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The oxidative metabolism of estrogens by mammalian liver Lazier, Catherine B. 1963

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THE OXIDATIVE METABOLISM OF ESTROGENS BY MAMMALIAN LIVER by Catherine B. Lazier B.A. The University of Toronto 1961 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Biochemistry We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May, 1963. i In' presenting th i s thesis in p a r t i a l fulf i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary shall , make i t free ly avai lable for reference and study. I further agree that per- . mission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives,, I t i s understood that copying, or p u b l i -cat ion of this thesis for f i n a n c i a l gain sha l l not be allowed without my written permission. Department of ^t^LJ-*^<^^>0^ The Univers i ty of B r i t i s h Columbia,. Vancouver 8, Canada. Date V 7 A ^ 7 , •  ABSTRACT The main problem of estrogen metabolism studied has been to determine the nature of the water-soluble products formed from es t rone-16-C^ by ra t l i v e r preparations. Comparative studies were ca r r i ed out i n the guinea p i g . Three types of water-soluble metabolites were demonstrated, namely, protein-bound de r iva t ives , glucosiduronate conjugates, and un iden t i f i ed products which were not bound to pro te in and were not hydrolysed by 2N HC1. The water-soluble metabolites formed on incubating ra t or guinea p ig l i v e r microsomes wi th es t rone-16-C^ i n the presence of NADPH and oxygen consisted of protein-bound mate r i a l , some unknown de r iva t ives , but v i r t u a l l y no simple conjugates. Incubation wi th the r a t l i v e r 8000 x g supernatant f r ac t ion resu l ted l a rge ly i n conversion of the estrogen to the unknown water-soluble end-products, w h i l e , i n contrast , th i s l i v e r f rac t ion from guinea p ig gave r i s e mainly to glucosiduronates. In the presence of UDPGA, both r a t and guinea p ig l i v e r micrdr somes converted estrone-16-cL4 to glucosiduronate conjugates, but th i s d id not occur wi th the r a t l i v e r 8000 x g supernatant f r a c t i o n . Es t r ad io l - i yp - lG-C 1 ^ - and s t i l b e s t r o l - C 1 4 behaved s i m i l a r l y to es t rone-16-C^. I n the r a t , i n v i v o , the b u l k o f t h e u r i n a r y 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 e s t r o n e were o f unknown n a t u r e , w h i l e i n t h e g u i n e a p i g , g l u c o s i d u r o n a t e . c o n j u g a t i o n p r e d o m i n a t e d . The p r o b l e m was a l s o s t u d i e d by a d i f f e r e n t a p proach. V a r i o u s compounds h a v i n g s t r u c t u r a l f e a t u r e s s i m i l a r t o e s t r o n e were t e s t e d f o r t h e i r a b i l i t y t o i n h i b i t t h e forma-t i o n 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 from t h i s e s t r o g e n by r a t l i v e r microsomes. I t was found t h a t 2 - h y d r o x y e s t r o n e , 2 - h y d r o x y e s t r a d i o l - 1 7 p and e q u i l e n i n were p o t e n t i n h i b i t o r s , w h i l e t h o s e e s t r o g e n s w h i c h had an oxygen f u n c t i o n a t C-6 or C-16, as w e l l as the 1 7 p - g l u c o s i d u r o n a t e s and n o n - p h e n o l i c s t e r o i d s t e s t e d were i n a c t i v e . The s y n t h e t i c e s t r o g e n s , s t i l b e s t r o l and hexe-s t r o l , b o t h i n h i b i t e d the r e a c t i o n , b u t t h e i r n o n - e s t r o g e n i c a n a l o g u e s had no e f f e c t . A group of benzoquinones, naphtho-quinones and o r t h o - and p a r a - h y d r o x y l a t e d p h e n o l s p r o v e d t o be p o w e r f u l i n h i b i t o r s , whereas a n t h r a q u i n o n e s and meta-hydroxy-l a t e d p h e n o l s showed no a c t i v i t y . I n k i n e t i c s t u d i e s , 2 - h y d r o x y e s t r o n e , e q u i l e n i n , and s t i l b e s t r o l appeared t o a c t as c o m p e t i t i v e i n h i b i t o r s , b u t menadione gave a mixed type o f i n h i b i t i o n . ACKNOWLEDGEMENTS The a u t h o r w i s h e s t o e x p r e s s her g r a t i t u d e t o Dr. P.H. J e l l i n c k f o r h i s h e l p and encouragement d u r i n g t h i s work, a l s o t o Dr. R.L. No b l e f o r t h e use o f the f a c i l i t i e s i n h i s l a b o r a t o r y . The a u t h o r i s v e r y g r a t e f u l f o r the a s s i s t a n c e o f M i s s J . Leon a r d , M i s s L. I r w i n and M i s s E. G r i s e d a l e i n the p r e p a r a t i o n and t y p i n g o f t h i s t h e s i s . TABLE OF CONTENTS Page INTRODUCTION 1 1. Inactivation of Estrogens . . 1 2. Ether-soluble Metabolites . 3 3. Glucosiduronate and Sulphate Conjugates 8 4. Water-soluble Metabolites other than Simple Conjugates 12 5. The Present Investigation 17 EXPERIMENTAL 17a I Materials and Methods 1. Materials 17a 2. Preparation of Tissue 19 3. Incubation and Extraction of Tissue Preparations 21 4. Determination of Radioactivity 22 5. Examination of the Ethereal Fraction 23 6. Examination of the Aqueous Fraction 23 7. I n h i b i t i o n Studies 27 8. In vivo Studies 29 I I Results 1. Optimal Conditions for the Formation of Water-soluble Metabolites from Estrone-16-C^ by Liver Preparations 30 2. Properties of the Ether-soluble Metabolites 32 3. Properties of the Water-soluble Metabolites 33 14 4. Metabolism of Estradiol -17p-16-C by Rat and Guinea Pig Liver Preparations 39 5. Metabolism of D i e t h y l s t i l b e s t r o l (monoethyl-l-C ) by Rat and Guinea Pig Liver Preparations 40 6. I n h i b i t i o n of the Formation of Water-soluble Metabolites from Estrone - 1 6-C^ by Rat Liver Microsomes 41 DISCUSSION 45 SUMMARY 63 BIBLIOGRAPHY . 66 TABLES Page I. Percentage d i s t r i b u t i o n of r a d i o a c t i v i t y a f t e r incubation of 10 \xg estrone-16-C with r a t l i v e r preparations (to face) 30 I I . Formation of protein-bound metabolites of estrone-16-C^ by r a t and guinea pig l i v e r preparations (to face) 34 I I I . Conjugate formation by ra t and guinea pig l i v e r preparations i n the presence and absence of UDPGA (to face) 35 IV. In vivo metabolism of estrone-16-Cl^ i n the r a t and guinea pig (to face) 39 V. Compounds tested as i n h i b i t o r s i n the formation of water-soluble products from estrone-16-C by r a t l i v e r microsomes. Group I (to follow) 42 VI. Compounds tested as i n h i b i t o r s i n the formation of water-soluble products from estrone-16-C by r a t l i v e r microsomes. Group I I (to follow Table V) VII. Compounds tested as i n h i b i t o r s i n the formation of water-soluble products from estrone-16-C by r a t l i v e r microsomes. Group I I I (to follow Table VI) Figures Page I. Chromatograms of ethereal fractions from incubation of estrone-16-C^ with l i v e r preparations (to face) 32 I I . Time curves for the formation of water-soluble metabolites and of protein-bound metabolites from estrone-16-C by-ra t l i v e r (to face) 33 I I I . Chromatograms of the aqueous f r a c t i o n from incubation of estrone-16-Cl^ with r a t l i v e r preparations (Legend to face) (to follow) 36 IV. Chromatograms of the aqueous f r a c t i o n from incubation of estrone-16-C with guinea pig l i v e r preparations (Legend to face) (to follow) 37 V. Chromatograms of the aqueous f r a c t i o n from incubations of estrone-16-C^ with guinea pig l i v e r preparations (to face) 38 VI. Lineweaver-Burk p l o t for the i n h i b i t i o n by 2-hydroxyestrone of the conversion of estrone-16-C-^ to water-soluble metabolites by r a t l i v e r microsomes (to follow) 44 VI I . Lineweaver-Burk p l o t for the i n h i b i t i o n by equilenin of the conversion of estrone-16-C^ to water-soluble metabolites by r a t l i v e r microsomes (to follow Figure VI) V I I I . Lineweaver-Burk p l o t for the i n h i b i t i o n by s t i l b e s t r o l of the conversion of estrone-16-C^ to water-soluble metabolites by r a t l i v e r microsomes (to follow Figure VII) IX. Lineweaver-Burk p l o t for the i n h i b i t i o n by menadione of the conversion of estrone-16-Cl4 to water-soluble metabolites by r a t l i v e r microsomes (to follow Figure VIII) - 1 -INTRODUCTION Inactivation of Estrogens. The o r i g i n a l observation that implicated l i v e r as a s i t e of estrogen metabolism was made i n 1934 by Zondek^", who reported that r a t l i v e r mince was capable of r a p i d l y i n a c t i v a t i n g estrone. Many workers confirmed and extended t h i s i n v i t r o r e s u l t , using loss of b i o l o g i c a l a c t i v i t y as a means of following hormone degradation. Thus, H e l l e r ^ showed that l i v e r was the major organ i n rats and rabbits involved i n the destruction of estrone, estradiol-17p and e s t r i o l , but that kidney was only s l i g h t l y a c t i v e , while spleen, heart, lung, placental tissue and uterus 3 were e n t i r e l y without e f f e c t . Twombly and Taylor demonstrated that human l i v e r preparations were also capable of i n a c t i v a t i n g estradiol-17p, but at a slower rate than either r a t or mouse l i v e r . In addition, beef^ and dog l i v e r ^ were shown to destroy estrogens i n v i t r o . 2 Heller found that cyanide i n h i b i t e d the i n a c t i v a t i o n of estrone and estradiol-17p by r a t l i v e r s l i c e s , and i t was shown by Levy that carbon monoxide and sodium azide were i n h i b i t o r s . This pointed to p a r t i c i p a t i o n of a heavy metal enzyme, si m i l a r to tyrosinase or the cytochrome system i n the i n a c t i v a t i n g system. De Meio e_t a l , i n 1948 showed that i n a c t i v a t i o n of estradiol-17p would not proceed i n the absence of oxygen, and - 2 -also found indications that an NAD-linked dehydrogenase was involved i n t h i s process. De Meio's r e s u l t s also suggested that the b i o l o g i c a l l y i n a c t i v e metabolites were not simple conjugates of the estrogens, but that oxidative degradation products were formed. Many i n vivo experiments have confirmed these i n v i t r o observations. Thus, Golden and Sevringhaus^ i n 1938 transplanted the ovaries of rats to the mesentery and to the a x i l l a e and found that estrus did not occur i n the animals bearing transplants i n the mesentery, where the blood carrying the estrogen would pass d i r e c t l y to the l i v e r . Biskind.9 implanted p e l l e t s of estrogen into the spleen of normal male rats and observed atrophy of the testes only when the blood flow from the spleen was prevented from passing d i r e c t l y into the hepatic p o r t a l system. In other experiments with rats i n which the l i v e r had been damaged, prolongation and enhancement of the response to endogenous and exogenous estrogens was observed^. Although t h i s work confirmed the idea that l i v e r performed an i n t e g r a l r o l e i n the i n a c t i v a t i o n of estrogens i n mammals, very l i t t l e evidence as to the nature of the metabolites was gained from these experiments and only with the introduction of , radioisotopic and chromatographic techniques did t h i s become possible. - 3 -Ether-soluble Metabolites. The f i r s t experiments using these more advanced techniques dealt with the interconversion of estrone and estradiol-17p. 11 12 Thus, Ryan and Engel ' i n 1952 using counter-current f r a c t i o n -ation and photo-fluorimetry demonstrated the interconversion of these two estrogens under both aerobic and anaerobic conditions, i n a v a r i e t y of normal and abnormal human tissues, while other workers 13 have observed i t i n a large number of di f f e r e n t species . The dehydrogenase catalyzing t h i s reaction was p u r i f i e d from placenta by Engel et a l . l ^ , and shown to require NAD or NADP as cofactor. The k i n e t i c s of the enzyme reaction have been studied i n v i t r o " ^ and Fishman-^ has shown that the equilibrium l i e s markedly i n favour of estrone i n vivo i n the human. From 1957 to 1960 a large number of di f f e r e n t enzymes involved i n the oxidative metabolism of estrogens were discovered i n l i v e r . The i s o l a t i o n of 2-methoxyestronel7 and of 2-methoxy-e s t r i o l ^ from the urine of patients who had been given C^-estrogens and of 2-methoxyestradiol from human pregnancy urine^O lead to the proposal by Kraychy and Gallagher that methoxylation was accom-plished i n two d i s t i n c t steps - namely hydroxylation and subsequent methyla t i o n ^ . This idea received strong support from the work of L. R. Axelrod^ 1, who infused 2-hydroxyestradiol into two post-menopausal women and detected 2-methoxy derivatives of e s t r a d i o l and e s t r i o l i n the i r urine. Fishman^ injected e s t r a d i o l - C ^ into a patient and recovered 12% of the r a d i o a c t i v i t y as 2-hydroxyestrone i n the urine so that i n man at le a s t , 2-hydroxy-l a t i o n appears to be quant i t a t i v e l y important. In v i t r o studies on the 2-hydroxylase were carried out by 23 24 King ' , who l o c a l i z e d the enzyme i n r a t and rabbit l i v e r microsomes, and found that oxygen and either NADH or NADPH was required for the reaction and that tetrahydrofolic acid was possibly involved. With regard to the proposed second step i n the formation 25 of the 2©methoxy estrogens, Knuppen et a l , observed enzymic methylation of 2-hydroxyestradiol i n the presence of human l i v e r s l i c e s and S-adenosylmethionine. I t was l a t e r shown that an O-methyl transferase which i s present i n the soluble f r a c t i o n of ra t l i v e r i s responsible for the methylation of the 2-hydroxy-estrogens and i t i s of in t e r e s t that the same enzyme methylates the catechol amine hormone, arterenol . There i s some evidence 27 for the presence of a demethylating enzyme i n l i v e r microsomes Another enzyme of importance i n the oxidative metabolism of estrogens i s a 6-hydroxylase. I t was f i r s t detected i n 1957 2 8 by Mueller and Rumney i n mouse l i v e r microsomes , and Breuer l a t e r observed the 6-hydroxylation of estrone, e s t r a d i o l and 29 e s t r i o  by r a t l i v e r s l i c e s i n the presence of NADPH and oxygen - 5 -Both 6a- and 6p-hydroxylases were present i n r a t l i v e r microsomes as w e l l as the corresponding dehydrogenases^. 