17α-ethinylestradiol cometabolism by bacteria degrading estrone, 17β-estradiol and estriol

17alpha-ethinylestradiol (EE2), the active compound of the contraceptive pill, is a recalcitrant estrogen, which is encountered at ng/l levels in wastewater treatment plant (WWTP) effluents and rivers and can cause feminization of aquatic organisms. The aim of this study was to isolate micro-organisms that could remove such low EE2 concentrations. In this study, six bacterial strains were isolated from compost that cometabolize EE2 when metabolizing estrone (E1), 17beta-estradiol (E2) and estriol (E3). The strains belong to the alpha, beta and gamma-Proteobacteria. All six strains metabolize E2 over E1, at mug/l to ng/l concentrations. In 4 days, initial concentrations of 0.5 mug E2/l and 0.6 mug EE2/l were degraded to 1.8 +/- 0.4 ng E2/l and 85 +/- 16 ng EE2/l, respectively. No other metabolites besides E1, E2, E3 or EE2 were detected, suggesting that total degradation and cleavage of the aromatic ring occurred. This is the first study describing that bacteria able to metabolize E2, can subsequently cometabolize EE2 at low mug/l levels.     

Solid-state NMR analysis of steroidal conformation of 17α- and 17β-estradiol in the absence and presence of lipid environment

Solid-state {(1)H}(13)C cross-polarization/magic angle spinning (CP/MAS) NMR spectroscopy has been applied to 17β-estradiol (E2) and 17α-estradiol (E2α), to analyze the steroidal ring conformations of the two isomers in the absence and presence of lipids at the atomic level. In the absence of lipid, the high-resolution (13)C NMR signals of E2 in a powdered form show only singlet patterns, suggesting a single ring conformation. In contrast, the (13)C signals of E2α reveal multiplet patterns with splittings of 20-300Hz, implying multiple ring conformations. In the presence of a mimic of the lipid environment, made by mixing 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC) in a molar ratio 3:1, E2 and E2α revealed multiplet patterns different from those seen in the absence of lipids, indicating that the two isomers adopt multiple conformations in the lipid environment. In this work, on the basis of chemical shift isotropy and anisotropy analysis, we demonstrated that E2 and E2α prefer to adopt multiple steroidal ring conformations in the presence of a lipid environment, distinct from that observed in solution phase and powdered form.     

Comparative studies of the 17β-estradiol receptors in rabbit liver,kidney and uterus

The cytosol 17β-estradiol receptors from rabbit kidney, liver and uterus, compared under identical experimental conditions, were similar in terms of their pH-activity profiles, dependence on incubation temperature, sensitivity to sulfhydryl reagents and steroid specificity. 17β-[³H]-Estradiol binding was saturable with all three tissues, having an apparent dissociation constant of 4 × 10⁻¹⁰ M. The binding of 17β-[³H]-estradiol in kidney, liver and uterus was inhibited by estrogens, including estrogen conjugates, but not by testosterone, progesterone or cortisol. The 17β-estradiol receptors of liver, kidney and uterus exhibited significant differences with respect to their Chromatographie behaviour on heparinSepharose. Furthermore, a comparison of their sucrose density gradient centrifugation patterns showed that the 17β-[³H]-estradiol-receptor complex of liver and kidney sedimented at 3-4 S in both low and high ionic strength media, while the uterine receptor sedimented at 7–8 S in low ionic strength media and at 4–5 S in high ionic strength media. When the liver and uterine cytosol fractions were combined the uterine receptor was altered and sedimented at 3–4 S in low ionic strength media.     

