The estrogen cytosol receptor of female ovine pituitary.

The biochemical parameters of estrone and estradiol binding to the cytosol fraction of ovine anterior pituitary were investigated. When increasing amounts of [3H]estrone or [3H]estradiol were incubated with the 105,000 g fraction from the pituitary, both hormones bound to a receptor with the same apparent KD (mean +/- S.E., estrone = 1.40 +/- 0.30 X 10(-10) M, estradiol = 1.03 +/- 0.11 X 10(-10)M) and the same concentration of binding sites (estrone = 3.22 +/- 0.58 X 10(-14) moles/mg protein, estradiol = 3.92 +/- 0.19 X 10(-14)). No conversion of [3H]estrone to [3H]estradiol under the experimental conditions used could be demonstrated. The receptor-estrogen complex exhibited identical sedimentation coefficients (7-8 S) with either hormone. The receptor was specific only for estrogens; neither 500-fold excess of testosterone nor progesterone affected binding. Competitive inhibition using increasing amounts of non-radioactive estrone or estradiol with [3H]estrone or [3H]estradiol resulted in parallel displacement of the radioactive hormone. These results strongly suggest that both hormones bind to the same pituitary cytosol receptor.     

Identification of 17 beta-estradiol as the estrogenic substance in Saccharomyces cerevisiae.

Saccharomyces cerevisiae possesses a high-affinity estrogen binding protein and an endogenous ligand that displaces [3H]estradiol from both the yeast binding protein and mammalian estrogen receptors. Semipurified preparations of this ligand have been shown to exhibit estrogenic activity in mammalian systems. We now describe the purification procedure and ultimate identification of the estrogenic substance in extracts of S. cerevisiae as 17 beta-estradiol. Organic solvent extracts of commercially obtained dried yeast were sequentially chromatographed on silica gel columns and then subjected to a series of reversed phase HPLC fractionations. Active ligand was monitored by [3H]estradiol displacement in a rat uterine cytosol assay. After seven chromatography steps, the purified and highly active ligand exhibited a single peak with retention times identical to those of 17 beta-estradiol on both HPLC and GC. The yeast material was identified as 17 beta-estradiol by its UV absorbance and mass spectrometric fragmentation pattern. In addition, radioimmunoassay confirmed the presence of approximately the same mass of 17 beta-estradiol (approximately equal to 800 ng/1.5 kg of yeast) as estimated both by a competitive binding assay with estrogen receptor and by mass spectrometry. Extraneous contamination by estradiol was excluded by repeat experiments with different batches of starting material and demonstration of estradiol by RIA in conditioned medium and cell pellets of laboratory-grown S. cerevisiae whereas non-conditioned medium did not possess the steroid. We conclude that 17 beta-estradiol is a yeast product.     

Effects of estrone and estradiol-17 beta on 25-hydroxycholecalciferol hydroxylase activities in female Japanese quail

Equimolar amounts of estrone and estradiol-17 beta injected into sexually immature female Japanese quail caused comparable increases and decreases in the renal activity of 25-hydroxycholecalciferol (25HCC) 1 alpha-hydroxylase and 25-hydroxycholecalciferol 24-hydroxylase, respectively. Peak activity of the former enzyme was induced by both estrogens within 6 hr and had declined by 24 hr. Plasma concentrations of estrone and estradiol following the injection were maximal within 3-6 hr of injecting the steroid i.m. Following [3H]estradiol injection, 25% of the radioactivity was located with estrone in the plasma following separation by TLC. Conversely, following [3H]estrone injection 30% of the radioactivity in plasma was located with estradiol. Thus, both estrone and estradiol may have physiological effects in quail, which in part may be due to their in vivo interconversion. Evidence that prolactin may be mediating the estrogenic stimulation of the 25HCC 1 alpha-hydroxylase is also presented.     