6-Hydroxylation 31 has been observed with human l i v e r , but as yet no 6-hydroxy estrogen has been detected i n human urine. In addition to the 2- and 6-hydroxylases, r a t l i v e r microsomes have been shown to contain an enzyme capable of hydroxylating estradiol-17p i n the 10p p o s i t i o n , giving r i s e to 17p-hydroxyestra-p-10p-quinol32. Again, the reaction requires oxygen and NADPH. In 1961 Hecker and Zayed^3 advanced an hypothesis to account for the formation of the 2-, 6-, and lo-hydroxylated derivatives of estradiol-17p. They postulated that r a t l i v e r microsomes contain an enzyme capable of removing hydrogen from the phenolic hydroxyl group of estradiol-17p and thereby giving r i s e to a phenoxyl r a d i c a l . I t was suggested that the unpaired electron i n t h i s reactive intermediate would tend to migrate to positions 2-, 6- and 10- of the ste r o i d molecule and int e r a c t with free hydroxyl r a d i c a l s to form corresponding hydroxylated metabolites. This a t t r a c t i v e theory for the NADPH-dependent formation of 2-, 6- or 10-hydroxylated estrogens awaits further i n v e s t i g a t i o n . In addition to rings A and B, rings C and D of the ste r o i d have also been shown to be hydroxylated i n the course of - 6 -estrogen metabolism. An 11-hydroxylase has recently been detected i n ox adrenal tissue though i t has not as yet been found i n l i v e r . However, l lp-hydroxyestrone i s metabolized by r a t l i v e r s l i c e s giving r i s e to ll-ketoestradiol -17p;-and. to l lp-hydroxyestradiol -17p . An 18-hydroxylase, apparently situated i n the adrenal cortex, can hydroxylate estrone, and 18-hydroxyestrone has been 35 i s o l a t e d from human pregnancy urine by L0ke, Marrian et a l Quantitatively the most important metabolites of estrone and e s t r a d i o l i n vivo are th e i r 16-oxygenated derivatives. As 36 early as 1942, Pincus demonstrated that estrone and estradiol - 1 7 p are converted to e s t r i o l i n large amounts i n vivo and Brown i n 37 1957 showed that 45% of injected estrone or estradiol - 1 7 p i s excreted by man as e s t r i o l i n the urine. Although i t was long thought that 16a-hydroxylation of estradiol-17p gave r i s e to 38 e s t r i o l , a recent experiment by Fishman et a l , has shown that estrone i s the more important precursor of e s t r i o l i n vivo i n humans. 3 9 Marrian i n 1957 i s o l a t e d 16-a hydroxyestrone from human pregnancy urine, and made the suggestion that 16a-hydroxyestrone and 16p-hydroxyestrone were the main precursors of the four epimeric e s t r i o l s . This hypothesis has been upheld both by the 3 8 i n vivo work of Fishman and also by numerous i n v i t r o experiments 40,41,43 by Breuer 16-ketoestradiol-17p has been shown by King and Levitz to a r i s e , i n vivo and i n v i t r o , from p a r t i a l oxidation of e s t r i o l , and 16-ketoestradiol-17p i t s e l f has i n turn been shown to be 44 metabolized to e s t r i o l , 1 7 - e p i e s t r i o l and to 16-ketoestrone. On the other hand, 16-ketoestrone, on i n j e c t i o n into man, gives r i s e to e s t r i o l and 1 6 - e p i e s t r i o l i n the u r i n e ^ , implicating 16-ketoestradiol-17p as an intermediate. In experiments with 46 human l i v e r s l i c e s , Breuer et a l 0 showed that 16-ketoestrone can be metabolized to 16a-hydroxyestrone, 16p-hydroxyestrone, 16-ketoestradiol-17p, 1 6 - e p i e s t r i o l , 1 7 - e p i e s t r i o l and 16,17 - e p i e s t r i o l . In addition, 16-ketoestradiol-17a has been 47 shown to give r i s e to both 17- and 16,17-epiestriol . In f a c t , the only possible compound of th i s type that has not been shown to be formed i n human l i v e r i s 16-ketoestradiol-17a and the formation of 16-ketoestrone from 16-ketoestradiol-17a has not yet been demonstrated. The complicated i n t e r r e l a t i o n s h i p s between the C-16 oxygen-ated estrogens can be summarized by saying that man and other mammal possess i n the l i v e r , estrone 16a- and 16(3-hydroxylases, 16a and 16p-hydroxysteroid dehydrogenases, as w e l l as 17a- and 17#-hydroxy-ste r o i d dehydrogenases. The formation of 16a-hydroxyestrone and i t s reduction to e s t r i o l are qua n t i t a t i v e l y the most important reactions. - 8 -Breuer and h i s colleagues have recently shown that human and r a t l i v e r contain a st e r o i d epoxidase, capable of forming 16a, 17a-epoxyestratrien-3-ol from estratetraen-3-ol^8. An epoxhydra-tase i s also present, capable of forming 16, 17 - e p i e s t r i o l from the a-epoxide and e s t r i o l from the p-epoxide^^. Glucosiduronate and Sulphate Conjugates. With the possible exception of 6-hydroxy or 6-ketoestriol, a l l of the aforementioned oxidative metabolites of estrone and e s t r a d i o l are water-insoluble and e t h e r - s o l u b l e ^ . However, i t has been known for some time that estrogens are often found i n the urine i n a water-soluble form, conjugated with either glucuronic or sulphuric acid. Cohen and Marrian 5^ i n 1936 f i r s t i s o l a t e d e s t r i o l monoglucosiduronate from pregnancy urine and more recently, Carpenter and RelUe 5-^ have obtained both estriol-16-glucosidu-ronate and estriol-17-glucosiduronate from t h i s source. Oneson 52 and Cohen have detected estrone glucosiduronate i n urine. In 1947 Crepy5-^ demonstrated the i n v i t r o formation of glucosiduronates of estrone, estradiol-17p and e s t r i o l , using rabbit and guinea pig l i v e r s l i c e s , but S c h i l l e r and Pincus 5^ f a i l e d to obtain evidence for conjugation of estrogen by male r a t l i v e r . Glucosiduronate conjugation of the "synthetic" estrogens, s t i l b e s t r o l , hexestrol and dienestrol has been observed i n vivo with r a t s 5 5 , r a b b i t s and c a t s 5 6 , and Zimmerberg5? - 9 -i n 1946 showed that r a t l i v e r s l i c e s conjugated s t i l b e s t r o l , probably as the glucosiduronate, whereas r a t l i v e r minces did not. 58 In 1958 Lehtinen et a l . found that r a t duodenal mucosa s l i c e s catalysed the formation of estradiol - 1 7 p-glucosiduronate from estradiol - 1 7 p . In these i n v i t r o studies the actual reaction products were not i s o l a t e d ; the experiments mainly involved the hydrolysis of the conjugate by p-glucuronidase or by acid with subsequent i d e n t i f i c a t i o n and recovery of the free steroids. Diczfalusy and his colleagues i n Sweden have recently reported a large series of experiments on estrogen conjugation i n humans. In studies on conjugation by i n t e s t i n a l t r a c t , they found that following i n j e c t i o n of e s t r i o l into an i s o l a t e d loop of the duodenum with the a r t e r i a l blood supply i n t a c t , large amounts of estriol - 1 6 ( 1 7 ? )-glucosiduronate appeared i n both the effluent venous blood and i n extracts of the i n t e s t i n a l w a l l . Evidence for the formation of a d i - or tri-glucosiduronate was also obtained, 59 and, i n addition, small amounts of estriol-3-sulphate were found . In a si m i l a r experiment, i n j e c t i o n of estradiol-l - 7 p gave r i s e to large amounts of estrone glucosiduronate, and some evidence for the presence of estradiol - 1 7 p-glucosiduronate and the 3 ,17-di-glucosiduronate was obtained*^. - 10 -Diczfalusy and his co-workers have also carried out extensive studies on estrogen metabolism i n the human foetus 61 and new-born . They showed that estriol-3~sulphate was the major conjugate of cord blood, and a minor component of amniotic f l u i d and of the urine of new-borns. In the l a t t e r two f l u i d s , estriol-16(17?)-glucosiduronate predominated. Three add i t i o n a l e s t r i o l conjugates were detected and p a r t i a l l y characterized i n these f l u i d s . Two of them appeared to be d i - or t r i - g l u c o -siduronates, and the t h i r d a double conjugate, possibly estriol-3-sulphate, 16(17?)-glucosiduronate or e s t r i o l - 3 -sulphoglucosiduronide . The sulphoglucuronide may represent an intermediate step i n a transconjugation reaction as suggested by Twombly and Levitz who is o l a t e d estrone glucosiduronate from the urine after administration of estrone sulphate (lab e l l e d i n the steroid moeity) to a patient. In addition, a trans-conjugation reaction may account for the observation by Diczfalusy that although sulphate conjugates are the major form of e s t r i o l i n cord blood, glucosiduronates make up most of the estrogen 6 2 conjugated material i n amniotic f l u i d and urine The biochemical mechanism of the formation of gluco-siduronates and of glucuronides was formulated from the work of several laboratories. Smith and M i l l s ^ i n 1954 is o l a t e d uridine diphospho glucuronic acid (UDPGA) from l i v e r , and showed - 11 -that i t acted as the glucuronic acid donor i n the formation of conjugates by l i v e r homogenates. Strominger^ l a t e r demonstrated that UDPGA was formed i n the soluble f r a c t i o n of the c e l l from UDPglucose under the influence of an NAD-linked dehydrogenase. The transfer of the glucuronic acid from the UDPGA to the aglycon acceptor was shown to take place i n the microsomes, being mediated by UDP-glucuronyl transferase^^. Isselbacher i n 1956^6 reported success i n s o l u b i l i z i n g the coupling system from guinea pig l i v e r and the preparation was active i n conjugating estradiol-17p, tetrahydrocortisone and thyroxine. More recently a better preparation of the enzyme has been obtained from rabbit l i v e r microsomes which i s active i n the synthesis of both ester and ethereal glucuronides . By way of confirmation of t h i s , Smith and Breur have shown that estrone, on incubation with a rabbit l i v e r microsomal preparation and UDPGA i s con-69 jugated as the monoglucosiduronate . As mentioned previously, estrogen sulphates are formed i n mammals. Thus, McKenna and co-workers^ have recently obtained estrone-3-sulphate from pregnancy urine and Menini and Diczfalusy have i s o l a t e d and i d e n t i f i e d estriol-3-sulphate i n human meconium^, and have also demonstrated i t s formation i n various tissues of the human foetus and new-born6^. The - 12 -The soluble f r a c t i o n from guinea pig placenta, but not from human placenta, has been shown to be capable of sulphurylating estrone-16-Q14 72 _ -j-n addition, Engel's group has i d e n t i f i e d estrone 73 sulphate as a major form of estrogen i n human pregnancy plasma The mechanism of sulphurylation has been investigated by De Meio and c o l l e a g u e s ^ , and they have demonstrated the form-ation of estrone and estradiol-17f3 sulphates by r a t and ox microsomes-free l i v e r supernatants, and by an ammonium sulphate p r e c i p i t a t e from the supernatant i n the presence of adenosine triphosphate (ATP) and magnesium ions. The requirements of the reaction are the same as those for the synthesis of sulphate esters of phenols, and i t i s postulated that, except for the sp e c i f i c active sulphate transferring enzyme, the process i s the same for estrogens as for simple phenols. Estrogen sulphatase has been demonstrated i n r a t and ox l i v e r microsomes and this enzyme i s present i n quantities comparable to the enzyme synthesising the estrogen sulphates^-*. Water-soluble Metabolites other than Simple Conjugates. I t has long been suggested that estrogens are converted i n mammals to water-soluble products other than sulphate or glucosiduronate conjugates. Beer and Gallagher i n 1955^ reported that extraction with ether f a i l e d to remove a l l of the - 13 -r a d i o a c t i v i t y from hydrolysed urine of human subjects who had been injected with C^-estrone or estradiol-17p. S i m i l a r l y , Valcourt et a l 7 7 administered estrone-16-C''"^ to rats and found that only one-third of the excreted i n b i l e and i n urine was extractable with ether. They also observed a small amount of r a d i o a c t i v i t y i n the. raieutral f r a c t i o n of the urine. In addition 78 Wotiz, Ziskind and Ringer' 0, i n perfusion experiments with radioactive estrone, found a large amount of chloroform-insoluble material containing i n the hydrolysed dialysates of r a t plasma. J e l l i n c k 7 9 i n 1959 reported that incubation of r a t and human l i v e r s l i c e s with estrone-16-C^ resulted i n the formation of water-soluble, ether-insoluble metabolites, which could not be made ether soluble by r e f l u x i n g with 77o ( w/v) hydrochloric acid for 3 hours. No radioactive carbon dioxide was evolved during the incubation, confirming an e a r l i e r i n vivo experiment 7 7 and showing that a least C16 of the steroid nucleus remains i n t a c t . Placental tissue or blood could not give r i s e to these water-soluble products. 8 0 Since Westerfeld e_t a l had shown that cyanide-sensitive plant phenol oxidases inactivate estrogens, and since cyanide i n h i b i t s the formation of water-soluble products from estrone by ra t l i v e r s l i c e s 7 9 , J e l l i n c k 0 ^ extended his studies to the - 13 -r a d i o a c t i v i t y from hydrolysed urine of human subjects who had been injected with C^-estrone or estradiol-17p. S i m i l a r l y , Valcourt e_t al.^7 administered estrone-16-C-'-^ to rats and found that only one-third of the excreted i n b i l e and i n urine was extractable with ether. They also observed a small amount of r a d i o a c t i v i t y i n the neutral f r a c t i o n of the urine. In addition Wotiz, Ziskind and Ringer' 0, i n perfusion experiments with radioactive estrone, found a large amount of chloroform-insoluble material containing i n the hydrolysed dialysates of r a t plasma. 79 J e l l i n c k i n 1959 reported that incubation of r a t and human l i v e r s l i c e s with estrone-16-C^ resulted i n the formation of water-soluble, ether-insoluble metabolites, which could not be made ether soluble by r e f l u x i n g with 77o ( w/v) > hydrochloric acid for 3 hours. No radioactive carbon dioxide was evolved during the incubation, confirming an e a r l i e r i n vivo experiment^ and showing that at least C-16 of the steroid nucleus remains i n t a c t . Placental tissue or blood could not give r i s e to these water-soluble products. Since Westerfeld et al.80 had shpwn that cyanide-sensitive plant phenol oxidases inactivate estrogens, and since cyanide i n h i b i t s the formation of water-soluble products from estrone by 79 81 r a t l i v e r s l i c e s , J e l l i n c k 0 e x t e n d e d his studies to the - 14 -comparatively simpler plant enzymes and found that mushroom tyrosinase preparations did i n fact y i e l d a high percentage of radioactive water-soluble products from estrone-16-C^. Evidence was presented that mushroom tyrosinase acts upon estrone i n the same manner as upon i t s normal phenolic substrates, i . e . that i t oxidizes Ring A of estrone giving a highly reactive o-quinonoid derivative, which can then undergo addition reactions and combine with protein or other acceptors to y i e l d highly stable complexes. In f a c t , 60-70% of the water-soluble r a d i o a c t i v i t y was shown to be bound to protein by a strong chemical bond. I t was therefore suggested that r a t l i v e r contained enzymes capable of acting on estrone i n a s i m i l a r way and, as discussed previously, the natural estrogens are indeed acted upon by l i v e r to give the o-hydroquinones. However, whether these are then oxidized to give the highly reactive o-quinonoid derivatives i s unknown, although estrogen-protein complexes that could have been formed by way of an o-quinone intermediate have been detected i n r a t 82 l i v e r . Thus Reigel and Mueller i n 1954 demonstrated the presence of an enzyme system i n r a t l i v e r homogenates which, i n the presence of NADPH and oxygen, bound a metabolite of 83 estradiol-17p to protein while Szego showed that l i v e r contained enzymes which bind estrone to the albumin f r a c t i o n of - 15 -homologous serum. J e l l i n c k , however, i n studies comparing the 14 tyrosinase and l i v e r s l i c e incubation products of estrone-16-C observed that only I0-157o of the water-soluble r a d i o a c t i v i t y formed by r a t l i v e r was bound to protein, while a much higher f r a c t i o n (60-70%) was bound by the phenol oxidase system. 