  could  or  testosterone.  132-286  5).  protein  incubation  of varying  binding  ( a p p . Kd=  kinetics  of microsomal  Effect  32 mg/ml  concentrations,  of microsomal  under  radioinert  no d e t e c t a b l e  binding  i n the binding  possibility  than  concentrations  high  possibility  the  hepatic  ratio  binding  while  testosterone  fractions,  molar greater  fmol/mg),  Differences  concentration  cytosol of varying  testosterone  relatively  the  excess  protein  cytosolic at  binding  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 n M ) , w i t h  Protein  at  male  ( 1 1 t o 74 mg/ml) was  100-fold  30-60  the protein  i n adult  without  Bmax=  testosterone  assay  (i)  4  of the c y t o s o l i c  two  would  make  unlikely.  and  rat liver  temperature  c y t o s o l and  microsomes Liver  cytosolic  mg/ml) w e r e were nM) at  prepared  separately with  as d e s c r i b e d  incubated  o r without  4 degrees  ( 4 0 mg/ml) a n d m i c r o s o m a l  i n the Methods  s e c t i o n and  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  100-fold  f o rvarying  f r a c t i o n s (1  excess  periods  radioinert  of time  testosterone,  ( 3 , 12 a n d 18  Table EFFECT  OF V A R Y I N G  TESTOSTERONE  6  INCUBATION  AND  DHT  CELLULAR  PERIOD  BINDING  ON  IN L I V E R  FRACTIONS  P o o l e d a d u l t m a l e r a t ( 2 8 0 - 3 0 0 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 w e r e 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 - 6 0 nM) f o r cytosol ( 4 0 mg/ml) a n d m i c r o s o m e s (1 m g / m l ) , o r t r i t i a t e d -DHT ( 2 - 6 0 nM) t o r m i c r o s o 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. T h e 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 determined 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 squares method t o r l i n e a r regression.  Cell  fraction used  Incubation period  Apparent  Binding  Kinetics  Testosterone  Cytosol  Microsomal  3  hours  Kd (nM)  Bmax (fmol/mg)  37  131  12  hours  54  40  18  hours  21  15  DHT Kd (nM)  Bmax (fmol/mg)  3  hours  101  173  5.7  205  12  hours  73  205  8.5  233  18  hours  48  106  7.5  162  hours).  In a p a r a l l e l  incubated 100-fold  in tritiated-DHT excess  conditions  as  testosterone 131  to  15  at  of  was  decrease nM  nM  (after  The studied  protein  without  4  binding using  hours  nM  of  varying  adult  (table  male  the  same  5.7  Bmax  In  tritiated-DHT for 3  and  and  to  f o r DHT  values  3 hours  to  incubation)  to  incubation  rat liver  18  7). C  molar  hours  temperature  was  which  unlabelled  the  eliminated  displaceable  incubation  fraction. conditions,  DHT  nM).  Increasing  cytosolic  101  remained  cytosol  ratio  from  microsomal  affinity 7.5  binding  appeared  (after  contrast,  to  hours  microsomal  Kd  nM  18  (range f o r  f o r c y t o s o l , and 48  from  contrast,  was  in tritiated-testosterone,  excess  degrees  In  incubation  app.  capacities  (after  binding  (range  f o r 3 hours  22  the  fmol/mg  to  same  fraction  incubation)  without  incubation  apparent  incubation)  unchanged  from  the  37  apparent  100-fold  to  from  were  cytosolic  6).  cytosolic  f o r microsomes.  using  competitor from  18  effect  incubated  106-205  binding  protein  relatively  was  or  the  the  hours  binding  The  3 hours  incubation)  18  (table  fmol/mg).  (after  binding  similar  somewhat,  21  C  of  i n the microsomal  binding  testosterone  using  decreased  to  degrees  6), yielded  162-233  3  with  t r i t i a t e d - t e s t o s t e r o n eor  protein  testosterone  DHT,  protein  tubes, microsomes  nM)  Incubation  after  4  test  (2-60  ratio  above.  fmol/mg  incubation  (table  molar  binding  respectively  binding  s e t of  the  both  temperature testosterone  other  the  or  testosterone  incubation  On  with  app.  hand, Kd  and  Table 7 EFFECT OF VARYING INCUBATION TEMPERATURE ON ADULT MALE RAT LIVER CYTOSOLIC AND MICROSOMAL TESTOSTERONE  BINDING  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 ) cytosolic (40 mg/ml) and m i c r o s o m a l (1 mg/ml) 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 in tritiated-testosterone (2-60 nM) w i t h o r 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 , for 3 hours at 4 o r 22 d e g r e e s C . 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 Scatchard analysis.  Cell  fraction used  Incubation temperature  Apparent Testosterone Binding Kinetics Kd (nM)  4 degrees C  Cytosol  22 d e g r e e s C  denotes  36  no b i n d i n g *  4 degrees C  27  268  22 d e g r e e s C  331  657  Microsomes  *  26  Bmax (fmol/mg)  no d e t e c t a b l e  displaceable  saturable binding  app.  Bmax  of the microsomal  were  increased  from  27 nM  degrees  C ) , and from  fmol/mg  ( a t 22 d e g r e e s  little the  likelihood  lack  dependent compared  C)  to cytosol  of  testosterone  binding  at  22 d e g r e e s  incubation saturation  were  done,  these  using  displacement  (iii) by and  various  Displacement competitors  microsomal  t o microsomes,  To f u r t h e r  support  and s p e c i f i c i t y  studies  testosterone  competitors.  of t r i t i a t e d - t e s t o s t e r o n e binding  i n adult  binding  fractions,  excess  progesterone, values  apparent  male  rat liver  was c o m p a r e d  using  whole  cytosol  molar  ratio  estradiol  Bmax  detectably  values  i n c y t o s o l and  a v a r i e t y of competitors  radioinert testosterone,  o r R1881 i n d i c a t e d  similar  Interestingly, triamcinolone ligand  from  DHT,  apparent  r e s p e c t i v e l y ) and  ( 1 3 9 , 1 4 4 , 9 9 , 105 a n d 101  displace  (table  o f t r i t i a t e d - t e s t o s t e r o n e by  ( 5 0 , 3 7 , 3 8 , 78 a n d 25 nM,  respectively). not  7).  studies  In c y t o s o l , displacement  100-fold  Kd  compared  and t h e l a c k  microsomes Testosterone  8).  based on:  in cytosol  f r a c t i o n s and comparing  by d i f f e r e n t  t o be  the time  i n microsomes;  (table  hypothesis,  seemed  contamination  binding  in cytosol  this  C) t o 657  There  3 hrs incubation;  no c h a n g e  ( a t 22  ( t a b l e 4) c o m p a r e d t o  i n testosterone  to relatively  protein  C ) t o 331 nM  respectively.  f  6) a f t e r  decrease  binding  ( a t 4 degrees  of i n t e r f r a c t i o n a l  (table  C  (at 4 degrees  268 f m o l / m g  o f DHT b i n d i n g  microsomes  testosterone  liver  fmol/mg,  acetonide d i d  cytosol.  Table 8 DISPLACEMENT OF TRITIATED-TESTOSTERONE FROM L I V E R CYTOSOLIC AND MICROSOMAL FRACTIONS BY VARIOUS COMPETITORS P o o l e d a d u l t m a l e r a t ( 2 8 0 - 3 0 0 g ; n= 12 a n i m a l s ) liver c y t o s o l (40 mg/ml) and m i c r o s o m e s ( l m g / m l ) w e r e 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 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 excess molar 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 radioactivity analysis. A p p a r e n t b i n d i n g k i n e t i c s were determined using Scatchard a n a l y s i s .  Apparent Testosterone Binding Kinetics Cytosol  Microsomes  Competitors  Kd (nM)  Bmax (fmol/mg)  Kd (nM)  Bmax (fmol/mg)  Testosterone  50  139  25  572  DHT  37  144  41  856  Progesterone  38  99  31  730  Estradiol  78  105  61  612  R1881  25  101  108  1351  119  1626  (methyltrienolone)  Triamcinolone acetonide  *  d e n o t e s no d e t e c t a b l e  no b i n d i n g  displaceable  *  and s a t u r a b l e  binding.  82 The under  microsomal  t h e same  equally  or  conditions  b y 100 f o l d  testosterone, 41,  testosterone  31 o r 61 nM 612 f m o l / m g  Kd= In  incubated  microsomal  experiment,  competitors were  o f 5 nM  of the high i e . Kd=  non-steroidal  ratios  chosen  t h e two c y t o s o l i c  protein,  cytosol  (tables  binding  and microsomes  were  oft r i t i a t e d - t e s t o s t e r o n e f o rmicrosomes),  (10 t o 1 0 0 0 - f o l d  9 A-D). since  testosterone  they  or  excess) of  These  ligand  were  within  binding  with  sites  the range  (table 4).  t r i t i a t e d - t e s t o s t e r o n e (within the  affinity-low  capacity  testosterone  binding  2.6 nM) b y a v a r i e t y o f s t e r o i d a l a n d  competitors  indicated  specificity  f o r testosterone  large  molar  ratio  hand,  incubation  profile,  an a p p .  In a d d i t i o n ,  testosterone  f o r c y t o s o l ; 1 o r 40 nM  various  with  1626 f m o l / m g ) .  concentrations  increasing molar  range  i n microsomes  1351 f m o l / m g .  a p p . Bmax=  in fixed  Displacement  radioinert  R1881 d i s p l a c e d  binding  displaced  concentrations  displaced  5 7 2 , 8 5 6 , 730  without  of  ratio  was  r e s p e c t i v e l y , a n d a p p . Bmax=  119 nM;  o r 40 nM  above,  incubated  ( a p p . Kd= 2 5 ,  respectively).  another  protein  or estradiol  108 nM a n d a p p . Bmax=  (app.  molar  DHT, p r o g e s t e r o n e  triamcinolone  (5  as described  excess  tritiated-testosterone Kd=  binding  relative  and p r o g e s t e r o n e  of 1000-fold  (table  a t 40 nM y i e l d e d  including  some  testosterone,  estradiol  a t a l l molar  ratios  flutamide  or flutamide  hydroxide  9A).  On  only  at the  the other  a non-selective  binding  DHT, p r o g e s t e r o n e a n d  tested  (table  9B).  d i d not produce  DES, appreciable  83  Table SINGLE  POINT BY  DISPLACEMENT VARIOUS  9A  STUDIES  COMPETITORS  AND  MICROSOMAL  OF  IN  TRITIATED-TESTOSTERONE  LIVER  CYTOSOLIC  FRACTIONS  P o o l e d a d u l t m a l e r a t ( 2 8 0 - 3 0 0 g ; n=8 a n i m a l s ) l i v e r cytosol (40 mg/ml) a n d m i c r o s o m e s (1 mg/ml) w e r e p r e p a r e d a s d e s c r i b e d i n M e t h o d s , a n d w e r e 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 without or with v a r y i n g excess molar r a t i o s (competitor:1igand) of competitor. C y t o s o l and m i c r o s o m e s w e r e t r e a t e d w i t h 2 . 5 % DCC t o s e p a r a t e b o u n d a n d free steroid fractions. Data were e x p r e s s e d as % bound r e l a t i v e to control. C o n t r o l c y t o s o l d i d not c o n t a i n competitor ( i e . 100%= no d i s p l a c e m e n t ) .  (A)  Cytosolic  fraction:  molar  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 b y r a t i o s of r a d i o i n e r t competitors.  Competitors  Molar  Ratio  varying  (competitor:ligand)  lOx  lOOx  100  100  100  Testosterone  88  92  91  Dihydrotestosterone  86  91  93  Progesterone  93  91  94  Estrad iol  89  90  100  DES  93  94  93  Aldosterone  100  100  R1881  100  99  100  100  100  100  100  100  100  Control  (methyltrienolone)  Flutamide Triamcinolone  acetonide  lOOOx  Table  (B)  Cytosolic  9B  fraction:  Tritiated-testosterone molar r a t i o s of competitor. experiments.  Competitors  ( 4 0 nM) d i s p l a c e d b y v a r y i n g D a t a r e p r e s e n t r e s u l t s o f two  Molar  Control  Ratio  (competitor:1igand)  lOx  lOOx  lOOOx  100  100  100  Testosterone  72-68  69-88  62-78  Dihydrotestosterone  75-89  70-88  62-78  Progesterone  79-85  68-83  68-83  Estradiol  63  70  DES  92  93  93  91  95  92  Triamcinolone Flutamide  acetonide  hydroxide  75  Table  9C  (C)  Microsomal  fraction:  molar  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 r a t i o s of d i f f e r e n t competitor.  Competitors  Molar  Ratio  by  varying  (competitor:ligand)  lOx  lOOx  lOOOx  100  100  100  Testosterone  89  81  81  Dihydrotestosterone  89  83  85  Progesterone  79  75  87  Estradiol  94  92  91  DES  87  90  95  Aldosterone  93  85  89  R1881  85  81  82  87  73  79  hydroxide  87  81  85  acetate  83  83  85  89  87  83  Androstenedione  86  81  80  Spironolactone  84  82  77  Control  (methyltrienolone)  Flutamide Flutamide Cyproterone Triamcinolone  acetonide  Table  (D)  Microsomal  9D  fraction:  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 b y v a r y i n g molar r a t i o s of 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 experiments.  Competitors  Molar  Control  Ratio  (competitor:1igand)  lOx  lOOx  lOOOx  100  100  100  Testosterone  46 -59  41--54  50'-69  Dihydrotestosterone  43 -57  42 -53  46 -62  Progesterone  40 -52  40- -48  4 5 .-59  Estradiol  48 -65  52 -61  49 -57  48  45  49 -61  52 -57  50 -55  51 -78  51 -73  50 -69  DES R1881  46 (methyltrienolone)  Flutamide Flutamide Cyproterone  hydroxide  51  51  48  acetate  50  49  53  52 -63  48 -60  50  60 -71  60 -65  67 -76  Triamcinolone Androstenedione  acetonide  87  displacement, 100-fold of  although  excess.  selective  equally  In microsomes,  binding,  and weakly  (between  t h e maximum m o l a r  since  displace  5-27% o f t o t a l  tritiated-testosterone displaced  there  ratio  was  little  a l l competitors  was  between  by a l l c o m p e t i t o r s  was  indication  ligand  a t 1 nM  displaced), while  40-60%  tested  only  seemed t o  the testosterone  bound  used  of total  (tables  a t 40  bound  nM  was  9C a n d 9D  respectively). Differences stability  i n the binding  of the microsomal  profile  compared  testosterone  binding  protein,  testosterone  binding  as a f u n c t i o n  indicates However,  the p r o t e i n  PMSF,  binding  metabolism  a known  protein  cytosol  with  may  of time  was  cytosolic or  temperature,  100-fold  excess  protease  inhibitor  preparation addition,  detectable  d i d n o t improve  f o r 3 o r 18 h o u r s protease  activity,  n o t a f a c t o r and p e r h a p s  mM  testosterone  rat liver  whole  o r absence of Addition  homogenate  the assay's  of male  of 1  of  radioinert testosterone.  incubation  Hide-powder  on t h e male  i n the presence  to the l i v e r  ligand or  The e f f e c t  adult  concentrations  tritiated-testosterone,  in cytosolic  inhibitor,  using  contamination.  by e i t h e r  or both.  protease studied  varying  decrease  be e x p l a i n e d  in vitro,  serine  binding  was  i e .decreased  dependent  relative  to the c y t o s o l i c  the 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  testosterone protein  and t h e  rat liver  of  during  consistency.^ whole  a t 37 d e g r e e s suggesting  this  In  cytosol  with  C yielded  no  that  ligand metabolism  proteolysis should  be  Table SEPARATION  OF T E S T O S T E R O N E USING  10 AND  THIN-LAYER  RELATED  METABOLITES  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) w e r e p r e p a r e d u s i n g diethyl ether or absolute ethanol. The s o l v e n t systems 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 (50:50) , a n d system I I - benzene: a b s o l u t e ethanol (95:5). 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 w e r e : UV d e n o t e s ultraviol l i g h t s e t a t 4 5 6 nm, DPH d e n o t e s dinitrophenylhydrazine r e a g e n t ( s e e M e t h o d s f o r p r e p a r a t i o n ) , a n d NPA d e n o t e s naphthoquinone 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 preparation). E a c h R f - v a l u e was c a l c u l a t e d a s t h e r a t i o 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 b y 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 front.  dry used  et  of  Rt-values  Standards  used  Solvent  systems  I  II  Visualization Techniques used  UV  NPA  x  x  Testosterone  0.35  0.43  5a-Dihydrotestosterone  0.39  0.28  x  x  5B-Dihydrotestosterone  0.39  0.47  x  x  5a-Androstan-3a,176-diol  0.33  0.35  x  5B-Androstan-3a,176-diol  0.20  0.26  x  5a-Androstan-3B,17B-diol  0.35  0.32  x  0.38  x  5B-Androstan-3B,176-diol Androstenedione  0.51  0.50  x  DPH  x  x  x  investigated.  3.  Testosterone Because  lack due  the previous  of consistency  (a) steroid  Separation standards layer  adequately and  beta-diol (table  results  and  and  visualized  separated  5 alphaOnly  the  assay  5 alphaand  light,  may  be  were  using  a t C3.  