Steroid sulfatase activity in human lung tissue and in endothelial pulmonary cells in culture

The conversion of tritium-labeled estrone sulfate to [3H]estrone was evaluated in human lung tissue in vitro. Under standardized conditions, the rate of hydrolysis of [3H] estrone sulfate to [3H]estrone was linear with time of incubation up to 4 h and with wet tissue weight up to 400 mg/ml. The apparent Km of sulfatase for estrone sulfate was 9 microM, and the maximum velocity was 1.4 nmol substrate hydrolyzed/100 mg lung . h. The lung tissue also metabolized the primary metabolite of [3H]estrone sulfate, [3H]estrone, to 17 beta-[3H]estradiol. The hydrolysis of [3H]dehydroisoandrosterone sulfate to [3H]dehydroisoandrosterone by human lung tissue was also measured. Sulfatase activity with this substrate was linear as a function of wet tissue weight up to 800 mg/ml. The apparent Km of sulfatase for dehydroisoandrosterone sulfate was 7 microM, and the maximum velocity was 1.0 nmol substrate hydrolyzed/100 mg lung . h. The highest specific activity of lung sulfatase for [3H]dehydroisoandrosterone sulfate was found in a microsomal fraction of lung homogenate. The primary metabolite, [3H]dehydroisoandrosterone, was metabolized further by lung tissue to [3H]androstenedione and [3H]5-androstene-3 beta, 17 beta-diol. Although isolated segments of human pulmonary arteries also metabolized both [3H] estrone sulfate and [3H]dehydroisoandrosterone sulfate, cultures of pulmonary arterial endothelial cells lacked sulfatase activity. The cell(s) source of sulfatase activity in human lung tissue and isolated arteries has not yet been identified. Our findings suggest that the metabolism of sulfated steroids by the lung should be considered in evaluating homeostasis.     

16-Hydroxylation of estrone-3-sulfate and estrone in the guinea pig in vivo.

[6,7-3H]Estrone-3-sulfate or [6,7-3H]-estrone of high specific activity was injected into adult female English Shorthair guinea pigs. Blood, liver, kidney, gall bladder bile, and urine were obtained and investigated for metabolites. Chromatographic procedures followed by enzymatic or solvolytic cleavage of conjugates and subsequent crystallization with appropriate carrier steroids revealed the pattern of metabolites formed. Injected estrone sulfate was partially hydrolyzed and reconjugated, resulting in the production of estrone and estradiol glucuronides. The main metabolites, however, were monosulfates of 16alpha-hydroxyestrone, 16-keto-17beta-estradiol, and estriol as well as disulfates of 16alpha-hydroxyestrone, estriol, and 16beta-hydroxyestrone. Particularly high amounts of these were found in urine. By far the main metabolites of injected estrone were glucuronides of estrone and estradiol, although the pattern of mono- and disulfated steroids was qualitatively similar to that found after estrone sulfate injection. It is concluded that the guinea pigs employed in the study hydroxylated estrogen in the 16alpha- and 16beta-configurations and that this activity was much more pronounced after injection of estrone sulfate than after estrone.     

3H]estradiol and its metabolites in the brain, pituitary gland, and reproductive tract of the male rhesus monkey. A combined autoradiographic and chromatographic study

Previous studies have shown that [3H]estradiol is the major nuclear metabolite of [3H]testosterone in the hypothalamus, preoptic area and amygdala of the male rhesus monkey, and it has been proposed that some of the stimulatory effects of testosterone on male sexual behavior may be mediated by estradiol-concentrating neurons. To map these neurons and identify metabolites, 4 castrated male rhesus monkeys were each injected with 0.47 mCi/kg [3H]estradiol, and the brains were removed 60 min later. Left halves were frozen for thaw-mount autoradiography, and right halves were used to isolate cell nuclei from different brain regions. A fifth animal was used to validate the methodology. Radioactive steroids were identified and measured by high performance liquid chromatography. Brain areas previously shown to contain labeled neurons after [3H]testosterone administration were also labeled after [3H]estradiol, including medial preoptic nucleus (n.), anterior hypothalamic area, bed n. of stria terminalis, ventromedial n., premammillary n., and corticomedial amygdala. Some areas not labeled after [3H]testosterone were labeled after [3H]estradiol, including lateral septal n., arcuate n., paraventricular n., claustrum, entorhinal cortex, and spinal cord. The pars distalis of the pituitary gland was heavily labeled. Most (83%) of the radioactivity in cell nuclei was [3H]estradiol while [3H]estrone was a major metabolite (25%) in supernatants. In the brain, the highest nuclear concentration of [3H]estradiol was in the hypothalamus (249.2 +/- 20.0 fmol/mg DNA), although in the previous experiments with [3H]testosterone, the highest nuclear concentrations of [3H]estradiol were found in the amygdala. We interpret these results to indicate that local metabolic differences in the brain may underlie some of the different behavioral effects of gonadal steroids in primates.     

