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Oxidative metabolism of estrogens Lewis, Joyce 1965

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i  THE OXIDATIVE METABOLISM OF ESTROGENS by  B.Sc,  JOYCE LEWIS The U n i v e r s i t y o f B r i t i s h Columbia, 1963.  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Biochemistry  We a c c e p t t h i s t h e s i s as conforming r e q u i r e d standard:  t o the  THE UNIVERSITY OF BRITISH COLUMBIA October, 1965  In p r e s e n t i n g the  this  Columbia,  available for mission  I agree that  for extensive  representatives.,  cation  of this  fulfilment of  Department o f  s h a l l make i t f r e e l y  I f u r t h e r agree that  copying o f t h i s  thesis  for  f o rf i n a n c i a l  that  scholarly  gain  copying o r p u b l i -  shall  permission.  f>\^^<AJI^/^>'L,^  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, C a n a d a  DU^V^LJ^.  per-  by t h e Head o f my D e p a r t m e n t o r by  I t i s understood  thesis  w i t h o u t my w r i t t e n  te  the Library  reference and.study.  p u r p o s e s may be g r a n t e d  D a  in partial  r e q u i r e m e n t s f o r an advanced d e g r e e a t t h e U n i v e r s i t y o f  British  his  thesis  Columbia  3  Ad  f<f/oSr-  n o t be a l l o w e d  ABSTRACT T h i s p r o j e c t was  undertaken to o b t a i n more i n f o r m a t i o n  the n a t u r e of the w a t e r - s o l u b l e  metabolites  about  formed by r a t l i v e r  s i n c e these had been shown to d i f f e r from the normal conjugates found i n other and  s p e c i e s and are i n f l u e n c e d by the sex of the animal  a l s o by compounds i n the s o l u b l e f r a c t i o n o f the A comparison was  made between these p r o d u c t s and  cell. those formed  from estrogens by mushroom phenolase under s i m i l a r c o n d i t i o n s , and i t was  found t h a t g l u t a t h i o n e had  the same e f f e c t as the' s o l u b l e  f r a c t i o n of the c e l l i n i n c r e a s i n g the y i e l d o f  water-soluble  metabolites  This tripeptide  i n the r a t l i v e r microsomal system.  a l s o competed e f f e c t i v e l y w i t h p r o t e i n - b i n d i n g and p r o p o s a l s account f o r t h i s r e a c t i o n have been put  forward.  A marked i n c r e a s e i n the y i e l d of w a t e r - s o l u b l e d e r i v a t i v e s was  a l s o observed when g l u t a t h i o n e was  estrogen  added to  phenolase system, and a l t h o u g h a d i f f e r e n t mechanism may i n v o l v e d , s i m i l a r types of p r o d u c t s were formed.  A  the  be  competitive  r e a c t i o n f o r p r o t e i n - b i n d i n g i n the presence of g l u t a t h i o n e again demonstrated.  t h i o l h i s t i d i n e had  systems, and  to be s p e c i f i c f o r g l u t a t h i o n e .  t h a t the s u l p h y d r y l group was  l i t t l e or no  effect  the r e a c t i o n t h e r e f o r e appears O x i d i z e d g l u t a t h i o n e was  i n i n c r e a s i n g the y i e l d of w a t e r - s o l u b l e  found to be  was  S u l p h y d r y l compounds such as c y s t e i n e , ergo-  t h i o n i n e , homocysteine and i n e i t h e r of the two  to  ineffective  metabolites i n d i c a t i n g  i n v o l v e d , and N-ethylmaleimide  strongly inhibitory.  was  iii.  The e f f e c t of estrogen o x i d a t i o n products on the i n v i t r o i n a c t i v a t i o n of gonadotropins was  also  investigated.  iv.  TABLE OF CONTENTS PAGE INTRODUCTION.. ...  .. ..  1. ' I n a c t i v a t i o n o f E s t r o g e n s . . . . . . . . . .  1  2. . E t h e r - S o l u b l e M e t a b o l i t e s . . . . . 3.  1  .  Water-Soluble M e t a b o l i t e s . . . . . . . . . . . . . . . . . . . . .  2 5  4. . Conjugates Other Than G l u c o s i d u r o n a t e s and Sulfates e . s . s . eec . eo. . . os . . s Q e. . . . . . o. . . o. o  6  5.  Other A s p e c t s o f E s t r o g e n M e t a b o l i s m . . . . . . . . .  9  6.  I n a c t i v a t i o n o f Pregnant Mare's Serum by  e  Quinones... ..  . • • «..  :  7.  The P r e s e n t  Investigation....'..........«.....12  EXPERIMENTAL..'.  .............  13  IA. M a t e r i a l s . . . . . . . . . . . . . . . . . . • •. • •'•............ e  IB. M e t h o d s  Q  11  « . . . . . . . . . . o . . . . . . . . . . . . . . . . . . . . o . . .  13 18  1.  Preparation of Tissue..  ••»•••.•••••••  18  2.  Preparation of Subcellular Fractions......  18  3.  I n c u b a t i o n and E x t r a c t i o n o f T i s s u e Preparations.........................••...  19  4.  Determination  20  5.  Examination o f the E t h e r e a l F r a c t i o n  21  6.  Examination o f the Aqueous F r a c t i o n . . . . . . .  21  Protein Precipitation.....  22  DxalySXS•oa«s..see»e.e..».e.e.............  22  of R a d i o a c t i v i t y .  V.  PAGE Paper Chromatography..... ....,...  22  Column Chromatography.., .................  23  Electrophoresis.... .'..v..................  24  NiTihydt xn. T6St« * • » e o'  25  1  :  7. RESULTS li  «•••»••«*  Assay of Tyrosinase A c t i v i t y . . . . . . . . . . . . .  25  Inactivation of Pregnant Mare's Serum by Estrogens i n v i t r o  26  .v.v  . .... .........  28  Optimal Conditions for the Formation of Water-Soluble Metabolites from 16-14C estradiol-17p by Mushroom Phenolase....»  28  2. . E f f e c t of Glutathione on the Y i e l d of WaterSoluble Estrogen Derivatives Formed by Rat Liver Microsomes and Phenolase.......... .  29  8  3.  Chromatography and Electrophoresis of the Aqueous F r a c t i o n . . . . . . . . . . . . . . . . . . . . . . . .  4. . Protective Action of Glutathione..... DISCUSSION. . .... ... . .V  .  ••  ......... ... ....  32 35 37  SUMMARY... . V . . . . . . . . . . . . . . . • . • • • ...... •....... BIBLIOGRAPHY.................. .... ....  .v  45 47  vi.  LIST OF TABLES  TABLE I  II  III  PAGE Optimum c o n d i t i o n s f o r the f o r m a t i o n o f w a t e r - s o l u b l e products from e s t r a d i o l - 1 7 p by mushroom phenolase i n the presence o f g l u t a t h i o n e ( t o f a c e ) . . . . .  28  E f f e c t of g l u t a t h i o n e on the s o l u b l e f r a c t i o n o f the l i v e r on the f o r m a t i o n o f w a t e r - s o l u b l e products from 16-l^C e s t r a d i o l - 1 7 p by r a t l i v e r microsomes and on t h e i r i n t e r a c t i o n w i t h p r o t e i n ( t o face)  29  E f f e c t o f g l u t a t h i o n e or the s o l u b l e f r a c t i o n o f the l i v e r on the f o r m a t i o n o f w a t e r - s o l u b l e products from 16-1 C e s t r a d i o l 17p by mushroom phenolase and on t h e i r i n t e r a c t i o n w i t h p r o t e i n ( t o face) 29 4  IV  V  VI  VII  E f f e c t o f o x i d i z e d g l u t a t h i o n e (GSSG) and N-ethylmaleimide (NEM) on the formation o f w a t e r - s o l u b l e products by mushroom phenolase ( t o f a c e ) . , . . . .  30  E f f e c t o f s u l p h y d r y l compounds on the y i e l d of w a t e r - s o l u b l e m e t a b o l i t e s by r a t l i v e r microsomes ( t o f o l l o w )  30  E f f e c t o f s u l p h y d r y l compounds on the y i e l d o f w a t e r - s o l u b l e m e t a b o l i t e s by mushroom phenolase ( t o f o l l o w ) .  30  I n a c t i v a t i o n o f PMS by e s t r o g e n o x i d a t i o n products and p r o t e c t i o n by g l u t a t h i o n e  vii. LIST OF FIGURES  FIGURE  PAGE 20  la.  P u l s e s p e c t r a o f S and H ( t o face)  lb.  The e f f e c t o f time on the f o r m a t i o n o f w a t e r - s o l u b l e p r o d u c t s from 16-l^C e s t r a d i o l - 1 7 p by mushroom phenolase i n the presence and absence o f g l u t a t h i o n e (to f o l l o w )  28  Paper chromatography o f the aqueous f r a c t i o n o b t a i n e d a f t e r i n c u b a t i o n o f v a r i o u s t h i o l s w i t h phenolase and 16-1 C e s t r a d i o l - 1 7 p i n the presence and absence o f NADPH2 ( t o face)  31  Column chromatography o f the aqueous f r a c t i o n o f phenolase and the microsomal system on Sephadex G-25 (to face)  32  Column chromatography o f the aqueous f r a c t i o n demons t r a t i n g c o m p e t i t i o n f o r p r o t e i n b i n d i n g i n the presence o f g l u t a t h i o n e ( t o f o l l o w )  32  Descending paper chromatography o f the aqueous f r a c t i o n s o f v a r i o u s i n c u b a t i o n mixtures c o n s i s t i n g o f 16-14c e s t r a d i o l - 1 7 p and 35s_g^ u t a t ] 1 j_ o n e ^ - b u t a n o l : a c e t i c acidrwater (to f a c e ) . .  33  6.  F r a c t i o n A o f the aqueous f r a c t i o n o f phenolase i n c u b a t i o n on Sephadex G-25 ( t o f o l l o w ) . . . . . . . .  33  7.  F r a c t i o n B o f the aqueous f r a c t i o n o f phenolase i n c u b a t i o n on Sephadex G-25 ( t o f o l l o w )  33  2.  3 5  3  4  3.  4.  5.  n n  8.  9.  - F r a c t i o n A o f the aqueous f r a c t i o n o f the microsomal i n c u b a t i o n on Sephadex G-25 ( t o f a c e ) . . . . .  34  Chromatography o f the aqueous f r a c t i o n o b t a i n e d a f t e r i n c u b a t i o n o f phenolase and 100,000 x g supernatant f r a c t i o n and 16-14c e s t r a d i o l - 1 7 p on Sephadex G-25 (to f o l l o w )  34  viii.  10,  11,  12,  Chromatography o f the aqueous f r a c t i o n o b t a i n e d a f t e r i n c u b a t i o n o f the microsomes and the 100,000 x g supernatant f r a c t i o n , NADPH? and 16-l^C estradiol-1763 on Sephadex G-25 ( t o f o l l o w )  34  E l e c t r o p h o r e s i s of the aqueous f r a c t i o n o b t a i n e d microsomal and phenolase i n c u b a t i o n s (to face)  35  from  . Proposed pathways f o r the f o r m a t i o n of w a t e r - s o l u b l e e s t r o g e n conjugates w i t h " g l u t a t h i o n e " and p r o t e i n by phenolase and r a t l i v e r microsomal enzymes ( t o face)  42  ix.  ACKNOWLEDGEMENTS The author wishes t o acknowledge the h e l p f u l a d v i c e , suggestions and c r i t i c i s m s o f Dr. P.H. J e l l i n c k d u r i n g the course o f t h i s work, . She i s a l s o v e r y g r a t e f u l t o Dr. R.L. Noble f o r the use o f l a b o r a t o r y  facilities.  The a s s i s t a n c e o f f e r e d by Miss T„ Dournovo i n the preparation  and t y p i n g o f t h i s t h e s i s i s a l s o g r a t e f u l l y  acknowledged.  -1INTRODUCTION I n a c t i v a t i o n of Estrogens Ever s i n c e Zondek's o r i g i n a l demonstration l i v e r was  (1) t h a t the  the main s i t e of estrogen i n a c t i v a t i o n , many workers  (2, 3, 4, 5) have s t u d i e d the metabolism  of these compounds, u s i n g  the l o s s of b i o l o g i c a l a c t i v i t y as c r i t e r i o n of hormone  metabolism.  More r e c e n t l y , by means of c o u n t e r - c u r r e n t d i s t r i b u t i o n and  fluoro-  m e t r i c a n a l y s i s , Ryan and E n g e l (6) found t h a t a f t e r i n c u b a t i o n of e s t r o n e and e s t r a d i o l w i t h r a t l i v e r m a t e r i a l was  s l i c e s , over 50% of the  starting  converted i n t o unknown m e t a b o l i t e s .  Very l i t t l e  i s known about the n a t u r e of these  inactivation  products of e s t r o g e n s , but t h e r e i s evidence t h a t e s t r o n e and e s t r a d i o l a r e a t t a c k e d by an o x i d a t i v e enzyme system.  Thus H e l l e r  (2) found t h a t cyanide i n h i b i t e d the i n a c t i v a t i o n of estrogens by l i v e r , s u g g e s t i n g t h a t an oxidase was  involved.  This finding  was  supported by the work o f W e s t e r f e l d (7) and Graubard and P i n c u s who  (8)  found t h a t oxidases from p l a n t sources were capable of i n a c t i v a -  ting estrogens. Levy  ( 5 ) , u s i n g r a t l i v e r homogenates demonstrated  inhibition  by cyanide, a z i d e and carbon monoxide suggesting t h a t a cytochrome system was  involved.  He a l s o observed t h a t the i n a c t i v a t i o n d i d not  proceed under a n a e r o b i c c o n d i t i o n s , and t h i s was De Meio (4) who  found i n h i b i t i o n w i t h cyanide or a z i d e , but none  w i t h malonate, i o d o a c e t a t e or f l u o r i d e . have shown t h a t e s t r a d i o l - 1 7 p was liver  l a t e r confirmed by  Twombly and T a y l o r (9)  i n a c t i v a t e d more s l o w l y by human  than by t h a t of the r a t or mouse; they a l s o examined a s e r i e s  -2of  human carcinoma  t i s s u e s , b u t c o u l d f i n d no r e l a t i o n between  the type o f carcinoma In  and i t s a b i l i t y t o m e t a b o l i z e e s t r a d i o l - 1 7 p .  a l l these experiments, no i n d i c a t i o n was g i v e n as t o the  extent to which the o r i g i n a l s t e r o i d molecule was degraded. However w i t h the more r e c e n t use o f r a d i o i s o t o p i c and chromatog r a p h i c techniques, some i n s i g h t i n t o the nature o f the compounds formed has been gained. The E t h e r - s o l u b l e M e t a b o l i t e s The  i s o l a t i o n o f a p h e n o l i c s t e r o i d p o s s e s s i n g a methoxy  group a t t a c h e d t o the s t e r o i d molecule a t C-2 was f i r s t r e p o r t e d by Kraychy and G a l l a g h e r (10} 11).  