Estrogenic action of estriol fatty acid esters

Recent studies suggest that, estriol, like estradiol, is biosynthetically esterified with fatty acids. We have synthesized the stearate estriol, at C-16 alpha, C-17 beta and the diester, C-16 alpha,17 beta and tested these D-ring esters for their estrogenic action both in vivo and in vitro, comparing them to estradiol, estriol and estradiol-17-stearate. None of the estriol esters bind to the estrogen receptor. They are only weakly estrogenic in a microtiter plate estrogen bioassay: stimulation of alkaline phosphatase in the Ishikawa endometrial cells. However, both estriol monoesters are extremely potent estrogens when injected subcutaneously (in aqueous alcohol) into ovariectomized mice. Compared to the free steroids, they produced a dramatically increased uterine weight with a greatly prolonged duration of stimulation. The 16 alpha,17 beta-diester also induced a protracted uterotrophic response, but the stimulation of uterine weight was comparatively low. Since the esters of estradiol and estriol do not bind to the estrogen receptor, their estrogenic signal must be generated through the action of esterase enzymes. These naturally occurring esters have the potential of being extremely useful pharmacological agents for long-lived estrogenic stimulation.     

Bioflavonoid interaction with rat uterine type II binding sites and cell growth inhibition

Competition analysis with a number of known bioflavonoids demonstrated that these compounds (luteolin, quercetin, pelargonin) compete for [3H]estradiol binding to cytosol and nuclear type II sites in rat uterine preparations. The inhibition of [3H]estradiol binding to type II sites was specific and these bioflavonoids did not interact with the rat uterine estrogen receptor. Since estradiol stimulation of nuclear type II sites in the rat uterus is highly correlated with cellular hypertrophy and hyperplasia, we assessed the effects of these compounds on the growth of MCF-7 human breast cancer cells in culture and on estradiol stimulation of uterine growth in the immature rat. The data demonstrated that addition of quercetin (5-10 micrograms/ml) to MCF-7 cell cultures resulted in a dose-dependent inhibition of cell growth (DNA/flask). This effect was reversible by removal of quercetin from the culture medium, or by the addition of 10 nM estradiol-17 beta to these cell cultures containing this bioflavonoid. Since estradiol-17 beta (10 nM) stimulated nuclear type II sites and proliferation of MCF-7 cells, we believe bioflavonoid inhibition of MCF-7 cell growth may be mediated through an interaction with nuclear type II sites. This hypothesis was confirmed by in vivo studies which demonstrated that injection of luteolin or quercetin blocked estradiol stimulation of nuclear type II sites in the immature rat uterus and this correlated with an inhibition of uterine growth (wet and dry weight). These studies suggest bioflavonoids, through an interaction with type II sites, may be involved in cell growth regulation.     

Effect of estrogens on phosphofructokinase activity of uterus, liver and kidney in rat and hamster

The effect of estradiol-17β on the phosphofructokinase (PFK) activity of uterus, liver and kidney in rat and hamster has been studied. 16 hours after a dose of 10 μg estradiol/100 g body weight, there are no differences in the uterotrophic responses of rats and hamsters and the increase in uterine PFK activity is similar in both animals. 48 hours after two doses of 100 μg estradiol/100 g body weight, the uterotrophic response is slightly higher in hamster than in rat, but rat uterus shows a greater increase of PFK activity than does hamster uterus.Hepatic and renal PFK activities in hamsters of both sexes are not modified 48 hours after two doses of 100 μg estradiol/100 g body weight. These results indicate that in hamster and rat, PFK is under estrogenic control in uterus and not in liver and kidney.     

Steroidal control of rat uterine 17β-hydroxysteroid dehydrogenase activity

The interconversion of estradiol-17β and estrone in the rat uterus is due to the action of 17β-hydroxysteroid dehydrogenase. Whole uteri or 800 × g supernatant fractions of the uteri were incubated in the presence of [³H] estradiol-17β and NAD at 37°C for 3 h or 1 h, respectively. In the mature rat uterus the oxidation of estradiol-17β and estrone was dependent on the stage of the estrous cycle, suggesting hormonal control. The 17β-hydroxysteroid dehydrogenase activity was highest at estrus (200 fmol estrone) and lowest at diestrus (80 fmol estrone). An enhancement of activity occurred when adult rats at each stage of the estrous cycle were administered estradiol-17β, while progesterone administration at each stage resulted in decreased enzyme activity. The uterine 17β-hydroxysteroid dehydrogenase activity of estradiol-17β treated ovariectomized rats was time and dose dependent but decreased when progesterone was administered with or without estradiol-17β administration. These results suggest that estradiol-17β caused an increase in enzyme activity that was inhibitable by progesterone in the rat uterus. The increased 17β -hydroxysteroid dehydrogenase activity may reflect a specific response of the rat uterus to estradiol-17β.     