Plasma sex steroids and tissue aromatization in hatchling zebra finches: implications for the sexual differentiation of singing behavior.

One of the best examples for sex hormone regulation of brain development is found in songbirds. In zebra finches, only males sing because of striking sex differences in the neural circuitry that controls songs. Because developing females treated with estradiol (E2) develop a masculine song system, E2 is considered the normal masculinizing hormone. However, questions about the role of E2 in male development persist, because E2 treatments that masculinize song can demasculinize other sexual behaviors, and there exists contradictory evidence for high levels of circulating E2 in developing males. We remeasured plasma steriods in zebra finches during the first 13 days after hatching. E2 circulated at low levels, and there were no sex differences in circulating E2, estrone, testosterone, androstenedione, or dihydrotestosterone. We also measured aromatase activity [( 3H]androstenedione conversion to [3H]estrone and [3H]E2) in gonad, adrenal, brain, and other tissues of hatchlings. Aromatase was abundant in ovary, but was not definitively detected in testes, adrenals, or other nonneural tissues of males. Aromatase was also found in diencephalon and in high amounts in telencephalon, but sex differences were not detected in whole brain or cellular subfractions of telencephalon. Because ovarian steroidogenesis is high, it may be involved in differentiation of the female zebra finch, as in nonpasserine birds. By contrast, the functional estrogen necessary for masculinization of song is most likely derived from brain, supplied with substrate from the adrenals. The puzzle remains why the song system is not masculinized in females, who possess high levels of aromatizable androgens and telencephalic aromatase.     

Characterization of an estrogen-binding protein in the yeast Candida albicans

An estrogen-binding protein (EBP) has been identified and characterized in the cytosol of the pathogenic yeast Candida albicans. Binding of [3H]estradiol was found to be optimal at pH 7.4 in the presence of 0.3 M KCl and was linearly related to protein concentration. Binding was very rapid, reaching maximal levels in about 30 min, and was reversible with a dissociation rate constant of 13.2 +/- 1.7 x 10(-4) sec-1. EBP binding was destroyed by treatment with proteolytic enzymes and by high temperatures. Scatchard analysis of the [3H]estradiol equilibrium binding data of C. albicans (strain stn-1) yielded an apparent dissociation constant of 12.3 +/- 2.1 nM and a maximal binding capacity of 753 +/- 145 fmol/mg protein. Binding competition experiments showed very high specificity and stereoselectivity of EBP, demonstrating the following order of potency in displacing [3H]estradiol: 17 beta-estradiol greater than estrone greater than estriol greater than 17 alpha-estradiol. Negligible competitive potency was found for other mammalian steroid hormones, diethylstilbestrol, tamoxifen, or fungal hormones. The abundance of EBP was 4- to 10-fold higher during the early logarithmic growth phase of yeast cells than during the stationary phase. The molecular size of EBP, measured by Sephacryl S-200 gel exclusion chromatography, yielded a Stokes radius of approximately 29 A. Sucrose density gradient sedimentation showed a sedimentation coefficient (S2020,W) of 4, with no ionic dependent aggregation of the [3H]estradiol-EBP complex. The apparent mol wt of the EBP is approximately 46,000, with an axial ratio of 1, indicating the symmetrical shape of the molecule. In summary, in addition to the previously described corticosterone-binding protein, a separate high affinity, stereospecific, estrogen-selective binder has been demonstrated in the cytosol of C. albicans.     

Dynamics of steroid biosynthesis during the luteal-placental shift in rhesus monkeys

