Effects of pinostrobin on estrogen metabolism and estrogen receptor transactivation.

The interaction between the estrogen receptor and 5-hydroxy-7-methoxyflavanone (pinostrobin) was studied in the presence or absence of estradiol or dehydroepiandrosterone sulfate (DHEAS), respectively, using a stably transfected human breast cancer cell line (MVLN). We also evaluated its action on the proliferation in estrogen-dependent (MCF-7) human breast cancer cells in the same conditions than the estrogen receptor assay. On the other hand pinostrobin was evaluated for their effects on the human placental aromatase, 3beta-hydroxysteroid dehydrogenase Delta(4)/Delta(5) isomerase and 17beta-hydroxysteroid dehydrogenase activities. Pinostrobin did not possess antiestrogenic activity but presented anti-aromatase activity and decreased the growth of MCF-7 cells induced by DHEAS and E(2). This study provides particularly evidence of the potential biological interest of pinostrobin among the flavonoids.

Aromatase and 17beta-hydroxysteroid dehydrogenase inhibition by flavonoids.

A method for estimating in the same assay both aromatase and 17beta-hydroxysteroid dehydrogenase activities in human placental microsomes using radiolabelled [1,2,6,7-3H]4-androstene-3,17-dione was proposed. In this assay, estrone (E1) and estradiol (E2) produced were separated by HPLC and estimated using a radioactive flow detector. Using this method, the inhibitory effect of various flavonoids, including flavone, flavanone and isoflavone, on the human placental aromatase and 17beta-hydroxysteroid dehydrogenase was studied. Flavonoids were shown to be potent inhibitors of both aromatase and 17beta-hydroxysteroid dehydrogenase activities. We found that 7-hydroxyflavone and apigenin are the most effective aromatase and 17beta-hydroxysteroid dehydrogenase inhibitors, respectively. Experiments showed that a hydroxyl group in position 7 was essential for anti-17beta-hydroxysteroid dehydrogenase activity. However, flavonoids with 7-methoxy or 8-hydroxyl groups on the A ring showed only anti-aromatase activity. Structure-activity relationships were discussed.

Estrogenic and antiproliferative activities on MCF-7 human breast cancer cells by flavonoids.

The interaction between the estrogen receptor and a variety of flavonoids was studied in the presence or absence of estradiol using a stably-transfected human breast cancer cell line (MVLN). On the other hand, flavonoids were evaluated for their effects on proliferation in estrogen-dependent (MCF-7) and independent (MDA-MB231) human breast cancer cells. We established a relationship structure-activity and determined regions and/or substituents essential for estrogenic or antiestrogenic activities. In contrast, we did not find the same relationship for cell proliferation. Among all flavonoids used, only 7-methoxyflavanone and 7,8-dihydroxyflavone at high concentrations (50 microM) possess antiestrogenic and antiproliferative activities. These results suggest that two hydroxyls (in positions 7 and 8) or 7-methoxy substituents are essential for the antiestrogenic activity of flavonoids. However, it seems that flavonoids at high concentrations exert their antiproliferative activity through other estrogen receptor-independent mechanisms.

Dexamethasone in resting and exercising men. I. Effects on bioenergetics, minerals, and related hormones.

A placebo and a low and a high dose of dexamethasone (Dex) were administered for 4.5 days, at 3-wk intervals, to 24 healthy men, following a double-blind, random-order, crossover procedure. After the last dose the subjects performed a maximal cycling exercise, during which respiratory exchanges, electrocardiogram, and blood pressures were monitored. Blood was sampled just before and after each exercise bout. Dex showed no significant effect on fitness, sleep, exhaustion during exercise, maximal O(2) consumption, ventilatory threshold, maximal blood lactate, or rest and exercise blood pressures. On the contrary, both doses of Dex significantly decreased heart rate at rest and during maximal exercise. Blood glucose at rest was higher after both doses of Dex than after placebo; the opposite was found during exercise. Blood levels of ACTH, beta-endorphin, cortisol, and cortisol-binding globulin were lowered by Dex at rest and after exercise. Dex stimulated the increase in atrial natriuretic factor during exercise and lowered rest and postexercise aldosterone. Finally, no difference between "fit or trained" and "less fit or untrained" subjects could be found with respect to Dex effects.