Following i n j e c t i o n s of  e s t r a d i o l - 1 7 p t o p a t i e n t s w i t h mammary carcinoma, i s o l a t e d r a d i o a c t i v e methoxyestrone from the u r i n e . was  they  This r e s u l t  l a t e r confirmed by E n g e l (12) who found t h a t these were by  no means minor m e t a b o l i t e s .  Fishman and G a l l a g h e r (13) l a t e r  d i s c o v e r e d 2-methoxyestriol a f t e r a d m i n i s t r a t i o n o f 1 6 - ^ C e s t r a d i o l - 1 7 p w h i l e Loke and M a r r i a n (14) i s o l a t e d 2-methoxyestrone from human pregnancy u r i n e .  I t was then suggested by E n g e l (12) t h a t  m e t h o x y l a t i o n was c a r r i e d out i n two separate stages, f i r s t o x i d a t i o n t o the 2-hydroxy compound and then m e t h y l a t i o n . King (15) has shown the requirement  o f reduced  pyridine  n u c l e o t i d e s f o r the 2-hydroxylase enzyme which i s l o c a t e d i n the microsomal  f r a c t i o n , NADP b e i n g more e f f i c i e n t than NAD. The  p a r t i c i p a t i o n o f a f o l i c a c i d d e r i v a t i v e i n t h i s r e a c t i o n has been i m p l i c a t e d b u t no d i r e c t evidence to support t h i s has been g i v e n . Knuppen e t a l (16) found t h a t S-adenosyl methionine was r e q u i r e d as the methyl group donor when 2-hydroxyestradiol-17§ was converted  -3to the methoxy d e r i v a t i v e by human l i v e r s l i c e s .  This reaction  i s b e l i e v e d to be mediated by an O-methyl t r a n s f e r a s e (17)  similar  to t h a t i s o l a t e d from the s o l u b l e f r a c t i o n o f the r a t l i v e r  by  A x e l r o d and Tomchick (18). The p h y s i o l o g i c a l importance of these C-2 m e t a b o l i t e s has not y e t been determined that methoxylation  s u b s t i t u t e d estrogen  although i t i s b e l i e v e d  serves mainly as a mechanism f o r i n a c t i v a t i n g  estrogens. I n c u b a t i o n o f 1 6 - C estradiol-17j3 w i t h mouse l i v e r microsomes 14  i n the presence  of oxygen and reduced NADP gave r i s e to a number of  r a d i o a c t i v e compounds more p o l a r than e s t r a d i o l 19).  (Mueller and Rumney,  These compounds were i d e n t i f i e d as 6 - o x o e s t r a d i o l , 6'p -hydroxy1  e s t r o n e and 6 '£3 - h y d r o x y e s t r a d i o l - 1 7 p . 1  Breuer  (20) showed t h a t  incubation of r a t l i v e r  s l i c e s w i t h estrone or e s t r a d i o l gave r i s e  to the same compounds.  These r e s u l t s i n d i c a t e d the presence  6a, and  6p hydroxylases  of  i n r a t l i v e r which were capable o f a t t a c k i n g  p h e n o l i c s t e r o i d s as w e l l as 6a and 6p hydroxy s t e r o i d dehydrogenases  (21).  Breuer  (22) has i s o l a t e d the hydroxylases  from the  microsomal f r a c t i o n and demonstrated an a b s o l u t e requirement NADPH2  as c o f a c t o r .  He has a l s o shown t h a t these m e t a b o l i t e s  be formed by human t i s s u e s .  for can  No p h y s i o l o g i c a l s i g n i f i c a n c e has been  a s s i g n e d to these m e t a b o l i t e s , but these r e a c t i o n s r e s u l t i n an i n c r e a s e i n water s o l u b i l i t y o f the p r o d u c t s . I n c u b a t i o n o f estrone w i t h ox a d r e n a l homogenate i n the of c i t r i c a c i d y i e l d e d 11-hydroxylated  estrogens.  Breuer  presence  and  Knuppen (17) have shown t h a t these s t e r o i d d e r i v a t i v e s were r e a d i l y  -4converted t o the 11-oxo compounds by r a t l i v e r  slices.  The c o n v e r s i o n o f estrone and e s t r a d i o l - 1 7 p t o estriol-16e*, 17p i n man was f i r s t demonstrated by Pincus and h i s c o l l e a g u e s (23). Experiments w i t h r a t l i v e r have shown t h a t the 16e$ hydroxylase i s l o c a l i z e d i n the microsomal f r a c t i o n and r e q u i r e s NADPHg as c o f a c t o r , but t h i s enzyme i s n o t c o n f i n e d t o the l i v e r .  E s t r i o l was the o n l y  C-16 s u b s t i t u t e d p h e n o l i c s t e r o i d known f o r years u n t i l M a r r i a n and Bauld  (24, 25) i s o l a t e d 1 6 - e p i e s t r i o l ( e s t r i o l 16, 17p) from human  pregnancy u r i n e .  T h i s was f o l l o w e d by the d i s c o v e r y o f 16a;-hydroxy-  estrone (26, 27) and 16p hydroxyestrone (26, 27) suggested  t h a t these compounds were i n t e r m e d i a t e s i n the  formation o f e s t r i o l and e p i e s t r i o l Breuer  (28, 2 9 ) . M a r r i a n and Layne  from e s t r o n e .  Knuppen and  (30) showed t h a t r a t l i v e r c o n t a i n e d a s t e r o i d epoxidase  as  w e l l as an epoxy hydratase which were r e s p o n s i b l e f o r the f o r m a t i o n of  16,17  epiestriol.  During t h e i r s t u d i e s on p r o t e i n b i n d i n g o f e s t r o g e n s , Hecker and M u e l l e r  (31) observed  t h a t when r a t l i v e r microsomes were  incubated w i t h 5,6,7,8 t e t r a h y d r o - 2 - n a p h t h o l  8-C-^, a p r o t e i n bound  d e r i v a t i v e and t e t r a l i n - p - q u i n o l were the major p r o d u c t s . r e a c t i o n s showed a b s o l u t e requirements  The  f o r oxygen and reduced NADP.  When t h i s r e a c t i o n was f u r t h e r i n v e s t i g a t e d u s i n g e s t r a d i o l - 1 7 p , the authors were a b l e t o d e t e c t 17p h y d r o x y e s t r a - p - l O p - q u i n o l , the f o r m a t i o n o f the e s t r a q u i n o l b e i n g s t r o n g l y i n h i b i t e d by p - c h l o r o mercuribenzoate. For the formation of 2- and 6-hydroxylated  estrogens and  e s t r a - p - q u i n o l , Hecker and Zayed (32) advanced a scheme i n which  -5r a t - l i v e r microsomes by an unknown mechanism remove the hydrogen of the p h e n o l i c h y d r o x y l group of estradiol-1765 to form a phenoxy radical.  T h i s v e r y r e a c t i v e i n t e r m e d i a t e c o u l d then  i t s e l f by conversion, i n t o mesomeric forms.  stabilize  Subsequent r e a c t i o n  w i t h f r e e r a d i c a l s from the a c t i v a t i o n process or the aqueous medium then g i v e r i s e to h y d r o x y l a t e d m e t a b o l i t e s .  Reaction  products were those c o n s i s t i n g mainly of h y d r o x y l groups i n o r t h o , para and b e n z y l p o s i t i o n s .  The authors however suggested  that  such an enxyme system p r o v i d e d a mechanism mainly f o r the i n a c t i v a t i o n of e s t r o g e n s . Water-soluble Metabolites E s t r o g e n s a r e e x c r e t e d mainly as conjugates of s u l p h u r i c  and  g l u c u r o n i c a c i d s , and these compounds a r e o f t e n r e f e r r e d to as the c l a s s i c a l e s t r o g e n conjugates.  As e a r l y as 1929,  Glimm and  Wadehn (33) showed t h a t estrogens were e x c r e t e d i n the u r i n e i n a form which c o u l d n o t be e x t r a c t e d by o r g a n i c s o l v e n t s and these conjugates were i s o l a t e d  later  (34) from pregnancy u r i n e .  The f o r m a t i o n of e s t r o g e n g l u c o s i d u r o n a t e s has been demonstrated i n v i t r o by Crepy  (35) L e h t i n e n (36) and D i c z f a l u s y  work o f these authors was  (37).  The  concerned mainly w i t h the h y d r o l y s i s o f  the conjugates w i t h p - g l u c u r o n i d a s e and i d e n t i f i c a t i o n of the steroid residues.  Hollman (38) proposed  t h a t the immediate donor  of g l u c u r o n i c a c i d i s the aglycone u r i d i n e diphosphate g l u c u r o n i c acid  (UDPGA) which a r i s e s from the o x i d a t i o n of u r i d i n e  glucose.  diphosphate  The f o r m a t i o n of these g l u c o s i d u r o n a t e s i s brought  about  by a t r a n s f e r a s e r e a c t i o n i n which UDPGA i s a donor r a t h e r than a  -6cofactor. the l i v e r .  T h i s enzyme i s l o c a t e d i n the microsomal f r a c t i o n o f I s s e l b a c h e r (39) i n d i c a t e d t h a t estrogens  can be  conjugated w i t h g l u c u r o n i c a c i d by the enzyme system d e s c r i b e d and t h i s was confirmed by Smith and Breuer  (40) who  estrone w i t h r a b b i t l i v e r microsomes i n the presence  incubated o f UDPGA and  i s o l a t e d e s t r o n e monoglucosiduronate. The potassium  s a l t o f estrone hydrogen s u l f a t e has been  i s o l a t e d by Schachter pregnant mare.  and M a r r i a n  (41) from the u r i n e o f the  De Meio e t a l (42) have demonstrated  of estrone and estradiol-17E3 w i t h microsome-free f r a c t i o n o f r a t and ox l i v e r i n the presence  sulfurylation  supernatant  o f ATP and Mg"*+.  De Meio proceeded to show t h a t the f o r m a t i o n , o f a r y l  sulfates  r e q u i r e s a s u l f a t e a c t i v a t i n g enzyme and a t r a n s f e r r i n g enzyme which s h i f t s the s u l f a t e from " a c t i v e " s u l f a t e t o the p h e n o l .  This  t r a n s f e r i s c a t a l y z e d by a group o f enzymes, the s u l f o k i n a s e s , so named by Lipmann (43, 4 4 ) , and corresponding t r a n s f e r r i n g enzymes o f De Meio.  to the s u l f a t e  E s t r o g e n sulphates can be  h y d r o l y s e d by s u l f a t a s e s (45) l o c a l i z e d i n the microsomes, and these a r e d i s t i n c t from a r y l s u l f a t a s e ( 4 6 ) . Conjugates other than g l u c o s i d u r o n a t e s and s u l f a t e s V a l c o u r t e t a l (47), on a d m i n i s t e r i n g 16-^ C estrone to r a t s 4  found t h a t even a f t e r enzymatic one  h y d r o l y s i s o f the conjugates,  only  t h i r d o f the r a d i o a c t i v i t y e x c r e t e d i n the b i l e and u r i n e  c o u l d be e x t r a c t e d w i t h e t h e r .  S i m i l a r l y Beer and G a l l a g h e r (48)  have shown t h a t e x t r a c t i o n w i t h ether f a i l e d t o remove a l l t h e r a d i o a c t i v i t y from the h y d r o l y s e d u r i n e o f p a t i e n t s i n j e c t e d w i t h  -7-  "C estrone on  1Z|  1 4  C  e s t r a d i o l ; and i n p e r f u s i o n experiments  w i t h r a d i o a c t i v e e s t r o n e , W o r t i z , Z i s k i n d and R i n g l e r (49) observed  a l a r g e amount o f c h l o r o f o r m i n s o l u b l e m a t e r i a l c o n t a i n i n g  r a d i o a c t i v i t y i n the h y d r o l y s e d d i a l y s a t e s o f r a t plasma. Szego (50) and Heusghem (51) have shown t h a t e s t r o n e can be converted t o w a t e r - s o l u b l e e t h e r - i n s o l u b l e m e t a b o l i t e s by mammalian liver  s l i c e s and a h i g h y i e l d o f these m e t a b o l i t e s was a l s o  o b t a i n e d by J e l l i n c k  (52) when e i t h e r r a t l i v e r  e x t r a c t s were used.  The r e s u l t s a t f i r s t suggested  mushroom enzyme a c t e d on estrogens  s l i c e s or mushroom t h a t the  i n the same way as on i t s normal  p h e n o l i c s u b s t r a t e s w i t h the f o r m a t i o n o f h i g h l y r e a c t i v e o - q u i n o i d d e r i v a t i v e s capable o f b i n d i n g w i t h p r o t e i n s or other a c c e p t o r s to give stable products.  F u r t h e r evidence  f o r the formation o f an  o - q u i n o i d estrogen d e r i v a t i v e by mushroom phenolase by J e l l i n c k  has been g i v e n  (53) u s i n g e t h y l e n e diamine as t r a p p i n g agent a f t e r  A x e l r o d and Rao (54) had shown t h a t the f r e e quinone was too unstable f o r d i r e c t i s o l a t i o n .  However, J e l l i n c k showed t h a t no  t r a c e o f an e t h y l e n e diamine condensation product c o u l d be d e t e c t e d when r a t l i v e r microsomes or the 8000 x g supernatant  fraction  (microsomes + s o l u b l e f r a c t i o n o f the c e l l ) was i n c u b a t e d estrogens  i n the presence  o f t h i s diamine and NADPHg.  suggested  t h a t the w a t e r - s o l u b l e products  with  He t h e r e f o r e  formed from estrone and  e s t r a d i o l by r a t l i v e r microsomes a r e n o t d e r i v e d from o - q u i n o i d i n t e r m e d i a t e s but t h a t they may a r i s e by way o f a phenoxy r a d i c a l . A r e a c t i v e i n t e r m e d i a t e o f t h i s k i n d i . e . a phenoxy r a d i c a l has been proposed by Hecker and Zayed (32) f o r the f o r m a t i o n o f  -82- and 6-hydroxy e s t r a d i o l - 1 7 p as w e l l as e s t r a - p - q u i n o l as previously described.  I t has a l s o been shown t h a t p e r o x i d a s e  which i s known to a c t by a f r e e r a d i c a l mechanism i s a good c a t a l y s t f o r the c o n v e r s i o n of estrogens i n the presence  to w a t e r - s o l u b l e  products  o f p r o t e i n s (53), amino a c i d s (55) and reduced  n i c o t i n a m i d e coenzymes ( 5 6 ) . R i e g e l and M u e l l e r rat  (57) succeeded i n i s o l a t i n g an enzyme i n  l i v e r homogenates which i n the presence  o f NADPH2 and oxygen  bound a m e t a b o l i t e o f e s t r a d i o l - 1 7 p to p r o t e i n , w h i l e Szego (58) showed t h a t l i v e r  c o n t a i n e d enzymes which b i n d e s t r o n e to the  albumin f r a c t i o n o f homologous serum.  