Investigating the membrane orientation and transversal distribution of 17β-estradiol in lipid membranes by solid-state NMR

17β-Estradiol (E₂) is a potent estrogen, which modulates many important cellular functions by binding to specific estrogen receptors located in the cell nucleus and also on the plasma membrane. We have studied the membrane interaction of E₂ using a combination of solid-state NMR methods. ²H NMR results indicate that E₂ does not cause a condensation effect of the surrounding phospholipids, which is contrary to the effects of cholesterol, and only very modest E₂ induced alterations of the membrane structure were detected. ¹H magic-angle spinning NMR showed well resolved signals from E₂ as well as of POPC in the membrane-lipid layer. Two-dimensional NOESY spectra revealed intense cross-peaks between E₂ and the membrane lipids indicating that E₂ is stably inserted into the membrane. The determination of intermolecular cross-relaxation rates revealed that E₂ is broadly distributed in the membrane with a maximum of the E₂ distribution function in the upper chain region of the membrane. We conclude that E₂ is highly dynamic in lipid membranes and may undergo rotations as it exhibits two polar hydroxyl groups on either side of the molecule.     

Solid-state ²H NMR shows equivalence of dehydration and osmotic pressures in lipid membrane deformation

Lipid bilayers represent a fascinating class of biomaterials whose properties are altered by changes in pressure or temperature. Functions of cellular membranes can be affected by nonspecific lipid-protein interactions that depend on bilayer material properties. Here we address the changes in lipid bilayer structure induced by external pressure. Solid-state 2H NMR spectroscopy of phospholipid bilayers under osmotic stress allows structural fluctuations and deformation of membranes to be investigated. We highlight the results from NMR experiments utilizing pressure-based force techniques that control membrane structure and tension. Our 2H NMR results using both dehydration pressure (low water activity) and osmotic pressure (poly(ethylene glycol) as osmolyte) show that the segmental order parameters (S(CD)) of DMPC approach very large values of ≈ 0.35 in the liquid-crystalline state. The two stresses are thermodynamically equivalent, because the change in chemical potential when transferring water from the interlamellar space to the bulk water phase corresponds to the induced pressure. This theoretical equivalence is experimentally revealed by considering the solid-state 2H NMR spectrometer as a virtual osmometer. Moreover, we extend this approach to include the correspondence between osmotic pressure and hydrostatic pressure. Our results establish the magnitude of the pressures that lead to significant bilayer deformation including changes in area per lipid and volumetric bilayer thickness. We find that appreciable bilayer structural changes occur with osmotic pressures in the range of 10-100 atm or lower. This research demonstrates the applicability of solid-state 2H NMR spectroscopy together with bilayer stress techniques for investigating the mechanism of pressure sensitivity of membrane proteins.     

Occurrence and Fate of Estrone, 17β-estradiol and 17α-ethynylestradiol in STPs for Domestic Wastewater

Estrone (E1), 17β-estradiol (E2) and 17α-ethynylestradiol (EE2) discharged from sewage treatment plants (STPs) into surface waters, are seen as a threat effecting aquatic life by its estrogenic character. Therefore, much research is conducted on the fate and removal of these compounds. Since these compounds are present in influents and effluents in the ng/l range, methods for detection deserve special attention. Most important processes that play a role in the removal of estrogens are: adsorption, aerobic degradation, anaerobic degradation, anoxic biodegradation and photolytic degradation. Halflifes tend to vary and are remarkably shorter when low initial concentrations are applied. In general anaerobic conditions result in longer halflifes then aerobic conditions. EE2 shows far most persistence of the compounds, thereby also the estrogenic effect in vitro is about 2–3-fold higher compared to E2. The three compounds show a higher affinity to sorb to sludge compared to other tested adsorption materials like sediment. Aerobic degradation is far the most efficient in removing these compounds, but adsorption seems to play a significant role in retaining the estrogens inside full-scale STPs. Removal rates in full scale plants depend on the HRT, SRT and loading rates, but lack of information on the exact dependency so far prevents an optimal design able to fully eliminate estrogens from wastewater.     

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.     

The monohydroxylation of 17β-estradiol at the 15α-, 16α-, and 16β-positions by bovine adrenal perfusion

17β-Estradiol-4-¹⁴C was perfused through bovine adrenal glands. The bulk (65%) of the estradiol was recovered, but several metabolites, all in low rates of conversion, were isolated. Four of these were identified as esterone, estriol, 16β-epiestriol and 15-αhydroxy-17β- estradiol.     

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.     

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.     

Interaction of 17β-estradiol and testosterone with the oviduct of the lizard Lacerta sicula

In the lizard, Lacerta sicula, oviduct is stimulated in spayed females by the administration of 17β-estradiol or testosterone. The experiments in vivo show that the oviduct retains radioactivity only in the animals injected with [³H]-17β-estradiol. In vitro, however, a specific binding of both hormones to oviduct-cytosol has been observed. It is not known if these sex hormones bind to the same macromolecule.     

17α- and 17β-boldenone 17-glucuronides: Synthesis and complete characterization by H and C NMR

Boldenone is an androgenic anabolic steroid intensively used for growth promoting purposes in animals destined for meat production and as a performance enhancer in athletics. Therefore its use is officially banned either in animals intended for consumption or in humans. Because most anabolic steroids are completely metabolized and usually no parent steroid is excreted, metabolite identification is crucial to detect the illegal use of anabolic steroids either in humans or in livestock. 17α- and 17β-boldenone 17-glucuronides were synthesized, purified and characterized in order to provide suitable standards for the identification and quantification of these metabolites.     