and  the l a t t e r ' s A l l of  3 beta,  either  (TLC)  3  I and 5  alpha,17  17 b e t a - d i o l  androstenedione  were  the method's  double  bond,  and 5 a l p h a -  sensitivity  and  acid  oxo- and h y d r o x y -  while 5  beta-DHT  colour  to the  the s t e r o i d s tested sulphonic  related  testosterone,  the dinitrophenylhydrazine  the naphthoquinone  contained  and  system  5 beta-AA-  C4-C5  were  oxo-groups  solvent  confirming  androstenedione,  confirming  and  5 beta-  to the conjugated  detected  testosterone  using  testosterone UV  of  androstenedione,  testosterone,  all  binding  t h i n - l a y e r chromatagraphy  chromatography  using  sensitivity  using  indicated that  the f o l l o w i n g experiments  detection  using  5 beta-DHT,  10).  reagent  had  out.  Thin  alpha-  studies  i n the testosterone  to ligand metabolism,  carried  II,  metabolism  colour groups  were  detected  reagent a t C3  since  or  C17  positions. Although (Rf=0.39) values  5 alpha-  using  using  solvent  solvent  respectively).  and  5 beta-DHT  system  system  In a d d i t i o n  II  had  I, both (Rf=0.28  t o UV  similar  had and  Rf  distinct  values Rf  Rf=0.47,  a n a l y s i s , the d i f f e r e n c e  90  in  Rf  values  beta-diol  between  was  testosterone  larger using  respectively)  than  in  system  system  respectively).  Furthermore,  solvent  when  systems  beta-diol; 0.35  (similar  system 17  this  II  was  beta-diol  (b)  had  quite or  for  0  silica  3  gel  extracts  from  using  4  sheets  (Rf  II), 5 system  a  Rf  and  alpha  but  of  from  3 alpha,  and  a  -A-  0.38  5  5  Rf=0.35  to  use  beta,  of  system 3  I  of  0.32  beta-A-  beta-A-  both  17  system  value  using  both  3  using Rf  17  Rf=0.35  advantageous  value  using  3  beta,  II;  this  alpha,  I  and  which  male  degrees  3  (Rf  =  either  of  C,  spots  0.37  17  using  17  rat  liver  were  values  The  material  testosterone  incubated  was for  11),  and  and  hrs  at  5  (40  beta-DHT  on  TLC these  including and  (Rf  0.45  using  value=  0.23  other  0.58-0.90).  not  detected 0  I  I I ) , and  did  or  e l u t i o n of  system  0-0.12  its  cytosol  dried  beta-diol  system  and  TLC  (table  using  alpha,  Testosterone were  using  chromatography.  endogenous  m o b i l i t i e s of beta-diol.  37  0.27  the  adult  testosterone  radioinert testosterone  values  beta-A-  of  extracts  number  elution  •cytosol  5  detection  of  or  for  material  17  value  and  with  endogenous  the  (Rf=0.43  testosterone).  cytosol  at  yielded  of  Rf  extracts  hrs  testosterone system  to  different  incubated to  i t was  a  alpha-A-  testosterone.  Ethereal mg/ml)  an  Separation  metabolites  had  II  5  (Rf=0.33  testosterone),  (dissimilar  beta-diol  value  to  I  identifying  steroid  and  seem 5  to  influence  beta-A-  in extracts 4  The  degrees,  3  alpha, of while  5  Table  11  SEPARATION OF ETHEREAL EXTRACTS TAKEN FROM L I V E R 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 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 M e t h o d s 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 radioinert testosterone (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 w e r e 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 b y 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 s y s t e m and w e r e i d e n t i f i e d u s i n g the 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 e a c h 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 systems used were: system 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 s y s t e m I I - b e n z e n e : absolute etnanol (95:5). pg= m i c r o g r a m s Cytosolic Extract Rf-values using s o l v e n t sytems  Incubation conditions Substrate  Time (hrs)  Temp. (deg.C)  I  II  Standard Rf-values I  II  ug T  0  4  0-0.1 0.39 0.56-0.70  0-0.19 0.43 0.37 0.55-0.80  0.45  (T)  2 ug T  3  4  0-0.1 0.24 0.57-0.76  0-0.19 0.27 0.23 0.57-0.78  0.27  (A)  37  0-0.20 0.23 O.bO-0.78  0-0.27 0.30 0.23 0.57-0.84  0.27  (A)  0-0.17 0.20 0.43-0.48  0-0.23 0.27 0.23 0.45-0.90  0.27  (A)  pg  2 p g DHT  3  37  no exogenous steroids added  3  4  0-0.12 0.58-0.90  beta-A-  3  cytosol  which  C.  In  was  from  degrees  reduction)  incubated  (Tomkins  beta-A-  3  hrs.  5  beta-A-3  a l . 1957).  carried  out  by  using  gas  gas  only  alpha, was  be  3 hours 17  results  or  with  (via 5  in  37  beta-diol  degrees  was  5  beta-DHT  of  beta-DHT  and  5  the  TLC  chromatography-mass  testosterone-  at  that  b e t a - d i o l , perhaps  dehydrogenase  of  4  suggest  in vitro  alpha,17  detected  at  incubated  Confirmation  Identification  derivatives  3  These  3 alpha-steroid et  (c)  5  after  i s metabolized  to  cytosolic  beta-diol could  c y t o s o l which  for  testosterone  was  17  addition,  detected 37  alpha-  by  beta-reductase experiments  spectrometry.  and  chromatography-mass  related  diol-  spectrometry  (GCMS). The  gas  powerful  tool  compounds its  et  and to  testosterone  are  by  the et  reflected  of  MSTFA  a l . 1974). separate  by  such  to  the  their  the  liquid  were  has  phase  on  and  been  derivatization the  stock  technique  of  solutions  of  prepared,  isomers of  a  of  and  trimethylsiloxy  rate  different  become  testosterone This  MOX  standard  TMS-androstanediol in  as  Following  (MO),  has  identification  and  androstane-diols  Differences  eluents  and  metabolites.  O-methyloxime  and  spectrometer  structures,  use  a_l. (1974),  derivatized  analysis.  separation  similar  (Bournot  testosterone  various  the  androstane-diol  aided  reagents Bournot  in  with  various  further  chromatograph-mass  retention  (TMS)  f o r GCMS  adsorption the  later  by  the  capillary times.  column  These  compounds of  m/z  were  further  values,  deflection  which  characterized  were  i n an e l e c t r o m a g n e t i c  Chromatography  retention  times  tested  were  varied  best  done  under  that  day.  The Rt v a l u e  min,  while  that  12.4 m i n .  testosterone  3 alpha,  reliably tested  3 alpha,  differ  data  17 b e t a with  (Rt values  those  range  to  poor  separation,  be  used  to characterize  The  latter  dioi  differences  3  a l p h a , 17  5 alpha-A-  3  alpha ,  5 alpha-A-  3  b e t a , 17  5 beta-A-  Separate androstane-diol yielded  3  that  the Rt values  by e x a m i n i n g  beta-diol beta-diol  mass  were  their  could  mass  (TMS) (TMS)  (TMS)  due not  spectra.  Rt=  9. 3 m i n  Rt=  9. 0 m i n  Rt=  9. 8 m i n  Rt=  9. 3 m i n  and t h e  analyzed  spectra  not  stereoisomers.  (TMS)  i n j e c t i o n s of testosterone  the following  for5  and t h a t  i n the Rt values  beta-diol  derivatives  rate  TMS-androstanediols  t o 9.8 m i n s ) ,  17 b e t a - d i o l  b e t a , 17  values,  a t a slower  the androstane-diol  was a c c o m p l i s h e d  5 beta-A-  i n Rt  (TMS)  (TMS; R t = 9.3 m i n ) d i d  9.0  of  (TMS).  of the other  from  conditions  17 b e t a - d i o l  eluted  17 b e t a - d i o l  of Rt  (MO-TMS) was 1 3 . 5  differences  indicated  comparison  running  3 alpha,  (MO-TMS) c o n s i s t e n t l y  following  beta-A-  basis;  f o r testosterone  daily  field.  (Rt) of the d e r i v a t i z e d  t h e same  f o r 5 beta-ADespite  5 beta-AThe  on a d a i l y  spectra  by t h e r e l a t i v e  compounds  values  than  determined  mass  of these  androgens  was  by t h e i r  values:  using  GCMS, a n d  (a)  testosterone m/z  (b)  (c)  (d)  values  5 beta-A-  weight  292)  m/z  values  436  346  ( 2 2 ) , 241  (m+,  3 alpha,  (molecular  weight  m/z  values  436  241  ( 2 0 ) , 256  values  214  ( 2 0 ) , 421  5 beta-A-  436  (m-15,  3 beta,  m/z  values  436  346  ( 1 8 ) , 241  17  (12)  (100),  (m-15,  256 ( 7 2 ) ,  4)  beta-diol  8 5 ) , 166  (100),  ( 8 ) , 346  129 ( 6 0 ) ,  ( 8 ) , 256  (8)  beta-diol  1 6 % ) , 129  (100),  346 ( 2 0 ) ,  (100),  256 ( 2 2 ) ,  18)  beta-diol  292)  (m+,  < 4 % ) , 129  (12)  testosterone tested  a t t h e C4-C5 while  17  17  (m+,  weight  position,  358  292)  (molecular  bond  8 % ) , 129  ( 1 0 ) , 331  beta,  m/z  (100),  292)  (m+,  weight  288)  beta-diol  (50),421  5 alpha-A-  5 alpha-A-3  weight  5 8 % ) , 153  (molecular  androstane-diols  C3  (m+  17  The  double  389  3 alpha,  (molecular  (e)  (molecular  molecule  differs  i n at least position  the l a t t e r  from  the  two w a y s :  the  and t h e e n o l  i s i n the reduced  conjugated  group form  a t the and  contains  a  hydroxy-group  differences  are  are  accentuated  further  instance, 389,  17  was  which  Of  the  16%  these  of  distinguished had  of  the  the  can  compared  to  alpha-A-  3  of  the beta,  alpha,17  256,  a  major  beta-Aion  3  molecular  taken  both  6A). was  molecular  weights,  5  a  from (40  This  the  base  m/z=  17  was by  major  the  Rf=  on  0.43  separation  the  analysis  yielded  the  for a  testosterone  ranging  lack 3  beta, 17  major (m/z=  17  5  from  5  ion  m/z=  beta-diol  beta-diol the  demethylated  and  5  m/z=  421  form  of  the  which  was  product.  ethereal  hr  mass  peak,  the  i t lacked  adult 0  their  characterized  base  of  4  between  of  was  from  stereoisomers.  (solvent of  varying  basis  the  beta,  since  m/z=  alpha/beta,  Differences  fragmentation  done  3  distinguished  beta-A3  A-  436),  or  the  of  436.  beta-diol  ion  For  ion  a l l contained  on  and  TMS-androstanediol  (m/z=  represent  incubated  as  the  peaks.  166  5  molecular  alpha/beta  alpha, m/z  a  of  ion  beta-diol  was  TLC  had  tested,  mg/ml)  identified  5  alpha-A-  zone  derivatization.  androstanediol  may  as  using  of  beta-diol  17  which  ion,  gel  cytosol  their  from  beta-diol  GCMS a n a l y s i s silica  These  determined  product.  alpha,  (m-15),  the  17  3  with  3  other  beta-A-  differed  be  alpha-A-  presence  as  position.  ions  molecular  relative  compounds 5  molecular  derivatives  their  spectra. by  by  C3  following  stereoisomers  beta-diols  quantities to  reflected  the  (TMS,MO)-testosterone  which  isomers  at  extract  system male  at  peak  4 at  389).  rat  II)  liver  degrees Rt=  of  C  13.5,  Other  whole (figure which  peaks  Figures  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  ( 2 8 0 - 3 0 0 g)  was p r e p a r e d  as  incubated at  different  radioinert  described  ether  (TLC).  TLC z o n e s  i n Methods s e c t i o n ,  (40  mg/ml)  and was  Steroids  were e x t r a c t e d  with  using  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 corresponding to testosterone  -androstan-3a,176-diol  (A) were e l u t e d and t h e  e x t r a c t was d e r i v a t i z e d p r i o r  L o w e r p a n e l : mass  and 56  resulting  Upper p a n e l :  D a t a were gas  spectrum.  Incubation Conditions:  (A)  0 hours at  hours at  and  (C)  4 degrees C ,  (T)  t o GCMS a n a l y s i s .  compared t o d e r i v a t i z e d s t a n d a r d s . chromatograph.  cytosol  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  testosterone.  diethyl  whole l i v e r  4 degrees C ,  3 h o u r s =at  (B)  37 d e g r e e s C .  3  figure  4°C,0h  30080-1  Etwid8tH  hi  . 00000H  -A  "> i r- • ' i 13 .fl 14 . 1 & . <j le.tf i P .0 I 8 . 8 1 9 .tf2 1  1111  1  figure i 98  -J  a  l  t  •ii• J• '> \  i t >  I.  4°C,3h ->>1M  -•88888*  c S88WH » 9 8 8 0 » - 'l|  30M09H  > • | I « I • | •  .i4  F i le  J;  Boh Mi I  9.W  8  13.8  14.8  I  1 &.8  • I | •< •  1 &.8  c kn 195 .?? t i n .  fyurc W.S-bN.I —.  I  • i i  48  1  I  I  I  I  I  i- L  1  tC  Mil.  S8 i 1  *-J  !•  L-i-  168  240  288  238  37°C,3h  'I 10000  •8.8  :B.5  ii  .1.8  .8  Sc ari 1'. I i  T-Dk ?t. T.S.S68 1138-,  -  .4 1:  346 30H  j  II  I  I I  ,• fliilliiii  43*  !• i JL . ---- --...1 . 1 . . f  rr  .  100  failed  t o show  r e c o g n i z a b l e mass  incubated  f o r 3 hours  alpha,  beta-diol  zone  17  Rf=  0.26  spectrum 241  ( 7 0 ) , 346  spectrum  solvent for  system  beta-diol  (30),  421  using  beta-diol  (d)  In  C,  min)  129  data  whole order  (100),  confirms  under  beta-diol,  further  under  t h e same  with  studies.  tritiated-testosterone  256  (100),  In a  ( R f = 0.26;  was  incubated  3 alpha,  (figure  17  6C)  m/z  ( 5 0 ) , 346 were n o t  were p r e - f r a c t i o n a t e d TLC  results  3 alpha,  experimental  that  17  conditions.  testosterone  metabolites  were  importance  3 alpha,  undertaken  tritiated-testosterone  c o n d i t i o n s as  binding  zone  compounds  to 5 beta-A-  experiments  incubated  t h e mass  in this  ( 9 0 ) , 241  the q u a n t i t a t i v e  of testosterone  was  3  i n TLC  spectra.  which  was  i_n v i t r o  to estimate  which  peaks  to 5 beta-A-  these  shows  436(m+,28),  the e a r l i e r  of r a d i o l a b e l l e d  cytosol  6B  product  256  they  5 beta-A-  5 beta-A-  or other  since  metabolism  testosterone  yielded  i s metabolized  in vitro,  cytosol  which  12.8 m i n  o f t h e same  as a major  zones  Formation  liver  from  Testosterone  These  testosterone  in  degrees  i n these  Figure  values=  analysis  (m+,10%),  TLC.  m/z  a t Rt=  r e c o g n i z a b l e mass  I I ) taken  (8).  peak II.  Cytosol  C yielded  A n a l y s i s of other  t o show  ( R t = 9.9  436  detected  product,  experiment,  3 h r s a t 37  values=  as a major  (70).  failed  different  at 4 degrees  ( s o l v e n t system  of t h i s  spectra.  TLC  those  used  f o r our  of the  17  using  cytosol  and t r e a t e d previous  A n a l y s i s of  of e t h e r e a l e x t r a c t taken  from  F i g u r e s 7 (A&B)  THIN-LAYER RADIOCHROMATOGRAM OF TESTOSTERONE METABOLISM IN VITRO IN ADULT MALE RAT LIVER WHOLE CYTOSOL  Cytosol  (40 mg/ml) was prepared  s e c t i o n , and was incubated  with  as d e s c r i b e d i n Methods 40 nM  t r i t i a t e d - t e s t o s t e r o n e , i n the absence l i n e ) or i n the presence  ( f i g . 7A; broken  ( f i g 7B; dotted  l i n e ) of 100-fold  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 .  Each  radiochromatogram was s e c t i o n e d i n t o 1-2 cm s t r i p s and analysed  for radioactivity.  The r a d i o a c t i v i t y  was p l o t t e d as a f u n c t i o n ot the Rt-value t h i n - l a y e r radiochromatogram s t r i p  f o r each  (abscissa).  peaks were i d e n t i f i e d using r a d i o i n e r t standard denotes t e s t o s t e r o n e ; A denotes  (ordinate)  The major s t e r o i d s (T  53-androstan-3a,176-dioi).  102  Figure  7A  CYTOSOLIC TESTOSTERONE METABOLISM 2 5 0  ( O)  no c o m p e t i t o r -  IN_ VITRO  added  T  3  2 0 0  D  W 0 -P X 0  E  1 5 0  H  ioo H  LT)  •  \ 0_  5 0  H  u /  o  i  0  10  \ I  1—  20 30 40 Rf values/100  5 0  60  Figure  CYTOSOLIC  250  o •  0  (/> 0  -]  7B  TESTOSTERONE  ( •)  with  METABOLISM  competitor-  IN  VITRO  added  200  150  +>  X u  E  100 H  in  • \  a. u  50 H  o  •  10  an  • 20 Rf  R  •  T  P  • • • • n - n  r-  30 40 values/100  50  60  the  protein  cytosol  bound  which  fraction  was  incubated  tritiated-testosterone fold  excess  at  degrees  4  0.38).  molar C,  These  in  ratio  formation  of  tritiated-  5  dependent  on  the  of  amount  mixture,  tritiated-testosterone Formation  beta-diol 100-fold data of  17  beta-A-  radioinert  order  avoid  to  this  chosen  4.  properly  in  (Rf=  beta-A-  alpha,  of for  0.21  the  beta-A-  metabolized of  must  be  the  a  for  studies.  a  relatively  was  added  to  of  8B).  major to  (figure  17  5  These fraction  beta-A-  for  data.  In  (R1881)  3  of  metabolism order  non-metabolizable  methyltrienolone  The  calculation  corrected binding  17  the  (figure  the  Rf=  7 A&B).  