Z i l l i g and M u e l l e r  (59)  s t u d i e d the i n t e r a c t i o n o f p r o t e i n and e s t r a d i o l d e r i v a t i v e s u s i n g rat  l i v e r microsomes and excluded p o s i t i o n s 2 and 4 from b e i n g  i n v o l v e d i n the b i n d i n g process because s u b s t i t u t i o n o f e i t h e r o f these p o s i t i o n s on the s t e r o i d w i t h f l u o r i n e d i d n o t a f f e c t combination  with p r o t e i n .  However,  s i n c e 2,'4-dif l u o r o e s t r a d i o l was  not t e s t e d , b i n d i n g a t one or the other of the two a l t e r n a t i v e p o s i t i o n s c o u l d s t i l l have o c c u r r e d . Thus the nature and mechanism of formation of the w a t e r - s o l u b l e d e g r a d a t i o n products o f e s t r o n e by mammalian t i s s u e s a r e l a r g e l y unknown, a l t h o u g h evidence has been g i v e n f o r the e x i s t e n c e o f protein-bound  water-soluble metabolites.  does n o t account  However p r o t e i n - b i n d i n g  f o r a l l the n o n - h y d r o l y s a b l e  water-soluble  m e t a b o l i t e s o f estrogens and the f o l l o w i n g types o f products have been suggested ( 4 0 ) :  -91)  unhydrolysed  conjugates  of an unknown n a t u r e  2)  p h e n o l i c s t e r o i d s w i t h t h e i r s t r u c t u r e i n t a c t but v e r y s o l u b l e i n water because of a l a r g e number of p o l a r h y d r o x y l groups  3)  d e g r a d a t i o n products  s i m i l a r to m a r r i a n o l i c and  doisynolic  a c i d i n which r i n g D i s o x i d i z e d to c a r b o x y l i c a c i d groups, or m e t a b o l i t e s formed by opening 4)  d e g r a d a t i o n products  of r i n g A o f the  steroid  i n which the s t e r o i d s t r u c t u r e has been  e x t e n s i v e l y m o d i f i e d and h i g h l y oxygenated. Not v e r y much can be s a i d about these compounds except i n the case of group #2 t y p i c a l example.  of which 6e&-hydroxyestriol can be c o n s i d e r e d a M a r r i a n and Sneddon (60) s t u d i e d the p a r t i t i o n  o f t h i s compound i n the systems e i t h e r r w a t e r and e t h y l a c e t a t e r t ^ O , and  found from the e q u i l i b r i u m constant t h a t the 6-hydroxyl  group  c o n f e r s a p p r e c i a b l e s o l u b i l i t y i n water. Other a s p e c t s of e s t r o g e n metabolism R e c e n t l y D i c z f a l u s y e_t a l have done e x t e n s i v e work on  the  metabolism o f e s t r o n e , estradiol-1763 and e s t r i o l i n human f e t u s e s and newborns (61, 62,  63, 64).  They concluded  t h a t the human  f e t u s p l a y e d an a c t i v e p a r t i n m e t a b o l i z i n g e s t r o g e n s .  After  i n j e c t i o n o f estradiol-17E3 or e s t r i o l i n t o the amnion, they c o u l d d e t e c t e l e v a t e d c o n c e n t r a t i o n s of conjugated and e s t r i o l i n the f e t a l organs. s u l f u r y l a t i n n was  e s t r o n e , estradiol-1763  The r e s u l t s i n d i c a t e d t h a t  the predominant type of c o n j u g a t i o n i n the  lungs, whereas both s u l f u r y l a t i o n and g l u c o s i d u r o n a t e o c c u r r e d i n l i v e r and  the i n t e s t i n e s .  formation  However, D i c z f a l u s y d i d  f i n d t h a t the metabolism of estrogen i n f e t u s and i n f a n t s  differed  -10from t h a t of a d u l t s i n t h a t e s t r i o l d i d not r e p r e s e n t stage of estrogen metabolism i n i n f a n t s . the i n t e r m e d i a r y metabolism of estrogen  According  the  final  to D i c z f a l u s y ,  i n the l a t t e r appears to  g i v e predominantly h i g h l y p o l a r compounds of an unknown n a t u r e . Attempts have been made to d e t e c t d i f f e r e n c e s i n the metabol i s m of estrogens by normal and a chemical breast  cancerous b r e a s t t i s s u e .  method, Ryan and E n g e l  (6) found t h a t s l i c e s of human  (during pregnancy), mammary fibro-adenomata and  adenocarcin-  omata of the b r e a s t , can convert estradiol-17(3 to estrone comparable r a t e s .  Using  at  An attempt to c o r r e l a t e the a b i l i t y of v a r i o u s  normal and malignant t i s s u e s to m e t a b o l i z e e s t r a d i o l - 1 7 p w i t h  the  l e v e l of o x i d a t i v e metabolism as measured by the oxygen uptake (QO2) has been made by Breuer and Nocke (65). mammary t i s s u e specimen showed higher v a l u e s examined. no  However, due  About 507. o f than other  the  tissues  to l a r g e v a r i a t i o n among i n d i v i d u a l v a l u e s ,  c l e a r c u t c o n c l u s i o n was  drawn.  Therefore  more work d i r e c t e d to i n v i t r o s t u d i e s was  i t was  felt  that  required.  Reports have been p u b l i s h e d which i n d i c a t e the presence of estrone,  e s t r a d i o l - 1 7 p and  Layne e t a l . . (67)) and  e s t r i o l i n b i r d s (Hurst e t a l . (66) ;  in fish  i s o l a t e d i n c r y s t a l l i n e form was hens.  T h i s was  (Wortiz  t h a t from the dropping of l a y i n g  a d i r e c t i n d i c a t i o n t h a t s t e r o i d a l estrogens are  a l s o produced by non-mammalian s p e c i e s . estradiol-17p with l i v e r Hobkirk (69)  ( 6 8 ) ) , but the o n l y estrogens  detected  A f t e r incubating  16-l^C  s l i c e s from l a y i n g hens, M i t c h e l l and  e s t r i o l ; and  t h i s s t e r o i d was  a l s o shown  to be formed by McRae and h i s workers (70) a f t e r i n j e c t i o n of  -1116- C estradiol-17(3.  16-l^C e s t r i o l was  14  r a d i o a c t i v e 16-oxoestradiol-17£3 and (71).  16-epiestriol  Conjugated estrogens were d e t e c t e d  while studying species.  found to g i v e r i s e to i n the  by I t t r i c h  (72)  the i n v i v o metabolism o f estradiol-17£3  in fish  in this  B i r d s were shown to possess a 16@j-hydroxylase i n the  l i v e r , and  a 16e? and  These r e s u l t s  16p  and  17£ hydroxy s t e r o i d  dehydrogenase.  c o n t r i b u t e to the f a c t t h a t s t e r o i d a l  are m e t a b o l i z e d i n a l i k e manner by a l l v e r t e b r a t e s . c e r t a i n whether a s i m i l a r invertebrates. one  urine  estrogens  I t i s not  type of metabolism takes p l a c e i n  Should t h i s be the case, t h i s problem c o u l d  of e v o l u t i o n a r y  i n t e r e s t . i n comparative e n d o c r i n o l o g y .  be -  I n a c t i v a t i o n o f Pregnant Mare's Serum Gonadotropin by Quinones As e a r l y as 1930, mare's serum (PMS) i t contained  was  C o l e and Hart (73)  an e x c e l l e n t source of gonadotropins, s i n c e  a f r a c t i o n which was  growth of the f o l l i c u l a r as FSH) The  and  fractions  essentially responsible 1  for  the  system i n the ovary (hormone b e t t e r known  a f r a c t i o n which was  lutenizing  showed t h a t pregnant  responsible  for lutenization  (LH).  hormone had no e f f e c t on the o v a r i e s a l t h o u g h both  produced o v u l a t i o n , optimum c o n d i t i o n b e i n g  on t h e i r r e l a t i v e Evans i n 1932  dependent  concentrations. (74) has  shown t h a t c e r t a i n  important  es e x i s t between the gonadotropic a c t i v i t i e s o f e x t r a c t s from the p i t u i t a r y and  from human pregnancy u r i n e .  differencobtained  U s i n g immature  r a t s , he n o t i c e d t h a t p i t u i t a r y e x t r a c t s caused a marked  increase  i n the weight o f the o v a r i e s whereas u r i n a r y e x t r a c t s produced only a l i m i t e d increase.  Furthermore, e x t r a c t s from the  pituitary  -12produced both f o l l i c u l a r growth and l u t e n i z a t i o n , whereas urinarye x t r a c t s r e s u l t e d m a i n l y i n l u t e n i z a t i o n . . T h e r e f o r e i t was suggested t h a t the gonadotropic hormone found i n the u r i n e i s not produced by the p i t u i t a r y , but i n the c h o r i o n i c Noble and Graham (75) have shown t h a t PMS  tissues.  lost i t s biological  a c t i v i t y when i n c u b a t e d w i t h c e r t a i n hydroquinones  or  quinones.  They suggested t h a t these compounds might be i n a c t i v a t i n g  circula-  t i n g gonadotropins i n the i n t a c t a n i m a l , thereby i n h i b i t i n g reproduction.  Beer and Noble  (76) showed t h a t the  hydroquinone  must f i r s t be o x i d i z e d to the corresponding quinone b e f o r e any i n a c t i v a t i o n of PMS  took p l a c e .  on the i n a c t i v a t i o n of PMS  The a c t i o n o f e s t r o g e n  quinones  however has not been i n v e s t i g a t e d .  The P r e s e n t I n v e s t i g a t i o n The main problem of i n t e r e s t was the w a t e r - s o l u b l e products- formed  to determine the n a t u r e of  from estrogens by r a t l i v e r  microsomes, and to compare these m e t a b o l i t e s to those formed mushroom phenolase under s i m i l a r c o n d i t i o n s .  I t was  by  a l s o important  to develop chromatographic methods and a double i s o t o p e technique to h e l p i d e n t i f y the unknown p r o d u c t s and to determine conditions for their formation.  the optimum  In a d d i t i o n , i t was c o n s i d e r e d  of i n t e r e s t to i n v e s t i g a t e the a c t i o n of e s t r o g e n o x i d a t i o n p r o d u c t s on the i n a c t i v a t i o n of  PMS.  -13EXPERIMENTAL M a t e r i a l s and Methods  IA .MATERIALS: Animals:  Male Sprague-Dawley r a t s about t h r e e months o f age and weighing approximately 250 g were o b t a i n e d from the  U n i v e r s i t y of B r i t i s h Columbia animal c o l o n y or  purchased from Guelph. r a t s used to demonstrate  The female Sprague-Dawley the i n a c t i v a t i o n o f pregnant  mare's serum (PMS) by estrogens were imported from W i s c o n s i n and v a r i e d i n age from 3-5 weeks. A l l animals were f e d Master L a b o r a t o r y cubes ad l i b i t u m . Phenolase:  Grade I I t y r o s i n a s e (mushroom phenolase) purchased from the Sigma Chemical Company, S t . L o u i s , M i s s o u r i , U.S.A. was used. Enzyme a c t i v i t y 1 unit =  . was 500-1000 units/mg.  O.D. o f 0.01/min a t 280 mo. a t pH 6.5 a t 25°C  i n 3 ml o f r e a c t i o n mixture c o n t a i n i n g L - t y r o s i n e . Radioactive estrogens: 16-l^C e s t r a d i o l - 1 7 p  (9.7mc/m mole) was purchased from  R a d i o c h e m i c a l C e n t r e , Amersham, E n g l a n d .  The estrogen  was maintained i n a s t o c k s o l u t i o n o f 1 mg/ml o f ethanol. 6,7- H e s t r a d i o l 3  (500 mc/m mole) was a l s o o b t a i n e d  from the same source.  -14Radioactive glutathione: •^•^-glutathione (0.95 mc/m mole) was purchased from Schwarz B i o r e s e a r c h Inc., Orangeburg, N.Y. Counting M a t e r i a l : The  scintillation  reagents:-  f l u i d was made up from the f o l l o w i n g  4 g  2,5-diphenyloxazole  100 mg l , 4 - b i s - 2 ( 5 - p h e n y l  (PPO) oxazolyl)-benzene (POPOP)  600 ml toluene 400 ml d i s t i l l e d PPO  ethanol  and POPOP and hyamine hydroxide  10-x were obtained  from the Packard Instrument Company, Inc., I l l i n o i s , USA. Buffers:  1)  0.1 M potassium  phosphate b u f f e r , pH 7.4 ( f o r i n c u b a t i o n s )  were made up as f o l l o w s : K HP0 2  4  . 3H 0  0.0802 m o l e / l i t r e  2  KH2PO4 2)  0.0198 m o l e / l i t r e  18.3 g 2.69 g  of H 0 2  0.2 M c i t r a t e b u f f e r , pH 5.0 ( f o r n i n h y d r i n reagent) C i t r i c acid  - 4.3 g 250 ml d i s t i l l e d  Na 3)  in1 litre  3  citrate  H 0 2  • 2H 0 - 8.7 g 2  G l a c i a l a c e t i c : p y r i d i n e : H 2 0 , pH 3.5 ( f o r e l e c t r o p h o r e s i s ) 100 ml g l a c i a l a c e t i c a c i d 10 ml p y r i d i n e 1 l i t r e d i s t i l l e d water  4)  0.067 M potassium 9.39  g K HP0  4  3.51  g KH P0  4  2  phosphate b u f f e r , pH 7.0 (PMS experiments)  • 3H 0 2  1 litre 2  H 0 2  -155)  0.06 M potassium phosphate b u f f e r 7.62 g K H P 0 2  4  • 3H 0 2  1 litre 3.6  g KH P0 2  (assay o f t y r o s i n a s e activity)  H 0 2  4  The f i n a l pH o f each b u f f e r was checked a g a i n s t a s t a n d a r d i z e d g l a s s electrode. N i n h y d r i n Reagent:  160 mg o f r e a g e n t grade S n C l » 2 H 0 2  2  dissolved i n  100 ml o f 0.2 M c i t r a t e b u f f e r pH 5.0 were added t o 4 gm o f 1,2,3 t r i k e t o h y d r i n d e n e h y d r a t e (1,2,3  Indantri-  one hydrate) d i s s o l v e d i n 100 ml o f methyl c e l l o s o l v e . The r e a g e n t was s t o r e d a t 4°C. The n i n h y d r i n was purchased from t h e Sigma Chemical Co., S t . L o u i s , M i s s o u r i , and the S n C l 2 H 0 from the B r i t i s h Drug Houses L t d . , P o o l e , o  2  2  England. Sephadex:  G-25 medium f o r column chromatography  and B l u e Dextran  2000 f o r d e t e r m i n a t i o n o f v o i d volume were purchased from Pharmacia, F i n e Chemicals I n c . , New York. N i c o t i n a m i d e Coenzymes:  Reduced t r i p h o s p h o p y r i d i n e n u c l e o t i d e  (NADPH)  was purchased from N u t r i t i o n a l B i o c h e m i c a l C o r p o r a t i o n , C l e v e l a n d , Ohio. F o l i n C i o c a l t e n Reagent:  T h i s reagent (a mixture o f sodium phospho-  molybdate and phosphotungstate) was o b t a i n e d from the B r i t i s h Drug Houses, Canada, L t d . . DOPA:  DLp3,4-dihydroxyphenylalanine was used f o r a s s a y i n g tyrosinase a c t i v i t y .  