Metabolites of estradiol-17β in guinea pig uterus late in pregnancy

The metabolism of estradiol-17β by the guinea pig uterus late in pregnancy was studied $\text{in vivo}$ and $\text{in vitro}$.Whole uteri were examined for estrogen metabolites one hour following an intravenous injection of [³H]-estradiol-17β or uterine sections were examined after incubation for one hour at 37°C in medium containing [³H]-estradiol-17β.In both instances uterine tissue metabolized estradiol-17g to five products: estrone, estrone-3-sulfate, 17β-estradiol-3-sulfate, estrone-3-glucuronide and 17β-estradiol-3-glucuronide. Of the total radioactive products 11 – 43% were glucuronides, 17 – 26% were sulfates and 4 – 17% was estrone. These results indicate that the guinea pig uterus actively transforms estradiol-17β into glucuronides and sulfates late in pregnancy.     

In vivo estrogenic and antiestrogenic activity of phenolphthalein and derivative compounds

The estrogenic activity of phenolphthalein and other related triphenylmethane dyes was evaluated in vivo in the immature rat uterus. Phenolphthalein behaved as a partial agonist of estradiol in stimulating the growth of rat uterus. Other specific estrogenic effects of the dye included an increase of the uterine DNA content, histological changes and induction of estrogen-modulated secretory proteins. The progressive introduction of side chains in the triphenylmethane skeleton concomitantly decreased the estrogenic activity. Triphenylmethanes competed with [3H]estradiol for the binding to the estrogen receptor in vitro, the relative binding affinity being correlated with the estrogenic potency observed in vivo. Phenolphthalein also showed antiestrogenic activity that could be overcome by increasing the dose of estradiol.     

Effect of estradiol and progesterone on phosphatidylinositol metabolism in the uterine epithelium of the mouse

The effect of estradiol and progesterone on uterine phosphatidylinositol (PtdIns) metabolism was examined in whole uteri and separated uterine luminal epithelium of ovariectomized mice. Incorporation of [3H]myo-inositol in vitro, into inositol-containing phospholipids extracted from whole uteri, increased in mice injected with estradiol, with maximal incorporation at 9-12 h. The breakdown of PtdIns to inositol polyphosphates was also stimulated in whole uteri by estrogen, with an abrupt increase between 6 and 9 h. Comparable increases in both processes occurred in the uterine epithelium after estrogen stimulation and were inhibited by progesterone pretreatment which by itself had little or no effect. These results suggest that PtdIns metabolism is involved in the stimulation of uterine epithelial cell proliferation by estrogens, and its inhibition by progesterone.     

A new class of long-acting hormonal steroid preparation: synthesis of oligomeric estradiol derivatives

A method for the synthesis of dimeric, trimeric and tetrameric estradiol esters is described. It is based on the esterification of the OH-groups of estradiol-17β or estradiol-17β-acetate with 3-acetoxy-estradiol-17β-hemisuccinate as carboxylic acid component in the presence of N, N'-carbonyldiimidazole. Preliminary biological tests have shown that these compounds possess a very protracted estrogenic activity when administered s.c. to the oophorectomized rat. A single injection of 40 μg/rat of the trimeric estradiol derivative e.g. brought about vaginal estrus for 120 days as compared to 80 days when estradiol undecylate was used.     

