The Oxidative Metabolism of Estrogens by Mammalian
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
in the Department
of
Biochemistry
We accept this thesis as conforming to the required standard
THE UNIVERSITY OF BRITISH COLUMBIA
May, 1963. i In' presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of
British Columbia, I agree that the Library shall, make it freely available 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,, It is understood that copying, or publi• cation of this thesis for financial gain shall not be allowed without my written permission.
Department of ^t^LJ-*^<^^>0^
The University of British Columbia,. Vancouver 8, Canada.
Date V7A^ 7, • ABSTRACT
The main problem of estrogen metabolism studied has been to
determine the nature of the water-soluble products formed from
estrone-16-C^ by rat liver preparations. Comparative studies were carried out in the guinea pig.
Three types of water-soluble metabolites were demonstrated, namely, protein-bound derivatives, glucosiduronate conjugates, and unidentified products which were not bound to protein and were not hydrolysed by 2N HC1.
The water-soluble metabolites formed on incubating rat or guinea pig liver microsomes with estrone-16-C^ in the presence of NADPH and oxygen consisted of protein-bound material, some unknown derivatives, but virtually no simple conjugates.
Incubation with the rat liver 8000 x g supernatant fraction resulted largely in conversion of the estrogen to the unknown water-soluble end-products, while,in contrast, this liver
fraction from guinea pig gave rise mainly to glucosiduronates.
In the presence of UDPGA, both rat and guinea pig liver micrdr
somes converted estrone-16-cL4 to glucosiduronate conjugates, but this did not occur with the rat liver 8000 x g supernatant fraction.
Estradiol-iyp-lG-C1^- and stilbestrol-C14 behaved similarly to estrone-16-C^. In the rat, in vivo, the bulk of the urinary water-soluble derivatives of estrone were of unknown nature, while in the guinea pig, glucosiduronate. conjugation predominated.
The problem was also studied by a different approach.
Various compounds having structural features similar to estrone were tested for their ability to inhibit the forma• tion of water-soluble metabolites from this estrogen by rat liver microsomes.
It was found that 2-hydroxyestrone, 2-hydroxyestradiol-17p and equilenin were potent inhibitors, while those estrogens which had an oxygen function at C-6 or C-16, as well as the
17p-glucosiduronates and non-phenolic steroids tested were inactive. The synthetic estrogens, stilbestrol and hexe- strol, both inhibited the reaction, but their non-estrogenic analogues had no effect. A group of benzoquinones, naphtho• quinones and ortho- and para-hydroxylated phenols proved to be powerful inhibitors, whereas anthraquinones and meta-hydroxy- lated phenols showed no activity.
In kinetic studies, 2-hydroxyestrone, equilenin, and stilbestrol appeared to act as competitive inhibitors, but menadione gave a mixed type of inhibition. ACKNOWLEDGEMENTS
The author wishes to express her gratitude to Dr. P.H. Jellinck for his help and encouragement during this work, also to Dr. R.L. Noble for the use of the facilities in his laboratory.
The author is very grateful for the assistance of Miss J. Leonard, Miss L. Irwin and Miss E. Grisedale in the preparation and typing of this thesis. 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. Inhibition Studies 27
8. In vivo Studies 29
II 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 Diethylstilbestrol (monoethyl-l-C ) by Rat and Guinea Pig Liver Preparations 40
6. Inhibition of the Formation of Water-soluble Metabolites from Estrone-16-C^ by Rat Liver Microsomes 41
DISCUSSION 45
SUMMARY 63
BIBLIOGRAPHY . 66 TABLES
Page
I. Percentage distribution of radioactivity after incubation of 10 \xg estrone-16-C with rat liver preparations (to face) 30
II. Formation of protein-bound metabolites of estrone-16-C^ by rat and guinea pig liver preparations (to face) 34
III. Conjugate formation by rat and guinea pig liver preparations in the presence and absence of UDPGA (to face) 35
IV. In vivo metabolism of estrone-16-Cl^ in the rat and guinea pig (to face) 39 V. Compounds tested as inhibitors in the formation of water-soluble products from estrone-16-C by rat liver microsomes. Group I (to follow) 42
VI. Compounds tested as inhibitors in the formation of water-soluble products from estrone-16-C by rat liver microsomes. Group II (to follow Table V)
VII. Compounds tested as inhibitors in the formation of water-soluble products from estrone-16-C by rat liver microsomes. Group III (to follow Table VI) Figures Page
I. Chromatograms of ethereal fractions from incubation of estrone-16-C^ with liver preparations (to face) 32
II. Time curves for the formation of water- soluble metabolites and of protein-bound metabolites from estrone-16-C by- rat liver (to face) 33
III. Chromatograms of the aqueous fraction from incubation of estrone-16-Cl^ with rat liver preparations (Legend to face) (to follow) 36
IV. Chromatograms of the aqueous fraction from incubation of estrone-16-C with guinea pig liver preparations (Legend to face) (to follow) 37
V. Chromatograms of the aqueous fraction from incubations of estrone-16-C^ with guinea pig liver preparations (to face) 38
VI. Lineweaver-Burk plot for the inhibition by 2-hydroxyestrone of the conversion of estrone-16-C-^ to water-soluble metabolites by rat liver microsomes (to follow) 44
VII. Lineweaver-Burk plot for the inhibition by equilenin of the conversion of estrone-16-C^ to water-soluble metabolites by rat liver microsomes (to follow Figure VI)
VIII. Lineweaver-Burk plot for the inhibition by stilbestrol of the conversion of estrone-16-C^ to water-soluble metabolites by rat liver microsomes (to follow Figure VII)
IX. Lineweaver-Burk plot for the inhibition by menadione of the conversion of estrone-16-Cl4 to water-soluble metabolites by rat liver microsomes (to follow Figure VIII) - 1 -
INTRODUCTION
Inactivation of Estrogens.
The original observation that implicated liver as a site
of estrogen metabolism was made in 1934 by Zondek^", who reported
that rat liver mince was capable of rapidly inactivating estrone.
Many workers confirmed and extended this in vitro result, using
loss of biological activity as a means of following hormone
degradation. Thus, Heller^ showed that liver was the major
organ in rats and rabbits involved in the destruction of estrone,
estradiol-17p and estriol, but that kidney was only slightly active, while spleen, heart, lung, placental tissue and uterus 3 were entirely without effect. Twombly and Taylor demonstrated
that human liver preparations were also capable of inactivating
estradiol-17p, but at a slower rate than either rat or mouse
liver. In addition, beef^ and dog liver^ were shown to destroy estrogens in vitro. 2 Heller found that cyanide inhibited the inactivation of
estrone and estradiol-17p by rat liver slices, and it was shown by Levy that carbon monoxide and sodium azide were inhibitors.
This pointed to participation of a heavy metal enzyme, similar
to tyrosinase or the cytochrome system in the inactivating
system. De Meio e_t al, in 1948 showed that inactivation of
estradiol-17p would not proceed in the absence of oxygen, and - 2 - also found indications that an NAD-linked dehydrogenase was involved in this process. De Meio's results also suggested that the biologically inactive metabolites were not simple conjugates of the estrogens, but that oxidative degradation products were formed.
Many in vivo experiments have confirmed these in vitro observations. Thus, Golden and Sevringhaus^ in 1938 transplanted the ovaries of rats to the mesentery and to the axillae and found that estrus did not occur in the animals bearing transplants in the mesentery, where the blood carrying the estrogen would pass directly to the liver. Biskind.9 implanted pellets 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 directly into the hepatic portal system.
In other experiments with rats in which the liver had been damaged, prolongation and enhancement of the response to endogenous and exogenous estrogens was observed^.
Although this work confirmed the idea that liver performed an integral role in the inactivation of estrogens in mammals, very little evidence as to the nature of the metabolites was gained from these experiments and only with the introduction of , radioisotopic and chromatographic techniques did this become possible. - 3 -
Ether-soluble Metabolites.
The first experiments using these more advanced techniques dealt with the interconversion of estrone and estradiol-17p. 11 12 Thus, Ryan and Engel ' in 1952 using counter-current fraction• ation and photo-fluorimetry demonstrated the interconversion of these two estrogens under both aerobic and anaerobic conditions, in a variety of normal and abnormal human tissues, while other workers 13 have observed it in a large number of different species . The dehydrogenase catalyzing this reaction was purified from placenta by Engel et al.l^, and shown to require NAD or NADP as cofactor.
The kinetics of the enzyme reaction have been studied in vitro"^ and Fishman-^ has shown that the equilibrium lies markedly in favour of estrone in vivo in the human.
From 1957 to 1960 a large number of different enzymes involved in the oxidative metabolism of estrogens were discovered in liver. The isolation of 2-methoxyestronel7 and of 2-methoxy- estriol^ 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 in two distinct steps - namely hydroxylation and subsequent methylation^. 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 estradiol and estriol in their urine. Fishman^ injected estradiol-C^
into a patient and recovered 12% of the radioactivity as
2-hydroxyestrone in the urine so that in man at least, 2-hydroxy-
lation appears to be quantitatively important.
In vitro studies on the 2-hydroxylase were carried out by 23 24
King ' , who localized the enzyme in rat and rabbit liver 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 in the formation 25 of the 2©methoxy estrogens, Knuppen et al, observed enzymic methylation of 2-hydroxyestradiol in the presence of human liver
slices and S-adenosylmethionine. It was later shown that an
O-methyl transferase which is present in the soluble fraction of rat liver is responsible for the methylation of the 2-hydroxy-
estrogens and it is of interest that the same enzyme methylates
the catechol amine hormone, arterenol . There is some evidence 27 for the presence of a demethylating enzyme in liver microsomes
Another enzyme of importance in the oxidative metabolism
of estrogens is a 6-hydroxylase. It was first detected in 1957 28 29 bestrioy Muellel byr anradt Rumneliver y slicein mouss ien livethe rpresenc microsomee ofs NADP, anH dan Breued oxyger n
later observed the 6-hydroxylation of estrone, estradiol and - 5 -
Both 6a- and 6p-hydroxylases were present in rat liver microsomes
as well as the corresponding dehydrogenases^. 6-Hydroxylation 31
has been observed with human liver , but as yet no 6-hydroxy
estrogen has been detected in human urine.
In addition to the 2- and 6-hydroxylases, rat liver
microsomes have been shown to contain an enzyme capable of
hydroxylating estradiol-17p in the 10p position, giving rise 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 rat liver microsomes contain an enzyme capable of removing hydrogen from
the phenolic hydroxyl group of estradiol-17p and thereby giving
rise to a phenoxyl radical. It was suggested that the unpaired
electron in this reactive intermediate would tend to migrate to
positions 2-, 6- and 10- of the steroid molecule and interact
with free hydroxyl radicals to form corresponding hydroxylated metabolites. This attractive theory for the NADPH-dependent
formation of 2-, 6- or 10-hydroxylated estrogens awaits further
investigation.
In addition to rings A and B, rings C and D of the
steroid have also been shown to be hydroxylated in the course of - 6 - estrogen metabolism. An 11-hydroxylase has recently been detected in ox adrenal tissue though it has not as yet been found in liver.
However, llp-hydroxyestrone is metabolized by rat liver slices giving rise to ll-ketoestradiol-17p;-and. to llp-hydroxyestradiol-17p .