I t was purchased from Sigma  Chemical Company, S t . L o u i s , M i s s o u r i , USA.  -16-  PMS:  Pregnant mare's serum (45 i n t e r n a t i o n a l u n i t s per m i l l i g r a m ) was  o b t a i n e d from A y e r s t , McKenna and  H a r r i s o n L t d . , M o n t r e a l , Canada. Inhibitors:  N-ethylmaleimide (NEM)  o b t a i n e d from Mann Research  Lab or a t o r y, I n c . - Potassium cyanide (KCN) D i v i s i o n , New  from the G e n e r a l Chemical  York, N.Y.,  USA  - Disodium e t h y l e n e diamine t e t r a - a c e t a t e (EDTA) was purchased from the F i s h e r S c i e n t i f i c Co., F a i r l a w n , N . J . Activators:  - O x i d i z e d and reduced g l u t a t h i o n e were purchased from the Sigma Chemical Co., S t . L o u i s , M i s s o u r i ; C y s t e i n e h y d r o c h l o r i d e was tific  o b t a i n e d from F i s h e r  Scien-  Co.;  DL-homocysteine  t h i o l a c t o n e h y d r o c h l o r i d e and bovine  serum albumin were o b t a i n e d from N u t r i t i o n a l B i o c h e m i c a l Corp., C l e v e l a n d , Ohio; a s c o r b i c a c i d from the B r i t i s h Drug Houses, Canada, L t d . ; L - e r g o t h i o n e i n e h y d r o c h l o r i d e d i h y d r a t e (A grade) and L - 2 - t h i o l h i s t i d i n e (A grade) from Calbiochem, Los Angeles. Reference Standards f o r Chromatography: g i f t s to Dr. P.H.  Many of these compounds were  Jellinck.  2-hydroxyestrone, 2 - h y d r o x y e s t r a d i o l - 1 7 p and  2-hydroxy-  e s t r i o l were o b t a i n e d from Dr. L. A x e l r o d ; 16-ketoestrone, 1 6 - k e t o e s t r a d i o l - 1 7 p and estradiol-17©  were o b t a i n e d  -17from the cancer therapy N a t i o n a l S e r v i c e Bethesda,  Md.  Estradiol-17f3-glucosiduronate California  Centre,  Corporation  Angeles, and from Dr. A.E.  was  obtained  for Biochemical  from  the  Research,  e s t r i o l - 1 7 p - g l u c o s i d u r o n a t e was  a  Los  gift  Kellie.  16&-hydroxyestrone.was donated by Dr. H. Breuer 6-keto e s t r a d i o l - 1 7 p and  16a-  17(3 were g i f t s from Dr. 0.  and  and  6p-hydroxyestradiol-  Wintersteiner.  -18I B . METHODS 1.  Preparation of Tissue:  The animals were k i l l e d by s u f f o c a t i o n  i n an atmosphere o f carbon d i o x i d e , the l i v e r s q u i c k l y e x c i s e d , washed w i t h i c e c o l d 0.25 M sucrose s o l u t i o n and weighed.  One  p a r t by weight o f t i s s u e i n one p a r t by volume o f 0.25 M sucrose was  then homogenized f o r t h r e e seconds i n a S e r v a l l c m n i - m i x e r .  One ml o f t h i s crude homogenate was d i l u t e d to 5 ml w i t h 0.25 M sucrose and f u r t h e r homogenized i n a P o t t e r Elvehjem  homogenizer  w i t h a t e f l o n p e s t l e , and the volume made up to 10 ml w i t h  sucrose  s o l u t i o n 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 50 mg l i v e r per ml o f sucrose 2.  solution.  Preparation of Subcellular Fractions: 8000 x g supernatant  fraction:  The l i v e r homogenate i n 0.25 M  sucrose s o l u t i o n was c e n t r i f u g e d a t 8000 x g f o r 15 minutes a t 2°C i n a Spinco Model L p r e p a r a t i v e u l t r a c e n t r i f u g e .  The supernatant  o b t a i n e d w i l l be r e f e r r e d t o i n t h i s t e x t as the 8000 x g supernatant fraction. 100,000 x g microsomal p e l l e t :  7-ml volumes o f the 8000 x g  supernatant f r a c t i o n were t r a n s f e r r e d t o c l e a n c e n t r i f u g e tubes and made up t o 10 ml w i t h 0.25 M s u c r o s e .  Microsomal  p e l l e t s were  o b t a i n e d by c e n t r i f u g i n g a t 100,000 x g f o r 60 minutes. natant f r a c t i o n  The super-  ( r e f e r r e d to as the 100,000 x g supernatant) was  decanted and t h e p e l l e t s washed w i t h 0.1 M potassium b u f f e r , pH 7.4, and resuspended  i n 4 ml o f b u f f e r .  phosphate The p e l l e t was  g e n t l y homogenized i n a P o t t e r Elvehjem homogenizer w i t h a t e f l o n p e s t l e u n t i l a homogenous s o l u t i o n was o b t a i n e d , and the volume  -19made up to 7 ml.  T h i s method y i e l d e d microsomes i n a c o n c e n t r a t i o n  of 50 mg/ml. 3.  I n c u b a t i o n and e x t r a c t i o n of t i s s u e p r e p a r a t i o n s The i n c u b a t i o n mixture c o n s i s t e d of 50 mg  of the 8000 x g  supernatant f r a c t i o n or the 100,000 x g microsomes or supernatant (1 m l ) , 1 mg  of NADPH, 10 (ig of r a d i o a c t i v e 1 6 - C e s t r a d i o l - 1 7 p 14  (20 |_ig of the s t e r o i d was used whenever i t was n e c e s s a r y to o b t a i n maximum y i e l d s ) . from 10"^ M t o 1 0 "  A c t i v a t o r s were added i n c o n c e n t r a t i o n s v a r y i n g 4  M and the r e a c t i o n was  c a r r i e d out i n a t o t a l  volume of 4 ml o f b u f f e r . Where t i s s u e p r e p a r a t i o n s were r e p l a c e d by mushroom phenolase, 1 mg/ml of the enzyme was used without reduced coenzyme, and the i n c u b a t i o n was  c a r r i e d out i n the same volume o f b u f f e r .  (When i t  was n e c e s s a r y to separate the w a t e r - s o l u b l e p r o d u c t s by paper chromatography, the b u f f e r was  d i l u t e d one hundred  fold.)  Oxygen was bubbled i n t o a l l tubes f o r 15 seconds b e f o r e they were immersed i n t o a water b a t h shaker maintained a t 37°C.  Tubes  were incubated" under an atmosphere of oxygen and were shaken a t 70 c y c l e s / m i n u t e f o r a d e s i g n a t e d p e r i o d o f time. f o r t i s s u e p r e p a r a t i o n s was preparations. mixture was  30 minutes  I n c u b a t i o n time  and 2 hours f o r the phenolase  The time of a d d i t i o n of s u b s t r a t e to r e a c t i o n  taken as zero time.  A t the end of the i n c u b a t i o n p e r i o d , the r e a c t i o n was by the a d d i t i o n of 1 ml 1NHC1, and the contents o f each e x t r a c t e d t h r e e times w i t h e q u a l volumes of e t h e r .  stopped  tube  When the  i n c u b a t i o n mixture had to be chromatographed on Sephadex  G-25,  Figure l a . Pulse spectra of 35s and ^H.  -20the a c i d was  omitted.  The aqueous f r a c t i o n and the ether f r a c t i o n  were t r e a t e d as r e q u i r e d . 4.  Determination of R a d i o a c t i v i t y : Measurement of r a d i o a c t i v i t y and Ethereal Fraction:  autoradiography  The ether f r a c t i o n was  evaporated to dryness  over an atmosphere o f n i t r o g e n and the r e s i d u e d i s s o l v e d i n 1 ml of  d i s t i l l e d ethanol.  The r a d i o a c t i v i t y was  determined by c o u n t i n g  0.1 ml of the e t h a n o l i c f r a c t i o n i n a Packard T r i c a r b L i q u i d S c i n t i l l a t i o n spectrometer.  The  sample was  f i r s t dissolved i n  1 ml of hyamine hydroxide i n a c o u n t i n g v i a l to which 9 ml of scintillation  f l u i d were added.  Aqueous f r a c t i o n :  A l l ether was removed w i t h n i t r o g e n from  the aqueous f r a c t i o n and 0.2 ml samples counted i n a s i m i l a r manner. No quenching  c o r r e c t i o n was  a p p l i e d s i n c e 85-95% of the added  r a d i o a c t i v i t y c o u l d be r e c o v e r e d c o n s i s t e n t l y from the e t h e r e a l p l u s aqueous f r a c t i o n s . volts  (tap s e t t i n g 6.0,  All  c o u n t i n g was  window s e t t i n g 10:60  c a r r i e d out a t  1150  on the r e d s c a l e r ) .  Double Isotope Counting Where the double i s o t o p e technique was counts per minute (cpm)  of  employed ( H and 3~*s) 3  S a t a g i v e n c o n c e n t r a t i o n were  obtained by v a r y i n g the v o l t a g e (V), and a graph of cpm v o l t a g e was  p l o t t e d a t d i f f e r e n t window s e t t i n g s  procedure was as above.  repeated for H 3  I t was  s e t t i n g 2.8)  versus  (Fig. 1).  This  and a graph drawn to the same s c a l e  found b e s t t o count  3 5  S  a t a v o l t a g e of 875  (tap  s i n c e there was v e r y l i t t l e i n t e r f e r e n c e w i t h H  this voltage.  at  3  3  H  v o l t a g e a t which  was 3 5  S  t  counted a t V = 1500  (tap s e t t i n g 9.2),  counts were the same a t t h a t as a t V =  a 875.  -21Therefore H counted a t V = 1500 gave the t o t a l H and 3  counts.  3  3 5  S  was counted a t V =875 where the counts from H 3  were n e g l i g i b l e .  Therefore counts at V = 1500 minus counts a t  V = 875 gave the true t r i t i u m counts. Autoradiography:-  Radioactive compounds were detected on the  chromatograms by autoradiography on Kodak no screen X-ray f i l m . A sample containing about 50,000 cpm was applied to the paper before running i n an appropriate solvent system.  An exposure  time of 4 days was required, and the radioactive spots on the paper were detected by corresponding darkened areas on the f i l m . By matching spots on the f i l m to the paper, r a d i o a c t i v e compounds could be cut out and r a d i o a c t i v i t y assayed by eluting or counting directly. 5.  Examination  of the Ethereal. Fraction:-  No extensive study  was c a r r i e d out with this f r a c t i o n since the main interest was i n the nature of the water-soluble metabolites.  However, when i t  was thought that radioactive metabolites had not been carried into the aqueous phase because the attached polar group may be small r e l a t i v e to the large non-polar nucleus, examination of the ether f r a c t i o n was carried out. The samples each containing about 50,000 cpm were spotted on a Whatman #1 paper and run on a modified Bush system . (benzene 19:ethyl acetate l:water 16:methanol 4) f o r two to three hours. autoradiography.  The radioactive compounds were located by  Non-radioactive estrogen standards were detected  by spraying the paper with F o l i n - C i o c a l t e u reagent. 6.  Examination  of the Aqueous F r a c t i o n :  -22Protein Precipitation:  A l l t r a c e s of ether were removed  from the a c i d i f i e d e t h e r - e x t r a c t e d aqueous f r a c t i o n e v a p o r a t i o n over n i t r o g e n and 3 ml of 20% acid  (TCA) were added.  (W/V)  (5 ml)  by  trichloroacetic  Tubes were allowed to stand f o r an hour  to ensure maximum p r e c i p i t a t i o n and the p r e c i p i t a t e s were spun down a t approximately 1000  x g f o r 10 minutes i n an  C l i n i c a l c e n t r i f u g e (Cenco Instruments).  The  supernatant  decanted and 0.2 ml counted f o r r a d i o a c t i v i t y . procedure, the p r e c i p i t a t e was  was  By the same  washed w i t h 1 ml of 20%  and 2 x 1 ml of d i s t i l l e d e t h a n o l . and  International  (W/V)  TCA  The washings were d i s c a r d e d  the p r e c i p i t a t e d i s s o l v e d i n 1 ml formamide and heated a t  150-180°C f o r 2 hours i n a d r y i n g oven.  0.1 ml o f the l i q u e f i e d  p r o t e i n was  then counted i n the u s u a l manner.  Dialysis:-  1 ml o f the aqueous f r a c t i o n c o n t a i n i n g a g i v e n number  o f counts was  p i p e t t e d i n t o a c e l l o p h a n e t u b i n g and  a g a i n s t r u n n i n g tap water f o r 24 hours.  dialysed  The volume was  measured  a t the end o f the d i a l y s i s and 0.1 ml of the contents o f the d i a l y s i s bag was  counted.  Paper chromatography:the aqueous f r a c t i o n was biodryer  A volume corresponding to 50,000 cpm evaporated down to 0.02  ( V i r t i s ) and s p o t t e d on Whatman #3 MM  of  ml i n a c e n t r i f u g a l  paper.  Where l a r g e  s c a l e i n c u b a t i o n s were c a r r i e d out, the contents of a l l tubes (18 tubes o f non r a d i o a c t i v e m a t e r i a l and 1 tube c o n t a i n i n g 20 \xg of 1 6 - C e s t r a d i o l - 1 7 p ) were p o o l e d , evaporated down t o a volume 14  o f 0.2 ml and s t r e a k e d a c r o s s the o r i g i n o f the chromatogram. A l l chromatograms were r u n i n nBu0H:HoAc: 1^0(50:25:25) f o r  12-15  -23hour s.  R a d i o a c t i v e g l u t a t h i o n e was. used as standard and a l l  r a d i o a c t i v e m e t a b o l i t e s were determined  by  autoradiography.  M e t a b o l i t e s were cut out from the paper as d e s c r i b e d p r e v i o u s l y and e l u t e d .  Both the e l u a t e and the paper were counted  i n the  s c i n t i l l a t i o n counter, thereby g i v i n g a q u a n t i t a t i v e e s t i m a t i o n of the m e t a b o l i t e s formed. Column Chromatography: Sephadex G-25  (medium) was  employed f o r t h i s  technique.  The method used f o l l o w e d c l o s e l y t h a t o f F l o d i n and P o r a t h 78), About 25 g of dry Sephadex powder was w i t h d i s t i l l e d water.  be a d j u s t e d .  s t i r r e d i n t o a beaker  A f t e r r e p e a t e d sedimentation and d e c a n t a t i o n  to remove f i n e p a r t i c l e s , the g e l was chromatographic  t r a n s f e r r e d to a  suitable  tube equipped w i t h a tap so t h a t flow r a t e c o u l d  G l a s s wool was  i n t r o d u c e d i n t o the column b e f o r e  a d d i t i o n of the g e l to prevent i t from seeping through. suspension was 2 to 5 cm was  added" to the tube  a f i l t e r paper was  or 0.1  The  The  (tap c l o s e d ) u n t i l a l a y e r of  formed, and then the tap was  a l l o w a slow stream of water.  