Comparable Attenuation of Aβ<Subscript>25–35</Subscript>-Induced Neurotoxicity by Quercitrin and 17β-Estradiol in Cultured Rat Hippocampal Neurons

In the present work, potential protective effects of quercitrin (a phytoestrogen) on Aβ-induced neurotoxicity in cultured rat hippocampal neurons were investigated in comparison with 17β-estradiol. Cell viability, oxidative status, and antioxidative potentials were used as comparative parameters. Co-exposure of cultured neurons to Aβ_25–35 with either quercitrin or 17β-estradiol (50–100 μM) for 72 h attenuated Aβ_25–35-induced neurotoxicity and lipid peroxidation, but not Aβ_25–35-induced ROS accumulation. However, only 17β-estradiol counteracted a reduction in glutathione content and only quercitrin counteracted a reduction in glutathione peroxidase activity. Both compounds displayed no effects on superoxide dismutase activity. A specific estrogen receptor antagonist, ICI 182780, did not abolish neuroprotective effects of quercitrin and 17β-estradiol. These findings suggested that quercitrin and 17β-estradiol attenuated Aβ_25–35-induced neurotoxicity in a comparable manner. Underlying neuroprotective mechanisms of both compounds were probably not related to estrogen receptor-mediated genomic mechanisms but might involve with their antioxidant and free radical scavenging properties.     

Targeted brain delivery of 17β-estradiol via nasally administered water soluble prodrugs

The utility of the nasal route for the systemic delivery of 17β-estradiol was studied using watersoluble prodrugs of 17β-estradiol. This delivery method was examined to determine if it will result in preferential delivery to the brain. Several alkyl prodrugs of 17β-estradiol were prepared and their physicochemical properties were determined. In vitro hydrolysis rate constants in buffer, rat plasma, and rat brain homogenate were determined by high-performance liquid chromatography. In vivo nasal experiments were carried out on rats. Levels of 17β-estradiol in plasma and cerebral spinal fluid (CSF) were determined with radioimunoassay using a gamma counter. The study revealed that the aqueous solubilities of the prodrugs were several orders of magnitude greater than 17β-estradiol with relatively fast in vitro conversion in rat plasma. Absorption was fast following nasal delivery of the prodrugs with high bioavailability. CSF 17β-estradiol concentration was higher following nasal delivery of the prodrugs compared to an equivalent intravenous dose. It was determined that water-soluble prodrugs of 17β-estradiol can be administered nasally. These prodrugs are capable of producing high levels of estradiol in the CSF and as a result may have a significant value in the treatment of Alzheimer's disease. Published: March 25, 2002.     

Neuroprotective Actions of the Synthetic Estrogen 17α-Ethynylestradiol in the Hippocampus

17α-Ethynylestradiol (EE2), a major constituent of many oral contraceptives, is similar in structure to 17β-estradiol, which has neuroprotective properties in several animal models. This study explored the potential neuroprotective actions of EE2 against kainic and quinolinic acid toxicity in the hippocampus of adult ovariectomized Wistar rats. A decrease in the number of Nissl-stained neurons and the induction of vimentin immunoreactivity in astrocytes was observed in the hilus of the dentate gyrus of the hippocampus after the administration of either kainic acid or quinolinic acid. EE2 prevented the neuronal loss and the induction of vimentin immunoreactivity induced by kainic acid at low (1 μg/rat) and high (10–100 μg/rat) doses and exerted a protection against quinolinic acid toxicity at a low dose (1 μg/rat) only. These observations demonstrate that EE2 exerts neuroprotective actions against excitotoxic insults. This finding is relevant for the design of new neuroprotective estrogenic compounds.     

Synergic Effects of β-Estradiol and Erythromycin on hERG Currents

The incidence rates of long QT syndrome (LQTS) and drug-induced torsades de pointes (TDP) are higher in women than men. Although gonadal steroids are assumed to play an important role in the gender-based differences in cardiac electrophysiological properties, the underlying mechanisms of the gender-based differences are not fully understood. In particular I _Kr, which comprises the repolarization phase of the action potential, has not been well understood in its modulation by sex hormones. To assess this, we examined the effects of the female sex hormone β-estradiol on the human ether-a-go-go-related gene (hERG)-encoded potassium current stably expressed in human embryonic kidney-293 (HEK) cells. We demonstrated that hERG currents were inhibited by β-estradiol maximally to 62% of control with an IC₅₀ of 1.3 μM and a Hill coefficient of 0.87, which might account for the sex-related differences in LQTS. We also examined whether estrogen modulated drug-induced blocking effects on hERG currents or not. With simultaneous application of 10 μM erythromycin, which is known to block hERG currents but not in low doses, the blocking effects of β-estradiol on hERG currents were enhanced. Namely, hERG currents were inhibited maximally to 45.8% of control with an IC₅₀ of 59 nM (P < 0.02) by β-estradiol with 10 μM erythromycin. We conclude here that a significant block of hERG currents by β-estradiol may account for the sex-related differences in LQTS and the synergic effects of β-estradiol and erythromycin indicate a higher risk of drug-induced TDP in women than men.     