Effects of phytoestrogens on aromatase, 3beta and 17beta-hydroxysteroid dehydrogenase activities and human breast cancer cells.

Isoflavones and others phytoestrogens have been suggested to be anticarcinogenic. Anti-aromatase, antiestrogenic or antiproliferative actions of these compounds have been postulated and related to the observation that there is a reduced incidence of breast cancer associated with diet. In this study, we explored some mechanisms by which they can exert cancer-preventive effects. Phytoestrogens were tested for estimating anti-aromatase, anti-3beta-hydroxysteroid dehydrogenase delta5/delta4 isomerase (3beta-HSD) and anti-17beta-hydroxysteroid dehydrogenase (17beta-HSD) activities in human placental microsomes. We found that isoflavonoids and compounds which presented the phenolic B ring in the 3 position on the pyran ring preferentially inhibited 3beta-HSD and/or 17beta-HSD activities than aromatase activity. We also evaluated their interactions with the estrogen receptor using a stably transfected human breast cancer cell line (MVLN). On the other hand phytoestrogens were evaluated for their effects on the proliferation in estrogen-dependent (MCF-7) and independent (MDA-MB231) human breast cancer cells. We established a relationship structure-activity and determined regions or/and substituents essential for these different activities. However, at high concentrations it seems that some phytoestrogens exert their protection against breast cancer through other estrogen-independent mechanisms.

Chalcones are potent inhibitors of aromatase and 17beta-hydroxysteroid dehydrogenase activities.

Chalcones were tested for estimating anti-aromatase, anti-3beta-hydroxysteroid dehydrogenase delta5/delta4 isomerase (3beta-HSD) and anti-17beta-hydroxysteroid dehydrogenase (17beta-HSD) activities in human placental microsomes. In the present study, we have demonstrated for the first time that chalcones are potent inhibitors of aromatase and 17beta-hydroxysteroid dehydrogenase activities: these enzymes being considered as important targets in the metabolic pathways of human mammary hormone-dependent cells. Our results showed that naringenin chalcone and 4-hydroxychalcone were the most effective aromatase and 17beta-hydroxysteroid dehydrogenase inhibitors with IC50 values of 2.6 and 16 microM respectively. In addition, inhibitory effects of some flavones and flavanones were compared to those of the corresponding chalcones. A structure-activity relationship was established and regions or/and substituents essential for these inhibitory activities were determined.

Estrogenic/antiestrogenic and scavenging properties of (E)- and (Z)-resveratrol.

Resveratrol, natural compound found in grapes and wine, has been reported to have a variety of health benefit properties. Based on the structural similarity to the synthetic estrogen diethylstilbestrol, we investigated estrogenic/antiestrogenic effects on human breast cancer cell lines, MCF-7 and MVLN, and scavenging properties using DPPH of both (E)- and (Z)-isomers. Both isomers increased the in vitro growth of MCF-7 cell lines at medium concentrations (10 and 25 microM) whereas the low concentrations (0.1 and 1 microM) had no effect and the high concentration (50 microM) decreased the cell growth and was cytotoxic. The 25 microM (E)-isomer alone was able to reduced the proliferation induced by the estradiol. Low concentrations of (E)- and (Z)-resveratrol (0.1 and 1 microM) and medium concentration 10 microM (Z)-resveratrol did not interfere with the estrogen receptor. In contrast, medium concentrations of (E)-resveratrol (10 and 25 microM) and (Z)-resveratrol (25 microM) functioned as superagonists of estradiol. Whatever the model used, MCF-7 or MVLN cell lines, (Z)-resveratrol was less effective than (E)-resveratrol. Extinction of DPPH and Fe(III) reduction experiments showed that both isomers of resveratrol could act as free radicals scavengers or pro-oxidant compounds. The properties of low concentrations of resveratrol raise the possibility that structure-function studies could lead to the development of more selective estrogen receptor agonists and antagonists, which could be useful as a therapeutic agent.