Radioautographic study of the effect of estradiol-17 , estrone, estriol, progesterone, testosterone and corticosterone on the in vitro uptake of 2,4,6,7- 3 H estradiol-17 by uterine eosinophils of the rat

The in vitro uptake of 2,4,6,7-tritiated estradiol-17beta in uterine eosinophils of the rat was inhibited by the presence of nonradioactive estradiol-17beta, estrone, and estriol, but not by progesterone, testosterone, or corticosterone. This action is attributed to competition between tritiated estradiol and the various estrogenic compounds for the same binding site. Compounds without any estrogenic activity do not compete. The proposal is made that the eosinophil binding system and the 8S-5S binding system are involved in different mechanisms of estrogen action. The parallelism between the doses of estradiol and estriol needed to promote certain estrogenic early effects in the uterus, and the affinity of these steroids for the eosinophil uptake sites, suggests that uterine eosinophils might be responsible for some of these early effects, such as water imbibition, histamine releasing activity, and estrogen priming effect.     

Influence of sex hormones on prostaglandin dehydrogenase activity in the rat uterus

The present experiments report the effects of estradiol or of progesterone on the activity of 15-prostaglandin-dehydrogenase (PGDH) in the uterus of spayed rats. When the substrate was PGF2 alpha the treatment with progesterone (4 mg X day-1, two days) or with estradiol-17-beta (0.5 ug + 1 ug) did not show any effect on the activity of the enzyme. On the contrary, uteri from ovariectomized rats injected with a higher dose of estradiol-17-beta (0.5 ug + 50 ug) exhibited a significant increment. When the substrate was PGE2, progesterone failed again to modify the enzyme activity, whereas estradiol, both at a low and at a high doses, enhanced significantly the uterine PGDH activity. The possibility of two different PGDHs for each PG and the role of estradiol in enhancing PGE2 catabolism into 15-keto-PGE2 as a mechanism subserving the effect of estrogens on the output of this PG in the rat uterus, are discussed.     

The metabolism of estradiol-17β by the pregnant guinea pig uterus in vitro

The in vitro metabolism of [³H estradiol-17β-by the uterus was studied in non-pregnant, prenant (day 30-term) and post-parturant guinea pigs. Following incubation of tissue sections for one hour is Krebs-Ringer phosphate buffer, five major metabolites could be extracted from the medium or tissue depending upon age of gestation: estrone-3-glucuronide, estrone-3-sulfate, estradiol-3-glucuronide and estradiol-3-sulfate. Both sulfated estrogens were detected at each age of gestation studied, whereas the glucuronides, mainly of estrone, were not detected until approximately day 50. Thereafter, as term (day 65–70) was approached, their percentage contribution to total radioactivity increased at the expense of estradiol and the sulfates. Following parturition, total metabolites of estradiol rapidly decreased, particularly the glucuronides. No conjugates were detected in uteri from nonpregnant guinea pigs. In addition, no conjugates were found in the pre-partum mouse, rat and hamster or in human endometrium obtained immediately after birth. The data suggest that, in the guinea pig, a biochemical factor in the termination of normal pregnancy is the control of tissue levels of active estrogen (estradiol) by conjugation with glucuronic acid.     

Characterization of estrogen binding sites in the liver of the newt Pleurodeles waltl (Urodela:Amphibia): demonstration of a sex-linked heterogeneity

Saturation analysis over a wide range of [3H]estradiol-17 beta concentrations (1-40 nM) in cytosols prepared from liver of the newt Pleurodeles waltl of both sexes revealed a sex-linked heterogeneity of the estradiol-17 beta binding sites. In females, one type of binding site has been identified as a classical receptor. It exhibited a high affinity for estradiol-17 beta (Kd = 9 X 10(-9) M), had a high specificity for estrogenic compounds and was stabilized by monothioglycerol. In males, in addition to the receptor found in females, a second estrogen binding component was detected, not observed in female cytosols. It exhibited a Kd of 4.8 X 10(-8) M for estradiol 17 beta, higher capacity and displayed the same highly specific estrogen binding as does the estrogen receptor. It was affected by monothioglycerol and its binding was found to be significantly increased on cytosol dilution, as well as by estrogen-treatment.     