An 18-hydroxylase, apparently situated in the adrenal cortex, can hydroxylate estrone, and 18-hydroxyestrone has been 35 isolated from human pregnancy urine by L0ke, Marrian et al
Quantitatively the most important metabolites of estrone and estradiol in vivo are their 16-oxygenated derivatives. As 36 early as 1942, Pincus demonstrated that estrone and estradiol-17p are converted to estriol in large amounts in vivo and Brown in 37
1957 showed that 45% of injected estrone or estradiol-17p is excreted by man as estriol in the urine. Although it was long thought that 16a-hydroxylation of estradiol-17p gave rise to 38 estriol, a recent experiment by Fishman et al, has shown that estrone is the more important precursor of estriol in vivo in humans. 39 Marrian in 1957 isolated 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 estriols. This hypothesis has been upheld both by the 38 in vivo work of Fishman and also by numerous in vitro experiments 40,41,43 by Breuer 16-ketoestradiol-17p has been shown by King and Levitz to arise, in vivo and in vitro, from partial oxidation of estriol, and 16-ketoestradiol-17p itself has in turn been shown to be 44 metabolized to estriol, 17-epiestriol and to 16-ketoestrone.
On the other hand, 16-ketoestrone, on injection into man, gives rise to estriol and 16-epiestriol in the urine^, implicating
16-ketoestradiol-17p as an intermediate. In experiments with 46
human liver slices, Breuer et al0 showed that 16-ketoestrone can be metabolized to 16a-hydroxyestrone, 16p-hydroxyestrone,
16-ketoestradiol-17p, 16-epiestriol, 17-epiestriol and
16,17-epiestriol. In addition, 16-ketoestradiol-17a has been 47 shown to give rise to both 17- and 16,17-epiestriol . In fact, the only possible compound of this type that has not been shown to be formed in human liver is 16-ketoestradiol-17a and the formation of 16-ketoestrone from 16-ketoestradiol-17a has not yet been demonstrated.
The complicated interrelationships between the C-16 oxygen• ated estrogens can be summarized by saying that man and other mammal possess in the liver, estrone 16a- and 16(3-hydroxylases, 16a and
16p-hydroxysteroid dehydrogenases, as well as 17a- and 17#-hydroxy- steroid dehydrogenases. The formation of 16a-hydroxyestrone and its reduction to estriol are quantitatively the most important reactions. - 8 -
Breuer and his colleagues have recently shown that human and rat liver contain a steroid epoxidase, capable of forming 16a, 17a- epoxyestratrien-3-ol from estratetraen-3-ol^8. An epoxhydra- tase is also present, capable of forming 16, 17-epiestriol from the a-epoxide and estriol from the p-epoxide^^.
Glucosiduronate and Sulphate Conjugates.
With the possible exception of 6-hydroxy or 6-ketoestriol, all of the aforementioned oxidative metabolites of estrone and estradiol are water-insoluble and ether-soluble^. However, it has been known for some time that estrogens are often found in the urine in a water-soluble form, conjugated with either glucuronic or sulphuric acid. Cohen and Marrian5^ in 1936 first isolated estriol monoglucosiduronate from pregnancy urine and more recently,
Carpenter and RelUe5-^ have obtained both estriol-16-glucosidu- ronate and estriol-17-glucosiduronate from this source. Oneson 52 and Cohen have detected estrone glucosiduronate in urine.
In 1947 Crepy5-^ demonstrated the in vitro formation of glucosiduronates of estrone, estradiol-17p and estriol, using rabbit and guinea pig liver slices, but Schiller and Pincus5^ failed to obtain evidence for conjugation of estrogen by male rat liver. Glucosiduronate conjugation of the "synthetic" estrogens, stilbestrol, hexestrol and dienestrol has been observed in vivo with rats55, rabbits and cats56, and Zimmerberg5? - 9 -
in 1946 showed that rat liver slices conjugated stilbestrol,
probably as the glucosiduronate, whereas rat liver minces did not. 58
In 1958 Lehtinen et al . found that rat duodenal mucosa slices
catalysed the formation of estradiol-17p-glucosiduronate from
estradiol-17p.
In these in vitro studies the actual reaction products were
not isolated; the experiments mainly involved the hydrolysis of
the conjugate by p-glucuronidase or by acid with subsequent
identification and recovery of the free steroids.
Diczfalusy and his colleagues in Sweden have recently
reported a large series of experiments on estrogen conjugation in humans. In studies on conjugation by intestinal tract, they found
that following injection of estriol into an isolated loop of the
duodenum with the arterial blood supply intact, large amounts of
estriol-16(17?)-glucosiduronate appeared in both the effluent venous blood and in extracts of the intestinal wall. Evidence
for the formation of a di- or tri-glucosiduronate was also obtained, 59
and, in addition, small amounts of estriol-3-sulphate were found .
In a similar experiment, injection of estradiol-l-7p gave rise to
large amounts of estrone glucosiduronate, and some evidence for
the presence of estradiol-17p-glucosiduronate and the 3,17-di-
glucosiduronate was obtained*^. - 10 -
Diczfalusy and his co-workers have also carried out extensive studies on estrogen metabolism in 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 fluid and of the urine of new-borns. In the latter two fluids, estriol-16(17?)-glucosiduronate predominated. Three additional estriol conjugates were detected and partially characterized in these fluids. Two of them appeared to be di- or tri-gluco- siduronates, and the third a double conjugate, possibly estriol-3-sulphate, 16(17?)-glucosiduronate or estriol-3- sulphoglucosiduronide . The sulphoglucuronide may represent an intermediate step in a transconjugation reaction as suggested by Twombly and Levitz who isolated estrone glucosiduronate from the urine after administration of estrone sulphate (labelled in 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 estriol in cord blood, glucosiduronates make up most of the estrogen 6 2 conjugated material in amniotic fluid and urine
The biochemical mechanism of the formation of gluco• siduronates and of glucuronides was formulated from the work of several laboratories. Smith and Mills^ in 1954 isolated uridine diphospho glucuronic acid (UDPGA) from liver, and showed - 11 -
that it acted as the glucuronic acid donor in the formation of
conjugates by liver homogenates. Strominger^ later demonstrated
that UDPGA was formed in the soluble fraction of the cell 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 in the microsomes, being mediated by UDP-glucuronyl transferase^^. Isselbacher in 1956^6 reported success in solubilizing the coupling system from guinea pig liver and the preparation was active in conjugating
estradiol-17p, tetrahydrocortisone and thyroxine. More recently a better preparation of the enzyme has been obtained from rabbit liver microsomes which is active in the synthesis of both ester and ethereal glucuronides . By way of confirmation
of this, Smith and Breur have shown that estrone, on incubation with a rabbit liver microsomal preparation and UDPGA is con- 69 jugated as the monoglucosiduronate .
As mentioned previously, estrogen sulphates are formed
in mammals. Thus, McKenna and co-workers^ have recently
obtained estrone-3-sulphate from pregnancy urine and Menini and Diczfalusy have isolated and identified estriol-3-sulphate
in human meconium^, and have also demonstrated its formation
in various tissues of the human foetus and new-born6^. The - 12 -
The soluble fraction 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 identified estrone 73 sulphate as a major form of estrogen in human pregnancy plasma
The mechanism of sulphurylation has been investigated by
De Meio and colleagues^, and they have demonstrated the form• ation of estrone and estradiol-17f3 sulphates by rat and ox microsomes-free liver supernatants, and by an ammonium sulphate precipitate from the supernatant in 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 it is postulated that, except for the specific active sulphate transferring enzyme, the process is the same for estrogens as for simple phenols.
Estrogen sulphatase has been demonstrated in rat and ox liver microsomes and this enzyme is present in quantities comparable to the enzyme synthesising the estrogen sulphates^-*.
Water-soluble Metabolites other than Simple Conjugates.
It has long been suggested that estrogens are converted in mammals to water-soluble products other than sulphate or glucosiduronate conjugates. Beer and Gallagher in 1955^ reported that extraction with ether failed to remove all of the - 13 - radioactivity from hydrolysed urine of human subjects who had been injected with C^-estrone or estradiol-17p. Similarly,
Valcourt et al77 administered estrone-16-C''"^ to rats and found that only one-third of the excreted in bile and in urine was extractable with ether. They also observed a small amount of radioactivity in the. raieutral fraction of the urine. In addition 78
Wotiz, Ziskind and Ringer'0, in perfusion experiments with radioactive estrone, found a large amount of chloroform-insoluble material containing in the hydrolysed dialysates of rat plasma.
Jellinck79 in 1959 reported that incubation of rat and human liver slices with estrone-16-C^ resulted in the formation of water-soluble, ether-insoluble metabolites, which could not be made ether soluble by refluxing with 77o (w/v) hydrochloric acid for 3 hours. No radioactive carbon dioxide was evolved during the incubation, confirming an earlier in vivo experiment77 and showing that a least C16 of the steroid nucleus remains intact. Placental tissue or blood could not give rise to these water-soluble products. 80 Since Westerfeld e_t al had shown that cyanide-sensitive plant phenol oxidases inactivate estrogens, and since cyanide inhibits the formation of water-soluble products from estrone by rat liver slices79, Jellinck0^ extended his studies to the - 13 - radioactivity from hydrolysed urine of human subjects who had been injected with C^-estrone or estradiol-17p. Similarly,
Valcourt e_t al.^7 administered estrone-16-C-'-^ to rats and found that only one-third of the excreted in bile and in urine was extractable with ether. They also observed a small amount of radioactivity in the neutral fraction of the urine. In addition
Wotiz, Ziskind and Ringer'0, in perfusion experiments with radioactive estrone, found a large amount of chloroform-insoluble material containing in the hydrolysed dialysates of rat plasma. 79 Jellinck in 1959 reported that incubation of rat and human liver slices with estrone-16-C^ resulted in the formation of water-soluble, ether-insoluble metabolites, which could not be made ether soluble by refluxing with 77o (w/v) > hydrochloric acid for 3 hours. No radioactive carbon dioxide was evolved during the incubation, confirming an earlier in vivo experiment^ and showing that at least C-16 of the steroid nucleus remains intact. Placental tissue or blood could not give rise to
these water-soluble products.
Since Westerfeld et al.80 had shpwn that cyanide-sensitive plant phenol oxidases inactivate estrogens, and since cyanide inhibits the formation of water-soluble products from estrone by 79 81 rat liver slices , Jellinck0extended his studies to the - 14 - comparatively simpler plant enzymes and found that mushroom tyrosinase preparations did in fact yield a high percentage of radioactive water-soluble products from estrone-16-C^. Evidence was presented that mushroom tyrosinase acts upon estrone in the same manner as upon its normal phenolic substrates, i.e. that it 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 yield highly stable complexes.