We studied the dynamics of steroid secretion during the luteal-placental shift of early pregnancy in rhesus monkeys. Daily blood samples were obtained from six pregnant rhesus monkeys during the cycle of conception and for the first 44 days of pregnancy to examine the relationships among aromatizable androgens, estrogens, progesterone (P), and CG in the peripheral circulation. To elucidate the biosynthetic mechanisms involved in the changes in hormonal patterns, minces of placentae and corpora lutea (CL) were incubated in vitro with [3H]pregnenolone ([3H]P5) and [3H]androstenedione ([3H]A), and steroid metabolites were isolated and identified by reverse isotope dilution. After a brief rise (corresponding to the approximate time of implantation of the blastocyst), serum P levels declined despite rising serum CG levels and reached a nadir at the time of maximal CG secretion. In contrast, serum androstenedione (A), testosterone (T), estrone (E1), and estradiol (E2) concentrations rose and fell in parallel with CG and reached peak levels near day 24 of pregnancy 2- to 10-fold higher than those during the luteal phase. The CL of early pregnancy produced less than one third as much P from [3H]P5 as did the CL of the cycle, but had a 9-fold greater capacity for estrogen biosynthesis. Total CL aromatase activity did not differ between the nonfertile cycle and early pregnancy. 17-Hydroxylase and C17-20 lyase activities increased in the CL during early pregnancy, since 17-hydroxyprogesterone and A (but not T) syntheses were 3- to 5-fold greater in the CL of early pregnancy than in the CL of the cycle. Although serum E1 and E2 concentrations during early pregnancy were similar, E1 was the predominant estrogen produced from [3H]P5 or [3H] A by luteal tissue in vitro. Placental tissue obtained on days 23-27 of pregnancy produced large amounts of P from [3H]P5, but only trace amounts of estrogens were formed from [3H]A. By day 45 of pregnancy, however, the placenta had acquired substantial amounts of aromatase activity. Neither estrogens nor androgens were formed from [3H]P5 by the placenta.(ABSTRACT TRUNCATED AT 400 WORDS)     

The intracellular control of aromatase activity by 5 alpha-reduced androgens in human breast carcinoma cells in culture

Four cell lines, each derived from a primary tumor from a patient with breast carcinoma, were grown to confluence in alpha-Minimum Essential Medium with 15% fetal calf serum and incubated for 24 h with [3H]androstenedione. The two lines (SA and PP) with the lowest formation of estrone and estradiol (less than 0.1% conversion) were the most active in the formation of the 5 alpha-reduced androgen metabolites androsterone (AND), 5 alpha-androstanedione (5 alpha-A-dione), and dihydrotestosterone (DHT). The two lines with the highest aromatase activity (DM and MD) had the lowest formation of 5 alpha-reduced metabolites. To determine if the 5 alpha-reduced androgen metabolites formed within the breast carcinoma cells could influence aromatase activity, the MD line was further studied. After 24-h preincubation with AND, DHT, or 5 alpha-A-dione at concentrations of 10(-6), 10(-7), and 10(-8) M, [3H]androstenedione was added to the culture medium, and aliquots were removed at 0, 4, 8, and 24 h. An 8-h incubation period was found to be optimum for inhibition studies. In comparison to control levels of estrone (2.5%) and estradiol (0.35%) formation, inhibition of aromatization was evident with all three compounds at 10(-8) M, with 5 alpha-A-dione producing the greatest inhibition (50%). At 10(-7) M, inhibition ranged from 45% (AND) to 70% (5 alpha-A-dione), and at 10(-6) M, inhibition was greater than 90% for each compound. 5 alpha-A-dione produced slightly greater inhibition than AND or DHT at each concentration tested. Since each of these compounds was capable of inhibiting aromatization, the cumulative effect of these 5 alpha-reduced metabolites could be an important factor in the intracellular regulation of aromatase activity.     

Differential hydroxylations of estrone and estradiol in man

We sought to test the hypothesis that in man, the fraction of estradiol (E2) that does not undergo oxidation to estrone (E1) in vivo is not available for subsequent hydroxylation at C-2 and C-16 alpha. Using radiometric methods, the extent of 17-oxidation of E2, 2-hydroxylation of E2 and E1, and 16 alpha-hydroxylation of E2 and E1 was determined in normal men. It was expected that administered E1 would transfer more of the tritium from C-2 and C-16 alpha into body water than would the corresponding dose of E2 and that the difference would be related to the fraction of E2 not oxidized at C-17. In eight of eight paired studies, the generation of tritiated water was substantially greater from [2-3H]E1 than from [2-3H]E2. By contrast, in six of seven paired studies, it was the administered [16 alpha-3H]E2 rather than [16 alpha-3H]E1 that underwent greater hydroxylation. No consistent relationship was found between the extent of 17-oxidation and the subsequent hydroxylation. We had previously hypothesized that the differences in the oxidative metabolism of E2 between men and women was due primarily to the lesser conversion of E2 to E1 in men and that the lesser extents of the subsequent hydroxylations at C-2 and C-16 alpha were secondary to this initial difference. The present work indicates that while this concept may be valid in reference to the decreased 2-hydroxylation in men, it does not account for the lesser extent of 16 alpha-hydroxylation.     