84 Z i l l i g and Mueller } using r a t l i v e r microsomes, carried out further studies on the i n t e r a c t i o n of estradiol-17p with protein. They purported to exclude positions 2 and 4 from being involved i n the protein-binding process, since substitution of one or the other of these positions on the steroid with f l u o r i n e did not a f f e c t combination with protein. Since the 2,4-di-fluoro-estradiol-17p was not tested, one of the two possible o-quinonoid derivatives could have formed i n each case, although the 3 94-hydroquinone of estrogens has never been detected i n b i o l o g i c a l systems. As i t stands, there i s therefore l i t t l e / evidence either for or against the quinonoid pathway of estrogen in t e r a c t i o n with protein or other acceptors. Another possible route for the formation of protein-bound metabolites of estrone resulted from studies on the i n t e r a c t i o n of horseradish peroxidase and radioactive estrogens. In t h i s case J e l l i n c k ^ showed that incubation of estrone-16-C^ with the plant enzyme i n the presence of protein, tryptophan, cysteine or reduced glutathione led to the formation of water-soluble products - 16 -i n high y i e l d s . Evidence was given that the horseradish enzyme was f i r s t acting as an aerobic oxidase, u t i l i z i n g protein or the amino acid to generate hydrogen peroxide i n the presence of manganous ions, and subsequently acting as a true peroxidase, converting estrone to highly reactive metabolites. These appeared to be phenoxyl r a d i c a l s which were able to combine with protein or amino acid acceptors, giving water-soluble end-product Hecker's theory33 f o r the formation of the 2-, 6- and 10-hydroxylated metabolites or e s t r a d i o l by r a t l i v e r microsomes, as outlined e a r l i e r i n t h i s discussion, can also be adapted as a possible free r a d i c a l mechanism for protein binding. In f a c t , he showed that the l a b e l l i n g of the t r i c h l o r o a c e t i c a c i d - p r e c i p i -table protein f r a c t i o n after aerobic incubation of estradiol-17p-with r a t l i v e r microsomes and NADPH for 15 minutes was preceded by the s l i g h t l y more rapid formation of 17p-hydroxyestra-p-l0p-33 quinol, which he suggested arises by free r a d i c a l mechanism 667o of the water-soluble metabolites were protein-bound and 347o of the t o t a l a c t i v i t y was i n the aqueous f r a c t i o n . Hence, the nature and mode of formation of the water-soluble degradation products of estrone by mammalian l i v e r are larg e l y unknown, although evidence has been given for the existence of protein-bound water-soluble derivatives. The l a t t e r do not, however, account for a l l of the non-hydrolysable water-- 17 -soluble metabolites of estrogens. The Present Investigation. The present investigation has been mainly concerned with the determination of the properties of the water-soluble metabolites formed from estrone-16-C"'"^' by r a t l i v e r preparations and two approaches have been taken. F i r s t , data on rate of formation of the water-soluble products under d i f f e r e n t experimental conditions has been obtained, followed by attempts to characterize the metabolites. Comparative studies were also carried out with guinea pig l i v e r preparations and some observations made on the oxidative metabolism of e s t r a d i o l - 1 7 p - 1 6 - C ^ and on d i e t h y l s t i l b e s t r o l -(monoethyl-l-C"^). The second approach has been to study the effe c t of i n h i b i t o r s i n order to gather more information on the mechanism of formation of these water-soluble products. A series of s t r u c t u r a l analogues of estrone as w e l l as c l a s s i c a l enzyme i n h i b i t o r s were tested for their i n h i b i t o r y power i n a r a t l i v e r microsomal system. - 17a -EXPERIMENTAL I Materials and Methods 1. Materials Animals: Female Wistar r a t s , each weighing about 150 gm were obtained from the University of B r i t i s h Columbia animal colony. Female guinea pigs, each weighing approximately 600 gm were obtained from the Cancer Research Centre animal colony. The animals were fed Master's Fox Chow ad l i b i t u m . In addition, the guinea pigs were fed fresh green vegetables. Radioactive Estrogens: Estrone-16-C^ 1 -, estradiol-17p-16-C''"^ and d i e t h y l s t i l b e s t r o l (monoethyl-l-C-'-^) were purchased from the Radiochemical Centre, Amersham, England. The estrogens were maintained i n a stock solution of 1 mg/ml. Counting Materials: The s c i n t i l l a t i o n f l u i d consisted of the following ingredients: 4 g 2,5-diphenyloxazole (PPO) 100 mg l,4-bis-2-(phenyloxazolyl)-benzene (P0P0P) 600 ml toluene 400 ml ethanol PPO, P0P0P and Hyamine hydroxide (1 M i n methanol) were obtained from the Packard Instrument Corporation, La Grange, I l l i n o i s . Buffers: 0.1 M Krebs phosphosaline buffer was made up as follows: NaCl (0.9%) 100 parts KC1 (1.15%) 4 parts MgS04.7H20 (3.84%) 1 part N a2P0 4.7H 2 o (2.68%) 30 parts - 18 -The pH was adjusted to 7.4 with 0.1 N HCl, and the solution was made up to 1 1. with d i s t i l l e d water. 0.1 M Potassium phosphate buffer, pH 7.4, contained the following materials: K 2HP0 4 - 0.0802 moles/1. KHoP0. - 0.0198 moles/1. ^ 4 Nicotinamide Nucleotides: NAD '(Grade 111) , NADH (Sigma Grade) , NADP (Sigma Grade) and NADPH (Type II) were obtained from the Sigma Chemical Company, St. Louis, Missouri. g-glucuronidase: B a c t e r i a l p-glucuronidase, Type I, containing 73,500 Fishman units/gm was obtained from the Sigma Chemical Company. One Fishman unit w i l l l i b e r a t e 1 [ig of phenolphthalein from phenolphthalein glucuronide/hr at pH 6.8 - 7.0 at 37°C. Uridine 5 1-diphosphoglucuronic acid: UDPGA as the ammonium s a l t was purchased from the Sigma Chemical Company. Catalase: Type C-100 catalase from the Sigma Chemical Company was used. Folin-Ciocalteu phenol reagent: This reagent (a mixture of sodium phosphomolybdate and phosphotungstate) was obtained from the B r i t i s h Drug Houses (Canada) Ltd. Chromatography Reference Standards: Many of these compounds were g i f t s to Dr. P.H. J e l l i n c k . 16a-hydroxyestrone was obtained from - 19 -Dr. H. Breuer, 6-ketoestradiol - 1 7 p , 16a and 6p-hydroxyestra-diol - 1 7 p from Dr. 0 . Wintersteiner and 2-hydroxyestrone, 2-hydroxyestradiol -17p and 2-hydroxyestriol from Dr. L. Axelrod. 16-ketoestrone, 1 6-ketoestradiol - 1 7 p and estradiol - 1 7 a were made available by the Cancer Chemotherapy National Service Center, Bethesda, Md. Estradiol- 1 7 p glucosiduronate was obtained from the C a l i f o r n i a Corporation for Biochemical Research, Los Angeles, and estriol - 1 7 p-glucosiduronate was a g i f t to Dr. P.H. J e l l i n c k from Dr. A.E. K e l l i e . Compounds tested as i n h i b i t o r s : 59 compounds were tested as i n h i b i t o r s of the r a t l i v e r estrone-metabolizing system; these are not l i s t e d but i n a l l instances, the compounds used were the purest r e a d i l y a v a i l a b l e . 1,4 benzoquinone, 1,2 naphthoquinone and 1,4 naphthoquinone, obtained from the Eastman-Kodak Company, were further p u r i f i e d by r e c r y s t a l l i z a t i o n from hot petroleum ether. 2. Preparation of Tissue Animals were k i l l e d by suffocation i n an atmosphere of carbon dioxide. Whole Homogenate: The l i v e r was r a p i d l y excised from the dead animal and one part of tissue was homogenized with three parts - 20 -of ice cold Krebs phosphosaline buffer i n a glass tissue grinder f i t t e d with a Teflon pestle. The concentration of the homo-genate was adjusted to 50 mg l i v e r (wet weight) /ml buffer. In experiments where the homogenate was to be further processed, 1 part of l i v e r was homogenized i n 3 parts of ice cold 0.25 M sucrose, and the f i n a l concentration was made up to either 50 mg l i v e r / m l sucrose or to 100 mg l i v e r / m l sucrose, depending upon the s p e c i f i c experiment. 8000 x g supernatant: The l i v e r homogenate i n 0.25 M sucrose was centrifuged at 8000 x g for 15 minutes at 2°C i n a Spinco Model L preparative ul t r a c e n t r i f u g e . (The supernatant obtained i s referred to throughout the text as the 8000 x g supernatant fraction.) The protein concentration was determined by the 8 6 micro-Kjeldahl method 100,000 x g p e l l e t : The 8000 x g supernatant of a 10% ( w/v) l i v e r homogenate obtained from 100 mg fresh tissue was recentrifuged at 100,000 x g for 60 minutes at 2°C. The supernatant was decanted and the p e l l e t suspended by homogenization i n the same volume of 0.25 M sucrose or 0.1 M potassium phosphate buffer at pH 7.4. This f r a c t i o n i s referred to throughout t h i s text as the 100,000 x g p e l l e t , or the microsomes, and i t was shown by electron microscopy to consist only of ribosomes attached to the membranes of the endoplasmic reticulum. The electron micrographs were kindly prepared by Dr. W. Chase. ^ - 21 -3. Incubation and Extraction of Tissue Preparations. When the whole homogentate was used, the incubation medium* consisted of 1 ml of the homogenized l i v e r preparation i n 0.1 M Krebs phosphosaline buffer, 1 mg of NAD or of NADP and 10 mg of nicotinamide i n 4 ml of buffer, and 0.01 ml (300,000 cpm) of a solution i n ethanol (1 mg/ml) of the radioactive estrogen i n 4 ml of buffer. The t o t a l volume of the mixture was 10 ml, and the incubations were carr i e d out i n 125 ml Erlenmeyer f l a s k s . When the 8000 x g supernatant or the 100,000 x g p e l l e t fractions were used, the incubation medium, unless otherwise indicated, consisted of 1 ml of the tissue preparation, pyridine nucleotide cofactor (1 mg) i n 1 ml of 0.1 M potassium phosphate buffer pH 7.4, and 10 ^ .g (300,000 cpm) of the radioactive estrogen i n 0.01 ml ethanol mixed with 1 ml of the phosphate buffer; t o t a l volume 3 ml. A l l incubations were carried out ir i 30 ml test tubes. Oxygen was bubbled through a l l incubation mixtures for 5 seconds p r i o r to immersion of the flasks or tubes i n a water bath shaker maintained at 37°C. The vessels were incubated under an atmosphere of oxygen, and were shaken at a constant rate of 70 cycles/min for a designated length of time. Zero time was taken as the moment of addition of the substrate to the incubation mixture. - 22 -In the case of the whole homogenate, immediately af t e r incubation the mixture was transferred to a centrifuge tube and spun i n a c l i n i c a l centrifuge for a short time i n order to pr e c i p i t a t e the tissue. The supernatant was decanted, and immediately extracted twice with 1.5 v o l . ether while the tissue was washed with 2 x 1 ml of 95% ethanol, dried and l i q u e f i e d by heating i n 1 ml of formamide at 150°C for 2 hours. The ether extracts were evaporated to dryness and the residue dissolved i n 1 ml of ethanol (ethereal f r a c t i o n ) . Aliquots of the four f r a c t i o n s , i . e . the l i q u e f i e d t i ssue, the ethanol washings, the aqueous f r a c t i o n and the ethereal f r a c t i o n were then assayed for r a d i o a c t i v i t y . Following incubation with the 8000 x g supernatant f r a c t i o n or the 100,000 x g p e l l e t suspension, 1 ml of N hydro-c h l o r i c acid was added i n order to stop the reaction, and the mixture was vigorously extracted with 2 x 1.5 volumes of ether. In certain cases, as indicated i n the text, the acid was omitted. The ether extracts and the aqueous fractions were then treated as above. 4. Determination of Radioactivity. The d i s t r i b u t i o n of r a d i o a c t i v i t y intthe various fractions obtained aft e r extraction of the incubation mixtures was determined by counting aliquots by means of a Packard T r i Carb Liquid - 23 -S c i n t i l l a t i o n Spectrometer. Either 0.1 or 0.2 m l of the f r a c t i o n to be counted or of standard estrogen were placed i n a counting v i a l and hyamine hydroxide (1 ml) and s c i n t i l l a t i o n f l u i d (9 ml) added. The hyamine served to neutralize acid samples, and to s o l u b i l i z e protein constituents of the aqueous samples. 5. Examination of the Ethereal Fraction The ether-soluble radioactive compounds formed from the C^-estrogen substrate during incubation with l i v e r preparations were studied mainly by descending paper chromatography. The samples were spotted on Whatman #1 paper and three d i f f e r e n t solvent systems were used to develop the chromatograms. These were the toluene-propylene g l y c o l system of J e l l i n c k 0 ^ , the methanol-oo heptane system of Dao° , and a modified Bush system (Benzene 9: 89 ethyl acetate 1: methanol 5: water 5) . The radioactive compounds separated on the chromatograms 79 were detected by autoradiography . Non-radioactive reference standards were detected by the Folin-Ciocalteu phenol spray and ammonia. 