Peripartal plasma concentrations of 15-ketodihydro-PGF2α, cortisol, progesterone and 17-β-estradiol in Martina Franca jennies

The transition from intra- to extrauterine environment represents a very delicate phase, in which the successful coordination of maturation is strictly connected with several hormonal changes during the last weeks of gestation and at parturition. While the peripartal endocrinology in the mare has been deeply investigated, the peripartal hormonal changes in the jenny need further evaluation. The aim of this study is to evaluate the mean 15-ketodihydro-PGF_2α (PGFM), cortisol (C), progesterone (P4), and 17β-estradiol (E2) levels during the peripartal period in this species. Ten Martina Franca jennies, with normal gestational length and parturition, were enrolled. From each jenny, blood was collected twice a day from 10 d before to 7 d after parturition and from the plasma obtained PGFM, C, P4 and E2 were analyzed by RIA. Higher, constant PGFM concentrations were observed in the pre-foaling days compared to the decreasing levels detected the days after delivery, as previously observed in the mare. During the whole period of observation no significant differences in plasma C levels were detected. In contrast to the mare, P4 has always been detectable and the highest level found at −2.5 days was significantly different compared to samples obtained between −10 and −4.5 days and between 1.5 and 7 days after foaling. Finally, E2 showed higher concentrations before foaling, with the highest values between −3 and −1.5 days, decreasing only one day before foaling. A positive correlation was found between PGFM and P4, during the last 4 days of gestation, while a positive correlation between PGFM and E2 was observed during the prepartum. Despite some similarities with the mare exist, differences have been found in P4 and E2 profiles, underlining once more the differences in the physiology of this two species.     

Apoptotic and anti-proliferative effects of 17β-estradiol and 17β-estradiol-like compounds in the Hep3B cell line

Since there is evidence for estrogen and estrogen-like compounds to have beneficial effect on the pathogenesis of hepatocellular carcinoma (HCC), this study was designed to investigative the apoptotic and anti-proliferative effects of these compounds on the human hepatoma Hep3B cell line. The Hep3B cells were treated with 17β-estradiol (E2), diethylstilbestrol (DES), tamoxifen, and genistein. After treatments of these compounds at the concentration of 10^-6 or 10^-8 M, the Hep3B cells were demonstrated to have significant DNA fragmentation, nucleus condensation, cytochrome-c leaking from the mitochondria and caspase-3 activation by DAPI and Western blotting. The cells were also observed to have declined proliferative potential by MTT assay, arrested cell cycle by flow-cytometry measurements. However, the cytochrome-c leaking from the mitochondria induced by E2 and E2-like compounds was blocked totally by ICI 182,780 treatment. These finding suggest that estrogen and the estrogen-like compounds may induce anti-proliferative and apoptotic effects in Hep3B cells, and the E2 and the E2-like compounds mediated apoptotic effect was estrogen receptor dependent. Among the drugs tested, E2, E2 agonists (DES and genistein) and partial antagonist (tamoxifen), all showed the stronger anti-tumor potential. The last two authors, Wei-Wen Kuo and Chih-Yang Huang, share equal contribution.     

Effects of 17β-estradiol and bisphenol A on the formation of reproductive organs in planarians

Planarians have a remarkable capacity for regeneration after ablation, and they reproduce asexually by fission. However, some planarians can also reproduce and maintain their sexual organs. During the regenerative process, their existing sexual organs degenerate and new ones develop. However, little is known about hormonal regulation during the development of reproductive organs in planarians. In this study, we investigated the effects of 17β-estradiol (a steroid) and bisphenol A (an endocrine disrupter) on the formation of sexual organs in the hermaphroditic planarian Dugesia ryukyuensis. Under control conditions, all worm tissues regenerated into sexual planarians with sexual organs within 4 weeks after ablation. However, in the presence of bisphenol A or 17β-estradiol, although they apparently regenerated into sexual planarians, the yolk glands, which are one of the female sexual organs, failed to regenerate even 7 weeks after ablation. These data suggest that planarians have a steroid hormone system, which plays a key role in the formation and maturation of sexual organs.     

Simultaneous radioimmunoassay of testosterone,dihydrotestosterone, 5α-androstane-3α, 17β-diol and 5α-androstane-3β, 17β-diol in the plasma of adult male rats

A single thin layer chromatography and three antibodies were used for the specific radioimmunoassay of four androgens in pooled rat plasma (Sprague-Dawley adult males). The following values were found (pg/ml ± SD). Testosterone : 3, 138 ± 173; dihydrotestosterone : 374 ± 20; 5α-androstane-3α 17β-diol : 284 ± 24; 5α-androstane-3β, 17β-diol : 223 ± 11.     