3 alpha,  that  hrs  and  fraction  i_n v i t r o  this,  3  beta-diol  of  bound  of  100  3 alpha,  17  22-32%  conclusion  analyse  cytosolic  absence  following addition  difficulty,  Hepatic  3  from  5  Because  further  or  testosterone  the  ligand concentration  concentrations  peaks 5  whole  tritiated-testosterone  present  i s being  liver  respectively (figure  tritiated-  beta-diol.  to  ranging  strengthen  rat  testosterone,  major  g r e a t l y enhanced  testosterone  free  was  of  excess  further  alpha,  in  was  presence  corresponded  testosterone  male  varying  radioinert  and  8A).  with  i n d i c a t e d two  peaks  incubation  adult  the  beta-diol  the  of  to  ligand  binding  assay Methyltrienolone non-metabolizable 1975,  1976).  To  and  (R1881) stable  determine  i s purported androgen  to  (Bonne  i t s properties  be  a  and  Raynaud  i n our  system,  we  Figures  8 (A&B)  THIN-LAYER  CHROMATOGRAPHIC  TESTOSTERONE Adult was  male  METABOLISM  r a t (280-300  prepared  incubated  testosterone, with  varying without  molar  treated  ratio  with  fractions.  diethyl  The bound  i n Methods  fraction  using  mixture  at  testosterone  bound  using  system  unbound  steroid  using  on TLC zones Rf=  I I , was p l o t t e d  i n the absence  circles  Rf= 0.21.  i n the presence  to the  the plot  of radioinert  concentration,  na  as a f u n c t i o n o f  F i g 8A s h o w s  on zone  denote  from  was  (5B-androstan-3a,  a n d R f = 0.21  radioactivity  denote  Cytosol  was e x t r a c t e d  contained  Rf= 0.38, w h i l e  squares  100-fold  thin-layer  on zone  a t each  fraction) or  o r "NSB" f r a c t i o n )  radioactivity  plotted;  Bound"  containing  (abscissa).  incubated  incubated  tritiated-  o f t r i t i a t e d - t e s t o s t e r o n e added  cytosol each  of  and m e t a b o l i t e s  (ordinate)  solvent  concentration  ( 4 0 mg/ml)  (TLC).  (or testosterone)  incubation  Bound  cytosol  s e c t i o n , a n d was  concentrations  a n d was a n a l y s e d  Radioactivity  -dioi)  whole  2 . 5 % DCC t o s e p a r a t e  ether  CYTOSOLIC  radioinert testosterone.  radiochromatography  the  liver  ( f i g 8A; " T o t a l  tritiated-testosterone  0.38  g)  ( f i g 8B; N o n - s p e c i f i c  excess  OF  IN VITRO  as d e s c r i b e d  with  ANALYSIS  of  testosterone;  denote  squares  denote  I n f i g 8B, c y t o s o l  of radioinert testosterone i s  tritiated-testosterone concentration,  radioactivity  radioactivity  on zone  on zone  Rf= 0.21, w h i l e  Rf= 0.38.  circles  106 Figure  Rod i o — c h r o m a t o g r a p h 15Q-.  < CO C•>  8A.  of" " T o t a l  "Testosterone" "DIDL" f o r m e d  Bound"  fraction  remaining  120 -  •  10  20  Free  30  Te added  4D  <nNO  50  107  Figure  Rodio—chromatograph 150  - i  (CO C •>  8B.  o f "NSB"  fraction  "Testosterone" remaining "•IOL" formed  120 a o o E D_ "D T3  C 0 0 CD  10  2D 3D F r e e Te added (nM)  40  conducted (a)  the  following  Effect  incubation  of  of  experiments.  protein  R1881  dilution  binding  and  in adult  time  male  course  rat  on  liver  whole  cytosol Cytosol and  was  was  prepared  incubated  at  (0.87-3.4  mg/ml) f o r  hrs)  degrees  at  4  capacity at  4  R1881  C.  concentrations,  between was  18  18  dependent  It  9  on  and  plots  to  inaccuracies  In  order  for  to  the  a  capacity  i s some  protein  of  time  10  18  hours  from  i t was  loss 1.5  of hours  completely variation  binding  concentration  on  At a l l  dependent  the  18  dependency  A-C.  experimental  plots,  for  incubation  component,  where  or  of  capacity  the  cytosol  10C). apparent  that  direct  involving multiple in  the  (or  displaceable  binding of  complication,  analysis  multiple  binding  graphical  determination  this  ( i ) computer  chemically  was  Scatchard  overcome  estimating  capacity  there  time  3  affinity-low  incubated  in figures  ( f i g 10C),  there  hour  became  Scatchard  pursued:  i s shown  hours  Although  (figures  effect  affinity-higher  the  high  of  The  binding  to  the  (1.5,  1.0).  protein  10A)  periods  cytosol  section,  concentrations  concentration  R1881  absent.  of  of  i n Methods  indicated protein  cytosol  (fig  time  Analysis  9;  lower  r=  protein  different  component  degrees,  described  various  (figure  the  as  using  binding blocking)  non-specific  sites  binding two  two  might  approaches  lower  binding  and  of lead  kinetics.  different  kinetics, the  analysis  were  methods  (ii)  affinity-higher sites,  using  Figure  9.  EFFECT R1881  OF  VARYING  BINDING  Cytosol  with  (0.05-2.0  nM)  ratio  Apparent  1.0)  ml to  cytosol.  incubation regression.  R1881,  Scatchard  cytosolic mixture,  i n Methods  or  of  presence  for  18  were  least  s e c t i o n , and  of  100-fold at  (expressed  analysis,  and  was  excess  4 degrees  i n fmoles)  were  directly  squares  (1-4  tritiated-R1881  hours  protein content  using  CYTOSOLIC  protein concentrations  sites  values  ON  LIVER  concentrations  binding  These  RAT  described  absence  radioinert  using  the  as  i n the  of  CONCENTRATION  A D U L T MALE  varying  number  estimated  PROTEIN  different  prepared  incubated  0.5  THE  containing  mg/ml) was  molar  IN  THE  expressed  C. were per  proportional  (mg/ml) method  added for  to  (r=  the  linear  110  Figure  9.  Figures  EFFECT  10  OF  VARYING  INTERVALS  Adult  ON  male  varying for  (A-C).  PROTEIN  CONCENTRATIONS  LIVER CYTOSOLIC  r a t (280-300 g)  cytosolic  different  protein  time  R1881  liver  100-fold  molar  1.64  mg/ml;  were  1.5  (fig  10C).  nM,  a n d was  regression.  used  ratio  were  (triangles)  hours  cytosol  p e r i o d s at 4 degrees  ( 0 . 0 5 - 2 . 0 nM)  concentrations  BINDING  radioinert  mg/ml.  ( f i g10A), 3 hours  Apparent  binding  determined  using  C,  incubated  using or presence  ( f i g  mg/ml;  10B) Kd  squares  was  of  Protein (squares)  I n c u b a t i o n time  affinity, least  prepared i n  a n d was  R1881.  0.87  TIME  PROTEIN  i n the absence  : (circles) 3.41  INCUBATION  was  concentrations,  tritiated-R1881 excess  AND  and  18  periods hours  expressed i n  method  for linear  Figure  TIME COURSE  • H D  1  10A.  C1.5 hi~s>  1  10 20 Specific  & PRDTEIN D I L U T I O N  1  3D Bound  1  1  40 50 (fmol/mg)  1  60  113 Figure  10B.  TIME CDURSE C3 h r s ) & PROTEIN  oH O  1  1  —i  10 20 30 S p e c i f i c Bound  1  DILUTION  1  40 50 (fmol/mg)  1  60  Figure  IOC.  TIME COURSE C18 h r s ) & PROTEIN DILUTION C O > CD) C A)  D. B 7 1. 6 4 3.41  mg/ml mg/ml mg/ml  KcfcO.15-025  1  0  1  1  10 2D 30 S p e c i f i c Binding  1  1  1  40 50 60 (fmol/mg)  Table COMPARISON  OF  LINEAR  ANALYSIS  12  AND OF  NON-LINEAR  BINDING  REGRESSION  DATA  A d u l t m a l e r a t l i v e r w h o l e c y t o s o l ( 1 - 3 mg/ml) was incubated with varying concentrations of tritiated-R1881 (0.05-2.0 nM), without o r with 100-fold excess molar r a t i o r a d i o i n e r t R 1 8 8 1 , 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 . Apparent binding 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 Scatchard analysis, or non-linear regression f o r saturation ( f r e e v s . bound) a n a l y s i s . T h e AMDAHL c o m p u t e r (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 used f o r these purposes. 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 constant (expressed i n n M ) , a n d Bmax i s t h e b i n d i n g c a p a c i t y (expressed i n fmol/mg). HALC d e n o t e s h i g h affinity-low c a p a c i t y a n d 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 binding proteins.  Estimates  HALC  Method  of a n a l y s i s  Non-1inear Regression using  LAHC  Bmax  Kd  Bmax  0. 22  5.6  12.2  138  0.25  5.3  10.0  135  Analysis  AMDAHL  Linear Regression using  Kd  f o r Binding  Apple  Analysis II  plus  Figures  11  COMPUTER  Adult was  3  ANALYSIS  male  hours  were  radioactivity determined  Bound) the  curve  b y AMDAHL  Scatchard  Specific denote for  100-fold  values  C.  by u s i n g  molar  Cytosol  cytosol a n d was  ratio was  computer,  f o r observed data.  data,  nM)  with with  R1881, f o r  with  2 . 5 % DCC  binding  kinetics  regression  R1881 added  o r by u s i n g  II-plus  incubated  (0.05-2.0  treated  ( f i g 11B; S p e c i f i c  by A p p l e  ( 1 - 3 mg/ml)  radioinert  Apparent  non-linear  ( f i g 11A; F r e e  computer-fitted  whole  of tritiated-R1881  was a n a l y s e d .  plot  Bound)  liver  DATA  i n Methods,  excess  a t 4 degrees  saturation  g)  as d e s c r i b e d  concentrations  without  and  OF R 1 8 8 1 B I N D I N G  r a t (280-300  prepared  varying or  (A&B).  of the  vs. Specific  linear  regression of  Bound/(Free  microcomputer. and squares  R1881) v s .  Circles  denote  values  1 1 7  Figure  NON-LINEAR  7 0 -i  CO) CD)  11A.  REGRESSION  ANALYSIS  Observed d a t a Computet—fitted  0) 6 0 E  \  r-l  0 50 E <+ TJ  Kd:122 nM  40 -  C D  0 30 -  OQ  U •ri  U- 2 0 u ai •_  10 0 -  -T  1  1  1  1  1  2  3  4  5  6  7  Free  R1881  CnM)  Figure 1 1 B .  LINEAR 4 0 -i  CO) CD)  REGRESSION  ANALYSIS  Observed data Microcomputei—fitted  20 -  •  Kd 10 nM  : l o Kd 025 nM  0  1  20  Specific  1  40  Bound  1  60  Cfmol  80  /mg)  100  excess  triamcinolone  (1983).  Liver  incubated had  for  provided  different  Both  Bmax=  (app.  should  the  of  same  set  10-12 that  when  using  the  a  data  a  the  time  required  program;  sake  computer  i e . the  (c)  Effect  combination whole  with  non-linear  of  Cortisol  the  data  on  R1881  were  figure  11  high nM;  app. capacity  fmol/mg).  program be  It  two  using  much  compared  attributed  to  computers.  utilizing  regression  and  a n a l y s i s was  may  the  analysed  triamcinolone  the  For  these AMDAHL  method.  acetonide  alone  binding  in rat  and  in  liver  cytosol Methyltrienolone  to  time,  were  for  this  the  conditions  12, a  regression  d i f f e r e n c e i n c a p a c i t i e s between  experimental  sites  135-138  Bmax=  and  period.  affinity-higher  non-linear  and  conditions  lower  app.  convenience  time  0.22-0.25  the  of  mg/ml  these  binding  results:  nM;  regression  1-3  et a l .  analysis, linear  (table  Kd=  Eagon  data  saturable  (app.  and  to  because  computer  of  by  convenient  similar  site  fmol/mg),  C,  binding  of  two  gave  noted  linear  and  methods  be  the  data  capacity  Kd=  described adjusted  degrees  a n a l y s i s of  methods  5.3-5.6  4  at  regression  using  as  c y t o s o l was  reliable  affinity-low  to  hrs  Two  A&B).  less  3  Computer  compared  site  whole  (b)  non-linear  acetonide  cytosolic  androgen  human p r o s t a t e  and  was  shown  and  progesterone  breast  to  bind  tissues  with  high  binding (Zava  et  affinity  proteins a l .  in  1979).  In  order  added  to separate  the synthetic  prostate block  these  progestin  c y t o s o l mixture,  the lower  affecting  Zava  binding  was u s e d  i n t h e measurement  receptor  by Eagon  et a l .  triamcinolone, (1983) the  added  studies,  triamcinolone following the  testosterone was  tritiated-R1881, excess C.  molar  were  Multiple  binding  triamcinolone high  app.  component  added  showed  plots  indicated  addition  used f o r  concentrations of  different  molar  ( a p p . Kd=  regression  138  or  without  two c o m p o n e n t s  a p p . Bmax=  o f t h e same  acetonide  mixture.  incubated  component  degrees  ratios  triamcinolone incubation  of  100-fold  f o r 3 hrs at 4  and a low a f f i n i t y - h i g h  12.2 nM;  linear  binding  as t h a t  o r absence  of c y t o s o l  capacity  Scatchard-type that  t o each  analysis  ( a p p . Kd=  In  the e f f e c t s of  o n R1881  various  of excess  or Cortisol  component  with  s e t of tubes,  5.6 f m o l / m g )  et a l .  cytosol mixture.  to test  r a d i o i n e r t R1881,  affinity-low  Bmax=  Single  also  androgen  In a d d i t i o n t o  and Aten  conditions  i n the presence  500 o r 1 0 0 0 - f o l d )  similar  studies.  incubated  ratio  Cortisol  a  binding  In a d i f f e r e n t  (100,  hepatic  and C o r t i s o l  incubation  A  and o t h e r s .  we d e c i d e d  acetonide  t h e same  Cytosol  to their  to the  without  of the hepatic  E i s e n f e l d e t a l . (1983)  Cortisol  following  (1983)  have  i s thought t o  component,  site.  approach  acetonide  which  R1881 b i n d i n g  affinity  e t a l . (1979)  triamcinolone  a technique  affinity  the high  effects,  (table 13), 0.22  nM;  capacity  fmol/mg).  analysis of  cytosolic  of triamcinolone  preparation  acetonide  (100-1000  EFFECT OF VARYING EXCESS MOLAR RATIOS OF TRIAMCINOLONE ACETONIDE OR CORTISOL ON R1881 BINDING IN ADULT MALE RAT L I V E R WHOLE CYTOSOL A d u l t male r a t ( 2 8 0 - 3 0 0 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 M e t h o d s s e c t i o n , and was incubated 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 tritiated-R1881 (0.05-2 nM), i n the absence or presence of excess 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 hours at 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 . Binding kinetics were d e t e r m i n e d u s i n g l i n e a r or 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. Excess molar Triamcinolone acetonide 0  ratio Cortisol  0  Apparent B i n d i n g Kd (nM)  Bmax  Kinetics (fmol/mg)  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  fold 500  excess) fold  excess)  following 0.27  +/-  values  alone  yielded  apparent 0.01 nM;  were  cytosol  acetonide  c h a n g e R1881 b i n d i n g .  be  noted  that  triamcinolone  acetonide  isotherm  binding  mixture  with  incubated  of c y t o s o l i c f o r 3 hours  kinetics  without  the  same  incubation  al.  (1980) and Ota e t a l . (1980),  effect the  of t h i s  following  (1-3  testosterone conditions  10 mM  cation  experiments  i n the usual  TED-supplemented  with  of  t h e TED p r e p a r e d  10  mM  potassium.  were  not  reason,  the  i e .  I t should with  excess  a single  t o those f o r 18  binding  binding  o f t h e same hours.  studies,  described  we  was  To t e s t t h e  i n hepatic  conducted.  used  by Sato e t  t h e TED b u f f e r  chloride.  o n R1881 b i n d i n g  mg/ml) was p r e p a r e d  incubated  yielded  as that  potassium  did  mixtures  triamcinolone  our previous  with  excess)  mixture.  similar  In  supplemented  containing  For this  the incubation  incubation  in  the addition of  ( 1 0 0 o r 500 f o l d  from  contained  triamcinolone  to cytosol  significantly  was o m i t t e d  of  these  of the high  component  Furthermore,  acetonide  with the  0.2 f m o l / m g ;  i n the absence  ( 1 0 0 o r 500 e x c e s s )  (100 o r  1 3 ) : a p p . Kd=  kinetics  triamcinolone  former  (table  R1881 b i n d i n g  incubated  Cortisol  isotherm  6.5 +/-  to the binding  or Cortisol.  Cortisol  kinetics  a p p . Bmax=  capacity  with  one b i n d i n g  binding  similar  affinity-low the  or i n combination  Whole  cytosol, cytosol  from  adult  male  manner  using  e i t h e r TED b u f f e r o r  10 mM  potassium.  c y t o s o l was  A l lcytosol  ratliver  A separate  incubated  a n d was  aliquot  i n the presence of  f r a c t i o n s were  incubated  with  Figures  EFFECT  12  OF  (A&B).  10  mM  POTASSIUM  BINDING  IN  Cytosol  (1-3  and  incubated  was  without  to  12A)  ratio  or  during  with  above,  presence  molar  ratio  cytosol  was  ( f i g 12B)  (circles)  preparation  potassium binding  of  (squares)  chloride  affinity  calculated squares  using  method  LIVER  CYTOSOLIC  described  no  was  mM  R1881  to  nM)  with  added;  TED  In  molar  buffer added  throughout  ( t r i a n g l e s ) 10  i n nM.  regression  or  absence  chloride  incubation.  regression  the  excess  maintained  expressed  linear  in  100-fold  potassium  Methods,  r a d i o i n e r t R1881.  potassium  and  non-linear for  (0.05-2.0  acetonide.  prior  in  incubated  of  10  cytosol  added  (Kd)  as  tritiated-R1881  excess  included:  incubation;  least  prepared  radioinert triamcinolone  mixture  were  the  ON  RAT  mg/ml) was  100-fold  addition (fig  ADULT,MALE  CHLORIDE  mM  Apparent  Binding  kinetics  ( f o r f i g 12A) (fig  12B).  