In fact, 60-70% of the water-soluble radioactivity was shown to be bound to protein by a strong chemical bond. It was therefore suggested that rat liver contained enzymes capable of acting on estrone in a similar way and, as discussed previously, the natural estrogens are indeed acted upon by liver to give the o-hydroquinones. However, whether these are then oxidized to give the highly reactive o-quinonoid derivatives is unknown, although estrogen-protein complexes that could have been formed by way of an o-quinone intermediate have been detected in rat 82 liver. Thus Reigel and Mueller in 1954 demonstrated the presence of an enzyme system in rat liver homogenates which, in the presence of NADPH and oxygen, bound a metabolite of 83 estradiol-17p to protein while Szego showed that liver contained enzymes which bind estrone to the albumin fraction of - 15 - homologous serum. Jellinck , however, in studies comparing the 14 tyrosinase and liver slice incubation products of estrone-16-C observed that only I0-157o of the water-soluble radioactivity formed by rat liver was bound to protein, while a much higher fraction (60-70%) was bound by the phenol oxidase system. 84
Zillig and Mueller } using rat liver microsomes, carried out further studies on the interaction of estradiol-17p with protein. They purported to exclude positions 2 and 4 from being involved in the protein-binding process, since substitution of one or the other of these positions on the steroid with fluorine did not affect 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 in each case, although
the 394-hydroquinone of estrogens has never been detected in biological systems. As it stands, there is therefore little / evidence either for or against the quinonoid pathway of estrogen interaction with protein or other acceptors.
Another possible route for the formation of protein-bound metabolites of estrone resulted from studies on the interaction of horseradish peroxidase and radioactive estrogens. In this case Jellinck^ showed that incubation of estrone-16-C^ with the plant enzyme in the presence of protein, tryptophan, cysteine or reduced glutathione led to the formation of water-soluble products - 16 - in high yields. Evidence was given that the horseradish enzyme was first acting as an aerobic oxidase, utilizing protein or the amino acid to generate hydrogen peroxide in the presence of manganous ions, and subsequently acting as a true peroxidase, converting estrone to highly reactive metabolites. These appeared to be phenoxyl radicals which were able to combine with protein or amino acid acceptors, giving water-soluble end-product
Hecker's theory33 for the formation of the 2-, 6- and
10-hydroxylated metabolites or estradiol by rat liver microsomes, as outlined earlier in this discussion, can also be adapted as a possible free radical mechanism for protein binding. In fact, he showed that the labelling of the trichloroacetic acid-precipi- table protein fraction after aerobic incubation of estradiol-17p- with rat liver microsomes and NADPH for 15 minutes was preceded by the slightly more rapid formation of 17p-hydroxyestra-p-l0p- 33 quinol, which he suggested arises by free radical mechanism
667o of the water-soluble metabolites were protein-bound and
347o of the total activity was in the aqueous fraction.
Hence, the nature and mode of formation of the water- soluble degradation products of estrone by mammalian liver are largely unknown, although evidence has been given for the existence of protein-bound water-soluble derivatives. The latter do not, however, account for all 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 rat liver preparations and two
approaches have been taken. First, data on rate of formation
of the water-soluble products under different experimental conditions
has been obtained, followed by attempts to characterize the metabolites. Comparative studies were also carried out with guinea pig liver preparations and some observations made on the oxidative metabolism of estradiol-17p-16-C^ and on diethylstilbestrol-
(monoethyl-l-C"^). The second approach has been to study the
effect of inhibitors in order to gather more information on the mechanism of formation of these water-soluble products. A series
of structural analogues of estrone as well as classical enzyme
inhibitors were tested for their inhibitory power in a rat liver
microsomal system. - 17a -
EXPERIMENTAL
I Materials and Methods
1. Materials
Animals: Female Wistar rats, each weighing about 150 gm were obtained from the University of British 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 libitum. In addition, the guinea pigs were fed fresh green vegetables.
Radioactive Estrogens: Estrone-16-C^1-, estradiol-17p-16-C''"^ and diethylstilbestrol (monoethyl-l-C-'-^) were purchased from the
Radiochemical Centre, Amersham, England. The estrogens were maintained in a stock solution of 1 mg/ml.
Counting Materials: The scintillation fluid 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 in methanol) were obtained from the Packard Instrument Corporation, La Grange, Illinois.
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
Na 2P04.7H2o (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 distilled water.
0.1 M Potassium phosphate buffer, pH 7.4, contained the following materials:
K2HP04 - 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: Bacterial p-glucuronidase, Type I, containing
73,500 Fishman units/gm was obtained from the Sigma Chemical
Company. One Fishman unit will liberate 1 [ig of phenolphthalein from phenolphthalein glucuronide/hr at pH 6.8 - 7.0 at 37°C.
Uridine 51-diphosphoglucuronic acid: UDPGA as the ammonium salt 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 British Drug Houses (Canada) Ltd.
Chromatography Reference Standards: Many of these compounds were gifts to Dr. P.H. Jellinck. 16a-hydroxyestrone was obtained from - 19 -
Dr. H. Breuer, 6-ketoestradiol-17p, 16a and 6p-hydroxyestra- diol-17p from Dr. 0. Wintersteiner and 2-hydroxyestrone,
2-hydroxyestradiol-17p and 2-hydroxyestriol from Dr. L. Axelrod.
16-ketoestrone, 16-ketoestradiol-17p and estradiol-17a were made available by the Cancer Chemotherapy National Service Center,
Bethesda, Md.
Estradiol-17p glucosiduronate was obtained from the
California Corporation for Biochemical Research, Los Angeles, and estriol-17p-glucosiduronate was a gift to Dr. P.H. Jellinck from Dr. A.E. Kellie.
Compounds tested as inhibitors: 59 compounds were tested as inhibitors of the rat liver estrone-metabolizing system; these are not listed but in all instances, the compounds used were the purest readily available. 1,4 benzoquinone, 1,2 naphthoquinone and 1,4 naphthoquinone, obtained from the Eastman-Kodak Company, were further purified by recrystallization from hot petroleum ether.
2. Preparation of Tissue
Animals were killed by suffocation in an atmosphere of carbon dioxide.
Whole Homogenate: The liver was rapidly excised from the dead animal and one part of tissue was homogenized with three parts - 20 - of ice cold Krebs phosphosaline buffer in a glass tissue grinder fitted with a Teflon pestle. The concentration of the homo- genate was adjusted to 50 mg liver (wet weight) /ml buffer. In experiments where the homogenate was to be further processed,
1 part of liver was homogenized in 3 parts of ice cold 0.25 M sucrose, and the final concentration was made up to either
50 mg liver/ml sucrose or to 100 mg liver/ml sucrose, depending upon the specific experiment.
8000 x g supernatant: The liver homogenate in 0.25 M sucrose was centrifuged at 8000 x g for 15 minutes at 2°C in a Spinco Model L preparative ultracentrifuge. (The supernatant obtained is referred to throughout the text as the 8000 x g supernatant fraction.) The protein concentration was determined by the 86 micro-Kjeldahl method
100,000 x g pellet: The 8000 x g supernatant of a 10% (w/v) liver 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 pellet suspended by homogenization in the same volume of 0.25 M sucrose or 0.1 M potassium phosphate buffer at pH 7.4. This fraction is referred to throughout this text as the 100,000 x g pellet, or the microsomes, and it 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 liver preparation in 0.1 M
Krebs phosphosaline buffer, 1 mg of NAD or of NADP and 10 mg of nicotinamide in 4 ml of buffer, and 0.01 ml (300,000 cpm) of a solution in ethanol (1 mg/ml) of the radioactive estrogen in
4 ml of buffer. The total volume of the mixture was 10 ml, and the incubations were carried out in 125 ml Erlenmeyer flasks.
When the 8000 x g supernatant or the 100,000 x g pellet fractions were used, the incubation medium, unless otherwise indicated, consisted of 1 ml of the tissue preparation, pyridine nucleotide cofactor (1 mg) in 1 ml of 0.1 M potassium phosphate buffer pH 7.4, and 10 ^.g (300,000 cpm) of the radioactive estrogen in 0.01 ml ethanol mixed with 1 ml of the phosphate buffer; total volume 3 ml. All incubations were carried out iri 30 ml test tubes.
Oxygen was bubbled through all incubation mixtures for
5 seconds prior to immersion of the flasks or tubes in 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 after incubation the mixture was transferred to a centrifuge tube and spun in a clinical centrifuge for a short time in order to precipitate the tissue. The supernatant was decanted, and immediately extracted twice with 1.5 vol. ether while the tissue was washed with 2 x 1 ml of 95% ethanol, dried and liquefied by heating in 1 ml of formamide at 150°C for 2 hours. The ether extracts were evaporated to dryness and the residue dissolved in
1 ml of ethanol (ethereal fraction). Aliquots of the four fractions, i.e. the liquefied tissue, the ethanol washings, the aqueous fraction and the ethereal fraction were then assayed for radioactivity.
Following incubation with the 8000 x g supernatant fraction or the 100,000 x g pellet suspension, 1 ml of N hydro• chloric acid was added in order to stop the reaction, and the mixture was vigorously extracted with 2 x 1.5 volumes of ether.
In certain cases, as indicated in the text, the acid was omitted.
The ether extracts and the aqueous fractions were then treated as above.
4. Determination of Radioactivity.
The distribution of radioactivity intthe various fractions obtained after extraction of the incubation mixtures was determined by counting aliquots by means of a Packard Tri Carb Liquid - 23 -
Scintillation Spectrometer. Either 0.1 or 0.2 ml of the fraction to be counted or of standard estrogen were placed in a counting vial and hyamine hydroxide (1 ml) and scintillation fluid (9 ml) added. The hyamine served to neutralize acid samples, and to solubilize 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 liver preparations were studied mainly by descending paper chromatography. The samples were spotted on Whatman #1 paper and three different solvent systems were used to develop the chromatograms. These were the toluene-propylene glycol system of Jellinck0^, 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 Precipitation and Dialysis: Protein was precipitated from the aqueous fraction by the addition of 3 ml of 207o (W/v) trichloroacetic acid (TCA) to 5 ml of the aqueous material. - 24 -
The precipitate was spun down at 1000 x g for 10 minutes in an
International Refrigerated Centrifuge, the supernatant decanted and the precipitate washed by resuspension in 2 ml of 20% (w/v)
TCA, followed by recentrifugation. By the same procedure the precipitate was washed with 2 x 3 ml of 95% ethanol. The TCA and ethanol washings were combined, and the precipitate was liquefied by heating in 1 ml of formamide at 150°C for 2 hours.
Samples of each of the liquefied precipitate, the washings and the supernatant fraction were counted in the liquid scintillation counter. The total recovery of C in the three fractions after TCA precipitation was 90-95%.
Dialysis of 1 ml samples of the aqueous fraction was carried out in cellophane tubing for 24 hours against running tap water. A portion of the indiffusible fraction was counted, and the loss of radioactivity by dialysis computed after 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 fraction from the liver incubations with radioactive estrogen were evaporated at 60-70°C under a stream of air, and the concentrated sample was streaked in a narrow band across a strip of Whatman 3 MM paper which was 3 cm wide. The chromatograms were developed in - 25 - either a solvent system consisting of butanol, acetic acid, water, 12 : 3 : 591, or in a solvent system of isopropanol, ammonia, water, 8 : 1 : 1°2. The radioactive compounds which separated on the chromatograms were detected by scanning in an Actigraph strip 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 fraction of 100, 000 x g microsomes preparation from
100 mg of liver, together with NADPH (1 mg) in 1 ml 0.1 M potassium phosphate buffer and 10 \ig of estrone-16-C^ in 1 ml of buffer were incubated for one hour under the conditions previously described. The incubation mixtures were then without further treatment transferred to lucite centrifuge tubes, and centrifuged at 100,000 x g for one hour at 2°C. The supernatant liquid was decanted and the pellets resuspended by homogenization in 5 ml of water. Samples from each fraction were then counted.