Gender dependent effects of testosterone and 17β-estradiol on bone growth and modelling in young mice

This study examined the effects of estrogen (17β-estradiol) and testosterone on the growth of long bones in male and female mice, with and without gonadectomy. Weight and nose-to-tail length were determined at 3 weeks of age at time of gonadectomy, 7 days later at the onset of hormone therapy, and throughout the treatment period. Gonadectomized mice exhibited an initial weight gain during the pretreatment period but length was unaffected. Hormone treatment altered weight gain in surgical and intact animals in a gender- and hormone-dependent manner. Estradiol enhanced weight gain in intact mice, but inhibited weight gain in ovariectomized mice. Lower doses of estradiol increased weight gain in orchiectomized mice at early time points. Testosterone increased weight in intact females and males, but not in gonadectomized mice. Estradiol increased nose-to-tail length in intact females at early time points, but inhibited length in ovariectomized females at later times, and it decreased length in intact males. Testosterone increased length in normal females and normal males. Serum Ca was unaffected by ovariectomy, but orchiectomy resulted in decreased levels. Estradiol reduced serum Ca in gonadectomized animals; serum Ca was increased by estradiol treatment in intact females. Changes in tibial bone weight, ash weight and mineral composition, and relative sizes of epiphyseal and metaphyseal bone were gender-, gonadectomy- and hormone-specific. Bone weight was greater in ovariectomized mice. Ash weight per bone was comparable, but there was an increase in Ca and P content with ovariectomy. Estradiol increased bone weight, ash content, and bone Ca and P in ovariectomized and intact females. Orchiectomy alone did not alter bone weight, ash content, or Ca and P, but orchiectomized mice were sensitive to estradiol; all parameters were increased in the orchiectomized animals treated with estradiol. Analysis of the ash content and Ca and P per mg bone, rather than per bone, demonstrated estradiol and testosterone alter net bone formation, but not the amount of mineral per unit bone. Ovariectomy increased hypertrophic cartilage. While estradiol did not alter tibial area in ovariectomized mice, it caused an increase in intact females. The total amount of growth plate cartilage in ovariectomized animals was decreased by estradiol to levels typical of intact animals due to a greater decrease in the hypertrophic cartilage in the ovariectomized mice, as well as a greater increase in metaphyseal bone area. Testosterone had no effect on these parameters in the females. Orchiectomy decreased the amount of growth plate cartilage, but increased the hypertrophic zone. Estradiol increased growth plate cartilage in intact male mice, but decreased it in orchiectomized mice. This difference was also seen in the hypertrophic zone. Total growth plate cartilage and hypertrophic cartilage were increased by testosterone in intact males, whereas metaphyseal and epiphyseal bone area were decreased. The results show for the first time that there is a gender-specific response in both male and female mice to both estradiol and testosterone, whether or not the animals have been gonadectomized. For many parameters, orchiectomized mice behave like females in response to both sex steroids, indicating that the male gonad is needed for mouse bone to exhibit the male phenotypic response to estradiol and testosterone.     

Effects of 17β-estradiol and antioxidant administration on oxidative stress and insulin resistance in ovariectomized rats

The prevalence of insulin resistance syndrome increases during menopause with the overproduction of reactive oxygen species and impairment of the free radical scavenger function. Therefore, we investigated the effects of 17β-estradiol (E(2)) and vitamin E, as an antioxidant, on lipid peroxidation and antioxidant levels in the brain cortex and liver of ovariectomized rats as well as on insulin resistance in those rats. Forty female Sprague-Dawley rats, 3?months of age and weighing 231.5?± 9.4 g, were divided into 4 groups: sham, ovariectomized (OVX), OVX treated with E(2) (40 μg/kg subcutaneously), and OVX treated with E(2) and vitamin E (100?mg/kg intraperitoneally). The 4 groups received the appropriate treatment every day for 8?weeks. Levels of glutathione, glutathione peroxidase, superoxide dismutase , catalase, and malondialdehyde in the brain cortex and liver of ovariectomized rats were measured. Also, fasting plasma insulin, glucose, and homeostatis model assessment of insulin resistance (HOMA-IR) were determined. Malondialdehyde increased and antioxidants (glutathione, glutathione peroxidase, catalase, superoxide dismutase) decreased in the brain cortex and liver of OVX rats. Also, fasting glucose, insulin, and HOMA-IR increased in OVX rats. E(2) and E(2) plus vitamin E decreased malondialdehyde and increased antioxidants in the brain cortex and liver of OVX rats. Moreover, they decreased fasting glucose, insulin, and HOMA-IR in ovariectomized rats. This study demonstrates that E(2) and E(2) plus vitamin E supplementation to OVX rats may improve insulin resistance, strengthen the antioxidant system, and reduce lipid peroxidation.     

