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Comparative effects of naturally occurring, synthetic and plant estrogens on uterine metabolism Kitts, David D. 1976

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THE COMPARATIVE EFFECTS OF NATURALLY OCCURRING, SYNTHETIC AND PLANT ESTROGENS ON UTERINE METABOLISM by  B.Sc,  David Dale K i t t s U n i v e r s i t y o f B r i t i s h Columbia,  1974  A THESIS SUBMITTED I N PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in  The F a c u l t y o f G r a d u a t e S t u d i e s (Department o f A n i m a l S c i e n c e )  We a c c e p t t h i s t h e s i s a s c o n f o r m i n g to the r e q u i r e d standard  THE UNIVERSITY OF B R I T I S H COLUMBIA September, 1976 ©  D a v i d D a l e K i t t s , 1976  In  presenting  this  an a d v a n c e d  degree  the  shall  I  Library  f u r t h e r agree  for  scholarly  by h i s of  this  written  thesis at make  It  for  for extensive  p u r p o s e s may be g r a n t e d  the  requirements  B r i t i s h Columbia,  it freely available  that permission  for  fulfilment of  the U n i v e r s i t y of  representatives. thesis  in p a r t i a l  I agree  r e f e r e n c e and copying of  this  f i n a n c i a l gain shall  that  not  copying  or  of  The U n i v e r s i t y o f B r i t i s h  Columbia  2075 W e s b r o o k P l a c e V a n c o u v e r , Canada V6T 1W5  Jefite/nier 2o  .  or  publication  permission.  Department  Date  thesis  be a l l o w e d w i t h o u t my  D a v i d D.  that  study.  by t h e Head o f my D e p a r t m e n t  is understood  for  Kitts  ABSTRACT  Various  estrogens  e s t r a d i o l - 1 7 3 were female on  rats  water  bestrol  to  administered  monitor  (DES)  were  Plant  the  stimulating  water  administration  was  shown t o  of  as  and  and  1.0  jig  and  in  immature  course  inducing  times  as  effects  diethystil-  coumestrol,  tissue  in  pure  potent  in  estradiol-173.  estradiol-173, the  time  estriol  estrogens  genistein  enhance  the  dose  a p p r o x i m a t e l y 10~3  The  by  relative  imbibition  to  i n t r a p e r i t o n e a l l y to  strongest  estrogens,  form were  indicated  equivalent  Estradiol-173,  crystalline  as  the  imbibition.  edema.  trol  biologically  genistein  permeability  d i f f u s i o n of  of  and  uterine  intravenously  coumesvasculature  infused  India  ink. Large immature and  DNA  cycle of  doses  female in  was  the  (5.0 rats  to  12  and  the  I t was  rate  observed  accumulation  hours  of  were  of  administered  synthesis  that  (P ^ 0.025) r e d u c e d  Net 24  estradiol-173  measure  significantly  after  of  uterus.  e s t r a d i o l - 1 7 3.  occur  .ug)  the  after  RNA  of  RNA  uterine  the  and  DNA  into  the  to  cell  administration was  shown  to  respectively. 3  The tissue  incorporation  was  estrogens six  hour  studied  by  of  [5,6-  administering  i n t r a p e r i t o n e a l l y to period  there  H]  was  a  uridine  natural,  immature  decreased  synthetic  female  uterine  uterine  rats. specific  and  plant  After  a  activity  3 of  [5,6-  control in  the  estriol  H]  uridine  animals. specific and  i n estrogen  treated  rats  Estradiol-17 3 produced activity  estradiol-17ot  of  the  hydrolyzed  produced i  the RNA  relatively  when  compared  greatest while smaller  to  reduction DES, reductions.  The  reduction  of  specific  activity  pulsing with estradiol-173 associated  was  s i x hours a f t e r i n  p r o b a b l y due  w i t h changes o c c u r r i n g  in cell  vivo  to a d i l u t i o n e f f e c t permeability.Genistein 3  and  coumestrol a l s o reduced  uridine  the  specific  s i m i l a r to that observed with Extracts  quantitatively  and  vivo p u l s i n g of  30  of a l f a l f a  hay  qualitatively  and  activity  H]  estriol. s o y b e a n m e a l were  for plant  minutes of v a r i o u s  C5,6-  of  estrogens.  e s t r o g e n s was  analyzed Short  in  employed  to  3  d e t e r m i n e t h e u p t a k e o f C5,6- HH u r i d i n e by t h e u t e r i n e t i s s u e . E s t r a d i o l - 1 7 3 , p u r i f i e d g e n i s t e i n , a l f a l f a and s o y b e a n e x t r a c t s 3  were f o u n d t o control  incorporate  groups.  L~5,6- Ej  uridine at greater  rates  than  TABLE OF CONTENTS Page Abstract  1  L i s t o f Tables  v  L i s t of F i g u r e s  V  L i s t of Appendix F i g u r e s  1  vii  Acknowledgements  viii  Introduction  1  L i t e r a t u r e Review  3  1. A n t i e s t r o g e n s 2. E f f e c t of Estrogen  3 on C e l l Metabolism  7  3. Phytoestrogens  . 17  Experimental Procedure  26  Experiment A : The Comparative E f f e c t s o f Estrogens and Phytoestrogens on Water I m b i b i t i o n and Hyperemia of the Rat Uterus  26  Introduction  26  M a t e r i a l s and Methods  28  Results  30  Discussion  31  Conclusions,  33  Experiment B : E f f e c t of Estrogen Uterine Tissue  on Hyperemia of 40  Introduction  40  M a t e r i a l s and Methods  40  Results  41  D i s c u s s i o n and Conclusions  41  Experiment C : E f f e c t s of E s t r a d i o l - 1 7 g on RNA and DNA S y n t h e s i s i n Immature U t e r i n e T i s s u e s . ... ... . . 4 5 Introduction 45 iii  Page M a t e r i a l s and Methods  46  Results  48  Discussion  50  Conclusions  52  E x p e r i m e n t D : E f f e c t s o^ E s t r o g e n s and P h y t o e s t r o g e n s on t h e I n c o r p o r a t i o n o f H U r i d i n e i n t o RNA b y t h e U t e r u s - S i x Hour In. V i v o  Pulsing  58  . . . :  Introduction  58  M a t e r i a l s and Methods  60  Results  62  Discussion  64 66  Conclusions E x p e r i m e n t E : E f f e c t o f E s t r o g e n s and P h y t o e s t r o g e n s o n t h e I n c o r p o r a t i o n o f T r i t i a t e d U r i d i n e I n t o RNA S h o r t In. V i v o P u l s i n g  70  Introduction  70  M a t e r i a l s and Methods  71  Results  74  Discussion  75  Conclusions  76  E x p e r i m e n t F : E a r l y E f f e c t s o f E s t r o g e n s and P l a n t E x t r a c t s on t h e I n c o r p o r a t i o n o f T r i t i a t e d U r i d i n e I n t o RNA b y U t e r i n e T i s s u e 80 Introduction  80  M a t e r i a l s and M e t h o d s  80  Results  82  Discussion  83  Conclusions  84  General Conclusions  89  Bibliography  92  Appendix  .  iv  102  L I S T OF  TABLES  Table 1  Page E f f e c t o f t i m e on w a t e r i m b i b i t i o n by r a t u t e r i n e t i s s u e s f o l l o w i n g a d m i n i s t r a t i o n of estradiol-173 (Expt. A-I) . .  37  E f f e c t o f d o s e on w a t e r i m b i b i t i o n by r a t u t e r u s t i s s u e s f o l l o w i n g a d m i n i s t r a t i o n of e s t r a d i o l - 1 7 3 ( E x p t . A-2)  38  E f f e c t s o f s t e r i o d and p l a n t e s t r o g e n s on w a t e r i m b i b i t i o n by r a t t u e r i n e t i s s u e s i x h o u r s f o l l o w i n g a d m i n i s t r a t i o n ( E x p t . A-3)  39  4  RNA and DNA c o n c e n t r a t i o n s i n h o m o g e n a t e s o f d i f f e r e n t immature r a t u t e r i n e w e i g h t s (Expt.  56  5  E f f e c t o f t i m e o n RNA u t e r i n e t i s s u e (Expt.  6  The i n v i t r o i n c o r p o r a t i o n o f r a d i o a c t i v i t y i n d i f f e r e n t f r a c t i o n s of r a t u t e r i , s i x hours f o l l o w i n g i n v i v o e s t r o g e n a d m i n i s t r a t i o n ( E x p t . D)  2  3  7  8  9  10  and DNA s y n t h e s i s by C ) . . . . .  C)  . .  rat  57  . .  68  RNA c o n t e n t and s p e c i f i c a c t i v i t y o f RNA extracted from r a t u t e r i s i x hours f o l l o w i n g estrogen adminis t r a t i o n ( E x p t . D)  69  Rf v a l u e s o f s t a n d a r d p h y t o e s t r o g e n s and t h o s e o b t a i n e d f r o m p l a n t e x t r a c t s a s m e a s u r e d by T h i n L a y e r C h r o m a t o g r a p h y ( E x p t . E)  79  The i n _ v i t r o i n c o r p o r a t i o n o f r a d i o a c t i v i t y i n d i f f e r e n t f r a c t i o n s o f r a t u t e r i , 30 m i n u t e s f o l l o w i n g i n v i v o e s t r o g e n a d m i n i s t r a t i o n ( E x p t . F) . .  87  I n c o r p o r a t i o n o f [5,6 H] u r i d i n e i n t o t h e h y d r o l y z e d RNA f r a c t i o n o f i m m a t u r e f e m a l e r a t u t e r i 30 m i n u t e s f o l l o w i n g e s t r o g e n a d m i n i s t r a t i o n ....  88  v  L I S T OF  FIGURES  Figure 1  Page Temporal sequence of m e t a b o l i c events i n u t e r i immature or o v a r i e c t o m i z e d r a t s a f t e r i n v i t r o a d m i n i s t r a t i o n of estrogen Structure  3  Wet u t e r i n e w e i g h t s i x h o u r s f o l l o w i n g a d m i n i s t r a t i o n ( E x p t . A-3)  4  E f f e c t o f t i m e on w a t e r i m b i b i t i o n i n r a t u t e r i n e t i s s u e s f o l l o w i n g a d m i n i s t r a t i o n of e s t r a d i o l - 1 7 3 (Expt. A-l) . . . " . .  36  The d i s t r i b u t i o n o f I n d i a i n k i n t h e u t e r i n e v a s c u l a t u r e o f c o n t r o l and e s t r a d i o l - 1 7 3 t r e a t e d r a t s ( E x p t . B) x 100  43  The d i s t r i b u t i o n o f I n d i a i n k i n t h e u t e r i n e v a s c u l a t u r e o f c o u m e s t r o l and g e n i s t e i n t r e a t e d r a t s ( E x p t . B) x 100  44  7  S t a n d a r d c u r v e s f o r RNA  53  8  RNA and DNA c o n c e n t r a t i o n i n h o m o g e n a t e s o f d i f f e r e n t i m m a t u r e r a t u t e r i n e w e i g h t s ( E x p t . C) 54  9  E f f e c t o f t i m e on RNA and DNA s y n t h e s i s i n r a t uterine tissue following estrogen administration ( E x p t . C)  55  Q u a l i t a t i v e examination of phytoestrogen by t h i n l a y e r c h r o m a t o g r a p h y  78  6  10 11  metabolism of estrogenic  Competitive binding  and  DNA  (Expt.  compounds . .  9  2  5  and  of  estrogen  C)  35  content  assay of phytoestrogens  vi  19  ( E x p t . E)  86  LIST 0F APPENDIX FIGURES :  Figure  Page  A  N u c l e i c A c i d E x t r a c t i o n Procedure  103  B  E x t r a c t i o n of Phytoestrogens From P l a n t Materials  104  Quench C o r r e c t i o n Curve f o r T r i t i u m  105  C  vii  AC KNOWLEDGEMENTS The  author wishes t o e x p r e s s h i s g r a t i t u d e t o t h e Department  o f A n i m a l S c i e n c e and t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a Committee f o r f i n a n c i a l  support of t h i s research.  d a t i o n p r o v i d e d by Dean W.D. Science i s g r a t e f u l l y I am  accommo-  the then chairman of Animal  acknowledged.  i n d e b t e d t o Dr. C R .  g e s t i o n s , w h i c h were v i t a l The  Kitts,  The  Research  Krishnamurti for his useful  i n the p r e p a r a t i o n of t h i s  a u t h o r w o u l d a l s o l i k e t o t h a n k Ms.  sug-  thesis.  F r a n c e s Newsome  f o r h e r t e c h n i c a l a s s i s t a n c e and h e r many u s e f u l s u g g e s t i o n s . S p e c i a l t h a n k s a r e e x t e n d e d t o M i s s E. R o s s i t e r f o r h e r a s s i s t a n c e and e n c o u r a g e m e n t d u r i n g t h e c o u r s e o f t h e s t u d y .  viii  1..'  INTRODUCTION  Following the of  operon  the  model  for  s t e r o i d hormone  areas  of  research  biology.  In  the  evidence  has  regulate  growth,  get  apparent  sequence  and  last  an  decades  suggesting  their  no  and  events  animal  RNA  be  of  new  of  species  specific  be  a  release  of  biogenic  of  mRNA w h i c h  proteins.  second  In  function  amines  of  that  the  earliest  associated  with  the  uterine  release.  Increases  membrane w e r e  in  observed  3J5' 15  amount  various  hormones  metabolism.  for  a  genes would  and  to  then  seconds  AMP  i n the  for there  hormones,  Szego  membrane  this most  this,  anabolic  date  trans-  code  to  tar-  activate  allow  known e f f e c t s o f  cyclic  To  The  hormone  addition  cell  in  a v a i l a b l e on  emerged.  most  of  activity  (i.e. histamine).  reported  popular  regulatory  has  certain functionally linked  of  mechanism  most  increasing  literature  would  (1961) the  the  metabolic  e f f e c t s on of  of  that  u n i f y i n g concept  of  to  were  two  Monod  expression,  i n endocrinology  appears  (1967)  gene  and  become one  i n c r e d i b l e amount  however  synthesis  has  Jacob  differentiation  subject,  cription  action  through  i s an  repress  regulating  accumulated  tissues  there  or  p u b l i c a t i o n by  and  Davis  estrogens  and  histamine  uterine  a f t e r exposure  the  to  cell  estradiol-  173. In  addition  genic  activity  plant  materials  and  Kitts,  presence  of  to  has  the been  (Guggolz  1964).  naturally occurring reported et  Bennett  phytoestrogens  aJL. et in  i n many 1961;  al.  estrogens,  forages  Beck,  (1946)  subterranean  and  1964;  first  estroother  Allison  reported  clover  the  (Trifolium  2.  subterraneum).  Since t h a t time p l a n t estrogens have been r e -  cognized  as agents r e s p o n s i b l e f o r the i n f e r t i l i t y  stock.  P l a n t c o n s t i t u e n t s which are regarded as e s t r o g e n i c  and  are r e s p o n s i b l e f o r many reported  animals g r a z i n g e s t r o g e n i c  forages  of grazing  cases o f i n f e r t i l i t y i n  and pasture.legumes are  the i s o f l a v o n e s , g e n i s t e i n , b i o c h a n i n A, d a i d z e i n , equol and formononetin  (Bradbury and White, 1954;  Cheng e t a l . 1954) and coumestrol B i c k o f f e t a l . 1957).  Cheng e t a l . 1953(b);  (Cheng e t al_. 1953 (a);  The e s t r o g e n i c p o t e n t i a l present i n  v a r i o u s p l a n t sources i s g e n e r a l l y much weaker than n a t u r a l l y o c c u r r i n g s t e r o i d o r s y n t h e t i c estrogens.  However, the l a r g e  amount o f phytoestrogens i n g e s t e d d u r i n g g r a z i n g may e l e v a t e the plasma l e v e l s o f estrogen with r e p r o d u c t i v e The  processes.  the time course and dose e f f e c t s o f e s t r a d i o l - 1 7 g  i t s v a r i o u s analogues on u t e r i n e t i s s u e metabolism and  secondly  t o employ an i n v i t r o bioassay  the i n i t i a l metabolic gen,  interfere  o b j e c t i v e s o f t h i s study were t o e s t a b l i s h the base  l i n e s regarding and  t o a degree which would  which would e l u c i d a t e  events o c c u r r i n g i n the c e l l a f t e r e s t r o -  phytoestrogen or p l a n t e x t r a c t a d m i n i s t r a t i o n .  3.  REVIEW OF LITERATURE 1.  Antiestrogens The word " a n t i e s t r o g e n " i s a widely used and g r o s s l y mis-  l e a d i n g term used t o d e s c r i b e compounds which e x i s t n a t u r a l l y (Cook and K i t t s , 1964; thetically  Chow e t a l . 1972) or a r e produced  ( C a l l a n t i n e e t a_l. 1968;  syn-  Davidson e t aJL. 1968;  Humphrey, 1968) and which decrease the response o f female r e p r o d u c t i v e organs t o estrogen comitantly.  administered  or i n j e s t e d con-  E s t r o g e n i c i t y o f a compound i s u s u a l l y assessed  by gross, h i s t o l o g i c and b i o c h e m i c a l c e r v i x and vagina o f o v a r i e c t o m i z e d  changes i n the u t e r u s , animals.  d e f i n i n g the degree o f e s t r o g e n i c i t y a r i s e  Difficulty in as a r e s u l t o f d i f -  f e r e n t t h r e s h o l d s o f the compound r e q u i r e d f o r e l i c i t i n g an e s t r o g e n i c response.  The dose, mode o f a d m i n i s t r a t i o n and  p h y s i o l o g i c a l s t a t u s o f the animal w i l l govern the a b s o r p t i o n and  r e t e n t i o n o f the hormone by a p a r t i c u l a r t a r g e t organ.  These v a r i a b l e s p l a y s i g n i f i c a n t r o l e s i n determining  an e s t r o -  genic response and t h e r e f o r e whether o r not a compound i s a n t i estrogenic . Kato e t a l . (1968) and E i s i n f e l d b i n d i n g o f estrogens  (1970) r e p o r t e d t h a t the  t o r e c e p t o r s i n the hypothalamus and  p i t u i t a r y as w e l l as other t a r g e t t i s s u e s c o n s t i t u t e s an import a n t mechanism r e g u l a t i n g r e p r o d u c t i o n .  The mechanism whereby  a n t i e s t r o g e n s i n h i b i t b i o l o g i c a l responses t o endogenous estrogens  i n v o l v e s the competition  components i n the uterus thalamus  f o r s p e c i f i c macromolecular  (Wyss e t al_. 1968) p i t u i t a r y and hypo-  (Kato and V i l l e e ,  1967);  L e a v i t t e t a l . 1973).  4. Emmens e t a l . (1960) r e p o r t e d t h a t s t e r o i d s and n o n s t e r o i d s a r e highly  a c t i v e when a d m i n i s t e r e d  injected  against estrogens.  antagonists  of estrogens  subcutaneously  Nonsteroids  o r when  locally  w h i c h a r e w e l l known  i n c l u d e DES and m e s o b u t e s t e r o l  a n d r o g e n s and p r o g e s t i n s a r e c o n s i d e r e d  classical  while  steroid  antagonists. The workers Kitts,  mode o f a c t i o n o f a n t i e s t r o g e n s (Emmens e t a l . 196 0;  1975) t o e n t a i l  i s b e l i e v e d by many  K a t o e t a l . 1968;  a successful competition  r e c e p t o r s as w e l l as t o c y t o s o l r e c e p t o r s . initiation  o f some r e l a t i v e l y  which a r e the r e s u l t  Rochefort  (1972) r e p o r t e d competing w i t h organ. the  o c c u r r i n g i n the c e l l  a n d / o r n u c l e a r membranes  and Capony, 1 9 7 2 ) .  that the i n h i b i t i o n  Rochefort  r e s u l t e d from  estradiol-173 f o r binding  site.  noncompetitively  formed o r p r e v e n t i n g  sites  Antiestrogens by c a u s i n g  altering  fying real  effect effect  and Capony  i n the target inhibition of the receptor  a reduction of the product  u p t a k e and r e t e n t i o n o f t h e hormone. steps  subsequent  h o r m o n a l s t i m u l u s may a l s o be r e s p o n s i b l e i n  t h e p h y s i o l o g i c a l a c t i o n o f an e s t r o g e n  Capony, 1 9 7 2 ) .  (Emmens  antiestrogens  may a l s o i n h i b i t  M o d i f i c a t i o n by a n t i e s t r o g e n s o f t h e m e t a b o l i c to the i n i t i a l  the nuclear  They p r e v e n t t h e  T h i s w o u l d be a n example o f c o m p e t i t i v e  receptor  site  e a r l y events  with  of modifications i n the transport of  e s t r a d i o l - 1 7 3 through c e l l u l a r e t a l . 1960;  Newsome and  In t h i s  case  ( R o c h e f o r t and  a n t i e s t r o g e n s w o u l d have a m o d i -  on t h e s e c o n d a r y r e s p o n s e o f t h e e s t r o g e n , on t h e p r i m a r y  b u t no  a c t i o n o f t h e hormone.  These a n t i e s t r o g e n s which reduce t h e c o n c e n t r a t i o n o f estrogen  a t the s i t e  o f t h e t a r g e t t i s s u e s may do so by  forming  5. a durable  complex of low b i o l o g i c a l a f f i n i t y w i t h  binding s i t e s  ( C a l l a n t i n e e t a l . 196 8).  estrogenic  A l t e r n a t i v e l y they  form a s h o r t - l i v e d complex w i t h the b i n d i n g s i t e s which are t r a n s i e n t to e x e r t any b i o l o g i c a l e f f e c t such as growth lation  (Rochefort and Capony, 1972).  Nevertheless,  l a b i l i t y of r e c e p t o r s i t e s f o r c i r c u l a t i n g estrogens Antiestrogens  i n h i b i t the b i n d i n g of estrogens  r e c e p t o r s i t e s i n v a r y i n g degrees major f a c t o r s governing estrogens  (Lee, 1974).  One  can too  stimu-  the a v a i i s reduced. to t h e i r of  the  the b i n d i n g a f f i n i t y of v a r i o u s  anti-  i s the s t r u c t u r e of the i n d i v i d u a l compounds.  Huggins 3  and Jenson  (1955) showed t h a t maximum c o m p e t i t i o n w i t h  estradiol-17g  H  f o r r e c e p t o r s i t e s i n the u t e r i n e c y t o s o l depends  s t r o n g l y on the presence of p h e n o l i c h y d r o x y l groups l o c a t e d on the 3' p o s i t i o n . and C-17  They r e p o r t e d t h a t s u b s t i t u e n t s a t the  p o s i t i o n i n the D-ring may  v a r i a t i o n from the 17-hydrcxyl reduced a f f i n i t y . of phytoestrogen  and  group of e s t r a d i o l r e s u l t e d i n  Shutt and Cox a c t i o n and  also a f f e c t binding,  C-16  (1972) s t u d i e d the mechanism  r e p o r t e d t h a t w i t h i n the i s o f l a v o n e  s e r i e s , the presence of a p h e n o l i c h y d r o x y l group i n both r i n g s A and B was  a s s o c i a t e d with the h i g h e s t r e l a t i v e  affinity.  