In addition, each fraction was extracted with 2x1.5 volumes of ether, and aliquots.from each of the extracted aqueous layers assayed for Cl4. In this way, the amount of radioactivity bound to microsomes after incubation was determined.
Enzymic Hydrolysis: The pH of a 3 ml sample of the aqueous fraction was adjusted to 6.8, and the sample incubated for 48 hrs - 26 - at 37°C with 75 units (1 mg) of bacterial p-glucuronidase,
50 jj,g of non-radioactive estradiol-17p-glucusiduronate and 1 drop of chloroform (to retard bacterial growth). A control tube, in 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 in
1 ml of 957o ethanol. Samples of the aqueous and ethereal 14 fractions were counted. Total recovery of C in this procedure was 95-1007c.
The ether-soluble material was chromatographed on the toluene-propylene glycol system and the developed chromatograms sprayed with Folin-Ciocalteu phenol reagent in 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 active.
The ether-extracted aqueous fractions from the control and p-glucuronidase incubations were concentrated by evaporation at 60-70/oC under a stream of air, and aliquots containing 4000 cpm were examined by ascending- paper chromatography in the butanol- acetic acid solvent system, followed by scanning for radioactivity in the Actigraph-recording scanner. Reference standards of estradiol-17p-glucosiduronate and of estriol-17p-glucosiduronate were detected with the Folin-Ciocalteu phenol reagent after - 27 -
chromatography.
Acid Hydrolysis: 8 ml of a diluted ether-extracted aqueous fraction were gently refluxed with 2 ml of concentrated hydro• chloric acid for 2 hours. After removal of a sample for counting, the acid-treated fraction was extracted with 2 x 1.5 volumes of ether and aliquots of this ethereal fraction as well as of the ether-extracted hydrolysed aqueous phase were counted in the
usual way. Total recovery of was 85-9570, and all values given are the mean of at least two experiments.
7. Inhibition Studies.
These investigations fall into two main categories. In the first, the effect of classical enzyme inhibitors such as cyanide on the conversion of estrone-16-C^ to water-soluble products by rat liver preparations was tested. The compounds were added in a small volume of ethanol or water to an incubation mixture consisting of 1 ml of rat liver 8000 x g supernatant fraction
(from 50 mg liver), 1 mg of NADPH in 1 ml of 0.1 M potassium 14 phosphate buffer and 10 |_ig of estrone-16-C in 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 acidified and extracted with ether. The distribution of radioactivity was determined as previously indicated. - 28 -
The second type of inhibition experiment involved
investigating the effect of structural analogues of estrone and
of related compounds on the metabolism of estrone-lG-C^ by rat
liver microsomes. In this case, the inhibitor (2.5 x 10~5M) was added to an incubation mixture consisting of 1 ml of a rat liver
100,000 x g pellet suspension (from 100 mg liver), 1 mg of NADPH 14 =1 in 1 ml buffer and 10 |ig of estrone-16-C (1.2 x 10"DK) in 1 ml buffer. An equivalent volume of the medium used to dissolve the inhibitor was added to a control incubation mixture. After exactly 15 minutes incubation, the reaction was stopped by acidification, the mixtures ether extracted, and the distri• bution of radioactivity in the various fractions determined as described previously.
In certain of these cases kinetic studies were carried out and the effect of a constant amount of inhibitor on the initial rates of formation of water-soluble products for different 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 initial velo• cities for the formation of water-soluble products at different substrate concentrations were determined, and the Lineweaver-Burk plots of the reciprocal of hie initial velocity (^) versus the - 29 - reciprocal of the substrate concentration (|) were made for the control and inhibited mixtures.
8. In vivo Studies.
In order to correlate the in vitro findings to the situation in the whole animals, estrone-16-C-^ (10° cpm in 25 |j,g) was given intraperitoneally to each of 2 rats and 2 guinea pigs. The urine from each animal was collected for 24 hours and the amount of radioactivity excreted by this route as well as the distribution 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 fluid 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 distribution of ascertained.
The urine samples (8 ml) were also subjected to acid hydrolysis by refluxing with cone. HCl (2 ml) for 2 hr and the amount of radioactivity removed by subsequent ether extraction determined. TABLE I
Percentage distribution of radioactivity after incubation* of 10 jig of estrone-16-C-^ with rat liver preparations.
Percentage of added radioactivity
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 liver 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 - - - liver 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 liver
100,000 x g from 60 mins _ 0.6 91.0 _ — 91.6 microsomal 100 mg NAD 9.7 82.5 - 92.2 pellet liver NADH 9.5 76.5 - 86.0 NADP 3.7 84.0 - - 87.7 NADPH 16.0 65.2 - - 81.2
* Conditions as described in text. ** Plus 10 mg nicotinamide. - 30 -
II Results
1. Optimal Conditions for the Formation of Water-soluble Metabolites from Estrone-16-C1^ by Liver Preparations.
Table I shows the distribution of radioactivity in the various
fractions following incubation of 10 \xg of estrone-16-Cl4- with rat liver preparations under the conditions listed.
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 effective, and NADP, although active with the 8000 x g
supernatant preparations, was inactive as cofactor with the microsomes. This is reasonable since NADP can be converted to
NADPH by enzymes in the 100,000 x g supernatant. The initial velocity for the conversion of estrone-16-C^ 4- in the presence
of NADPH and the 100,000 x g pellet preparation was 0.085 y,moles/
1/min, whereas with either NAD or NADH it 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 this value therefore resulted in no further stimula•
tion of the reaction.
The optimum; pH for the conversion was found to be about
7.4, a lower yield of water-soluble metabolites being obtained - 31 - when the incubation was carried out at pH 6.0 or 8.0.
The conversion proceeded more rapidly at 37°C than it 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 in an evacuated Thunberg tube, no radioactivity was incorporated into the aqueous fraction and chromatography (toluene-propylene glycol system) of the ethereal fraction showed that estradiol-17p was the only product formed under these conditions.
The 100,000 x g supernatant fraction of rat liver was unable to effect the conversion of estrone to water-soluble products, and the nuclei or mitochondria were also inactive in this respect.
Wiiih guinea pig liver preparations, good incorporation of radioactivity from estrone-16-Cl^- was observed on incubating the estrogen with the whole homogenate, the 8000 x g supernatant fraction or with the 100,000 x g microsomes. Addition of NAD
or NADPH increased the yield to 50-60?o, although even in 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 activity. FIGURE I
CHROMATOGRAMS OF ETHEREAL FRACTIONS FROM INCUBATION OF 14 ESTRONE-16-C WITH LIVER PREPARATIONS Toluene-Propylene glycol Solvent System
Rat Rat Guinea Pig Guinea Pig 8000 x g 100,000 x g Reference 8000 x g 100,000 x g supernate microsomes Standards supernate microsomes
1. Estriol, 2-hydroxyestriol 6. Estradiol-17a 2. 2-hydroxyestradiol-17p 7. 6a-hydroxyestradiol-17p 3. 6-ketoestradiol-17p 6p-hydroxyestradiol-17g 4. 2-hydroxyestrone 8.i16a-hydroxyes trone \_ 5. estradiol-17p 9. 16-ketoestradiol-17p 16-ketoestrone - 32 -
2. Properties of the Ether-soluble Metabolites.
In this 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 typical resolution of ether-soluble metabolites obtained by means of the toluene-propylene glycol system. Two other solvent system (methanol-heptane, modified Bush) were used, but the only metabolite that could be identified in all three systems was estradiol-17p. This compound was formed by rat liver preparations, and also by the guinea pig liver 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 similar to 6-ketoestradiol in two of the solvent systems mentioned, but a mixture of the unknown and the reference standard showed some separation in the toluene-propylene glycol system. Another ether-soluble metabolite was chromatographically similar to 16ct-hydroxyestrone, but again not identical with it and a fourth metabolite, or group of metabolites, for which good separation was not achieved, behaved very much like estriol in 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 liver 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 Rat liver 100,000 x g microsomes (0.7 mg protein) •H 4-» O «>-J(
w 3 O <1) cr <
O 10 20 30 40 50 60 70 80 90 o Time (minutes)
* Incubation mixtures consisted of 1 ml of tissue preparation from 100 mg liver, 1 mg NADPH in 1 ml 0.1 M potassium phosphate buffer, and 10 |ig estrone-16-C1^ in 1 ml buffer. Total volume 3 ml. - 33 -
2-Hydroxy estradiol-17p was detected in the modified Bush solvent system, but this result was not always reproducible.
2-Hydroxyestrone was not observed to be formed from estrone-16- 14 C by rat or guinea pig liver preparations, nor could
2-hydroxyestriol be identified.
Although the guinea pig liver microsomes produced ether- soluble metabolites similar to those formed by the rat liver preparations, the guinea pig liver 8000 x g supernatant did not.
In this case, only compounds with Rf values similar to estriol, and possible 6- or 2-hydroxyestradiol-17p were detected.
3. Properties of the Water-soluble Metabolites.
(i) Protein Binding: Figure II depicts the rate of formation of water-soluble products and of TCA-precipitable protein-bound metabolites from estrone-16-C"^ by the rat liver 8000 x g supernatant fraction and by the 100,000 x g microsomes. In the first case, the initial rate of conversion of estrone-16-C^ to protein-bound derivatives was 0.007 (x moles/l/min, whereas in the second case it was 0.05 \± moles/l/min. In each instance the enzyme preparation was derived from 100 mg liver, 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 inability of the radioactive metabolites to dialyse across a cellophane membrane TABLE II
FORMATION OF PROTEIN-BOUND METABOLITES OF ESTRONE-16-C14
BY RAT AND GUINEA PIG LIVER PREPARATIONS*.
Percentage of water-soluble C^4 precipitated by TCA
Rat liver Guinea pig liver 8000 x g supernatant 12.5% 8000 x g supernatant 11.2% Rat liver Guinea pig liver 100,000 x g microsomes 62.7% 100,000 x g microsomes 78.7%
Percentage of water-soluble C^4 non-dialysable
Rat liver Guinea pig liver 8000 x g supernatant 23.5% 8000 x g supernatant 23.2%
Rat liver Guinea pig liver 100,000 x g microsomes 76.8% 100,000 x g microsomes 77.0%
Percentage of water-soluble C^ bound to microsomes**
Rat liver Guinea pig liver 8000 x g supernatant 15.0% 8000 x g supernatant 4.5%
Rat liver Guinea pig liver 100,000 x g microsomes 61.9% 100,000 x g microsomes 49.0%
* Each incubation mixture consisted of 1 ml of liver preparation from 100 mg liver, 1 mg NADPH in 1 ml. 0.1 M potassium phosphate buffer, 10 p,g estrone-16-C14 in 1 ml. buffer: total volume 3ml.
** The mixtures were not acidified prior to extraction with ether. - 34 - within 24 hours, was shown by this technique to account for a similar proportion of the water-soluble metabolites as did TCA precipitation. Table II gives these results for both rat and guinea pig liver preparations.