Stimulation of phosphatidylcholine synthesis by 17β-estradiol in fetal rabbit lung

We investigated the mechanism by which estrogen stimulates pulmonary surfactant production in the fetal rabbit. Maternal administration of 17β-estradiol (5–75 μg) on day 25 of gestation resulted in a greater than twofold increase in the rate of choline incoporation into phosphatidylcholine in fetal lung slices on day 26 (full term = 31 days). Estrogen administration increased the activity of fetal lung cholinephosphate cytidylyltransferase by 62%. It had no effect on the liver enzyme. When assayed in the presence of phosphatidylglycerol fetal lung cholinephosphate cytidylyltransferase activity was increased 4.6-fold but it was not influenced by estrogen under these conditions. These findings suggest that estrogen stimulates cholinephosphate cytidyltransferase by increasing the activity of existing enzyme (possibly by increasing the amount of phosphatidylglycerol or other acidic phospholipid in the tissue) rather than by increasing the amount of enzyme-protein. Stimulation of fetal lung cholinephosphate cytidylyltransferase by estrogen as well as by glucocorticoids (Rooney, S.A., Gobran, L.I., Marino, P.A., Maniscalco, W.M., and Gross, I. (1979) Biochim. Biophys, Acta 572, 64–76) suggest that this enzyme may be rate-regulatory in the de novo biosynthesis of phosphatidylcholine.Estrogen administration also resulted in a 26% increase in the activity of pulmonary lysolecithin acyltransferase, an enzyme involved in the synthesis of disaturated, surface-active phosphatidylcholine. Lung choline kinase was slightly decreased following estrogen treatment bu ethanolaminephosphate cytidylyltransferase, cholinephosphotransferase, phosphatidate phosphatase and lysolecithin : lysolecithin acyltransferase were unaffected.     

Ovariectomy and 17β-estradiol replacement in rats and mice: a visual demonstration

Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are commonly ovariectomized in order to ablate the rapidly cycling hormone production, replacing the 17β-estradiol exogenously. There is, however, lack of consensus regarding how the hormone should be administered to obtain physiological serum concentrations. This is crucial since the 17β-estradiol level/administration method profoundly influences the experimental results. We have in a series of studies characterized the different modes of 17β-estradiol administration, finding that subcutaneous silastic capsules and per-oral nut-cream Nutella are superior to commercially available slow-release pellets (produced by the company Innovative Research of America) and daily injections in terms of producing physiological serum concentrations of 17β-estradiol. Amongst the advantages of the nut-cream method, that previously has been used for buprenorphine administration, is that when used for estrogen administration it resembles peroral hormone replacement therapy and is non-invasive. The subcutaneous silastic capsules are convenient and produce the most stable serum concentrations. This video article contains step-by-step demonstrations of ovariectomy and 17β-estradiol hormone replacement by silastic capsules and peroral Nutella in rats and mice, followed by a discussion of important aspects of the administration procedures.     