The  binding  presence of an a d d i t i o n a l 5' h y d r o x y l group i n  genistein increased i t s a f f i n i t y f o r binding. to b i o c h a n i n A there was I t appears from these  a sharp decrease i n b i n d i n g  r e p o r t s t h a t to be capable  w i t h endogenous estrogens  Upon  methylation affinity.  o f competing  f o r s i m i l a r p r o t e i n subunits an  anti-  estrogen must be s t r u c t u r a l l y s i m i l a r to the n a t u r a l l y o c c u r r i n g estrogens. The degree of hydrophobic bonding i s important  i n regard  6. to the b i n d i n g a f f i n i t i e s of v a r i o u s bestrol  (DMS)  counterparts  and DES  meso-butestrol and  differ  binding  The  i n t h e p l a s m a may  affinities.  Dimethylstil-  f r o m t h e i r more p o t e n t  m e s o - h e x o e s t r o l o n l y by  hydrophobic hydrocarbon chain. of a n t i e s t r o g e n s  estrogens.  the  s i z e of  their  dose o r c i r c u l a t o r y  level  a l s o h a v e an e f f e c t on  C a l l a n t i n e cat a l _ . (1968) r e p o r t e d  maximum r e s p o n s e o f a hormone i s c h a r a c t e r i z e d by receptor  sites.  The  The  of estrogen  saturated  i s a necessary step  p r o t e i n complex.  The  Interference with various  estrogens,  has  f a i l u r e due  been documented and  e t a l _ . 1957;  Braden, 1974).  reported  t h a t c l o m i p h e n e , an a n t i e s t r o g e n p o s s e s s i n g  function  D a v i d s o n e t a l . (1968) r e p o r t e d  s e c r e t i o n o f l u t e n i z i n g hormone.  T h i s was  of estrogen  as w e l l as p r o g e s t e r o n e .  (1958) d e m o n s t r a t e d t h a t c l o m i p h e n e was v e n t e d i m p l a n t a t i o n by by d a m a g i n g t h e  i t s e f f e c t on  zygote at biochemical  t h e y p r e v e n t e d i m p l a n t a t i o n by  the an  optimum Segal  b l a s t o t o x i c and  et a l . pre-  t h e endometrium as w e l l levels.  (1965) s u g g e s t e d t h a t most a n t i e s t r o g e n s but  and  that  f o u n d t o have  s u c c e s s f u l i m p l a n t a t i o n w h i c h r e q u i r e s an  concentration  and  anti-  d a i l y d o s e s o f c l o m i p h e n e i n h i b i t e d e s t r a d i o l - 1 7 g and  e f f e c t on  anti-  K a t o e t a l . (1968)  c h a r a c t e r i s t i c s suppressed the p i t u i t a r y  gonadotrophin release.  associated  Leavitt  1965;  an  saturation.  t o t h e e f f e c t o f numerous  Wright,  fertility  Cox  p h y s i o l o g i c a l events,  (Engle  for  presence of  a n t i e s t r o g e n would r e s u l t i n the r e d u c t i o n of estrogen  with reproductive  a  steroid-protein interaction in  t h e c y t o p l a s m i s a random p r o c e s s and formation  that  degree of response i s thus p r o p o r t i o n a l  t o t h e amount i n j e c t e d .  the  the  as  Prasad et a l .  were not  zygotoxic,  t h e i r e f f e c t s on  the  maternal  7. environment. C a r t e r e t a i . (1955) w o r k i n g w i t h p h y t o e s t r o g e n s demons t r a t e d t h a t g e n i s t e i n when f e d t o m i c e a t 0.02%  of d i e t induced  the premature  o p e n i n g o f t h e c e r v i x and r e s u l t e d i n f e w e r  being  S i m i l a r studies regarding maternal  born.  a l t e r a t i o n s , due and Humphrey deciduomata  to antiestrogens  (1968).  were r e p o r t e d  Implantation  environment by F i n n  mice are s t r o n g l y depend-  e n t on o p t i m u m e s t r o g e n and p r o g e s t e r o n e l e v e l s .  than 2.  Finn  d e c i d u a l i z a t i o n i s e v e n more d o s e  (1966)  dependent  implantation. E f f e c t o f E s t r o g e n on C e l l Estrogenic  Metabolism  hormones e x e r t an i n f l u e n c e on t h e m e t a b o l i s m  o f many t a r g e t o r g a n s by b i n d i n g e f f e c t i v e l y t o components.  intracellular  There e x i s t s a s t r o n g , r e v e r s i b l e a s s o c i a t i o n  b e t w e e n t h e c i r c u l a t o r y hormone and i t s r e c e p t o r G o r s k i , 1966;  J e n s o n e t a l . 1969;  O ' M a l l e y and S c h r a d i e r , step  (1966)  and t h e i n d u c t i o n o f  i n i n t a c t or ovariectomized  demonstrated t h a t  litters  1976).  site  (Toft  Means and O ' M a l l e y ,  and  1972;  This a s s o c i a t i o n i s the primary  i n u t e r o t r o p h i c p r o c e s s e s and i s g o v e r n e d by two  main  criteria: 1.  The  u p t a k e o f t h e hormone i s n o t s a t u r a t e d  i n d e p e n d e n t o f any h y p e r p h y s i o l o g i c a l 2.  The  and i s  level.  r e t e n t i o n o f t h e hormone i s s a t u r a t e d and i s  dependent  on t h e d o s e w h e t h e r  or not i t exceeds  the p h y s i o l o g i c a l l e v e l . Hamilton  (1963) d e m o n s t r a t e d t h a t t h e r a t  p o s s e s s e s the c a p a c i t y under e s t r o g e n i c  stimulus  uterus to  synthesize  8. RNA  i n v i v o and  viously  i n v i t r o by m e c h a n i s m s s i m i l a r t o t h o s e  reported  i n the  events f o l l o w i n g the initiated level  at the  Two  a d m i n i s t r a t i o n of  cellular  resulting  p r o t e i n and  rat liver nuclei.  i n the  l e v e l and  and  progresses to the the  cellular  Gorski  t o e x p l a i n t h e mode o f a c t i o n o f e s t r o g e n s .  binding  and  cellular  The  i n the  "Sustained  n u c l e u s and  same e f f e c t on  uterus.  on  i n contrast  e s t r a d i o l - 1 7 3.  o f an  the  the  continued  estrogen  of estrogenic The binding  and  of  Gorski  and  e a r l y events.  hours e s t r i o l 300%  increase  had  the  sustained  l i t t l e or  over the  output of  e s s e n t i a l f o r the  full  some  rat  no  c o n t r o l caused presence  critical  physiological effects  r e s p o n s e o f a hormone i s i t s n o n c o v a l e n t  hormone t o t h e  i n the  estriol  hours) of the  to a s e r i e s of c a r r i e r p r o t e i n s responsible  which are  eukaryotic  important  i n the  genome o f  t r a n s p o r t of the  for trans-  i t s respective target  organism are  two  t r a n s p o r t and  Serum p r o t e i n a l b u m i n i s t h e the  (2-4  the  hormones.  initial  p o r t i n g the Present  to the  later  presence  These w o r k e r s s u g g e s t e d t h a t the  f a c t o r s seems t o be  in  (cytosol  Output Theory" d e s c r i b e s  e a r l y responses  H o w e v e r , a t 18-24  effect,  (1974)  "Domino  (1974) d e m o n s t r a t e d t h a t b o t h e s t r a d i o l - 1 7 3 and  have the  by  Raker  induced protein-RNA sythesis) which set o f f  l a t e r e v e n t s w h i c h a r e d e p e n d e n t on  Raker  The  RNA,  (Fig. 1).  and  the e a r l y responses of estrogen  events.  estrogen  of  genome  synthesis of  division  t h e o r i e s h a v e b e e n p r o p o s e d by  Theory" d e s c r i b e s  sequence  estradiol-17g is  s t i m u l a t i o n of  e v e n t u a l l y DNA  The  pre-  types of  organ.  proteins  b i n d i n g o f e s t r a d i o l - 1 7 3.  principal binding protein  s t e r o i d to i t s t a r g e t organ.  involved The  UTERINE CELL  METABOLISM  36 SEC.  72 SEC.  F_ig.  1.  360 1 SEC. HOUR  5 HOURS  10-20 HOURS  Temporal sequence o f m e t a b o l i c events i n U t e r i o f immature o r o v a r i e c t o m i z e d r a t s a f t e r i n vivo administration o f estrogen  30 HOURS  10. circulating  steroids are also present  glycoprotein with This  the carbohydrate  association with  conjugated  before  1973).  The s e c o n d  glucuronic acid  i t can enter binding  of the steroid  Talwar  moiety b e i n g  the c e l l  albumin  and i t s s p e c i f i c  hypothesized Gorski,  steroids penetrate  t o be a f a c i l i t a t e d 1975).  After entry  adsorbed  by s p e c i f i c  receptor  protein binds  greater  (Chamness,  carrier  up-  1972). linkage  receptor  i n the  t h a n t h e serum  action with  sedimentation  profiles  reported  the steroid  extracellular  receptor  -steroid  Toft  mech-  i s made, t h e hormone i s high  affinities.  a n d DES w i t h  , progesterone  affinity and  a term used t o des-  i n t h e t i s s u e and whose  and G o r s k i  explicit  gradients,  spontaneously  (200,000  salt  response,  (1966) u s i n g  and sucrose  p r o t e i n t o f o r m a 8S  inter-  w e i g h t o f 100,000  hormone a s s o c i a t e s  Higher  The  higher  i n a hormone i n d u c e d  of low s a l t  complex.  The  (Katzenellenbogen  a minimum m o l e c u l a r  1974).  sites  membrane i s  diffusion  protein present  a macromolecule w i t h  that  the c e l l  1969).The r e c e p t o r ,  results  receptor  t h e hormone.  than estradiol-17a  the steroid  and Raker,  onto  estradiol-17B  (Terenius,  a particular  possess  proteins possessing  specifity  testosterone  the  and DeSombre,  i n i t s specificity  o f r e c o g n i z i n g and b i n d i n g  anism by which  (Gorski  i s  binding.  capable  is  acid.  protein involves the selective  Target tissues of estradiol-17g  cribe  (Jenson  e t a l . (1968) c h a r a c t e r i z e d t h e n o n c o v a l e n t  t a r g e t o r g a n t o be g r e a t e r  and  glucuronic  i s r e f e r r e d t o as t h e  by t h e t a r g e t o r g a n  between t h e s t e r o i d  and  involving a  form o r i n a c t i v e f o r m o f t h e hormone and c l e a v a g e  required  take  i n a complex  with  daltons)  concentrations  and t h e  use  of  They the  urea  resulted in various  suggested native  also  that  estrogen  reported  receptor nucleus  was  that  Jenson  low  temperatures  various  (1969) under  an  al.  these  (1971),  Solof  affinity  f o r the  a  extraction  living  5S  nuclear  and  unlike  and  affinity  volving nucleus,  a  (1971) cells  the  i n the  dilute and  protein the  presence  tritiated the  of  and  a l .  Jenson  receptor  8S  are  DeSombre bound  i n the  receptor  steroid.  acid  receptor  to  complex  the  the  possessing  protein  after Williams  nucleus.  i t differs et  al.  protein with They  respective  form.  portion of  site,  placed  (1973) d i s c l o s e d  to  Anderson  nuclear  i s bound  5S  to  chloride solution.  and  a  showed  nuclear  such  et a l .  complex,  i t in this steroid  high  protein  degrade  the  to  1971).  potassium  estradiol-173.  estrogen  the  et  to  inhibitors  to  sites  a  enter  enzymes,  sediments  i s found  the  (1972)  steroid sensitive  specific (King  with,  nucleus,  cytoplasmic  from  a l .  will  Jenson  binding  and  reagents  et  the  i s very  d i s s o c i a t e and  nucleus  the  cytoplasmic  proteolytic  1972)  interfered  change which with  Gorski  firmed  I f not  present  that  estradiol-173  the  conditions will  subunits.  t r a n s f e r r e d to  confirmed  a l . 1971),  (Chamness,  established that  undergoes  in  et  (1974)  estradiol-173  transportation of  sulphydryl blocking  et  Once  and  (Puca  Means  was  synthesis.  (1973)  f o r the  and  cytoplasmic  was  species.  (45,000 d a l t o n s )  p r o t e i n - s t e r o i d complex  concentrations  Raynaud  4S  The  and  RNA  4S  O'Malley  classical  specific  necessary  nucleus.  as  8S  DeSombre  the  salt  the  sedimentation  subunit,  unit.  i t stimulated  and  was  smaller  binding  estrogen  where  binding  a  slower  The  the in  (1972)  chromatin i t s  within  receptor  size  have  experiments that  that  con-  inthe  protein  which  i s smaller  steroid  than  The  the  moiety  both  receptors.  i s an  the  termed  ability  of  formation  et  nuclei  specificity.  position The  in  the  hypothesis  estrogen the  the  concentration of  plasma  a  property  of  as  binding  well sites  as  i t i s the  required  proposes  sulfhydryl  to  a  trans-  receptor  chromatin  t h a t the  cell  the  would on and  the  is  blocking  receptor  is  structure of  determined  the  by  the  genome. receptor  pro-  circulating  estrogen  level  A f t e r ovariectomy  receptor  i n the  be  and  an  nuclear  total  ovariectomy  the  i s decreased.  that a reduction  after  This  estradiol-173 receptor  acceptor  nuclear  a decrease  receptor  explain for i t s additive  a l . 1972).  the  occurred  recep-  a nuclear to  receptors  cytoplasmic  that  t h a t the  function of  (Solof et  to  cytoplasmic  a _ l . (1971) d e m o n s t r a t e d  sites  the  sensitive  responsive  locus of  a p p e a r s t o be  acceptor et  of  of  smaller  protein  chromatin.  d e p e n d e n t and  This  an  nuclear  the  receptor  pro-  disappears  a  attach onto  i s complimentary to the  of  The  with  cytoplasmic  a h i s t o n e , which would  acceptor  nuclei  the  They a l s o suggested  possibly  effect  i n the  The  association with  s t e r o i d - p r o t e i n complex  precursors  from cytoplasmic  temperature  immun-  binding properties in  to transform  then  the  have v a r i o u s  f o l l o w i n g the  affinity. are  the  ajl. (1971) h a v e p r o p o s e d  i t can  reagents.  receptors  part of  reappears  greater  the  form where  teins  essential  "neo-receptors" King  for  p h y s i c a l p r o p e r t i e s i n common.  p r o t e i n , the  but  tors.  also  and  in i t s affinity  receptor protein.  cytoplasmic  c y t o s o l and  specificity  greater  After a period  cytoplasmic  from  and  chemical  tein  and  cytoplasmic  nuclear  ological,  the  in size  i n 4S  amount o f or  King  receptor:: cytoplasmic  hypophysectomy  was  performed.  T h i s w o u l d i n d i c a t e t h a t t h e number o f c y t o p l a s m i c  r e c e p t o r s a n d e s p e c i a l l y t h e 4S o v a r i a n and p i t u i t a r y Ten  component i s c o n t r o l l e d b y  hormones.  minutes a f t e r the a d m i n i s t r a t i o n o f an e s t r o g e n i c  pound a p a r t i c u l a r RNA i s s y n t h e s i z e d induced  protein  by N o t i d e s  (IP)  estrogens  ( G o r s k i e t aJL. 1 9 7 5 ) .  and G o r s k i  Katzenellenbogen  (1966),  and G o r s k i  made  (1970) a n d  (1972) h a v e l e d t o t h e c o n c e p t  that  are responsible for theinduction of thesynthesis of  a f t e r 4 0-60  i s very  This protein f r a c t i o n  minutes i s e s s e n t i a l f o r t h e increase i n  a c t i v i t i e s o f thetissue.  detectable metabolic  P u r i f i c a t i o n o f I P , h a s shown t h a t  s i m i l a r i n c h a r a c t e r i s t i c s t o ovalbumin w i t h a b i o -  logical half l i f e t h a t the  estrogen  Observations  Barnea and G o r s k i  a particular uterine protein.  it  for a specific  com-  o f nine hours.  Ruh e t a _ l . (1973) d e m o n s t r a t e d  s y n t h e s i s o f I P c a n be r e g u l a t e d o n l y b y t h o s e  which possess e s t r o g e n i c p r o p e r t i e s and which w i l l uterine estrogen  receptor proteins.  bind t o the  Among s e v e r a l  estrogenic  compounds t e s t e d e s t r a d i o l - 1 7 3, DES, e s t r i o l a n d e s t r o n e e f f e c t i v e i n IP synthesis i nthat order.  compounds  Progesterone  were and  t e s t o s t e r o n e h a d no e f f e c t . Recently  G o r s k i e t a l . (1975) r e p o r t e d t h a t t h e  o f 40-60 m i n u t e s was t h e t i m e n e c e s s a r y and  moved f r o m t h e n u c l e u s  f o r mRNA t o be  t o the cytoplasm,  t o induce  s y n t h e s i s o f RNA a s a p r i m a r y  a c t i o n o f estrogens  Estrogen i s  t h e s y n t h e s i s o f mRNA w h i c h i n  t u r n i s e s s e n t i a l f o r s y n t h e s i s o f an induced Though t h e  processed  where i t a s s o c i a t e s  w i t h the polysomes, t h e s i t e o f p r o t e i n s y n t h e s i s . therefore considered  lag period  protein. event i ne a r l y  has been e s t a b l i s h e d , t h e mechanism by w h i c h  14.  estrogen It  influences  i s not  effect  on  1967),  RNA  transport  the  the  template  chromatin  polymerase of  RNA  .1968),  RNA  polymerase  the  nucleoplasm  or  autography by  dependent in  the  a  hours  all  types  of  RNA  the  capacity addition  to  Several hours  after  (Pepe  and  first  173  on  RNA  influencing  the  events  a  the  Yochim,  1971)  Gorski,  1963)  estrogen  workers  to  synthesis  uterine  and  cytosol  rate  Mg  of and  a  the  Mn was  synthesis  (1964)  uterine ability  to  ribosomes.  shown t o  are  among  the  demonstrate  an  in  uterine  isolated  in vitro  estradiol-17 3  phospholipid  changes  et_ a l . effect  nuclei. and  1-2  polymerase  initial  Raynaud  proteins  increase  Glucose, RNA  is  regulate  of  of  of  ribosomes  synthesis  activity  success-  demonstrated  synthesize  the  observed  administration  to  been  + +  synthesis  ribosomes  have  poly-  The  accelerated  the  and  radio-  1967).  Kenny  of  of  and  protein  et  dependent  dependent  estrogen  have  injection.  with  use  the  nucleolus  nucleoplasm  the  activity  functional  the  DNA  the  nucleolus,  and  also  from  Hamilton  Greenman the  of  following  estrogens  the  (Hamilton  By  in  and  enzymes  uterus. lowers  cytoplasm factors.  an  Warren,  the  estradiol-17 3 administration.  single  the  (Maul  increased  metabolic  (Noteboom and a  these  that of  hours  rat  that  ovariectomy suggested  4.0  and  from  1967),  stimulation  immediately  i n the  and  calf  of  evidence  occurring  after  by  results  (Barker  isolated  i n the  question.  Hamilton,  cells.  occurring  polymerase  to  to  and  a l l these  been  eukaryotic  sequential  stimulation  in  of  have  subject  stimulation  (Maul  of  been  activity  nucleus  combination  the 2.0  the  enzymes  ive  that  from  has  hormonal  activity  ovariectomized  provide  synthesis  c l e a r whether  al.  merase  RNA  adding  I noticed  (1971) of By  were  estradiolincubating  isolated  nuclei  they  demonstrate specific RNA  assayed a  f o r RNA  successful  interaction  receptor site.  polymerase  polymerase  An  activity  was  template  of  specific. from  the In  noted  uterine The  template  same a n i m a l  was  specific.  enhanced  as  f o r DNA  template  extent that  than  as  2 minutes  occurring  after  ment H a m i l t o n RNA  was  hour  of  RNA  although  achieved  until  as  h i g h as  was  8-12  hours  that  that  levels  do  not  hours  series  of  events  of  newly  This  until  tissue  6 and  12  there exists  s y n t h e s i z e d RNA  from  the  RNA  and  protein  in vivo  rate  of  with  the  between  nucleus  to  24  nuclear  control.  hours  The  RNA  transfer the  one  not  reports and  protein These  of  a  part  cytoplasm.  increased accumulation 2 and  treat-  elevated after  total  a  as binding  estrogen  respectively.  that  serve  greater  c o n t e n t was  the  to  (1967) o b s e r v e d  after  suggest  i s i n accordance  a  ( M e u l l e r e t _ a l . 1958 ).Other  e t a l . 1969(b) i n d i c a t e rise  to  also  hours,  w i t h maximal  the  RNA  was  chromatin  500-600% o f  (Billings  tissue  chromatin  f o r 12  chromatin  significantly  increase in uterine  is  effect  Hamilton  Twenty minutes  polymerase  an  the  i t s administration  was  increase in  lung  polymerase  to uterine  Warren  estrogen.  uterine  and  a l . (1968) n o t i c e d  synthesis i n vivo  activity  RNA  Teng  bound  of  and i t s  of  i n an  and  specific,  ability  dependent  8 hours.  et  liver  to  dependent and  injection  incubation in vitro  the  after  of  able  steroid  s y n t h e s i s which  e l e v a t e d by  tissue  estradiol-173.  estradiol-173  early  not  After  were  Barker  resulted  f o r RNA  capacity  to being  the  well.  intravenous  chromatin  estradiol-173 a  as  to ovariectomized rats  addition  steroid  between  and  i n c r e a s e i n n u c l e a r DNA  (1967) r e p o r t e d t h a t a s i n g l e estradiol-173  activity  without  of any  whole  detectable  increase  i n t h e RNA/DNA a n d p r o t e i n / D N A r a t i o s o f t h e  nuclei. On t h e b a s i s o f h i s t o l o g i c a l e v i d e n c e , M e u l l e r demonstrated t h a t increased  nucleolar  e t a _ l . (1958)  size, greater  abundancy o f  endoplasmic r e t i c u l u m and i n c r e a s e d  basophilic character  uterine c e l l  24 h o u r s a f t e r e s t r o g e n  c y t o p l a s m were n o t i c e d  a d m i n i s t r a t i o n and t h e s e were c o r r e l a t e d w i t h duction  a n d a c c u m u l a t i o n o f RNA a n d e n h a n c e d p r o t e i n  Hamilton twice  increased  (1963) r e p o r t e d  that the uterus  of the  pro-  synthesis.  