The affinity of catechol estrogens for estrogen receptors in the pituitary and anterior hypothalamus of the rat.

The purpose of these experiments was to evaluate the potential for interaction between 2-hydroxyestrone and 2-hydroxyestradiol and estrogen receptors in rat pituitary and anterior hypothalamus. The 150,000 X g supernatant fractions of these tissues were prepared, the estrogen receptor-site concentration was measured, and the relative abilities of unlabelled estradiol, estrone, 2-hydroxyestradiol and 2-hydroxyestrone to compete with [3H]estradiol for estrogen binding sites was determined. From these results, and the previously determined association constant for [3H]estradiol, 10(10)M-1, the association constants of the other estrogens were calculated. The introduction of the 2-hydroxy group caused only a modest reduction in the affinity of these estrogens for the receptors. The association constants of the 2-hydroxy derivatives were within one order of magnitude of those of the parent compounds. These results demonstrate the potential for interaction between catechol estrogens and estrogen receptor in rat brain and pituitary of a magnitude which could be biologically significant.     

Comparison of results from in situ and in vitro perfusions of rabbit uterus with labeled estrone and estradiol.

The interconversion of labeled estrone (E1) and estradiol (E2) in rabbit uterus was studied in vivo and in vitro, in order to evaluate the influence of incubation conditions upon the preferred direction of the reversible conversion of E2 to E1. In a typical experiment, one uterine horn was perfused intraluminally in situ with a mixture of [3H]E2 and [14C]E1, while the other horn was dissected and used for in vitro perfusions with the same tracers, both intraluminally and on tissue slices. The ratios of rate constants corresponding to the oxidative and reductive reactions were found to be similar under the three perfusion conditions, as calculated from the isotopic data. The rate constant of conversion of E1 to E2 was 10-20 times larger than the rate constant of the opposite reaction. Equal E2 nuclear saturation levels were obtained by perfusion of the uterine tissue with excess [3H]E2 in situ and in vitro after slicing.     

The cyclic relationship of estrogen sulfurylation to the nuclear receptor level in human endometrial curettings.

Human endometrial curettings were selected principally from patients undergoing tubal ligations for elective sterilization. Specimens were analyzed for the metabolism of 17beta-[6,7-3H]estradiol and Na2 35SO4; assayed for the nuclear receptor content with 17beta-[6,7-3H]estradiol; and examined for the cytosol receptor content with 17beta-[2,4,6,7-3H]estradiol. The results of these experiments demonstrated that a) estrogen sulfotransferase activity is greatly stimulated during the secretory phase; b) although estrogen dehydrogenase is active throughout the menstrual cycle, the formation of estrone is elevated in concert with sulfurylation; and c) this increased metabolism of 17beta-estradiol is accompanied by a decreased nuclear uptake of the estrogen receptor complex. The importance of this endometrial estrogen metabolism in the maintenance of a secretory tissue is discussed.     

In situ subcellular distribution and metabolism of progesterone, estradiol and androstenedione in the vascularly separated and isolated hypothalamus of the female rhesus monkey

A neurosurgical procedure has been developed for the vascular isolation of the hypothalamus-thalamus region of the rhesus monkey brain. The circulation to the left and right halves of the hypothalamus was also isolated and each half of the hypothalamus was perfused simultaneously, but separately, with a dextran-blood solution which contained radioactive gonadal steroids. The hypothalamus in situ efficiently converted [3H]androstenedione to [3H]estrone and this aromatization was inhibited by the presence of androsta-1,4,6-triene-3,17-dione (ATD) in the perfusate. [3H]Progesterone was metabolized predominantly to 5 alpha-pregnane-3,20-dione (5 alpha-DHP) and 20 alpha-hydroxypregn-4-ene-3-one (20 alpha-OHP). Subcellular fractionation of the hypothalamus after the in situ perfusion with [3H]-progestin or [3H]estradiol to the hypothalamus of estrogen-treated ovariectomized monkeys or oil-treated ovariectomized monkeys, respectively, indicated that the retention of [3H]estradiol in the nucleus was a saturable, limited-capacity phenomenon. No saturable subcellular distribution of [3H]progesterone or [3H]R 5020 was observed. This latter observation might be attributable to the presence of a progesterone receptor in too small a concentration to be detected by the methods used.     

The prosence of estrogen receptor in kidneys from normal and androgen-insensitive tfm/y mice.