17β-estradiol attenuates exercise-induced neutrophil infiltration in men

17β-estradiol (E2) attenuates exercise-induced muscle damage and inflammation in some models. Eighteen men completed 150 eccentric contractions after random assignment to placebo (Control group) or E2 supplementation (Experimental group). Muscle biopsies and blood samples were collected at baseline, following 8-day supplementation and 3 h and 48 h after exercise. Blood samples were analyzed for sex hormone concentration, creatine kinase (CK) activity and total antioxidant capacity. The mRNA content of genes involved in lipid and cholesterol homeostasis [forkhead box O1 (FOXO1), caveolin 1, and sterol regulatory element binding protein-2 (SREBP2)] and antioxidant defense (SOD1 and -2) were measured by RT-PCR. Immunohistochemistry was used to quantify muscle neutrophil (myeloperoxidase) and macrophage (CD68) content. Serum E2 concentration increased 2.5-fold with supplementation (P < 0.001), attenuating neutrophil infiltration at 3 h (P < 0.05) and 48 h (P < 0.001), and the induction of SOD1 at 48 h (P = 0.02). Macrophage density at 48 h (P < 0.05) and SOD2 mRNA at 3 h (P = 0.01) increased but were not affected by E2. Serum CK activity was higher at 48 h for both groups (P < 0.05). FOXO1, caveolin 1 and SREBP2 expression were 2.8-fold (P < 0.05), 1.4-fold (P < 0.05), and 1.5-fold (P < 0.001) and higher at 3 h after exercise with no effect of E2. This suggests that E2 attenuates neutrophil infiltration; however, the mechanism does not appear to be lesser oxidative stress or membrane damage and may indicate lesser neutrophil/endothelial interaction.     

BODIPY-17α-ethynylestradiol conjugates: Synthesis,fluorescence properties and receptor binding affinities

In vivo imaging of estrogen receptor (ER) densities in human breast cancer is a potential tool to stage disease, guide treatment protocols and follow-up on treatment outcome. Both positron emission tomography (PET) and fluorescence imaging have received ample attention to detect ligand-ER interaction. In this study we prepared BODIPY-estradiol conjugates using 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) as fluorescent probe and estradiol derivatives as ligand and established their relative binding affinity (RBA) for the ERα. The synthesis of the conjugates involves attachment of a BODIPY moiety to the C17α-position of estradiol using Sonogashira or click reactions of iodo-BODIPY or aza-BODIPY with various 17α-ethynylestradiol (EE2) derivatives. The highest RBA for the ERα was observed with the EE2-BODIPY conjugate (7) featuring a linear eight carbon spacer chain. Cell uptake studies and in vivo imaging experiments in an ER-positive mouse tumor model are in progress.     

Protective effect of 17β-estradiol on oxidative stress and liver dysfunction in aged male rats

In aging liver oxidative stress increases due to the decrease in antioxidant bio-molecules such as estrogens which can be modified by hormonal replacement therapy (HRT). With this in mind, we hypothesized that age-related decline in steroidogenesis may be associated with the impairment of the antioxidant defense cells in liver, the increase in lipid peroxidation, hepatic dysfunction and histological changes; estrogens prevent all these changes induced by aging. 17beta-estradiol treatment was initiated in 12 month-old Wistar rats, and continued until 18 months of age. Our results showed that 17beta-estradiol (E2) level in the serum of the aged untreated rats was reduced by -32% in 18 month-old rats compared to the young animals (4-month-old). The superoxide dismutase (SOD), catalase (CAT), and gluthatione peroxidase (GPX) activities were reduced by -47, -46, and -29% respectively in old rat liver. In addition, the TBARs in liver and hepatic dysfunction parameters in plasma such as gamma-glutamyl transferase (GGT), phosphatase alkalin (PAL) as well as bilirubin level increased significantly in old rats, and histological changes were investigated. In E2-treated rats, protective effects were observed. Indeed, 17beta-estradiol attenuates all changes induced by aging. The 17beta-estradiol level was higher in old E2-treated rats compared to the control rats. Moreover, the SOD, CAT and GPX activities were higher by +28, +15, and +11% respectively. This anti-aging effect of estrogens was clarified by a lower level of lipid peroxidation and liver dysfunction parameters as well as by histological observation.     