i n a normal r a t contains  t h e amount o f RNA a n d s i x t i m e s t h e q u a n t i t y o f p r o t e i n  than i n the ovariectomized during  estrus  than during  contained  rat.  The u t e r u s  obtained  from a r a t  0.3 t o 6 t i m e s t h e amount o f p r o t e i n  diestrus.  I n a d d i t i o n t o t h e e f f e c t on RNA a n d p r o t e i n  synthesis,  e s t r o g e n s h a v e a l s o b e e n shown t o s t i m u l a t e many enzymes i n t h e uterus the  (Pepe a n d Y o c h i m , 1 9 7 1 ;  Hall,  1973).  The i n c r e a s e i n  a c t i v i t y o f many enzymes i s r e f l e c t e d i n h i g h  o f 14 C a c e t a t e  a n d 14 C g l u c o s e i n t o l i p i d s  3 3 1963(a) , H u r i d i n e a n d H - c y t i d i n e  incorporation  (Noteboom a n d G o r s k i ,  i n t o RNA  ( M i l l e r a n d Emmens,  14 1967;  B i l l i n g e t a l _ . 1969 ( a ) ) a n d  ( M e u l l e r e t a_l. 1958). and  Increases  C-glycine  into protein  i n phospholipid,  glycogen  l a c t a t e m e t a b o l i s m i n t h e r a t endometrium have a l s o been  attributed to estradiol-173 Estradiol-173  (Walaas e t a l . 1 9 5 2 ) .  and i t s analogues a r e a l s o a s s o c i a t e d  c h a n g e s i n t h e u t e r i n e membrane a n d h i s t a m i n e L i e b e r m a n e t a l . (1963) r e p o r t e d  increased  with  release.  l e v e l s o f 3',5'-  c y c l i c AMP i n t h e u t e r i n e membrane 15 s e c o n d s a f t e r t h e a d m i n i s tration of estradiol-173.  Spaziani  et a l .  (1958)demonstrated  t h a t t h e r e l e a s e o f h i s t a m i n e f r o m t h e u t e r u s was r e s p o n s i b l e for  i m b i b i t i o n o f water which  estradiol-17$  on t h e c e l l .  d e t e c t a b l e 1-2 h o u r s uptake  i s one o f t h e e a r l y e f f e c t s o f Hyperemia o f u t e r i n e t i s s u e s i s  a f t e r e s t r o g e n a d m i n i s t r a t i o n and f l u i d  i n t o t h e uterus reaches  (Astwood,. .1938).  a maximum a f t e r  Increases i n d r y weight  a f t e r estrogen a d m i n i s t r a t i o n a r e accounted p r o t e i n content of the uterus f i r s t hours  (Billing  e t a l . 1969(b)).  6 hours  of uterine tissues f o r by i n c r e a s e i n  s e e n a t a p p r o x i m a t e l y 18  I n c r e a s e s i n t h e r a t e o f DNA  (Kaye e t a l . 1972) a n d h i s t o n e s y n t h e s i s ( W i l l i a m s a n d G o r s k i , 1971;  A n d e r s o n e t a l . 1972) b e g i n a t a b o u t 18 h o u r s  estrogen treatment. which et 3.  begins  a l . 1972;  T h i s i s f o l l o w e d by c e l l u l a r  i n the uterus a t approximately  division  24 h o u r s  (Kaye  L e e , 1974).  Phytoestrogens The  e f f e c t s of phytoestrogens  on l i v e s t o c k r e p r o d u c t i o n  have b e e n r e c o g n i z e d f o r a l o n g t i m e et  after  a l . 1964;  ( B a r t l e t t e t a l . 1948; O c h i  Cayen e t a l . 1965; Cox and Braden, 1974).  The  p o t e n t i a l e c o n o m i c l o s s may be a s c r i b e d t o t h e i r a n t a g o n i s m t h e b i o c h e m i c a l and p h y s i o l o g i c a l r e s p o n s e s a d d i t i o n t o e s t r o g e n i c substances  t o estrogen.  In  o t h e r compounds p r e s e n t i n  c e r t a i n s p e c i e s o f p l a n t s have a l s o been found t o a f f e c t ductive processes.  to  repro-  F o r e x a m p l e , compounds e x t r a c t e d f r o m t h e  ponderosa pine needles  ( P i n u s p o n d e r o s a ) a r e b e l i e v e d t o be  r e s p o n s i b l e f o r weak c a l v e s a t b i r t h a s w e l l a s a b o r t i o n i n grazing animals (Cook a n d K i t t s ,  i n the western  p a r t o f U.S.A. a n d C a n a d a  1964; Chow e t a l . 1 9 7 2 ; S t e v e n s o n  e t a l . 1972).  18. The  various f a c t o r s i n f l u e n c i n g t h e presence o f phyto-  estrogens  and t h e i r e f f e c t s on animal  s t u d i e d b y many w o r k e r s .  The f i r s t  reproduction  report of estrogenic  Fellner\  who r e p o r t e d  estrogenic characteristics  e x t r a c t s o f o v a r i e s o f thewater rose sequently  many e s t r o g e n  coumestrol  and o t h e r coumestans  i s o f l a v o n e s , formononetin,  have been  activity  (benzofuranocoumarins) and t h e  equol.  p r e s e n c e o f e s t r o g e n i c compounds i n f o r a g e s was d i s -  i n subterranean  clover  B r i t i s h and American p a s t u r e  subterranean  (1951)  isoflavone, biochanin A (5,7,  from soybean o i l meal  reported  together  n e t i n i nboth subterranean e q u o l , was f i r s t  (Cheng e t a l . 1 9 5 3 ( a ) ) .  iso-  dihydroxy  Pope  i n soybean  w i t h b i o c h a n i n A and formono-  and r e d c l o v e r s .  The i s o f l a v o n e ,  e x t r a c t e d and i s o l a t e d by M a r r i a n  from a p h e n o l i c  1962).  G e n i s t e i n has a l s o been  t h a t g e n i s t e i n was n o t o n l y p r e s e n t  meal b u t a l s o o c c u r r e d  (•*" c i t e d  Wong a n d F l u x ,  i s o l a t e d t h e i s o f l a v o n e g e n i s t e i n from  H-methoxy-isoflavone) from r e d c l o v e r .  (1932)  from e x t r a c t s o f  c l o v e r a n d a l f a l f a , w h i l e Pope e t a l . (1953)  l a t e d another estrogenic  Later  plants, i nparticular red clover  ( T r i f o l i u m p r a t e n s e t ) , (Pope e t a]L. 1 9 5 3 ; Bradbury and White  on t h e e s t r o g e n i c  ( T r i f o l i u m subterraneum).  many e s t r o g e n i c compounds h a v e b e e n i d e n t i f i e d  isolated  identified  These i n c l u d e t h e s t e r o l s ,  b y B e n n e t t e t a l . (194 6 ) , who r e p o r t e d  covered  Sub-  b i o c h a n i n A, d a i d z e i n , g e n i s t e i n a n d  t h e most r e c e n t l y i s o l a t e d The  from t h e  and w i l l o w c a t k i n s .  analogues i n forages  r e l a t e d t o reproductive problems.  (1954)  sub-  i n p l a n t s was made i n 1926 b y D o h r n e t a l . Loewe e t a l . a n d  stances  and  have been  fraction of ether-soluble.  f r o m B r a d b u r y a n d W h i t e , 1954)  and Haselwood  Fig.  2.  S t r u c t u r e and metabolism of e s t r o g e n i c compounds  20. e x t r a c t s o f p r e g n a n t mare's u r i n e s u g g e s t i n g t h a t t h i s may  have o r i g i n a t e d  from the metabolism o f the i n g e s t e d f o r a g e .  S i n c e t h e n i t has b e e n i s o l a t e d  f r o m c a t t l e and f o w l  samples  ( K l y n e and W r i g h t , 1959;  logical  s i g n i f i c a n c e of which i s discussed  The  (1954) who  urine  Cayen e t a l . 1965), t h e b i o later.  e s t r o g e n i c a c t i v i t y o f t h e i s o f l a v o n e s was  by Chang e t al_.  compound  demonstrated  f e d them t o i m m a t u r e m i c e  m e a s u r e d t h e e f f e c t on u t e r i n e w e i g h t .  Results of  and preliminary  b i o l o g i c a l t e s t s showed t h a t g e n i s t e i n had an e s t r o g e n i c -5 o f a p p r o x i m a t e l y 10  times that of estrone.  activity  Formononetm  was  f o u n d t o be i n a c t i v e a s a s s e s s e d by t h e A l l e n - D o i s y m e t h o d w h i c h i s b a s e d on t h e e s t i m a t i o n o f t h e c o r n i f i c a t i o n o f r a t v a g i n a . The m e t a b o l i s m o f v a r i o u s p h y t o e s t r o g e n s by t h e rumen m i c r o b e s h a s b e e n s t u d i e d by many w o r k e r s . Nilsson  (1961 a and b) and  In a s e r i e s of experiments  (1962) d e m o n s t r a t e d t h a t t h e rumen  microorganisms demethylated the c o m p a r a t i v e l y weaker  isoflavones,  f o r m o n o n e t i n and b i o c h a n i n - A t o y i e l d more p o t e n t e s t r o g e n i c compounds s u c h as d a i d z e i n and g e n i s t e i n e t a l . (1967)  (Fig. 2).  f o u n d t h a t i n sheep t h e i s o f l a v o n e s ,  Braden genistein,  b i o c h a n i n A, and f o r m o n o n e t i n w e r e a l l e s t r o g e n i c when a d m i n i s t e r e d i n t r a r u m i n a l l y , b u t had o n l y 5% o f t h e a c t i v i t y when g i v e n intramuscularly.  B i o c h a n i n A i s m e t h y l a t e d a t t h e 4'  position  on t h e C - r i n g and a f t e r m i c r o b i a l m e t a b o l i s m , t h e m e t h y l i s c l e a v e d and g e n i s t e i n i s p r o d u c e d and B r a d e n , 1 9 7 4 ) .  (Braden e t a l .  Cox  Formononetin which i s a l s o methylated at  t h e 4' p o s i t i o n i s c o n v e r t e d t o d a i d z e i n a f t e r of the m i c r o f l o r a .  1967;  group  similar  action  Daidzein i n turn i s metabolized to equol,  w h i c h i s a v e r y p o t e n t e s t r o g e n a n a l o g u e and i s b e l i e v e d t o be  the c h i e f p l a n t c o n s t i t u e n t r e s p o n s i b l e f o r "Clover ( L i g h t f o o t and Wroth, 1974). in alfalfa.  Coumestrol i s found  Disease" predominantly  I n t h e f o w l i t i s degraded a l o n g pathways q u i t e  d i f f e r e n t from those  of genistein, despite the fact that the  b i o s y n t h e t i c pathway o f c o u m e s t r o l of the i s o f l a v o n e s Phytoestrogens  i n the p l a n t resembles  that  (Cayen e t a l . 1 9 6 5 ) . have been r e p o r t e d t o a c t l i k e e s t r a d i o l - 1 7 ^ .  a f t e r t h e i r c o n s u m p t i o n a n d s u b s e q u e n t m e t a b o l i s m by t h e g r a z i n g animal  (Braden e t a l . 1967;  Endogenous e s t r o g e n s them i n a c t i v e . absorption the  liver  S h u t t £t a l . 1 9 6 7 ;  circulate  Phytoestrogens,  and c i r c u l a t e  form which  renders  with glucuronic acid i n  i n t h e plasma i n t h e form o f i n a c t i v e  A f t e r t h e i r i n t e r a c t i o n with the receptor  proteins the covalent  conjugation with glucuronic acid i s s p l i t  by t h e a c t i o n o f g l u c u r o n i d a s e rate conjugation phytoestrogens  (Cox and B r a d e n , 1974) .  The  and t h e m e t a b o l i s m w i l l g o v e r n t h e e f f e c t o f  on t h e r e p r o d u c t i v e p e r f o r m a n c e o f g r a z i n g  D i f f e r e n c e s have been o b s e r v e d i n t h e m e t a b o l i s m o f  phytoestrogens  b e t w e e n s h e e p and c a t t l e .  reported that c i r c u l a t i n g phytoestrogens conjugated  1967).  a f t e r t h e i r m e t a b o l i s m and  i n the gut, are conjugated  glucuronates.  animals.  i n a conjugate  Lindner,  B r a d e n e t a l . (1971) w e r e more  i n c a t t l e than i n sheep, r e n d e r i n g  c e p t i b l e to the action of  efficiently  them l e s s  sus-  phytoestrogens. - 3 - 5  Phytoestrogens estradiol-173  a r e 10  t o 10  i n t h e l a b o r a t o r y mouse  times  (Bradbury  C a r t e r e t all955).However, w i t h continuous r e l a t i v e l y high  intake of phytoestrogens  b i o l o g i c a l e f f e c t s ( B a r r e t , 1961).  as e s t r o g e n i c as and W h i t e , 1951;  grazing the can induce  significant  R e s u l t s on t h e d e g r e e o f  estrogenicity primarily  of  because  solubility  by  mouse u t e r i n e (1959).  u n i t weight  of  macromolecular methylated  free  receptor.  estrogen There  have  or  and  plants  Sanger  and  tility  i s temporary five  material  effect  their  to  s i x weeks  i s stopped  1974;  variables.  Stage  of  1959),  mineral  depends  maturity deficiency  and  The  did  not  suggests  i n biochanin  A  and  interaction  with  animals  grazing  estrogenic  et  a l . 1948; In  Pope,  for a  many  Wroth, when  the  1954;  many c a s e s  of  the  1974).  The  of  return  estro-  Permanent  ewes h a v e  number  infer-  conception  consumption  1974). on  for  on  result  Marshall,  potency  cytosol.  This  of  effects  of  clover pastures  units  profound  ( L i g h t f o o t and may  70  affinity  binding.  the  to  estrogenic  r e c y c l i n g and  after  et a l .  formononetin  1974).  and  were  approximately  uterine  effective  (Bartlett  Kitts  that  binding  and  of  Marshall,  phytoestrogens  trace  A  low  semi-quantitative  by  The  i n the  reports  i n nature  the  their  equivalent  genistein.  located  (clover disease)  Wroth,  of  was  reproduction  1960;  estrogenic  and  reported  groups methylated  pastures  Bell,  infertility  foot  of  b e e n many  lactation  grazing  (1971)  using  developed  e s s e n t i a l f o r the  forage  genic  method  estradiol-173  the  normal  (1962)  isoflavones, biochanin  are  to  Flux  components  formononetin  growth,  activities  i s related to  hydroxyl  the  estrogenic  units  that  contradictory,  Relative  a l .  inhibit  been  with  assay  significantly  have  associated  estradiol-173  175  phytoestrogens  compounds  errors  Wong a n d  weight  and  plant  the  Shemesh e t  coumestrol of  of  i n water.  investigated  one  various  been  years  magnitude  (Lightof  the  environmental  defoliation  (Kitts  ( R o s s i t e r , 1970),  et a l .  route  of  administration  (Ostrovsky  e t a i . 197 2) and  and  Kitts,  i n forage  I n f e r t i l i t y r e s u l t i n g from the be m a n i f e s t e d  1961; on  sperm p r o d u c t i o n  ( G e o r g e and  (1974) r e p o r t e d  viability  a reduction  and  were a l s o r e p o r t e d  Bell,  t o be  pastures  1960;  e f f e c t s of  Barrett,  phytoestrogens  i n rams h a v e b e e n shown t o  Ovulation  L i g h t f o o t and  be  Wroth  i n ewes  r a t e s o f ewes on  reduced, thereby  cycle.  t h e p a s s a g e o f t h e egg  successful f e r t i l i z a t i o n of  (Saba  plants.  i n the onset of e s t r u s  normal l e n g t h of the estrous fer  The  T u r n b u l l , 1966).  grazing estrogenic pastures. pastures  pasture  ( S a n g e r and  Wroth, 1974). and  and  i n g e s t i o n of estrogenic  i n many f o r m s  L i g h t f o o t and  negligible  disease  i n d i v i d u a l p l a n t p a r t s d e t e r m i n e t h e amount o f  estrogenic m a t e r i a l present  may  1962), f o l i a r  similar  a f f e c t i n g the  D i f f e r e n c e s on ovum t r a n s -  through the  F a l l o p i a n tube f o r  have a l s o been a t t r i b u t e d t o t h e e f f e c t  phytoestrogens. The  establishment  o f a p o o l o f sperm i n t h e c e r v i x i m m e d i a t e -  l y a f t e r mating i s important ( R e s t a l l and  W a l e s , 1966;  for successful f e r t i l i z a t i o n  Smith, 1970).  L i g h t f o o t and  (1974) c o l l e c t e d e g g s f r o m ewes f e d e s t r o g e n i c t h a t t h e y had  l o w e r numbers o f s p e r m a t o z o a p r e s e n t  p e l l u c i d a , which reduced the p r o b a b i l i t y of No  e v i d e n c e has  estrogenic pastures.  on  and the  Wright  e f f e c t o f p h y t o e s t r o g e n s was  zona  abnormalities  O b s t and  Semark  (197 0)  (196 5) r e p o r t e d to i n h i b i t the  of  reported  to functional  changes i n the corpus luteum of a n i m a l s g r a z i n g v a r i o u s L e a v i t t and  found  c o r t e x as a c o n s e q u e n c e  f l u c t u a t i n g l e v e l s o f p l a s m a p r o g e s t e r o n e due  pastures.  Wroth  fertilization.  been e s t a b l i s h e d s u g g e s t i n g  i n the ovary, p i t u i t a r y or adrenal g r a z i n g on  forages  i n sheep  t h a t the  r e l e a s e of  clover principal gona-  dotrophic  hormones by  s e r v o m e c h a n i s m on t h e  hypothalamus.  A greater incidence of i r r e g u l a r estrous c y c l e s i n d a i r y cows f e d e s t r o g e n i c f o r a g e s was Barrett and  r e p o r t e d by A l d e r  (1961) f o u n d a c o r r e l a t i o n b e t w e e n f e r t i l i t y of.' t h e  s i z e of macroscopic c y s t s seen a t autopsy.  observed i n c a t t l e grazing subterranean of uterus and  (1965).  and  o v a r i e s and  Other changes  c l o v e r i n c l u d e the  u d d e r e v e n i n n o n - p r e g n a n t and  increased c l i n i c a l  cystic  P a t h o l o g i c a l con-  d i t i o n s c h a r a c t e r i z e d by c y s t i c g l a n d u l a r h y p e r p l a s i a o f e n d o m e t r i u m w e r e a l s o n o t i c e d i n ewes.  N o v a k and  of  lesions described  by  (1941) i n p o s t m e n o p a u s a l women w e r e  observed i n the e p i t h e l i a l epithelial  the  V a r i a b l e degrees  stroma c e l l s resembling  Richardson  size  n o n l a c t a t i n g cows,  incidence of swollen v u l v a s ,  h y p e r e m i c mucous membranes.  h y p e r p l a s i a i n the  cow  l i n i n g was  l i n i n g of the c y s t i c glands.  c h a r a c t e r i z e d as b e i n g  flat  and  The  devoid  of  all mitotic activity. The  incidence of a b o r t i o n f o l l o w i n g the  n e e d l e s by c a t t l e  i s not  e n t i r e l y known.  sponsible for abortions i n cattle, needles i s a teiterpene  i n g e s t i o n of  The  substance r e s -  sheep o r deer g r a z i n g  (Glycyrrhetmic acid).  c o n s u m i n g p i n e n e e d l e s t h a t f e t a l d e v e l o p m e n t had u t e r i were r e p o r t e d  was  i n the process  Cook and  Kitts  t o be h y p e r e m i c pr  of reabsorption.  (1964) r e p o r t e d  nodular  A l l e n and  r e s u l t e d i n embryonic m o r t a l i t y .  y e l l o w p i n e have been r e p o r t e d istics.  animals,  been a r r e s t e d . and  Kitts  the  fetus  (1961)  t h e p r e s e n c e o f an a g e n t i n  needles which suppressed growth of the uterus and  pine  Chow e t a l .  (1972) r e p o r t e d a f t e r e x a m i n i n g t h e u t e r u s o f p r e g n a n t  The  pine  and  pine  of immature mice  Aqueous e x t r a c t s from  to possess estrogenic  character-  Attempts have been made to reduce the i n c i d e n c e o f r e p r o d u c t i v e d i s o r d e r s i n animals consuming  estrogenic  plant  m a t e r i a l s by a l t e r i n g the metabolism o f phytoestrogens i n animals and by immunologic methods.  The former r e q u i r e s a b e t t e r  under-  standing o f the chemical changes to these compounds by the a c t i o n of the rumen microorganisms and subsequent metabolism.  Cox  e t a l . (1972) r e p o r t e d t h a t there i s a c o v a l e n t attachement of a n t i g e n i c p r o t e i n w i t h s u i t a b l e phytoestrogens r e s u l t i n g i n haptens.  Sera o b t a i n e d from sheep p o s s e s s i n g a n t i b o d i e s a g a i n s t  v a r i o u s phytoestrogens r e a c t e d s p e c i f i c a l l y w i t h p l a n t e s t r o g e n s . Antibody t i t r e s were r e p o r t e d t o p e r s i s t i n sheep f o r about one year, w i t h no e f f e c t on the normal e s t r o u s c y c l e of the animal.  Experiment  A  : The and and  Comparative E f f e c t s of Estrogens P h y t o e s t r o g e n s on W a t e r I m b i b i t i o n Hyperemia o f the Rat Uterus.  Introduction  Estrogenic metabolism probably lation that  hormones  i n many  target  secondary  of  exert  components  estrogen  induced  estrogen  receptor  in  of  There  estrogens,  been  vaginal  the  past  the  occur  metabolic  events  Astwood during  earlier  more  vaginal  and  are  six-hour  the  stages  late  the  the  female  may  in  reproductive in  response  laboratory of  the  frequently  1964)  .  animals  uterine  selected  cornification  than  character-  others.  responses  and  to  inhibition  These  accumulation  of  various  test, water  in  increased  certain vascular  nonspecific  estrogen  in  quantitatively  difference  (Martin,  later  of  many  results  general  s e n s i t i v e than  water  possible  on  uterus  are  stimu-  particular  differ  of  epithelial  also  i s also  the  is a  species  reactions  effects  initial  effect  of  action.  techniques  considerably  initial  growth  Responses most  (1938) m e a s u r e s very  marked  relatively  reduction  responses  by  level  genome a c t i v a t i o n and  growth  assay  tissues.  were  tetrazolium  promote  an  In  hormonal  uterine  also  on  cell.  the  these  It  c o m p o u n d s w h i c h may  estrogen  based  and  The  the  to  are  separate  some b e i n g  Standard have  of  capacity  tract. to  a  on  of  primary  histamine  i n t e r a c t i o n and  estrogenic  their  one  target  of  Most  process.  than  i n the  represent  Stimulation istic  more  release  influence  r e s u l t i n g from  biochemical  cellular  therefore  an  tissues.  phenomena  some k e y  estrogens  exert  and  estrogens.  developed in  the  administration.  by  uterus  S p a z i a n i e t a _ l . (1958) f i r s t r e p o r t e d water i n the uterus  f o l l o w i n g estrogen  o n d a r y t o h y p e r e m i a and r e s u l t of estrogen The  per  the  and  to estrogen.  