In order to ascertain whether the radioactivity was bound to microsomal or to soluble protein, incubation mixtures were, prior 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 II compares the values obtained for radioactivity bound to microsomes with those obtained for protein binding TCA precipitation and dialysis. It can be seen that for the rat liver preparations the percentage of
precipitated by TCA is approximately equal to that sedimented at 100,000 x g, while with the guinea pig somewhat more radio• activity is precipitated by TCA than is sedimented by spinning at 100,000 x g. This may indicate that some of the C^ is bound to "soluble" protein in the latter case.
(ii) Conjugate Formation in vitro: Table III records the percentage incorporation of radioactivity into the aqueous fraction following incubation of estrone-16-0-^ with rat and guinea pig liver preparations in the presence and absence of UDPGA. The table also depicts the extent to which treatment of the various TABLE III
CONJUGATE FORMATION BY RAT AND GUINEA PIG LIVER
PREPARATIONS* IN THE PRESENCE AND ABSENCE OF UDPGA.
% of C^-4 of the aqueous fraction remaining water- soluble after treatment
% of total C14 Liver UDPGA in aqueous Incubation with Refluxed preparation 50 mg fraction 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 liver preparation from 100 mg liver, 1 mg of NADPH and 1 mg of NAD in 1 ml of 0.1 M potassium phosphate buffer, 10 pg of estrone-16-C14 in 1 ml buffer, and some UDPGA where indicated. Total volume: 3 ml.
After incubation for 60 minutes at 37°C under 02, the mixtures were extracted with ether without prior acidification. - 35 - aqueous fractions with p-glucuronidase or refluxing with 20% (v/v)
HCl rendered the radioactive components of the fractions soluble in ether. NAD was added to each incubation mixture in order to show that the lack of glucosiduronate formation in 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 fraction or 100,000 x g microsomes of rat liver caused a slight increase in the yield of water-soluble metabolites in the first case, and a more marked increase in the latter case. When UDPGA was omitted from either rat liver 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 in the presence of UDPGA did give rise to water-soluble metabolites which were hydrolysed by acid or p-glucuronidase and thus made soluble in ether. The rat liver 8000 x g supernatant fraction in 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 liver 8000 x g supernatant incubations did not produce a significant increase in the formation of water-soluble products from estrone-lG-C-^, but the extent of hydrolysis of the aqueous fraction by acid or - 36 -
enzyme treatment was somewhat increased. However, presence of
UDPGA in the incubation of the guinea pig liver 100,000 x g microsomes resulted in a marked increase in the percentage of radioactivity from estrone-16-C-*-4 incorporated into the aqueous
fraction, and also a large increase in the proportion of hydrolysable conjugates was observed.
Chromatograms of aqueous fractions which had been incubated,
in the presence or absence of p-glucuronidase for 48 hours at
37°C were developed in the butanol-acetic acid-water solvent
system, as described previously. Chromatograms of aqueous
fractions which had been stored at 2°C were also developed in
this system. The separation of radioactive compounds on the
chromatograms as recorded on the Actigraph scanner is depicted
in Figures III and IV. It is important to note that in each
case, 4§00 cpm of the concentrated aqueous fractions were applied
to the chromatograms. This means that in those instances where
hydrolysis of the fraction was carried out with p-glucuronidase,
the hydrolysed samples were applied in higher concentrations
than their controls, and hence the peaks in the hydrolysed
samples are exaggerated in comparison to those in the controls.
Illustrations la and 2a, Figure III show that compounds with values of 0(XQ) , 0.5(XQ 5) and 0.9 (XQ.O,) were formed by
the rat liver fractions, XN r being found in greater quantity by LEGEND FOR FIGURE III
Treatment of aqueous Illustration Liver fraction prior to number preparation UDPGA chromatography**
la 8000 x g Stored at 2° for 48 hrs and supernatant extracted with ether after spinning at 100,000 x g.
lb 8000 x g Incubated at 37° for 48 hrs supernatant and extracted with ether.
lc 8000 x g Incubated with p-glucuronidase supernatant for 48 hrs at 37° and extracted with ether.
2a 100,000 x g Stored at 2° for 48 hrs and microsomes extracted with ether after spinning at 100,000 x g.
2b 100,000 x g Incubated at 37° for 48 hrs microsomes and extracted with ether.
2c 100,000 x g Incubated with p-glucuronidase microsomes for 48 hrs at 37° and extracted with ether.
3b 8000 x g + Incubated at 37° for 48 hrs supernatant and extracted with ether.
3c 8000 x g Incubated with p-glucuronidase supernatant for 48 hrs at 37 and extracted with ether. 4b 100,000 x g Incubated at 37° for 48 hrs microsomes and extracted with ether.
4c 100,000 x g Incubated with p-glucuronidase microsomes for 48 hrs at 37° and extracted with ether.
* Complete incubation mixture as given on Table III.
** 4000 cpm of the treated aqueous material was applied to each chromatogram. FIGURE III
CHROMATOGRAMS OF THE AQUEOUS FRACTION FROM INCUBATION OF
ESTRONE-16-C^ WITH RAT LIVER PREPARATIONS
la lb
lc
2b
2c
Butanol-Acetic Acid-Water V 3b Solvent System
3c
LEGEND TO FACE 4b 1
Reference standards 1. Estriol-178-glucosiduronate 2. Estradiol-17$-glucosiduronate 9°i - 37 - the 8000 x g supernatant fraction than by the microsomes.
Illustrations 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 it appears from the chromatograms that they were changed to more polar derivatives, which remained at the origin.
The results depicted in Figure III show that a peak with
similar to that of estradiol-17p-glucosiduronate was formed only by the 100,000 x g microsomes in the presence of UDPGA.
This peak was abolished by p-glucuronidase treatment. In illustration 4c the peak at the origin is exaggerated with reference to the control (4b), since the hydrolysed sample applied contained about twice as much material as the unhydrol• ysed control. No significant amount of a product with an
Rf similar to estradiol-17p-glucosiduronate was present in the aqueous fractions from incubation of the 8000 x g super• natant fraction in the presence or absence of UDPGA.
Illustrations la and lb (Fig. IV) show that the major compound formed on incubation of the guinea pig liver 8000 x g 14 supernatant fraction with estrone-16-C had an Rf similar to that of estradiol-17p-glucosiduronate, that this 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
Treatment of aqueous Illustration Liver fraction prior to number preparation UDPGA chr oma t o gr ap hy**
la 8000 x g Stored at 2° for 48 hrs and supernatant extracted with ether after spinning at 100,000 x g.
lb 8000 x g Incubated at 37° for 48 hrs supernatant and extracted with ether.
lc 8000 x g Incubated with p-glucuronidase supernatant for 48 hrs at 37° and extracted with ether.
2a 100,000 x g Stored at 2° for 48 hrs and microsomes extracted with ether after spinning at 100,000 x g.
2b 100,000 x g Incubated at 37° for 48 hrs microsomes and extracted with ether.
2c ./: 100,000 x g Incubated with p-glucuronidase microsomes for 48 hrs at 37° and extracted with ether.
3b 8000 x g + Incubated at 37° for 48 hrs supernatant and extracted with ether.
3c 8000 x g + Incubated with p-glucuronidase supernatant for 48 hrs at 37° and extracted with ether.
4b 100,000 x g + Incubated at 37° for 48 hrs microsomes and extracted with ether.
4c 100,000 x g + Incubated with p-glucuronidase microsomes for 48 hrs at 37° and extracted with ether.
* Complete incubation mixture as given on Table III.
** 4000 cpm of the treated aqueous material was applied to each chromatogram. FIGURE IV
CHROMATOGRAMS OF THE AQUEOUS FRACTION FROM INCUBATION OF
ESTRONL-16-C14 WITH GUINEA PIG LIVER PREPARATIONS
Butanol-Acetic Acid-Water Solvent System 3b
3c
LEGEND TO FACE 4b
4c
Reference standards Estriol-17£-gluco s idurona te Estradiol-17$-glucosiduronate 9? FIGURE V
CHROMATOGRAMS OF THE AQUEOUS FRACTION FROM
INCUBATIONS* OF ESTRONE-16-C14 WITH GUINEA PIG
LIVER PREPARATIONS.
Ammonia-Isopropanol"solvent system Liver \ preparation
8000 x g supernatant J ^^^^ ^^^f ^^^^ ^^^^^^v^
100,000 x g microsomes J Reference o o Standards
1. Estriol-17(^-glucosiduronate 2. Estradiol-17£-glucosiduronate
* Complete incubation mixture as given on Table III. - 38 - incubation with the UDPGA-fortified 8000 x g supernatant fraction
(illustrations 3b and 3c).
Very small amounts of the compound with Rf 0.5 previously observed in fractions from rat liver incubations were detected in the hydrolysed samples (lc and.3c).
Illustrations 2b and 2c show that the guinea pig 100,000 x g microsomes give rise to very little estradiol-17p-glucosid- uronate-like material, unless UDPGA is added to the incubation medium (illustrations 4b and 4c).
Chromatograms of the various aqueous fractions developed in the ammonia-isopropanol water solvent system did not show good separation of material from the rat systems, but it was effective for the "glucosiduronate" product from the guinea pig 8000 x g supernatant incubation (Figure V). It appears that estriol-17p- glucosiduronate as well as the estradiol-17p derivative was formed in this system.
It was also shown by chromatography in the toluene-propylene glycol and methanol-heptane solvent systems that substantial amounts of estradiol-17p are released from the guinea pig aqueous fractions after hydrolysis with p-glucuronidase. Smaller amounts of estrone, and of metabolites with similar mobility to estriol and its epimers were also released by the enzyme. This data, together with that obtained from the chromatograms of the aqueous fractions, points to TABLE IV
IN VIVO METABOLISM OF ESTRONE-16-C
IN THE RAT AND GUINEA PIG
Estrone-16(10^ cpm in 25 (j,g) given I.P. to 2 animals; urine collected for 24 hours
% of C^ remaining in urine after treatment
Treatment of urine RAT GUINEA PIG (141,000 cpm in urine) (572,000 cpm in 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 - 39 -
estradiol-17p-glucosiduronate as being the major conjugate formed by the guinea pig 8000 x g supernatant fraction from estrone.
Other conjugates were undoubtedly also present. Rat and guinea pig microsomes fortified with UDPGA appeared to produce estradiol-
17p-glucosiduronate from estrone, but the rat 8000 x g supernatant did not form this conjugate under any circumstances.
Conjugate Formation in vivo: Table IV depicts the fate of
estrone-16-C^1- in the rat and guinea pig following intraperitoneal
injection of 25 \xg of the estrogen (10 cpm) . Whereas 5770 of the injected C^ was excreted in the urine of the guinea pig, only 147o of the injected radioactivity was found in rat urine. Ether extraction, however, showed that a substantial part of the C^"4 was water-soluble in both cases.
Incubation of the rat urine at pH 6.8 with p-glucuronidase
failed to render any more radioactivity extractable with ether than
in the untreated control and even after acid hydrolysis the bulk of the remained in the aqueous fraction. In contrast, the guinea pig urine contained a very large percentage of acid and enzyme-hydrolysable conjugates.