Effects of castration on aggression and levels of serum sex hormones and their central receptors in mandarin voles (Microtus mandarinus)

Aggression in socially monogamous mandarin vole (Microtus mandarinus) was observed after castration. Levels of serum sex hormones and their central receptors were also measured using enzyme-linked immunosorbent assay and immunohistochemistry methods. The data indicate that adult males showed higher levels of aggression after castration. However, castration significantly reduced levels of serum testosterone, and the number of androgen receptor immunoreactive neurons in the anterior hypothalamus, bed nucleus of the stria terminalis, medial amygdaloid nucleus (P < 0.01) and lateral septal nucleus (P < 0.05). In addition, levels of estrogen receptor β in the anterior hypothalamus and medial amygdaloid nucleus (P < 0.05), bed nucleus of the stria terminalis and lateral septal nucleus (P < 0.01) declined to varying degrees at weekly intervals. In contrast, serum 17β-estradiol concentrations were up-regulated by castration and castration did not change levels of estrogen receptor α in the medial amygdaloid nucleus and lateral septal nucleus, but increased it in the anterior hypothalamus 3 weeks after castration (P < 0.05). We suggest that higher levels of aggression induced by castration may be independent of serum testosterone and androgen receptors, and may be associated with high serum 17β-estradiol concentrations, stable estrogen receptor α immunoreactive neurons in some brain regions and the relative ratio of the two estrogen receptors.     

Estrogen alleviates neuropathic pain induced after spinal cord injury by inhibiting microglia and astrocyte activation

Neuropathic pain after spinal cord injury (SCI) is developed in about 80% of SCI patients and there is no efficient therapeutic drug to alleviate SCI-induced neuropathic pain. Here we examined the effect of estrogen on SCI-induced neuropathic pain at below-level and its effect on neuroinflammation as underlying mechanisms. Neuropathic pain is developed at late phase after SCI and a single dose of 17β-estradiol (100, 300?μg/kg) were administered to rats with neuropathic pain after SCI through intravenous injection. As results, both mechanical allodynia and thermal hyperalgesia were significantly reduced by 17β-estradiol compared to vehicle control. Both microglia and astrocyte activation in the lamina I and II of L4-5 dorsal horn was also inhibited by 17β-estradiol. In addition, the levels of p-p38MAPK and p-ERK known to be activated in microglia and p-JNK known to be activated in astrocyte were significantly decreased by 17β-estradiol. Furthermore, the mRNA expression of inflammatory mediators such as Il-1β, Il-6, iNos, and Cox-2 was more attenuated in 17β-estradiol-treated group than in vehicle-treated group. Particularly, we found that the analgesic effect by 17β-estradiol was mediated via estrogen receptors, which are expressed in dorsal horn neurons. These results suggest that 17β-estradiol may attenuate SCI-induced neuropathic pain by inhibiting microglia and astrocyte activation followed inflammation.     

A Bipartite Recombinant Yeast System for the Identification of Subtype-Selective Estrogen Receptor Ligands

Since the estrogen receptor α (ERα) and β (ERβ) are thought to mediate different biological effects, there is intense interest in designing or screening subtype-selective ER ligands. Here, we constructed a biosensor identified as bipartite recombinant yeast system (BRYS) to screen and evaluate subtype-selective ER ligands. Uniform design and immunoblotting was used to determine an optimal dose of copper which control the expression of ERs through a copper inducible metallothionine promoter (CUP1). Some chemicals including natural estrogen (17β-estradiol), specific estrogen receptor agonist (PPT and DPN), and phytoestrogens (genistein) were tested to validate this system. There was obvious and anticipative discrimination between the agonistic activities when these chemicals were identified and characterized. Furthermore, antagonist (ICI 182,780), which could antagonize the transactivation of estrogen, and chromatin immunoprecipitation (ChIP) were used to confirm that the agonistic effects were mediated through ERs. Comparative studies showed that this system was reproducible and sensitive. 4.7 pM (1.3 ng/l) estrogen was the lowest concentration that could be detected to ERα and 0.12 nM (33.5 ng/l) for ERβ. The results from our study showed that in vitro screening for subtype-selective ER ligands could be conducted in a simple yeast system that is rapid, sensitive, reliable, and quantitative. An erratum to this article can be found at     

Activation of estrogen receptor β-dependent nitric oxide signaling mediates the hypotensive effects of estrogen in the rostral ventrolateral medulla of anesthetized rats

Background  

Apart from their well-known peripheral cardiovascular effects, emerging evidence indicates that estrogen acts as a modulator in the brain to regulate cardiovascular functions. The underlying mechanisms of estrogen in central cardiovascular regulation, however, are poorly understood. The present study investigated the cardiovascular effects of 17β-estradiol (E2β) in the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons are located, and delineated the engagement of nitric oxide (NO) in E2β-induced cardiovascular responses.     