F i n n and  cell  least three d a i l y  response u n t i l  s i n g l e dose of e s t r o g e n  estrogen The  72 h o u r s and  and As  dif-  an  was  given.  at  response.  increase and  The  the  required  in cell 28  division  hours a f t e r a  accelerated  more m a r k e d i n t h e e p i t h e l i u m t h a n t h e  cellular stroma  of  These f i n d i n g s suggest t h a t t i s s u e growth i s f o r the  notable  increase  i n u t e r i n e dry weights  of  t r e a t e d animals over c o n t r o l groups. o b j e c t of the present  s e r i e s o f e x p e r i m e n t s was  d e t e r m i n e t h e mode o f a c t i o n o f v a r i o u s e s t r o g e n s w i t h ticular  and  i n response  i n j e c t i o n whereas  maximum m i t o t i c a c t i v i t y b e t w e e n 24  responsible  stimulated  r e s p o n s e t o e a c h was  i n j e c t i o n s f o r maximal  Kaye e t a_l. (1972) r e p o r t e d  myometrium.  (1973)  l u m i n a l e p i t h e l i u m showed m a x i m a l m i t o s i s a f t e r  g l a n d s showed l i t t l e  d i v i s i o n was  Martin  c e l l s o f mouse u t e r u s  24 h o u r s i n r e s p o n s e t o a s i n g l e d a i l y  and  the  p r o l i f e r a t i o n i n both  d i v i s i o n i n b o t h t y p e s was initial  sec-  r e f l e c t s the p h y s i o l o g i c a l  luminal e p i t h e l i a l  However, t h e  The  on  of  histamine.  (Hinshaw, 1959).  e f f e c t of estrogen  found t h a t c e l l  ferent.  t h a t i n c r e a s e d p e r m e a b i l i t y was  cent of water i n general  glandular  a d m i n i s t r a t i o n was  induced release of  s t a t e of the uterus s t u d i e d the  t h a t the accumulation  reference  to the e a r l y biochemical  t h e r o l e o f p h y t o e s t r o g e n s i f any, a p r e l i m i n a r y step  mechanisms  i n modifying  to par-  involved  the  same.  i n t h i s d i r e c t i o n E x p e r i m e n t A was  taken t o t e s t the potency of v a r i o u s  under-  e s t r o g e n i c compounds  compare t h e i r t i m e c o u r s e e f f e c t s on t h e b a s i s o f w a t e r  and  inhibition  28.  of  immature r a t u t e r i n e  tissues.  M a t e r i a l s and Methods Animals Immature f e m a l e were p u r c h a s e d Ontario.  (35-40 g ) , d e r i v e d f r o m W i s t a r s t o c k  f r o m UBC C o l o n y  and Woodlyn L a b o r a t o r i e s ,  Guelph,  They w e r e h o u s e d i n a i r c o n d i t i o n e d q u a r t e r s w i t h  uniform exposure Animals  rats  t o 12 h o u r s o f l i g h t  had f r e e a c c e s s t o p e l l e t e d  a n d 12 h o u r s o f d a r k n e s s .  food  (Purina Laboratory  Chow) a n d w a t e r . Materials E s t r a d i o l - 1 7 3 ( e s t r - 1 , 3 , 5 ( 1 0 ) - t r i e n e - 3 ,17c* d i o l ) , E s t r i o l ( e s t r - 1 , 3 , 5 ( 1 0 ) - t r i e n e 3,16 , 1 7 3 - t r i o l ) , E s t r o n e ( e s t r - 1 , 3 , 5 ( l O ) . t r i e n e 3-01-17-one) , . DES (a , a , - d i m e t h y l - s t i l b e n e - 4 - 4 ' - d i o l ) , E s t r a d i o l 17a ( e s t r - 1 , 3 , 5, ( 1 0 ) - t r i e n e - 3 - 1 7 , ) ; d i o l ) , a n d T e s t o s t e r o n e (173 H y d r o x y - 4 - a n d r o s t e n - 3 - o n e ) w e r e o b t a i n e d f r o m S i g m a C h e m i c a l s Co. The f o l l o w i n g p l a n t e s t r o g e n s w e r e d o n a t e d by D r . A.B. B e c k o f C.S.I.R.O., W e s t e r n A u s t r a l i a , D e p t . o f Agriculture: Formononetin (7-hydroxy-4'methoxyisoflavone), B i o c h a n i n - A ( 4 ' m e t h o x y - 5 - 7 - h y d r o x y i s o f l a v o n e ) and C o u m e s t r o l (7-hydroxy-coumarono-coumarins). P l a n t e x t r a c t s w e r e made from a l f a l f a by t h e method o f F r a n c i s and M i l l i n g t o n (1965).  A d m i n i s t r a t i o n o f e s t r o g e n s and P l a n t In Experiment  A - I , e i g h t groups  group) w i t h comparable of  estrogens of animals  (4 a n i m a l s /  body w e i g h t s were g i v e n a s i n g l e  injection  1.0 mg o f e s t r a d i o l - 1 7 3 i n t r a p e r i t o n e a l l y t o s t u d y t h e t i m e  c o u r s e e f f e c t o f e s t r o g e n on w a t e r tissues.  Experiments  imbibition  by r a t u t e r u s  A-2 a n d A - 3 w e r e d e s i g n e d t o s t u d y t h e  e f f e c t o f v a r y i n g c o n c e n t r a t i o n s o f e s t r a d i o l - 1 7 3 and t h e r e l a t i v e e f f e c t s o f v a r i o u s e s t r o g e n s on water A stock solution  imbibition.  o f e s t r a d i o l - 1 7 3 was made b y d i s s o l v i n g  5.0 mg i n 2.0 m l o f 95% e t h a n o l .  T h i s was b r o u g h t  t o a volume  of 5.0 ml w i t h  0.9% NaCI t o g i v e a c o n c e n t r a t i o n o f 1 0 0 ug/0.1 ml.  A 1 0 0 u l a l i q u o t of t h i s s o l u t i o n was made up t o a volume o f 1 0 . 0 ml w i t h  0.9% NaCI t o g i v e an a d m i n i s t r a t i o n c o n c e n t r a t i o n o f  1.0 ug/0.1 ml.  S o l u t i o n s of DES, e s t r i o l and t e s t o s t e r o n e  were made i n s i m i l a r c o n c e n t r a t i o n s . were prepared i n c o n c e n t r a t i o n s  E s t r a d i o l - 1 7 d and estrone  o f 2.0 ug/0.1 ml and 6.0 ug/0.1  ml r e s p e c t i v e l y . The  c o n c e n t r a t i o n o f phytoestrogens was made t o correspond  to e s t r a d i o l - 1 7 3 i n terms o f u t e r o t r o p h i c a c t i v i t y Lidner, 1 9 7 0 ) .  (Perel and  Coumestrol s o l u t i o n s were made by d i s s o l v i n g  3.0 mg o f p u r i f i e d coumestrol i n 1 0 . 0 ml o f propylene g i v i n g a c o n c e n t r a t i o n o f 9 0 . 0 ug / 0 . 3 ml.  glycol  Genistein  ( 8 . 0 mg)  was d i s s o l v e d i n 3.0 ml o f propylene g l y c o l t o g i v e a f i n a l c o n c e n t r a t i o n o f 0.8 mg/0.3 ml. biochanin-A  Formononetin  (100 mg/ml) and  (100 mg/ml) stock s o l u t i o n s were made up i n t o l u e n e .  An a l i q u o t from both was taken and evaporated t o dryness under a stream o f n i t r o g e n .  The r e s i d u e was d i s s o l v e d i n propylene  g l y c o l t o g i v e a c o n c e n t r a t i o n o f 1 0 0 mg/0.1 ml. To t e s t f o r u t e r o t r o p h i c a c t i v i t y the compounds were i n j e c t e d i n t r a p e r i t o n e a l l y i n t o immature female r a t s . animals were given i n corresponding After appropriate  Control  i n t r a p e r i t o n e a l i n j e c t i o n s o f 0.5% a l c o h o l  volumes o f 0.9% NaCI o r propylene  glycol.  time i n t e r v a l s the animals were s a c r i f i c e d by  p l a c i n g them i n a j a r f i l l e d w i t h carbon d i o x i d e . were e x c i s e d and s t r i p p e d o f adhering  Their  uteri  f a t and mesentery.  U t e r i n e t i s s u e s were b l o t t e d d r y and weighed on a m i c r o p r e c i s i o n torque balance.  T i s s u e s were then t r a n s f e r r e d t o an oven and  d r i e d t o a constant  weight a t 90°C f o r 12 hours.  of water i m b i b i t i o n was-.'calculated  The degree  from the per cent  moisture.  30. All  statistical  a n a l y s i s ' was  done - by  Student's  t-test.  Results  In hours  Experiment  A-I  gradually,  reaching  levels  a f t e r s i x hours  slight  decrease  hours.  that  a t 24  creases  i n uterine  content  noticed  reached  a t s i x hours.  a t s i x hours  content  (50-1000  Significant noticed In  was  ng)  increased  Experiment  doses  and were  given  i n a dose  Table  3)  A-3  12  a  and  24  significantly  corresponded Increases  with i n i n moisture as  performed  to study  the increase  i n water  noticed  increases doses  of  estrogens  to different  that  increases  (P< 0 . 0 2 5 )  ones  those  of  i n weight greater  at  ng)  dry weights  due  to  lower  (Table were  were  administered  in  of estradiol-173 The  various standards  results  ( F i g . 3,  e f f e c t s of the steroid estrogens  estradiol-17 3  , DES,  estradiol-17  a  were and  estrone. The were  effects of plant  compared  2).  estrogen.  o f l*0.^ig i n t r a p e r i t o n e a l l y .  of e s t r i o l ,  doses  (5000-50,000  i n uterine  to the e f f e c t  show t h e r e l a t i v e  the order  between  by  not as great  i n higher  compared  control  however,  i n response  than  over  followed  1).  few  imbibition i n -  to increase  (Table  significantly  (P< 0.025)  with  content  the f i r s t  were,  I t was were  water  was  hours which  dry weights  A-2  estradiol-17 3 . moisture  This  found  30  and  at 30 hours  Experiment imbibition  (Fig.4).  i m b i b i t i o n was  again  during  a maximum o f 4 0.6%  i n tissue moisture  Water  (P < 0 . 0 2 5 )  in  noticed  after estradiol-17 3 administration,  creased  doses  i t was  estrogens  t o e s t r a d i o l - 1 7 3 and  on u t e r i n e testosterone  water  imbibition  ( 1 . 0 p.g I . P . )  31. standards.  Coumestrol  and g e n i s t e i n d i s p l a y e d s i g n i f i c a n t  (P 1 0.025) e f f e c t s o v e r c o n t r o l s water  i n regard t o t h e i r degree o f  i m b i b i t i o n , b u t a t much l a r g e r d o s a g e s  Formononetin  (Table 3 ) .  a n d b i o c h a n i n - A d i d n o t show a n y s i g n i f i c a n t  increase i n water  (P>0.025)  imbibition. Discussion  Results of Experiments  A - l , 2, 3 i n d i c a t e t h a t t h e d e g r e e  of  water  imbibition varies with various estrogens.  of  an e s t r o g e n t o p r o d u c e t h i s e f f e c t i s r e l a t i v e l y  governed  The  ability  constant,  m a i n l y by t h e t i m e and dose l e v e l s c h a r a c t e r i s t i c o f  the e s t r o g e n .  Among t h e e a r l i e s t known e f f e c t s o f e s t r o g e n s  a r e t h o s e a s s o c i a t e d w i t h t h e u t e r i n e membrane a n d h i s t a m i n e release  ( S p a z a n i e t al_. 1958;  Experiment  Szego e t a l . 1967).  1-A d e m o n s t r a t e d  r e s u l t s i n accumulation of f l u i d  t h a t a s i n g l e dose o f e s t r o g e n i n u t e r i n e t i s s u e s which  be a t i t s maximum a t s i x h o u r s .  The p e r c e n t m o i s t u r e  will  reached  a maximum a t t h i s p a r t i c u l a r t i m e .  This result  i s i n accord-  ance w i t h t h e o b s e r v a t i o n o f Hinshaw  (1959) who r e p o r t e d t h a t  t h e g a i n i n w e i g h t d u r i n g t h e f i r s t s i x h o u r s was m a i n l y due to  the accumulation of f l u i d  decrease hours its  i n t h e lumen o f t h e u t e r u s .  i n per cent moisture content o f the t i s s u e a f t e r s i x  i s p r o b a b l y due t o p a r t i a l  reabsorption of fluid  e n t e r i n g i n t o t h e u t e r i n e .growth  suggested In  The  by Hinshaw  Experiment  prior to  p h a s e a t 12-14 h o u r s a s  (1959).  A-2 t h e amount o f a n e s t r o g e n a d m i n i s t e r e d  o v e r a n d a b o v e 50 ng d i d n o t p r o d u c e a n y f u r t h e r g a i n i n w e t u t e r i n e weight or per cent moisture.  A c c o r d i n g t o Hinshaw  (1959)  the a  decreases  i n uterine  single large  effects  weight which occurs  dose o f e s t r a d i o l - 1 7 3  because greater  hours a f t e r estrogen  uterine  weights occurred  s u c h a n e f f e c t may b e a f f e c t e d  this  of estrogens  experiment,  abilities in  weight,  estradiol-173 This  estriol  ( 1 9 5 3 ) who r e p o r t e d  six  that  effect.  g e n i s t e i n was r e q u i r e d  of  coumestrol. use o f uterine  Results  activity obtained  ment w i t h (Bickoff  reports  possessed  To o b t a i n  , estrone  i n  similar  a 32%  and  i n promoting uterine was i n t h e o r d e r  coumestrol  about an estrogenic  of  estrogen  following the adminis-  increase by  testosterone.  i n 24-27 d a y o l d r a t s , t h e r e l a t i v e  intraperitoneally eight  The  t o produce  water  imbibition  of estriol,  estrone.  capable o f bringing istered  ability  was t h e most e f f e c t i v e , f o l l o w e d  Among t h e p h y t o e s t r o g e n s  The  and e s t r i o l  hours a f t e r administration and  i m b i b i t i o n was  t h e f i n d i n g s o f Szego and Roberts  o f estrogens  estradiol-173  and four  From t h e d a t a o b t a i n e d  , DES, e s t r a d i o l - 1 7 a  i s i n agreement w i t h  effectiveness  by t h e time  estradiol-173 this  that water  though t h e i r  and t h e dose.  i n producing  uterine  three  inhibitory  administration.  common r e s p o n s e o f a l l e s t r o g e n s  tration  following  was n o t due t o s e l f  I n E x p e r i m e n t A-3 i t was e v i d e n t a  s i xhours  has y i e l d e d  response.  t o t e n times  to elicit  weight  and g e n i s t e i n  the  a response  increases conflicting  When  similar t o that  f o r the assay  v a r i a b l e s w h i c h must be t a k e n  o f phyto-  r e s u l t s i n the past.  of phytoestrogen uterotrophic 1962;  admin-  concentration  i n E x p e r i m e n t A-3 a r e n o t i n c o m p l e t e  e t a l . 1962, N i l s s o n ,  were  agree-  activity  Braden e t a l . 1967).  i n t o a c c o u n t when  evaluating  such d i f f e r e n c e s are the r a t e of a b s o r p t i o n , r a t e of  metabolism  and method o f a d m i n i s t r a t i o n o f t h e e s t r o g e n i c compounds.  This  has l e d t o c o n f l i c t i n g r e p o r t s i n t h e l i t e r a t u r e r e n d e r i n g the proper assessment  of uterine weight bioassays d i f f i c u l t .  I n v i e w o f t h i s i t was  c o n s i d e r e d d e s i r a b l e t o u n d e r t a k e more  a c c u r a t e experiments which would  d e s c r i b e the e a r l y  f o l l o w i n g the a d m i n i s t r a t i o n of e s t r o g e n i c  events  compounds.  Conclusions These s e r i e s o f e x p e r i m e n t s were d e s i g n e d t o e s t a b l i s h  an  o p t i m a l t i m e c o u r s e and d o s e e f f e c t o f e s t r o g e n s f o r u s e i n subsequent  experiments.  o f w a t e r due  The p u r p o s e o f i n c r e a s e d  t o e s t r o g e n i s n o t e n t i r e l y known, h o w e v e r i t  a p p e a r s t o be i m p o r t a n t due t o i t s r e l a t i v e l y dynamic  imbibition  early  and  effect.  A s i x h o u r t i m e c o u r s e p r o v e d t o be o p t i m a l t i m e a f t e r s t a n d a r d 1.0 eally.  ug d o s e o f e s t r a d i o l - 1 7 3 was  given  D u r i n g t h i s t i m e and a t t h i s d o s e ,  t h e t i s s u e r e a c h e d i t s maximum a b i l i t y t o I n E x p e r i m e n t A-3  intraperiton-  i t was  found  that  respond.  r e s u l t s o b t a i n e d ( F i g . 3, T a b l e  showed v a r i o u s e s t r o g e n s h a v e t h e c a p a c i t y t o i n d u c e imbibition;  a  3)  water  however dose l e v e l s and t i m e a f t e r dose were  c h a r a c t e r i s t i c of p a r t i c u l a r estrogens. From t h e s e r e s u l t s i t may a r e more e f f e c t i v e  be c o n c l u d e d t h a t some e s t r o g e n s  i n enhancing water  i m b i b i t i o n by t h e u t e r u s  w h i l e o t h e r s a r e more a c t i v e i n t h e p r o m o t i o n o f growth.  E s t r a d i o l - 1 7 3, t h e m o s t p r o m i n e n t  endogenously,  uterine  estrogen produced  i s the s t r o n g e s t n a t u r a l estrogen f o r growth  and  water uptake by the u t e r u s even a t low doses. Phytoestrogens a l s o demonstrated some degree o f  estrogenic  a c t i v i t y i n regard t o water i m b i b i t i o n , although i t was t h a t the c a r r y i n g v e h i c l e and route o f a d m i n i s t r a t i o n important c r i t e r i a i n i n f l u e n c i n g t h e i r response.  felt  were  WET  UTERINE  WEIGHT  (MG)  CD CD i—I I  CD  CD  CN]  I  CD  •cr  CONTROL  E S T R A D I O L - 1 7 / ^ ( 1 , 0 JUG)  ESTRADIOL-17 ^(2.0 C  DES  ( 1 . 0 JUG)  ESTRONE  ( 6 . 0 JUG)  ESTRIOL  ( 1 . 0 JUG)  TESTOSTERONE  COUMESTROL  GENISTEIN  BIOCH-A  (l.OjJG)  (90 JUG)  (0,87TSG)  ( 1 0 0 JUG)  FORMONONETIN  g. 3.  JUG)  (100 JUG)  Wet u t e r i n e weight s i x hours f o l l o w i n g estrogen a d m i n i s t r a t i o n (Expt. A-3)  36.  70  r  Fig.  4.  E f f e c t of time on water i m b i b i t i o n i n r a t u t e r i n e t i s s u e s f o l l o w i n g administ r a t i o n of e s t r a d i o l - 1 7 3 (Expt. A-I)  Table 1 : E f f e c t o f time on water i m b i b i t i o n by r a t u t e r i n e t i s s u e s f o l l o w i n g a d m i n i s t r a t i o n o f e s t r a d i o l - 1 7 3 (Expt. A-I) Animal C h a r a c t e r i s t i c s Time (hours) following administration of e s t r a d i o l - 1 7 3  Number of Animals  Body Weight (g)  C o n t r o l (0)  4  37.6 ± 0.60  19.0 ± 0.01  ( a )  4.0 ± 0.01  ( a )  2  4  37.6 ± 0.90  22.0 ± 0.01  ( b )  4.2 ± 0.0.1  ( b )  3  4  37.5 ± 1.20  22.0 ± 0.02  ( b )  4.2 ± 0.01  6  4  37.5 ± 0.70  32.1 ± 0.02  ( c )  12  4  37,. 4 ± 0.60  28.1 ± 0.01  14  4  36.7 ± 1.20  24  4  30  4  U t e r i n e Wet Weight (mg)  U t e r i n e Dry Weight (mg)  Moisture Content o f Uterus (%) 79.4 *  a )  80.1  ( a )  ( b )  81.3  ( b )  4.6 ± 0.01  ( c )  85.2  ( d )  ( C )  5.0 ± 0.01  ( d )  82.1  ( b )  28.0 ± 0.02  ( c )  5.1 ± 0.01  ( d )  81.0  ( b )  37.5 ± 1.70  32.0 ± 0.02  ( d )  5.5 ± 0.01  ( e )  82.2  ( b )  37.2 ± 0.60  37.0 ± 0.02  ( e )  5.9 ± 0.02  ( f )  83.7  ( C )  (a,b,c,d,e,f) Means w i t h d i f f e r e n t s u b s c r i p t s are s i g n i f i c a n t l y d i f f e r e n t  (P < 0.025)  Table  2: E f f e c t o f d o s e o n w a t e r i m b i b i t i o n b y r a t u t e r u s following administration of estradiol-17$ (Expt.  Animal  D o s a g e (ng) of estradiol-17 3 injected  Number of Animals  Body Weight (g)  tissues A-2)  Characteristics  U t e r i n e Wet Weight (mg)  U t e r i n e Dry Weight (mg)  Moisture Content of Uterus (%)  4  37.6  ±  1.6  19.0  ± 1.10  (  a  )  4.0  ± 0.10  (  a  )  79.0  (  a  )  50  4  38.6  ±  2.1  32.6  ± 0.80  (  b  )  4.6  ± 0.10  (  b  )  86.0  (  b  )  100  4  41.6  ±  3.6  33.6  ± 0.40  (  c  )  4.8  ± 0.10  (  c  )  85.7  (  b  )  500  4  44.1  ±  2.1  33.1  ± 0.20  (  b  )  4.9  ± 0.05  (  c  )  85.2  (  b  )  1000  4  42.9  ±  1.8  33.8  ± 0.20  (  c  )  5.0  ± 0.10  (  C  )  85.2  (  b  )  5000  4'  43.7  ±  1.2  32.8  ± 0.90  (  b  )  5.2  ± 0.05  (  C  )  84.2  (  C  )  4  42.8  ±  1.1  32.9  ± 1.20  (  b  )  5.1  ± 0.01  (  c  )  84.2  (  C  )  Control  (0)  50000 (a,b,c)  Means w i t h  different ' superscripts  are significantly  different  (P <  0.025)  Table  3 : E f f e c t s o f s t e r o i d and p l a n t e s t r o g e n s on w a t e r i m b i b i t i o n by r a t u t e r i n e t i s s u e s i x hours f o l l o w i n g a d m i n i s t r a t i o n (Expt. A - 3 )  Animal Estrogens injected and d o s e -Conto 1  ( 0 ug)  Body Weight (g)  Number of Animals  Characteristics  (%)  4  37.2  ±  1.30  23.1  ±  3.10  (  a  •  )  Estradiol-173  ( 1 . 0 ug)  4  42.9  ±  0.50  33.8  ±  1.0  (  c  )  Estradiol-17a  ( 2 . 0 ug)  4  43.1  ±  1.30  30.5  ±  0.8  (  b  )  4  46.9  ±  0.80  .33.1  ±  0.6  (  C  )  DES  ug)  (1.0  Estrone  ( 6 . 0 ug)  4  48.9  ±  1.00  34.1  ±  1.6  (  c  )  Estriol  ( 1 . 0 ug)  4  47.2  ±  1.00  36.9  ±  1.2  (  d  )  4  36.5  ±  1.30  23.6  ±  1.1  (  a  )  4  43.9  ±  1.50  33.9  ±  1.2  (  c  )  4  47.1  ±  1.90  32.7  ±  1.5  (  C  )  4  48.2  ±  1.30  24.7  ±  1.5  (  a  )  4  47.5  ±  1.90  25.3  ±  1.3  (  a  )  Testosterone Coumestrol Genistein  ( 1 . 0 ug)  ( 9 0 . 0 ug) ( 0 . 8 mg)  B i o c h a n i n A ( 1 0 0 ug) Formononetin  ( 1 0 0 ug)  (a,b,c,d) Means w i t h d i f f e r e n t  Moisture Content o f Uterus  Uterine Dry Weight (mg)  U t e r i n e Wet Weight (mg)  79.8  (  a  )  85.2  (  b  )  83.9  (  c  )  85.0  (  b  )  85.1  (  b  )  85.6  (  b  )  )  80.1  (  a  )  a  )  86.3  (  b  )  (  C  )  85.6  (  b  )  0.10  (  C  )  80.5  (  a  )  0.70  (  a  )  82.8  (  C  )  4.6.  ±  0.10  (  a  )  5.0  ±  0.40  (  b  )  4.8  ±  0.10  (  C  )  4.9  ±  0.10  (  C  )  5.0  ±  0.20  (  b  )  4.8  ±  0.10  (  C  )  4.7  ±  0.10  (  C  4.6  ±  0.30  (  4.7  ±  0.60  4.8  ±  4.3  ±  s u b s c r i p t s are s i g n i f i c a n t l y d i f f e r e n t  (P <: 0.025)  40.  Experiment B : E f f e c t o f E s t r o g e n on Hyperemia o f Uterine Tissue Introduction I n E x p e r i m e n t B, c h a n g e s i n t h e u t e r i n e v a s c u l a t u r e studied  i n immature female r a t s by i n t r o d u c i n g  the v a s c u l a r  were  India i n k into  system s i x hours a f t e r t h e a d m i n i s t r a t i o n o f  e s t r a d i o l - 1 7 3 , genistein or coumestrol. Materials Immature f e m a l e r a t s  and Methods  ( 2 5 - 3 0  g) w e r e e m p l o y e d .  m a i n t a i n e d and housed a s d e s c r i b e d 173,  g e n i s t e i n and c o u m e s t r o l  They w e r e  i n E x p e r i m e n t A.  Estradiol-  ( 1 . 