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.     

Solid-state NMR analysis of interaction sites of curcumin and 42-residue amyloid β-protein fibrils

Aggregation of 42-residue amyloid β-protein (Aβ42) plays a pivotal role in the etiology of Alzheimer's disease (AD). Curcumin, the yellow pigment in the rhizome of turmeric, attracts considerable attention as a food component potentially preventing the pathogenesis of AD. This is because curcumin not only inhibits the aggregation of Aβ42 but also binds to its aggregates (fibrils), resulting in disaggregation. However, the mechanism of interaction between curcumin and the Aβ42 fibrils remains unclear. In this study, we analyzed the binding mode of curcumin to the Aβ42 fibrils by solid-state NMR using dipolar-assisted rotational resonance (DARR). To improve the quality of 2D spectra, 2D DARR data were processed with the covariance NMR method, which enabled us to detect weak cross peaks between carbons of curcumin and those of the Aβ42 fibrils. The observed (13)C-(13)C cross peaks indicated that curcumin interacts with the 12th and 17-21st residues included in the β-sheet structure in the Aβ42 fibrils. Interestingly, aromatic carbons adjacent to the methoxy and/or hydroxy groups of curcumin showed clear cross peaks with the Aβ42 fibrils. This suggested that these functional groups of curcumin play an important role in its interaction with the Aβ42 fibrils.     

Regulation of Akt expression and phosphorylation by 17β-estradiol in the rat uterus during estrous cycle

Background

Ablation of the low-affinity receptor subunit for leukemia inhibitory factor (LIFR) causes multi-systemic defects in the late gestation fetus. Because corticosterone is known to have a broad range of effects and LIF function has been associated with the hypothalamo-pituitary-adrenal axis, this study was designed to determine the role for LIFR in the fetus when exposed to the elevated maternal glucocorticoid levels of late gestation. Uncovering a requirement for LIFR in appropriate glucocorticoid response will further understanding of control of glucocorticoid function.

Methods

Maternal adrenalectomy or RU486 administration were used to determine the impact of the maternal glucocorticoid surge on fetal development in the absence of LIFR. The mice were analyzed by a variety of histological techniques including immunolabeling and staining techniques (hematoxylin and eosin, Alizarin red S and alcian blue). Plasma corticosterone was assayed using radioimmunoassay.

Results

Maternal adrenalectomy does not improve the prognosis for LIFR null pups and exacerbates the effects of LIFR loss. RU486 noticeably improves many of the tissues affected by LIFR loss: bone density, skeletal muscle integrity and glial cell formation. LIFR null pups exposed during late gestation to RU486 in utero survive natural delivery, unlike LIFR null pups from untreated litters. But RU486 treated LIFR null pups succumb within the first day after birth, presumably due to neural deficit resulting in an inability to suckle.

Conclusion

LIFR plays an integral role in modulating the fetal response to elevated maternal glucocorticoids during late gestation. This role is likely to be mediated through the glucocorticoid receptor and has implications for adult homeostasis as a direct tie between immune, neural and hormone function.     

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.     

Bisphenol A and 17β-estradiol promote arrhythmia in the female heart via alteration of calcium handling

Background

There is wide-spread human exposure to bisphenol A (BPA), a ubiquitous estrogenic endocrine disruptor that has been implicated as having potentially harmful effects on human heart health. Higher urine BPA concentrations have been shown to be associated with cardiovascular diseases in humans. However, neither the nature nor the mechanism(s) of BPA action on the heart are understood.

Methodology/Principal Findings

The rapid (<7 min) effects of BPA and 17β-estradiol (E2) in the heart and ventricular myocytes from rodents were investigated in the present study. In isolated ventricular myocytes from young adult females, but not males, physiological concentrations of BPA or E2 (10⁻⁹ M) rapidly induced arrhythmogenic triggered activities. The effects of BPA were particularly pronounced when combined with estradiol. Under conditions of catecholamine stimulation, E2 and BPA promoted ventricular arrhythmias in female, but not male, hearts. The cellular mechanism of the female-specific pro-arrhythmic effects of BPA and E2 were investigated. Exposure to E2 and/or BPA rapidly altered myocyte Ca²⁺ handling; in particular, estrogens markedly increased sarcoplasmic reticulum (SR) Ca²⁺ leak, and increased SR Ca²⁺ load. Ryanodine (10⁻⁷ M) inhibition of SR Ca²⁺ leak suppressed estrogen-induced triggered activities. The rapid response of female myocytes to estrogens was abolished in an estrogen receptor (ER) β knockout mouse model.

Conclusions/Significance

Physiologically-relevant concentrations of BPA and E2 promote arrhythmias in a female-specific manner in rat hearts; the pro-arrhythmic actions of estrogens are mediated by ERβ-signaling through alterations of myocyte Ca²⁺ handling, particularly increases in SR Ca²⁺ leak. Our study provides the first experimental evidence suggesting that exposure to estrogenic endocrine disrupting chemicals and the unique sensitivity of female hearts to estrogens may play a role in arrhythmogenesis in the female heart.     

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.