In vitro and in vivo binding of [3H]estradiol to cytosol proteins and nuclei was studied in kidneys from normal and androgen-insensitive tfm/y mice. The use of tfm/y mice permitted study of the estrogen-receptor complex in the absence of androgen receptor. A high affinity, [3H]estradiol-labeled, 8S molecule was demonstrated in low salt sucrose gradients. This macromolecule sedimented more slowly in gradients containing KC1 (0.5 M). Binding of [3H]estradiol was inhibited by 100-fold excess estrone, estradiol, or diethylstillbestrol but was not affected by testosterone, androstenedione, or progesterone. Studies of equilibrium-binding kinetics for estradiol indicated a Kd of 1.4 X 10(-9) M and 4.4 X 10(-14) mol of binding sites/mg cytosol protein. Furthermore, the binder was an acidic, heat-labile protein (pI = 4.8) which could be precipitated from cytosol with 33% ammonium sulfate, and needed sulfhydryl groups for activity. The demonstration of an estradiol-binding protein in vitro was correlated with specific [3H]estradiol uptake by tfm/y kidney nuclei in vivo. We concluded that the mouse kidney contains an estradiol-binding protein, distinct from that for androgens, which has many of the characteristics of a steroid receptor. The presence of an estrogen receptor in both normal and tfm/y mice indicates that a genetic defect in one receptor does not influence the properties of another. We concluded that androgen and estrogen receptors are under independent genetic control.     

Human fetal liver estrogen 16 alpha-hydroxylase: precursor specificity, kinetic parameters, and in vitro regulation

The properties of human fetal liver (HFL) estrogen 16 alpha-hydroxylase (16 alpha-OHase) were studied in microsomal preparations and hepatocytes maintained in culture. A specific assay was developed for the determination of estrogen 16 alpha-OHase activity based on the enzymatic release of tritium from the C-16 alpha position of stereospecifically labeled 16 alpha-3H-labeled C18-steroids, viz. 17 beta-[16 alpha-3H]estradiol, 17 beta-[16 alpha-3H]estradiol 3-sulfate, [16 alpha-3H]estrone, and [16 alpha-3H]estrone sulfate. The percentage of tritium at the C-16 alpha position of 17 beta-[16 alpha-3H]estradiol was 92.5%. There was no kinetic isotope effect in the 16 alpha-hydroxylation of 17 beta-[16 alpha-3H]estradiol. HFL hepatocyte and microsomal 16 alpha-OHase activity was linear with incubation time for at least 2 h, with a hepatocyte number up to 6.7 X 10(6) cells/ml and a microsomal protein concentration up to 1 mg/ml. The apparent Km of 16 alpha-OHase for estrone sulfate (E1S) was greater than that for either 17 beta-estradiol (E2) or estrone (E1; 2.9-6.4 microM vs. 0.70-0.84 microM). The maximum velocity of HFL 16 alpha-OHase also was greater with E1S or 17 beta-estradiol 3-sulfate than with either E1 or E2, and E1S was the most efficient substrate. The apparent temperature optimum for the microsomal enzyme was 37 C, and the apparent pH optimum was 7.0. 16 alpha-Hydroxylation of E1S by HFL microsomes was inhibited noncompetitively by E1. The biosynthesis of estriol from E2 by fetal liver microsomes does not require an intermediate oxidation step of E2 to E1 as appears to be the case in vivo in the human adult; this was demonstrated by the formation of [17 alpha-3H]estriol from [17 alpha-3H] E2 in incubations with HFL microsomes. A number of growth factors, hormones, and xenobiotics were preincubated with hepatocytes for 24 or 72 h to test for stimulation/inhibition of 16 alpha-OHase activity. 16 alpha-OHase activity was stimulated by dexamethasone, forskolin, (Bu)2cAMP, cholera toxin, 1,2-benzanthracene, and phenobarbital. Diethylstilbestrol, E2, and progesterone did not alter hepatocyte 16 alpha-OHase activity, except when very high concentrations of E2 and progesterone were used, when they became inhibitory; other hormones and growth factors did not alter the basal levels of the enzyme.     