or  Figure 1 2 A .  EFFECT OF KCI  ON  CYTOSOLIC R1881  BINDING  120 -i  m GO  100  80 H  0 . 2 3 - 0 . 2 9  60  40 H  20 H  1  O  20 40 60 80 S p e c i f i c Bound (fmol/mg)  100  Figure  EFFECT  OF K C I  12B.  ON C Y T O S O L I C  HALC  R1881  BINDING  120 -|  CD  OD  1D0  cr  CD  cu L LL S  80 H  CD  •  60 H  T) C  D 0  CO  u  Kd:0.14-0.24  40 H  •ri  u•ri  u ai o.  20 H  LO  1  1  T  T  20 40 60 80 S p e c i f i c B o u n d Cfmol/mg)  100  tritiated-R1881, radioinert contained  R1881.  were  Rigorous  excess  treated  and were  or without  A parallel  100-fold  mixtures buffer  with  with  2 . 5 % DCC  centrifuged  before  using  absence  of triamcinolone  of  ligand concentrations  affinity-low binding  capacity  proteins.  molar  acetonide.  and lower  A l l  i n the appropriate  cytosol  analysis.  incubated  was  used  ratio  tubes  radioactivity  acetonide  Despite  excess  s e t of incubation  triamcinolone  a n a l y s i s of data  free  100-fold  limited  i nthe  by t h e number  to determine  the high  affinity-higher  capacity  this,  Scatchard  a n a l y s i s f o r two  saturable  binding  sites  indicated that  a d d i t i o n o f 10 mM  potassium  t o TED b u f f e r  d i d not affect  R1881  (figures binding of  +/-  molar  0.24 nM;  ratio  binding  i n hepatic  omitted  from  In  0.02  10 mM  r a t whole a high  100-fold  cytosol  does  acetonide  100-fold  ( a p p . Kd= 0.14  These  studies  n o t i n f l u e n c e R1881  in vitro,  on t h e b a s i s  and t h e r e f o r e  could  molar  capacity  two R1881  site  be  site  29 f m o l / m g ,  ratio  excess  ( a p p . Kd= n=8).  adult  binding  ( a p p . Kd=  5.61 +/*- 0.97 f m o l / m g ,  capacity  131 +/-  of saturation studies,  cytosol contains  affinity-low  a p p . Bmax=  Bmax=  containing  17-22 f m o l / m g ) .  potassium  liver  affinity-higher app.  mixture  o r absence  the assay.  summary,  nM;  c a p a c i t y R1881  by t h e p r e s e n c e  triamcinolone  a p p . Bmax=  that  sites:  affinity-low  KCI i n t h e i n c u b a t i o n  suggest  male  The h i g h  p r o t e i n was n o t a l t e r e d  10 mM  excess  12 A & B ) .  binding  0.22  n=8) a n d a  1 0 . 5 +/-  1.2  +/-  lower nM;  The a d d i t i o n o f  triamcinolone  acetonide  to a l l  incubation a  mixtures  convenient  (d) R1881  means  of assaying  the former  site  point  of  tritiated-R1881  using  various  or without  varying  of  steroid  Displacement  of  tritiated-R1881  affinity-low  capacity  100-fold  were  done  tritiated-Rl881 acetonide, capacity  binding  sites,  tritiated-R1881 binding  and  were  and  done  molar  done  they  binding  using  ratios  from  by  the  using  high  cytosol  cytosol  was  which of  produced by  incubated  with  affinity-higher  concentrations values  Similar  triamcinolone  the lower  t h e Kd  cytosol  (50-1000  acetonide.  ligand  site,  competitors.  (1 nM)  i n the absence  These  of  8  competitors  affinity-higher 14).  (including  vitro.  of  the  detectable the various  were  chosen  respective  degrees  of  competitors  from  sites.  various  (table  provided  cytosolic  triamcinolone  displacement  Displacement by  using  approximated and  was  t o measure  component. they  site  (8 nM)  i n order  because  the  in  t h e R1881  or non-steroid  excess  by  of  experiments  excess)  studies  steroidal  the s p e c i f i c i t y  screening  with  containing  from  and  competitors  determine  incubated fold  site,  sites  non-steroidal  single  the l a t t e r  Displacement  binding  To  eliminated  nM  indicated  capacity  Furthermore,  progestins,  anti-androgens)  t r i t i a t e d - R l 8 8 1 from  binding of  the  liver  that  the  lower  site  was  non-selective  17  competitors  tested  g l u c o c o r t i c o i d s , estrogens,  using  molar  cytosol  ratios  from  50  to  androgens 1000-fold  128  Table  14  SINGLE POINT DISPLACEMENT STUDIES OF 8 nM T R I T I A T E D - R 1 8 8 1 BY VARIOUS COMPETITORS IN L I V E R CYTOSOLIC FRACTIONS A d u l t male r a t ( 2 8 0 - 3 0 0 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 (competitor:ligand) of competitor. 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 fractions. D a t a were e x p r e s s e d as % bound r e l a t i v e t o control. 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 ) . Competitors  Molar Ratio  (competitor: ligand)  50x  lOOx  500x  1000X  100  100  100  100  80  79  81  73  96  88  73  69  80  78  83  86  Cortisol  77  78  78  75  Estradiol  88  82  75  71  DES  91  82  77  73  100  100  100  95  99  100  94  95  70  65  70  66  84  78  68  65  100 100  96 92  90 96  82 86  94  92  81  72  97 100 100 100  92 99 95 100  87 100 97 95  85 95 87 85  Control R1881  (methyltrienolone)  Progesterone Triamcinolone  acetonide  Flutamide Flutamide  hydroxide  Cyproterone  acetate  Testosterone 5a-Dihydrotestosterone 53-Dihydrotestosterone Androstenedione 5a-Androstan-3a,178-dioi 56-Androstan-3a,178-diol 5a-Androstan-3B,178-diol 5B-Androstan-33,178-diol  Table  SINGLE  POINT  DISPLACEMENT BY V A R I O U S  15  S T U D I E S OF  COMPETITORS  CYTOSOLIC  InM  IN  TRITIATED-Rl881  LIVER  FRACTIONS  A d u l t m a l e r a t ( 2 8 0 - 3 0 0 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 a s d e s c r i b e d i n M e t h o d s , a n d 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 a n d 100 nM t r i a m c i n o l o n e acetonide, without o r with varying excess molar r a t i o s (competitor:1igand) of competitor. 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 b o u n d a n d 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 . Control 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 ) .  Competitors  Molar  Control R1881  (methyltrienolone)  Progesterone Triamcinolone Cortisol  Ratio  (competitor:ligand)  50x  100  500  1000  500(  100  100  100  100  100  78  81  83  79  80  99 100 100  100 100 100  93 99 100  83 100 100  Estradiol DES  100 100  96 100  78 98  76 99  Flutamide  100  100  99  98  100  91  ' 81  77  77  88  83  77  76  79  84 81 100 89 87 98 95 100 99 97 99  75 73 100 84 76 100 94 100 100 98 94  76 73 91 78 75 91 76 85 97 96 92  76 78 87 81 73 87 78 85 97 94 92  Flutamide Cyproterone  acetonide  hydroxide acetate  Testosterone 5a-Dihydrotestosterone 5 3-Dihydrotestosterone Androstenedione 5 a - A n d r o s t a n - 3 a , 17 3 - d i o l 53-Androstan-3a,173-diol 5 a - A n d r o s t a n - 3 3,17 3 - d i o l 53-Androstan-36,173-diol RU23908 RU2956 Spironolactone  73  80 80 83  excess,  5  beta-A-  flutamide bound in  hydroxide  R1881;  the  competitors  (or  65%  total  50-1000  fold.  which  bound  Displacement affinity-low  (table  ratios  radioinert alpha-A-  hydroxide The  bound  fraction). required Other potent  17  of  R1881  (or  for  higher 5  competitors  yielded  or  flutamide  by  equal  75-80% molar  total  ratio  and DES,  analogue),  5  total and  bound  Cortisol  R1881 at  from  this  1000-fold  high  spectrum  of  ratio,  R1881,  while  excess  androstenedione, 3  beta,  and  more  17  flutamide  this than  ligand. 20-25%  remaining  (1000-fold  5  in  excess)  of the  was  beta-diol.  f l u t a m i d e , RU23908  (a  highly  RU2956  Even  competitors  from  17  the of  of  3 alpha,  displacers  ligand.  the  500-fold  bound  beta-A-  rest  varying  molar  displace was  total remained  the  different  excess  to  the  bound  ratios  acetate  displacement  and  group  of  or  displaceable binding  5 alpha-A-  to maximally  properties),  latter  a  alpha-DHT,  amti-androgen of  35%  radioinert  cyproterone  such  total  at molar  beta-diol,  beta-DHT  to  of  to  fifty-fold  than  needed  range  A  up  displaced  estradiol,  maximum  total  At  greater  were  5-6%  up  t r i t i a t e d - R 1 8 8 1 from  testosterone, 5  beta-diol,  only  i s i n c o n t r a s t to  yielded  1 nM  15).  3 alpha,  flutamide  remaining)  of  was  beta-diol,  94-95%  This  capacity site  tritiated-R1881 molar  words,  fraction.  the  17  displaced  in other  bound  results  3 alpha,  (a R1881  s p i r o n o l a c t o n e were at  these  molar  insignificantly site.  did  analogue  not  poor  ratios,  displaced  Triamcinolone displace  with  the  the of  acetonide  ligand  from  the  high On  the  affinity the  high  basis  more  affinity-higher flutamide  rigorous  to  displacement  focus  on  the  that  fractions  i n our  protamine  (adsorption These  the  R1881  testosterone DCC  two  2.5%  binding binding  concentration  other  protein assay assay,  (0.5%)  different  was  the  were  etc.  compared.  and  ease  of  A the  studies.  that  (DCC  column  following  possibility  tested.  unbound  rapidity  concentrations  To  principles  gradient,  to  decision  techniques  eg.  compared  our  and  techniques  binding  These  site.  separation  used,  testosterone  cytosolic  to  and  More  later.  bound  of  flutamide)  site.  support  capacity  advantage  was  HALC  respectively)  density DCC  of  described  separates  employing  to  sucrose  conditions  the  suitably  lower  Furthermore,  the  lend  precipitation,  of  for  are  preparation,  concentration  lower  from  studies,  demonstrated  the  metabolite  affinity-low  compared  did  active  high  the  site  site.  studies  have  screening  (LAHC)  described  techniques  as  than  R1881  sulphate)  fractionation,  (HALC)  just  DCC  and  application,  since  (the  displaced  studies  preliminary  capacity  capacity  reconfirm  and  these  specificity  hydroxide  flutamide  binding  of  affinity-low  considerably  not  site.  However,  are of of  same  required the  using  a  lower  Table DIFFERENT AND  METHODS  FREE  16 TO S E P A R A T E  STEROID  BOUND  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 m a l e r a t ( 2 8 0 - 3 0 0 g ) w h o l e l i v e r , a n d 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 l 8 8 1 ( 0 . 2 5 - 2 . 0 nM) a n d 1 0 0 - f o l d excess molar r a t i o r a d i o i n e r t triamcinolone acetonide, with or without 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 sulphate, 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 sulphate 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 regression.  Separation  Techniques  A p p a r e n t R1881 Binding Kinetics  Bmax (fmol/mg)  Kd (nM) A.  Dextran  coated  charcoal  no  0.5%  B.  Protamine  sulphate  2.90  0.10  non-prec i p i t a b l e  denotes  detectable binding *  fraction  precipitable  *  2.94  0.23  2.5%  no d i s p l a c e a b l e o r s a t u r a b l e  no  detectable binding *  binding  (e) steroid  Separation  cytosol  triamcinolone 100-fold  with  acetonide,  excess  molar  separation  using  different  three  binding  component  16).  cytosol  2 . 5 % DCC  This  which  results affinity (app. not  sulphate  t o t h e 2 . 5 % DCC  Bmax=  and d e c a n t i n g  binding.  2.9  fmol/mg;  treated The 2.5%  r a p i d and assay  (including  time).  and y i e l d e d  a much  a slightly binding  The c y t o s o l i c  fraction  produced  longer  similar  and a s i m i l a r  sulphate  found  specific  binding.  required  with  was  of c y t o s o l  a p p . Bmax=  was r e l a t i v e l y  technique,  using  steroid  t o t h e 0 . 5 % DCC  90 m i n ) ,  2.9 f m o l / m g ) .  by  done  sulphate.  Analysis  0.23 nM;  p r e c i p i t a t e d by p r o t a m i n e  displaceable  s t e r o i d s was  vs. free  precipitation  0.1 nM)  Following  precipitation  15 m i n p e r b i n d i n g  (approximately  ( a p p . Kd=  free  i n no d i s p l a c e a b l e  technique  approximately  time  ratio  indicated a displaceable  i s i n contrast  of  o r absence of  protamine  o f bound  m i n a n d 3-5 m i n s a m p l i n g  assay  from  conditions:  resulted  Protamine  i n the usual  r a d i o i n e r t R1881.  o f bound  ( a p p . Kd=  precipitation  required 10  ratio  2 . 5 % DCC, b u t n o t 0 . 5 % DCC. with  molar  i n the presence  separation  treated  table  and f r e e  concentrations  excess  o r 2 . 5 % DCC, o r b y u s i n g Adequate  DCC  varying  and 1 0 0 - f o l d  incubation,  using  bound  ( 1 - 3 mg/ml) was p r e p a r e d  and incubated  tritiated-Rl881  0.5%  protein  fractions  Whole manner  of c y t o s o l i c  higher  capacity  no  which  was  Both  methods,  precipitation, high-affinity  2.5%  could  DCC  be  or protamine  used  low c a p a c i t y  f o r the a n a l y s i s of the  R1881  for  the purpose  of our s t u d i e s ,  the  convenience  of decreased  (f) binding  Optimization  the b a s i s  experiments, measurement protein.  of  (pH  varying  was  7.4).  excess  was  however,  chosen  due  to  time.  cytosolic  DCC,  followed C.  placed  analysis. least  described  protocol  was  cytosolic  prepared  adjusted The  molar  competitor,  steroid  the  was  sampling  androgen  (1-3 mg/ml),  diluted of  f r a c t i o n s were by  An into  (0.5 ml)  scintillation  Apparent squares  binding  method  of  kinetics  and  TED  with  ( 0 . 0 5 t o 5 nM) acetonide, C.  x g  Bound  and  at 4  decanted  for radioactivity were  estimated  regression.  or  2.5%  f o r 10 m i n s was  and  with  by a d d i t i o n o f  supernatant  vials  for linear  ice cold  incubated  at 4 degrees  c e n t r i f u g a t i o n . a t 1500  aliguot  using  triamcinolone  separated  binding  protein  c y t o s o l was  hours  f o r the  homogenization  tritiated-R1881  ratio  for 3  androgen  The  preliminary  adopted  following  centrifugation steps.  free  and  total  the p r e v i o u s l y  concentrations  fold  degrees  of  the hepatic  Cytosol  concentration  without  protein;  the former  the hepatic  the f o l l o w i n g  differential  100  binding  assay  On  buffer  of  sulphate  using  5.  Characterization  affinity-low  capacity  (a)  In  (i)  Displacement  whole  of  vitro  the  hepatic  (HALC) a n d r o g e n  of  HALC  R1881  studies  binding  indicated  androstenedione,  hydroxide  500-fold  amounts ratio  (at  of  molar  tritiated-R1881  r a d i o i n e r t R1881.  quantitative competitors  for  the  inhibition  purpose,  molar  as  of  or  and  competitor, (%  SB  added.  calculated  by by  regression. calculated app.  of  i n male  rat  liver  for  3  using The  =  according  (IC50)  similar  by to  100-fold gain  were  hrs  used.  and  4  of  value (%SB  of  and  this  sub-maximal was  with  1  acetonide,  a  C.  The  each  equilibrium  the  to  1000  inhibition of  competitor  method  nM in  (0.25  function  remaining)  least-sguares  more  For  incubated  as  these  Cytosol  degrees  molar  more  concentrations  at  flutamide  excess  protein,  maximal  plotted  logit  a  conducted.  caused  (inhibitor  the  displaced  specificity  were  IC50  the  Ki  using Ki  a  and  triamcinolone  was  The  using  nM  radioinert  estradiol  binding  varying  remaining)  competitor  and  R1881  earlier,  100  absence  that  the  displacement  tritiated-R1881 presence  of  which  described  DHT,  order  studies  ratios  tritiated-R1881 prepared  HALC  that  ratio)  as  In  appreciation  rigorous  plot,  protein  cytosol  testosterone,  curve  binding  high  experiments:  Preliminary  nM)  cytosolic  free was  vs.  log  for  linear  constant)  (F)  was  equation: /  (1  to Weiland  + and  L/app.  Kd),  Molinoff  (1981),  where  IC50  (F)  Figures  13  (A&B)  SPECIFICITY  STUDIES  AFFINITY-LOW  Adult was  male  rat  ratio  presence  multiplied  with  R1881,  of  1  R1881 by  logarithm  the  incubation  of  the  and  of  3 hours  This free  re-plotted  was  (Flut-OH);  reference.  molar  to  in  Fig  C.  for (T),  R1881 a  the bound,  function  of  added  to  shows  radioinert  R1881  androstenedione (DHT).  (CA),  R1881  the  Percentage  total  13A  excess  ratio  concentration  displacement  acetate  HIGH  mg/ml)  100-fold  p l o t t e d as  (abscissa). curves  (1-3  c a l c u l a t e d as  5a-dihydrotestosterone  hydroxide for  was  competitor  for  and  4 degrees  testosterone  curves  cytosol  acetonide,  relative  value  r a d i o i n e r t cyproterone  flutamide  liver  at  CYTOSOLIC  PROTEIN  excess  (ordinate)  inhibition  inhibition  LIVER  tritiated-R1881  100-fold  mixture  (methyltrienolone),  by  whole  displaced  100.  tritiated-R1881  shown  RAT  BINDING  g)  nM  for  binding  the  (A-dione)  R1881  (280-300  absence  specific  fraction  ADULT MALE  radioinert triamcinolone  or  radioinert (%)  CAPACITY  incubated  molar  FOR  of  In  f i g 13B  is  tritiated-R1881  estradiol  inhibition  (E)  and  curve  was  137  Figure  INHIBITION j QQ _  CURVE C CO C •) ( A) C V)  • \ •  FQR  13A  C Y T O S O L I C HALC  R1B81  BINDING  R1881 CN-0 Testosterone <N~) Androstenedione CN") D i h y d r o t e s t o s t e r o n e CN")  8 0 CO C •i-i  V  c  6 0 -  m  dione  u •rt t+-  0 4 0)  0  0_  tn  2 0 R1881  — i — i II i u i |  - 1  1—rr  • Log  if—i " rrrfp" r e n^frrrn 1  (Competitor  2 added;nM)  3  138  Figure  INHIBITION  CURVE  Log  FOR  13B  CYTOSOLIC  (Competitor  HALC  RIBB1  added;nM)  BINDING  Table  17  I N H I B I T I O N STUDY OF L I V E R CYTOSOLIC R1881 BINDING BY VARIOUS COMPETITORS IN ADULT MALE RAT A d u l t male r a t ( 2 8 0 - 3 0 0 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 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 the presence or absence of d i f f e r e n t molar r a t i o s of 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 hours at 4 degrees 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 w e r e 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 the e q u a t i o n d e s c r i b e d i n t e x t . Data were e x p r e s s e d i n n M . I n some c a s e s , e x p e r i m e n t s w e r e r e p e a t e d and t h e d a t a w e r e 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 .  Apparent K i (nM)  Competitors R1881  (methyltrienolone)  0.06  Testosterone  0.12  Androstenedione  0.12  5oi-Dihydrotestosterone  0.07  Estradiol  0.63  Flutamide hydroxide Cyproterone  acetate  77.0 0.38  +/-  0.02  (n=3)  +/-  64.0  (n=3)  140  for L  each  competitor  i s the free  apparent value for  i s estimated  ligand  each  estimated cytosol  Analysis apparent  dissociation  s e p a r a t e l y by d o i n g  of the displacement (table  displacement  profile  the  o f R1881  binding and  protein  5 alpha-DHT  higher  affinity  (0.38), In  from  t h e HALC  i s the  T h e a p p . Kd  a saturation  curves  indicated  of competitors ( 0 . 0 6 +/-  to this  protein  (0.63)  study  the  0.02)  than  R1881  testosterone  competitors  have a  do c y p r o t e r o n e hydroxide  of the competitors  site,  and  following  t o t h e HALC  These  ineffective  13 A&B)  ( t h e a p p . K i , i n nM):  or flutamide  relatively binding  (figure  to androstenedione,  (0.07 t o 0.12).  contrast to the rest was  17)  i s similar  estradiol  hydroxide  constant.  curve,  preparation.  Ki values  affinity  the i n h i b i t i o n  c o n c e n t r a t i o n , a n d t h e a p p . Kd  equilibrium  was  from  ( 7 7 +/- 6 6 ) .  tested,  flutamide  in displacing  and produced  acetate  highly  R1881  variable  results.  (ii) The  R1881  saturation  previously described  relative  specificity  binding  site.  binding  t o t h e same  conducted. incubated 100-fold absence  study  with excess  different  inhibition  of the competitors  To d e t e r m i n e  Cytosol  using  site,  i f these  tested  indicated  t o t h e R1881  competitors  the following  ( 1 - 3 mg/ml) was  studies  competitors  were  experiments  prepared  were  as u s u a l , and  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 triamcinolone acetonide,  of 500-fold  excess  molar  ratio  i n the presence  competitor,  except  or  Table BINDING  KINETICS  OF  PROTEIN  LIVER BY  18 CYTOSOLIC  VARIOUS  R1881  BINDING  COMPETITORS  A d u l t m a l e r a t ( 2 8 0 - 2 3 0 g) l i v e r w h o l e c y t o s o l (1-3 mg/ml) was prepared 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 100-fold excess molar r a t i o triamcinolone acetonide, without or with 500-fold excess molar r a t i o competitor, f o f 3 hours at 4 d e g r e e s C. In c o n t r a s t to the o t h e r c o m p e t i t o r s , 100-lold 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 used. C y t o s o l was t r e a t e d w i t h 2.5% DCC; radioactivity was analysed. 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 regression. Data were e x p r e s s e d a s 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 .  Apparent Competitors  Rlb81  used  (methyltrienolone)  Kd  Binding  (nM)  Bmax  Kinetics (fmol/mg)  0.26  +/-  0.08  5.42  +/-  0.65  Mibolerone  0.12  +/-  0.04  4.45  +/-  1.77  (3)  ns  Testosterone  0.29  3.8  5a-Dihydrotestosterone  0.32  3.41  Androstenedione  0.17  2.9  Estradiol  0.82  +/-  3.61  (3)  ns  Cyproterone acetate  0.35  Flutamide  +/-  0.41  ;  8.19  (3)  2.00  hydroxide  3  hrs.  incubation  18  hrs.  incubation  no  ns  d e n o t e s no competitor  significant (p> 0.05).  *  denotes  detectable  no  detectable  specific  d i f f e r e n c e compared  displaceable  or  to  binding  R1881  saturable  *  as  binding  for  r a d i o i n e r t R1881  molar  ratio  androgen al.  was  which  1973).  indicated  used.  Analysis that  the  compounds  of  total  which  was  2.9  were  to  potent  for  synthetic  plots  8.2  (table 5  to  fmol/mg), the  alpha-DHT, had  0.35)  and  suggesting  same  hydroxide 18  hrs  Scatchard  Tritiated-Mibolerone  (at  500  failed  that  binding  fold  to  et  18)  estradiol  0.17  to  (Liao  tritiated-Rl881 binding  3 or  ( i e . no  100-fold  in prostate  and  Kd=  binding  flutamide  binding  (iii)  (app.  a  testosterone,  acetate  displaceable  incubated  saturable  mibolerone,  Bmax=  bound),  is a  Scatchard-type  affinities  (app.  having  of  i n which  metabolized  cyproterone  capacities  Despite  Mibolerone  R1881,  binding  latter  mibolerone,  i s minimally  androstenedione, similar  or  site. (6-20%  excess)  demonstrate  plot).  binding  in adult  male  rat  cytosol The  previous  displaced and  study  tritiated-R1881  i t was  binding  to  Unfractionated usual, 0.05  to  molar  was  incubated  10  nM)  in  the  that  the  HALC  androgen  (1-3  with  presence  excess  Following  incubation,  radioactivity (figure  14)  binding  or  absence A  was  of  as (from  500-fold set  excess  of  tubes  acetonide.  treated  Scatchard incubated  site,  site.  prepared  separate  triamcinolone  analysis.  binding  tritiated-mibolerone  c y t o s o l was  which  HALC R1881  mg/ml) was  radioinert mibolerone.  100-fold  r a d i o i n e r t mibolerone  from  cytosol  contained  cytosol  the  and  ratio  before  indicated  in  with  2.5%  analysis the  DCC, of  absence  of  Figure  14  SCATCHARD PLOT  FOR  A D U L T MALE  TRITIATED-MIBOLERONE  Adult was  male  BINDING  r a t (280-300  incubated  with  g)  varying  RAT  liver  presence  excess  in  In a d d i t i o n  the absence  100-fold  excess  acetonide. linear  of  Data  were  regression.  nM),  cytosol  molar  ratio  ratio  or presence radioinert  analysed  Apparent  using  Kd was  ( 1 - 3 mg/ml)  of  i n the absence  t o the above,  (circles)  molar  whole  concentrations  (0.05-2.0  mibolerone.  CYTOSOLIC  PROTEIN  tritiated-mibolerone of 500-fold  HEPATIC  or  radioinert c y t o s o l was of  incubated  (squares)  triamcinolone  least  squares f o r  expressed  i n nM.  144 Figure  RAT  •  _|  O  LIVER  1  14.  MIBOLERONE  1  1  2 4 6 Specific Binding  1  BINDING  1  B 10 (fmol/mg)  1  12  triamcinolone saturable Bmax= site may  acetonide  mibolerone  14.2 f m o l / m g ; analysis  points  a t lower  addition  binding last  failed  be p r e s e n t ,  i n d i c a t e d a d i s p l a c e a b l e and  ligand  excess  (app.  0.54 nM;  results R1881  of this  (b)  yielded  t h e HALC  results.  support  ratio  site more  hand,  triamcinolone  the e a r l i e r  isotherm  consistent  R1881 b i n d i n g  with  protein.  conclusion  t o t h e same  binding  reguire  the other  3.5 f m o l / m g ) ,  are binding androgen  would On  binding  Another  a s i n g l e HALC  o f t h e HALC  study  and m i b o l e r o n e  presumably  molar  1.9 nM; a p p .  Multiple  estimations  a p p . Bmax=  kinetics  used).  concentrations.  to the mixture  binding  ( a p p . Kd=  reliable  but accurate  acetonide  the  5 points  to give  of 100-fold  Kd=  site,  The  that  site,  protein.  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  animal  models In  our previous  enzyme  testosterone-responsive metabolizing  enzymes  studies,  hepatic  involved  i n the r e g u l a t i o n of these  to  the hormonal  enzymes.  experiments  were  then  cytosolic  i t should  environment  In order  shown  drug  and  are s e n s i t i v e to the animal's  I f the hepatic  activities,  have  microsomal  status.  enzyme  we  to test  done.  androgen  this  hypothesis,  hormonal  dependent  t h e same  as the h e p a t i c  steroid  receptor i s  androgen  share  that  sensitivity  microsomal the following  Figure  SEX  15.  DIFFERENCES  IN  ADULT  RAT  HEPATIC  CYTOSOLIC  R1881  BINDING  PROTEIN  Liver  whole  age-matched Methods  cytosol adult  presence Circles adult  of  of  denote  adult  rat  triamcinolone  ratio  Note adult  and  were  male  liver  in  molar  and  the  female  presence  of  was  no  rat  liver  with  described  the  ratio  denote  presence  of  in  absence  or  r a d i o i n e r t R1881.  denote adult  from  varying  incubated  t r i a n g l e s denote  in  the  adult  female  absence  male rat  100-fold  and  of the  liver  excess  molar  acetonide.  displaceable  R1881  c y t o s o l which  triamcinolone  as  open-upright  squares  radioinert triamcinolone  : there  prepared  rats  incubated  cytosol  triangle  incubated  female  excess  acetonide;  filled-inverted  mg/ml) w e r e  tritiated-Rl881, in  100-fold  female  cytosol  male  s e c t i o n , and  concentrations  (1-3  acetonide.  binding was  detected  incubated  in  in the  147 Figure  SEX  DIFFERENCES  IN RAT  15.  LIVER CYTOSOLIC  R1BB1  BINDING  50 -i  •  20 Specific  40 60 60 100 B i n d i n g (fmol/mg)  Table  19  AGE DIFFERENCES BETWEEN MALE AND FEMALE RAT L I V E R 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 r o m a d u l t m a l e (70 d a y s ; 2 8 0 - 3 0 0 g) and i m m a t u r e male and t e m a l e (24 d a y s 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 M e t h o d s 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 or w i t h 1 0 0 - l o l d excess molar r a t i o R1881, t o r 3 hours at 4 degrees 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 of animals used. Apparent Binding K i n e t i c s A n i m a l model used  Kd (nM)  Bmax  A d u l t male  0.27  +/(3)  0.10  Immature m a l e  0.96  +/-  0.55  ns  (fmol/mg)  8.00  +/(3)  2.28  1.96  +/-  1.07  (3) Immature t e m a l e  (4) no  detectable Scatchard  **  (4)  *  : significant difference a d u l t m a l e (p< 0 . 0 5 )  compared  to  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 a d u l t m a l e (p>0.05)  **  : no d e t e c t a b l e  displaceable  to  and s a t u r a b l e  binding  (i) and  Age and s e x d i f f e r e n c e s  female  ratliver  Hepatic the  adult  post  liver  figure  present  studies)  binding rats  When  male  male  (table  to adult  three  male  male;  and s a t u r a b l e  Although  t h e HALC  i t had a s i g n i f i c a n t l y  that  t h e HALC  characteristics metabolizing  similar  enzymes  experiments  lower  cytosol  which  previous female  taken  from the  HALC R 1 8 8 1  HALC  detectable  ( i e . no  ( a p p . Kd=  capacity  R1881 b i n d i n g  Scatchard  0.55  ( a p p . Bmax=  1.96  male.  drug  male  0.96 +/-  protein  to the hepatic  male  binding  i n t h e immature  to the adult  conducted  our  added  Of t h e 4 immature  i n the r a t , i e .  were  167  was  and immature  o n e h a d no  affinity  a  component  with  but detectable  component  nM),  indicate  a n HALC  rats.  while  only  acetonide  R1881 b i n d i n g  had a s i m i l a r b i n d i n g  1.07 f m o l / m g ) c o m p a r e d  Liver  notably,  a p p . Bmax=  o r no d e t e c t a b l e  had lower  to the adult  1 3 . 9 nM;  that  R1881  yielded  (consistent  19).  r a t had lower  compared  male and  acetonide;  female  i n the adult  rats  Other  Both adult  triamcinolone  i t became c l e a r  cytosol  displaceable plot).  ( a p p . Kd=  i n the adult  tested,  compared  +/-  (24 d a y s o l d ; 8 d a y s  rats.  the adult  was n o t p r e s e n t  liver  immature  15).  was m e a s u r e d i n  demonstrated displaceable  from  isotherm  the cytosol,  rat  cytosol  taken  binding  fmol/mg;  was  and female  protein  i n the absence of triamcinolone  cytosol  single  o l d ) o r immature  male  ratliver  binding  to  (70 d a y s  i n male  cytosol  c y t o s o l i c R1881 b i n d i n g  weaning)  female  i n R1881 b i n d i n g  These share  results  some  and s t e r o i d  sex and age dependency.  to further  characterize the  Table EFFECT  OF C A S T R A T I O N  CYTOSOLIC  AND  20  TESTOSTERONE  HIGH A F F I N I T Y - L O W  CAPACITY  R E P L A C E M E N T ON R1881 BINDING  LIVER  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 ) o n a d u l t m a l e a n d 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 c o m p o n e n t 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 a n d 18 h r s t o 10 d a y 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) a s 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 d o n e 24 h o u r s after last injection. 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 . 0 5 - 2 . 0 nM) a n d 1 0 0 - f o l d e x c e s s m o l a r r a t i o triamcinolone acetonide, without or with 100-fold 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 analysed. 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 regression. D a t a w e r e e x p r e s s e d a s 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 . Apparent  model  used  Kd  Intact  adult  male  0.17 +/-  0.03  7.50 +/-  1.11 ( 4 )  0. 24 0. 18 0. 25  +/+/+/-  0. 07 0. 04 0. 06  5.86 7.75 4.76  +/+/+/-  0.88 ( 4 ) 1.62 ( 4 ) 1.77 ( 4 )  0. 16  +/-  0. 03  4.27  +/-  0.76 ( 4 )  no  detectable displacement  (4) *  day-Ovariectomized (Ovx) a d u l t f e m a l e  no  detectable displacement  (4) *  Testosterone enanthatet r e a t e d 10 d a y s Ovx a d u l t f e m a l e  no  detectable displacement  (4) *  10  *  adult  (fmol/mg)  male  Testosterone enanthatet r e a t e d (10 d a y s ) Gx a d u l t m a l e Intact  Bmax  Kinetics  Animal  C a s t r a t e d (Gx) a d u l t 18 h o u r s 4 days 10 d a y s  (nM)  Binding  female  : no d e t e c t a b l e  displaceable  and s a t u r a b l e  binding  HALC  R1881  (ii) liver  binding  Effect  cytosol Because  mainly of  cytosol  adult  male  female  t h e R1881  rats.  r a t s were  10 d a y s  R1881  days  compared  from  Some  20).  was  drug  metabolizing these  under  (endogenous  (testosterone)  cytosolic  Wistar r a t .  HALC  o r the absence  R1881  binding  results, female  replacement R1881  Unlike  f o r 10  binding  the hepatic  enzymes,  these  conditions,  or estradiol) or  s t e r o i d s do n o t seem  ( i n the male)  to  adult  experimental  testosterone  4 or  cytosolic  compared  o n HALC  and  (5  f o r 18 h o u r s ,  i n the intact enanthate  male  enanthate  our e a r l i e r  and s t e r o i d  factors  liver  male,  with  effects  using  adult  on t h e h e p a t i c  i n adult  effect  o r 10 d a y c a s t r a t e d  i n e i t h e r sex.  gonadal  the  effect  significant  that,  presence  4  the  was m e a s u r e d  Castration  testosterone  suggest  the  This  specificity  i n males,  testosterone  absent  and female r a t  h a d shown  only  hours,  with  results  exogenous  found  Consistent  to the c o n t r o l s  microsomal  18  protein  protein  no  protein  10 d a y - g o n a d e c t o m i z e d  treated  Furthermore, produced  a n d was  male  protein  of i n t e r e s t .  