Altered phosphorylation and distribution status of vimentin in rat seminiferous epithelium following 17β-estradiol treatment

Vimentin, type III intermediate filament, has stage-specific localization in the Sertoli cell. In the rat, during stages I–V and XI–XIV of the seminiferous epithelium, vimentin is localized in the perinuclear area with filaments projecting into the apical region toward the developing germ cells. These filaments decrease in length at stages VI–VII with perinuclear staining in stages VIII–IX, when spermiation occurs. Our earlier studies following 17β-estradiol treatment to adult male rats demonstrated an increase in germ cell apoptosis, spermiation failure and disruption of Sertoli cell microfilaments and microtubules. The present study was undertaken to determine the stage-specific distribution of vimentin and its involvement in spermiation failure and germ cell apoptosis. Immunofluorescence studies revealed that in contrast to the perinuclear localization with small extensions in control stages VII–IX, long extensions radiating apically to the spermatids in deep recess were observed in the treated group. Immunoprecipitation studies showed marked absence of phosphorylated vimentin in stages VII–VIII in the treated group. Further, localization of plectin, cytoskeletal linker protein, showed decrease in all the stages of spermatogenesis following estradiol treatment. Interestingly, for the first time the localization of plectin in the tubulobulbar complex was observed. In conclusion, the study suggests that estradiol treatment leads to an effect on vimentin phosphorylation, which could have inhibited the disassembly of vimentin leading to retention of apical projection in stages VII–VIII. These effects could be presumably due to a decrease in plectin, affecting the reorganization of vimentin and therefore the apical movement of spermatids, leading to spermiation failure.     

A comparative study of the effect of 17β-estradiol and estriol on peripheral pain behavior in rats

Although estradiol has been reported to influence pain sensitivity, the role of estriol (an estradiol metabolite and another widely used female sex hormone) remains unclear. In this study, pain behavior tests, whole-cell patch clamp recording and Western blotting were used to determine whether estriol plays a role in pain signal transduction and transmission. Either systemic or local administration of 17β-estradiol produced a significant rise of mechanical pain threshold, while estriol lacked this effect in normal and ovariectomized (OVX) rats following estriol replacement. Local administration of 17β-estradiol or estriol significantly decreased ATP-induced spontaneous hind-paw withdrawal duration (PWD), which was blocked by an estrogen receptor antagonist, ICI 182, 780. However, systemic application of estriol in normal or OVX rats lacked this similar effect. In cultured dorsal root ganglion neurons, estriol attenuated α,β-methylene ATP-induced transient currents which were blocked by ICI 182, 780. In complete Freund's adjuvant treated (CFA) rats, systemic application of 17β-estradiol or estriol decreased the mechanical pain threshold significantly, but did not change the inflammatory process. Similar effects were observed after estriol replacement in OVX rats. The expression of c-fos in lumbosacral spinal cord dorsal horn (SCDH) was increased significantly by administration of 17β-estradiol but not estriol, and not by estriol replacement in OVX rats. These results suggest that 17β-estradiol but not estriol plays an anti-hyperalgesic role in physiological pain. However, both peripheral 17β-estradiol and estriol play anti-hyperalgesic roles in ATP-induced inflammatory pain. Systemic application of estriol as well as 17β-estradiol plays hyperalgesic roles in CFA-induced chronic pain.     

Complex effects of 17β-estradiol on mitochondrial function

Existing literature on estradiol indicates that it affects mitochondrial functions at low micromolar concentrations. Particularly blockade of the permeability transition pore (PTP) or modulation of the enzymatic activity of one or more complexes of the respiratory chain were suspicious. We prepared mitoplasts from rat liver mitochondria (RLM) to study by single-channel patch-clamp techniques the PTP, and from rat astrocytes to study the potassium BK-channel said to modulate the PTP. Additionally, we measured respiration of intact RLM. After application of 17β-estradiol (βE) our single-channel results reveal a transient increase of activity of both, the BK-channel and the PTP followed by their powerful inhibition. Respiration measurements demonstrate inhibition of the Ca(2+)-induced permeability transition, as well, though only at higher concentrations (≥30μM). At lower concentrations, we observed an increase of endogenous- and state 2-respiration. Furthermore, we show that βE diminishes the phosphorylating respiration supported by complex I-substrates (glutamate/malate) or by the complex II-substrate succinate. Taken together the results suggest that βE affects mitochondria by several modes, including partial inhibition of the activities of ion channels of the inner membrane and of respiration. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).