Simultaneous radioimmunoassay of 5alpha-androstane-3alpha, 17beta-diol and 5alpha-androstane-3beta, 17beta-diol unconjugated and conjugated in human serum.

The simultaneous determinations of both 3alpha and 3beta epimers of 5alpha-androstane-3,17beta-diol as their glucuronides, sulfates and in their unconjugated forms are described. The diol estimation is carried out by radioimmunoassay with two specific immune sera after purification of the serum by use of chromatography on Sephadex LH-20. The values obtained (mean +/- S.D.) in pg/ml for the unconjugated 3alpha and 3beta epimers were, respectively, 267 +/- 67 and 816 +/- 76 for men; 114 +/- 33 and 515 +/- 177 for women; 142+/- 77 and 779 +/- 200 for hirsute women. Among the conjugates, the most important were the sulfoconjugates, their rates being, respectively (men +/- S.D. in ng/ml 41.6 +/- 9.5 and 103+/- 40 for men; 12.4 +/- 3.1 and 51.2 +/- 14.9 for women and 36 +/- 22 and 72 +/- 36 for hirsute women. Differences in the conjugation of both epimers were also noticed.

Aromatase in synovial cells from postmenopausal women.

Peripheral aromatization of androgens exerts estrogenic actions in many tissues. In this study, osteoarthritis synoviocytes were examined to clarify the possible action of adrenal androgen on synovial cell. Synoviocytes from postmenopausal women are able to express aromatase mRNA. By sequence analysis, the PCR fragment (485 bp) was determined to be 100% identical to that of human placental aromatase cDNA. Moreover, this study demonstrates that adrenal androgen, androstenedione, is converted to estrone (E(1)) and estradiol (E(2)) in synoviocytes by aromatase which is positively regulated by glucocorticoids such as dexamethasone. E(2) production reduced significantly IL-6 secretion. These data provide preliminary evidence that in situ estrogen production by synoviocytes may have a role in OA susceptibility. However the role of E(2) in OA is not clear and remains to be determined.

Conversion of dehydroepiandrosterone sulfate at physiological plasma concentration into estrogens in MCF-7 cells.

Metabolism of dehydroepiandrosterone (DHEA), its sulfate (DHEAS), and androstene-3,17-dione (delta(4)) was performed at their physiological plasma concentrations in MCF-7 cell cultures (1 microM, 10 and 2 nM, respectively). Final metabolic products of these steroids were separated by HPLC-radioactive flow detection and identified by LC/MS or MS/MS. Typical and specific mass fragmentation spectra identified the presence of estrone (E(1)), 17beta-estradiol (E(2)), delta(4), DHEA, 5-androstene-3beta,17beta-diol (delta(5)), and testosterone as principal DHEAS metabolites. Other steroids, such as androstenedione, androsterone, and DHEA fatty acid esters at very low concentrations (from pM to nM), were also obtained after steroid incubation. This highly specific method allowed us to conclude whether a metabolite and enzymatic activity of interest were present in MCF-7 cells or not. We also showed that DHEAS at its physiological plasma concentration may be converted into estrogens and estrogen-like compounds in breast cancer cells. The estrogenic action of DHEAS on breast cancer cells was also measured by bioluminescence in a stably transfected human breast cancer MCF-7 cell line with a reporter gene that allowed expression of the firefly luciferase enzyme under the control of an estrogen regulatory element.

C19 steroids estrogenic activity in human breast cancer cell lines: importance of dehydroepiandrosterone sulfate at physiological plasma concentration.