disturbance.  (77,  c a r e f u l l y opened to  When a l l the g e l g r a i n s had  settled,  p l a c e d on top of the bed to prevent i t s column was  washed w i t h e i t h e r d i s t i l l e d water  N NaCl as i n d i c a t e d i n the t e x t .  A t no time was  the  level  o f water or e l e c t r o l y t e allowed to f a l l below the l e v e l of the bed.  T h e . s i z e of the column bed was  u s u a l l y 1.8  cm x 36  cm.  B e f o r e samples were a p p l i e d to the. column, the e l u a n t  was  allowed to d r a i n to the l e v e l of the upper s u r f a c e of the column  -24and  the t e s t s o l u t i o n  1.5  - 1.8  ml)  sample was and  (the aqueous f r a c t i o n evaporated down to  a p p l i e d by p i p e t t e to the top o f the bed.  a l l o w e d to d r a i n to the  a larger quantity  l e v e l o f the f i l t e r  The paper  o f e l u a n t added to the top o f the  from a connecting r e s e r v o i r to s t a r t the e l u t i o n .  column,  3 ml  fractions  were c o l l e c t e d i n a f r a c t i o n c o l l e c t o r (Research S p e c i a l t i e s and  0.2  ml of each f r a c t i o n counted i n the  Peaks were a l s o determined w i t h n i n h y d r i n The  v o i d volume (Vo)  of Blue Dextran 2000 on top d i s t i l l e d water.  The  was  counter.  reagent.  o b t a i n e d by p l a c i n g a narrow zone  of the column and  v o i d volume was  In c e r t a i n cases, the  scintillation  column was  eluting with  approximately 33 not  but a l s o as a means o f p u r i f i c a t i o n . The  ml.  only used f o r  separation  e l u t i o n o f compounds  from paper u s u a l l y gave r i s e to l a r g e amounts of i m p u r i t i e s were removed by p a s s i n g through the column. afforded  Co.)  which  T h i s technique a l s o  a good method o f d e s a l t i n g , a step t h a t was  necessary f o r  subsequent paper chromatography as w e l l as paper  electrophoresis.  Electrophoresis:  were o b t a i n e d  The-samples f o r e l e c t r o p h o r e s i s  by e i t h e r e l u t i n g c e r t a i n areas of the paper chromatograms  (as  i n d i c a t e d i n the t e x t ) and  small  e v a p o r a t i n g t h i s down to a v e r y  volume on the c e n t r i f u g a l b i o d r y e r  or by e v a p o r a t i o n o f f r a c t i o n s  o b t a i n e d d i r e c t l y o f f the column.  The  samples were a p p l i e d  the middle o f the paper by means of c a p i l l a r y tubing and ly dried.  The  at  thorough-  samples were then washed i n t o a s t r a i g h t l i n e a t  the o r i g i n w i t h a c e t i c a c i d : p y r i d i n e : w a t e r chromatograms were run  b u f f e r , pH  3.5.  i n t h i s same b u f f e r a t 3000 v o l t s f o r  The  -253 hours on a Shandon Model E l e c t r o p h o r e s i s Apparatus and the compounds on the d r i e d e l e c t r o p h o r e t o g r a m l o c a t e d by a u t o r a d i o graphy. Ninhydrin Test:  N i n h y d r i n reagent was used f o r the d e t e c t i o n  of o x i d i z e d and reduced g l u t a t h i o n e , amino a c i d s , p r o t e i n s and p e p t i d e s , u s i n g the method d e s c r i b e d i n E x p e r i m e n t a l . B i o c h e m i s t r y (79) .  To 0.5 ml o f the sample to be tested,.1.5 ml of n i n h y d r i n  reagent was added.  The contents o f each tube was mixed t h o r o u g h l y  and p l a c e d i n t o a b o i l i n g water bath f o r 20 minutes. i n g , 8 ml o f 50% aqueous,n-propanol  After  cool-  were added t o each tube, and  a f t e r shaking, the mixture was allowed to stand f o r 10 minutes to develop maximum c o l o u r .  The b l a n k was .prepared i n a s i m i l a r  manner except t h a t 0.5 ml o f d i s t i l l e d water was used i n p l a c e o f the sample.  Four assay tubes c o n t a i n i n g 10, 25, 50 and 100 u.g o f  t e s t m a t e r i a l were u s u a l l y used as standards.  The o p t i c a l d e n s i t y  of the standard and t e s t s o l u t i o n s were r e a d a t 520-580 mjj, (using a green f i l t e r ) a g a i n s t the blank i n a Klett-Summerson P h o t o e l e c t r i c Colorimeter. Assay o f T y r o s i n a s e A c t i v i t y :  T y r o s i n a s e a c t i v i t y was measured  by the a b i l i t y o f the enzyme t o c o n v e r t DL-dopa t o melanin i n the presence o f oxygen as d e s c r i b e d by S e k e r i s and Mergenhagen (80) .  Known c o n c e n t r a t i o n s o f t y r o s i n a s e were i n c u b a t e d w i t h  5 ml o f 0.02% DL-dopa i n 0.06 M potassium phosphate b u f f e r , pH 7.0 a t 20°C f o r 60 minutes.  Oxygen was bubbled i n t o each tube  b e f o r e i n c u b a t i o n which.was c a r r i e d out i n an atmosphere o f oxygen.  A t the end o f t h e i n c u b a t i o n , 1 ml o f the mixture was  -26d i l u t e d w i t h 10 ml o f 0.06 and  M potassium phosphate b u f f e r pH  the o p t i c a l d e n s i t i e s r e a d a t "480  mu. i n a Coleman  photometer u s i n g a 445-505 mu. f i l t e r . o b t a i n e d , the c o n c e n t r a t i o n s  7.0,  spectro-  From the standard curve  of t y r o s i n a s e  i n the  various  f r a c t i o n s o b t a i n e d a f t e r chromatography on Sephadex c o u l d  be  estimated. The  I n a c t i v a t i o n of Pregnant Mare's Serum by Estrogens i n v i t r o 150  i . u . of PMS  ( i n 3.3  potassium phosphate b u f f e r , pH  mg)  were d i s s o l v e d i n 0.067M  7.0,  and  incubated i n an  of oxygen a t 37°C f o r 2 hours w i t h 1 mg of e s t r a d i o l - 1 7 p  glutathione  on PMS  as o t h e r . r e a c t a n t s .  C o n t r o l tubes were  p r o t e c t i v e a c t i o n of also  (10" M) added a t the same time, 4  weeks o l d were used f o r the experiment, each animal r e c e i v i n g  i n g 25 i . u . of PMS.  and  u.g  Female Sprague-Dawley r a t s approximately  a s i n g l e subcutaneous dose of 0.5  by  The  60  i n a c t i v a t i o n by o x i d i z e d estrogens was  i n v e s t i g a t e d w i t h the g l u t a t h i o n e  3-5  of phenolase and  i n a t o t a l volume of 3 ml.'  incubated without s t e r o i d or enzyme.  atmosphere  ml of the t e s t s o l u t i o n  contain-  A f t e r t h r e e days, a l l the animals were k i l l e d  s u f f o c a t i o n i n C02>'and the o v a r i e s and u t e r i c a r e f u l l y e x c i s e d f i x e d i n 10% n e u t r a l formal s a l i n e f o r 4-6  hours.  t i s s u e s were l a t e r dehydrated i n 50% a l c o h o l o v e r n i g h t , d r i e d and weighed on a R o l l e r - S m i t h The  standard e r r o r  f o l l o w i n g formula  (S.E.) was  blotted,  t o r s i o n balance. c a l c u l a t e d by means o f  (81): S.E.  All  = Standard  Deviation  the  -27Standard d e v i a t i o n (S.D.) was  SD =  \/  d e r i v e d from the e x p r e s s i o n  (x-x)  2  N Where N = no. o f animals employed i n the experiment "x = mean o v a r i a n weight x = observed o v a r i a n weight  TABLE 1  Optimum c o n d i t i o n s f o r the formation  of w a t e r - s o l u b l e  products  from 1 6 - C e s t r a d i o l - 1 7 p by mushroom phenolase i n the presence i4  of glutathione.  Phenolase (mg)  16= C e s t r a d i o l - 1 7 p (M<g) 14  Glutathione Concentration  Incubation time(hrs)  % added radioactivity i n aqueous fraction  0.5  20  10" M  4  34.0  1.0  10  10 "  4  M  4  81.6  1.0  20  10 "  4  M  4  71.9  1.0  40  10" M  4  30.4  1.0  80  10 "  4  11.8  1.0  20  4  35.3  1.0  20  10"  M  4  36-2  1.0  20  10" M  2  69-4  1.0  10  10" M  2  88.9  C o n d i t i o n s as d e s c r i b e d i n t e x t .  4  4  4  M  3 x 10-5M 5  4  4  -28II 1.  RESULTS  Optimal C o n d i t i o n s f o r the Formation from  B s t r a d i o l - 1 7 p by Mushroom  T a b l e I shows the percentage  o f Water-Soluble  Metabolites  Phenolase.  o f added r a d i o a c t i v i t y which  remains i n the aqueous f r a c t i o n when 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 p and g l u t a t h i o n e were incubated w i t h mushroom for different  time  phenolase  intervals.  S i n c e t h e r e was n e g l i g i b l e  d i f f e r e n c e between i n c u b a t i n g  10(ig o f e s t r a d i o l f o r 2 hours and 4 hours, a 2-hour i n c u b a t i o n p e r i o d was chosen as the time r e q u i r e d t o g i v e optimum y i e l d o f water-soluble products.  1 mg o f the phenolase  was chosen as the  o p t i m a l enzyme c o n c e n t r a t i o n . U n l i k e w i t h the microsomal system which had an a b s o l u t e requirement in  f o r NADPR2 (82), the r e a c t i o n c a t a l y z e d by  the presence  t h i s , no reduced  phenolase  o f g l u t a t h i o n e d i d n o t r e q u i r e N A D P H g , and f o r coenzyme was added to the phenolase  system.  The  optimum pH f o r the c o n v e r s i o n o f e s t r o g e n t o w a t e r - s o l u b l e m e t a b o l i t e s was found  to be 7.4 s i n c e i n c u b a t i o n s i n a more a c i d i c  more b a s i c (pH 8.0) s o l u t i o n was an a b s o l u t e requirement  decreased  (pH 4.2) or  the y i e l d markedly.  Oxygen  f o r the r e a c t i o n ( 8 2 ) .  F i g . I shows the e f f e c t o f i n c u b a t i o n time on the formation of w a t e r - s o l u b l e products  i n the presence  and absence o f g l u t a t h i o n e .  Maximum amount o f r a d i o a c t i v i t y i n the aqueous f r a c t i o n was o b t a i n e d w i t h i n the f i r s t  2 hours.  With g l u t a t h i o n e there i s a l i n e a r  i n c r e a s e w i t h time d u r i n g the f i r s t 20 minutes with a more g r a d u a l i n c r e a s e up to 2 hours.  With enzyme and s u b s t r a t e o n l y , there was  Figure lb.  The effect of time on the formation of water-soluble products from 16-^-estradiol-17p (10 Hg) by mushroom phenolase (1 mg). Separate incubation carried out In the presence and absence of reduced glutathione (lO" * M). 2  20  <&r-^-  40  60  Glutathione  ~ ® — N o  80 100 TIME (MINUTES)  (]Q"^  Glutathione  M)  120  140  TABLE I I E f f e c t of g l u t a t h i o n e or the s o l u b l e f r a c t i o n of the l i v e r formation of w a t e r - s o l u b l e l i v e r microsomes and  products  from 16-^C  on t h e i r i n t e r a c t i o n  Activator  +  -  the  e s t r a d i o l - 1 7 p by r a t  with p r o t e i n .  % o f added C i n aqueous f r a c t i o n a f t e r e x t r a c t i o n w i t h ether 1 4  Microsomes  on  (99)  7c i n aqueous f r a c t i o n bound to p r o t e i n  12.5  62.4  Glutathione (0.1 lpM)  49.6  4.9  +  Glutathione (0P1 uiM)  30.6  16.6  +  Soluble liver  f r a c t i o n of  37.2  17.5  mm  Soluble liver  f r a c t i o n of  1.2  Microsomes from 50 mg TENH (0.3 uM)  and  liver  incubated w i t h i ^ C e s t r a d i o l  soluble f r a c t i o n  (from 50 mg  (10[xg) .  l i v e r ) or g l u t a t h i o n e .  1  -29a l i n e a r i n c r e a s e d u r i n g the f i r s t 10 minutes f o l l o w e d by a g r a d u a l i n c r e a s e f o r the l e n g t h o f time t h a t the experiment was  carried out. Optimal c o n d i t i o n s f o r the formation  metabolites  of water-soluble  by the microsomal system have been worked out p r e v i o u s -  l y by J e l l i n c k and Lazier.--(82)... NADPH and oxygen were r e q u i r e d f o r t h i s system, the optimal- c o n c e n t r a t i o n o f NADPH being 1 mg i n 4 ml of i n c u b a t i o n m i x t u r e .  The optimum pH was a l s o found t o be 7.4.  '. E f f e c t o f g l u t a t h i o n e on t h e y i e l d o f w a t e r - s o l u b l e  estrogen  d e r i v a t i v e s formed by r a t l i v e r microsomes and phenolase. T a b l e I I shows the e f f e c t o f g l u t a t h i o n e and the s o l u b l e f r a c t i o n o f the c e l l microsomal system.  (100,000 x g supernatant  f r a c t i o n ) i n the  "When t h i s t r i p e p t i d e was omitted  from the  r e a c t i o n , v e r y l i t t l e r a d i o a c t i v i t y c o u l d be found i n the aqueous f r a c t i o n , most o f i t b e i n g  bound t o p r o t e i n .  In the presence o f  g l u t a t h i o n e however, the r a d i o a c t i v i t y i n the aqueous f r a c t i o n i n c r e a s e d p r o p o r t i o n a l l y w i t h the c o n c e n t r a t i o n o f g l u t a t h i o n e present.  The s o l u b l e f r a c t i o n o f the l i v e r had a s i m i l a r  activa-  t i n g e f f e c t but was i n e f f e c t i v e when the i n c u b a t i o n was c a r r i e d out without  the microsomes.  the y i e l d of w a t e r - s o l u b l e  I t i s of interest products  t o note t h a t as  i n c r e a s e s w i t h added glutathione  or s o l u b l e f r a c t i o n , the amount o f r a d i o a c t i v i t y bound t o p r o t e i n  that i s a c t u a l l y  decreases.  S i m i l a r r e s u l t s were obtained when the microsomal system was  r e p l a c e d by mushroom phenolase (Table I I I ) . A g a i n g l u t a t h i o n e  and  the c e l l sap showed a marked a b i l i t y t o i n c r e a s e the y i e l d o f  TABLE I I I E f f e c t o f g l u t a t h i o n e or the s o l u b l e f r a c t i o n of the l i v e r on the formation  of water-soluble  p r o d u c t s from  phenolase and on t h e i r i n t e r a c t i o n  with p r o t e i n .  % added C i n the aqueous f r a c t i o n a f t e r e x t r a c t i o n w i t h ether 1 4  Activator  e s t r a d i o l by mushroom  % C i n aqueous f r a c t i o n bound to protein  33.5 Glutathione  79.6  Soluble f r a c t i o n of l i v e r  79.3  Bovine serum albumin  71.1  74.1  Bovine serum albumin + glutathione  78.6  2.9  Phenolase (1 mg) incubated w i t h 1 6 - C e s t r a d i o l - 1 7 p (10 |ig) and/or g l u t a t h i o n e (0.1 uM), bovine serum albumin (5 mg) and the s o l u b l e f r a c t i o n (from 50 mg of r a t l i v e r ) . 1  TABLE IV E f f e c t o f o x i d i z e d g l u t a t h i o n e and N-ethylmaleimide (NEM) on the formation  of water-soluble  products  by mushroom phenolase.  14 7o added C i n aqueous fraction after extraction w i t h ether  Compounds  33,5 GSSG ( o x i d i z e d g l u t a t h i o n e )  37,7  GSSG + NEM  32.2  GSH (reduced  85.2  glutathione)  30.2  GSH + NEM  Phenolase incubated w i t h 16- ^C (0.1 uM) and 10" M NEM. L  2  e s t r a d i o l - 1 7 p (10 (ig) g l u t a t h i o n e  -30water-soluble  metabolites.  Bovine serum albumin was  added source of p r o t e i n s i n c e the c o n c e n t r a t i o n employed was reaction.  used as  an  of t y r o s i n a s e  inadequate to demonstrate p r o t e i n p r e c i p i t a t i o n  When g l u t a t h i o n e was  omitted,  most of the r a d i o a c t i v i t y  c o u l d be accounted f o r by protein-bound d e r i v a t i v e s , but i n i t s presence, most of the r a d i o a c t i v i t y i n the aqueous f r a c t i o n found i n unknown m e t a b o l i t e s The  not a s s o c i a t e d w i t h p r o t e i n .  involvement of the amino group and/or the  group were suspected,  and  with oxidized glutathione  sulphydryl  f o r t h i s r e a s o n , phenolase was  incubated  (10" M) and reduced g l u t a t h i o n e  (10~ M)  4  4  i n the presence and absence of N-ethylmaleimide (10" M).  The  2  r e s u l t as shown i n T a b l e i s probably  was  IV i n d i c a t e s t h a t the s u l p h y d r y l group  i n v o l v e d s i n c e o x i d i z e d g l u t a t h i o n e had no e f f e c t  on  i n c r e a s i n g water s o l u b i l i t y and a l s o N-ethylmaleimide which binds s u l p h y d r y l groups i n h i b i t e d the r e a c t i o n . The  e f f e c t of other  y i e l d of w a t e r - s o l u b l e mal  s u l p h y d r y l compounds on i n c r e a s i n g the  metabolites  and phenolase systems, and  T a b l e s V and V I .  was  t e s t e d i n both the microso-  the r e s u l t s are i n d i c a t e d i n  W i t h the microsomal system, c y s t e i n e had  e f f e c t w h i l e homocysteine gave o n l y a s l i g h t i n c r e a s e and t h i o n e i n e and  t h i o l h i s t i d i n e i n h i b i t e d the r e a c t i o n .  phenolase i n c u b a t i o n s however, a s i m i l a r p a t t e r n was w i t h c y s t e i n e and homocysteine, but e r g o t h i o n e i n e  In  aqueous and  ether  ergothe  observed  and  a l s o produced an i n c r e a s e i n the y i e l d of w a t e r - s o l u b l e though to a l e s s e r extent  no  thiolhistidine products  than g l u t a t h i o n e . . Chromatography of  f r a c t i o n s o f b o t h systems i n which c y s t e i n e ,  the  TABLE V E f f e c t o f s u l p h y d r y l compounds on the y i e l d o f w a t e r - s o l u b l e m e t a b o l i t e s by r a t l i v e r microsomes.  Activator  -  % added r a d i o a c t i v i t y i n aqueous f r a c t i o n  34.5  Cysteine  33.5  Homocysteine  43.8  Ergothioneine  17.7  Thiolhistidine  14.8  Glutathione  67.5  S u l p h y d r y l compounds (10" M) incubated w i t h 16- C e s t r a d i o l (10 |ig), NADPH (1 mg) and r a t l i v e r microsomes (from 50 mg o f l i v e r ) . Conditions described i n text.  TABLE VI  E f f e c t o f s u l p h y d r y l compounds on the y i e l d o f w a t e r - s o l u b l e m e t a b o l i t e s by mushroom  phenolase.  Activator  7o added r a d i o a c t i v i t y i n aqueous f r a c t i o n  21.9 Cysteine  20.8  Homocysteine  39.6  Ergothioneine  52.6  L-2-thiolhistidine  49.4  Glutathione  85.2  S u l p h y d r y l compounds (10' incubated w i t h 1 6 - C e s t r a d i o l and mushroom phenolase (1 mg). 14  (10 p,g)  F i g u r e 2. t P a p l r c h r o m a t o g r a p h y o f t h e a q u e o u s f r a c t i o n o b t a i n e d a f t e r I n c u b a t i o n o f v a r i o u s t h i o l s w i t h p h e n o l a s e (1 mg), and 1 6 - ' ^ - e s t r a d l o ! - 1 7 ( 3 (10 H g ) I n t h e p r e s e n c e and a b s e n c e o f NAOPH2. ( M o d i f i e d 8ush.)  PHSK  Abbreviations:  PHCH  EftC  f*0  KASPM2  PHEW P « « H  '  T  T  PHEX  H.CYST J  T  "  M  F  H  2  n*» C  «  T  CYST  ••Hi  M  "MM'  „  HAOPNJ  PHEN • p h e n o l a s e (1 mg) CYST - c y s t e i n e h y d r o c h l o r i d e (10-4 M) H.CYST - h o m o c y s t e i n e h y d r o c h l o r i d e (10*4 M) ERGO e r g o t h l o n e l n e (10-4 M) a  -31homocysteine, e r g o t h i o n e i n e and way  t h i o l h i s t i d i n e were added i n no  i n d i c a t e d a c o n j u g a t i o n r e a c t i o n between the s u b s t r a t e and  these compounds.  The aqueous f r a c t i o n s , developed  a c e t i c acid-water  system (descending)  on the b u t a n o l -  d i d not g i v e o n l y d i s c r e t e  r a d i o a c t i v e spots of the type formed w i t h g l u t a t h i o n e but i n s t e a d s t r e a k s near the s o l v e n t f r o n t .  Chromatography o f the e t h e r e a l  f r a c t i o n i n the M o d i f i e d Bush System i n d i c a t e d t h a t the  products  i n t h i s f r a c t i o n c o n s i s t e d mainly of 2-hydroxyestradiol-17p, estrone and unreacted was  estradiol  (Fig. 2).  The  some  2-hydroxyestradiol  d e t e c t e d by comparison w i t h standard 2 - h y d r o x y e s t r a d i o l which  i n t u r n was  d e t e c t e d by F o l i n C i o c a l t e u r e a g e n t .  r e s u l t e d i n an i n c r e a s e d p r o d u c t i o n of  A d d i t i o n of TPNH  2-hydroxyestradiol-17f3.  The p o s s i b i l i t y a l s o e x i s t s t h a t i f these s u l p h y d r y l compounds other than g l u t a t h i o n e , became conjugated w i t h e s t r a d i o l ,  they  might be dragged i n t o the ether f r a c t i o n because of t h e i r  small  r e g i o n of p o l a r i t y r e l a t i v e to the r e s t of the non-polar molecule.  F o r t h i s r e a s o n , the ether f r a c t i o n was  b u t a n o l - a c e t i c acid-water r u l e d out any  steroid  a l s o r u n on  the  system, but the a u t o r a d i o g r a p h i c r e s u l t s  such r e a c t i o n .  T h e r e f o r e i t was  f e l t t h a t the r e a c t i o n was  i n some way  s p e c i f i c f o r g l u t a t h i o n e , p r o b a b l y because of i t s h i g h redox potential  (Eo'pH 7.0  + 0.03)  compared to the other t h i o l compounds.  A p o s s i b l e c o n j u g a t i o n between the s u l p h y d r y l group o f the t i d e and the s u b s t r a t e was  suggested.  With t h i s i n mind, the remaining determine whether t h e r e was  tripep-  experiments were designed  a c t u a l combination  to  w i t h g l u t a t h i o n e or  Figure 3.  Column chromatography of the aqueous f r a c t i o n s of phenolase and the microsomal system of Sephadex G-25. Each system was Incubated with ^ H - e s t r a d l o l 17p (10 ug) and 35s glutathione (10 * M). Column size = 39 x 1.8 cm; eluant d i s t i l l e d H2O. Conditions described In t e x t . -1  3  EFFLUENT  (ML)  -32whether t h i s t r i p e p t i d e was e x e r t i n g i t s e f f e c t by some type o f a c t i v a t i o n mechanism. Chromatography and E l e c t r o p h o r e s i s : Because o f the l a r g e degree o f c r o s s l i n k a g e , Sephedex G-25 columns were used f o r the s e p a r a t i o n and i s o l a t i o n o f m e t a b o l i t e s i n the aqueous f r a c t i o n . Incubations  o f the phenolase and microsomes gave s i m i l a r  e l u t i o n p a t t e r n s as can be observed from F i g . 3. determined by t r i t i u m and 5 g assay; 3  obtained  two major peaks were  i n e i t h e r case, n e i t h e r o f which corresponded to ~>S3  g l u t a t h i o n e or S - o x i d i z e d J J  assay.  Peaks were  g l u t a t h i o n e as i n d i c a t e d by  J  S  (The o x i d i z e d and the reduced forms o f the r a d i o a c t i v e  g l u t a t h i o n e were d e t e c t e d by the a d d i t i o n o f n o n - r a d i o a c t i v e g l u t a t h i o n e , the p o s i t i o n s o f which were determined by n i n h y d r i n color.)  However, the l a r g e s t peak obtained  i n both cases appears  s h o r t l y a f t e r t h a t o f unchanged g l u t a t h i o n e . When the microsomal p r e p a r a t i o n was incubated c o n d i t i o n s w i t h g l u t a t h i o n e omitted,  under  identical  most o f the r a d i o a c t i v i t y was  found i n a pro t e i n peak emerging a t the f r o n t o f the column, b u t some w a t e r - s o l u b l e  products o f e s t r a d i o l i n a n i n h y d r i n  negative  r e g i o n behind g l u t a t h i o n e was a l s o observed. With the phenolase system ( F i g . 4 ) , bovine serum albumin was added t o the i n c u b a t i o n mixture t o demonstrate t h i s t i o n f o r p r o t e i n b i n d i n g when g l u t a t h i o n e was p r e s e n t .  competiAgain i t  can be seen t h a t i n the absence o f g l u t a t h i o n e , most o f the r a d i o a c t i v i t y was a s s o c i a t e d w i t h p r o t e i n , b u t on a d d i t i o n o f the t r i p e p t i d e , there was a s h i f t o f r a d i o a c t i v i t y from the p r o t e i n  Figure  Column chromatography of the aqueous fraction demonstrating competition for protein binding In the presence of glutathione. (Sephadex G-25; column size " 38 x 1.8 cm; eluant - d i s t i l l e d water.)  16  14  12  10  o. °  8  6  k  2  0 EFFLUENT  (ML 1  Figure 5 .  Descending paper chromatography o f t h e aqueous f r a c t i o n s o f the f o l l o w i n g incubation mixtures in n-butanol: a c e t i c a c i d : H 0 (12-15 h o u r s ) . 2  PHEN GS*H  GS*JH  PHEN  PHEN  PHEN  DIOL GS^Trl  OIOL* gib*.  DIOL* GSH  PHEN DIOL*  0 Q 0 Q  Q  2331^  5065  Ratio o f  15W  12834  0  Abbreviations:  55T  Phen = p h e n o l a s e (I mg) G*SH = 35S g l u t a t h i o n e ( 1 0 * M) DIOL = e s t r a d i o l - 1 7 8 (10 Hg) -1  R a t i o o f l^C. = 8 . 5 35 S  - 3 3 f r a c t i o n to some m e t a b o l i t e  or group of m e t a b o l i t e s  having  a  m o b i l i t y s i m i l a r to g l u t a t h i o n e . Paper chromatography o f the aqueous f r a c t i o n i n b u t a n o l a c e t i c acid-water was there was  any  glutathione.  c a r r i e d out i n order  to determine whether  t r i t i u m a s s o c i a t e d w i t h compounds c o n t a i n i n g A c o n s i d e r a b l e amount of t r i t i u m was  ~*S-  3  found i n the  same r e g i o n as reduced g l u t a t h i o n e , but another m e t a b o l i t e t r i t i u m l a b e l l e d e s t r a d i o l associated with T h i s compound had an  v a l u e of 0.68.  was  the  a l s o found.  Both the microsomal  the phenolase systems gave s i m i l a r r e s u l t s except t h a t compound which appears to be a s s o c i a t e d w i t h reduced was  of  and  the  glutathione  the major product i n the microsomal system. The r a t i o of -*S 3  to H 3  i n the spots was  determined by  chromatography of the aqueous f r a c t i o n f o l l o w e d by e l u t i o n of r a d i o a c t i v e areas and t h a t the r a t i o of H 3  r a t i o i n spot A, and i n t h i s system.  i s o t o p e assay ( F i g . 5 ) . to  i n spot B i s twice as l a r g e as  seen this  t h a t l i k e o x i d i z e d g l u t a t h i o n e , A runs slower  T h i s procedure was  u s i n g ^ C - e s t r a d i o l and 4  I t can be  repeated  on a l a r g e s c a l e  spots A and B were e l u t e d  separately.  The u l t r a v i o l e t s p e c t r a of both areas gave no c l e a r peaks but blank i t s e l f c o n t a i n e d much a b s o r b i n g  the  material i n this region.  . When paper chromatographic f r a c t i o n s A and B of the phenolase system were run s e p a r a t e l y on Sephadex columns, i t was f r a c t i o n A c o n s i s t e d of 3 peaks (A]_, A , 2  A3)  found t h a t  ( F i g . 6 ) , the l a r g e s t  peak g i v i n g the most p o s i t i v e n i n h y d r i n t e s t . . F r a c t i o n B however was  found to be homogeneous and a l s o n i n h y d r i n p o s i t i v e ( F i g . 7 ) .  Figure 6.  Fraction A ( F i g . 5) of the aqueous f r a c t i o n of phenolase incubation on Sephadex G-25. (Column size ° Wi x. 1.8 cm; eluant - d i s t i l led w a t e r . )  EFFLUENT (ML)  Figure 7.  Fraction B ( F i g . 5) of the aqueous f r a c t i o n of phenolase Incubation In Sephadex G-25. (Column size - 38 x 1.8 era; eluant - d i s t i l l e d water.)  [0.2k  0.20 a E  'o LTV  ul  EFFLUENT (ML)  Figure 8.  