6. Examination of the Aqueous Fraction Protein P r e c i p i t a t i o n and D i a l y s i s : Protein was pr e c i p i t a t e d from the aqueous f r a c t i o n by the addition of 3 ml of 207o ( W/v) t r i c h l o r o a c e t i c acid (TCA) to 5 ml of the aqueous material. - 24 -The p r e c i p i t a t e was spun down at 1000 x g for 10 minutes i n an International Refrigerated Centrifuge, the supernatant decanted and the p r e c i p i t a t e washed by resuspension i n 2 ml of 20% ( w/v) TCA, followed by recentrifugation. By the same procedure the pr e c i p i t a t e was washed with 2 x 3 ml of 95% ethanol. The TCA and ethanol washings were combined, and the p r e c i p i t a t e was l i q u e f i e d by heating i n 1 ml of formamide at 150°C for 2 hours. Samples of each of the l i q u e f i e d p r e c i p i t a t e , the washings and the supernatant f r a c t i o n were counted i n the l i q u i d s c i n t i l l a t i o n counter. The t o t a l recovery of C i n the three fractions a f t e r TCA p r e c i p i t a t i o n was 90-95%. D i a l y s i s of 1 ml samples of the aqueous f r a c t i o n was carried out i n cellophane tubing for 24 hours against running tap water. A portion of the i n d i f f u s i b l e f r a c t i o n was counted, and the loss of r a d i o a c t i v i t y by d i a l y s i s computed aft e r allowing for volume changes. Paper chromatography: Two solvent systems were used for ascending paper chromatography of the water-soluble metabolites of the estrogens. Samples of the ether-extracted aqueous f r a c t i o n from the l i v e r incubations with radioactive estrogen were evaporated at 60-70°C under a stream of a i r , and the concentrated sample was streaked i n a narrow band across a s t r i p of Whatman 3 MM paper which was 3 cm wide. The chromatograms were developed i n - 25 -either a solvent system consisting of butanol, acetic acid, water, 12 : 3 : 591, or i n a solvent system of isopropanol, ammonia, water, 8 : 1 : 1°2. The radioactive compounds which separated on the chromatograms were detected by scanning i n an Actigraph s t r i p recorder. Non-radioactive reference standards chromatographed by these systems were detected with Folin-Ciocalteu phenol reagent. Ultracentrifugation: Tubes containing 1 ml of an 8000 x g supernatant f r a c t i o n of 100, 000 x g microsomes preparation from 100 mg of l i v e r , together with NADPH (1 mg) i n 1 ml 0.1 M potassium phosphate buffer and 10 \ig of estrone-16-C^ i n 1 ml of buffer were incubated for one hour under the conditions previously described. The incubation mixtures were then without further treatment transferred to l u c i t e centrifuge tubes, and centrifuged at 100,000 x g for one hour at 2°C. The supernatant l i q u i d was decanted and the p e l l e t s resuspended by homogenization i n 5 ml of water. Samples from each f r a c t i o n were then counted. In addition, each f r a c t i o n was extracted with 2 x 1 . 5 volumes of ether, and aliquots.from each of the extracted aqueous layers assayed for Cl4. In this way, the amount of r a d i o a c t i v i t y bound to microsomes after incubation was determined. Enzymic Hydrolysis: The pH of a 3 ml sample of the aqueous f r a c t i o n was adjusted to 6.8, and the sample incubated for 48 hrs - 26 -at 37°C with 75 units (1 mg) of b a c t e r i a l p-glucuronidase, 50 jj,g of non-radioactive estradiol-17p-glucusiduronate and 1 drop of chloroform (to retard b a c t e r i a l growth). A control tube, i n which the enzyme was omitted, was also incubated. The tubes were then extracted with 2 x 1.5 volumes of ether, the extracts evaporated to dryness and the residues dissolved i n 1 ml of 957o ethanol. Samples of the aqueous and ethereal 14 fractions were counted. Total recovery of C i n t h i s procedure was 95-1007c. The ether-soluble material was chromatographed on the toluene-propylene g l y c o l system and the developed chromatograms sprayed with Folin-Ciocalteu phenol reagent i n order to detect any estradiol-17p that may have been released from the added non-radioactive estradiol-17p-glucosiduronate. This was to show that the p-glucuronidase preparation was ac t i v e . The ether-extracted aqueous fractions from the control and p-glucuronidase incubations were concentrated by evaporation at 60-70/oC under a stream of a i r , and aliquots containing 4000 cpm were examined by ascending- paper chromatography i n the butanol-acetic acid solvent system, followed by scanning for r a d i o a c t i v i t y i n the Actigraph-recording scanner. Reference standards of estradiol-17p-glucosiduronate and of estriol-17p-glucosiduronate were detected with the Folin-Ciocalteu phenol reagent af t e r - 27 -chromatography. Acid Hydrolysis: 8 ml of a dilu t e d ether-extracted aqueous fr a c t i o n were gently refluxed with 2 ml of concentrated hydro-c h l o r i c acid for 2 hours. After removal of a sample for counting, the acid-treated f r a c t i o n was extracted with 2 x 1.5 volumes of ether and aliquots of th i s ethereal f r a c t i o n as w e l l as of the ether-extracted hydrolysed aqueous phase were counted i n the usual way. Total recovery of was 85-9570, and a l l values given are the mean of at least two experiments. 7. I n h i b i t i o n Studies. These investigations f a l l into two main categories. In the f i r s t , the e f f e c t of c l a s s i c a l enzyme i n h i b i t o r s such as cyanide on the conversion of estrone-16-C^ to water-soluble products by r a t l i v e r preparations was tested. The compounds were added i n a small volume of ethanol or water to an incubation mixture consisting of 1 ml of r a t l i v e r 8000 x g supernatant f r a c t i o n (from 50 mg l i v e r ) , 1 mg of NADPH i n 1 ml of 0.1 M potassium 14 phosphate buffer and 10 |_ig of estrone-16-C i n 1 ml of the buffer. An equivalent amount of the ethanol or water was also added to a control incubation mixture and the solutions after incubation for 60 minutes were a c i d i f i e d and extracted with ether. The d i s t r i b u t i o n of r a d i o a c t i v i t y was determined as previously indicated. - 28 -The second type of i n h i b i t i o n experiment involved investigating the effe c t of s t r u c t u r a l analogues of estrone and of related compounds on the metabolism of estrone-lG-C^ by r a t l i v e r microsomes. In this case, the i n h i b i t o r (2.5 x 10~5M) was added to an incubation mixture consisting of 1 ml of a r a t l i v e r 100,000 x g p e l l e t suspension (from 100 mg l i v e r ) , 1 mg of NADPH 14 =1 i n 1 ml buffer and 10 |ig of estrone-16-C (1.2 x 10"DK) i n 1 ml buffer. An equivalent volume of the medium used to dissolve the i n h i b i t o r was added to a control incubation mixture. After exactly 15 minutes incubation, the reaction was stopped by a c i d i f i c a t i o n , the mixtures ether extracted, and the d i s t r i -bution of r a d i o a c t i v i t y i n the various fractions determined as described previously. In certain of these cases k i n e t i c studies were carried out and the ef f e c t of a constant amount of i n h i b i t o r on the i n i t i a l rates of formation of water-soluble products for d i f f e r e n t concentrations of estrone substrate was determined. Incubations were usually carried out for 5, 10, 15 and 30 minutes at estrone-16-C^1" concentrations of 2, 5, 10 and 15 [ig/3 ml of incubation mixture. From the time curves, the i n i t i a l velo-c i t i e s for the formation of water-soluble products at d i f f e r e n t substrate concentrations were determined, and the Lineweaver-Burk plots of the r e c i p r o c a l of hie i n i t i a l v e l o c i t y (^ ) versus the - 29 -r e c i p r o c a l of the substrate concentration (|) were made for the control and i n h i b i t e d m i x t u r e s . 8. In vivo Studies. In order to correlate the i n v i t r o findings to the s i t u a t i o n i n the whole animals, estrone-16-C-^ (10° cpm i n 25 |j,g) was given i n t r a p e r i t o n e a l l y to each of 2 rats and 2 guinea pigs. The urine from each animal was collected for 24 hours and the amount of r a d i o a c t i v i t y excreted by t h i s route as w e l l as the d i s t r i b u t i o n of C-^ between the ethereal and aqueous phases was determined. For hydrolysis by p-glucuronidase, the pH of the urine samples was adjusted to 6.8 and 3 ml of the f l u i d were incubated for 48 hours with 2.5 mg (187 units) of the enzyme at 37°C inv the presence of one drop of chloroform. These samples were then extracted with ether and the d i s t r i b u t i o n of ascertained. The urine samples (8 ml) were also subjected to acid hydrolysis by r e f l u x i n g with cone. HCl (2 ml) for 2 hr and the amount of r a d i o a c t i v i t y removed by subsequent ether extraction determined. TABLE I Percentage d i s t r i b u t i o n of r a d i o a c t i v i t y after incubation* of 10 jig of estrone-16-C-^ with rat l i v e r preparations. Percentage of added r a d i o a c t i v i t y Liver Incubation Cofactor Aqueous Ethereal Ethanolic Tissue Total preparation Amount period added (1 mg) Fraction Fraction Fraction Fraction Recovei Whole 50 mg 180 mins _ 6.1 58.3 13.8 7.8 86.0 homogenate l i v e r NAD*" 15.9 48.3 10.2 10.5 84.9 • NADP** 28.8 39.2 7.9 9.0 84.9 8000 x g from 60 mins _ 2.0 87.0 _ _ 89.0 supernatant 50 mg NAD 5.8 - - -l i v e r NADH 6.2 - - -NADP 17.7 63.1 - 80.8 NADPH 20.6 64.9 - 85.5 8000 x g from 60 mins NADPH 21.2 75.2 96.4 supernatant 100 mg l i v e r 100,000 x g from 60 mins _ 0.6 91.0 _ — 91.6 microsomal 100 mg NAD 9.7 82.5 - 92.2 p e l l e t l i v e r NADH 9.5 76.5 - 86.0 NADP 3.7 84.0 - - 87.7 NADPH 16.0 65.2 - - 81.2 * Conditions as described i n text. ** Plus 10 mg nicotinamide. - 30 -I I Results 1. Optimal Conditions for the Formation of Water-soluble  Metabolites from Estrone-16-C 1^ by Liver Preparations. Table I shows the d i s t r i b u t i o n of r a d i o a c t i v i t y i n the various fractions following incubation of 10 \xg of estrone-16-Cl4- with r a t l i v e r preparations under the conditions l i s t e d . With the whole homogenate, 8000 x g supernatant, or 100,000 x g microsomes preparations NADPH was required for optimum formation of water-soluble products. NADH or NAD was less e f f e c t i v e , and NADP, although active with the 8000 x g supernatant preparations, was inactive as cofactor with the microsomes. This i s reasonable since NADP can be converted to NADPH by enzymes i n the 100,000 x g supernatant. The i n i t i a l v e l o c i t y for the conversion of estrone-16-C^ 4- i n the presence of NADPH and the 100,000 x g p e l l e t preparation was 0.085 y,moles/ 1/min, whereas with either NAD or NADH i t was 0.025 |amoles/l/min. The optimal concentration of NADPH cofactor was found to be about 1 mg/3 ml of incubation mixture (4.5 x 10"^ M). Increasing t h i s value therefore resulted i n no further stimula-ti o n of the reaction. The optimum; pH for the conversion was found to be about 7.4, a lower y i e l d of water-soluble metabolites being obtained - 31 -when the incubation was carried out at pH 6.0 or 8.0. The conversion proceeded more ra p i d l y at 37°C than i t did at room temperature. Oxygen was shown to be an absolute requirements for the formation of the water-soluble metabolites from estrone-16-Cl^. When the incubation was carried out i n an evacuated Thunberg tube, no r a d i o a c t i v i t y was incorporated into the aqueous f r a c t i o n and chromatography (toluene-propylene g l y c o l system) of the ethereal f r a c t i o n showed that estradiol-17p was the only product formed under these conditions. The 100,000 x g supernatant f r a c t i o n of r a t l i v e r was unable to effe c t the conversion of estrone to water-soluble products, and the nu c l e i or mitochondria were also inactive i n th i s respect. Wiiih guinea p i g l i v e r preparations, good incorporation of r a d i o a c t i v i t y from estrone-16-Cl^- was observed on incubating the estrogen with the whole homogenate, the 8000 x g supernatant f r a c t i o n or with the 100,000 x g microsomes. Addition of NAD or NADPH increased the y i e l d to 50-60?o, although even i n the absence of nicotinamide nucleotide, 307o conversion was observed with the homogenate or the 8000 x g supernatant. The 100,000 x g supernatant, however, was completely without a c t i v i t y . FIGURE I CHROMATOGRAMS OF ETHEREAL FRACTIONS FROM INCUBATION OF 14 ESTRONE-16-C WITH LIVER PREPARATIONS Toluene-Propylene g l y c o l Solvent System Rat 8000 x g supernate Rat 100,000 x g microsomes Reference Standards Guinea P ig 8000 x g supernate Guinea P i g 100,000 x g microsomes 1. E s t r i o l , 2-hydroxyes t r io l 2. 2-hydroxyestradiol-17p 3. 6-ketoestradiol-17p 4. 2-hydroxyestrone 5. es tradiol-17p 6. Estradiol - 1 7 a 7. 6a-hydroxyestradiol -17p 6p-hydroxyestradiol -17g 8.i16a-hydroxyes trone \_ 9. 16-ketoestradiol -17p 16-ketoestrone - 32 -2. Properties of the Ether-soluble Metabolites. In t h i s work, observations on the formation of water-soluble metabolites from estrone-16-C^ were emphasized, however, certain properties of the ether-soluble metabolites were noted. Figure I shows the t y p i c a l resolution of ether-soluble metabolites obtained by means of the toluene-propylene g l y c o l system. Two other solvent system (methanol-heptane, modified Bush) were used, but the only metabolite that could be i d e n t i f i e d i n a l l three systems was estradiol-17p. This compound was formed by ra t l i v e r preparations, and also by the guinea pig l i v e r micro-somes. In addition, these tissue preparations converted estrone-16-C^ to at least three other ether-soluble metabolites, which were more polar than estradiol-17p. Quantitatively, the most important compound had Rf values si m i l a r to 6-ketoestradiol i n two of the solvent systems mentioned, but a mixture of the unknown and the reference standard showed some separation i n the toluene-propylene g l y c o l system. Another ether-soluble metabolite was chromatographically si m i l a r to 16ct-hydroxyestrone, but again not i d e n t i c a l with i t and a fourth metabolite, or group of metabolites, for which good separation was not achieved, behaved very much l i k e e s t r i o l i n the three solvent systems. On occasion, another compound, with an Rf corresponding to that of FIGURE II TIME CURVES FOR THE FORMATION OF WATER-SOLUBLE METABOLITES AND OF PROTEIN-BOUND METABOLITES FROM ESTRONE-16-C14 BY RAT LIVER* Rat l i v e r 8000 x g supernatant (2.0 mg protein) c o •U o cO u w O a; cr < r-i u o 10 20 30 40 50 60 Time (minutes) c o •H 4-» O «J >-( w 3 O <1) cr < O o Rat l i v e r 100,000 x g microsomes (0.7 mg protein) 10 20 30 40 50 60 Time (minutes) 70 80 90 * Incubation mixtures consisted of 1 ml of tissue preparation from 100 mg l i v e r , 1 mg NADPH i n 1 ml 0.1 M potassium phosphate buffer, and 10 |ig estrone-16-C 1 ^ i n 1 ml buffer. T o t a l volume 3 ml. - 33 -2-Hydroxy estradiol-17p was detected i n the modified Bush solvent system, but t h i s r e s u l t was not always reproducible. 2-Hydroxyestrone was not observed to be formed from estrone-16-14 C by r a t or guinea p i g l i v e r preparations, nor could 2-hydroxyestriol be i d e n t i f i e d . Although the guinea pig l i v e r microsomes produced ether-soluble metabolites s i m i l a r to those formed by the r a t l i v e r preparations, the guinea pig l i v e r 8000 x g supernatant did not. In t h i s case, only compounds with Rf values si m i l a r to e s t r i o l , and possible 6- or 2-hydroxyestradiol-17p were detected. 