The estrogenic action of C19 steroids on breast cancer cells was measured by bioluminescence in stably transfected human breast cancer MCF-7 and T47D cell lines with a reporter gene that allows expression of the firefly luciferase enzyme under control of an estrogen regulatory element. The "estrogenic activity" of C19 steroids, such as dehydroepiandrosterone (DHEA) and its sulfate (DHEAS), androst-5-en-3 beta,17 beta-diol, androst-4-en-3,17-dione, dihydrotestosterone, testosterone, and 5 alpha-androstan-3 beta,17 beta-diol was studied. This showed that DHEAS, at concentration observed in physiological conditions (10(-6) M), had a high "estrogen-like effect" in MCF-7 and T47D cell lines. Other C19 steroids, at physiological plasma concentration, alone or together did not have any significant effect on the luciferase activity. Moreover aminoglutethimide, an inhibitor of the aromatase enzyme, in the presence of C19 steroids, partially decreased the luciferase activity. These results suggest that MCF-7 and T47D cell lines could convert DHEAS to estrogen-like compounds by different enzymatic systems.

Dehydroepiandrosterone sulfate estrogenic action at its physiological plasma concentration in human breast cancer cell lines.

Estrogen receptor activation in hormone dependent cells (MCF-7 and T47D) and hormone independent cells (MDA-MB-231) was measured by bioluminescence in stably transfected human breast cancer MCF-7, T47D and MDA-MB-231 cell lines with a reporter gene which allows expression of the firefly luciferase enzyme under the control of an estrogen regulatory element. Estrogen receptor activation by estrogens and steroid sulfates such as estrone 3-sulfate (E1S), estradiol 3-sulfate (E2S), estriol 3-sulfate (E3S) and dehydroepiandrosterone sulfate (DHEAS) were studied and compared in these cell lines. DHEAS, at a physiological plasma concentration only (1 microM), had a high "estrogen-like" effect in MCF-7 and T47D cells. In contrast, estrogen sulfates did not have any effect on the luciferase activity at their physiological plasma concentration. These results suggest that MCF-7 and T47D cells could convert DHEAS to estrogens by aromatase and/or to estrogen-like compounds by other enzymatic systems. This is the first demonstrahon of the steroid sulfates action through their metabolites on the estrogen receptor. Therefore, at the concentrations observed in physiological conditions, DHEAS could contribute to the pool of compounds having estrogenic activities in human breast cancer hormone dependent cell lines.

Use of a biotinyl-estradiol derivative to demonstrate estradiol-membrane binding sites on adherent human breast cancer MCF-7 cells.

A biotinyl-derivative of 17 beta-estradiol has been used to demonstrate a site of recognition and binding of estradiol located on the plasma membrane of human breast cancer MCF-7 cells by using the biotin/avidin-FITC system. The specificity of this binding has been shown by a displacement of the fluorescent label by 17 beta-estradiol. No displacement was observed when testosterone was added. Quantification of this phenomenon has been shown by laser scanning cytometry while preserving the cells adhesiveness to their growth support as well as their membrane integrity. An analysis by confocal laser scanning microscopy suggested that the fluorescence distribution on MCF-7 cells treated with estradiol-biotin was on the cell periphery. The results obtained are in favour of the recognition and binding site of 17 beta-estradiol located on the plasma membrane of MCF-7 cells and they would indicate that the biological activity of estradiol, among others, could be initiated by an interaction with the membrane.

Chalcones: structural requirements for antioxidant, estrogenic and antiproliferative activities.

Flavonoids are largely studied for their biological properties and particularly for their scavenging and antioxidant activities. In the present study, we first evaluated the antioxidant and the estrogenic actions of chalcones, then we tested their effects on MCF-7 cell proliferation. Chalcones are unique in the flavonoids family in lacking a heterocyclic C ring. We tested substituted chalcones with different numbers and different positions of the hydroxy groups: 2'-hydroxychalcone, 4'-hydroxychalcone, 4-hydroxychalcone, 2',4-dihydroxychalcone, isoliquiritigenin, 2',4'-dihydroxychalcone, phloretin and naringenin chalcone. For the antioxidant tests we established the importance of the alpha-beta double bond and the 6'-hydroxy group. The establishment of the structure-activity relationship for the estrogenic properties showed a correlation between the antioxidant and the estrogenic properties. The importance of conformation and hydroxy group positions observed for chalcones, having antioxidant and estrogenic properties, was also observed on MCF-7 cell growth with the same structure-activity relationship. The role of electron and hydrogen transfer in the correlation between these three biological activities was discussed.