Fraction A of the aqueous f r a c t i o n of the microsomal Incubation on Sephadex G-25. (Size of colunai - hi x 1.8 cm; eluant - d i s t i l l e d w a t e r . )  -34-  A r u n o f standard o x i d i z e d and reduced  glutathione indicated that  any unreacted m a t e r i a l as w e l l as the o x i d i z e d t r i p e p t i d e o c c u r r e d f u r t h e r towards the f r o n t than A 3 or B, suggesting t h a t the major peak may  be the one a s s o c i a t e d w i t h g l u t a t h i o n e , and the other  two peaks were some other type of w a t e r - s o l u b l e d e r i v a t i v e s o f estradiol. A r u n o f f r a c t i o n A o f the microsomal i n c u b a t i o n ( f r a c t i o n B was  n o t t e s t e d s i n c e i t appeared to be a minor m e t a b o l i t e i n  t h i s system) l e d to a s i m i l a r type of r e s u l t  (Fig. 8).  I t has been shown e a r l i e r i n t h i s t h e s i s t h a t the 100,000 x g supernatant  fraction reacted l i k e glutathione i n increasing 1  the y i e l d o f w a t e r - s o l u b l e products and d e c r e a s i n g the of those bound to p r o t e i n .  percentage  Chromatography of the aqueous f r a c t i o n  r e s u l t i n g from an i n c u b a t i o n w i t h added c e l l sap gave one major p r o d u c t having an system.  v a l u e o f 0.55  acid-water  T h i s r e g i o n of r a d i o a c t i v i t y d i d not correspond  spots A or B formed from -^C tography  i n n-butanol-acetic  e s t r a d i o l by phenolase,  to e i t h e r  but chroma-  of t h i s r e g i o n on Sephadex gave r i s e to.one major peak  s i m i l a r i n p o s i t i o n to peak A 3 o f the microsomal system ( F i g . 9 ) . Smaller areas a- and b were found to be bound mainly to p r o t e i n . A s i m i l a r type of r e s u l t was  o b t a i n e d when the supernatant  was  added to the microsomes ( F i g . 10). The  l a s t experiment to determine the a c t i o n of g l u t a t h i o n e  i n these systems was  to s u b j e c t the aqueous f r a c t i o n s to e l e c t r o -  p h o r e s i s and to compare the m o b i l i t i e s of the products w i t h t h a t o f g l u t a t h i o n e added as standard  ( F i g . 11).  E l e c t r o p h o r e s i s was  Figure 9.  Chromatogrephy of the aqueous f r a c t i o n obtained after Incubation of phenolase (1 mg) and the 100.000 x g supernatant (from 50 mg of l i v e r ) , and l6-' *C estrad i o l -178 (10 H9> ° " Sephadex G-25. (Size of column = ^3 x 1.8 cm; elu»nt - d i s t i l l e d water.) /  28-  EFFLUENT (ML)  Figure 10. Chromatography of the aqueous fraction obtained after Incubation of microsomes and 100,000 x g supernatant (each f r a c t i o n obtained from 50 mg of t i s s u e ) , NADPH2 (1 rag) and 16-1^-estradlol-17B (10 H9) on Sephadex G-25, (Size of column • ^ x 1.8 cm; eluant " d i s t i l l e d water.)  30  50  To~ EFFLUENT  70 (ML)  K>0~  Figure 11. Electrophoresis of the aqueous f r a c t i o n s . o f the f o l l o w i n g Incubat ions: 1.  STANDARD G35SH  2.  PHENOLASE (I mg) incubated with GSH (10-^ M) and 16^ C - e s t r a d i o l - 1 7 p (10 ug)  3.  MICROSOMES (50 mg) incubated with GSH (lO'^M) NADPH2 (1 mg) and l 6 - ' 4 c estradiol-178 (10 ug)  4.  STANDARD l 6 - C - e s t r a d i o l - 1 7 p ,/+  -35c a r r i e d out a t pH 3.5 i n which reduced  g l u t a t h i o n e migrates t o  the anode, i n d i c a t i n g t h a t i t i s n e g a t i v e l y charged a t t h i s pH. A number o f w a t e r - s o l u b l e products were shown up by t h i s method, but more m e t a b o l i t e s c o u l d be d e t e c t e d a f t e r i n c u b a t i o n w i t h phenolase  than w i t h the microsomes, a f t e r i n c u b a t i n g  w i t h e i t h e r phenolase  or l i v e r microsomes.  product formed i n e i t h e r system appeared  1 4  C-estradiol  However, the major  t o be i d e n t i c a l .  With microsomes, a l l t h e m e t a b o l i t e s were n e g a t i v e l y charged a t pH 3.5 w h i l e phenolase  was capable o f m e t a b o l i z i n g the s u b s t r a t e  i n t o b o t h n e g a t i v e l y and p o s i t i v e l y charged  d e r i v a t i v e s a t t h i s pH.  E s t r a d i o l a l s o c a r r i e s a p o s i t i v e charge a t pH 3.5. The a d d i t i o n o f t h e 100,000 x g supernatant f r a c t i o n i n these i n c u b a t i o n s was n o t c a r r i e d f u r t h e r s i n c e l a r g e amounts o f l i p i d c o n t a i n i n g m a t e r i a l made i t extremely d i f f i c u l t t o p u r i f y the aqueous f r a c t i o n s s u f f i c i e n t l y to c h a r a c t e r i z e the p r o d u c t s . Protective A c t i o n of Glutathione:I f estrogens a r e o x i d i z e d to r e a c t i v e i n t e r m e d i a t e s which can i n t e r a c t w i t h p r o t e i n , they might a l s o i n a c t i v a t e  gonadotropins  i n t h i s manner, and t h i s i n a c t i v a t i o n should be r e v e r s e d by glutathione i f conjugation with t h i s peptide occurs.  The p r o t e c t i v e  a c t i o n o f g l u t a t h i o n e on Pregnant Mare's Serum (PMS) i n a c t i v a t i o n by o x i d i z e d estrogens can be observed  from T a b l e V I I .  With PMS  o n l y , t h e r e was a marked i n c r e a s e i n the s i z e o f the o v a r i e s over those o f the c o n t r o l .  When PMS was incubated w i t h  estradiol,  t h e r e was no i n a c t i v a t i o n s i n c e there was no enzyme p r e s e n t to o x i d i z e the s u b s t r a t e t o m e t a b o l i t e s capable o f b r i n g i n g about the  TABLE V I I I n a c t i v a t i o n o f PMS by estrogen o x i d a t i o n products and p r o t e c t i o n by g l u t a t h i o n e . Immature female r a t s were each i n j e c t e d subcutaneously w i t h PMS (25 i . u . ) incubated w i t h phenolase (0.167 mg) and estrogen (10 |xg) . C o n d i t i o n s as d e s c r i b e d i n t e x t .  Compound  Phenolase  Control (buffer) PMS  PMS + e s t r a d i o l  PMS + 2-hydroxy estradiol  PMS + e s t r a d i o l + glutathione (0.5 mM)  *Mean + Standard  Error  No. o f r a t s  Ovarian wt.*  -  13  23.3  +  2.0  14  118.3  +  5.7  +  8  114.2  +  4.0  -  4  116.2  +  8.9  +  14  47.4  ±  2.4  -  6  115.7  +  11.4  +  6  63.7  +  6.3  +  8  109.7  +  15.5  -36inactivation process.  However, i n t r o d u c t i o n o f phenolase l e d to  a marked decrease i n the a c t i v i t y o f PMS even though the enzyme by i t s e l f had no i n a c t i v a t i n g  effect.  2 - H y d r o x y e s t r a d i o l was found to be no more a c t i v e than e s t r a d i o l i n t h i s r e g i o n even though i t might be expected to be more r e a d i l y converted t o the quinone . Attempts  than the p a r e n t e s t r o g e n .  to i n h i b i t . g o n a d o t r o p i c a c t i v i t y by estrogen  "quinones" i n v i v o have so f a r proved u n s u c c e s s f u l b u t these o x i d a t i v e p r o d u c t s may o n l y be a c t i v e i f produced  i n situ.  -37DISCUSSION Mammalian l i v e r has the a b i l i t y to c o n v e r t n a t u r a l estrogens to a v a r i e t y o f products which can be b r o a d l y c l a s s i f i e d  into  w a t e r - s o l u b l e and e t h e r - s o l u b l e m e t a b o l i t e s . r ; V a r i o u s workers (15, 20) have shown t h a t l i v e r microsomes c o n t a i n a number of hydroxylases and dehydrogenases which c a t a l y z e the c o n v e r s i o n o f the s t e r o i d s u b s t r a t e to these The  products.  e t h e r - s o l u b l e m e t a b o l i t e s c o n s i s t mainly o f r i n g D  h y d r o x y l a t e d p r o d u c t s , 2-methoxy (10, 11) and 2-hydroxy d e r i v a t i v e s as w e l l as s m a l l amounts of 6e$ and  663 hydroxy s t e r o i d have been  identified. The w a t e r - s o l u b l e m e t a b o l i t e s of estrogens may s u b d i v i d e d i n t o protein-bound  be f u r t h e r  d e r i v a t i v e s , s u l f u r i c and g l u c u r o n i c  a c i d conjugates and a t h i r d type the n a t u r e of which i s y e t unknown but which seem, to be u n u s u a l l y r e s i s t a n t to chemical  and  enzymatic h y d r o l y s i s . Jellinck  (83) has r e c e n t l y shown t h a t a marked sex d i f f e r e n c e  e x i s t s i n the r a t e of c o n v e r s i o n of estrogens to w a t e r - s o l u b l e p r o d u c t s by r a t l i v e r microsomes. estrone and e s t r a d i o l were found  The products  formed from  to be devoid o f e s t r o g e n i c  a c t i v i t y suggesting t h a t t h i s r e a c t i o n which occurs i n the male r a t l i v e r may  be of p h y s i o l o g i c a l importance i n i n a c t i v a t i n g  endogenous e s t r o g e n s . observed  J e l l i n c k and L a z i e r (84) have a l s o  a marked s p e c i e s d i f f e r e n c e i n estrogen, metabolism  between the r a t and  the guinea p i g both i n v i v o and i n i n c u b a t i o n s  involving subcellular fractions.  They found t h a t a d m i n i s t r a t i o n  -38of  l ^ C e s t r o n e to r a t s gave n e g l i g i b l e q u a n t i t i e s of  sulfate  and g l u c o s i d u r o n a t e i n the u r i n e , and a l t h o u g h l i v e r microsomes had the a b i l i t y to conjugate r e a c t i o n was  estrogen with glucuronic a c i d ,  a b o l i s h e d i f the s o l u b l e f r a c t i o n o f the c e l l  this was  a l s o present... On the other hand, l a r g e amounts of normal estrogen conjugates were formed by the guinea p i g both i n v i v o and  in vitro. In the work undertaken,  optimum c o n d i t i o n s f o r then formation  of water s o l u b l e products by the phenolase the r e a c t i o n had a b s o l u t e requirements not a f f e c t the r e a c t i o n to any e x t e n t .  system were worked out;  f o r oxygen but NADPrL? d i d The requirements  for this  c o n v e r s i o n by the microsomal system were found to be i n g e n e r a l agreement w i t h the work o f J e l l i n c k and L a z i e r (84). Reduced g l u t a t h i o n e or the s o l u b l e f r a c t i o n o f the  cell  showed a marked a b i l i t y to i n c r e a s e the y i e l d of w a t e r - s o l u b l e m e t a b o l i t e s i n both the microsomal and phenolase  systems.  i n c r e a s e i n water s o l u b i l i t y p a r a l l e l e d a decrease  This  i n the p r o t e i n  b i n d i n g r e a c t i o n i n e i t h e r system, i n d i c a t i n g t h a t a r e a c t i o n which competes w i t h p r o t e i n b i n d i n g of estrogen m e t a b o l i t e s must be o c c u r r i n g when the t r i p e p t i d e or the s o l u b l e f r a c t i o n of the c e l l i s present. I t was  decided to determine the mechanism i n v o l v e d i n the  " g l u t a t h i o n e r e a c t i o n " s i n c e i t was  f e l t that t h i s sulphydryl  compound might be i n c r e a s i n g water s o l u b i l i t y by a c o n j u g a t i o n reaction. As f a r as the phenolase  system i s concerned,  there i s e v i -  -39dence t h a t t h i s oxygenase c a t a l y z e s the aromatic h y d r o x y l a t i o n o f r i n g A o f the s t e r o i d a t C-2 f o l l o w e d by subsequent o x i d a t i o n o f the h y d r o x y l a t e d compound t o a " q u i n o i d " d e r i v a t i v e .  Since  g l u t a t h i o n e c o n t a i n s an SH group, and quinones a r e known t o r e a c t r e a d i l y w i t h SH compounds (85), i t was concluded combination  t h a t t h e r e was  between the q u i n o i d i n t e r m e d i a t e and the t r i p e p t i d e .  T h i s theory was supported by the f a c t t h a t o x i d i z e d g l u t a t h i o n e had no e f f e c t i n i n c r e a s i n g the y i e l d o f w a t e r - s o l u b l e m e t a b o l i t e s and N-ethylmaleimide  which binds s u l p h y d r y l compounds was  found  to be a s t r o n g i n h i b i t o r o f the r e a c t i o n . F i g g e and A l l e n  (86) have e a r l i e r demonstrated t h a t  g l u t a t h i o n e completely i n h i b i t e d the f o r m a t i o n o f melanin by t y r o s i n a s e , but when estrone and g l u t a t h i o n e were added i n equimolar  q u a n t i t i e s t o a t y r o s i n e - t y r o s i n a s e mixture,  the e s t r o n e  r e l e a s e d the enzyme from g l u t a t h i o n e i n h i b i t i o n .  The r e a c t i o n s  i n v o l v e d were n o t e l u c i d a t e d but i t was suggested  t h a t the enzyme  o x i d i z e d r i n g A o f e s t r o n e to a quinone which then r a p i d l y o x i d i z e d g l u t a t h i o n e and thus r e l i e v e d the i n h i b i t i o n . . However, t h i s does n o t appear t o be the mechanism o c c u r r i n g i n the formation o f w a t e r - s o l u b l e products from estrogens  s i n c e such a r e a c t i o n would  l e a d t o an i n c r e a s e d p r o d u c t i o n o f 2-hydroxy d e r i v a t i v e s ,  thereby  d e c r e a s i n g the y i e l d o f w a t e r - s o l u b l e m e t a b o l i t e s and i n c r e a s i n g the percentage  of  i n the e t h e r e a l f r a c t i o n .  I t i s more  l i k e l y t h a t c o n j u g a t i o n between g l u t a t h i o n e and the estrogen quinone had o c c u r r e d . C y s t e i n e had no e f f e c t on the r e a c t i o n , b u t t h i s may be due  -40to i t s higher redox p o t e n t i a l which would make i t more e a s i l y o x i d i z a b l e than g l u t a t h i o n e . . C a l v i n (87) has g l u t a t h i o n e may  suggested t h a t  not be a simple s t r u c t u r e w i t h a f r e e c y s t e i n e  mercaptan group, but i s i n v o l v e d i n some type o f o f the mercaptan; t h i s p r o c e s s does not compounds.  