3. Properties of the Water-soluble Metabolites. (i ) Protein Binding: Figure I I depicts the rate of formation of water-soluble products and of TCA-precipitable protein-bound metabolites from estrone-16-C"^ by the r a t l i v e r 8000 x g supernatant f r a c t i o n and by the 100,000 x g microsomes. In the f i r s t case, the i n i t i a l rate of conversion of estrone-16-C^ to protein-bound derivatives was 0.007 (x moles/l/min, whereas i n the second case i t was 0.05 \± moles/l/min. In each instance the enzyme preparation was derived from 100 mg l i v e r , and the 8000 x g supernatant preparation contained 2.0 mg protein, whereas the 100,000 x g microsomes contained 0.7 mg protein. Protein binding, as measured by the i n a b i l i t y of the radioactive metabolites to dialyse across a cellophane membrane TABLE I I FORMATION OF PROTEIN-BOUND METABOLITES OF ESTRONE-16-C 1 4 BY RAT AND GUINEA PIG LIVER PREPARATIONS*. Percentage of water-soluble C ^ 4 p r ec ip i t a t ed by TCA Rat l i v e r Guinea p ig l i v e r 8000 x g supernatant 12.5% 8000 x g supernatant 11.2% Rat l i v e r Guinea p ig l i v e r 100,000 x g microsomes 62.7% 100,000 x g microsomes 78.7% Percentage of water-soluble C ^ 4 non-dialysable Rat l i v e r Guinea p ig l i v e r 8000 x g supernatant 23.5% 8000 x g supernatant 23.2% Rat l i v e r Guinea p ig l i v e r 100,000 x g microsomes 76.8% 100,000 x g microsomes 77.0% Percentage of water-soluble C ^ bound to microsomes** Rat l i v e r Guinea p ig l i v e r 8000 x g supernatant 15.0% 8000 x g supernatant 4.5% Rat l i v e r Guinea p ig l i v e r 100,000 x g microsomes 61.9% 100,000 x g microsomes 49.0% * Each incubation mixture consisted of 1 ml of l i v e r preparation from 100 mg l i v e r , 1 mg NADPH i n 1 m l . 0.1 M potassium phosphate buffer , 10 p,g es t rone-16-C 1 4 i n 1 ml . buffer: t o t a l volume 3ml. ** The mixtures were not a c i d i f i e d p r i o r to ext rac t ion wi th ether. - 34 -within 24 hours, was shown by th i s technique to account for a sim i l a r proportion of the water-soluble metabolites as did TCA p r e c i p i t a t i o n . Table I I gives these r e s u l t s for both r a t and guinea pig l i v e r preparations. In order to ascertain whether the r a d i o a c t i v i t y was bound to microsomal or to soluble protein, incubation mixtures were, pr i o r to ether extraction, centrifuged at 100,000 x g for 60 minutes, as described previously. The amounts of water-soluble metabolites associated with microsomes and with the 100,000 x g supernatant were determined, and Table I I compares the values obtained for r a d i o a c t i v i t y bound to microsomes with those obtained for protein binding TCA p r e c i p i t a t i o n and d i a l y s i s . I t can be seen that for the r a t l i v e r preparations the percentage of precipitated by TCA i s approximately equal to that sedimented at 100,000 x g, while with the guinea pig somewhat more radio-a c t i v i t y i s prec i p i t a t e d by TCA than i s sedimented by spinning at 100,000 x g. This may indicate that some of the C ^ i s bound to "soluble" protein i n the l a t t e r case. ( i i ) Conjugate Formation i n v i t r o : Table I I I records the percentage incorporation of r a d i o a c t i v i t y into the aqueous f r a c t i o n following incubation of estrone-16-0-^ with r a t and guinea pig l i v e r preparations i n the presence and absence of UDPGA. The table also depicts the extent to which treatment of the various TABLE I I I CONJUGATE FORMATION BY RAT AND GUINEA PIG LIVER PREPARATIONS* IN THE PRESENCE AND ABSENCE OF UDPGA. % of C^ -4 of the aqueous f r a c t i o n remaining water-soluble after treatment % of t o t a l C 1 4 Liver UDPGA i n aqueous Incubation with Refluxed preparation 50 mg fr a c t i o n 3-glucuronidase with 20% HCl RAT 8000 x g supernatant - 25.2 94.8 83.3 8000 x g supernatant + 32.2 92.9 62.7 100,000 x g microsomes - 12.3 91.5 92.3 100,000 x g microsomes + 23.0 48.7 48.9 GUINEA PIG 8000 x g supernatant - 68.5 37.1 38.8 8000 x g supernatant + 71.3 21.4 22.1 100,000 x g microsomes - 46.0 94.1 77.3 100,000 x g microsomes + 64.2 26.9 31.8 * The incubation mixture consisted of 1 ml of l i v e r preparation from 100 mg l i v e r , 1 mg of NADPH and 1 mg of NAD i n 1 ml of 0.1 M potassium phosphate buffer, 10 pg of estrone-16-C 1 4 i n 1 ml buffer, and some UDPGA where indicated. Total volume: 3 ml. After incubation for 60 minutes at 37°C under 0 2, the mixtures were extracted with ether without p r i o r a c i d i f i c a t i o n . - 35 -aqueous fractions with p-glucuronidase or re f l u x i n g with 20% ( v/v) HCl rendered the radioactive components of the fractions soluble i n ether. NAD was added to each incubation mixture i n order to show that the lack of glucosiduronate formation i n certain instances was not due to lack of the cofactor required for UDPG oxidation to UDPGA65. The additon of UDPGA to the incubation mixtures containing the 8000 x g supernatant f r a c t i o n or 100,000 x g microsomes of ra t l i v e r caused a s l i g h t increase i n the y i e l d of water-soluble metabolites i n the f i r s t case, and a more marked increase i n the l a t t e r case. When UDPGA was omitted from either r a t l i v e r system, the water-soluble products formed could not be made ether extractable by acid or p-glucuronidase treatment. However, incubation of the 100,000 x g microsomes i n the presence of UDPGA did give r i s e to water-soluble metabolites which were hydrolysed by acid or p-glucuronidase and thus made soluble i n ether. The rat l i v e r 8000 x g supernatant f r a c t i o n i n the presence of UDPGA catalysed the formation of some acid-hydrolysable conjugated material but none that could be hydrolysed by p-glucuronidase. Addition of UDPGA to the guinea pig l i v e r 8000 x g supernatant incubations did not produce a s i g n i f i c a n t increase i n the formation of water-soluble products from estrone-lG-C-^, but the extent of hydrolysis of the aqueous f r a c t i o n by acid or - 36 -enzyme treatment was somewhat increased. However, presence of UDPGA i n the incubation of the guinea pig l i v e r 1 0 0 , 0 0 0 x g microsomes resulted i n a marked increase i n the percentage of r a d i o a c t i v i t y from estrone-16-C-*-4 incorporated into the aqueous f r a c t i o n , and also a large increase i n the proportion of hydrolysable conjugates was observed. Chromatograms of aqueous fractions which had been incubated, i n the presence or absence of p-glucuronidase for 48 hours at 37°C were developed i n the butanol-acetic acid-water solvent system, as described previously. Chromatograms of aqueous fractions which had been stored at 2°C were also developed i n t h i s system. The separation of radioactive compounds on the chromatograms as recorded on the Actigraph scanner i s depicted i n Figures I I I and IV. I t i s important to note that i n each case, 4 § 0 0 cpm of the concentrated aqueous fractions were applied to the chromatograms. This means that i n those instances where hydrolysis of the f r a c t i o n was carried out with p-glucuronidase, the hydrolysed samples were applied i n higher concentrations than th e i r controls, and hence the peaks i n the hydrolysed samples are exaggerated i n comparison to those i n the controls. I l l u s t r a t i o n s l a and 2a, Figure I I I show that compounds with values of 0 (XQ) , 0 . 5 (XQ 5) and 0 . 9 (XQ.O,) were formed by the r a t l i v e r f r a c t i o n s , X N r being found i n greater quantity by LEGEND FOR FIGURE I I I I l l u s t r a t i o n Liver number preparation UDPGA Treatment of aqueous f rac t ion p r i o r to chromatography** l a lb l c 8000 x g supernatant 8000 x g supernatant 8000 x g supernatant Stored at 2° for 48 hrs and extracted wi th ether after spinning at 100,000 x g. Incubated at 37° for 48 hrs and extracted wi th ether. Incubated wi th p-glucuronidase for 48 hrs at 37° and extracted wi th ether. 2a 2b 100,000 x g microsomes 100,000 x g microsomes Stored at 2° for 48 hrs and extracted wi th ether after spinning at 100,000 x g. Incubated at 37° for 48 hrs and extracted wi th ether. 2c 100,000 x g microsomes Incubated wi th p-glucuronidase for 48 hrs at 37° and extracted wi th ether. 3b 3c 4b 8000 x g supernatant 8000 x g supernatant 100,000 x g microsomes + Incubated at 37° for 48 hrs and extracted wi th ether. Incubated wi th p-glucuronidase for 48 hrs at 37 and extracted wi th ether. Incubated at 37° for 48 hrs and extracted wi th ether. 4c 100,000 x g microsomes Incubated wi th p-glucuronidase for 48 hrs at 37° and extracted wi th ether. * Complete incubation mixture as given on Table I I I . ** 4000 cpm of the treated aqueous mater ia l was appl ied to each chromatogram. FIGURE I I I CHROMATOGRAMS OF THE AQUEOUS FRACTION FROM INCUBATION OF ESTRONE-16-C^ WITH RAT LIVER PREPARATIONS l a lb l c 2b 2c Butanol-Acet ic Acid-Water Solvent System V 3b 3c LEGEND TO FACE 4b 1 Reference standards 1. Estr iol -178-glucosiduronate 2. Estradiol-17$-glucosiduronate 9°i - 37 -the 8000 x g supernatant f r a c t i o n than by the microsomes. I l l u s t r a t i o n s lb and 2b show that XQ g and XQ ^  were decomposed after 48 hours incubation at 37°C. These unstable compounds were not converted to ether-soluble products, and i t appears from the chromatograms that they were changed to more polar derivatives, which remained at the o r i g i n . The r e s u l t s depicted i n Figure I I I show that a peak with s i m i l a r to that of estradiol-17p-glucosiduronate was formed only by the 100,000 x g microsomes i n the presence of UDPGA. This peak was abolished by p-glucuronidase treatment. In i l l u s t r a t i o n 4c the peak at the o r i g i n i s exaggerated with reference to the control (4b), since the hydrolysed sample applied contained about twice as much material as the unhydrol-ysed control. No s i g n i f i c a n t amount of a product with an Rf sim i l a r to estradiol-17p-glucosiduronate was present i n the aqueous fractions from incubation of the 8000 x g super-natant f r a c t i o n i n the presence or absence of UDPGA. I l l u s t r a t i o n s l a and lb (Fig. IV) show that the major compound formed on incubation of the guinea pig l i v e r 8000 x g 14 supernatant f r a c t i o n with estrone-16-C had an Rf simi l a r to that of estradiol-17p-glucosiduronate, that t h i s compound was stable to prolonged incubation, but hydrolysed by p-glucuroni-dase. The same picture was observed for the material from the LEGEND FOR FIGURE IV I l l u s t r a t i o n Liver number preparation UDPGA Treatment of aqueous fr a c t i o n p r i o r to chr oma t o gr ap hy** l a lb l c 2a 2b 8000 x g supernatant 8000 x g supernatant 8000 x g supernatant 100,000 x g microsomes 100,000 x g microsomes Stored at 2° for 48 hrs and extracted with ether a f t e r spinning at 100,000 x g. Incubated at 37° for 48 hrs and extracted with ether. Incubated with p-glucuronidase for 48 hrs at 37° and extracted with ether. Stored at 2° for 48 hrs and extracted with ether after spinning at 100,000 x g. Incubated at 37° for 48 hrs and extracted with ether. 2c ./: 100,000 x g microsomes 3b 8000 x g supernatant 3c 8000 x g supernatant 4b 100,000 x g microsomes 4c 100,000 x g microsomes Incubated with p-glucuronidase for 48 hrs at 37° and extracted with ether. + Incubated at 37° for 48 hrs and extracted with ether. + Incubated with p-glucuronidase for 48 hrs at 37° and extracted with ether. + Incubated at 37° for 48 hrs and extracted with ether. + Incubated with p-glucuronidase for 48 hrs at 37° and extracted with ether. * Complete incubation mixture as given on Table I I I . ** 4000 cpm of the treated aqueous material was applied to each chromatogram. FIGURE IV CHROMATOGRAMS OF THE AQUEOUS FRACTION FROM INCUBATION OF ESTRONL-16-C 1 4 WITH GUINEA PIG LIVER PREPARATIONS Butanol-Acetic Acid-Water Solvent System LEGEND TO FACE Reference standards E s t r i o l - 1 7 £ - g l u c o s idurona te Estradiol-17$-glucosiduronate 3b 3c 4b 4c 9? FIGURE V CHROMATOGRAMS OF THE AQUEOUS FRACTION FROM INCUBATIONS* OF ESTRONE-16-C 1 4 WITH GUINEA PIG LIVER PREPARATIONS. Ammonia-Isopropanol"solvent system Liver \ preparation 8000 x g supernatant 100,000 x g microsomes Reference Standards * Complete incubation mixture as given on Table I I I . J ^^^^ ^^^f ^^ ^^  ^^ ^^ ^^ v^  J o o 1. Estriol-17(^-glucosiduronate 2. Estradiol-17£-glucosiduronate - 38 -incubation with the UDPGA-fortified 8000 x g supernatant f r a c t i o n ( i l l u s t r a t i o n s 3b and 3c). Very small amounts of the compound with Rf 0.5 previously observed i n fractions from r a t l i v e r incubations were detected i n the hydrolysed samples ( l c and.3c). I l l u s t r a t i o n s 2b and 2c show that the guinea pig 100,000 x g microsomes give r i s e to very l i t t l e estradiol-17p-glucosid-uronate-like material, unless UDPGA i s added to the incubation medium ( i l l u s t r a t i o n s 4b and 4c). Chromatograms of the various aqueous fractions developed i n the ammonia-isopropanol water solvent system did not show good separation of material from the r a t systems, but i t was e f f e c t i v e for the "glucosiduronate" product from the guinea p i g 8000 x g supernatant incubation (Figure V). I t appears that estriol-17p-glucosiduronate as w e l l as the estradiol-17p derivative was formed i n t h i s system. I t was also shown by chromatography i n the toluene-propylene g l y c o l and methanol-heptane solvent systems that substantial amounts of estradiol-17p are released from the guinea pig aqueous fractions a f t e r hydrolysis with p-glucuronidase. Smaller amounts of estrone, and of metabolites with similar mobility to e s t r i o l and i t s epimers were also released by the enzyme. This data, together with that obtained from the chromatograms of the aqueous f r a c t i o n s , points to TABLE IV IN VIVO METABOLISM OF ESTRONE-16-C IN THE RAT AND GUINEA PIG E s t r o n e - 1 6 ( 1 0 ^ cpm i n 25 (j,g) given I.P. to 2 animals; urine collected for 24 hours % of C^ remaining i n urine after treatment Treatment of urine RAT GUINEA PIG (141,000 cpm i n urine) (572,000 cpm i n urine) Extracted with ether 74.8 63.4 Incubated with p-glucuronidase and extracted with ether 67.8 10.8 Refluxed with 20% HC1 and extracted with ether 51.8 9.6 - 3 9 -estradiol-17p-glucosiduronate as being the major conjugate formed by the guinea pig 8000 x g supernatant f r a c t i o n from estrone. Other conjugates were undoubtedly also present. Rat and guinea pig microsomes f o r t i f i e d with UDPGA appeared to produce e s t r a d i o l -17p-glucosiduronate from estrone, but the r a t 8000 x g supernatant did not form t h i s conjugate under any circumstances. Conjugate Formation i n vivo: Table IV depicts the fate of estrone-16-C^1- i n the r a t and guinea pig following intraperitoneal i n j e c t i o n of 25 \xg of the estrogen (10 cpm) . Whereas 5770 of the injected C ^ was excreted i n the urine of the guinea p i g , only 147o of the injected r a d i o a c t i v i t y was found i n r a t urine. Ether extraction, however, showed that a substantial part of the C^ "4 was water-soluble i n both cases. Incubation of the r a t urine at pH 6.8 with p-glucuronidase f a i l e d to render any more r a d i o a c t i v i t y extractable with ether than i n the untreated control and even after acid hydrolysis the bulk of the remained i n the aqueous f r a c t i o n . In contrast, the guinea pig urine contained a very large percentage of acid and enzyme-hydrolysable conjugates. 