4. Metabolism of Estradiol-17p-16-C14 by Rat and Guinea Pig Liver Preparations
•Estradiol-17p-16-C^ behaved very much like estrone when incubated with rat or guinea pig liver preparations and exhibited the same 40 - specificity towards the nicotinamide nucleotide cofactor in forming water-soluble metabolites.
Chromatograms of the ethereal fractions obtained after 14 incubation of estradiol-17p-16-C with rat liver microsomes showed that only small amounts of estrone were formed, and that the major ether-soluble metabolites were relatively polar compounds with R.£ values similar to estriol and to 2- and 6-hydroxyestradiol-17p.
In contrast to incubations with estrone-16-C^, a metabolite with an similar to that of 6-ketoestradiol-17p was not formed from estradiol-17p-16-C^. The toluene-propylene glycol and methanol- heptane chromatographic systems were used in these studies.
Acid and p-glucuronidase hydrolysis experiments showed that estradiol-17p-16-C"'"4 is not metabolized to simple glucosiduronate conjugates by rat liver microsomes in the absence of added UDPGA, nor did guinea pig liver microsomes catalyse the formation of substantial amounts of the conjugates. The guinea pig liver
8000 x g supernatant fraction did however give rise 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 rat and guinea pig liver. 5. Metabolism of Diethylstilbestrol (monoethyl-l-C^4) by Rat and Guinea Pig Liver Preparations. Stilbestrol-C^4 appeared to be metabolized by rat and guinea pig - 41 - preparations in a manner similar to estrone-16-Cl4 and estradiol-
17p-16-cl4. 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 rat or guinea pig liver microsomes in the absence of added UDPGA, but they were formed by the guinea pig 8000 x g supernatant fraction. In the latter case, stilbestrol-C^-4 itself, as well 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 fraction of rat liver was bound to protein.
6. Inhibition of the Formation of Water-soluble Metabolites from Estrone-16-C-*-4 by Rat Liver Microsomes.
(i) Classical Enzyme Inhibitors: Potassium cyanide at a concetr- ation of 10"^ M produced 74% inhibition in the formation of water- soluble metabolites from estrone-16-C"'-4 by rat liver 8000 x g 3 supernatant fraction. 65% inhibition was observed with 10 M KCN, and 38% inhibition with 10"4 M KCN.
N-ethylmaleimide, a sulphydryl group inhibitor, at a con• centration of 10"^ M or 10~3 M produced 90% inhibition of the reaction(s) and inhibitor concentration of 10~4 M reduced the yield of water-soluble products by 57%. p-chloromercuribenzoate, another strong sulphydryl group inhibitor, inhibited the conversion to the same extent. It was shown by chromatography in the toluene-propylene glycol system that KCN did not inhibit the formation of estradiol-
17f3 from radioactive estrone, whereas the sulphydryl group
inhibitors, as expected, completely abolished this dehydrogenase- mediated reaction. 2 10 M sodium fluoride did not affect the estrone metabolizing
system and the enzyme catalase, at a concentration of 67 [xg/ml produced very slight inhibition in the formation of the water-
soluble metabolites ( 1570) .
(ii) Inhibition Studies with Structural Analogues of Estrone: A
large number of compounds having structural features similar to
those of estrone were tested for their ability to inhibit the
formation of water-soluble metabolites from estrone-16-C-'-4 by rat
liver microsomes. The inhibitors were added at twice the molar
concentration of the substrate and, after incubation, as described previously, the effect of the added compound on the incorporation of radioactivity from estrone-16-C^4 into the aqueous fraction was determined. Table V lists those compounds that produced
over 757o inhibition, Table VI those compounds which gave 50-60%
inhibition and Table VII the compounds tested that were without appreciable inhibitory activity.
It can be seen from Table V that whereas 2-hydroxyestrone and 2-hydroxyestradiol were both potent inhibitors, 2-hydroxy- TABLE V
COMPOUNDS TESTED AS INHIBITORS IN THE FORMATION OF WATER-SOLUBLE
PRODUCTS FROM ESTRONE-16-C14 BY RAT LIVER MICROSOMES.
GROUP I (over 75%' inhibition)
Added substance* Inhibition (%)
2-hydroxyestrone 85
2-hydroxyestradiol 85
equilenin 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- di-t-butyl hydroquinone 78
tetramethyl hydroquinone 83
pyrogallol (1,2,3-trihydroxybenzene) 89
* Inhibitors (2.5 x 10 M) were added at twice the molar concentration of estrone. TABLE VI
COMPOUNDS TESTED AS INHIBITORS IN THE FORMATION OF WATER-SOLUBLE
PRODUCTS FROM ESTRONE-16-C14 BY RAT LIVER MICROSOMES.
GROUP II (50-60% inhibition)
Added substance* Inhibition (%)
estradiol-17a 52
estradiol-17p 52
equilin 57
2-hydroxyestriol 60
estrone sulfate 50
estrone benzoate 59
ethynyl estradiol 57
diethylstilbestrol 59
hexestrol 59
dienestrol 50
catechol 56
p-naphthol 59
2,5-dihydroxyphenylacetic acid 59
* Inhibitors (2.5 x 10°M) were added at twice the molar concentration of estrone.
0 TABLE VII
COMPOUNDS TESTED AS INHIBITORS IN THE FORMATION OF WATER-SOLUBLE
PRODUCTS FROM ESTRONE-16-C14 BY RAT LIVER MICROSOMES.
GROUP III (less than 20% inhibition)
Added substance*
6a-hydroxyestradiol 1.2- dimethylanthraquinone
6p-hydroxyestradiol 1.3- dimethylanthraquinone
6-ketoestrone 1.4- dimethylanthraquinone
6-ketoes tradiol resorcinol
16ct-hydroxyestrone 3.5- dihydroxytoluene
16-ketoestradiol 1,3,5-trihydroxybenzene
17p-hydroxyestra-p-quinol hexahydroxybenzene
16-ketoestrone 3,4-dimethylphenol estriol** tyrosine estradiol-17p-glucosiduronate tryptophan estriol-17p-glucosiduronate progesterone** diethylstilbestrol glucosiduronate testosterone dihydroxyhexestro1 cortisone dicarboxyhexestrol Cortisol thyroxine p-hydroxypropiophenone adrenaline dehydroepiandrosterone
* Inhibitors (2.5 x 10"5M) were added at twice the molar concentration of estrone.
** Produced 277o inhibition, - 42a -
estriol was only an inhibitor of intermediate strength (Table VI),
and the 6-hydroxy and 6-keto estrogens, as well as the 16-
hydroxy derivatives showed no inhibition (Table VII). Estradiol-
17p, estradiol-17a, ethynyl estradiol, estrone sulphate, and
estrone benzoate gave 50-607o inhibition, but the glucosiduronate conjugates of the estrogens were inactive. The naphtholic
estrogen equilenin was a potent inhibitor, and equilin retained
intermediate activity while the synthetic estrogens, diethylstil-
bestrol, hexestrol and dienestrol also fell in 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
inactive.
A distinct group of non-steroidal compounds which
included benzoquinones, naphthoquinones, and o- and p-hydroxyl-
ated phenols were shown to be as potent inhibitors 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 in the
yield of water-soluble metabolites from estrone-C^4 and menadione
(10"^ M) produced 60% inhibition. In contrast to this group,
various anthraquinones were completely without activity as
inhibitors, as were m-hydroxylated phenols such as resorcinol, - 43 -
3,4-dihydroxytoluene and 1,3,5-trihydroxybenzene.
Chromatograms of the ethereal fractions obtained from
incubations in the presence of inhibitors failed to show any
consistent changes in the ether-soluble metabolites that could
be correlated with the ability of a compound to inhibit the
conversion of estrone to water-soluble material. However, an
increased amount of C^4 estradiol was formed in the presence of non-radioactive estradiol-17p or -17a, 2-hydroxyestradiol-17a or
any estradiol derivative, including the non-inhibitory ones such as 6-ketoestradiol-17p.
Lineweaver-Burk Plots: Since zero order kinetics appeared to be operating during the first 15 minutes of the reaction in 14 which estrone-16-C was being converted to water-soluble material by the rat liver microsomes, kinetic studies with some
inhibitors were carried out and the classical Lineweaver-Burk
analysis for types of inhibition was applied to the kinetic data.
The inhibitors tested were 2-hydroxyestrone, equilenin, stilbestrol,
and menadione, and the Lineweaver-Burk plots for these inhibitors
are given in Figures VI, VII, VIII and IX respectively.
The plots suggest that 2-hydroxyestrone, equilenin and
stilbestrol act as competitive inhibitors at a rate limiting step
involved in the formation of the water-soluble products from
-4 t ie estrone-16-C-' . In each case the maximum velocity (V max) °f ^ - 44 - reaction was unchanged in the presence of inhibitor, whereas the Km was distinctly altered, fitting the requirements for competitive inhibition.
Menadione, on the other hand, appears to produce a mixed
type of inhibition since both the Vmax and the Km were changed in the presence of the inhibitor. 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 / 2-HYDROXYESTRONE V 0/ (5.9 nM) 40 -
/1/min)"1 / ^^^^ \ x / • \ 20 • y CONTROL ;
;—i———i L-4- t 0 0.2 0.4 0.8 -1 1 (VLM) S
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
3 Vmax 0.17 umol.es/1/myn
Km = 5.6 uM
Kt =2.4 FIGURE VIII
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-C14 TO WATER-SOLUBLE METABOLITES
BY RAT LIVER MICROSOMES.
-*"•
80 - \
60 - . MENADIONE (1.9 uM) ,. : • ^ •
• ' ' -\ 1 V 40 • '.X /. • . • • (p.moles/1/min) ^ \.S •: , . • ' - •' • V , ••• ' .- ' \ • • ' • •
20 - 1. ****
CONTROL ; )
- : 1 0 0.2 0.4 \ 0.6 —1 \ 1 • — 1 (uM)"1 S • . /
vmax (control) = 0.16 M-moles/l/min
vmax (inhibited) - 0.06 umoles/l/min - 45 -
DISCUSSION
The natural estrogens are metabolized by mammalian liver to a variety of products. Liver microsomes contain estrogen
2-hydroxylase^^ as well as 16a and 16p9^, 6a and 6p^°, and 10p
hydroxylases^} and each of these enzymes requires molecular oxygen and NADPH. The corresponding dehydrogenases are also present in the microsomes.
A significant proportion of the metabolites of estrogens formed by liver consists of products which are soluble in water, and insoluble in ether, a solvent in which these steroids and their simple mono-hydroxylated metabolites are readily 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 liver 100,000 x g supernatant fraction74, while Breuer and Smith^ have recently shown that estrone is conjugated as the monoglucosiduronate on incubation with rabbit liver microsomes fortified with UDPGA. However, water-soluble metabolites that are not hydrolysed by sulphatase,
v p-glucuronidase or 207o ( /v) HC1 have also been discovered.
Mueller and other workers have described a metabolite formed by rat liver from estradiol which is strongly bound to protein82,83 while Jellinck0^, working with rat liver slices, showed that - .46 -
10-15% of the water-soluble metabolites of estrone existed in 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 liver.
The first type includes protein-bound metabolites, the second includes simple conjugates such as glucosiduronates and sulphates, and a third the unknown compounds.