He  stabilization  occur i n other  thiol  f e l t t h a t t h i s enhanced s t a b i l i t y might be brought  about by an i n t e r a c t i o n between the c a r b o x y l oxygen of the  y-  g l u t a m y l r e s i d u e and  the  the SH group o f the c y s t e i n e r e s i d u e ,  r e s u l t being a t h i a z o l i d i n e d e r i v a t i v e . precedes n u c l e a r may  I f t h i s type o f r e a c t i o n  s u b s t i t u t i o n , then the r e a c t i o n w i t h  glutathione  w e l l i n v o l v e a s t a b i l i z a t i o n r e a c t i o n i n which g l u t a t h i o n e i s  protected  from o x i d a t i o n .  GSSG A l t h o u g h Barron and  Singer  (88)  i s not a s u l p h y d r y l enzyme, no one  have shown t h a t phenolase  has  claimed  t h a t t h i o l groups  are completely absent from the enzyme.  I t has been concluded  t h a t i f there a r e t h i o l groups p r e s e n t ,  they are not i n v o l v e d i n  the f i x a t i o n of the copper which i s the p r o s t h e t i c group of the enzyme.  Kubowitz (90) and  K e i l i n and Mann (91) have shown  (89)  -41t h a t the metal group o f the enzyme i s i n the b i v a l e n t s t a t e and t h a t the c a t a l y t i c a c t i v i t y o f phenolase i s based on a.cupriccuprous v a l e n c y ( C U + + )  change.  2-enzyme + O-dihydroxyphenol = (Cu+) 2-enzyme + 0 - q u i n o n e + 2H+  (cu ) +  e n z  2  y  m e  •+ i /  2  °2  +  2  H  = (Cu  +  4 +  )2-  e n z  y  m e  +  J o l l e y and Mason (92) have shown by means o f e l e c t r o p h o r e s i s t h a t v a r i o u s s p e c i e s o f mushroom t y r o s i n a s e c o n t a i n isoenzymes which a r e t o a c e r t a i n degree i n t e r c o n v e r t a b l e depending on the pH, i o n i c s t r e n g t h and p r o t e i n c o n c e n t r a t i o n . 5-6 d i f f e r e n t ratios  E q u i l i b r i u m among the  isoenzymes a r e c h a r a c t e r i z e d by d e f i n i t e  o f c r e s o l a s e a c t i v i t y and c a t e c h o l a s e a c t i v i t y .  concentration They  suggested v a r i o u s reasons f o r the m u l t i p l i c i t y o f t y r o s i n a s e s . 1)  v a r i o u s degrees o f p o l y m e r i z a t i o n  2)  v a r i o u s combinations o f u n l i k e  3)  conformational  4)  combination o f these The  subunits  changes o f a s i n g l e p r o t e i n three.  e x i s t e n c e o f u n l i k e s u b u n i t s , one l a r g e l y w i t h c r e s o l a s e  a c t i v i t y and the other w i t h c a t e c h o l a s e a c t i v i t y combining i n s e v e r a l p r o p o r t i o n s may e x p l a i n the observed d i f f e r e n c e s among the m u l t i p l e forms o f t y r o s i n a s e towards mono- and d i - p h e n o l s , and r e p o r t s o f a c t i v e enzymes w i t h low molecular c o u l d be a f f e c t i n g  weights.  Glutathione  t h i s r a t i o d i r e c t l y or i n d i r e c t l y .  In the case o f the microsomes which c o n s i s t o f a number o f enzyme systems, the s i t u a t i o n  i s more complex.  King  t h a t the l i v e r microsomal system i s a b l e t o c o n v e r t and  other estrogens  to ortho-hydroxylated  (15) has shown estradiol-17p  d e r i v a t i v e s , and the  PROPOSED PATHWAYS FOR THE FORMATION OF WATER-SOLUBLE ESTROGEN CONJUGATES WITH "GLUTATHIONE" AND PROTEIN BY PHENOLASE AND RAT LIVER MICROSOMAL ENZYMES  -42p o s s i b i l i t y t h e r e f o r e e x i s t s t h a t c o n j u g a t i o n may way  o f an o r t h o - q u i n o i d i n t e r m e d i a t e .  take p l a c e by  On the-other hand, the  r e a c t i o n might i n v o l v e the f r e e r a d i c a l mechanism proposed  by  Hecker (32) to account f o r the formation of the 2- and 6-hydroxyl a t e d estrogens and e s t r a - p - q u i n o l . r e q u i r e s NADPH2 and molecular  T h i s second r e a c t i o n a l s o  and a c t s by forming the phenoxy  r a d i c a l of the e s t r o g e n which tends to s t a b i l i z e i t s e l f c o n v e r s i o n i n t o mesomeric forms.  by  T h i s r a d i c a l can then r e a c t  w i t h f r e e hydroxy1 groups to y i e l d the observed hydroxy l a t e d products  (see T i g . 12).  However, the estrogen i n t e r m e d i a t e s should  a l s o be a b l e to combine w i t h s u l p h y d r y l groups which are known to be a c t i v e agents i n "mopping up" f r e e r a d i c a l s Isenberg  (93, 94).  (95) has claimed t h a t f r e e r a d i c a l s are e s s e n t i a l  i n t e r m e d i a t e s i n many m e t a b o l i c pathways, and p a r t i a l l y r e d u c i b l e but s t a b l e i n t e r m e d i a t e s can e x i s t between an o r g a n i c molecule and i t s f u l l y reduced form. o f e l e c t r o n s p i n resonance i n o x i d a t i v e metabolism,  H i s work f a c i l i t a t e d by the technique has shown the presence o f f r e e r a d i c a l s  and i t i s now  known t h a t they do occur i n  a number o f e n z y m a t i c a l l y c a t a l y z e d r e a c t i o n s . Another p o s s i b i l i t y i n v o l v e s epoxide f o r m a t i o n and and Sims (96) have claimed t h a t the s o l u b l e r a t l i v e r  Boyland  supernatant  can c a t a l y z e c o n j u g a t i o n of epoxides w i t h g l u t a t h i o n e . From a l l t h e o r i e s put forward and the r e s u l t s o b t a i n e d i n t h i s t e x t , c o n j u g a t i o n appears.to be the most f e a s i b l e r e a c t i o n occurring.  R e s u l t s w i t h column chromatography have shown t h a t the  major peak o b t a i n e d i n e i t h e r system d i d n o t t r a v e l f a s t e r  than  -43g l u t a t h i o n e as a n t i c i p a t e d but appeared behind these compounds. (97)  However G e l o t t e result.  He  has put  forward a theory  to e x p l a i n such a  observed t h a t aromatic and h e t e r o c y c l i c compounds  tend to be absorbed more s t r o n g l y to Sephadex than other of substances, and g l u t a t h i o n e and  t h e r e f o r e one would expect t h a t conjugates of  e s t r a d i o l would t r a v e l more s l o w l y than reduced  glutathione i t s e l f .  The  f a c t t h a t the p r o t e i n peak emerging a t  the f r o n t of the column i n the absence of g l u t a t h i o n e when the t r i p e p t i d e i s p r e s e n t The t i o n s of  J J  types  p o i n t s to a p o s s i b l e  determination  o f theviratio of ^S  S-glutathione  and  3  J  H  and  3  H  disappears conjugation.  after  incuba-  estrogens w i t h t y r o s i n a s e  or  l i v e r microsomes have i n d i c a t e d t h a t a d i - s u b s t i t u t e d as w e l l as a mono-substituted product might be formed, the former predominant.  One  would expect spot A to be  being  the d i s u b s t i t u t e d  d e r i v a t i v e as i n d i c a t e d by r a d i o a c t i v e assay s i n c e t h i s compound would have a m o b i l i t y s i m i l a r to t h a t o f g l u t a t h i o n e .  The  d i s u b s t i t u t e d product appears to be v i r t u a l l y the o n l y one  formed  i n the microsomal system. . E l e c t r o p h o r e s i s d i d not g i v e v e r y c l e a r cut r e s u l t s about the type o f r e a c t i o n t a k i n g p l a c e , a l t h o u g h i t " i s  q u i t e obvious  t h a t the major p r o d u c t i n e i t h e r system i n the presence o f g l u t a t h i o n e i s the same. glutathione  A t pH  3.5  which was  i s n e g a t i v e l y charged, and  used f o r t h i s  one would expect t h a t i t s  conjugates would a l s o be n e g a t i v e l y charged, and towards the anode.  technique,  therefore t r a v e l  However, not a l l n e g a t i v e l y charged  are n e c e s s a r i l y a s s o c i a t e d w i t h g l u t a t h i o n e .  No product  metabolites coincided  -44w i t h g l u t a t h i o n e but t h i s t r i p e p t i d e tagged t o a l a r g e nonp o l a r s t e r o i d molecule would p r o b a b l y have a d i f f e r e n t m o b i l i t y from the o r i g i n a l compound.  ( I t was a l s o observed t h a t e s t r a d i o l  17(3 migrated towards the cathode i n t h i s system.) S i n c e the r e a c t i o n p r o d u c t s formed i n the presence o f the c e l l sap appear to be s i m i l a r to t h a t formed w i t h g l u t a t h i o n e , i t was suggested t h a t g l u t a t h i o n e or some compound or group o f compounds w i t h s i m i l a r s t r u c t u r e a r e p r e s e n t f r a c t i o n o f the c e l l and a r e r e s p o n s i b l e type o f r e a c t i o n .  Glutathione  i s present  i n the supernatant  f o r b r i n g i n g about t h i s i n the c e l l i n r e l a t i v e -  l y l a r g e amounts (98) and i t i s found i n the s o l u b l e f r a c t i o n o f the c e l l . a f t e r c e n t r i f u g a t i o n .  However, paper chromatography has  shown t h a t one major product i s formed i n t h i s system, and t h a t the r e a c t i o n may i n v o l v e a compound other Turning  t o the q u e s t i o n  than  glutathione.  o f PMS i n a c t i v a t i o n by the estrogen  products formed i n the presence o f t y r o s i n a s e , i t was found t h a t hydroquinone must be o x i d i z e d t o benzoquinone b e f o r e i n a c t i v a t e PMS and  ( 7 6 ) . Since estradiol-17(3  i s converted t o 2 - h y d r o x y e s t r a d i o l  i t can f u l l y  i s a phenolic s t e r o i d  both i n v i v o and i n v i t r o ,  i t ©ould be f u r t h e r o x i d i z e d t o the "O-quinoid" d e r i v a t i v e as mentioned p r e v i o u s l y .  Mason (85) has e s t a b l i s h e d t h a t  thiols  r e a c t r e a d i l y w i t h quinones and g l u t a t h i o n e p r o b a b l y e x e r t s i t s p r o t e c t i v e a c t i o n a g a i n s t estrogen i n a c t i v a t i o n o f PMS i n t h i s manner.  T h i s type o f r e a c t i o n supports p r e v i o u s  conjugation  evidence f o r  between e s t r o g e n and g l u t a t h i o n e under the c o n d i t i o n s  o f the experiment.  -45SUMMARY The pathways i n v o l v i n g the formation metabolites  of e s t r a d i o l - 1 7 p may  three main types.  One  gates w i t h s u l f u r i c and  be b r o a d l y  of  water-soluble  c l a s s i f i e d into  pathway i n v o l v e s the formation  of  conju-  g l u c u r o n i c a c i d s , the r e a c t i o n being  c a t a l y z e d by microsomal enzymes, a second c o n s i s t i n g o f p r o t e i n bound d e r i v a t i v e s , a r e a c t i o n which takes p l a c e i n the microsomes a l s o and r e q u i r e s NADPrL? and  oxygen, and a t h i r d pathway, the  mechanism and p r o d u c t s of which are y e t unknown.  Oxygen  and  NADPrL? were a l s o found to be a b s o l u t e requirements f o r t h i s reaction. A d d i t i o n of reduced g l u t a t h i o n e to the r a t microsomal system r e s u l t e d i n a marked i n c r e a s e i n w a t e r - s o l u b l e  metabolites,  but  the compound(s) formed tends to mediate the t h i r d pathway. i s known however, t h a t g l u t a t h i o n e p a r t i c i p a t e s i n the o f mercapturic  of estrogen  formation  a c i d s to which c e r t a i n c y c l i c compounds g i v e r i s e  i n the animal body (94), and way  It  i t i s p o s s i b l e t h a t t h i s may  be  one  inactivation in rat liver tissues.  The mechanism by which g l u t a t h i o n e r e a c t s i s not c l e a r cut a l t h o u g h i t does appear t h a t c o n j u g a t i o n  quite  takes p l a c e ,  a r e a c t i o n i n v o l v i n g the s u l p h y d r y l group of the t r i p e p t i d e and a r e a c t i v e "o-quinoid" of e s t r a d i o l - 1 7 p .  i n t e r m e d i a t e r e s u l t i n g from the  oxidation  There i s some doubt however, as to whether  t h i s i s the r e a c t i o n o c c u r r i n g i n the microsomal system. .Evidence f o r the involvement of the s u l p h y d r y l group been p r o v i d e d by the f a c t t h a t N-ethylmaleimide i s a  strong  has  -46i n h i b i t o r of the r e a c t i o n i n both the microsomal and  phenolase  system, a n d . o x i d i z e d g l u t a t h i o n e had no e f f e c t on the r e a c t i o n . The  s p e c i f i c i t y o f g l u t a t h i o n e i n t h i s r e a c t i o n i s questioned  s i n c e other sulphydryIs were of l i t t l e or no  effect.  F u r t h e r i n v e s t i g a t i o n for. a c o n j u g a t i o n r e a c t i o n has been o b t a i n e d through serum (PMS)  the a c t i o n of g l u t a t h i o n e on pregnant mare's  gonadotropins.  However, attempts to i n h i b i t gonado-  t r o p i c a c t i v i t y by estrogen quinones i n v i v o have so f a r proved u n s u c c e s s f u l , but these s t e r o i d o x i d a t i o n products may a c t i v e i f produced i n s i t u .  Such a r e a c t i o n i f demonstrated c o u l d  then p r o v i d e a mechanism f o r the "feedback" gonadotropins  by  estrogens.  o n l y be  i n h i b i t i o n of  -47-  BIBLIOGRAPHY Skand. A r c h . P h y s i o l . 7 0 .  J 1934.  1.  Zondek, B.  2.  H e l l e r , C.G.  3.  E n g e l , P., Rosenberg, E .  4.  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