Effect of estradiol on estrogen receptor expression in rat uterine cell types

In rodent uterus, both up- and down-regulation of estrogen receptor alpha (ERalpha) messenger ribonucleic acid (mRNA) and protein levels by estradiol has been demonstrated; however, it is not known which of the uterine compartments (endometrial epithelium, stroma, myometrium) respond to estradiol with autoregulation of ERalpha. The purpose of the present study was to investigate and compare the kinetics and cell type-specific effects of estradiol on uterine ERalpha expression in immature and adult rats. Ovariectomized female rats were injected s.c. with sesame oil or estradiol-17beta. Uteri were collected and analyzed for changes in ERalpha mRNA using RNase protection assays (RPA) and in situ hybridization using radiolabeled probes specific for ERalpha. Immunohistochemical analysis was performed with a polyclonal antibody specific to ERalpha. Expression of ERalpha in the uterine epithelial cells decreased at 3 and 6 h after estradiol administration to immature and adult rats, respectively. At 24 h, ERalpha mRNA levels in the immature and mature rat uterus were higher than pretreatment levels but returned to baseline by 72 h. Pretreatment with cycloheximide did not block the 3-h repressive effect of estradiol, suggesting that the estradiol-induced decrease in ERalpha mRNA occurs independent of new protein synthesis. A decrease in ERalpha mRNA and protein was also observed in uterine epithelia at 3 and 6 h after an estradiol injection to immature and adult rats, and intensity of both the in situ hybridization signal and the immunostaining in the epithelium increased at 24 and 72 h. However, the periluminal stromal cells in the adult uterus and the majority of stromal cells of the immature uterus appeared to have increased ERalpha expression. The results indicate that down-regulation of ERalpha in the epithelia and up-regulation of stromal ERalpha play a role in early events associated with estradiol-induced cell proliferation of the uterine epithelia.     

High affinity estrogen binding by rabbit liver

Macromolecular binding components for [3H]estradiol-17beta are present to cytosol prepared from rabbit liver. When cytosol from sexually mature male liver was incubated with [3H]estradiol and analyzed for binding on low ionic strength sucrose gradients, two peaks of binding activity were detected. One peak had a sedimentation coefficient of 4--5 S and the other had a sedimentation coefficient of 8--9 S. The two components differed from each other regarding steroid specificity and various physiocochemical parameters. [3H]estradiol binding to the 4--5 S component was not inhibited by estrogens, 5alpha-dihydrotestosterone, progesterone or cortisol. Binding to this component did not appear to be saturable and label was rapidly stripped from it by charcoal. Estradiol binding to the 8--9 S component was estrogen specific, saturable and of high affinity. The specific binder dissociates on high ionic strength sucrose gradients and sediments as a 4--5 S moiety. The specific binding protein has a Kd of 3.05 . 10(-10) M and a dissociation half-time of 33 h and there are 35.2 fmol of binding sites/mg cytosol protein. Estrogen binders are also present in liver cytosol from sexually mature female and sexually immature male rabbits. During prolonged incubation of [3H]estradiol with mature male liver cytosol at 0--5 degrees C polar metabolites of estradiol are produced.     

Selective turnover of the essential fatty acid ester components of estradiol-17 beta lipoidal derivatives formed by human mammary cancer cells in culture

The properties of fatty acyl coenzyme A: estradiol-17 beta acyl transferase in microsomes derived from pooled human mammary cancer tissue have been examined. A pH optimum of 5.5 was found and addition of long-chained fatty acyl CoAs increased estradiol-17 beta (E2) 17-monoacyl ester synthesis; the apparent Km for E2 being 8 microM when oleoyl CoA, linolenoyl CoA or palmitoyl CoA were employed. Testosterone, dehydroepiandrosterone, and 5-androsterone-3 beta, 17 beta-diol acted as competitive inhibitors with Ki values of 36, 36 and 46 microM, respectively. The composition of E2 fatty acyl esters (E2-L) formed by incubation of [3H]E2 with human mammary cancer tissue and human mammary cancer cell lines has been determined by HPLC. Although the composition of E2-L in estrogen receptor negative cell lines (MDA-MB-231 and MDA-MB-330) was generally similar to that found for MCF-7 cells (estrogen receptor positive) and pooled human mammary cancer tissue, the former cell lines contained a 3-fold higher relative concentration of E2-17 beta stearate. MCF-7 cells were exposed to 30 nM [3H]E2 and the composition of the isolated [3H]E2-L fraction studied at various time intervals. At 0.5 h, the intracellular concentration of E2-L was 1.8 +/- 0.4 (SEM) pmol/mg DNA which increased to values of 3.6 +/- 0.6 and 4.3 +/- 0.5 at 4 h and 16 h, respectively. In the subsequent 3 h following transfer to medium lacking [3H]E2, the concentration of E2-L declined to 3.7 +/- 0.3 pmol/mg DNA. The subfraction of E2-L composed of E2-17 beta arachidonate, linolenate and docosahexaenoate, was seen to decline in relative abundance after 0.5 h and to reach significantly lower relative levels at 16 h, and again in the 3 h period following estrogen withdrawal. The data suggests that these components, derived from essential fatty acids, are more metabolically active. This may then provide a new lead to link these novel estrogen derivatives with the established relationship between unsaturated fatty acids and an increased mammary cancer incidence.     