0 u g , 8 0 . 0 mg a n d 9 0 . 0 u g  r e s p e c t i v e l y ) were a d m i n i s t e r e d , i n t r a p e r i t o n e a l l y . were p r e p a r e d f o r p e r f u s i o n  by o p e n i n g t h e t h o r a c i c w a l l and  exposing the thoracic aorta.  The e s o p h a g u s was s e v e r e d a n d a l l  c o n n e c t i v e t i s s u e was r e m o v e d . 0 . 6 7 " )  Animals  Polyethylene  tubing  ( 0 . 4 7 "  was t h r e a d e d i n t o a P a s t e u r p i p e t t e w h i c h i n t u r n was  f i t t e d t o an adaptor  (Leur-Lok T u o h y - ^ .  a d a p t o r was a t t a c h e d  t o a 2 6 G n e e d l e w h i c h was i n s e r t e d  the  artery.  A Polystaltic perfusion  was e m p l o y e d t o i n f u s e warm  0 . 9 %  The o t h e r e n d o f t h e  pump ( B u c h l e r  saline  (37°C,  rate o f 3 . 2 ml min ^ through t h e polyethylene duration the  of the perfusion  pH  into  Instr.)  7 . 2 )  tubing.  at a The  was d e t e r m i n e d b y t h e a p p e a r a n c e o f  v i s c e r a and u s u a l l y l a s t e d 3 . 5 minutes depending on t h e  size of the artery. u t e r u s t o o much d u r i n g ink  x  ( 1 0 - 2 0  C a u t i o n was t a k e n n o t t o m a n i p u l a t e t h e the perfusion.  cm ) was i n t r o d u c e d  Warm, u n d i l u t e d  into the tubing  India  b y means o f a  hypodermic  syringe.  Following t r a c t was  the p e r f u s i o n w i t h I n d i a i n k , the  r e m o v e d w i t h a m i n i m a l amount o f h a n d l i n g  r i n s e d i n warm 0.9%  normal s a l i n e .  The  placed  95%  was  e x t r a uterine  plexus  on m i c r o s c o p e s l i d e s f o r an e x a m i n a t i o n o f t h e  mesometrial region. 24  and  u t e r i n e t i s s u e s were  e x c i s e d t o form open sigments c l o s e t o t h e and  reproductive  h o u r s and  U t e r i w e r e f i x e d w i t h 10%  then dehydrated s u c c e s s i v e l y w i t h  alcohol.  Following  f i x a t i o n and  anti-  formalin  for  50,  80,  and  dehydration,  the  tissues  were c l e a r e d w i t h x y l e n e  f o r 5 minutes.  The  prepared  w e r e o b s e r v e d u n d e r 100X  power u s i n g a p h a s e c o n t r a s t  2 X  slides micro-  scope. Results From F i g . 5 and possessed the  6 i t may  be  noted that  estradiol-173  g r e a t e s t a b i l i t y to induce hyperemia i n  immature female r a t u t e r u s .  G e n i s t e i n and  the  coumestrol  also  e n h a n c e d u t e r i n e v a s c u l a r p e r m e a b i l i t y when c o m p a r e d t o c o n t r o l tissues.  The  d e g r e e o f h y p e r e m i a i n d u c e d by  coumestrol d i d not  a p p e a r t o be  even though r e l a t i v e l y  l a r g e r doses were  Discussion The ing  use  as d i s t i n c t  and  of t h i s technique  has  d e f i n i t e drawbacks i n The  To m i n i m i z e  made t o t r e a t t h e  study-  difficulties  of u t e r i n e t i s s u e s  t h e c o n s e q u e n t damage.  d i s a d v a n t a g e s e v e r y e f f o r t was  estradiol-173,  Conclusions  are a s s o c i a t e d w i t h the manipulation and  and  administered.  e s t r o g e n i c p o t e n c y o f v a r i o u s compounds.  preparation  as  genistein  during  these  specimens  the same way i n c l u d i n g the i n j e c t i o n o f s a l i n e a t constant  rate,  temperature and the p r e p a r a t i o n o f s e c t i o n s with the same f i x i n g and  c l e a r i n g reagents.  C a p i l l a r i e s i n any given r e g i o n were  found t o vary i n diameter though almost a l l specimens were f i l l e d with I n d i a ink.  Some appeared as exceedingly  fine  lines  under a m a g n i f i c a t i o n o f 100X. By means o f v i s u a l comparison o f v a r i o u s treatments w i t h c o n t r o l s , i t was noted t h a t the blood v e s s e l s o f the uterus r e f l e c t a g r a d i e n t o f response t o estrogen  concentration.  C a p i l l a r i e s around the u t e r i n e horns become an e a s i l y able f e a t u r e o f v a s c u l a r a r c h i t e c t u r e o f the uterus the i n j e c t e d specimens.  G e n i s t e i n and coumestrol  recogniz-  as seen i n (0.8 mg and  90. ug, r e s p e c t i v e l y ) had some e f f e c t on the v a s c u l a r permeab i l i t y o f the u t e r i n e c e l l .  P e r e l and L i d n e r  (1970) r e p o r t e d  s i m i l a r responses o c c u r r i n g w i t h hyperemia a f t e r the i n j e c t i o n of coumestrol and g e n i s t e i n (80 ;ug, 0.62 mg r e s p e c t i v e l y ) . These phytoestrogens d i d not provide response as d i d e s t r a d i o l - 1 7 3  t h a t same magnitude o f  (1.0 u g ) .  43.  CONTROL  F i g . 5.  The d i s t r i b u t i o n o f I n d i a i n k i n the u t e r i n e v a s c u l a t u r e of c o n t r o l and e s t r a d i o l - 1 7 8 t r e a t e d r a t s (Expt. B) x 100.  COUMESTROL  GENISTEIN  F i g . 6.  The d i s t r i b u t i o n of India ink i n the u t e r i n e v a s c u l a t u r e of coumestrol and g e n i s t e i n t r e a t e d r a t s (Expt. B) x 100  E x p e r i m e n t C : E f f e c t s o f E s t r a d i o l - 1 7 3 o n RNA andDNA S y n t h e s i s i n Immature U t e r i n e T i s s u e s . Introduction The  effect of estrogenic  i n t h e mammalian u t e r u s O'Malley  (1972).  of estrogens, antibiotic  synthesis  h a s b e e n r e v i e w e d i n d e t a i l b y Means a n d  In order  response o c c u r r i n g  hormones o n RNA a n d DNA  t o study the e a r l i e s t  i n the uterine c e l l  detectable  after the administration  a t t e m p t s h a v e b e e n made t o s t u d y t h e e f f e c t s o f  i n h i b i t o r s o f RNA a n d DNA s y n t h e s i s .  As a r e s u l t  of t h e s e s t u d i e s a c h r o n o l o g i c a l sequence o f b i o c h e m i c a l occurring  i n v i v o a f t e r t h e a d m i n i s t r a t i o n o f e s t r o g e n s has  been e s t a b l i s h e d  ( G o r s k i and K a t z e l l e n b o g e n ,  1975).  From s t u d i e s based on t h e i n c o r p o r a t i o n o f v a r i o u s precursors,  t h e r a t e o f RNA a n d DNA s y n t h e s i s  m i n e d b y many w o r k e r s . an  increased  Gorski  and N i c o l e t t e  r a t e i n RNA s y n t h e s i s  occurring  hour a f t e r e s t r a d i o l - 1 7 3 a d m i n i s t r a t i o n . regarding  events  the early action of estradiol-173  has been (1963)  labelled deter-  reported  i n t h e n u c l e u s one  Additional  information  o n RNA s y n t h e s i s  obtained  by Noteboom a n d G o r s k i  (1963^b)) who r e p o r t e d  activity  i n t h e RNA p o l y m e r a s e enzyme a s e a r l y a s one t o f o u r  hours a f t e r estrogen a d m i n i s t r a t i o n . activity  i s s i g n i f i c a n t l y elevated  t r a t i o n o f e s t r o g e n an i n c r e a s e not  achieved u n t i l  newly s y n t h e s i z e d but  8-12 h o u r s  increased  A l t h o u g h RNA p o l y m e r a s e  one h o u r a f t e r  the adminis-  i n n e t u t e r i n e RNA c o n t e n t i s  ( B i l l i n g e t al.1969(a) ) .  RNA a p p e a r s t o r e p r e s e n t  i s p r i m a r i l y ribosomal  an  was  The  a l l t y p e s o f RNA,  RNA a n d may d i f f e r i n c o m p o s i t i o n  from t h a t produced i n t h e absence o f e s t r o g e n s as i n d i c a t e d by  DNA-RNA of  hybridization studies  ribosomes  senger These  RNA  to  polysomes  into  the  of  events  to  synthesis  in vitro  i n messenger  indicates  nucleus  synthesize  peptides  alterations  that  promote of  new  was  also  injection  (Gorski  histone  division after  after were  view  the  et  of  to  nucleic  procedure  i n the  for  followed  estradiol-173  by on  of  determine  and  DNA  reflect This  produce  hours  and  about  18  These  events  at  hours  Materials  (Meuller  mes-  in  the  of  effect  in  et  polysomes.  an  increase  i n the after  in  rate  prior  ai.  24  to  1958). observed  A,  studies  cell.  on The  standardized.  the  the  cell  hours  estradiol-17g  of  of  estrogen  weights  uterine  by  the  subsequent  occur  dry  e f f e c t s of  popu-  earlier  sequence  i n Experiment  synthesis  and  the  approximately  e x t r a c t i o n was  DNA  new  after estradiol-17 3  Increases  quantitative determination RNA  conversion  1972).  cytoplasmic to  16  estrogen the  of  O'Malley,  in uterine  a c i d metabolism  RNA  on  uterus  increases  entry  i t s primary  1974).  begin  A  a c t i v a t i o n and  reported  Raker,  administration  and  exerts  administration  the  the  production.  a l . 1972).  begins  initiated  overall  was  and  estradiol-173 In  to  should  i n t o DNA,  synthesis  (Kaye which  which  RNA  a _ l . 1'968).  quantitatively different  protein molecules  incorporation  treatment  a  estrogen  thymidine  and  due  s e l e c t i v e gene  Estradiol-173  DNA  occurs  et  cytoplasm,(Means  polyribosomes  lation  (Hahn  This  effects  uterine  of  tissues.  Methods  Animals Immature They  were  female  housed  and  rats  (40-50  maintained  g) as  were  used  previously  for  this  described  study. under  Experiment  A.  Materials E s t r a d i o l - 1 7 3 was  o b t a i n e d from Signa Chemicals.  RNA  ( y e a s t , S i g m a C h e m i c a l s ) and s-RNA (A g r a d e , C a l b i o c h e m ) by Dr. B.C. a s RNA  S u n g , D e p t . o f N e u r o l o g i c a l S c i e n c e s , UBC  standards.  L a b o r a t o r i e s was  C a l f thymus DNA  u s e d a s DNA  (TCA)  were used  o b t a i n e d f r o m Mann R e s e a r c h  standards.  o b t a i n e d u s i n g s-RNA and c a l f t h y m u s DNA acid  donated  S t a n d a r d c u r v e s were i n KOH-Trichloroacetic  and HCIO^ r e s p e c t i v e l y .  Methods 1.  S t a n d a r d i z a t i o n o f RNA  Initial leic  and DNA  Extraction  Procedures  e x p e r i m e n t s were p e r f o r m e d t o s t a n d a r d i z e t h e  acid extraction procedures.  e x t r a c t i n g RNA  and DNA  -as d e s c r i b e d b e l o w .  T h i s was  nuc-  a c c o m p l i s h e d by  from i n c r e a s i n g w e i g h t s o f u t e r i n e  tissue  A n i m a l s w e r e s a c r i f i c e d by e x p o s i n g them  t o c a r b o n d i o x i d e i n a s e a l e d j a r and u t e r i n e t i s s u e s w e r e p o o l e d i n i c e c o l d 0.9%  NaCl.  b l o t t e d and w e i g h e d respectively.  Wet  t i s s u e s l i c e s were randomly  i n q u a n t i t i e s o f 40.0,  N u c l e i c a c i d c o n t e n t was  methods o f Schmidt-Thannhauser  f o r 5 minutes. were added.  the  (1955)  ml o f i c e c o l d  u s i n g a t i s s u e homogenizer  To t h e homogenate 7.0 The  e x t r a c t e d by  mg  with  i n A p p e n d i x , F i g . A.  u t e r i w e r e p o o l e d i n 4.0  w a t e r and h o m o g e n i z e d  and 100.0  (1945) and C e r i o t t i  m i n o r m o d i f i c a t i o n s as s u m m a r i z e d Three whole  50.0  selected,  deionized  (ASCO I n d u s t r i e s )  m l o f i c e c o l d 10%  TCA  t u b e s w e r e a l l o w e d t o s t a n d f o r 10 m i n u t e s  on  i c e , c e n t r i f u g e d , and t h e p r e c i p i t a t e r e s u s p e n d e d and w a s h e d w i t h 10 ml o f i c e c o l d 95% e t h a n o l .  Following  centrifugation  48. the  r e s u l t i n g p r e c i p i t a t e was s u s p e n d e d i n 2.0 m l o f I N KOH f o r  16 h o u r s a t 37°C. and  A f t e r a d d i t i o n o f 0.4 m l o f 6N HC1,  DNA w e r e p r e c i p i t a t e d w i t h c o l d 5% TCA.  n a t a n t f r a c t i o n was e s t i m a t e d  by r e a d i n g  RNA  The p r e c i p i t a t e o f DNA  i n 2.5 m l o f 10% H C 1 0  i n the super-  the absorbance a t  260 nm i n a n U n i c a m 800 s p e c t r o p h o t o m e t e r a g a i n s t KOH-TCA b l a n k .  protein  an  appropriate  and p r o t e i n was  suspended  a n d h e a t e d a t 70-80°C f o r 25 m i n u t e s .  4  T h i s m a t e r i a l was c e n t r i f u g e d was m e a s u r e d a t 260 nm u s i n g  and t h e DNA  i n the supernatant  an a p p r o p r i a t e  HCIO^  blank.  S t a n d a r d c u r v e s w i t h y e a s t RNA o r p u r e s-RNA and c a l f  thymus  DNA w e r e u s e d t o d e t e r m i n e t h e n u c l e i c a c i d c o n t e n t o f t h e extracts. 2.  A n a l y s i s o f d a t a was done by S t u d e n t ' s  Time C o u r s e E f f e c t s o f E s t r a d i o l - 1 7 3 S y n t h e s i s by t h e u t e r i n e t i s s u e .  t-test.  on N u c l e i c  Acid  A f t e r s t a n d a r d i z i n g t h e e x t r a c t i o n p r o c e d u r e o f RNA and DNA, DNA  a t i m e c o u r s e e f f e c t o f e s t r a d i o l - 1 7 3 on RNA and synthesis  b y t h e u t e r i n e t i s s u e was s t u d i e d .  d i v i d e d i n t o groups o f three an  R a t s were  a n i m a l s and e a c h a n i m a l was  i n t r a p e r i t o n e a l i n j e c t i o n o f 5 jug o f e s t r a d i o l - 1 7 g  animals received  .  given Control  i n j e c t i o n s o f e q u a l v o l u m e s o f 0.9% NaCI c o n -  t a i n i n g 1% e t h a n o l .  They w e r e s a c r i f i c e d a f t e r 0,6,12,24,4 8  and  72 h o u r s by p l a c i n g them i n a j a r o f c a r b o n d i o x i d e .  and  DNA were', e x t r a c t e d  RNA  from t h e u t e r i n e t i s s u e s t o determine  t h e t i m e when maximum e f f e c t o f e s t r o g e n o n n u c l e i c a c i d  syn-  t h e s i s was m a n i f e s t e d . Results 1.  Standardization  o f RNA a n d DNA E x t r a c t i o n P r o c e d u r e s  Due t o t h e i m p u r i t i e s i n t h e y e a s t RNA  i t was f o u n d  neces-  sary  to standardize  purified  s-RNA.  preparation  The m o l a r  was d e t e r m i n e d buffer,  this  tion  coefficient  this  value,  s-RNA  i n complete  o f 18 O.D.^^Q  i t was p o s s i b l e  the  corresponding  yeast  and  DNA  i n Fig.  are given  approximate  immature (Table of  rats  5).  uterine  was  sessing  o f RNA  concentrations 176.1±2.1,  respectively  estradiol-173  (Table  .  was n o t i c e d  The  effect  showed  increases estrogen  Maximal  48 h o u r s  per uterus  f o r DNA  o f RNA  of  RNA  232+11, 515+9,  4). on N u c l e i c  t o immature synthesis  synthesis  following  540.7+2.5 y g  were  a single  o f RNA  48 a n d 72 h o u r s ,  Acid  rats  was  pos-  seen  after  i n t h e immature r a t injection of  i n the uterine but i tnever  o f e s t r a d i o l - 1 7 ^ o n t h e DNA  a delayed  administration. were  curves  of  tissue  reached  (Fig. 9).  i n net uterine  centrations  obtaining  i n 4 0 . 0 , 5 0 . 0 a n d 1 0 0 . 0 mg  n e t RNA  between  level  absorp-  the concentration  a n d DNA  was a d m i n i s t e r e d  declined  control  After  Standard  ( F i g . 8, T a b l e  The c o n c e n t r a t i o n  uterus  - 1  223.2±1.9,  values  body w e i g h t s  5).  slightly  acetate  7.  estradiol-173.  its  s-RNA  filtrate.  corresponding  equivalent  hours  uterus  RNA  o f 0.1 M  s o l u t i o n had a molar  Time C o u r s e E f f e c t o f E s t r a d i o l - 1 7 3 S y n t h e s i s by t h e u t e r i n e t i s s u e  When  12  X mg  o f s-RNA  1 1 2 . 0 ± 1 0 p.g a n d 2 3 7 ± 1 1 . 0 y g r e s p e c t i v e l y  T h e RNA  respectively;  2.  coefficient  t o determine  content  t i s s u e s were  1 0 2 3 ± 1 5 2 jag  absorption  known s o l u t i o n s o f  b y d i s s o l v i n g i t i n 0.5 mg/ml  pH 5 . 0 .  The  against  noticed  response DNA  which  lasted  d i d not occur  Slight  content  a f t e r 48 h o u r s .  longer.  until  decreases  of the  48 h o u r s  i n uterine A  Maximum  steady  DNA  after con-  decrease  in  t h e RNA/DNA r a t i o n s was n o t e d  the decrease levels.  b e t w e e n 24 a n d 72 h o u r s , b u t  i n RNA/DNA r a t i o s d i d n o t r e t u r n t o c o n t r o l  A l l d a t a was s t a t i s t i c a l l y  a n a l y z e d by s t u d e n t ' s  t-test.'  Discussion The l e v e l s o f n u c l e i c a c i d  i n t h e u t e r u s o f immature  rats  a r e i n agreement w i t h t h e r e s u l t s o b t a i n e d by G o r s k i and Katzellenbogen  (1975).  RNA when e x p r e s s e d lower  i n t e r m s o f c e l l u l a r RNA w e r e  i n t h e u t e r u s o f immature r a t s t h a n  The RNA/DNA r a t i o s corresponds  5.0 i n l i v e r Reports  dose.  i n other  tissues.  i n t h e i m m a t u r e r a t u t e r u s was 0.47 w h i c h  (1975).  T h i s i s i n c o n t r a s t t o r a t i o s o f 2.0  and E - c o l i  respectively.  b y Kaye e t a _ l . (1972) 3  c o r p o r a t i o n o f (Me-H) t h y m i d i n e and  comparatively  t o t h e r a t i o o f 0.4 r e p o r t e d b y G o r s k i a n d  Katzellenbogen and  They r e p o r t e d t h a t c o n c e n t r a t i o n s o f  I n r a t s l e s s than  have r e v e a l e d t h a t t h e i n -  i n t o DNA i s d e p e n d e n t o n a g e  15 d a y s o l d , s i n g l e  injections of  e s t r a d i o l - 1 7 3 d i d n o t r e s u l t i n an i n c r e a s e i n t h e u t e r i n e weight, old  RNA c o n t e n t , o r r a t e o f DNA s y n t h e s i s .  r a t s , o n t h e o t h e r h a n d , showed  t h e r a t e o f RNA a n d DNA s y n t h e s i s .  significant  Twenty d a y increases i n  Amounts o f e s t r a d i o l - 1 7 3  a s l o w a s 50 p g t o 20 d a y o l d r a t s , w e i g h i n g  33 grams  a l s o r e p o r t e d t o i n c r e a s e t h e r a t e o f DNA s y n t h e s i s . t h i s work i n mind immature female  were With  r a t s were employed i n t h i s  study and a r e l a t i v e l y  l a r g e d o s e o f e s t r a d i o l - 1 7 3 (5 ug) was  administered t o e l i c i t  a definite  R e s u l t s from t h i s experiment  response. indicate that theeffects of  e s t r a d i o l - 1 7 3 on t i s s u e growth a r e mediated  by changes i n  uterine  RNA  (1969(b)) content  and  DNA.  reported  as  Hamilton  by  chemical  m e t h o d s was  significant  increase  seven  after  administration  ment  i n the  estradiol-173 the  enzyme  early  RNA  stages  cursor  pool  expressed these  increased estrogen  on of  estrogen  that  of  to  results  agree  from The  et  over  the  12  this  al.  RNA  hours  experi-  increase  in  administration  increased  of  at  with  (1969(b));  a  on  DNA  increases  in cell  following  estrogen  data.  net  of  synthesis  rRNA  content  of et  of  rate  the  of  al.  could  that  the  et  using  tritiated  thymidine  and  found  increases  i n mitotic  indexes  al. who  occurred noticed  administration.  the  RNA  be  sub-  the  response  cell,  to  i.e.  tissue  the  reported after only  were  content.  (1958);  autoradiographic mouse  to  action  ,  Similarly,  of  in  pre-  at  mode o f  ascribed  uterine  the  in  have  requires  of  the  affected  the  species.  (1972),  be  (1975)  greatly  and  during  changes  functions  than  Meuller  hypertrophy  number  and  programming  and  slower  noticed  They  nuclear  the  mRNA  those  cellular  lag  was  i n nature  various  Kaye  time  suggested  experimental  i n the  occur  significant  uterine  observed  Katzellenbogen  have  general  specific  Increases  the  synthesis  They  more  of  and  RNA  dependent  noticed  the  Data  administration  Gorski  activity was  was  following  attributed to  Billing  noticed  findings.  tissue  basis  i s more  by  production  al.  the  times.  estrogens  these  in  and  polymerase.  sizes,  early  increase  estradiol-173 .  with  been  doubts  stantiated  et  of  uterine  has  However,  of  a  i s i n accordance  synthesis  (1968)  though  first  no  al.  that  measured hours,  et  six after  These  Billing that  though  hours, 24  hours  Epifanova  (1966)  techniques,  uterine  epithelium  42-24  hours  following  estrogen  fold  shortening  of the cell  cell  generation  time  stages and  which  DNA The  time  activity  et  al.  occurred  gap between  organelles  (1975)  mRNA n e e d e d  r e s u l t i n g i n 1.5  time.  a t t h e expense  This  reduction  of  o f t h e G-^ a n d  of preparation  f o r DNA  synthesis  respectively. the uptake  o f the polysomes,  thesizing  generation  r e f e r t o t h e time  replication  administration  o f water  particularly  of the cells  t o be t h e t i m e  f o rthe synthesis  rRNA,  has been  required  a n d t h e maximum the protein  a t t r i b u t e d by  f o r the production  o f induced  syn-  protein  Gorski of  ."(I.P.).  Conclusions  Experiment and  DNA  course  extraction  DNA.  RNA  rat  uterus  were  From cluded  ratios  noticed  and 237±21  RNA  estrogen  pattern  after  24 h o u r s  12 h o u r s .  A, B a n d C  administration  metabolism.  