In the present investigation the general requirements for the conversion of estrone-16-C-'-4 to water-soluble products by rat and guinea pig liver preparations were determined. The results, localizing the enzymes involved in the microsomes and showing that NADPH and oxygen are required, were found to be in agreement with published data. The guinea pig liver prepara• tions were more active than the rat liver 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 rat liver preparations.
An investigation of the properties of the water-soluble 14 metabolites of estrone-16-C showed that both the rat and guinea pig liver preparations were capable of binding the estrogen or its metabolite to protein. However, a marked difference in the proportion of protein-bound metabolites was observed on incubating 14 estrone-16-C with different tissue preparations. The initial - 47 - rate of formation of protein-bound metabolites by the 100,000 x g rat liver microsomes was 0.05 u. moles/l/min whereas it was only
0.007 |i moles/l/min for the 8000 x g supernatant fraction, each tissue preparation being derived from 100 mg of liver. Similarly, the amount of radioactivity bound to protein accounted for about 14
707o of the C in the aqueous fraction after incubation with rat or guinea pig 100,000 x g microsomes, but represented only about
157o of the C"''4 in the aqueous fraction obtained after incubation with the rat or guinea pig 8000 x g supernatant fraction. This is a striking difference in view of the fact that the 8000 x g supernatant preparation contains 2.0 mg protein, whereas; the
100,000 x g pellet fraction contains only 0.7 mg protein. It appears, therefore, that in the presence of the 100,000 x g supernatant, a pathway for estrone metabolism other than: protein binding predominates, whereas in its absence protein binding becomes the preferred metabolic reaction. A possible reason for this is given in later discussion.
Each of the three techniques used to measure protein binding gave results that were in reasonable agreement with one another. The fact that the same percentage of water-soluble products from the rat liver incubations was precipitated by TCA as sedimented on centrifugation at 100,000 x g for 60 minutes suggests that the radioactivity was actually 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
100,000 x g was somewhat less than the amount precipitated by TCA,
it is possible that the radioactivity is bound to soluble as well as to microsomal protein.
It 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. Little is known outside the fact that the bond between the estrogen moiety and the protein is very strong.
A proposed mechanism for the formation of such metabolites is that
the estrogen is first converted to a ring A o-hydroxylated deri• vative which is then oxidized to the o-quinone. This highly reactive
substance can now combine with protein acceptors to yield stable
complexes. However, although mushroom tyrosinase has been shown
81 to catalyse estrogen interaction with protein in this manner
there is little evidence that such a process takes place in rat
or guinea pig liver. The present studies failed to confirm the
formation of detectable quantities of 2-hydroxyestrone or 2-hydroxy-
estriol in systems which were rapidly producing the protein-bound
derivatives. Occasionally traces of a compound with an Rf value
similar to 2-hydroxyestradiol were detected, but this was not
consistently reproducible. It could, of course, be argued that
o-hydroquinones are in fact formed by the liver systems, but are - 49 - then oxidized so rapidly to quinones which in turn Interact with protein that they cannot be obtained as stable intermediates.
However, the isolation of relatively 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 in fact formed was recently obtained by Jellinck^4. Estrone-16-C"'"4 was incubated with rat liver microsomes or with mushroom tyrosinase in the presence of ethylene diamine. A dihydropyrazine derivative of the estrone was formed in the plant enzyme incubation mixture, and this derivative was shown to be the adduct of the estrogen o-quinone with the ethylene diamine. Such a derivative was not formed in the rat liver incubation mixture, even though high yields of water-soluble metabolites were obtained, suggesting that o-quinonoid intermediates of estrone are not formed in this animal system.
An alternative 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 in the introduction^ .
The estrogen free radicals formed in the process could, instead of being converted to the hydroxylated derivatives, interact with certain reactive groups on protein to form a stable complex. - 50 -
Evidence for this type of interaction has been obtained from the studies on the effect of horseradish peroxidase on estrone- Jellinck0^ found that this enzyme, in the presence of protein or certain amino acids, converted estrone-16-
to water-soluble metabolites in high yields. The enzyme appeared to be acting initially as an aerobic oxidase, and could utilize protein or amino acid to generate hydrogen peroxide.
Subsequently, acting as a true peroxidase the enzyme converted estrone to highly reactive metabolites, probably phenoxyl radicals, which were then able to combine with the protein or amino acid. Catalase was a potent inhibitor of this system as would be expected for a reaction involving hydrogen peroxide.
However, it was found in the present studies with liver prepa• rations that catalase, even at high concentrations, only slightly inhibited the formation of protein-bound and other water-soluble estrone-16-cl4 derivatives. This would seem to rule out a peroxidative type of reaction for the formation of the protein- bound derivatives by liver, though it has been suggested by
95
Posner e_t al B that hydrogen peroxide generated within the microsomes may not be affected by catalase.
Estradiol-17p was also shown to be metabolized to protein-bound derivatives by liver, and it is likely that the mechanism is the same as for estrone. Diethylstilbestrol also - 51 -
gave rise to protein-bound derivatives and again, one or other
of the two theories proposed above could apply in this case.
The formation of estrogen conjugates by rat and guinea pig liver was also investigated and the results shown that in the absence of added UDPGA rat liver preparations do not give rise
to products that can by hydrolysed by p-glucuronidase or hydro•
chloric 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 well^.
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^,
it was first thought the Wistar rats used in these experiments did not possess the enzyme which transfers glucuronic acid to
the aglycon acceptor. However, this was shown not to be the case,
since incubation of the rat liver microsomes with excess UDPGA and estrone-16-Cl^ resulted in increased incorporation of radio• activity into the aqueous fraction, and in the formation of a metabolite chromatographically identical with estradiol-17p- 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 in water-soluble radioactivity. It seems, therefore, that Wistar rat liver does not lack glucuronosyl transferase, but for some reason or other is without an adequate supply of available UDPGA. The picture, however, is complicated by the observation that in the case of the 8000 x g supernatant fraction of rat liver 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. It is possible that a complex conjugate such as' a sulphoglucuronide, which would likely be only partially hydrolysed by p-glucuroni- C 62 dase under the conditions used , is formed. It is possible that the soluble portion of the rat liver cell contains some factor which inhibits the formation of simple estrogen glucosiduronates, even in the presence of ample glucuronic acid donor, or that an alternative type of conjugation occurs more readily under these conditions.
In experiments with the guinea pig, it was shown that glucosiduronate conjugates of estrone are formed in vivo, and that the 8000 x g supernatant fraction of the liver produces glucosiduronates of estrone, estradiol-17p and stilbestrol, with or without added UDPGA. Estrone was shown to be metabolized by the 8000 x g supernatant fraction to give compounds which were - 53 - chromatographically similar to estradiol-17p-glucosiduronate and to estriol-17p-glucosiduronate. The liver microsomes, however, on incubation with estrone in the absence of UDPGA did not give rise to appreciable amounts of glucuronic, acid conjugated material, which was to be expected since UDPGA is produced only in the soluble portion of the liver cell^. Addition of the glucuronic acid donor to an incubation mixture containing the microsomes resulted in substantial amounts of a compound similar to estradiol-17p-glucosiduronate being formed from estrone.
Thus, there appears to be a marked species difference in the ease with which glucosiduronate conjugates of estrogens are formed in vitro by the rat and the guinea pig. In fact, exo• genous estrogen is excreted in the urine of the guinea pig mainly as the glucuronic acid conjugate, whereas in the rat, although it is excreted largely in a water-soluble form, most of it is not bound as a simple glucosiduronate.
It is unlikely that any quantity of the simple sulphate conjugates of estrogens was formed by the liver preparations since the amount of hydrolysis produced by acid corresponded to the amount that occurred with p-glucuronidase, and this enzyme preparation was free of sulphatase activity under the conditions used, since sulphatase is markedly inhibited by 0.1 M phosphate 97 buffer . The one exception to this, where an acid-hydrolysable - 54 -
conjugate which was insensitive to p-glucuronidase was produced
from estrone by the 8000 x g supernatant of rat liver fortified
with UDPGA, has already been discussed.
So far, discussion of the possible identity of the water-
soluble estrogen metabolites which are not protein-bound or
conjugated with glucuronic or sulphuric acid has been neglected.
This type of compound in fact accounts for about 857Q of the
water-soluble material produced from estrone, estradiol-17p and
stilbestrol by the rat liver 8000 x g supernatant preparations,
and for about 3070 of the metabolites formed by the rat and guinea
pig liver microsomes. With the guinea pig 8000 x g supernatant,
or whole homogenate, and also in vivo, these compounds are not
formed in appreciable amounts since glucuronic acid conjugation
predominates. However, these unidentified compounds do appear
to be major metabolites of estrogen both in vitro and in vivo in
the rat.
This material formed from estrone-16-cl4 in incubations with liver preparations is by no means homogenous. Chromato•
graphy of various aqueous fractions, from which the microsomes
had been removed by centrifugation, showed that several radio•
active compounds were present in each fraction. In the butanol-
acetic acid system an unstable compound with an Rf of 0.5 (XQ 5) was the major metabolite from the rat 8000 x g supernatant - 55 - incubation, although compounds with R£ values of 0.9 (XQ g) and
0 (XQ) were also detected. Similar metabolites were formed by the rat liver microsomes but the relative amount of compound
XQ was smaller. Guinea pig microsomes gave rise to XQ and
small amounts of XQ 9, XQ ^ being detected only in highly conc• entrated hydrolysed samples. Hence, at least three different metabolites of estrone, which are not protein bound and are not simple conjugates must be considered. Formation of large amounts of XQ ^ by rat liver seems to depend on a factor in the 100,000 x g supernatant fraction and together with XQ g, XQ 5 was shown to be converted after incubation at 37°C for 48 hours to material which did not move from the origin. Unfortunately, no chromatographic system has been found that was suitable for separating these very polar products.
At present, very little is known about the nature or mode of formation of these water-soluble estrogen metabolites, though several speculations can be made. The two theories put forward to account for formation of the protein-bound metabolites of estrogens both involve highly reactive unstable intermediates.
In one case, an o-quinoid derivative, and in the other a phenoxyl radical type of intermediate was postulated. It is possible that one or other of these intermediates is formed by the micro• somes, and that in the absence of the soluble portion of the liver - 56 - cell, these interact mainly with microsomal protein. In the presence of the soluble portion of the cell the reactive inter• mediate could combine with other acceptors not present in such large amounts in the microsomal fraction. These could be amino acids or small polypeptides. This theory would explain why a much higher proportion of the radioactivity remaining in the ether-extracted aqueous fraction is bound to protein after incuba• ting liver microsomes with estrone-16-C^4 than after incubation with the 8000 x g supernatant fraction.
There are also other possibilities for the identity of these water-soluble metabolites. It has been suggested that they are intact phenolic estrogen derivatives which are water-soluble no by virtue of a large number of hydroxyl groups . 6-hydroxy- estriol for instance is relatively water-soluble, having a partition coefficient in an ether-water system of 0.12, whereas QQ that for estriol is 7.7 . There is evidence, however, that such simple polyhydroxylated derivatives are not formed to any great extent by rat liver preparations, since Jellinck''"^ showed that acetylation or methylation of the aqueous fraction from a rat liver incubation failed to make a significant amount of the products soluble in ether.