binding  binding  binding  significant  (table  on a d u l t  binding  s c . , 10 d a y s ) .  h a d no  controls  rat.  was taken  micromoles/kg  this  R1881  f o r androgens  liver  in vivo.  of gonadectomy  HALC  gonadectomy  HALC  component  to influence  ( i n the female) of  protein  i n the adult  152  Table EFFECT  OF HYPOPHYSECTOMY AND LIVER  CYTOSOLIC R1881  21 TESTOSTERONE  HIGH A F F I N I T Y - L O W BINDING  REPLACEMENT  ON  CAPACITY  PROTEIN  The e l f e e t o f h y p o p h y s e c t o m y a n d 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 ) a d m i n i s t r a t i o n o n a d u l t m a l e 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 a n d 1 7 - d a y s hypophysectomized (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 . Experiments w e r e d o n e 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 a s d e s c r i b e d i n M e t h o d s s e c t i o n , a n d 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) a n d 1 0 0 - t o l d e x c e s s m o l a r ratio radioinert triamcinolone acetonide, without or with 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 squares 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 d e n o t e numbers of animals used.  Apparent  Animal  model  used  Intact  adult  male  Kd  (nM)  Binding  Bmax  Kinetics  (fmol/mg)  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)  Testosterone enanathatet r e a t e d Hypox a d u l t male  0.18 +/-  0.03  5.84  +/-  0.35 (9)  (iii) enanthate HALC  Effect  administration  R1881 In  binding  addition  testosterone differences rat,  protein  was  which  To  affinity-low  capacity  adult  males  treated  with  ( f o r 10 d a y s )  days)  testosterone  21.  significant male  liver  control  enanthate  o f two s u c h  Hypophysectomy effect  administration  male  rat.  to  o i l vehicle  is  binding  rat injected  was m e a s u r e d  control or  or without  treated  indicate that  not s e n s i t i v e to removal  R1881  liver  ( f o r 10 o r 17 s c . , 10  days). in  h a d no  kinetics  to the intact  o n t h e HALC  High  using  a r e summarized  binding  Furthermore,  with  hypophysectomized  (5 m i c r o m o l e s / k g  R1881  protein  s c . , 10 d a y s ) .  in adult  sham-operated  testosterone  to the hypophysectomized  effect  results  male  binding  hormones and  f o r 10 o r 17 d a y s  o n HALC  significant  These  t h e R1881  experiments  c y t o s o l , compared  adult  the corn  male  i n the  testosterone  pituitary  binding  from  serum  decreased sex  o f t h e HALC  (5 m i c r o m o l e s / k g R1881  and  e t a l . 1979)  the s e n s i t i v i t y  taken  table  hormones  by  i n the hypophysectomized  results  cytosol  enzyme m e t a b o l i s m  (Kramer  testosterone,  enanthate  Pooled  rat liver  also  not reversed  of c i r c u l a t i n g  testosterone  cytosol  microsomal  was  test  i n serum  measured  male  hypophysectomy  e t a l . 1 9 8 2 ) o r DHT  to removal  reduction  on a d u l t  testosterone  to deleting pituitary  i n hepatic  replacement.  and  protein  levels,  an e f f e c t  (Bellward  of hypophysectomy  animal  h a d no  site  compared  hypophysectomized  control.  t h e HALC  binding  enanthate  R1881  of p i t u i t a r y  binding  hormones,  protein and  confirm on  the e a r l i e r  serum  testosterone  (iv)  Effect  treatment male  on  of flutamide  testosterone  administration  active  testosterone  hepatic  levels.  effect  with  and w i t h o u t  binding  10 d a y s ) . animals  flutamide  liver male  using  control Flutamide  HALC  (table  R1881  binding  capacity  BPH serum  hydroxide  R1881  protein  hydroxide  (the  from the  in vitro.  was  male  To  a s c . dose  rats  Castration  and  untreated  of either  were  done  f o r 10 d a y s  22) c o n f i r m i n g  compared  with  to castrates  Kd v a l u e s  without  +/-  to the  our e a r l i e r  0.20  or  effect  on  intact  results.  significantly  affecting nM;  f o r 10 24 h r s  h a d no s i g n i f i c a n t  binding  on  treated  (25 m i c r o m o l e s / k g )  Experiments  ( a p p . Kd=0.81  tested  (5 m i c r o m o l e / k g , s c .  enanthate-treated  hydroxide  R1881  adult  enanthate  treatment,  apparent  flutamide  binding  administration  lowered  shown t o  affecting  displaced  adult  protein  microsomal  r a t , without  castrated  injection.  cytosolic  binding  hepatic  injected with  testosterone  castrated  two phenomena a r e r e l a t e d , t h e  the experiment.  the l a s t  without  R1881  testosterone  also  hydroxide  previously  and f l u t a m i d e  or flutamide  before  was  testosterone  Some  were  male  these  of flutamide  R1881  R1881  Furthermore,  HALC  whether  HALC  HALC  of flutamide)  cytosolic  determine  and f l u t a m i d e  dependent  i n the adult  metabolite  i s not dependent  enanthate-treated  Flutamide  testosterone  the l a t t e r  levels.  cytosolic  activities  after  that  rat liver  decrease  days  results  the  a p p . Bmax=  4.27  Table  22  EFFECT OF FLUTAMIDE AND FLUTAMIDE HYDROXIDE ADMINISTRATION ON L I V E R 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 m a l e 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 binding protein. 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 w e r e 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 M e t h o d s s e c t i o n t r o m 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 ( 2 8 0 - 3 0 0 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 acetonide, without or w i t h 1 0 0 - r o l d excess molar r a t i o r a d i o i n e r t R1881, f o r 3 hours at 4 degrees 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 ot a n i m a l s u s e d . D i f f e r e n c e s b e t w e e n g r o u p 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 u s i n g t h e Duncan m u l t i p l e r a n g e 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 b e t w e e n g r o u p s were i n d i c a t e d b y common l e t t e r s . Apparent Binding K i n e t i c s A n i m a l model used  Kd (nM)  Bmax  (fmol/mg)  0 . 20 + / - 0 . 0 4 (4)  4 . 50 + / (4)  0 .07  0 . 24 + / - 0 . 0 3 ( 4 ; a)  3 . 90 + / (4)  1. 26  Testosterone enanathatet r e a t e d Gx a d u l t male  0 . 29 + / - 0 . 0 6 (4)  3 . 24 + / - 0 . 40 (4;a)  Flutamide-treated Gx a d u l t m a l e  0 . 81 + / - 0 . 2 0 (4;a)  4 . 27 + / - 0 . 92 (4;b)  Testosterone enanthate plus flutamide-treated Gx a d u l t m a l e  0 . 53 + / - 0 . 1 1 (3)  0 . 89 + / - 0 . 52 (8;ab)  Intact adult Gx a d u l t  male  male  Testosterone enanthate 0 . 3 4 +/plus tlutamide hydroxide(3) t r e a t e d Gx a d u l t m a l e  0.17  1.52 +/(4)  0.57  +/-  0.92 f m o l / m g ) ,  Kd=  0.24 +/-  compared  0.03 nM;  Duncan's m u l t i p l e testosterone days).  Bmax=  range  enanthate  enanthate  treated  alter  treatment  treated  displaceable  binding  fmol/mg,  n=8) c o m p a r e d  controls  ( a p p . Bmax=  Duncan's  multiple  testosterone  demonstrated hydroxide binding  since  flutamide  (v)  range  administration  binding  Effect  HALC  treated  (PZI) treatment  binding  i n the adult  serum  controls.  the  detectable  0.89 +/-  p<  castrated  0.52 castrated  0.05  using  flutamide-  animals  R1881 b i n d i n g . t o reduce  compared  Flutamide  50% o f t h e  analysis  were  HALC  Flutamide androgen  on l i v e r  of variance  not determined  castrated  of streptozotocin  Streptozotocin  testosterone  h y d r o x i d e and t e s t o s t e r o n e  however,  insulin  decreasing  treated,  s c . , 10  affinity  fmol/mg;  About  appeared  kinetics  hydroxide  0.4  by  d i d not  or eliminated  test).  enanthate  male;  rats  ( a p p . Bmax=  3.24 +/-  no d i s p l a c e a b l e  adult  to  to the testosterone-treated  i n the flutamide  castrated  male  castrated  capacity  was r e v e r s e d  ( b o t h 25  R1881 b i n d i n g  reduced  using  (5 m i c r o m o l e s / k g  administration  t h e HALC  significantly  effect  hydroxide  adult  c o n t r o l (app.  1*26 f m o l / m g ,  This  replacement  castrated  the testosterone  done  test).  s c . , 10 d a y s )  significantly  and  3.90 +/-  Flutamide or flutamide  micromoles/kg  to  to the castrated  adult  treated was n o t  on t h e  male r a t .  (STZ) and protamine cytosolic  zinc  HALC R 1 8 8 1  male r a t i s a d i a b e t o g e n , which  testosterone  and p r o s t a t e  i n addition to androgen  receptor  Table  23  EFFECT OF STREPTOZOTOCIN AND PROTAMINE ZINC I N S U L I N REPLACEMENT ON L I V E R 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 ( P Z I ) 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 m a l e r a t ( 2 8 0 - 3 0 0 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 d o s e was 60 mg/kg i v s i n g l e d o s e 4 d a y s 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 P Z I d o s e 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 M e t h o d s 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 excess molar 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 or w i t h 1 0 0 - t o l d excess molar r a t i o r a d i o i n e r t R1881, f o r 3 hours at 4 degrees 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 . Binding 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 d e n o t e numbers o f a n i m a l s u s e d . Apparent Binding K i n e t i c s A n i m a l model used Control  a d u l t male  Streptozotocin treated  adult  (STZ)  *  Bmax  (fmol/mg)  0.22  +/(4)  0.02  5.71  +/(4)  0.97  0.24  +/(7)  0.08  2.53  +/(8i  0.47  *  0.21  +/(4)  0.04  2.23  +/(4)  0.11 ns  male  Streptozotocin plus PZI-treated  Kd (nM)  adult  male denotes s i g n i f i c a n t d i f f e r e n c e c o n t r o l untreated adult male.  (p<  0.05)  compared  n s 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 c o m p a r e d s t r e p t o z o t o c i n treated adult male.  to  to  158  levels BPH  (Tesone  et  activities A  al.  1980),  (Warren  1982)  single  i v dose  of  significantly  reduced  the  binding 0.24 to  +/-  the  Bmax= the  protein 0.08  i n the  nM;  untreated 5.71  +/-  0.97  protamine effects R1881  of  zinc  binding  R1881  but  liver  Bmax=  2.53  +/-  (app.  Kd=  table  tested  binding. (10  had  of  the  HALC  (app.  fmol/mg)  +/-  0.02  R1881 Kd=  compared  nM;  app.  Furthermore,  no  Daily  mg/kg)  cytosol  23).  units/kg) no  (60  0.47  0.22  microsomal  male r a t .  capacity  male  showed  protein.  adult  adult  fmol/mg;  insulin  STZ,  binding  animals  HALC  i n the  streptozotocin  control  8 STZ-treated  displaceable  app.  decreases hepatic  one  of  detectable sc  i n j e c t i o n s of  reversed  significant  the  effect  glucosuric on  the  HALC  DISCUSSION  1.  Age  steroid  and  with  and  sex  dependent  metabolism  varying  dependent  activities  decrease  Testosterone  effects.  1977;  These  Kramer  The  are  (5  are  and  with  and  and  partially  testosterone  suggest  flutamide,  adult  male  the  androgens  from  act a  involve  with by  the  the  cytosolic the  at  of  10  days; et  competitive androgen of  10  days  increase  female  lvels. (5  these  those  of  and  sex  in  other  Parker  and  age  secretion. doses  treated  Peets  adult  enanthate  that  the  hypothesis  alteration  an  prostate/BW  rats  sc.,  results  with  P-450  the  reduced  Gurtoo  related  to  using  with  1980;  models  Sex  reversed  consistent  and  animals  towards  and  agrees may  male,  drug  4).  testosterone  Bellward  micromoles.kg  metabolite  not  using  i n androgen-dependent  consistent others,  drug  cytochrome  shown  a l . 1979),  post-castrated  enanthate  does  effects  et  adult  days)  anti-androgen  decrease  day  10  (Gontovnick  differences  a  sc.,  been  and  activities  replacement  micromoles/kg,  TR  significantly  the  i n BPH  different  ( f i g u r e s 3 and  have  d i f f e r e n c e s were  TR,  workers  microsomal  microsomal  using  microsomal  c a s t r a t i o n of  a  hepatic  status  following and  in hepatic  shown  in hepatic  BPH  these  was  hormonal  differences  rat;  differences  metabolism  Age steroid  sex  used,  ratios  with  figure  2).  receptor.  10  This  The  is  and  flutamide  displacement  serum  in  testosterone  a_l. (1974) that  caused  or  its  of effect  testosterone  levels  since  these  treated  values  with  testosterone  Flutamide 10  days)  BPH  (figure  (1976)  (at  who  3).  rats,  authors,  enanthate greater  in  These  showed  unchanged  a  10  similar  differ  since  and  than  day  results  with  15  flutamide  activity  at  the  (table  adult  with  Brown  of  hepatic  results  1). sc.,  hepatic  in vitro  doses  male  micromoles/kg,  post-castrated  agree  other  castrated  flutamide  inhibition  N-demethylase  but  i n the  testosterone-stimulated  activity  ethylmorphine male  doses  inhibited  microsomal rats  remained  by  used  et a l .  in  the  male  microsomal prepubertal  same  i n our  study i  failed  to  have  prostate/BW flutamide  a  significant  ratios  effect  in  in  the  the  compensatory  increases  suggested  Clark  by  microsomal  BPH  antagonized  Schriefers  (1981)  significant  the  male  study age  hypothesis  adult  serum  a l .  showed on  the  rat.  male  same  involved (day  that  concentrations  21).  flutamide in  to  males  as  of  of  not  as  and  have  a  DHT-treated  adults, were  due  to  since  while  male may  rat  our  the  castrated  is consistent  puberty  to  TR  Lax  is likely  the  due  flutamide  4). did  which  of  hepatic  testosterone,  difference  during  be  levels  of  the  or  lack  r a t may  with  (figure  exposure  androgens  The  inhibition  castrated  The  rat.  doses  discrepancy  adult  previous of  male  exposure  rats  activities  male  As  activities  This  BPH  testosterone  (1981).  that  TR  in pubertal  involved  earlier  in  castrated  effect  differences  latter  intact  on  adult  testosterone-dependent in  study  intact  activities,  activities  castrated  et  effect  at  with  to  induce  high a  an the  "classical" Schriefers In  receptor  contrast  to  flutamide  hepatic  microsomal  female  active  in  or  the  female  hepatic  male  or  may  resulted  stimulation  be  acting  in  the  3  and upon  be  to  due  receptor,  effect  as  of  results  male  rat.  castrated  flutamide  very  reverses  low  flutamide  flutamide  hydroxide  independent by  to  Brown  the  of  the  et a l .  castrated  metabolism  and  adult  a  testosterone  metabolism  suggest  flutamide  that  androgen  Further  animals  effects  are  and  activity  still  present  experiments  would  may  using  have  to  be  on  these  done  hypothesis.  the  prostate,  BP  synthesized  sensitive hepatic  the  of  microsomal  adrenally adult  proposed  flutamide  inhibition  These  of  i t s actions  testosterone  on  flutamide  The  anti-androgenic  rat  activities  both.  this  male  TR  adequate  in  The  of  the  but  of  4).  test  lack  hydroxide,  small rat  on  sham-operated  lack  and  to  a  female  The  the  flutamide  caused  adult  in  effect  the  adrenalectomized order  and  Schriefers  either flutamide's  hepatic  castrated  activities  levels. be  and  significant  However  of  Lax  to  i n an  of  TR  of a  flutamide  Administration  (figures  (Lax  due  to  or  androgen  (1976).  of  r a t may due  have  5).  female  hydroxylation, on  not  stimulation  female  efficacy,  results  BPH  (figure  control  the  the  did  metabolite  significant above  is flutamide-sensitive  1981).  (1981),  adult  which  similar are  the  of  microsomal to  of  enzyme  with  the  on  rat  in  action  hypothesis  testosterone  two  systems  i t s anti-androgenic  consistent  effects  flutamide  that liver  microsomal cytosolic hepatic  androgen  ligands  microsomal  fractions  Yamada  al.  binding  table  binding  no d e t e c t a b l e  and would  require  fraction.  