4. Metabolism of Estradiol-17p-16-C 1 4 by Rat and Guinea Pig Liver  Preparations •Estradiol-17p-16-C^ behaved very much l i k e estrone when incubated with r a t or guinea pig l i v e r preparations and exhibited the same 40 -s p e c i f i c i t y towards the nicotinamide nucleotide cofactor i n forming water-soluble metabolites. Chromatograms of the ethereal fractions obtained after 14 incubation of estradiol-17p-16-C with r a t l i v e r microsomes showed that only small amounts of estrone were formed, and that the major ether-soluble metabolites were r e l a t i v e l y polar compounds with R.£ values si m i l a r to e s t r i o l and to 2- and 6-hydroxyestradiol-17p. In contrast to incubations with estrone-16-C^, a metabolite with an s i m i l a r to that of 6-ketoestradiol-17p was not formed from estradiol-17p-16-C^. The toluene-propylene g l y c o l and methanol-heptane chromatographic systems were used i n these studies. Acid and p-glucuronidase hydrolysis experiments showed that estradiol-17p-16-C"'"4 i s not metabolized to simple glucosiduronate conjugates by r a t l i v e r microsomes i n the absence of added UDPGA, nor did guinea pig l i v e r microsomes catalyse the formation of substantial amounts of the conjugates. The guinea pig l i v e r 8000 x g supernatant f r a c t i o n did however give r i s e to large amounts of conjugated radioactive material. 14 Estradiol-17p-16-C was converted to protein-bound derivatives to the same extent as estrone-16-C"'"4 by r a t and guinea pig l i v e r . 5. Metabolism of D i e t h y l s t i l b e s t r o l (monoethyl-l-C^ 4) by Rat and  Guinea Pig Liver Preparations. S t i l b e s t r o l - C ^ 4 appeared to be metabolized by r a t and guinea pig - 41 -preparations i n a manner similar to estrone-16-Cl 4 and e s t r a d i o l -17p-16-cl 4. Oxygen and NADPH were required for maximal formation of water-soluble metabolites. As with the natural estrogens, simple conjugates were not fommed from stilbestrol-C-'- 4 by r a t or guinea pig l i v e r microsomes i n the absence of added UDPGA, but they were formed by the guinea pig 8000 x g supernatant f r a c t i o n . In the l a t t e r case, s t i l b e s t r o l - C ^ - 4 i t s e l f , as w e l l as several more polar ether-soluble materials were conjugated with glucuronic acid. Approximately 20% of the water-soluble products formed during incubation of stilbestrol-C-'- 4 with the 8000 x g supernatant f r a c t i o n of r a t l i v e r was bound to protein. 6. I n h i b i t i o n of the Formation of Water-soluble Metabolites from  Estrone-16-C-*-4 by Rat Liver Microsomes. (i) C l a s s i c a l Enzyme In h i b i t o r s : Potassium cyanide at a concetr-ation of 10"^ M produced 74% i n h i b i t i o n i n the formation of water-soluble metabolites from estrone-16-C"'-4 by r a t l i v e r 8000 x g 3 supernatant f r a c t i o n . 65% i n h i b i t i o n was observed with 10 M KCN, and 38% i n h i b i t i o n with 10" 4 M KCN. N-ethylmaleimide, a sulphydryl group i n h i b i t o r , at a con-centration of 10"^ M or 10~3 M produced 90% i n h i b i t i o n of the reaction(s) and i n h i b i t o r concentration of 10~ 4 M reduced the y i e l d of water-soluble products by 57%. p-chloromercuribenzoate, another strong sulphydryl group i n h i b i t o r , i n h i b i t e d the conversion to the same extent. I t was shown by chromatography i n the toluene-propylene g l y c o l system that KCN did not i n h i b i t the formation of e s t r a d i o l -17f3 from radioactive estrone, whereas the sulphydryl group i n h i b i t o r s , as expected, completely abolished this dehydrogenase-mediated reaction. 2 10 M sodium fl u o r i d e did not a f f e c t the estrone metabolizing system and the enzyme catalase, at a concentration of 67 [xg/ml produced very s l i g h t i n h i b i t i o n i n the formation of the water-soluble metabolites ( 1570) . ( i i ) I n h i b i t i o n Studies with Structural Analogues of Estrone: A large number of compounds having s t r u c t u r a l features s i m i l a r to those of estrone were tested for t h e i r a b i l i t y to i n h i b i t the formation of water-soluble metabolites from estrone-16-C-'-4 by r a t l i v e r microsomes. The i n h i b i t o r s were added at twice the molar concentration of the substrate and, after incubation, as described previously, the eff e c t of the added compound on the incorporation of r a d i o a c t i v i t y from estrone-16-C^ 4 into the aqueous f r a c t i o n was determined. Table V l i s t s those compounds that produced over 757o i n h i b i t i o n , Table VI those compounds which gave 50-60% i n h i b i t i o n and Table VII the compounds tested that were without appreciable i n h i b i t o r y a c t i v i t y . I t can be seen from Table V that whereas 2-hydroxyestrone and 2-hydroxyestradiol were both potent i n h i b i t o r s , 2-hydroxy-TABLE V COMPOUNDS TESTED AS INHIBITORS IN THE FORMATION OF WATER-SOLUBLE PRODUCTS FROM ESTRONE-16-C 1 4 BY RAT LIVER MICROSOMES. GROUP I (over 75%' i n h i b i t i o n ) Added substance* I n h i b i t i o n (%) 2-hydroxyestrone 85 2-hydroxyestradiol 85 equi lenin 78 1,2-naphthoquinone 81 1,4-naphthoquinone 88 menadione (2-methyl-l,4-naphthoquinone) 84 1,4-benzoquinone 78 1.4- toluquinone 80 2.5- dimethyl-l,4-benzoquinone 77 hydroquinone 77 1.4- toluhydroquinone 78 2.5- d i - t - b u t y l hydroquinone 78 tetramethyl hydroquinone 83 p y r o g a l l o l (1,2,3-trihydroxybenzene) 89 * Inh ib i to r s (2.5 x 10 M) were added at twice the molar concentration of estrone. TABLE V I COMPOUNDS TESTED AS INHIBITORS IN THE FORMATION OF WATER-SOLUBLE PRODUCTS FROM ESTRONE-16-C 1 4 BY RAT LIVER MICROSOMES. GROUP I I (50-60% i n h i b i t i o n ) Added s u b s t a n c e * I n h i b i t i o n (%) e s t r a d i o l - 1 7 a 52 e s t r a d i o l - 1 7 p 52 e q u i l i n 57 2 - h y d r o x y e s t r i o l 60 e s t r o n e s u l f a t e 50 e s t r o n e b e n z o a t e 59 e t h y n y l e s t r a d i o l 57 d i e t h y l s t i l b e s t r o l 59 h e x e s t r o l 59 d i e n e s t r o l 50 c a t e c h o l 56 p-naphthol 59 2 , 5 - d i h y d r o x y p h e n y l a c e t i c a c i d 59 * I n h i b i t o r s (2.5 x 10°M) were added a t t w i c e the molar c o n c e n t r a t i o n o f e s t r o n e . 0 TABLE V I I COMPOUNDS TESTED AS INHIBITORS IN THE FORMATION OF WATER-SOLUBLE PRODUCTS FROM ESTRONE-16-C 1 4 BY RAT LIVER MICROSOMES. GROUP I I I ( l e s s than 20% i n h i b i t i o n ) Added s u b s t a n c e * 6 a - h y d r o x y e s t r a d i o l 6 p - h y d r o x y e s t r a d i o l 6 - k e t o e s t r o n e 6-ketoes t r a d i o l 16ct-hydroxyestrone 1 6 - k e t o e s t r a d i o l 1 7 p - h y d r o x y e s t r a - p - q u i n o l 1 6 - k e t o e s t r o n e e s t r i o l * * e s t r a d i o l - 1 7 p - g l u c o s i d u r o n a t e 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 d i e t h y l s t i l b e s t r o l g l u c o s i d u r o n a t e d i h y d r o x y h e x e s t r o 1 d i c a r b o x y h e x e s t r o l t h y r o x i n e a d r e n a l i n e 1.2- d i m e t h y l a n t h r a q u i n o n e 1.3- d i m e t h y l a n t h r a q u i n o n e 1.4- d i m e t h y l a n t h r a q u i n o n e r e s o r c i n o l 3.5- d i h y d r o x y t o l u e n e 1,3,5-trihydroxybenzene hexahydroxybenzene 3 , 4 - d i m e t h y l p h e n o l t y r o s i n e t r y p t o p h a n p r o g e s t e r o n e * * t e s t o s t e r o n e c o r t i s o n e C o r t i s o l p-hydroxypropiophenone d e h y d r o e p i a n d r o s t e r o n e * I n h i b i t o r s (2.5 x 10" 5M) were added a t t w i c e t h e molar c o n c e n t r a t i o n o f e s t r o n e . ** P r o d u c e d 277o i n h i b i t i o n , - 42a -e s t r i o l was only an i n h i b i t o r of intermediate strength (Table VI), and the 6-hydroxy and 6-keto estrogens, as w e l l as the 16-hydroxy derivatives showed no i n h i b i t i o n (Table V I I ) . E s t r a d i o l -17p, estradiol-17a, ethynyl e s t r a d i o l , estrone sulphate, and estrone benzoate gave 50-607o i n h i b i t i o n , but the glucosiduronate conjugates of the estrogens were in a c t i v e . The naphtholic estrogen equilenin was a potent i n h i b i t o r , and e q u i l i n retained intermediate a c t i v i t y while the synthetic estrogens, d i e t h y l s t i l -b e s t r o l , hexestrol and dienestrol also f e l l i n the intermediate category. On the other hand, dihydroxyhexestrol and dicarbo-xyhexestrol, which are not estrogenic, were non-inhibitory. Thyroxine, adrenaline and various non-phenolic steroids were inac t i v e . A d i s t i n c t group of non-steroidal compounds which included benzoquinones, naphthoquinones, and o- and p-hydroxyl-ated phenols were shown to be as potent i n h i b i t o r s as 2-hydroxy-estrone and 2-hydroxyestradiol-17p. With the exception of catechol and homogentisic acid, these compounds at concentrations as low as 2.5 x 10"^ M produced a decrease of over 757o i n the y i e l d of water-soluble metabolites from estrone-C^ 4 and menadione (10"^ M) produced 60% i n h i b i t i o n . In contrast to th i s group, various anthraquinones were completely without a c t i v i t y as i n h i b i t o r s , as were m-hydroxylated phenols such as r e s o r c i n o l , - 43 -3,4-dihydroxytoluene and 1 ,3,5-trihydroxybenzene. Chromatograms of the ethereal fractions obtained from incubations i n the presence of i n h i b i t o r s f a i l e d to show any consistent changes i n the ether-soluble metabolites that could be correlated with the a b i l i t y of a compound to i n h i b i t the conversion of estrone to water-soluble material. However, an increased amount of C^4 e s t r a d i o l was formed i n the presence of non-radioactive estradiol - 1 7 p or -17a, 2-hydroxyestradiol -17a or any e s t r a d i o l derivative, including the non-inhibitory ones such as 6-ketoestradiol - 1 7 p . Lineweaver-Burk Plo t s: Since zero order k i n e t i c s appeared to be operating during the f i r s t 15 minutes of the reaction i n 14 which estrone-16-C was being converted to water-soluble material by the r a t l i v e r microsomes, k i n e t i c studies with some i n h i b i t o r s were carried out and the c l a s s i c a l Lineweaver-Burk analysis for types of i n h i b i t i o n was applied to the k i n e t i c data. The i n h i b i t o r s tested were 2-hydroxyestrone, equilenin, s t i l b e s t r o l , and menadione, and the Lineweaver-Burk plots for these i n h i b i t o r s are given i n Figures VI, VII, VIII and IX respectively. The plots suggest that 2-hydroxyestrone, equilenin and s t i l b e s t r o l act as competitive i n h i b i t o r s at a rate l i m i t i n g step involved i n the formation of the water-soluble products from estrone-16-C-'-4. In each case the maximum v e l o c i t y (V m a x ) °f t^ i e - 44 -reaction was unchanged i n the presence of i n h i b i t o r , whereas the Km was d i s t i n c t l y a l t e r e d , f i t t i n g the requirements for competitive i n h i b i t i o n . Menadione, on the other hand, appears to produce a mixed type of i n h i b i t i o n since both the V m a x and the Km were changed i n the presence of the i n h i b i t o r . FIGURE VI LINEWEAVER-BURK PLOT FOR THE INHIBITION BY 2-HYDROXYESTRONE OF THE CONVERSION OF ESTRONE-16-C14 TO WATER-SOLUBLE METABOLITES BY RAT LIVER MICROSOMES. / 80 • \ 60 • 1 V 40 -/ 2-HYDROXYESTRONE 0/ (5.9 nM) /1/min)" 1 / ^^^^ \ x / • \ 20 • y CONTROL ; ; — i — — — i L-4- t 0 0.2 0.4 1 (VLM) S 0.8 -1 vmax= 0 16 (imoles/1/min Km = 5.0 |iM K. = 2.5 nM FIGURE VII LINEWEAVER-BURK PLOT FOR THE INHIBITION BY EQUILENIN OF THE 14 CONVERSION OF ESTRONE-16-C TO WATER-SOLUBLE METABOLITES BY RAT LIVER MICROSOMES. / S V m a x 3 0.17 umol.es/1/myn Km = 5.6 uM Kt = 2 . 4 FIGURE V I I I LINEWEAVER-BURK PLOT FOR THE INHIBITION BY STILBESTROL OF THE CONVERSION OF ESTRONE-16-C14 TO WATER-SOLUBLE METABOLITES BY RAT LIVER MICROSOMES. FIGURE IX LINEWEAVER-BURK PLOT FOR THE INHIBITION BY MENADIONE OF THE CONVERSION OF ESTRONE-16-C 1 4 TO WATER-SOLUBLE METABOLITES BY RAT LIVER MICROSOMES. 80 - \ -*" • 60 - . MENADIONE (1.9 uM) ,. : • ^ • • ' ' -\ 1 V 40 • (p.moles/1/min) ^  ' . X / . • . • • \.S •: , . • ' - •' • V , ••• ' .- ' \ • • ' • • 20 -- : 1 1 . **** CONTROL ; ) — 1 \ 1 • — 0 0.2 0.4 \ 0.6 1 (uM)" 1 S • . / vmax (control) = 0.16 M-moles/l/min vmax ( inhib i ted) - 0.06 umoles/l /min - 45 -DISCUSSION The natural estrogens are metabolized by mammalian l i v e r to a va r i e t y of products. Liver microsomes contain estrogen 2-hydroxylase^^ as w e l l as 16a and 1 6 p 9 ^ , 6a and 6 p ^ ° , and 10p hydroxylases^ } and each of these enzymes requires molecular oxygen and NADPH. The corresponding dehydrogenases are also present i n the microsomes. A s i g n i f i c a n t proportion of the metabolites of estrogens formed by l i v e r consists of products which are soluble i n water, and insoluble i n ether, a solvent i n which these steroids and their simple mono-hydroxylated metabolites are r e a d i l y soluble. In some cases conjugation can account for the water-soluble metabolites. De Meio, for instance, has described the formation of estrogen sulphates by an ox l i v e r 100,000 x g supernatant f r a c t i o n 7 4 , while Breuer and Smit h ^ have recently shown that estrone i s conjugated as the monoglucosiduronate on incubation with rabbit l i v e r microsomes f o r t i f i e d with UDPGA. However, water-soluble metabolites that are not hydrolysed by sulphatase, p-glucuronidase or 207o ( v/ v) HC1 have also been discovered. Mueller and other workers have described a metabolite formed by r a t l i v e r from e s t r a d i o l which i s strongly bound to protein82,83 while J e l l i n c k 0 ^ , working with r a t l i v e r s l i c e s , showed that - .46 -10-15% of the water-soluble metabolites of estrone existed i n a protein bound form. The remaining 85-90% was not characterized. There are, therefore, at least three types of water-soluble products formed from estrone under various conditions by l i v e r . The f i r s t type includes protein-bound metabolites, the second includes simple conjugates such as glucosiduronates and sulphates, and a t h i r d the unknown compounds. In the present investigation the general requirements for the conversion of estrone-16-C-'-4 to water-soluble products by r a t and guinea pig l i v e r preparations were determined. The r e s u l t s , l o c a l i z i n g the enzymes involved i n the microsomes and showing that NADPH and oxygen are required, were found to be i n agreement with published data. The guinea pig l i v e r prepara-tions were more active than the r a t l i v e r fractions when incuba-ted under the same conditions. About twice as much estrone-16-C-'-4' was converted to water-soluble metabolites by the guinea pig fractions as was by the r a t