[Effect of beta-adrenergic receptors blockade on auricular natriuretic factor, aldosterone and renine blood activity during physical exercise]. / Effet du blocage des récepteurs beta-adrénergiques sur le facteur natriurétique auriculaire, l'aldostérone et l'activité rénine plasmatique, au cours d'un exercise physique.

Physical exercise stimulates the renin-angiotensin-aldosterone system. However several factors affect the control of mineralocorticoid secretion. In this study, eight healthy volunteers performed maximal exercise on cycle ergometer after being pretreated for 3 days with placebo (P) or with a non selective beta-blocker (B) (pindolol 15 mg/day). Plasma reinin activity (PRA), aldosterone (ALD), atrial natriuretic factor (ANF), and kalemia (K+) were measured at rest (R) and during exercise until exhaustion (E). (table; see text) These results confirm the role of beta-adrenoceptor activation in the increased PRA during exercise. It appears an exercise-induced increase in plasma ANF which was more elevated in subjects treated with pindolol, but which had no inhibitory effect on ALD secretion in theses conditions. K+ rose during exercise and this hyperkalemia tended to be higher with a beta-blocker. It is suggested that K+ elevation counterbalance both PRA decrease and ANF increase to be responsible for the absence of change in plasma ALD during beta-blockade.

[Atrial natriuretic factor: one of the mechanisms of action of the phlebology bath at Barbotan]. / Le facteur natriurétique atrial: un des mécanismes d'action du bain de phlébologie à Barbotan.

UNLABELLED: The effect of thermal baths on oedema of the lower limbs might be explained by physical mechanisms of hydrostatic pressure resulting from the use of a deep bath and a centripetal underwater jet, by which the veins and lymph ducts are drained every day. The purpose of this experiment is to demonstrate the existence of hormonal mechanisms which would account for the diuretic effect of thermal baths. One of the effects observed with hydrotherapy is the physiological diuresis that follows each bath, this diuresis would appear to depend at least in part on the atrial natriuretic factor (ANF). The criteria by which assessment can be made essentially biological: ANF level and its biological effects on blood and urine; aldosterone level; plasma renin activity (PRA); creatinine clearance; hematocrit; proteinemia; and blood and urine electrolyte balance. The inclusion criteria are: subjects selected at random and willing cooperate. The criteria for exclusion are disease states which modify ANF kinesis: congestive heart failure, cardiac rhythm disorders, decompensated cirrhosis of liver, obesity, treatment antihypertensive drugs. METHODS: Thirty patients were put through the same experimental sequence, as follows: emptying of the bladder and ingestion of 200 cc of water; seated rest fort 30 mn, after which (to): blood sample; urine sample; ingestion of 200 cc of water; deep bath for 20 mn, i.e. the basic hydro treatment in phlebology at Barbotan. The deep bath is specific to Barbotan and the patient is subjected to maximum immersion in water at a mesothermal temperature of 34.5 degrees C, followed by (t1): blood and urine sample; ingestion of 200 cc of water; supine rest for 90 mn, followed by (t2): blood and urine sample. RESULTS: Data from twenty-eight patients were usable. In this protocol, we use variance analysis with repeated measurements and a 95% confidence limit. The mean value of the principal parameters studies are set out in the following table; these value are accompanied by the degree of significance of the modification at (t1) and (t2). Our experimentation with thirty patients showed that the big thermal bath at Barbotan produces a highly significant increase in ANF secretion, resulting in the diuresis observed after the use of the bath. The antagonist effect of AFN on the renin--angiotensin-aldosterone system was corroborated: we found decreased aldosterone, PRA and creatinine clearance, and increased diuresis and natriuresis. The renal and cardiovascular effects observed after extended immersion in the Barbotan bath (increased diuresis, tachycardia and hypotension, transitory venous vasoplegia and ephemeral vasodilatation of the surface capillaries) are the result of increased ANF secretion. [formula: see text] Supine rest immediately after the bath is essential. This sustains the enhanced ANF and thus reinforces its renal effects, while reducing adverse cardiovascular effects such as the orthostatic hypotension and venous vasoplegia that are normally observed after use of the bath. Moreover, by reducing venular and lymphatic pressure, clinostatism facilitate interstitial to intravascular tissue fluid exchanges and thus helps to drain oedema from the legs. It is striking to note that the hydrotherapy prescribed at Barbotan les Thermes has always included the three most potent factors for ANF release: deep immersion in the big bath, immediate supine rest, and walking. Physiological diuresis has thus been induced empirically as an essential part of the treatment of lower limb phlebopathies.