The l a t t e r  binding  incubation  protein  simply  (table  after  cytosolic hepatic  of  the hepatic  reflected  (Sato  steroid  that  either  concentrations  in  the c y t o s o l i c  concentrations f o r from  the microsomal  u n l i k e l y based  on d i f f e r e n c e s  (table  d i f f e r e n c e s under  various  7 ) , and t h e o b s e r v e d  binding  to the microsomal receptor  cytosolic  DHT  low i n b i n d i n g  protein  4), binding  systems  of p r o t e i n ,  e t a l . 1980).  t o Roy  displaceable  the a d d i t i o n of triamcinolone  compared  and  et a l .  in contrast  protein  contamination  much l o w e r c o n c e n t r a t i o n s (Powell-Jones  high  seemed  temperatures  differences  was  cytosolic  workers  However,  5) s u g g e s t e d  as  binding  high  (>30 mg/ml;  a n d DHT)  saturable  i n hepatic  other  1982).  was  and  6-8) w i t h  with  developed  (testosterone  of very  o r was  Other  detected  (tables  a  (Roy e t a l . 1974; S a t o e t  Displaceable  was  at a  hypothesis,  was  requirement  detection,  the  assay  (table 4).  capacity  DHT  this  cytosol  testosterone  in  by antagonism  i n the l i v e r  The vitro  protein  consistent  there  test  androgens  and M i y a j i  (1974),  To  of others  natural  binding  characteristics  et  binding  and c o m p e t i t o r s .  testosterone  metabolism  receptor.  the protocol  1980) u s i n g  1980;  and s t e r o i d  androgen  following al.  drug  The poor  testosterone  non-specificity (tables  acetonide  fraction  a r e known e g . 1-3  9A a n d 9 B ) .  (table 8). to require  mg/ml,  displacement binding  to  profile  protein These  system results  are  in contrast  protein from  reported  their  cytosolic reported  by  to  laboratory, displaced  Ota  et  25-30%  a l .  underscoring  the  (i)  than  to  success.  requirement  Two  of  cited  workers,  by  these  reproducibility binding.  At was  suggesting activity,  addition  of  these reduced  et  hepatic  (1980) from  same estradiol  50%  a l .  involved  or  The of  that  less (1980),  in measuring  and  detection  this  with  the  conditions  ( i i ) inconsistent  high  concentrations,  protein  saturable  concentration degradation  protease the  proteolytic  with  protein  and  in a  met  encountered:  displaceable  in addition, of  were  were  cytosolic  consistent  possibility a  results  problems  ligand metabolism  former PMSF,  these  high  mg/ml),  either protein  consistency; for  Sato  major  very  (40-60  vitro.  did  the  nor  testosterone.  reported  reproduce  concentrations  binding  (1980)  a l .  that  by  the  testosterone  from  reported  displaced  variability  attempts  partial the  displace  conclusive of  et  results  protein.  Our only  not  (1980)  et  binding  specificity Sato  binding  However,  neither  p r o t e i n , while  that  testosterone  (1980).  provided  did  of  Ota  testosterone  receptor.  a l .  Furthermore,  a l .  suggest  estradiol binding  et  have  testosterone that  hepatic  Sato  evidence  androgen  binding  the  laboratory  consistent  the  with  was  testosterone testosterone  dependent by  may  proteolytic be  less  inhibitor,  manner,  occurring  likely did  not  since improve  R e m a z o l b r i l l i a n t Blue activity  indicated  in  no  method  detectable section not  a  protease  i i i ) .  activity  These  f a c t o r and  in vitro  results  perhaps  suggest  that  (see that  Results  2b  proteolysis  ligand metabolism  was  was  a  possibility.  2.  Testosterone TLC  to  5  studies  beta-A-3  using  the  high  conditions protein  addition  enzymatic the  the  law  of  of  to  mass  and  In  those  for  binding  and  the  metabolism:  cofactors  words, the  the  would  reactions,  beta-DHT  alpha,17  addition  to  testosterone  concentration  testosterone  metabolism  overall  5  cytosolic  mixture  toward  in vitro,  ( l i g a n d and  of  the  favour  following  ^  ^ >  beta-diol excess would Km  radioinert increase  value  thereby  radioinert)  the  for  leading  to  testosterone  metabolism. These  results  inaccuracies  would  testosterone  ligand  lead be  one  made  to  conclude  in estimating  concentrations  (i)  (NADPH),  action: >  8),  protein.  testosterone  hydrogenation  testosterone  increased  metabolized  testosterone  necessary  unlabelled  beta-Androstan-3  other  as  was  (figures 7  testosterone  soluble  the  testosterone 5  in vitro  testosterone  favour  and  excess  reduction  testosterone  conditions  concentrations, enzymes,  vitro  that  hepatic  tend  in  beta-diol  incubation  of  metabolizing (ii)  indicated  alpha,17  same  measurement These  metabolism  used  that the for  gross free saturation  studies, more  and thus  suitable  significantly  would  ligand  i n v a l i d a t e Scatchard  h a d t o be c h o s e n ,  metabolized  under  these  Methyltrienolone  ( R 1 8 8 1 ) was u s e d  is  androgen  a very  potent  demonstrated  minimal  (Decker  and L e v i n s o n  Raynaud  1976; P o u s e t t e  3.  indicated  in vitro,  using  liver (Bonne and  1979).  capacity  o f R1881 i n a d u l t  two b i n d i n g  Scatchard  sites  cytosolic  (Kd= 0.22 +/-  0.02 nM; lower  10.4  +/-  131 +/-  1.2 nM;  Bmax=  site.  Measurement  excess  triamcinolone results  Eagon  e t a l . (1983),  Most  are consistent Decker  androgen  recently,  androgen  with  fmol/mg,  capacity  n=8)  the recent  (Kd=  binding  by a d d i t i o n o f  abstracts of  (1983) and  used  similar  conditions i n  "receptor"  binding  studies.  a controversy  has a r i s e n  l o c a t i o n of the steroid  Greene  and Welshons  histocytochemical  0.97  13 a n d f i g u r e 1 2 B ) .  and L e v i n s o n  who  cytosol  determined  5.61 +/-  was b l o c k e d  (table  whole  affinity-low  29 f m o l / m g ,  intracellular (1984)  high  affinity-higher  acetonide  e t a_l. (1983),  hepatic  Bmax=  of the l a t t e r  These  liver  11B), as  analysis: a specific  and a n o n s p e c i f i c  Eisenfeld  male  (figure  n=8)  their  since i t  protein  Incubation  using  purpose,  e t a l . 1979), and  Characterization of the hepatic  binding  conditions.  1983) o r p r o s t a t i c c y t o s o l et a l .  A  was n o t  binding  f o r this  (Raynaud  metabolism  which  analysis.  regeptor.  e t a l . (1984)  approaches,  have  regarding  using  challenged  King  the and  different the  hypothesis  166 that  unoccupied  cytoplasm  of  estrogen  estrogen-responsive  absent.  King  receptor  antibody  indicated both  in  the  the  Welshons  et  and  of  or  a l . (1984) cells  loss  estrogen  former  group,  in  nucleus.  by  of  of  Both  that  estrogen groups  receptor  suggest  estrogen  receptor  radioligand  exchange  technigues,  preparation,  centrifugation nuclear during  earlier  cited  light  does  receptors  importance  not, and  the  (1984)  our  become  an  complete of  the  conventional  artefact  of  homogenization  may  be  partitioned  not  noting  loosely  into  interesting  that  techniques,  distinguish in  the  continues; of  an  hormone  the  and  bound  cytosol  nucleus  or  this  receptors involve  presence  of  the  HALC  on  was  the  based  perinuclear  unfilled the  does  in cell a  r e b u t t a l to  evidence  that  between  however  experiments  their  and  characterization studies  in vivo  a  conclusion  using  the  receptors  offered  papers,  located  controversy  whether  which  microscopy  staining  These  during  pituitary  disruption.  Schrader  on  steps.  receptors cell  occurring  of  unfilled  measurement be  binding,  resides primarily  that  may  estrogen  tissues  nearly  the  is  steroid.  enucleation caused  the  steroid  specific  estrogen  confirming  cytoplasmic  cytosol  that  the  exogenous  cytochalasin B  the  when  nuclear  absence  receptors,  that  found  primarily in  estrogen-responsive  only  found  reside  cells  (1984)  staining  presence  GH3  the  Greene  presence  derived of  receptors  estrogen  nucleus.  not  discount  physiology.  nuclear  The  contaminant  (specificity,  the Thus, or  saturability  component) would  still  be  valid, could  and t h e p r e s e n c e still  enzyme  be r e l a t e d  induction.  the  above  the  very  Of  The  high showed  (R1881)  and n a t u r a l  metabolites)  relative  the  sub-nanomolar  adult  male  are  serum  20  approximately  1980).  Flutamide  non-saturable Bouton  range  nM  binding  androgen  hydroxide The present compared  had a v e r y hepatic  rapid  HALC  i n the intact  by D e c k e r  (Patsch  i n the were levels  et at.  d i s p l a c e a b l e but  i s consistent  e t a l . (1979) concluded  that  or ovariectomized  measuring flutamide  p r o t e i n was n o t adult  female  20), consistent with  and L e v i n s o n  with  constant.  binding  (table  within  t h e known  estrogen  showed  This  off-rate  androgen  t o the a d u l t male  observations  however,  who  were  within  serum  hydroxide,  Both the  constants  i n range  and Raynaud  receptor,  15).  concentrations  18).  binding  and n o n - s t e r o i d a l  of sex hormones  1), while  (table  intracellular  and f l u t a m i d e  17), well  testosterone  hydroxide  t o answer  i t s related  (table  range  subnanomolar  e t a l . (1981)  prostate  acetate  (table  utilizing  f o r the synthetic  inhibitory  (table  hepatic  protein.  and the s t e r o i d a l  and  help  protein  (HALC) a n d r o g e n  specificity  concentration  rat?  approximately  capacity  may the  binding  for estradiol  dissociation  physiological  regarding  cyproterone  interestingly,  apparent  techniques  ( t e s t o s t e r o n e and  androgens,  anti-a"ndrogens,  binding  f u r t h e r experiments  androgen  affinity-low  protein  and  course,  question  o f t h e HALC  androgen  to testosterone-dependent  histocytochemical important  location  o f a n HALC  (1983).  Further,  the i t was  not  present  immature  i n t h e immature  male;  though  female  rats  i n the l a t t e r ,  affinity  adult  male  (table  adult  male  but not i n the female, would  and  during  puberty. this  19).  birth  i n the l i v e r  pseudohermaphroditic genetic  neonatal  defects  imprinting  Castration binding (table in  20).  androgen  reported male  This  levels  system  may  a marked  by a s l o w  b y d a y 8-12  This  influence would  secondary  cytosol  t o LH  of  the absence of  by  adequate  receptor  alter  HALC  synthesis. androgen male  d i d not influence  The m a i n t e n a n c e  1973).  ( 8 0 % ) i n DHT  increase  during  suggests that a l t e r a t i o n s  in vivo.  after  before  t o the sham-operated  and S t r o t t  decrease  occur  as  be d u e t o a t e s t o s t e r o n e  1-4  o f an  independent These  binding  workers  in adult  days c a s t r a t i o n ,  and r e s t o r a t i o n t o above  control  post-castration. (10-17  the hepatic  suggest  may  be e x p l a i n e d  as t e s t o s t e r o n e  (Sullivan  Hypophysectomy not  may  androgen  of change such  suggest the  to the lack  compared  characteristics  r a t prostate  followed  male  i n the  of the  r a t , which  leading  lack  receptor  induction  cytosol  had a  to the  protein  reported  d i d not s i g n i f i c a n t l y  secretions  binding  (1983)  o r improper  i n the adult  gonadal  the  male  protein  p h a s e ) , as w e l l  and L e v i n s o n  to the  compared  s e c r e t i o n which  (imprinting  Decker  capacity  The p r e s e n c e o f t h i s  of t e s t o s t e r o n e  protein  either  binding  t h e HALC  lower  involvement  and lower  compared  that  levels  days) HALC  neither  of the adult  androgen  male  protein  t h e serum  rat did  (table 21).  testosterone,  nor the presence of other  pituitary  factors levels  are responsible of this  Sullivan and  adrenalectomy  and  binding  later  increase authors  was  protein,  speculated  maintained  some  male  metabolite,  rats  thereby vitro,  since  HALC  r a t was  and  our experiments  effect  to  Since  o n HALC  hydroxide lower  hours  of this  or flutamide  binding,  competitively testosterone  binding  hydroxide,  compared This  R1881  personnel  the hypothesis  to  i n vivo,  binding  hydroxide  the  be u n l i k e l y .  that  Moreover,  affinities  i n the flutamide-treated  in  last h a d no  flutamide binding,  t r a n s l o c a t i o n , and a r e d u c t i o n  would  in  testosterone  androgen  synthesis,  was  communication),  24 h r s a f t e r o f serum  the  adult  effect  accumulation  of flutamide  inhibited  or  castrated  22).  apparent  the reduction  may  f o r development.  hydroxide  done  as a  tissue concentration  (table  (Buxbaum,  These  receptor  differentiation,  half-life  were  following  secretions.  androgen  controls  t h e serum  the  injection.  receptor  to testosterone-treated  to decreased  5-6  the disappearance  or adrenal  a pre-set  due t o f l u t a m i d e  leading  that  prostatic  to e i t h e r the  of flutamide  decreased  likely  alter  to  response  embryonic  testosterone-treated not  hypophysectomy  unknown s t i m u l i  Administration active  that  the presence  with  during  by  showed  of the androgen  that  protein  determined  similar  suggesting  outflow  cellular  were  who  not a secondary  of  results  d i d not s i g n i f i c a n t l y  in pituitary  constitutive  These  (1973),  reappearance  castration  be  protein.  and S t r o t t  androgen  f o r the maintenance  reduced  castrated  leading i n HALC  binding  male,  and  lack  of  effect  and  i n the hypophysectomized  pituitary-independent  receptor  (STZ) treatment  i n the adult  were  found  male  r a t hepatic  et  using  a l . 1984).  in prostate  diabetic  male  HALC  et a l .  gonadal-  androgen  that  account  protein  restoration  (1980),  who  to untreated  f o r reduced reverse  to differences  sensitivity,  disagree  in  the  receptor.  latter  Insulin  effect  (table  23).  who  showed  a  on t h e  This  isin  partial  levels.  regimen,  However,  i n serum  male  or tissue differences  STZ-treated  with  the i n h i b i t o r y  i n the i n s u l i n  (Rodway  an 86%  decreases  to control  adult  those  controls.  androgen  (1980)  (43% of c o n t r o l )  with  protein  STZ m e d i a t e d  et a l .  effects  levels  reported  binding  i n the STZ-treated  t o Tesone  androgen  Similar  receptor  are consistent  compared  conclusion  23).  HALC  o f STZ a n d m e a s u r i n g  estrogen  androgen  to significantly  contrast  due  rats  reduced  the c a s t r a t i o n studies  testosterone failed  dose  Our r e s u l t s  decrease  workers'  f o r HALC  r a t (table  cytosolic  by Tesone  from  male  t h e same  reported  results  mechanisms  supports  synthesis.  Streptozotocin binding  male  This  may  be  insulin  i n the receptor  synthetic  pathway.  The  role  o f t h e HALC  testosterone-dependent correlating  androgen  drug  t h e enzyme d a t a  binding  data.  protein  correlate well  binding  metabolism with  c a n be d i s c u s s e d  the i n v i t r o  Sex and a g e - d i f f e r e n c e s with  protein in  t h e same  i n HALC  and  by  i n vivo  androgen  differences  i n BPH a n d  TR  activities.  these to  hepatic  (Colby  protein  model.  microsomal  That  the adult  cytochrome forms  metabolizing  male  P-450  BPH  different rats.  from  microsomal  neonatally  activities female  of c o n s t i t u t i v e rat is  P-450's  binding  a  of  o f t h e HALC levels  apparently  (Lu and West  protein  present  testosterone-dependent  the c o n s t i t u t i v e  Presence  testosterone-responsivity determined  the absence  of cytochrome androgen  enzyme  i n the adult  i n the female  i s stimulating  and h e p a t i c  by  Maintenance  i s , t h e HALC  of female  protein  drug  of the response  of t e s t o s t e r o n e  to the m u l t i p l i c i t y  1979).  