l i v e r preparations. An investigation of the properties of the water-soluble 14 metabolites of estrone-16-C showed that both the r a t and guinea pig l i v e r preparations were capable of binding the estrogen or i t s metabolite to protein. However, a marked difference i n the proportion of protein-bound metabolites was observed on incubating 14 estrone-16-C with d i f f e r e n t tissue preparations. The i n i t i a l - 47 -rate of formation of protein-bound metabolites by the 100,000 x g rat l i v e r microsomes was 0.05 u. moles/l/min whereas i t was only 0.007 |i moles/l/min for the 8000 x g supernatant f r a c t i o n , each tissue preparation being derived from 100 mg of l i v e r . S i m i l a r l y , the amount of r a d i o a c t i v i t y bound to protein accounted for about 14 707o of the C i n the aqueous f r a c t i o n a f t e r incubation with r a t or guinea pig 100,000 x g microsomes, but represented only about 157o of the C"''4 i n the aqueous f r a c t i o n obtained after incubation with the r a t or guinea p i g 8000 x g supernatant f r a c t i o n . This i s a s t r i k i n g difference i n view of the fact that the 8000 x g supernatant preparation contains 2.0 mg protein, whereas; the 100,000 x g p e l l e t f r a c t i o n contains only 0.7 mg protein. I t appears, therefore, that i n the presence of the 100,000 x g supernatant, a pathway for estrone metabolism other than: protein binding predominates, whereas i n i t s absence protein binding becomes the preferred metabolic reaction. A possible reason for t h i s i s given i n l a t e r discussion. Each of the three techniques used to measure protein binding gave r e s u l t s that were i n reasonable agreement with one another. The fact that the same percentage of water-soluble products from the r a t l i v e r incubations was precipitated by TCA as sedimented on centrifugation at 100,000 x g for 60 minutes suggests that the r a d i o a c t i v i t y was a c t u a l l y bound to microsomal - 48 -protein, and not to soluble protein. With the guinea pig prepara-tions, since the amount of water-soluble spun down at 1 0 0 , 0 0 0 x g was somewhat less than the amount prec i p i t a t e d by TCA, i t i s possible that the r a d i o a c t i v i t y i s bound to soluble as w e l l as to microsomal protein. I t would be of interest to know the mechanism of protein binding and the nature of the protein(s) to which the estrogen derivatives are bound. L i t t l e i s known outside the fact that the bond between the estrogen moiety and the protein i s very strong. A proposed mechanism for the formation of such metabolites i s that the estrogen i s f i r s t converted to a r i n g A o-hydroxylated d e r i -vative which i s then oxidized to the o-quinone. This highly reactive substance can now combine with protein acceptors to y i e l d stable complexes. However, although mushroom tyrosinase has been shown 81 to catalyse estrogen i n t e r a c t i o n with protein i n t h i s manner there i s l i t t l e evidence that such a process takes place i n r a t or guinea pi g l i v e r . The present studies f a i l e d to confirm the formation of detectable quantities of 2-hydroxyestrone or 2-hydroxy-e s t r i o l i n systems which were r a p i d l y producing the protein-bound derivatives. Occasionally traces of a compound with an Rf value s i m i l a r to 2-hydroxyestradiol were detected, but t h i s was not consistently reproducible. I t could, of course, be argued that o-hydroquinones are i n fact formed by the l i v e r systems, but are - 49 -then oxidized so r a p i d l y to quinones which i n turn Interact with protein that they cannot be obtained as stable intermediates. However, the i s o l a t i o n of r e l a t i v e l y large amounts of the 2-2? 18 . -hydroxylated and 2-methoxylated estrogens from urine speaks against such a sequence of reactions. Good evidence that the o-quinones are not i n fact formed was recently obtained by J e l l i n c k ^ 4 . Estrone-16-C"'"4 was incubated with r a t l i v e r microsomes or with mushroom tyrosinase i n the presence of ethylene diamine. A dihydropyrazine derivative of the estrone was formed i n the plant enzyme incubation mixture, and th i s derivative was shown to be the adduct of the estrogen o-quinone with the ethylene diamine. Such a derivative was not formed i n the r a t l i v e r incubation mixture, even though high yie l d s of water-soluble metabolites were obtained, suggesting that o-quinonoid intermediates of estrone are not formed i n t h i s animal system. An a l t e r n a t i v e theory to account for the formation of the protein-bound derivatives of estrogens can be drawn from Hecker's hypothesis for the mechanism of formation of the 2-, 6- and 10-hydroxylated estrogens outlined i n the introduction^ . The estrogen free r a d i c a l s formed i n the process could, instead of being converted to the hydroxylated derivatives, interact with certai n reactive groups on protein to form a stable complex. - 50 -Evidence for t h i s type of in t e r a c t i o n has been obtained from the studies on the ef f e c t of horseradish peroxidase on estrone- J e l l i n c k 0 ^ found that t h i s enzyme, i n the presence of protein or certain amino acids, converted estrone -16-to water-soluble metabolites i n high y i e l d s . The enzyme appeared to be acting i n i t i a l l y as an aerobic oxidase, and could u t i l i z e protein or amino acid to generate hydrogen peroxide. Subsequently, acting as a true peroxidase the enzyme converted estrone to highly reactive metabolites, probably phenoxyl r a d i c a l s , which were then able to combine with the protein or amino acid. Catalase was a potent i n h i b i t o r of t h i s system as would be expected for a reaction involving hydrogen peroxide. However, i t was found i n the present studies with l i v e r prepa-rations that catalase, even at high concentrations, only s l i g h t l y i n h i b i t e d the formation of protein-bound and other water-soluble estrone - 1 6-cl 4 derivatives. This would seem to r u l e out a peroxidative type of reaction for the formation of the protein-bound derivatives by l i v e r , though i t has been suggested by 95 Posner e_t a l B that hydrogen peroxide generated within the microsomes may not be affected by catalase. Estradiol - 1 7 p was also shown to be metabolized to protein-bound derivatives by l i v e r , and i t i s l i k e l y that the mechanism i s the same as for estrone. D i e t h y l s t i l b e s t r o l also - 51 -gave r i s e to protein-bound derivatives and again, one or other of the two theories proposed above could apply i n t h i s case. The formation of estrogen conjugates by r a t and guinea pig l i v e r was also investigated and the re s u l t s shown that i n the absence of added UDPGA r a t l i v e r preparations do not give r i s e to products that can by hydrolysed by p-glucuronidase or hydro-c h l o r i c acid under conditions known to bring about the complete hydrolysis of the mono- or diglucosiduronates of natural estrogens^^. The conditions of acid hydrolysis used were more than adequate to hydrolyse any sulphate conjugates as w e l l ^ . In addition, the urine of rats which had been injected with estrone-16-C"^4 contained water-soluble estrogen metabolites which could not be rendered ether-soluble by treatment with acid or p-glucuronidase. Since certain strains of rats had been reported to have a congenital lack of glucuronosyl transferase enzyme9^, i t was f i r s t thought the Wistar rats used i n these experiments did not possess the enzyme which transfers glucuronic acid to the aglycon acceptor. However, th i s was shown not to be the case, since incubation of the r a t l i v e r microsomes with excess UDPGA and estrone-16-Cl^ resulted i n increased incorporation of radio-a c t i v i t y into the aqueous f r a c t i o n , and i n the formation of a metabolite chromatographically i d e n t i c a l with estradiol - 1 7 p -glucosiduronate, which was hydrolysed by acid and by p-glucuro-nidase. The amount of the conjugated metabolite formed - 52 -corresponded to the UDPGA-promoted increase i n water-soluble r a d i o a c t i v i t y . I t seems, therefore, that Wistar r a t l i v e r does not lack glucuronosyl transferase, but for some reason or other i s without an adequate supply of available UDPGA. The picture, however, i s complicated by the observation that i n the case of the 8000 x g supernatant f r a c t i o n of r a t l i v e r the addition of excess UDPGA did not provide conditions suitable for the form-ation of p-glucuronidase-hydrolysable material from estrone-IS-C''"4 though an acid-hydrolysable conjugate was produced. I t i s possible that a complex conjugate such as' a sulphoglucuronide, which would l i k e l y be only p a r t i a l l y hydrolysed by p-glucuroni-C 62 dase under the conditions used , i s formed. I t i s possible that the soluble portion of the r a t l i v e r c e l l contains some factor which i n h i b i t s the formation of simple estrogen glucosiduronates, even i n the presence of ample glucuronic acid donor, or that an alter n a t i v e type of conjugation occurs more r e a d i l y under these conditions. In experiments with the guinea p i g , i t was shown that glucosiduronate conjugates of estrone are formed i n vivo, and that the 8000 x g supernatant f r a c t i o n of the l i v e r produces glucosiduronates of estrone, estradiol - 1 7 p and s t i l b e s t r o l , with or without added UDPGA. Estrone was shown to be metabolized by the 8000 x g supernatant f r a c t i o n to give compounds which were - 53 -chromatographically si m i l a r to estradiol - 17p-glucosiduronate and to estriol - 1 7 p-glucosiduronate. The l i v e r microsomes, however, on incubation with estrone i n the absence of UDPGA did not give r i s e to appreciable amounts of glucuronic, acid conjugated material, which was to be expected since UDPGA i s produced only i n the soluble portion of the l i v e r c e l l ^ . Addition of the glucuronic acid donor to an incubation mixture containing the microsomes resulted i n substantial amounts of a compound simi l a r to estradiol - 1 7 p-glucosiduronate being formed from estrone. Thus, there appears to be a marked species difference i n the ease with which glucosiduronate conjugates of estrogens are formed i n v i t r o by the r a t and the guinea p i g . In f a c t , exo-genous estrogen i s excreted i n the urine of the guinea pig mainly as the glucuronic acid conjugate, whereas i n the r a t , although i t i s excreted lar g e l y i n a water-soluble form, most of i t i s not bound as a simple glucosiduronate. I t i s u n l i k e l y that any quantity of the simple sulphate conjugates of estrogens was formed by the l i v e r preparations since the amount of hydrolysis produced by acid corresponded to the amount that occurred with p-glucuronidase, and t h i s enzyme preparation was free of sulphatase a c t i v i t y under the conditions used, since sulphatase i s markedly i n h i b i t e d by 0.1 M phosphate 97 buffer . The one exception to t h i s , where an acid-hydrolysable - 54 -conjugate which was in sens i t i ve to p-glucuronidase was produced from estrone by the 8000 x g supernatant of r a t l i v e r f o r t i f i e d wi th UDPGA, has already been discussed. So fa r , discussion of the poss ible i d e n t i t y of the water-soluble estrogen metabolites which are not protein-bound or conjugated wi th glucuronic or sulphuric ac id has been neglected. This type of compound i n fact accounts for about 857Q of the water-soluble mater ia l produced from estrone, estradiol-17p and s t i l b e s t r o l by the r a t l i v e r 8000 x g supernatant preparations, and for about 3070 of the metabolites formed by the r a t and guinea p ig l i v e r microsomes. With the guinea p i g 8000 x g supernatant, or whole homogenate, and also i n v i v o , these compounds are not formed i n appreciable amounts since glucuronic ac id conjugation predominates. However, these un iden t i f i ed compounds do appear to be major metabolites of estrogen both i n v i t r o and i n v ivo i n the r a t . This mater ia l formed from estrone-16-cl 4 i n incubations wi th l i v e r preparations i s by no means homogenous. Chromato-graphy of various aqueous f rac t ions , from which the microsomes had been removed by cent r i fugat ion , showed that several r ad io -act ive compounds were present i n each f r a c t i o n . In the butanol-ace t ic ac id system an unstable compound wi th an Rf of 0.5 ( X Q 5) was the major metabolite from the r a t 8000 x g supernatant - 55 -incubation, although compounds wi th R£ values of 0.9 ( X Q g) and 0 ( X Q ) were a lso detected. S imi la r metabolites were formed by the ra t l i v e r microsomes but the r e l a t i v e amount of compound X Q was smaller . Guinea p ig microsomes gave r i s e to X Q and small amounts of X Q 9, X Q ^ being detected only i n h igh ly conc-entrated hydrolysed samples. Hence, at leas t three d i f fe ren t metabolites of estrone, which are not prote in bound and are not simple conjugates must be considered. Formation of large amounts of X Q ^ by ra t l i v e r seems to depend on a factor i n the 100,000 x g supernatant f r ac t ion and together wi th X Q g, X Q 5 was shown to be converted after incubation at 37°C for 48 hours to mater ia l which d id not move from the o r i g i n . Unfortunately, no chromatographic system has been found that was su i tab le for separating these very polar products. At present, very l i t t l e i s known about the nature or mode of formation of these water-soluble estrogen metaboli tes, though several speculations can be made. The two theories put forward to account for formation of the protein-bound metabolites of estrogens both involve h ighly reac t ive unstable intermediates. In one case, an o-quinoid de r iva t ive , and i n the other a phenoxyl r a d i c a l type of intermediate was postulated. I t i s poss ib le that one or other of these intermediates i s formed by the micro-somes, and that i n the absence of the soluble por t ion of the l i v e r - 56 -c e l l , these in te rac t mainly wi th microsomal p ro t e in . In the presence of the soluble por t ion of the c e l l the reac t ive i n t e r -mediate could combine wi th other acceptors not present i n such large amounts i n the microsomal f r ac t i on . These could be amino acids or small polypeptides. This theory would expla in why a much higher proport ion of the r a d i o a c t i v i t y remaining i n the ether-extracted aqueous f rac t ion i s bound to pro te in after incuba-t ing l i v e r microsomes wi th estrone-16-C^ 4 than after incubation wi th the 8000 x g supernatant f r a c t i o n . There are also other p o s s i b i l i t i e s for the i d e n t i t y of these water-soluble metaboli tes . I t has been suggested that they are in t ac t phenolic estrogen der ivat ives which are water-soluble no by v i r t u e of a large number of hydroxyl groups . 