water  a t 12 h o u r s  Maximum  o f f o f RNA  female  i t may  be  con-  t o immature  rats  retention  a ts i x  and a c c e l e r a t e d  a t 24 h o u r s .  proliferation cellular  after administration  a levelling  i n t h e immature  o f RNA  ug r e s p e c t i v e l y .  proliferation  indicate cellular  o f RNA  the time  on t h e s y n t h e s i s  of increased  synthesis  and c e l l u l a r  which  indicating DNA  following  increased  gradually  concentrations  112±10  t h e method  t i s s u e s and determine  of estradiol-173  a n d DNA  i s a distinct  metabolism  uterine  to standardize  the r e s u l t s o f Experiments  that  hours,  in  from  of the effect  and  there  C was d e s i g n e d  of  DNA  RNA/DNA  increased proliferation  was  estradiol-173,  production  and an  increase  RNA AND DNA C O N C E N T R A T I O N  Fig.. 7.  (uG/ML)  Standard curves f o r RNA and DNA  (Expt. C)  54.  F i g . 8.  RNA and DNA c o n c e n t r a t i o n i n homogenates o f d i f f e r e n t immature r a t u t e r i n e weights (Expt. C)  500  12  24  48 TIME  Fig.  9.  72  (HOURS)  E f f e c t o f t i m e o n RNA a n d DNA synthesis in rat uterine tissue following estrogen administration (Expt. C)  Table 4 : RNA and DNA c o n c e n t r a t i o n s i n homogenates of d i f f e r e n t immature r a t u t e r i n e weights (Expt.  Wet weight of Uterus (mg)  RNA DNA c o n c e n t r a t i o n s c o n c e n t r a t i o n s RNA/DNA (ug) .  (ug).  40.0  176.1 ± 2.1  232.0 ± 11.0  0.75  50.0  223.3 ± 1.9  515.6 ±  9.03  0.4.3  100.0"  540.7 ± 2.5  1023.6 ± 1.52  0.52  Table  5 : E f f e c t o f t i m e on RNA and DNA s y n t h e s i s by r a t u t e r i n e t i s s u e ( E x p t . C)  Animal Time ( h o u r s ) following administration of e s t r a d i o l - 1 73  RNA concentration (ug/uterus)  DNA concentration (ug/uterus)  112 ± 10  (  a  )  237 ± 21  (  a  )  0. 47  )  117 ± 12  (  a  )  232 ± 20  (  a  )  0. 50  C  )  200 ± 19  (  b  )  253 + 29  (  a  )  0. 79  (  d  )  357 ± 37  (  C  )  271 ± 25  (  a  )  1.30  (  e  )  452 ± 29  (  d  )  402 ± 31  (  c  )  1.12  (  C  )  302 ± 19  (  c  )  309 ± 28  (  b  )  0.97  U t e r i n e Wet Weight (mg)  Number of Animals  Body Weight (g)  9  4 3 + 2.0  25.7 + 3.0  (  a  )  6  9  45 ± 3.0  31.1 ± 5.0  (  b  12  9  44 ± 2.5  44.5 ± 2.5  (  24  9  43 ± 3.0  66.1 ± 3.9  48  9  46 ± 1.5  75.3 ± 4 . 8  72  9  ,44 ± 1.8  C o n t r o l (0)  (a,b,c,d,e)  Means w i t h d i f f e r e n t  ,47.2 ± 6.9  Charcteristics  subscripts are s i g n i f i c a n t l y d i f f e r e n t  RNA/DNA  (P ^ 0.025)  58.  E x p e r i m e n t D : E f f e c t s o f E s t r o g e n s and P h y t o e s t r o g e n s o n t h e I n c o r p o r a t i o n o f 3H U r i d i n e i n t o R N A by t h e U t e r u s - S i x Hour I n V i v o P u l s i n g . Introduction A net synthesis  o f R N A by t h e u t e r u s  i n response to estrogen  a d m i n i s t r a t i o n has been d e m o n s t r a t e d i n t h e p r e v i o u s  experiment.  O t h e r w o r k e r s h a v e a l s o shown t h e i n c o r p o r a t i o n o f v a r i o u s cursors  i n t o d i f f e r e n t t y p e s o f R N A ( H a m i l t o n e t a l . 1968;  Billing  e t a l _ . 1969 (B) ) . However,  isotopic tracer studies  t o m o n i t o r t h e r a t e o f u t e r i n e RNA s y n t h e s i s flicting  results.  Billing  adenosine t o e q u i l i b r a t e w i t h the u t e r i n e adenosine pool  designed  have y i e l d e d  e t a l . (1969(b)) a l l o w e d  pre-  con-  labelled nucleotide  p r i o r t o and f o r a l i m i t e d t i m e a f t e r e s t r a d i o l - 1 7 3  a d m i n i s t r a t i o n , and n o t i c e d  that  incorporation of l a b e l l e d  adenosine i n t o u t e r i n e RNA i n c r e a s e d initial hours.  only  s l i g h t l y during  the  p h a s e o f t h e r e s p o n s e and was n o t s u b s t a n t i a l u n t i l On t h e c o n t r a r y ,  that during  H a m i l t o n e t a l . (1968) h a v e 3  a 10 m i n u t e p u l s e  into uterine nuclear  i n v i v o , 5- H u r i d i n e  5  reported  incorporation  R N A was m a x i m a l 20 m i n u t e s a f t e r  estradiol-173  administration. The e x t e n t not  only  activity of  of isotope  by t h e r a t e o f R N A s y n t h e s i s associated  incorporation.  has  pool  with  i t s nucleotide  In regard  precursors  to the uterus,  specific  at the time  estrogen  a c t i v i t i e s of  treatment various  by i n c r e a s i n g t h e v a s c u l a r i t y ( S z e g o , 1967), o r  permeability sizes  i n t o RNA i s governed  b u t a l s o by t h e  b e e n shown t o i n f l u e n c e t h e s p e c i f i c  RNA p r e c u r s o r s the  incorporation  of precursors  (Mueller  ( B i l l i n g cat al.  e t a l . 1958).  Gorski  1 9 6 9 ( b ) ) and  e t a l . (1975)  have  drawn  attention  experiments Katzman  the  difficulties  involving precursor  (1971)  poration  to  employing  preceded  by  an  an  i n drawing  conclusions  incorporation.  in vitro  in vivo  system  Munns  of  and  precursor  administration  of  from  incor-  estradiol-173  14 reported of  that  methylated  advantage  of  nucleotide  labelled  in vivo  et  a l . 1975). due  to  clear  rRNA).  useful  The  precursor  apparent  on  fluctuating  sizes. have  in vitro et  a  i t s independence  not  al.  been have  assays (1972)  encountered employed  (Kaye  et  favored  incorporation  vitro  the  the In  Katzellenbogen  review cut  The  of  the  evidence  into  RNA  and  methods  of  early  and  with  thymidine  tritiated  a l _ . 1972;  the  use  better  of  thymidine Carter  in  recovery  of  tritiated  following of  estrogenic to  data  phytoestrogens  nucleoside,  the in  uridine.  increases  in  the  vitro rates  of  or  various  comparative inducing  the  was  phytoestrogen A,  B  of  existed. to  study into  in RNA  administration. and  C  estrogens,  Experiment  e f f e c t s of  steroid  incorporation  an  that  incorporation  incorporation  i n Experiments  from  workers  treatment  experiments  uridine  estrogen  obtained  activity  study  various  following  degree  concluded  by  estradiol-173  the  (1975)  employed  to  of  view  Gorski  due  purpose  uterus  initiated and  and  greater  . .  precursors  the  and  was  H-thymidine.  extensive  by  was  pool  Kaye  Recently  no  methionine  Numerous w o r k e r s  both  systems  label  differences  DNA.  C]  ( i . e . tRNA  1  precursor  in  3  RNA s this  Similar  of  L-[methyl-  of  regarding D  was  estrogens  the  isotopic  60. Materials  and  Methods  Animals Immature study. were  rats  In addition  also  tained  female  used  in a  (4 0-50  to these  g)  were  animals  two  few e x p e r i m e n t s .  as d e s c r i b e d  i n Experiment  mostly  used  groups  in  this  of rats  They were  housed  (60 g) and main-  A.  Materials 3 [5,6obtained defined It  was  uridine  from  (specific  Amersham  biological  (Sigma).  activity,  Searle.  m e d i u m was  supplemented  albumin from  H]  4 4 . 5 Ci/;m m o l e ..was  TC medium  199,  o b t a i n e d from  with  1.0  mM  glutamine  Yeast  RNA  and c a l f  Difco  Laboratories.  (Difco)  thymus  S i g m a c h e m i c a l s a n d Mann R e s e a r c h  chemically  Lab.  DNA  and  2%  were  bovine  obtained  respectively.  Methods Administration Immature steroid  rats  were  given  e s t r o g e n s and p h y t o e s t r o g e n s  Experiment test  female  of Estrogens  A.  Control  vehicles,  animals  alcohol-saline  i n vivo  single  injections  i n doses  received  mentioned  similar  or propylene  of the in  volumes o f the  glycol.  The  incor-  3 poration  of  H - u r i d i n e by  rat uteri  was  determined  as  described  below. _3 Incorporation in vitro Animals of  cold  The 0.9%  connective  uterine NaCI.  tissue  [5,6  sacrificed  e s t r o g e n s by p l a c i n g  dioxide. ice  were  of  them  H]  s i x hours in a  tissues Tissues  and p l a c e d  uridine  were  after  sealed  were  by  the uterus  the  administration  j a r containing  removed  carbon  and t r a n s f e r r e d  stripped  of adhering  i n a prewarmed  (37°C) f i v e  to  f a t and ml  stoppered  b o t t l e c o n t a i n i n g 1.0 m l o f TCM 199 a n d a p p r o x i m a t e l y  3  1.6 juCi o f  H-uridine.  Uterxne  a t m o s p h e r e o f 95%0 -5%CC> 2  a shaking  2  t i s s u e s were i n c u b a t e d under an  f o r 1.0 h o u r i n a w a t e r b a t h  (37°C) a t  s p e e d o f one s t r o k e p e r s e c o n d .  A f t e r t h e i n c u b a t i o n t h e m e d i a was removed a n d t h e u t e r i p l a c e d on d r y i c e t o t e r m i n a t e t h e r e a c t i o n . t i s s u e s were r i n s e d t h r e e t i m e s  Whole u t e r i n e  successively with i c e cold  d i s t i l l e d w a t e r t o remove t h e e x t r a c e l l u l a r  radioactivity.  T h i s f r a c t i o n r e p r e s e n t i n g t i s s u e wash was p o o l e d taken  f o r determining  the r a d i o a c t i v i t y .  w e r e a d d e d t o 10 m l o f PCS s c i n t i l l a t i o n and  counted  o n an I s o c a p  (Nuclear Chicago). as d e t e r m i n e d  300 l i q u i d  a n d an a l i q u o t  Duplicate fluid  aliquots  (Amersham  scintillation  Searle)  counter  The e f f i c i e n c y o f t h e c o u n t i n g was 3 8 - 4 1 %  by t h e c h a n n e l  ratio  method.  b l o t t e d d r y , w e i g h e d a n d s t o r e d a t 20°C u n t i l  T i s s u e s were RNA a n d DNA  e x t r a c t i o n p r o c e d u r e s were c a r r i e d o u t . D e t e r m i n a t i o n o f S p e c i f i c A c t i v i t y o f RNA in Uterine Tissues Three u t e r i from each group were p o o l e d genates were p r e p a r e d  and t i s s u e  homo-  as d e s c r i b e d i n Experiment C (Appendix F i g .  The h o m o g e n a t e s w e r e f i r s t washed w i t h 7.0 m l o f c o l d 1 0 % TCA. The r e s u l t i n g p r e c i p i t a t e was r e s u s p e n d e d i n 7.0 m l o f c o l d 5% TCA and t h e two w a s h e s p o o l e d .  D u p l i c a t e a l i q u o t s o f t h e TCA  a c i d s o l u b l e f r a c t i o n w e r e a d d e d t o 10 m l o f PCS a n d c o u n t e d i n the l i q u i d  scintillation  counter.  u n d e r t h e s e c o n d i t i o n s was 3 3 . 5 % .  The e f f i c i e n c y o f c o u n t i n g The r a d i o a c t i v i t y  i n this  f r a c t i o n r e p r e s e n t s t h e e x t e n t o f p r e c u r s o r uptake which has n o t y e t b e e n i n c o r p o r a t e d i n t o RNA. by  Student's  t-test.  A n a y l s i s o f d a t a was done  Following precipitate to  remove  the c o l l e c t i o n  was  resuspended  the greater  This  was  allowed  3500  RPM  on a desk  supernatant  no  neutralized  with  cold  TCA.  Under these  form  of mononucleotides  to  HC1  as the h y d r o l y z e d  duplicate  50  The  ditions  28-33%.  used  was  The  the solutions  RNA  will  RNA  taken  remaining  resulting  determination  which  washes  counting  by  fractions  levels.  were  be p r e s e n t  portion  at  precipitated  for liquid  of tritium  o f t h e RNA  fraction.  beyond background  fraction.  The  ethanol  centrifuged  were  the  will were  with  i n the be  referred  pooled  and  scintillation under  these  of the fraction  conwas  spectrophotometry. was  expressed  a s DPM  per  RNA. DNA  on  activity  then  i n the supernatant  were  95%  minutes.  a n d DNA  conditions  efficiency  for quantitative  Specific ug  and p r o t e i n  u l aliquots  counting.  for five  hydrolysis,  RNA  fraction,  of the f a t soluble  radioactivity  alkaline  soluble  of i c e cold  f o r 10 m i n u t e s  centrifuge  showed  Following  i n 10 m l  portion  to stand  of the acid  extracted  i n hot perchloric  the spectrophotometer  isotope  radioactivity.  background The checked  was  recorded  efficiency by  expressing  adding  acid  and a l i q u o t s No  radioactivity  in this  also  taken  quantitated  to  determine  significantly  above  fraction.  of the overall  extraction  the r a d i o a c t i v i t y  i t as a p e r c e n t  were  was  procedure  i n individual  of total  was  fractions  radioactivity  and  administered.  Results The 85-90%  extraction  recovery  procedure  used  of the i s o t o p i c a l l y  i n Experiment labelled  D accounted f o r  uridine  nucleoside.  In  general  fractions controls the  RNA  was  less  (Table  case of  estrone RNA  rats  unit  treated  of  of  had  i n the  to  s h o w no  be  rats.  of  treated  difference  of  0.05)  lower  (Table  rats.  Estrone  from the  In phytoestrogen t r e a t e d  in  the  the  control.  incorporation In  the  control of  i n DES, treated  of  rats  incorporation  RNA  and  testos-  i n the  uteri  (P<0.05)  Similar trends  were observed  the  expressed  significantly  7).  in  testosterone  the  specific activity  (Table  i n the  7).  i n the  was  was  in  estradiol-17g  estradiol-17a  estradiol-17g  RNA  of  uteri  rats  lower  f r a c t i o n was  lower than  The  control  activity  estradiol-17a  to  and  than  control  (P<  the  radioactivity in  higher  RNA  in  uridine  exception  uteri  control  estriol  with  only  the  weight the  ones.  treated  specific  of  significantly  DES,  treated  lower than  The  RNA  than  w h o l e u t e r u s was  slightly  that  wet  l a b e l appeared  rats  7).  r a d i o a c t i v i t y i n the  uterine  with  from the  r a d i o a c t i v i t y by  resembled  rats  treated  terone  tritiated  of  animals where the  l a b e l than those of  the  incorporation  (Table  treated  the  When t h e  of  animals  The  rats  hydrolyzed  estrogen treated  f r a c t i o n was  uptake of  treated  the  i n the  6).  treated  hydrolyzed  per  mean r a d i o a c t i v i t y i n t h e  f r a c t i o n obtained  estrogen  The  the  in  estriol animals  the  and appeared  controls. groups  (Table 7),  coumestrol  and  _3 genistein hydrolyzed  treated RNA  treated  with  similar  to  influence  rats  incorporated  f r a c t i o n per estradiol-17g,  control  and  uterus estriol  testosterone  [5,6 at  H]  uridine  l e v e l s higher  and  DES,  treated  of  the  weaker phytoestrogens,  biochanin-A,  the  incorporation  and  at  groups.  formononetin  into than  the those  rates Under and  -3 rate  of  [5,6  H]  uridine  into  the  the  hydrolyzed  the  steroid estrogens.  in  RNA  weight,  formononetin  induced  a lesser  The  and c o n t r o l  specific  phytoestrogen trol  and t e s t o s t e r o n e  specific  activity  and  coumestrol  incorporation  statistically  promote uptake  with  treated  significant  estriol  lower  i n their  than  fraction i n  than  than  as compared  (P<0..05). to  Genistein  stimulate  Biochanin  the control  i n this  i n the conthe low  rats  ability  o f t h e l a b e l and resembled  DES a n d e s t r a d i o l - 1 7 $  biochanin  of uridine  RNA  lower  of the isotopic precursor.  formononetin were a l s o  expressed  genistein,  animals. P a r t i c u l a r l y  i n genistein  resembled  with  groups.  of the hydrolyzed  treated  was  incorporation  r a t s was g e n e r a l l y  o f RNA  t h e c o n t r o l was  coumestrol,  treated  activity  treated  observed  When t h e r a d i o a c t i v i t y  uterine  testosterone  to  similar to that  terms o f u n i t  and  to  f r a c t i o n was  A and  i n their  ability  the trends  observed  respect.  Discussion Experiment ferent  D was u n d e r t a k e n  estrogens  precursor  i n enhancing  by t h e r a t u t e r i n e  Experiment A that after  estrogen  this  experiment  fore  greater  supply  to allow  tissue.  Though one would  Having  this  time  of the c e l l  to find  dif-  established i n  interval  s i x hours  was c h o s e n i n  i m b i b i t i o n and  membrane t o a n  there-  exogenous  nucleoside. have expected  an i n c r e a s e  o f RNA a f t e r t h e a d m i n i s t r a t i o n  surprising  of  o f an i s o t o p i c  imbibition occurred  f o rmaximal water  permeability  of the labelled  activity  the incorporation  maximum w a t e r  administration  t o compare t h e a b i l i t y  that  both  i n the specific  of estrogens  i t was  t h e s t e r o i d and phytoestrogens d i d  in  fact  being  reduce  incorporated  through panded The  an u r i d i n e  specific  size  and l e s s  the control  version  a n d owes  o f RNA  estradiol-173 monal  i n rats  of labelled  estradiol-173,  estriol  Katzman  found  short  (1971)  periods  uterus  To  noticeable  treated  with  i n the cas  The  i n estrone  solely  1969).  absence  treated  rats  weak  t o i t s con-  The h i g h e r  estradiol-17a  i n d i c a t e s the importance  a n d Emmens  corporation  the  t o reduce the  i t i s a comparatively  (Terenius,  s i x hours  to the  and e s t r i o l .  activity  et a l  specifi  than  of configuration  on  hor-  effects. Miller  of  that  noticed  sufficient  o f RNA  its biological  to estradiol-173  activity  s o i n DES  i s ex-  by M u e l l e r  be a s c r i b e d  pool  pass  of which  o f RNA  i s particularly  activity  suggests  may  Before  u r i d i n e must  as suggested  of estrogens  This  s i x hours.  the size  activity  of the nucleotide  activity.  after RNA,  pool,  of estrogens  i n the specific  estrogen  the uterine  i n the specific  estradiol-173  change  activity  nucleotide  administration  increased  over  into  by t h e a c t i o n  reduction  after  of  the specific  estrogens uptake  pulsing  I f the duration  whether  studies  Comparable uridine  exposure  the uptake  and l a b e l l e d  uptake  were  an i n c r e a s e  i n t h e mouse  and estrone  attempted  Munns a n d i n vivo  even f o r  of tritiated  uridine  by t h e  between  was  later  r e s u l t s on t h e e f f e c t by t h e r a t u t e r u s  i n -  to estrogen  the  too long  by t h e u t e r i n e  or not this  i n the  following  treatment.  u r i d i n e was  of radioactivity  determine  observed  uridine  that  enhanced  i n vitro.  (1967)  true,  administration a decrease i n  t i s s u e s was short  noticed.  i n vivo  on i n E x p e r i m e n t  E.  of phytoestrogens  are not available  on  i n the  literature.  As a l r e a d y i n d i c a t e d the s o l u b i l i t y of phyto-  estrogens i n v a r i o u s s o l v e n t s should be c o n s i d e r e d w h i l e a s s e s s i n g t h e i r e s t r o g e n i c potency. ienced i n d i s s o l v i n g  In t h i s study d i f f i c u l t y was  exper-  coumestrol.  The low s p e c i f i c a c t i v i t y of RNA  i n the u t e r i of r a t s  t r e a t e d w i t h the phytoestrogens, b i o c h a n i n A and formononetin i s of i n t e r e s t i n the l i g h t of t h e i r r e l a t i v e l y poor a b i l i t y t o i n c r e a s e water i m b i b i t i o n as shown i n Experiment t i c u l a r the s p e c i f i c a c t i v i t y of RNA b i o c h a n i n A and formononetin was  A.  In par-  i n r a t s treated with  even lower than i n those  t r e a t e d w i t h e s t r a d i o l - 1 7 3 and DES,  two potent e s t r o g e n s .  How-  ever, r e l a t i v e l y h e a v i e r (60 g) r a t s were i n c l u d e d i n the group t r e a t e d w i t h b i o c h a n i n A and formononetin. i n c r e a s e d u t e r i n e weight and RNA these animals may  On the b a s i s of the  content i t i s p o s s i b l e t h a t  have entered the e a r l y phase o f the e s t r o u s  c y c l e and the c i r c u l a t i n g endogenous estrogens may  have supple-  mented the a c t i o n of the phytoestrogens. Conclusions T h i s experiment  was  i n i t i a t e d t o study the r e l a t i v e  of e s t r o g e n i c s t e r o i d s and phytoestrogens  i n terms of t h e i r  a b i l i t y i n v i t r o to s t i m u l a t e i n c o r p o r a t i o n of l a b e l l e d i n the RNA  effects  uridine  e x t r a c t e d from r a t u t e r i s i x hours a f t e r a d m i n i s t e r i n g  v a r i o u s estrogens i n v i v o . t a i n e d i n t h i s experiment  Contrary t o e x p e c t a t i o n s data showed t h a t estrogen t r e a t e d  had a lower s p e c i f i c a c t i v i t y of RNA  a d m i n i s t r a t i o n was  uteri  than c o n t r o l t i s s u e s .  i s p o s s i b l e t h a t the p e r i o d of s i x hours  ob-  f o l l o w i n g estrogen  too l o n g f o r the i n v i t r o e f f e c t s t o be  It  manifested. increased trends order  The  low s p e c i f i c a c t i v i t y  size of the uterine  nucleotide  i n the s p e c i f i c a c t i v i t y o f DES  £ estriol  ( 1 . 0 ;jg) a n d g e n i s t e i n  (P<  0.05)  and  ( P < 0.1)  o f RNA  < estradiol-17a  's  may  ( 0 . 8 mg)  also  be due t o t h e  pool.  