The uptake of radioactivity by the uterus of the rabbit following the sytemic administration of (3H) estradiol-17beta and (3H) estrone and the identification and subcellular localization of radiometabolites in the uterus

In an effort to determine the relevance of the uterine oxido-reduction of estrogens to their action in the rabbit uterus, the uterine uptake of radioactivity administered subcutaneously as [³h] estradiol-17β or [³H]estrone and the subcellular distribution of radio-metabolites in the uterine tissue were studied. The animals were killed 20 min, 1, 3 and 9 hr after the administration of 0.1 μg tritiated steroid and the relative proportions of radioactive estradiol-17β and estrone in plasma and in ‘cytosol’, ‘mitochondrial/microsomal’ and ‘nuclear’ fractions of the uterine homogenates were studied. Despite the presence of a high proportion of estrone in chloroform extract of plasma, very little was found in the fractions from uterine tissue irrespective of the steroid administered. Highest levels of uterine estrone were found in the ‘mitochondrial/microsomal’ preparation. There was no apparent difference in the pattern of uptake of radioactivity administered as [³H] estradiol-17β or [³H] estrone. The presence of high levels of 17β-hydroxysteroid dehydrogenase activity in the rabbit uterus may be responsible for the apparent difference between these results and those of similar experiments using the rat.     

An estrogen-dependent esterase activity in MCF-7 cells

The presence of steroidal esters in hormonally sensitive tissues lends importance to the esterases which convert the biologically inactive adducts to the parent potent forms. Accordingly, esterase-activities were studied in a human breast cancer model--the MCF-7 cell line. Tritiated estradiol esters- estradiol-17-acetate (EA), estradiol-17-valerate (EV) and estradiol-17-stearate (ES) were tested systematically, but 3 beta-ol esters of androgens, and phorbol diesters were also investigated. All compounds tested, except the phorbol diesters were hydrolyzed either when added to growing cultures or to the 28,000 g supernate of homogenized MCF-7 cells. Among the estrogens, the relative rates of hydrolysis were EA greater than EV greater than ES. The esterase for EA was different as it was not inhibited by saturating concentrations of EV or ES, and unlike the others its activity was stimulated by the addition of estradiol to the culture medium. The antiestrogen keoxifene,[(6-Hydroxy-2-(4-hydroxyphenyl)benzo less than b greater than thien-3-yl greater than less than 4- less than 2-(1-piperidinyl)ethoxy greater than phenyl greater than methanone], negated the stimulatory effect. Other major classes of steroids did not influence EA esterase activity. Results of inhibition experiments indicated that the esterases are of the serine active-site types. The significance of the estrogen-dependent esterase activity can be assessed when the natural substrate(s) for the enzyme is elucidated.     

Metabolism of progesterone in subcellular fractions of rat uterus

The metabolism of progesterone and 5α-pregnane-3,20-dione was studied in subcellular fractions of uterus from untreated and estradiol-17β treated immature rats. The reduction of progesterone to 5α-pregnane-3, 20-dione took place in all the particulate fractions of the uterus. The nuclear 5α-reductase accounted for the greatest fraction of enzymatic activity and was stimulated by estradiol treatment in vivo. The 5α-reductase activity in the mitochondrial and microsomal fractions was not increased after estradiol treatment. The reduction of 5α-pregnane-3,20-dione to 3α-hydroxy-5α-pregnan-20-one occurred mainly in the soluble fraction and was only slightly stimulated by estradiol. It proceeded much more rapidly than the reduction of progesterone to pregnanedione. Progesterone was also reduced to 20α-hydroxy-4-pregnen-3-one by a soluble enzyme whose activity was increased after estradiol-17β treatment.