6-hydroxy-e s t r i o l for instance i s r e l a t i v e l y water-soluble , having a p a r t i t i o n coe f f i c i en t i n an ether-water system of 0.12, whereas QQ that for e s t r i o l i s 7.7 . There i s evidence, however, that such simple polyhydroxylated der ivat ives are not formed to any great extent by r a t l i v e r preparations, since J e l l i n c k ' ' " ^ showed that ace ty la t ion or methylation of the aqueous f rac t ion from a r a t l i v e r incubation f a i l e d to make a s i g n i f i c a n t amount of the products soluble i n ether. Yet another p o s s i b i l i t y i s that the water-soluble metabolites - 57 -are degradation products i n which Ring D has been oxid ized to give a marr ianol ic ac id or further oxid ized compounds. Evidence against th i s i s that no C^40"2 i s evolved during incubations wi th estrone-IS-C''-4 and a lso that marr ianol ic ac id and re la ted compounds have never been detected i n n a t u r e ^ . Much of the information on the oxida-t i ve formation of water-soluble products i n fact points to changes i n Ring A as being of primary importance and i t s extensive oxidat ion to y i e l d an open structure wi th free carboxyl groups must a lso be considered. More de f in i t e knowledge about the nature of the water-soluble estrogen metabolites that are not bound to p ro te in or conjugated wi th sulphuric or glucuronic a c i d awaits the i r i s o l a t i o n and chemical charac te r iza t ion . Pre l iminary studies on the i r separation by paper chromatography has been discussed i n th i s work. Due to methodological d i f f i c u l t i e s i n the charac te r iza t ion of the water-soluble end-products of estrone metabolism by l i v e r , another approach to the problem was taken. I t was considered that information on the mode of formation of the end-products could be obtained from studies on the i n h i b i t i o n of the i r formation by various compounds. The observation that KCN and the sulphydryl group i n h i b i t o r s N-ethylmaleimide and p-chloromercuribenzoate i n h i b i t the formation of water-soluble products from estrone by r a t - 58 -l i v e r preparations confirms the work of others. KCN could be inac t iva t i ng a copper or i ron-conta ining oxidase, while the other two i n h i b i t o r s would be i n a c t i v a t i n g a sulphydryl group containing enzyme, poss ib ly a dehydrogenase. Mushroom tyrosinase and horse-radish peroxidase, both of which metabolize estrone to water-soluble products, are i nh ib i t ed by KCN but not by N-ethylmal-eimide, showing that a more complex ser ies of react ions must be occurring wi th l i v e r . The studies on the i n h i b i t i o n of formation of water-soluble products from estrone by compounds having s t r uc tu r a l features s imi l a r to those of estrone have y ie lded some in t e re s t ing data. I t was reasoned that th i s type of study could give some clues about the nature of the groups that are of importance i n the pathway leading to the formation of water-soluble products from estrone. Some ideas have been obtained, though the data i s some-times hard to in te rp re t , due to ce r ta in l i m i t a t i o n s i n the experiments themselves. Thus, since the enzyme preparation used (the microsomes) represent a heterogeneous system, the p o s s i b i l i t y ex is t s that the compound which was tested as an i n h i b i t o r was not i n h i b i t o r y per se, but was f i r s t metabolized to give the ac tua l i n t e r f e r ing substance. A l s o , i t i s d i f f i c u l t to ascer ta in whether the various i n h i b i t o r s were a f fec t ing the same step i n - 59 -the conversion of estrone to water-soluble metaboli tes, since t h i s , i n a l l l i k e l i h o o d , i s a mult i -s tage process. Another d i f f i c u l t y i n in te rpre ta t ion ar i ses i n the k i n e t i c s tudies . Cer ta in ly zero-order k i n e t i c s appear to be operating i n the f i r s t stages of the process by which water-soluble metabolites are formed, but the p o s s i b i l i t y ex is t s that several d i f fe ren t rates of reac t ion combine to give what only appears to be zero-order k i n e t i c s . Even considering these l i m i t a t i o n , however, the r e su l t s obtained appear to j u s t i f y th i s c l a s s i c a l approach, i f only to point out further work of in te res t that could be ca r r i ed out when p u r i f i e d preparation of the enzyme(s) involved are obtained, and when the i d e n t i t y of the metabolites i s ascer tained. A group of estrogens and estrogen der iva t ives were shown to be potent i n h i b i t o r s of estrone conversion to water-soluble metabolites by r a t l i v e r microsomes. 2-Hydroxyestrone, 2-hydroxy-est radiol-17p, and equi lenin gave over 7570 i n h i b i t i o n whi le 2-hydroxyes t r io l , es t radio l -17p, es t rad io l -17a , e q u i l i n , ethynyl e s t r a d i o l and estrone sulphate and benzoate gave 50-6070 i n h i b i t i o n . Since the sulphate and the benzoate esters were shown to be hydrolyzed by the microsomal enzymes, the effect of these compounds can be a t t r ibu ted to a d i l u t i o n of the es t rone-16-C 1 4 substrate. In contrast to these r e s u l t s , the 6-hydroxy-, 16-keto- and 10-hydroxy- der iva t ives d id not exh ib i t any i n h i b i t o r y ac t i on . - 60 -Non-phenolic s ter iods were also i nac t i ve , as were the 17p-gluco-siduronate conjugates of the phenolic s t e ro ids . At t h i s stage i t appears that a phenolic Ring A i s required for i n h i b i t o r y a c t i v i t y by a s t e ro id , and that 2-hydroxy subs t i tu -ents grea t ly enhance th i s e f fec t , whereas subs t i tu t ion on the C-16 or C-6 pos i t i ons , or glucosiduronate conjugation at C-17 has the opposite e f fec t . I t i s i n t e res t ing to note that the non-s tero ida l synthet ic estrogens, d i e t h y l s t i l b e s t r o l , hexest rol and d ienes t ro l i n h i b i t , whereas the i r non-estrogenic der iva t ives dihydroxyhexestrol and dicarboxyhexestrol do not . The substi tuents i n these l a s t two compounds are on the e thy l side chains. In the Lineweaver-Burk p l o t s , s t i l b e s t r o l , 2-hydroxyestrone and equi len in a l l appeared to act as competitive i n h i b i t o r s i n the conversion of estrone to water-soluble products, implying that the i r s tructure or that of the i r metaboli tes, i s s u f f i c i e n t l y s imi l a r to that of estrone or an intermediate of estrone, to be able to combine wi th the same ac t ive s i t e ( s ) on the enzyme. A further in te rp re ta t ion , e spec ia l ly wi th regard to the i n h i b i t i o n by the 2-hydroxy compounds, i s that the added i n h i b i t o r i s i d e n t i c a l w i th an intermediate formed from the estrone-16-C 1 4 substrate, and that i n h i b i t i o n occurs as a r e s u l t of C 1^ d i l u t i o n or e lse by a feed-- 61 -back type of mechanism. From th i s point of view i t might be sa id that 2 -hydroxyla t ion i s an e s sen t i a l part of the mechanism for the formation of water-soluble metabolites of estrone, whereas 6-, 10- or 16-hydroxylat ion, or glucosiduronate formation i s not involved . However, the i n h i b i t o r y ac t ion of the naphtholic estrogen, equ i l en in , and of the non-s teroid , s t i l b e s t r o l , does not f i t e a s i l y in to th i s scheme, and there i s no d i r e c t evidence that 2 -hydroxylat ion i s an e s sen t i a l step i n forming water-soluble metabolites, as has been discussed p rev ious ly . Although the i n h i b i t o r y estrogens and the i r der iva t ives appear to act compet i t ively , benzoquinones, naphthoquinones and the i r reduced forms do not act i n t h i s way i f the r e su l t s obtained wi th menadione can be taken to apply to the whole group. The Lineweaver-Burk p l o t for i n h i b i t i o n by menadione showed a mixed type of competitive and non-competitive a c t i o n . I t i s worth noting that meta-hydroxylated phenols, which of course cannot be oxidized to a quinonoid form, were inac t ive as i n h i b i t o r s , as were the anthraquinones tes ted. The mechanism of ac t ion of these i n h i b i t o r s i s very much open to specula t ion. Their common features make one think that they may i n some way in te r fe re wi th an oxidat ion-reduct ion process i n estrogen metabolism. Molecular oxygen i s an absolute requi re-ment for the formation of water-soluble products from estrone by - 62 -ra t l i v e r , and i s u n l i k e l y to be u t i l i z e d d i r e c t l y , but rather by way of an e lec t ron transport system. Menadione, and re la ted compounds could poss ib ly act by i n h i b i t i n g e lec t ron transport , although i t was noted that these i n h i b i t o r s d id not in te r fe re wi th the formation of ether-soluble metabolites from estrone, which are presumably hydroxylated de r iva t ives . I t can only be sa id that the mechanism by which both the s t e ro ida l and non-s tero idal compounds i n h i b i t the conversion of estrone to water-soluble products i s very complex and i t w i l l be of in te res t to repeat these studies wi th p u r i f i e d enzymes. A l l attempts by the author to render soluble the microsomal enzymes have so far been unsuccessful. - 63 -SUMMARY Three main pathways i n l i v e r by which estrone may be converted to water-soluble metabolites have been demonstrated. By one such pathway, estrone or i t s der iva t ives are s t rongly bound to p ro te in , and th i s process, which i s mediated by microsomal enzymes, requires NADPH and oxygen. A second route gives r i s e to glucosiduronate conjugates, the transfer of glucuronic a c i d from UDPGA to the s te ro id being catalysed by a microsomal enzyme. A t h i r d pathway for estrone metabolism i n l i v e r was demonstrated, but the nature of the products formed by i t i s l a rge ly unknown. Oxygen and NADPH are required, and the enzymes are probably i n the microsomes, although a factor(s) i n the 100,000 x g supernatant f rac t ion of the c e l l i s of importance. The metabolites formed by th i s route are not bound to p ro te in , nor are they hydrolysed by r e f lux ing wi th 20% ( V / v ) HC1 or incubating wi th p-glucuronidase, and at leas t three d i f fe ren t products have been detected. In add i t ion , t h i s "unknown" pathway i s i n h i b i t e d by cyanide and by sulphydryl group i n h i b i t o r s . S p e c i f i c a l l y , i t was shown that when r a t l i v e r microsomes were incubated wi th NADPH and es t rone-16-C 1 4 about 70% of C 1 4 became bound to p ro te in , and no conjugation wi th glucuronic or sulphuric ac id occurred. I f , however, UDPGA were added to the - 63 -SUMMARY Three main pathways i n l i v e r by which estrone may be converted to water-soluble metabolites have been demonstrated. By one such pathway, estrone or i t s der iva t ives are s t rongly bound to p ro te in , and th i s process, which i s mediated by microsomal enzymes, requires NADPH and oxygen. A second route gives r i s e to glucosiduronate conjugates, the transfer of glucuronic ac id from UDPGA to the s te ro id being catalysed by a microsomal enzyme. A t h i r d pathway for estrone metabolism i n l i v e r was demonstrated, but the nature of the products formed by i t i s l a rge ly unknown. Oxygen and NADPH are required , and the enzymes are probably i n the microsomes, although a factor(s) i n the 100,000 x g supernatant f rac t ion of the c e l l i s of importance. The metabolites formed by th i s route are not bound to p ro te in , nor are they hydrolysed by r e f lux ing wi th 20% ( v / v ) HCl or incubating wi th p-glucuronidase, and at l eas t three d i f fe ren t products have been detected. In add i t ion , t h i s "unknown" pathway i s i n h i b i t e d by cyanide and by sulphydryl group i n h i b i t o r s . S p e c i f i c a l l y , i t was shown that when r a t l i v e r microsomes were incubated wi th NADPH and es t rone-16-C 1 4 about 70% of the water-soluble C l 4 was bound to p ro t e in , and no conjugation wi th glucuronic or sulphuric ac id occurred. I f , however, UDPGA were added to the - 64 -incubation mixture, estrogen glucosiduronates were formed i n addi t ion to the other products. When the r a t l i v e r 8000 x g supernatant f rac t ion was incubated wi th NADPH and estrone-16-C^ 4 , protein-bound metabolites accounted for only 10-157o of the water-soluble r ad io -a c t i v i t y , the remainder being formed by the unknown route . Addi t ion of UDPGA to the incubation medium promoted the formation of an a c i d -hydrolysable conjugate which, however, d id not behave as a simple glucosiduronate. Injected estrone- 16-C^"4 was excreted i n the urine of ra ts mainly as water-soluble metabolites which could not be hydrolysed by HCl or p-glucuronidase, and the "unknown"pathway therefore appears to predominate i n v ivo i n th i s species. When guinea p ig l i v e r microsomes were incubated under 14 aerobic conditions wi th NADPH and estrone-16-C , most of the water-soluble metabolites formed were bound to pro te in (707o) . Addi t ion of UDPGA to the incubation mixture, however, promoted the formation of large amounts of glucosiduronate conjugates of the s t e ro id , at the expense of prote in bound ma te r i a l . Incubation of the guinea p ig l i v e r 8000 x g supernatant f r ac t ion wi th NADPH and estrone-16-C 1" 4 resul ted i n the conversion of the rad ioac t ive substrate mainly to glucosiduronate conjugates, both i n the presence and absence of UDPGA. In the guinea p ig i n v i v o , glucosiduronate conjugation was - 65 -shown to be the major route for the metabolism of estrone. Es t rad io l-17p-16 -C 1 4 and s t i l b e s t r o l (monoethyl- l -C 1 4 ) are metabolized by ra t and guinea p ig l i v e r preparations i n a pat tern s imi la r to estrone-16-C-*-4. In order to gain further information about the nature of the unknown water-soluble products formed from estrone by r a t l i v e r microsomes the effect on th i s reac t ion by compounds having s t ruc t -u r a l features s im i l a r to the rad ioac t ive substrate was s tudied. 2-Hydroxyestrone, 2-hydroxyestradiol-17p and equi lenin were found to be very potent i n h i b i t o r s , whi le estradiol-17p and -17a, s t i l b e s t r o l and hexes t ro l showed intermediate a c t i v i t y . A l l the 6- or 16-hydroxylated der iva t ives of the na tura l estrogens, as w e l l as the 17p-glucosiduronates and non-phenolic s teroids tested were without a c t i v i t y . A group of benzoquinones, naphthoquinones and the i r reduced forms were found to be very strong i n h i b i t o r s , but anthraquinones and meta-hydroxylated phenols were i n a c t i v e . 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