Estrogen receptors in cultured rat uterine cells: induction of progesterone receptors in the absence of estrogen receptor processing

When cultured rat uterine cells were treated for up to 6 h with 5 nM 17 beta-estradiol, no decrease in the [3H] estradiol-binding capacity of the cells was observed (i.e. no processing). This was true whether the cells were treated directly with 5 nM [3H]estradiol or with 5 nM unlabeled 17 beta-estradiol followed by homogenization and exchange with [3H]estradiol in vitro. In additional experiments, intact cells were treated with medium containing 5 nM [3H]estradiol for 30 min, and then that medium was removed and replaced with medium containing 5 nM unlabeled 17 beta-estradiol. Receptor-bound estradiol in intact cells was totally exchangeable with estradiol in the culture medium (t1/2, approximately 90 min). Six-hour treatment of cells with 5 nM 17 beta-estradiol led to a 50% increase in the [3H]progesterone-binding capacity of the cells, while no loss of estrogen-binding capacity occurred. These results indicate that progesterone receptors can be induced by estrogen in the rat uterus in the absence of estrogen receptor processing.     

Radioimmunoassay of plasma equilin and estrone in postmenopausal women after the administration of premarin

A RIA for the measurement of plasma equilin (3-hydroxy-1,3,5-(10)7-estratetraen-17-one) and estrone in postmenopausal women and other estrogen-deficient women on exogenous equine estrogen replacement therapy is described. Antiserum against estriol-3,16,17-trihemisuccinate-HSA and high specific activity [2,4-3H]equilin and [6,7-3H]estrone were used in the assay procedure. Specificity of the assay was achieved by separation of equilin from estrone by chromatography on micro-Celite partition columns using silver nitrate as the stationary phase. The sensitivity of the standard curves for both equilin and estrone was 10-20 pg, and the smallest amount of estrone and equilin that could be measured accurately in the plasma was 250 pg/ml for both steroids. The coefficient of variation for both steroids ranged from 1-8%, over a range of 250-5000 pg/ml plasma. The interassay coefficients of variation for equilin and estrone were 4.6% and 2.4%, respectively. After the administration of 10 mg Premarin iv, maximum concentrations of 4 and 11.2 ng/ml for equilin and estrone, respectively, were obtained after 20 min. Thereafter, both steroids disappeared from the plasma gradually. When 10 mg Premarin were administered orally, equilin and estrone appeared in the blood gradually, and maximum levels of 560 and 1400 pg/ml were reached after 3 and 5 h for equilin and estrone, respectively. Equilin gradually disappeared, and by 24 h, only small amounts (125 pg/ml) were detectable. The levels of estrone declined more rapidly, though it was still detectable after 24 h. These preliminary results indicate that equilin sulfate is converted to circulating unconjugated equilin in a manner similar to the conversion of circulating estrone sulfate to estrone.     

Catechol estrogen formation in MCF-7 cell culture and effects of bromoestrogen inhibitors

Agonist and antagonist activities have been reported for several catechol estrogens given exogenously. Since the metabolic clearance rate for catechol estrogens in the body is very rapid, catechol estrogens produced at other tissues will have minimal effect on breast tissue. Information of the extent of catechol estrogen formation within cells is critical in assessing the overall importance of these estrogen metabolites. Investigations of the conversion of estrogens to catechol estrogens were performed in the MCF-7 human mammary carcinoma cell culture system. Reverse-phase high-performance liquid chromatography (HPLC) analysis demonstrated that very little metabolism of estradiol occurs after 48 h, with only small amounts of estrone, 2-hydroxyestradiol, 2-hydroxyestrone, and estriol being observed. The total amount of 2-hydroxyestrogen products formed from 1 microM estradiol was 406.2 pmol (SD = 60.9) per 3 x 10(7) cells in 48 h. Similar results were obtained using the simpler radiometric assay for estrogen 2-hydroxylase, which measures the release of 3H2O from [2(-3)H]estradiol. The effects of inhibitors of estrogen 2-hydroxylase were also examined in MCF-7 cells. 2-Bromoestradiol, 4-bromoestradiol, and 2,4-dibromoestradiol effectively block estrogen 2-hydroxylase in a dose-dependent manner in MCF-7 cultures, with ED50 of approximately 1 microM for each inhibitor. Furthermore, these bromoestrogens bind poorly to estrogen receptors in MCF-7 cells and do not alter cell growth. Thus, in MCF-7 mammary cell cultures, metabolism of estradiol occurs to only a minor degree, and it is unlikely that the levels of catechol estrogens would reach physiologically relevant concentrations in the intact breast cancer cells.(ABSTRACT TRUNCATED AT 250 WORDS)