[A liquid chromatography technique for routine determination of urinary estriol in human pregnancy (author's transl)]. / Détermination de l'estriol urinaire durant la grossesse par chromatographie liquide haute performance.

A liquid chromatography procedure for routine determination of urinary estriol concentrations is described. After enzymic hydrolysis of a 1 ml sample, automatic extraction with chloroform/ethyl acetate, purification by chromatography on a disposable "minicolumn" of AG 1-X2, urinary estrogens are separated with normal phase column and isocratic elution with isooctane/ethyl acetate/methanol/water and the optical density at 280 nm is measured. The specificity is assessed by comparison of liquid chromatography with fluorometry and gas liquid chromatography. The technique is simple, rapid and quite suitable for routine determination.

[Analysis of 17-oxosteroids by gas liquid chromatography after purification of the urinary extract by Girard's T reagent (author's transl)]. / Analyse des 17-oxostéroïdes par chromatographie gaz-liquide après purification de l'extrait urinaire par le réactif T de Girard.

The authors describe a method of fractionation of urinary 17-oxosteroids after purification of the urinary extract by Girard's T reagent. After solvolysis, enzyme hydrolysis and purification by Girard's T reagent, the 17-oxosteroids were estimated in the form of trimethyl silyl ethers by gas liquid chromatographye on OV 225 using cholesterol acetate for standardisation. The improvement in the quality of the fraction using this technic, the analytical characteristics of which are studied in this work, is of definite interest, in particular after stimulation by ACTH, HCG, metopirone and in the case of pigmentation of the urine.

[Assay of plasma dehydroepiandrosterone sulfate by gas chromatography with electron-capture detection]. / Dosage du sulfate de déhydro-épiandrostérone plasmatique par chromatographie en phase gazeuse avec détection par capture d'électrons.

A sensitive and specific method for the determination of plasma S-DHA by gas chromatography (GC), with fused silica capillary column (stationary phase SE 54) and electron capture, after solvolysis of the sample and purification by high performance liquid chromatography (HPLC) is described. The interest of the new internal standard (5 alpha androst-9(11)-ene-3 beta ol-17-one) for the determination of the DHA by GC is shown. The analytical characteristics of this method as well as a comparative study of the values obtained by this method and by radioimmuno-assay are described.

[Excretion of androsterone and etiocholanolone related to plasma testosterone binding in three male pseudohermaphrodites (author's transl)]. / Influence de la liaison de la testostérone plasmatique sur l'excrétion urinaire de l'androstérone et de l'étiocholanolone dans trois cas de testicule féminisant.

The authors studied the relation of plasma testosterone and Sex-Hormone-Binding-Globulin levels to the urinary excretion of androsterone (A) and etiocholanolone (E) in three patients with the syndrome of testicular feminization. They found levels of SHBG in the upper female range, testosterone concentration in the normal male range and urinary excretion rate of A and E in the normal female range.