the lack  1 9 8 0 ) c a n be e x p l a i n e d  in this  hepatic  due  in  microsomal  p h y s i o l o g i c a l doses  rat  of  Furthermore,  o f t h e HALC  levels  androgen  binding  enzyme  i n the adult  ( i e . neonatal  m a l e may  be  imprinting,  Chung e t a l .  1975) . Age-related explained presence  by  androgen  BPH  decrease  i n enzyme  testosterone  our  hypothesis.  enzyme  activity, This  by d e c r e a s e d  leading  present  activities  i n the growing  kinetics.  c a n be e x p l a i n e d  receptor  following  receptor  binding  levels  a n d TR  c a n be  s e c r e t i o n of testosterone  l e d to decreased  testosterone  support  the pubertal  i n t h e HALC  dichotomy  This  i n BPH  of the androgen  Castration change  changes  to decreased  and d e c r e a s e d  activity  replacement,  young  male.  b u t no  apparent  "agonist"  or  occupancy enzyme  i s readily  and t h e  serum  of the  synthesis.  reversible  an e f f e c t  which  tends  to  Flutamide  administration  activities  in  rat,  flutamide  while  doses)  were  action  of  two  found  different at  agonist  androgen  effect  on  leading  decreased  and  synthesis. involve  a  hepatic  ( i i ) by  the  are  likely  microsomal  induction  which  occupancy  of  reduced  the  to  not  adult  treatment,  demonstrated  activities  towards  HALC  androgen  "pituitary  factors"  the  hypophysectomy  al.  1979).  sensitive synthesis.  of  an  hepatic  in  depend  thereby  male,  on  to  the  enzyme both  or  yet  no  TR  observations androgen drug  testosterone  change seem are  was  that  receptor  not in  metabolizing  BPH found  in  other  involved  activity do  or  The  microsomal  hormone  and  enzyme  presence  without  I t would  BPH  underlying  the  hepatic  growth  microsomal  synthesis  mechanisms,  receptor.  with  protein.  hepatic  reducing  protein  metabolizing  solely  levels,  these  of  enzyme  mechanisms  decreased  on  least  occupancy.  androgen  effect  at  translocated  these  male  The  displacement  binding  steroid  as  by  BPH  same  testosterone-dependent  other  and  such  However,  involvement  of  be  female  binding  explained  agonist  receptor  hepatic  hypophysectomized  of  the  binding.  level,  HALC  adult  (at  testosterone-dependent  drug  may  be  receptor  differences in  hydroxide  androgen  can  microsomal  castrated  competitive  reduction  decrease  HALC  amounts  Despite  There  reduce  ( i ) by  or  nucleus,  flutamide  hydroxide  synthesis, to  and  to  ways: the  hepatic  testosterone-treated  flutamide  androgens the  the  reduced  (Kramer  discount  in et the  testosterone enzyme  In BPH  contrast,  activities  reduced  HALC  reduced  serum  HALC  in adult  androgen  androgen  binding  receptor  reduced  BPH  the  effect  Further  understanding metabolism  In may  be  process, complex  may  as by  concentrations,  1982),  an h e p a t i c  synthetic  to gain  a  to  reverse  i t d i d not  o f t h e HALC  protein.  better  o f STZ o n h e p a t i c binding  protein.  cytosolic  androgen  s e e m s t o be o n l y  pathway.  to  t r a n s l o c a t i o n , and  (Warren  androgen  by  leading  activities  the presence  as  decreased  i s known  are required  this  as w e l l  be e x p l a i n e d  i n the testosterone-dependent  however,  microsomal  insulin  a n d t h e HALC  involved  1982),  as w e l l  reduced  of the mechanism  conclusion,  hepatic  Although  influence  experiments  This  levels  protein  occupancy,  o n BPH  (Warren  binding.  synthesis.  significantly  males  testosterone  reduced  STZ  s t r e p t o z o t o c i n reduced  BPH  one s t e p  drug  receptor induction in a  very  174  FUTURE EXPERIMENTS  The f o l l o w i n g e x p e r i m e n t s characterize  the h e p a t i c  and i n v i t r o . strengthen basis  Results  are  androgen  intended to  b i n d i n g p r o t e i n iin v i v o  from these experiments  our c o n c l u s i o n s  as  further  well  as  would  provide a  greater  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 a n d r o g e n  binding  protein.  1.  B a s e d on t h e s a t u r a t i o n  studies,  we have r e p o r t e d  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 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 unchanged compared this  p r o t e i n was  pituitary saturation  to i n t a c t  insensitive  hormones.  other  studies  nM t r i t i a t e d - R 1 8 8 1  varying concentrations  the  protein  is  the hepatic  insensitive  environment,  then i t s  should  incubated with  of  From t h i s  and one  could  t h e HALC p r o t e i n p r e s e n t i n adult  cytosolic  male r a t  liver  HALC a n d r o g e n  to the a l t e r a t i o n of  1  ratio  competitors.  c o u l d be c a l c u l a t e d  s p e c i f i c i t y of  If  cytosol  studies  i n the presence or absence  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 tractions.  and  our  inhibition  of d i f f e r e n t  K i values  that  h o w e v e r , were b a s e d on  and 100 f o l d e x c e s s ; m o l a r  triamcinolone acetonide,  the app.  liver  relatively  and s u g g e s t e d  to strengthen  s u c h as  liver  l o s s of gonadal  These r e s u l t s ,  be done u s i n g a d u l t male r a t  determine  to the  s t u d i e s and i n o r d e r  conclusions,  data,  controls,  that  its  s p e c i f i c i t y would remain  the  cytosol  binding  hormonal relatively  unchanged compared t o that present i n t a c t a d u l t male c o n t r o l s . i n c l u d i n g the determination  i n the sham-operated  Other c h a r a c t e r i z a t i o n s t u d i e s of the sedimentation  c o - e t t i c i e n t s may be u s e f u l i n supporting Eagon et a l . (1980) r e p o r t e d  our c o n c l u s i o n s .  a s h i f t i n the sedimentation  c o - e t t i c i e n t o f the estrogen b i n d i n g  p r o t e i n from 10 S t o 4  S, i n the 30 day c a s t r a t e d a d u l t male compared t o the i n t a c t control.  I f 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 b i n d i n g p r o t e i n are a l t e r e d i n the c a s t r a t e d or hypophysectomized animal models compared to c o n t r o l s , then time course s t u d i e s of these e t t e c t s should  be done.  be u s e f u l i n c o n f i r m i n g  In a d d i t i o n , these techniques would other  conclusions  t h a t ontogeny i s  important t o the presence of the HALC androgen  binding  protein.  2.  Our s t u d i e s i n d i c a t e t h a t the presence of the HALC  androgen b i n d i n g  p r o t e i n i s dependent on the animal's age,  i e . absent or i n low q u a n t i t i e s i n the immature male r a t compared t o the a d u l t male. gonadal f a c t o r s  In order  ( i e . testosterone)  to study the r o l e of  and p i t u i t a r y f a c t o r s on  the r e g u l a t i o n of t h i s age-dependent p r o t e i n , the HALC p r o t e i n c o u l d be measured i n the p r e p u b e r t a l l y c a s t r a t e d or hypophysectomized a d u l t male r a t .  Continued absence or  presence of low l e v e l s of the HALC p r o t e i n i n these animals compared t o sham-operated c o n t r o l s would suggest the importance of hormonal s e c r e t i o n s d u r i n g  maturity  on t h i s  androgen b i n d i n g p r o t e i n . normal a d u l t following  prepubertal castration  the ontogenic  that  The p r e s e n c e  enanthate  reversed  treatment  secretion is  involved  enanthate  mechanism i s  in  treated  castrated  treated  involved  a d u l t male and  ovariectomized  adult  imprinting.  i n sex d i f f e r e n c e s  in  hepatic  (Skett  1979;  i n HALC a n d r o g e n  Colby 1980).  If  b i n d i n g p r o t e i n are  due t o n e o n a t a l  imprinting,  c a s t r a t i o n would lead to the  i n the a d u l t male.  castration hypothesis,  Gustafsson  then  loss of t h i s  protein  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  i f the hypothesis  s h o u l d be r e v e r s e d  measured  sex d i f f e r e n c e s  and  A  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  Furthermore,  is  true,  by t e s t o s t e r o n e  and t h r o u g h o u t m a t u r i t y . the presence  of  then t h i s  treatment  out.  effect  following  To f u r t h e r t e s t  this  t h e HALC p r o t e i n c o u l d be  i n the 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 female  was t r e a t e d  with testosterone  during maturity.  f o l l o w i n g gonadectomy  In a d d i t i o n to the b i n d i n g  these animal models,  i n order to c o r r e l a t e  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 BPH i n d u c t i o n .  and  using  the presence  dependent  which  studies,  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 m e a s u r e d  microsomal  it  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 i n  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  neonatal  to  or hypophysectomy, then  testosterone  absence i n the s i m i l a r l y  similar  are  process.  the t e s t o s t e r o n e its  these e f f e c t s  l e v e l s by t e s t o s t e r o n e  w o u l d be l i k e l y  3.  If  hepatic  of  177  4.  Flutamide treatment  reduced both the presence of  the  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 and t e s t o s t e r o n e dependent  hepatic  the t e s t o s t e r o n e enanthate If  the r e d u c t i o n i n the  m i c r o s o m a l BPH a c t i v i t i e s  treated  levels  of  castrated  then r e v e r s a l increased  of  hepatic  BPH a c t i v i t i e s . increases  the agonist  Increases  should lead  accompanied  hypothesis.  by s t i m u l a t i o n o f without  i n BPH a c t i v i t y  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  Reversal  by a number o f w a y s : the presence of  decreased  to  i n HALC p r o t e i n l e v e l s  i n BPH a c t i v i t y , o r i n c r e a s e s  without increases  and  of the f l u t a m i d e e f f e c t (1)  adult males.  following  the  last  may be  Experiments  s h o u l d be done 24  in tlutamide-treated the e f f e c t  in vitro;  (2)  a d u l t males;  to reverse  the e f f e c t s  BPH l e v e l s  i n the a d u l t male,  periods  insulin  of  treatment.  of  hrs  a  a time  t h e HALC course  treatment  i n a d u l t male r a t s w h i c h  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 . be u s e d  (3)  of d i s c o n t i n u i n g flutamide  HALC p r o t e i n and BPH l e v e l s  on  of  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  study of  tested  flutamide-treated  i n j e c t i o n to avoid carry-over  flutamide or tlutamide metabolites  protein  the  a flutamide dose-response study  t h e HALC p r o t e i n i n  castrated  to  m i c r o s o m a l BPH i n d u c t i o n ,  the tlutamide e f f e c t  HALC p r o t e i n l e v e l s  rat.  t h e HALC p r o t e i n l e a d s  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 e s t o s t e r o n e dependent  a d u l t male  in  on  are  A s i m i l a r approach  may  STZ on HALC p r o t e i n and  using different  doses or  time  5.  P u r i f i c a t i o n studies  binding  protein  different  techniques  precipitation, and  column  purified whole  would  of  have may  sucrose  be  cytosol.  would  t o be used,  density  chromatography.  protein  the hepatic done.  cytosolic For  s u c h as  gradient  this  androgen  purpose,  ammonium  sulphate  ultracentrifugation,  Characterization  studies  enable comparison to those  of  of the  the  SUMMARY  The  present  testosterone  studies  induced  hepatic  metabolism  i s an  conclusion  i s based  microsomal  cytochrome  hydroxylase different altered  support  androgen  (BPH)  on  drug  P-450  modulated  dependent and  whose  steroid  process.  This  hepatic  benzo(a)pyrene  androgen hormonal  p h y s i o l o g i c a l and  that  and  r e l a t i o n s h i p s between  models  following  hypothesis  microsomal  receptor  activity  animal  the  binding status  kinetics,  has  in  been  pharmacological  manipulations.  1.  Testosterone  hepatic  cytosolic  complicated earlier  by  metabolism  studies  indicated  testosterone  binding  protein  ligand  studies  using  metabolism  in vitro.  testosterone  as  a  that  assay  the was  Conclusions  ligand  are  from  therefore  questionable.  2.  Methyltrienolone  measurement  of  the  ( R 1 8 8 1 ) was  hepatic  a  cytosolic  useful  tool  androgen  for  the  binding  protein.  3.  The  androgen in  the  range)  adult  male  binding  HALC  non-specific  liver  components:  subnanomolar or  rat  a  range)-low  androgen  lower  cytosol  binding  contained  specific capacity protein,  affinity-higher  high  affinity  ( i n the and  capacity  two  a  (Kd  fmol/mg relatively  (LAHC)  androgen  binding  4.  component.  T h e HALC  specificity and  to natural  androstenedione),  (including  R1881  specificity hydroxide protein DES,  androgen  binding  protein  as w e l l  to estradiol,  as s y n t h e t i c  cyproterone  5  lower  acetate  and  of flutamide).  significantly  triamcinolone  alpha-DHT,  androgens  b u t had a  (the active metabolite  progesterone,  high  (including testosterone,  and m i b o l e r o n e ) ,  d i d not bind  had a  to other  acetonide,  flutamide This  s t e r o i d s such Cortisol  as  or  flutamide.  5. and  T h e LAHC bound  t o many  progesterone, and  binding  component  different  Cortisol,  androstenedione,  was  steroid  estradiol,  relatively classes  DES,  non-specific  including  testosterone,  but not to flutamide  or  DHT  flutamide  hydroxide.  6.  Addition  cytosol  eliminated  analysis. analysis  7.  of triamcinolone  This  t h e LAHC  procedure  o f t h e HALC  Saturation  androstenedione,  allowed  binding  kinetics  binding  t o t h e same  or a  to the l i v e r  component  f o r easy  binding  using  cyproterone  similar  binding  androgen  studies  acetonide  suggesting similar  during  Scatchard  protein.  testosterone,  acetate  5  alpha-DHT,  and e s t r a d i o l that  high  whole  these  affinity  produced  steroids site.  were  Flutamide  hydroxide,  consistent  8.  saturable  binding  Tritiated-mibolerone  tritiated-Rl881 binding  9.  site  T h e HALC  androgen being  i n the adult  low  quantities  with  10.  Although  remained without  present  female.  rat.  be  yield  plots).  substituted for o f t h e HALC  protein  was  i n the adult  Furthermore,  These  hepatic  male  androgen  s e x - and  male  i t was  hepatic  rat liver detected  and a b s e n t  characteristics  microsomal  t o c a s t r a t i o n , an e f f e c t replacement,  relatively testosterone  testosterone  in  i n the  correlate  microsomal  but  BPH  well  activities  activities  which  was  androgen  following  (10-17  days),  were  r e v e r s i b l e by binding  protein  c a s t r a t i o n with  with  or  or  without  d i d not s i g n i f i c a n t l y  of the adult  binding  BPH  treatment.  administration,  kinetics  androgen  t h e HALC  unchanged  Hypophysectomy  binding  to  rat.  testosterone  HALC  binding  i n t h e immature  female  sensitive  11.  could  sex and age-dependent  the  failed  ( i e . no S c a t c h a r d  f o r the measurement  not  immature  hand,  in vitro.  age-dependent,  in  on t h e o t h e r  protein.  male  r a t hepatic  alter  cytosolic  the  12.  Administration  of flutamide  to the  testosterone-treated  and u n t r e a t e d  reduced  dependent  testosterone  activities. had  no s i g n i f i c a n t  treatment  with  flutamide) enzyme's  13.  Administration  15. of for  to reduce  HALC  HALC  These t h e HALC  androgen  the l a t t e r  dependent  to control  binding  suggest binding  that  hepatic  of  microsomal  BPH  female  however  decrease  of  in this  levels.  flutamide, to the adult  male r a t  binding.  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