Decreasing  were n o t i c e d  i n the  < estrone.  Estradiol-  produced  a significant  respectively decreasing  e f f e c t on t h e  -3 incorporation fraction. treated  of  However,  r a t s was  Both g e n i s t e i n capacity In  rats  activity the  [5,6  H]  uridine  into the hydrolyzed  the s p e c i f i c a c t i v i t y  not s i g n i f i c a n t l y  and c o u m e s t r o l  lower  resembled  t o promote the i n c o r p o r a t i o n treated o f RNA  with  biochanin  estrogenic  very  of the animal  p o t e n t i a l of these  than  i n coumestrol  control  estriol  animals.  i n their  of labelled uridine.  A or formononetin the s p e c i f i c  i n t h e u t e r u s was  physiological state  o f RNA  RNA  l o w w h i c h may rather  phytoestrogens.  than  be due t o  to the  true  Table 6 : The i n v i t r o i n c o r p o r a t i o n of r a d i o a c t i v i t y uterl~7 s i x hours f o l l o w i n g i n v i v o estrogen  Distribution Treatment  Groups of animals (n)  i n d i f f e r e n t f r a c t i o n s of r a t a d m i n i s t r a t i o n (Expt. D)  of r a d i o a c t i v i t y  Medium and t i s s u e wash  Cold TCA soluble fraction  i n different  fractions  Hydrolyzed RNA fraction  (DPM)  Recovery of administered l a b e l (%)  Control  4  2 893886  Estrone  1  2859439  283670  93,771  89. 5  Estriol  1  2917317  245712  55,199  88.9  DES  1  2946 730  284026  58,259  90.9  Estradiol-17ct  1  2829438  2 8.0 0 9 8  78,138  88.1  Estradiol-17g  4  2810321  Testosterone  1  2872281  Genistein  3  2848505  ±  Coumestrol  2  2 811235  ±. 6 4 , 2 4 9  1  2919000  144268  67,419  1  2924519  11697.3  43,351  Biochanin  A  Formononetin  +  ±  107,990  100,010  20 4 5 3 1  285316  ±  ±  26,205  18520  220133 215,441  89,146  54,299  ±  ±  14,780  88.1  87.7  10,270  90,477  ±  4.1  ±  3.5  ±  6.5  88.0  164578  ±  12,158  78,527  +  10,290  85.4  183639  ±  35,663  87,871  ±  2,54 3  85.2 86.5 .  85.3  Table  7 : RNA c o n t e n t a n d s p e c i f i c a c t i v i t y o f RNA e x t r a c t e d f r o m r a t u t e r i s i x h o u r s f o l l o w i n g e s t r o g e n a d m i n i s t r a t i o n ( E x p t . D)  RNA Mean Body Weight (g)  Treatment  ug  RNA  uterus  Radioactivity i n hydrolyzed  content ug mg  uterus  uterus  ± 0.2 29715 ± 4928  DPM  DPM  DPM  RNA  RNA  ug  mg u t e r i n e w e i g h t 933.9 ± 1.5  6.9 ± 1.2  Control  42.0  ± 8.3 134.0  ± 7 . 1 4.3  Estrone  41.0  134.8  3.9  31257  930. 3  6.9  Estriol  39. 4  133  3.9  18399  541.2  4.0  DES  46. 8  143  3.8  19419  516.5  3.6  Estradiol-17 g  38.3 ± 5.1 143.2 ± 3.2 4.1 ± 0.4 17659 ± 1583  Estradiol-17 a  49.1  161.0  5.2  26659  851.2  5.2  Testosterone  35.6  109.0  3.5  30159  985.4  9.0  Genistein  50.3 ± 3.7 156.5 ± 4.7 4.2 ± 0.1 26916 ± 1400  Coumestrol  50.3  Rinrhanin  A  ± 4 . 7 141.5 ± 0.9 3.5 ± 0.3 29306 ± 1498  59. 3  196. 8  4.4  59 7  176. 5  3.9  ((a,b,c,d)  . .  501.7 ± 4419  725.7 ± 106 741.9 ±. 3.7.9  ( a )  ( b )  ( c )  ( C )  RNA  3.5 ± 0.3  ( a )  ( C )  4.6 ± 0.7  ( b )  5.2 ± 0.3  ( a )  22473  510.7  2.5  144 50  580.3  3.2  Means w i t h d i f f e r e n t s u b s c r i p t s a r e s i g n i f i c a n t l y d i f f e r e n t (P < 0.05).  70.  E x p e r i m e n t E : E f f e c t o f E s t r o g e n s and P h y t o e s t r o g e n s on t h e I n c o r p o r a t i o n o f T r i t i a t e d U r i d i n e I n t o RNA - S h o r t i n v i v o P u l s i n g  Introduction The  very  material  of phytoestrogens i n plant  and t h e i r weak e s t r o g e n i c  difficult further  low c o n c e n t r a t i o n  p o t e n c y h a v e made i t  t o study t h e i r b i o l o g i c a l  complicated  effects.  by the d i f f i c u l t i e s  The problem i s  associated with  e x t r a c t i o n p r o c e d u r e s w h i c h do n o t p e r m i t q u a n t i t a t i v e of  the plant  estrogens.  suitable bioassays estrogenic  be employed t o t e s t  p o t e n c y and t o p r o v i d e  a c t i o n h a s b e e n hampered.  activity  their  k n o w l e d g e on t h e i r mode o f  Recently  techniques,  measure t h e b i o l o g i c a l  the i n t r o d u c t i o n o f competi-  w h i c h a r e s e n s i t i v e enough t o of very  small  quantities of  p h y t o e s t r o g e n s h a s p a r t l y overcome t h e l i m i t a t i o n s o f The  sensitivity  increased  recovery  Consequently t h e development o f  which could  tive protein binding  various  of bioassay  p r o c e d u r e s has a l s o been  bioassays. greatly  by t h e development o f t e c h n i q u e s i n which t h e i n c o r -  poration  of a l a b e l l e d precursor  in vitro  i s determined q u a n t i t a t i v e l y a f t e r the a d m i n i s t r a t i o n  of e s t r o g e n i c The vitro  by u t e r i n e  cellular  organelles  compounds i n v i v o .  r e s u l t s o f E x p e r i m e n t D have i n d i c a t e d t h a t  administration  of t r i t i a t e d  the i n  uridine to the uterine  tissue  s i x hours a f t e r i n j e c t i o n o f e s t r o g e n s i n v i v o has n o t r e s u l t e d in  large  that  incorporation  t h e s i x hour d u r a t i o n  e s t r o g e n s was t o o l o n g the  of the l a b e l  tissues.  as e x p e c t e d .  after i n vivo  I t i s possible  administration of  t o observe t h e e a r l y e f f e c t s produced i n  Miller occurred 173  (1964) r e p o r t e d  that reduction of tetrazolium  salts  as e a r l y as 2 8 minutes a f t e r i n j e c t i o n o f e s t r a d i o l -  and suggested t h a t t h i s s h o r t d u r a t i o n i s c r i t i c a l i n  observing  the e a r l y e f f e c t s o f estrogens.  Shutt  (1967)  employed a s i m i l a r technique as an index o f the b i o l o g i c a l e f f e c t of g e n i s t e i n .  Subsequently i s o t o p i c p r e c u r s o r s  have  been w i d e l y used w i t h d i f f e r e n t degrees of success i n s t u d y i n g the e a r l y a c t i o n o f estrogen. t h a t the extent  (1971) observed  of incorporation of l a b e l l e d precursors  when the l e v e l was administered estradiol-173.  Munns and Katzman  was high  30 minutes a f t e r i n j e c t i o n o f  In the l i g h t o f these experiments t h i s  experi-  ment was undertaken t o study the very e a r l y e f f e c t s o c c u r r i n g i n the u t e r i n e c e l l a f t e r a b r i e f exposure to d i f f e r e n t e s t r o gens using  the b a s i c technique of Munns and Katzman  Phytoestrogens were f i r s t e x t r a c t e d  (1971).  from p l a n t m a t e r i a l s .  The  e a r l y e f f e c t s which these e x t r a c t s produced on the u t e r i n e t i s s u e were then compared w i t h those caused by e s t r a d i o l - 1 7 3 . Estrogenic  a c t i v i t y o f c e r t a i n p l a n t m a t e r i a l s has been  a t t r i b u t e d t o the presence of n o n s t e r o i d a l capable of competing w i t h e s t r a d i o l - 1 7 3 s i t e s l o c a t e d i n the u t e r i n e c y t o s o l .  phytoestrogens  for specific  binding  T h i s study presents the  r e s u l t s o f q u a l i t a t i v e and q u a n i t a t i v e d e t e r m i n a t i o n s o f v a r i o u s phytoestrogens and t h e i r a b i l i t y f o r the u t e r i n e  binding  protein. M a t e r i a l s and Methods Materials F i r s t c u t t i n g s o f orchard  grass hay (Dactylus  glomerata)  and a l f a l f a hay Research  (Medicago s a t i v a ) were o b t a i n e d from A g a s s i z  Station.  Buckerfields.  [2,4,6,7 (n)  96 Ci/mmole) was was f rom  Soyabean  ( G l y c i n e max)  meal was  obtained  H ] estradiol-173 (specific  o b t a i n e d from Amersham/Searle.  o b t a i n e d from Pharmacia Chemicals  from  activity,  Dextran  T-40  and a c t i v a t e d c h a r c o a l  S igma.  Methods E x t r a c t i o n and I d e n t i f i c a t i o n of Phytoestrogens  Phytoestrogens  were e x t r a c t e d from 2.0  g samples of oven  d r i e d ground o r c h a r d grass hay, a l f a l f a hay and soyabean meal by t r e a t i n g them s u c c e s s i v e l y w i t h a b s o l u t e a l c o h o l and f r e e ether was  ( F r a n c i s and M i l l i n g t o n 1965).  The  peroxide-  f i n a l extract  concentrated to dryness under a stream o f n i t r o g e n and  a b s o l u t e a l c o h o l was u n t i l ready f o r use.  added.  Samples were then s t o r e d a t -2 0°C  The e f f i c i e n c y o f e x t r a c t i o n was  checked  by adding known c o n c e n t r a t i o n s of g e n i s t e i n t o those samples and by c a l c u l a t i n g the per cent recovery. Q u a l i t a t i v e d e t e r m i n a t i o n of phytoestrogens was d i r e c t i o n a l t h i n l a y e r chromatography u s i n g G-25 plates  (D.C. F e r t i g p l a t t e n - M a c h e r a y - N a g e l Co.).  done on  s i l i c a gel The p l a t e s  were a c t i v a t e d a t 100°C f o r one hour p r i o r to use and 10-20 a l i q u o t s of p l a n t e x t r a c t s were p l a c e d on each p l a t e . m i c r o l i t e r a l i q u o t s of formononetin,  ul  Fifty  g e n i s t e i n , coumestrol  biochanin-A d i s s o l v e d i n a l c o h o l were used as standards. d e v e l o p i n g s o l v e n t systems used were  two  and The  chloroform-methanol  (91:9,V/V) i n the f i r s t d i r e c t i o n and ammonia s a t u r a t e d c h l o r o form-methanol  (91:9 ,V/V) i n the second. T  allowed to dry a t room temperature  The p l a t e s were  f o r approximately  15 minutes  b e f o r e running them i n the second d i r e c t i o n .  Developed  chroma-  tograms were observed under u l t r a v i o l e t l i g h t f o r the d e t e c t i o n of formononetin,  d a i d z e i n and coumestrol.  I d e n t i f i c a t i o n of  these compounds was made by comparing t h e i r Rf v a l u e s w i t h standards and by t h e i r v i s u a l c h a r a c t e r i s t i c s as seen under ultraviolet light.  Areas r e p r e s e n t i n g the phytoestrogens were  c i r c l e d w i t h a p e n c i l and the p l a t e s t r a n s f e r r e d t o a fume hood where they were sprayed w i t h a s o l u t i o n o f c o l d 1.0N a c i d c o n t a i n i n g 10%Na CO 2  3  and 4.5% NaN0  2  sulphanillic  f o r the d e t e c t i o n of  the n o n f l u o r e s c e n t compounds, g e n i s t e i n , biochanin-A and e q u o l . The former two compounds were i d e n t i f i e d by comparing t h e i r Rf v a l u e s w i t h known standards. Q u a n t i t a t i v e Determination of Phytoestrogens The c o m p e t i t i v e p r o t e i n b i n d i n g assay o f Korenman(1968) was employed t o determine quantitatively.  the c o n c e n t r a t i o n o f phytoestrogens  U t e r i n e c y t o s o l was o b t a i n e d from a s i x day  pregnant r a b b i t and homogenized a t 4°C i n t h r e e volumes of T r i s buffer  (pH,4.0 W/V)  u s i n g a Waring blender  (Sorvall).  The homo-  genate was c e n t r i f u g e d a t 7000 g f o r 15 minutes a t 0°C. natant f r a c t i o n was removed and r e c e n t r i f u g e d  (Beckman, u l t r a -  c e n t r i f u g e L5-65) a t 100,000 g f o r 90 minutes a t 0°C. a t e l y f o l l o w i n g u l t r a c e n t r i f u g a t i o n the second  The super-  Immedi-  supernatant  fraction  c o n t a i n i n g the c y t o s o l p r o t e i n s was c o l l e c t e d by Pasteur p i p e t t e and s t o r e d i n l i q u i d n i t r o g e n u n t i l ready f o r use. Standard curves f o r c o m p e t i t i v e p r o t e i n b i n d i n g assays were obtained u s i n g d i f f e r e n t c o n c e n t r a t i o n s of p u r i f i e d genistein.  A l i q u o t s o f i n c r e a s i n g c o n c e n t r a t i o n of p u r i f i e d  g e n i s t e i n were added t o t e s t tubes and d r i e d under a stream o f nitrogen.  To each sample were added s u c c e s s i v e l y 100 LII of  T r i s b u f f e r , pH 8.0, 50 u l o f ^ H - e s t r a d i o l - l V ^ a n d 100 u l o f rabbit uterine cytosol.  The t u b e s w e r e m i x e d a f t e r t h e  a d d i t i o n o f t h e u t e r i n e c y t o s o l , samples were a l l o w e d t o s t a n d at  room t e m p e r a t u r e  f o r 30 m i n u t e s a t t h e e n d o f w h i c h  of  d e x t r a n c o a t e d c h a r c o a l was a d d e d t o e a c h t u b e .  1.0 m l  The  tubes  w e r e t h e n m i x e d , k e p t a t 4°C f o r 15 m i n u t e s a n d c e n t r i f u g e d o n a desk c e n t r i g u g e .  The s u p e r n a t a n t was c o l l e c t e d w i t h a  P a s t e u r p i p e t t e a n d an a l i q u o t t a k e n f o r t h e d e t e r m i n a t i o n o f radioactivity.  Based on t h e c o m p e t i t i o n f o r b i n d i n g  sites  between e s t r a d i o l - 1 7 3 and t h e added g e n i s t e i n t h e r a d i o a c t i v i t y in  the supernatant w i l l  be d i r e c t l y p r o p o r t i o n a l t o t h e added  genistein. P l a n t e x t r a c t s were d r i e d i n a stream concentration of t o t a l phytoestrogens t i t i v e p r o t e i n b i n d i n g assay of  o f n i t r o g e n and t h e  was m e a s u r e d b y t h e compe-  and t h e v a l u e s e x p r e s s e d  i n terms  estrogenic a f f i n i t y of genistein. Results Q u a l i t a t i v e a n a l y s i s o f o r c h a r d g r a s s h a y e x t r a c t s on two  d i m e n s i o n a l t h i n l a y e r c h r o m a t o g r a p h y d i s c l o s e d no spots corresponding t o phytoestrogens. f i e d phytoestrogens  standards  identifiable  The R f v a l u e s o f p u r i -  are given i n Table  8.  In the  a l f a l f a hay e x t r a c t s spots c o r r e s p o n d i n g t o c o u m e s t r o l , n o n e t i n and g e n i s t e i n were o b s e r v e d  (Table 8 ) .  s t a n d a r d s o f e q u o l w e r e u s e d a weak s p o t to  formo-  Although  no  corresponding  t h e R f v a l u e o f e q u o l was n o t i c e d i n t h e a l f a l f a  extract.  Both g e n i s t e i n and d a i d z e i n were p r e s e n t i n t h e soyabean extract The  (Fig. 10). standard curve f o r the competitive p r o t e i n b i n d i n g  assay o f p u r i f i e d g e n i s t e i n i s shown i n F i g . 11.  When known  q u a n t i t i e s o f g e n i s t e i n was added t o o r c h a r d grass hay, the e x t r a c t i o n procedure gave a recovery o f 59% as determined by the competitive p r o t e i n binding The hay  concentration  assay.  o f phytoestrogens i n the o r c h a r d grass  e x t r a c t was very low and e q u i v a l e n t  u n i t s per 2.0 g o f the sample. extracts  t o 1.5-2.0 ug g e n i s t e i n  A l f a l f a hay and soyabean meal  c o n t a i n e d much l a r g e r c o n c e n t r a t i o n s of phytoestrogens  equivalent  t o 70 and 126 rig r e s p e c t i v e l y o f g e n i s t e i n  units.  Discussion The  presence o f e s t r o g e n i c  a c t i v i t y i n forages has been  reviewed by many r e s e a r c h e r s working i n t h i s f i e l d al.  1957; Kohler and B i c k o f f , 1961).  Beck e t a l . (1964) reported the  i s o l a t i o n and subsequent i d e n t i f i c a t i o n o f v a r i o u s gens by t h i n l a y e r chromatography. currently believed  to contribute  (Bickoff et  The p l a n t  to estrogenic  plant  estro-  constituents a c t i o n are the  isoflavongs, g e n i s t e i n , biochanin-A, f o r m o n o n e t i n ^ d a i d z e i n and equol as w e l l as the coumarin d e r i v a t i v e , coumestrol.  The  i s o l a t i o n and i d e n t i f i c a t i o n o f coumestrol, formononetin and g e n i s t e i n i n the a l f a l f a e x t r a c t i n t h i s study support the f i n d i n g s o f K o h l e r and B i c k o f f  (1961) who r e p o r t e d  s t r o l accounts f o r the m a j o r i t y  o f the b i o l o g i c a l  a l f a l f a w h i l e the i s o f l a v o n e s activity.  are responsible  t h a t coumeactivity i n  f o r the remaining  The i d e n t i f i c a t i o n o f g e n i s t e i n i n soyabean meal  e x t r a c t a l s o agrees w i t h Cheng e t a l . (1953(b) who r e p o r t e d  high  l e v e l s o f g e n i s t e i n i n soyabean meal. Phytoestrogens have s i n c e been r e p o r t e d  t o b i n d on s p e c i f i c  uterine proteins  (Shutt and Cox, 1972) and a c t i n v a r i o u s  of c e l l metabolism  (Noteboom and G o r s k i , 1963Xa)•  I t was a l s o evident was present  that s u f f i c i e n t estrogenic  activity  i n v a r i o u s p l a n t e x t r a c t s s t r o n g enough t o d i s p l a c e  estradiol-173  from u t e r i n e b i n d i n g p r o t e i n s .  Though no e v i -  dence f o r the presence o f phytoestrogens i n orchard could be d e t e c t e d very  areas  g r a s s hay  by the TLC method, i t was p o s s i b l e t o d e t e c t  small q u a n t i t i e s o f phytoestrogen a c t i v i t y when the s e n s i -  t i v e competitive  protein binding  assay method was used.  l o g i c a l t o expect t h a t f o l l o w i n g the i n i t i a l b i n d i n g  It is  o f the  phytoestrogen t o u t e r i n e c y t o s o l p r o t e i n s a d d i t i o n a l e s t r o g e n mediated responses would occur s e q u e n t i a l l y l a t e r . t h i s h y p o t h e s i s i t was decided  To t e s t  t o undertake f u r t h e r  studies  i n v o l v i n g the i n v i t r o i n c o r p o r a t i o n of l a b e l n u c l e o s i d e  by the  u t e r i n e t i s s u e a f t e r a b r i e f i n v i v o exposure t o estrogens. Conclusions In Experiment E the q u a n t i t a t i v e and q u a l i t a t i v e determinat i o n of the phytoestrogen content o f v a r i o u s  forages was made.  Q u a l i t a t i v e s t u d i e s o f the a l f a l f a e x t r a c t r e v e a l e d the presence o f coumestrol, g e n i s t e i n and formononetin.  From  v i s u a l observations  extracts,  o f the chromatograms from a l f a l f a  the i s o f l a v o n e s appeared t o be i n a much s m a l l e r q u a n t i t y coumestrol.  than  In t h e soyabean e x t r a c t , g e n i s t e i n and d a i d z e i n  were i s o l a t e d and although no standards f o r d a i d z e i n were a v a i l a b l e , i d e n t i f i c a t i o n c o u l d be made from i t s c h a r a c t e r i s t i c fluorescence  under u l t r a v i o l e t l i g h t .  According  t o the v i s u a l  observations  made from the soyabean meal chromatograms,  g e n i s t e i n appeared t o be i n g r e a t e r c o n c e n t r a t i o n than d a i d z e i n . Q u a l i t a t i v e examination  o f the o r c h a r d g r a s s hay e x t r a c t d i d not  d i s c l o s e any spot corresponding  t o phytoestrogens.  Q u a n t i t a t i v e l y , e s t r o g e n i c a c t i v i t y was observed  i n the  g e n i s t e i n s p i k e d hay and a l f a l f a hay and soyabean meal e x t r a c t s . A very s m a l l amount of e s t r o g e n i c a c t i v i t y of  (1.5-2.0 ug) per 2.0g  sample was recorded i n the o r c h a r d grass hay e x t r a c t .  hay and soyabean meal e x t r a c t s possessed activity  Alfalfa  a s u b s t a n t i a l amount o f  of 70 ug and 126 ug r e s p e c t i v e l y .  78.  FORMONONETIN  Q  o o  EQUOL  GENISTEIN  COUMESTROL  ALFALFA  HAY  GENISTEIN  (MEDICAGO  SATIVA)  DAIDZEIN  o  COUMESTROL  SOYBEAN  Fig.  10.  MEAL  (GLYCINE  MAX)  Q u a l i t a t i v e examination o f phytoestrogen c o n t e n t by t h i n l a y e r chromatography A = Chloroform-methanol  (91:9 v / v )  B = Ammonia S a t u r a t e d C h l o r o f o r m - m e t h a n o l (91  Table  Sample U s e d Standard Alfalfa  Hay  Soybean Meal Orchard G r a s s Hay  Coumestrol  8 : R f v a l u e s o f s t a n d a r d p h y t o e s t r o g e n s and t h o s e o b t a i n e d from p l a n t e x t r a c t s as m e a s u r e d by T h i n L a y e r C h r o m a t o g r a p h y ( E x p t . E)  Genistein  Formononetin  0.41  (0.85)  0.58  (0.91)  0.74  (0.74)  0.47  (0.87)  0.56  (0.94)  0.70  (0.82)  0.51  (0.83)  0.63  (0.89)  -  -  -  - Figures o u t s i d e brackets correspond - Figures w i t h i n brackets correspond  Daidzein  Equol  -  0.7 6 0.62  0.54  Biochanin A  (0.53)  (0.79)  -  (0.14)  -  -  -  -  -  t o Rf v a l u e s i n f i r s t  solvent  t o Rf v a l u e s i n second s o l v e n t  80 .  Experiment F : E a r l y E f f e c t s o f Estrogens and P l a n t E x t r a c t s on the I n c o r p o r a t i o n o f T r i t i a t e d U r i d i n e Into RNA by U t e r i n e T i s s u e . Introduction In the l i g h t o f . q u a n t i t a t i v e and q u a l i t a t i v e  determination  of phytoestrogen a c t i v i t y i n p l a n t e x t r a c t s made i n Experiment E f u r t h e r e f f o r t s were made t o o b t a i n i n f o r m a t i o n of a c t i o n of e s t r o g e n i c for binding  compounds.  