Yet another possibility is that the water-soluble metabolites - 57 -
are degradation products in which Ring D has been oxidized to give
a marrianolic acid or further oxidized compounds. Evidence against
this is that no C^40"2 is evolved during incubations with estrone-IS•
C''-4 and also that marrianolic acid and related compounds have never
been detected in nature^. Much of the information on the oxida•
tive formation of water-soluble products in fact points to changes
in Ring A as being of primary importance and its extensive oxidation
to yield an open structure with free carboxyl groups must also be
considered.
More definite knowledge about the nature of the water-
soluble estrogen metabolites that are not bound to protein or
conjugated with sulphuric or glucuronic acid awaits their isolation
and chemical characterization. Preliminary studies on their
separation by paper chromatography has been discussed in this work.
Due to methodological difficulties in the characterization
of the water-soluble end-products of estrone metabolism by liver,
another approach to the problem was taken. It was considered that
information on the mode of formation of the end-products could be
obtained from studies on the inhibition of their formation by various compounds.
The observation that KCN and the sulphydryl group
inhibitors N-ethylmaleimide and p-chloromercuribenzoate inhibit the formation of water-soluble products from estrone by rat - 58 - liver preparations confirms the work of others. KCN could be inactivating a copper or iron-containing oxidase, while the other two inhibitors would be inactivating a sulphydryl group containing enzyme, possibly a dehydrogenase. Mushroom tyrosinase and horse• radish peroxidase, both of which metabolize estrone to water- soluble products, are inhibited by KCN but not by N-ethylmal- eimide, showing that a more complex series of reactions must be occurring with liver.
The studies on the inhibition of formation of water- soluble products from estrone by compounds having structural features similar to those of estrone have yielded some interesting data.
It was reasoned that this type of study could give some clues about the nature of the groups that are of importance in the pathway leading to the formation of water-soluble products from estrone. Some ideas have been obtained, though the data is some• times hard to interpret, due to certain limitations in the experiments themselves. Thus, since the enzyme preparation used
(the microsomes) represent a heterogeneous system, the possibility exists that the compound which was tested as an inhibitor was not inhibitory per se, but was first metabolized to give the actual interfering substance. Also, it is difficult to ascertain whether the various inhibitors were affecting the same step in - 59 -
the conversion of estrone to water-soluble metabolites, since
this, in all likelihood, is a multi-stage process.
Another difficulty in interpretation arises in the kinetic
studies. Certainly zero-order kinetics appear to be operating in
the first stages of the process by which water-soluble metabolites
are formed, but the possibility exists that several different rates
of reaction combine to give what only appears to be zero-order
kinetics. Even considering these limitation, however, the results
obtained appear to justify this classical approach, if only to
point out further work of interest that could be carried out when
purified preparation of the enzyme(s) involved are obtained, and when the identity of the metabolites is ascertained.
A group of estrogens and estrogen derivatives were shown to
be potent inhibitors of estrone conversion to water-soluble metabolites by rat liver microsomes. 2-Hydroxyestrone, 2-hydroxy-
estradiol-17p, and equilenin gave over 7570 inhibition while
2-hydroxyestriol, estradiol-17p, estradiol-17a, equilin, ethynyl
estradiol and estrone sulphate and benzoate gave 50-6070 inhibition.
Since the sulphate and the benzoate esters were shown to be
hydrolyzed by the microsomal enzymes, the effect of these compounds
can be attributed to a dilution of the estrone-16-C14 substrate.
In contrast to these results, the 6-hydroxy-, 16-keto- and
10-hydroxy- derivatives did not exhibit any inhibitory action. - 60 -
Non-phenolic steriods were also inactive, as were the 17p-gluco- siduronate conjugates of the phenolic steroids.
At this stage it appears that a phenolic Ring A is required for inhibitory activity by a steroid, and that 2-hydroxy substitu• ents greatly enhance this effect, whereas substitution on the C-16 or C-6 positions, or glucosiduronate conjugation at C-17 has the opposite effect.
It is interesting to note that the non-steroidal synthetic estrogens, diethylstilbestrol, hexestrol and dienestrol inhibit, whereas their non-estrogenic derivatives dihydroxyhexestrol and dicarboxyhexestrol do not. The substituents in these last two compounds are on the ethyl side chains.
In the Lineweaver-Burk plots, stilbestrol, 2-hydroxyestrone and equilenin all appeared to act as competitive inhibitors in the conversion of estrone to water-soluble products, implying that their structure or that of their metabolites, is sufficiently similar to that of estrone or an intermediate of estrone, to be able to combine with the same active site(s) on the enzyme. A further interpretation, especially with regard to the inhibition by the
2-hydroxy compounds, is that the added inhibitor is identical with an intermediate formed from the estrone-16-C14 substrate, and that inhibition occurs as a result of C1^ dilution or else by a feed- - 61 - back type of mechanism. From this point of view it might be said that 2-hydroxylation is an essential part of the mechanism for the formation of water-soluble metabolites of estrone, whereas
6-, 10- or 16-hydroxylation, or glucosiduronate formation is not involved. However, the inhibitory action of the naphtholic estrogen, equilenin, and of the non-steroid, stilbestrol, does not fit easily into this scheme, and there is no direct evidence that 2-hydroxylation is an essential step in forming water-soluble metabolites, as has been discussed previously.
Although the inhibitory estrogens and their derivatives appear to act competitively, benzoquinones, naphthoquinones and their reduced forms do not act in this way if the results obtained with menadione can be taken to apply to the whole group. The
Lineweaver-Burk plot for inhibition by menadione showed a mixed type of competitive and non-competitive action. It is worth noting that meta-hydroxylated phenols, which of course cannot be oxidized to a quinonoid form, were inactive as inhibitors, as were the anthraquinones tested.
The mechanism of action of these inhibitors is very much open to speculation. Their common features make one think that they may in some way interfere with an oxidation-reduction process in estrogen metabolism. Molecular oxygen is an absolute require• ment for the formation of water-soluble products from estrone by - 62 -
rat liver, and is unlikely to be utilized directly, but rather by way of an electron transport system. Menadione, and related compounds could possibly act by inhibiting electron transport, although it was noted that these inhibitors did not interfere with the formation of ether-soluble metabolites from estrone, which are presumably hydroxylated derivatives.
It can only be said that the mechanism by which both the steroidal and non-steroidal compounds inhibit the conversion of estrone to water-soluble products is very complex and it will be of interest to repeat these studies with purified enzymes. All attempts by the author to render soluble the microsomal enzymes have so far been unsuccessful. - 63 -
SUMMARY
Three main pathways in liver by which estrone may be converted to water-soluble metabolites have been demonstrated.
By one such pathway, estrone or its derivatives are strongly bound to protein, and this process, which is mediated by microsomal enzymes, requires NADPH and oxygen. A second route gives rise to glucosiduronate conjugates, the transfer of glucuronic acid from
UDPGA to the steroid being catalysed by a microsomal enzyme.
A third pathway for estrone metabolism in liver was demonstrated, but the nature of the products formed by it is largely unknown.
Oxygen and NADPH are required, and the enzymes are probably in the microsomes, although a factor(s) in the 100,000 x g supernatant fraction of the cell is of importance. The metabolites formed by this route are not bound to protein, nor are they hydrolysed by
V refluxing with 20% ( /v) HC1 or incubating with p-glucuronidase, and at least three different products have been detected. In addition, this "unknown" pathway is inhibited by cyanide and by sulphydryl group inhibitors.
Specifically, it was shown that when rat liver microsomes were incubated with NADPH and estrone-16-C14 about 70% of C14 became bound to protein, and no conjugation with glucuronic or sulphuric acid occurred. If, however, UDPGA were added to the - 63 -
SUMMARY
Three main pathways in liver by which estrone may be
converted to water-soluble metabolites have been demonstrated.
By one such pathway, estrone or its derivatives are strongly bound
to protein, and this process, which is mediated by microsomal
enzymes, requires NADPH and oxygen. A second route gives rise to
glucosiduronate conjugates, the transfer of glucuronic acid from
UDPGA to the steroid being catalysed by a microsomal enzyme.
A third pathway for estrone metabolism in liver was demonstrated, but the nature of the products formed by it is largely unknown.
Oxygen and NADPH are required, and the enzymes are probably in the microsomes, although a factor(s) in the 100,000 x g supernatant
fraction of the cell is of importance. The metabolites formed by
this route are not bound to protein, nor are they hydrolysed by
v refluxing with 20% ( /v) HCl or incubating with p-glucuronidase,
and at least three different products have been detected. In
addition, this "unknown" pathway is inhibited by cyanide and by
sulphydryl group inhibitors.
Specifically, it was shown that when rat liver microsomes
were incubated with NADPH and estrone-16-C14 about 70% of the water-
soluble Cl4 was bound to protein, and no conjugation with glucuronic
or sulphuric acid occurred. If, however, UDPGA were added to the - 64 -
incubation mixture, estrogen glucosiduronates were formed in addition
to the other products. When the rat liver 8000 x g supernatant
fraction was incubated with NADPH and estrone-16-C^4, protein-bound metabolites accounted for only 10-157o of the water-soluble radio•
activity, the remainder being formed by the unknown route. Addition
of UDPGA to the incubation medium promoted the formation of an acid-
hydrolysable conjugate which, however, did not behave as a simple
glucosiduronate.
Injected estrone- 16-C^"4 was excreted in the urine of rats mainly as water-soluble metabolites which could not be hydrolysed by
HCl or p-glucuronidase, and the "unknown"pathway therefore appears to predominate in vivo in this species.
When guinea pig liver microsomes were incubated under 14 aerobic conditions with NADPH and estrone-16-C , most of the water-
soluble metabolites formed were bound to protein (707o) . Addition
of UDPGA to the incubation mixture, however, promoted the formation
of large amounts of glucosiduronate conjugates of the steroid, at
the expense of protein bound material. Incubation of the guinea pig
liver 8000 x g supernatant fraction with NADPH and estrone-16-C1"4
resulted in the conversion of the radioactive substrate mainly to
glucosiduronate conjugates, both in the presence and absence of
UDPGA. In the guinea pig in vivo, glucosiduronate conjugation was - 65 -
shown to be the major route for the metabolism of estrone.
Estradiol-17p-16-C14 and stilbestrol (monoethyl-l-C14) are metabolized by rat and guinea pig liver preparations in a pattern
similar to estrone-16-C-*-4.
In order to gain further information about the nature of the unknown water-soluble products formed from estrone by rat liver microsomes the effect on this reaction by compounds having struct• ural features similar to the radioactive substrate was studied.
2-Hydroxyestrone, 2-hydroxyestradiol-17p and equilenin were found to be very potent inhibitors, while estradiol-17p and -17a, stilbestrol and hexestrol showed intermediate activity. All the
6- or 16-hydroxylated derivatives of the natural estrogens, as well as the 17p-glucosiduronates and non-phenolic steroids tested were without activity. A group of benzoquinones, naphthoquinones and
their reduced forms were found to be very strong inhibitors, but anthraquinones and meta-hydroxylated phenols were inactive.
In kinetic studies, 2-hydroxyestrone, equilenin, and
stilbestrol appeared to be acting as competititve inhibitors in the conversion to water-soluble metabolites, while menadione produced a mixed type of inhibition.
Finally, the significance and limitations of these studies were discussed. -66-
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