on the mode  Based on the competition  s i t e s between e s t r a d i o l - 1 7 3 and the v a r i o u s  phyto-  estrogens i t was f e l t t h a t a d d i t i o n a l experiments designed s p e c i f i c a l l y t o study the uptake o f a r a d i o a c t i v e  nucleoside  i n t o the RNA f r a c t i o n by u t e r i n e t i s s u e s would be i n f o r m a t i v e i n d e f i n i n g t h e mode o f a c t i o n of e s t r o g e n i c plant extracts.  compounds i n v a r i o u s  In Experiment D the s i x hour d u r a t i o n between  the i n v i v o a d m i n i s t r a t i o n o f estrogen and i n v i t r o uptake of l a b e l l e d nucleoside  by the u t e r i n e t i s s u e r e s u l t e d i n a lower  s p e c i f i c a c t i v i t y o f RNA. the d u r a t i o n before  Therefore  i t was decided  f o l l o w i n g estrogen a d m i n i s t r a t i o n  t o shorten  t o 30 minutes  undertaking i n v i t r o uptake s t u d i e s . M a t e r i a l s and Methods  Animals Immature female rats-(33-43 g) were used i n t h i s They were housed and maintained as d e s c r i b e d  study.  i n Experiment A.  Materials P l a n t e x t r a c t s o f orchard  grass hay, a l f a l f a hay and soya-  bean meal were prepared as d e s c r i b e d  i n Experiment E.  Addi-  81. t i o n a l m a t e r i a l s r e q u i r e d f o r i n v i t r o s t u d i e s were the same as d e s c r i b e d i n Experiment  D.  Methods A d m i n i s t r a t i o n of Estrogens i n v i v o Immature female r a t s were g i v e n s i n g l e i n j e c t i o n s of e s t r a d i o l - 1 7 3, g e n i s t e i n , a l f a l f a hay  e x t r a c t and  e x t r a c t i n doses of 1.0  14.5  tively.  6 0.0  ug,  E s t r a d i o l - 1 7 3 , g e n i s t e i n and  administered A l f a l f a hay of 50%  ug,  i n t r a p e r i t o n e a l l y i n 0.2 e x t r a c t was  propylene g l y c o l .  volumes of 1% e t h a n o l  administered  soyabean meal  ug and  40.0  }ig respec-  soyabean meal e x t r a c t ml of aqueous 1%  ethanol.  i n t r a p e r i t o n e a l l y i n 0.2  ml  C o n t r o l animals r e c e i v e d s i m i l a r  in saline. _3  I n c o r p o r a t i o n of [5,6 H] U r i d i n e Uterus In V i t r o  By  Animals were s a c r i f i c e d 30 minutes a f t e r the  administration  of estrogens by p l a c i n g them i n a s e a l e d j a r c o n t a i n i n g  CG^.  The whole u t e r i were removed w i t h i n one minute, f r e e d o f f a t and connective  t i s s u e and p l a c e d i n i c e c o l d i s o t o n i c s a l i n e .  Two  to three groups per treatment c o n t a i n i n g three whole u t e r i group were g e n t l y b l o t t e d and t r a n s f e r r e d t o prewarmed 5 ml 3  i n c u b a t i o n v i a l s c o n t a i n i n g 2.5  H-uridine f o r 1.0  and  1.0  mM  glutamine.  ml of TCM  hour a t 37°C i n a water bath at a shaking  A f t e r i n c u b a t i o n the medium was 0.5  ml of 0.6  N HCIO^ was  10.0  U t e r i n e t i s s u e s were  s t r o k e per second under an atmosphere of 95%  and  199,  uCi of incubated one  CG^.  removed by Pasteur  added to terminate  (37°C)  speed of  02~5%  per  pipette  the r e a c t i o n .  Whole u t e r i n e t i s s u e s were r i n s e d w i t h i c e c o l d d i s t i l l e d water  82. t o remove e x t r a c e l l u l a r r a d i o a c t i v i t y . representing  This f r a c t i o n  t i s s u e wash was p o o l e d a n d p r o c e s s e d a s  i n E x p e r i m e n t D.  Tissues  described  were b l o t t e d , weighed and s t o r e d a t  -20°C u n t i l RNA a n d DNA e x t r a c t i o n s w e r e c a r r i e d o u t . radioactivity described  i n the c e l l u l a r  u n d e r E x p e r i m e n t D.  The  f r a c t i o n was d e t e r m i n e d a s Recovery r a t e s of the isotope  w e r e a l s o made t o d e t e r m i n e t h e e f f i c i e n c y o f t h e e x t r a c t i o n procedure. students'  A l l r e s u l t s were a n a l y s e d  s t a t i s t i c a l l y by t h e  t-test. Results  The  p e r c e n t o f t o t a l r a d i o a c t i v i t y w h i c h was r e c o v e r e d i n  t h e d i f f e r e n t f r a c t i o n s amounted t o 7 9 - 9 1 % was a s l i g h t l y g r e a t e r the hydrolyzed  (Table  RNA f r a c t i o n i n u t e r i o f e s t r a d i o l - 1 7 3  i n c r e a s e was h o w e v e r n o t s i g n i f i c a n t students'  There  incorporation of t r i t i a t e d uridine i n  estrogen t r e a t e d r a t s than i n c o n t r o l animals.  the  9).  t-test.  This  and p h y t o apparent  (P < 0.05) when a n a l y z e d  The r a d i o a c t i v i t y  i n the hydrolyzed  f r a c t i o n when e x p r e s s e d p e r u n i t o f u t e r i n e w e t w e i g h t was ficantly  (P  <  0.05) h i g h e r  i n the u t e r i of estradiol-173  g e n i s t e i n treated r a t s than c o n t r o l s  (Table  and  soyabean meal e x t r a c t s a l s o i n c r e a s e d  the  l a b e l l e d precursor  i n t o the hydrolyzed  by  10).  RNA signi-  ahd p u r e  A l f a l f a hay  the incorporation of RNA f r a c t i o n p e r u n i t  o f u t e r i n e w e t w e i g h t , h o w e v e r t h i s i n c r e a s e was n o t s i g n i f i c a n t . The  s p e c i f i c a c t i v i t y o f RNA i n t h e e s t r a d i o l - 1 7 3  e s t r o g e n t r e a t e d r a t u t e r i was s i g n i f i c a n t l y than the controls also significantly  (Table  10).  and p h y t o -  (P < 0.05) g r e a t e r  The a l f a l f a a n d s o y a b e a n  (P < 0.05) i n c r e a s e d  extracts  the s p e c i f i c a c t i v i t y of  RNA i n t h e i m m a t u r e f e m a l e r a t u t e r i when c o m p a r e d t o c o n t r o l  83.  animals  (Table 10) . Discussion  Having e s t a b l i s h e d i n Experiment E t h a t d i f f e r e n t phytoestrogens were present  i n a l f a l f a hay and soyabean, and t h a t  they were capable o f b i n d i n g onto s p e c i f i c u t e r i n e p r o t e i n s , Experiment F was i n i t i a t e d t o determine whether or not they were potent  enough t o enhance the e a r l y i n c o r p o r a t i o n of l a b e l l e d  u r i d i n e i n t o RNA o f u t e r i n e t i s s u e s .  To determine t h i s  short  i n v i v o p u l s i n g o f the e s t r o g e n i c compound i n q u e s t i o n was employed. The  i n c r e a s e d uptake o f the l a b e l l e d u r i d i n e observed i n  t h i s experiment i s i n agreement w i t h t h a t r e p o r t e d by Munns and Katzman (19 71).  I t i s p a r t i c u l a r l y noteworthy t h a t the e s t r a -  d i o l - 1 7 3 or phytoestrogen t r e a t e d r a t u t e r i had a h i g h e r  specific  a c t i v i t y o f RNA u n l i k e i n Experiment D where they had a lower s p e c i f i c a c t i v i t y than the c o n t r o l s in  [5, 6  (Tables 6 and 9 ) .  Increases  H] u r i d i n e i n c o r p o r a t i o n i n t o RNA during the e a r l y  phase o f estrogen  and phytoestrogen a c t i o n may be a t t r i b u t e d t o  the i n c r e a s e d p e r m e a b i l i t y o f the u t e r i n e c e l l membrane. F u r t h e r , during the r e l a t i v e l y long d u r a t i o n a f t e r i n v i v o e s t r a d i o l - 1 7 3 a d m i n i s t r a t i o n i n Experiment D, t h e r e may be an i n c r e a s e d i n f l u x o f endogenous supply  o f n u c l e o s i d e s which would  cause a r e d u c t i o n i n the u t e r i n e s p e c i f i c a c t i v i t y as suggested by Munns and Katzman (1971).  Hence the d u r a t i o n time o f 30  minutes chosen f o r i n v i v o p u l s i n g with e s t r a d i o l - 1 7 3 and v a r i o u s phytoestrogens i n Experiment F appeared t o r e s u l t i n higher  s p e c i f i c a c t i v i t i e s o f RNA.  84. The  doses o f v a r i o u s  estrogenic  compounds a d m i n i s t e r e d  chosen on t h e b a s i s o f t h e e s t r o g e n i c  activity  competitive  Despite  protein binding  assays.  t h e s e compounds w e r e a d m i n i s t e r e d  radioactive precursor.  determined by the fact that  i n different quantities this  d i d n o t appear t o i n f l u e n c e t h e s p e c i f i c porated  were  a c t i v i t y of the incor-  When t h e p h y t o e s t r o g e n  activity  o f t h e e x t r a c t s was e x p r e s s e d i n t e r m s o f g e n i s t e i n u n i t s , alfalfa  and soyabean e x t r a c t s  were s i m i l a r  ( 1 4 . 5 u g a n d 40.0 u g r e s p e c t i v e l y )  to estradiol-176  in their ability  (1.0 ug) a n d g e n i s t e i n (60.0 ug)  t o enhance i n c o r p o r a t i o n o f t r i t i a t e d u r i d i n e .  E s t r a d i o l - 1 7 3 has been r e p o r t e d DNA d e p e n d e n t RNA p o l y m e r a s e enzyme  to increase the a c t i v i t y of ( M a u l a n d H a m i l t o n 196 7 ) .  A l t h o u g h no e x p e r i m e n t s w e r e done t o d e t e r m i n e w h e t h e r o r n o t v a r i o u s phytoestrogens a r e capable o f i n c r e a s i n g polymerase activity,  i t seems r e a s o n a b l e  that the greater uridine  t i o n observed i n the hydrolyzed greater  induced  RNA f r a c t i o n may b e due t o  a c t i v i t y o f t h i s enzyme.  required before  conclusions  polymerase  incorpora-  More e x p e r i m e n t a l  c a n be made r e g a r d i n g  data i s  phytoestrogen  activity.  Based on t h e e f f e c t s on e a r l y events o c c u r r i n g i n c e l l u l a r m e t a b o l i s m due t o a s i n g l e i n j e c t i o n o f p h y t o e s t r o g e n s  obtained  from a s m a l l q u a n t i t y o f p l a n t m a t e r i a l i t i s i n t e r e s t i n g t o speculate grazing  t h e magnitude o f e f f e c t s w h i c h c o u l d be p r o d u c e d i n  animals i n j e s t i n g  large quantities of  phytoestrogens.  Conclusions I n E x p e r i m e n t F t h e o b j e c t i v e was t o e x a m i n e t h e e s t r o g e n i c p o t e n t i a l of d i f f e r e n t forages  by e s t a b l i s h i n g a b i o a s s a y  useful  85. in  studying  an e s t r o g e n i n d u c e d r e s p o n s e , namely t h e  tion of a specific  isotopic  incorpora-  precursor.  E x p o s u r e o f u t e r i n e t i s s u e t o e s t r o g e n s f o r 30 m i n u t e s i n v i v o was and  phytoestrogens.  were o b t a i n e d in  e f f e c t s of estradiol-17 3  s u f f i c i e n t t o study the i n i t i a l  S i g n i f i c a n t l y greater  (P < 0.05)  differences  b e t w e e n e s t r o g e n t r e a t e d r a t s and c o n t r o l  animals  regard  to incorporation of t r i t i a t e d  u r i d i n e i n t o the hydro-  l y z e d RNA  f r a c t i o n of the u t e r i n e c e l l .  I t i s probable that  shorter pulsing period quantities  and e x p o s u r e o f u t e r i n e t i s s u e s t o l a r g e r  (10 u C i ) o f i s o t o p i c p r e c u r s o r  Experiment D enabled the i n i t i a l  t h a n were used i n  e f f e c t s o f e s t r a d i o l - 1 7 3 and  p h y t o e s t r o g e n s t o be o b s e r v e d . More e x p e r i m e n t a l  a  data regarding  t h e s p e c i f i c mode o f  a c t i o n o f p h y t o e s t r o g e n s on u t e r i n e c e l l increased  polymerase a c t i v i t y )  regarding  p h y t o e s t r o g e n m e d i a t e d RNA  metabolism ( i . e .  are required  before  synthesis  conclusions  c a n be made.  11.  Competitive Binding Assay P h y t o e s t r o g e n s ( E x p t . E)  of  Increasing concentrations of g e n i s t e i n w e r e a d d e d t o 100 u l t r i s buffer; 50 u l H E s t r a d i o l - 1 7 B and 100 r i l p r e g n a n t r a b b i t u t e r i n e c y t o s o l to determine the G e n i s t e i n Standard Curve. 3  4 6  S E M I - L O G A R I T H M I C 4 CYCLES  X  7 0 DIVISIONS  KEUFFEL  a ESSER  6 0 1 0  86a.  H»0£ IK U . S . A .  CO.  Ul  GENISTEIN ADDED TO ASSAY TUBE (NG)  CD N CO 13 O  Table 9 : The i n v i t r o i n c o r p o r a t i o n o f r a d i o a c t i v i t y i n d i f f e r e n t f r a c t i o n s of r a t u t e r i , 30 minutes f o l l o w i n g i n v i v o e s t r o g e n a d m i n i s t r a t i o n (Expt. F)  Distribution  Treatment  Groups of Animals  Medium and t i s s u e wash  of r a d i o a c t i v i t y  i n different  fractions  Cold TCA soluble f r a c t i o n  Hydrolyzed RNA f r a c t i o n  (DPM)  Recovery o f administered label %  Control  2  22692616 ± 66,578  2248490 ± 179538  929984 ± 82521  91 ± 2%  Estradiol-17 3  2  22158070 ± 321857  2507680 ± 194458  1186117 ± 22805  91 * 1%  Genistein  3  18844267 ± 232915  2672025 ± 508445  1033235 ± 17193  79 i 1%  A l f a l f a hay extract  2  19836720 ± 105723  2351821 ± 51510  1178381 ± 32684  82 ~ 1%  Soyabean meal extract  2  20929731 ± 148367  2484358 ± 313876  1128354 ± 31858  86 i 3%  Table 10 : I n c o r p o r a t i o n of [5,6 H] u r i d i n e i n t o the hydrolyzed RNA f r a c t i o n of immature female r a t u t e r i 30 minutes f o l l o w i n g estrogen a d m i n i s t r a t i o n  R a d i o a c t i v i t y i n hydrolyzed  Treatment  Groups of Animals (n)  DPM  uterus  DPM  RNA DPM  mg u t e r i n e weight  ug RNA  Control  2  309,994 ± 27,507  8,732 ± 774  ( a )  69.3 ± 6.2  ( a )  Estradiol-173  2  395:,372 ± 10,750  11,526 ± 221  ( b )  92.5 ± 1.4  ( b )  89.2 ± 6.2  ( b )  91.1 ± 2.5  ( b )  87.8 ± 2.5  ( b )  -  Genistein  3  329,411 ± 17,477  11,124 ± 802  ( b )  Alfalfa  2  392,793 ± 10,894  11,385 ± 315  ( a )  2  376,118 ± 10,619  11,062 ± 220  ( a )  extract  Soyabean e x t r a c t  (a,b)  Means w i t h d i f f e r e n t s u b s c r i p t s are s i g n i f i c a n t l y d i f f e r e n t (P < 0.05)  89 .  GENERAL CONCLU SIONS E s t r a d i o l - 1 7 3, the major c i r c u l a t i n g estrogen of mammalian  s p e c i e s was chosen as the standard  estrogens were compared.  i n majority  w i t h which  other  I n i t i a l experiments i n v o l v i n g dose  l e v e l s and s e q u e n t i a l time i n t e r v a l s f o l l o w i n g e s t r a d i o l - 1 7 3 a d m i n i s t r a t i o n were designed t o e s t a b l i s h b a s e l i n e regarding  values  t a r g e t organ metabolism. .  S i x hours f o l l o w i n g the i n t r a p e r i t o n e a l a d m i n i s t r a t i o n single i n j e c t i o n of estradiol-173 was observed.  The  ability  of a  (1.0 ug) maximal t i s s u e edema  o f other  steroid,  s y n t h e t i c and  p l a n t estrogens t o produce s i m i l a r water i m b i b i t i o n was a l s o studied.  E s t r i o l and DES were e q u i v a l e n t  i n d u c i n g water i m b i b i t i o n .  to estradiol-173 i n  The p l a n t estrogens, g e n i s t e i n and  coumestrol were capable of i n d u c i n g -3  tissue  edema however they  were only 10  times as potent as e s t r a d i o l - 1 7 3 i n doing so.  Administration  of e s t r a d i o l - 1 7 3, g e n i s t e i n and coumestrol were  shown to enhance u t e r i n e v a s c u l a r p e r m e a b i l i t y their administration  s i x hours a f t e r  as i n d i c a t e d by I n d i a i n k p e r f u s i o n  experi-  ments . Studies  on the metabolism of RNA and DNA were c a r r i e d out  by experiments designed t o monitor the s y n t h e s i s o f n u c l e i c acids following estradiol-17 3 administration.  I t was found t h a t  e s t r a d i o l - 1 7 3 s i g n i f i c a n t l y shortened the u t e r i n e c e l l c y c l e . Net  accumulation o f RNA and DNA was n o t i c e d t o occur 12 and 2 4  hours r e s p e c t i v e l y f o l l o w i n g the a d m i n i s t r a t i o n o f e s t r a d i o l - 1 7 3 . I t was f e l t t h a t these s e r i e s o f data would be u s e f u l i n future i s o t o p i c precursor nucleoside  experiments.  The f i r s t l a b e l l e d  experiment employed the data obtained  from  earlier  90.  experiments i n v o l v i n g a s i x hour estradiol-17 3 or i t s equivalent  (iri v i v o )  p u l s i n g p e r i o d of  f o l l o w e d by a one hour i n v i t r o  exposure t o a l a b e l l e d p r e c u r s o r .  The e s t r o g e n  t r e a t e d groups d i s p l a y e d lower s p e c i f i c a c t i v i t i e s than c o n t r o l groups.  With e s t r a d i o l - 1 7 3 having the lowest s p e c i f i c  i t was f e l t t h a t the data a c t u a l l y s i g n i f i e d those  activity,  compounds  which possessed the g r e a t e s t a f f i n i t y t o a l t e r the c e l l membrane and by doing so r e s u l t e d i n t h e d i l u t i o n o f the t r a c e r by  s i m i l a r endogenous p r e c u r s o r s .  s p e c i f i c a c t i v i t y , the g r e a t e r stronger  the estrogen.  Therefore  compound  the lower the  the d i l u t i o n o f the t r a c e r and t h e  E s t r a d i o l - 1 7 3 s i g n i f i c a n t l y reduced t h e  s p e c i f i c a c t i v i t y of a l a b e l l e d precursor.  Plant  g e n i s t e i n and coumestrol a l s o showed r e d u c t i o n s a c t i v i t i e s o f the l a b e l l e d p r e c u r s o r , trend noticed with e s t r i o l .  estrogens,  i n the s p e c i f i c  and were s i m i l a r t o the  The p h y s i o l o g i c a l s t a t u s o f the  animals was seen to be an important f a c t o r as the r e s u l t s o f b i o chanin-A and formononetin were m i s l e a d i n g  due t o the suspected  onset o f e s t r u s o f these t r e a t e d animals. The  e a r l y a c t i o n o f e s t r a d i o l - 1 7 3, g e n i s t e i n and v a r i o u s  p l a n t e x t r a c t s was s t u d i e d i n the f i n a l experiment aimed a t l o o k i n g a t the comparative a b i l i t y of each t o i n c o r p o r a t e an i s o t o p i c precursor. tions of estrogenic  Q u a n t i t a t i v e and q u a l i t a t i v e determinaa c t i v i t y o f the p l a n t e x t r a c t s were made  p r i o r t o employing a s h o r t in_ v i v o p u l s i n g method used t o prevent the d i l u t i o n o f the r a d i o a c t i v e t r a c e r .  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E n d o c r i n o l . Copeh. 1 1 : 6 1 - 7 9 , 1 9 5 2 . W i l l i a m s , D. a n d G o r s k i , J . A New A s s e s s m e n t o f S u b c e l l u l a r D i s t r i b u t i o n o f Bound E s t r o g e n i n t h e U t e r u s . Biochem. B i o p h y s . R e s . Comm. 45:258 : 264, 1 9 7 1 . Wong, E. a n d F l u x , D.S. The E s t r o g e n i c A c t i v i t y o f Red C l o v e r I s o f l a v o n e s a n d Some o f T h e i r D e g r e d a t i o n P r o d u c t s . J . E n d o c r i n o l . 24:341:348, 1962. Wyss, R.H., K a r s z n i a , R., H e i n r i c h s , W.L. a n d Hermann, W.L. I n h i b i t i o n o f Uterine Receptor Binding o f E s t r a d i o l - 1 7 3 by a n t i e s t r o g e n s . J . C l i n . E n d o c r i n o l . 28:1824-1828, 1968.  102 .  APPENDIX  103. Three. Whole U t e r i n e T i s s u e s - H o m o g e n i z e i n 2.0 m l i c e c o l d d e m i n e r a l i z e d water f o r approx. 5 minutes - a d d 7.0 m l i c e c o l d 1 0 % TCA - s t a n d 10 m i n u t e s  and c e n t r i f u g e Pellet  Supernatant  r e s u s p e n d i n 95% ETOH a t 0°C  - TCA, a c i d S o l u b l e Fraction  Centrifuge Supernatant  Pellet - a d d 2.0 m l 1 N KOH - s t a n d f o r 16 h r s . , 37°C - a d d 0.4 m l 6 N HC1 - a d d 5% TCA a t 0°C - mix and p l a c e i n i c e f o r 10 m i n u t e s -  centrifuge Supernatant  Pellet add  2.5 m l 1 0 % HCIO4  h e a t t o 70-85°C f o r 25 m i n ,  H y d r o l y z e d RNA  Fraction  - r e a d a t 260 nM  C o o l - s t a n d f o r 10 m i n . Centrifuge Supernatant  Pellet Protein - hydrolyze i n 2.0 m l NaOH  Appendix  F i g . A.  - DNA F r a c t i o n - read a t 260 nM  Nucleic Acid Extraction  Procedure  104. 2.0  Oven D r i e d Sample.of P l a n t M a t e r i a l s 1. g r i n d , add 3.0 ml 0.01 stand 20 minutes 2. add  3.0  ml Abs.  M  HCI  ethanol  Reflux 10 minutes Decant  L i q u i d Phase 1  Residue Reflux 10 ml ETOH Decant  L i q u i d Phase 2  Residue - discard  - p o o l phases 1 & 2 - reduce volume - filter  i n t o e x t r a c t i o n tubes  Filtrate  Residue  - add 15 ml D i s t i l l e d L i g r o i n e Ether - s i t 10 min. a t 45°  - discard  angle  - suction o f f ether - add 10 ml peroxide f r e e e t h y l ether - shake 20 min. Freeze i n Dry  Ice  L i q u i d Phase 1  Solid  - c o l l e c t e t h e r and evaporate under N (40°)  - add 10 ml peroxide f r e e e t h y l ether Refreeze  2  L i q u i d phase 2  S o l i d phase  - add to L i q u i d phase 1 - evaporate  under  N  2  add 0.5 ml Absolute E t h a n o l wait 20 min. and s t o r e at 4°C.  Appendix F i g . B.  E x t r a c t i o n of Phytoestrogens From P l a n t M a t e r i a l s  - discard  70-  Appendix  F i g . C.  Quench C o r r e c t i o n Tritium  Curve  for  

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