Sex steroid-induced inhibition of food intake in sea bass (<Emphasis Type="Italic">Dicentrarchus labrax</Emphasis>)

This study was conducted to test the sensitivity to gonadal steroids of the systems regulating food intake in sea bass. Animals were treated with silastic implants containing 17-β-estradiol or testosterone. Self-feeding was recorded for 31 days using computerized demand feeders and unfed-pellet recovery systems. Both steroids strongly decreased self-feeding levels, feed efficiency and specific growth rates. The linear growth of fish treated with testosterone was higher than in 17-β-estradiol treated fish. In the second experiment, fish were treated with lower 17-β-estradiol doses and 11-keto-androstenedione, a precursor of the main fish androgen (11-keto-testosterone). The results demonstrated a dose–response effect of estrogen and no effect of non-aromatizable androgens on food intake or growth performance. The inhibitory effect of testosterone on food intake seems to be mediated by its aromatization to estradiol, while linear growth promotion is mediated by the androgen per se. Data suggest that gonadal steroids may be involved in the seasonal feeding pattern of sea bass. The results demonstrate the sensitivity of the mechanisms regulating food intake to estrogenic compounds and point to the risk of including feed containing estrogenic substances in fish diets as well as the risk involved in exposure to “estrogenic environments”.     

Role of mitochondria in tamoxifen-induced rapid death of MCF-7 breast cancer cells

Tamoxifen (Tam) is widely used in chemotherapy of estrogen receptor-positive breast cancer. It inhibits proliferation and induces apoptosis of breast cancer cells by estrogen receptor-dependent modulation of gene expression, but recent reports have shown that Tam (especially at pharmacological concentrations) has also rapid nongenomic effects. Here we studied the mechanisms by which Tam exerts rapid effects on breast cancer cell viability. In serum-free medium 5–7 μM Tam induced death of MCF-7 and MDA-MB-231 cells in a time-dependent manner in less than 60 min. This was associated with release of mitochondrial cytochrome c, a decrease of mitochondrial membrane potential and an increase in production of reactive oxygen species (ROS). This suggests that disruption of mitochondrial function has a primary role in the acute death response of the cells. Accordingly, bongkrekic acid, an inhibitor of mitochondrial permeability transition, was able to protect MCF-7 cells against Tam. Rapid cell death induction by Tam was not associated with immediate activation of caspase-9 or cleavage of poly (ADP-ribose) polymerase. It was not blocked by the caspase inhibitor z-Val-Ala-Asp-fluoromethylketone either. Diphenylene ionodium (DPI), an inhibitor of NADPH oxidase, was able to prevent Tam-induced cell death but not cytochrome c release, which suggests that ROS act distal to cytochrome c. The pure antiestrogen ICI 182780 (1 μM) could partly oppose the effect of Tam in estrogen receptor positive MCF-7 cells, but not in estrogen receptor negative MDA-MB-231 cells. Pre-culturing MCF-7 cells in the absence of 17β-estradiol (E₂) or in the presence of a low Tam concentration (1 μM) made the cells even more susceptible to rapid death induction by 5 or 7 μM Tam. This effect was associated with decreased levels of the anti-apoptotic proteins Bcl-X_L and Bcl-2. In conclusion, our results demonstrate induction of a rapid mitochondrial cell death program in breast cancer cells at pharmacological concentrations of Tam, which are achievable in tumor tissue of Tam-treated breast cancer patients. These mechanisms may contribute to the ability of Tam therapy to induce death of breast cancer cells.     

Genistein and daidzein induce neurotoxicity at high concentrations in primary rat neuronal cultures

Summary It is known that estrogen can protect neurons from excitotoxicity. Since isoflavones possess estrogen-like activity, it is of interest to determine whether isoflavones can also protect neurons from glutamate-induced neuronal injury. Morphological observation and lactate dehydrogenase (LDH) release assay were used to estimate the cellular damage. It is surprising that, contrary to estrogen, isoflavones, specifically genistein and daidzein, are toxic to primary neuronal culture at high concentration. Treatment of neurons with 50 μM genistein and daidzein for 24 h increased LDH release by 90% and 67%, respectively, indicating a significant cellular damage. Under the same conditions, estrogen such as 17β-estradiol did not show any effect on primary culture of brain cells. At 100 μM, both genistein and daidzein increased LDH release by 2.6- and 3-fold, respectively with a 30-min incubation. Furthermore, both genistein and daidzein at 50 μM increased the intracellular calcium level, [Ca²⁺]_i, significantly. To determine their mode of action, genistein and daidzein were tested on glutamate and GABA_A receptor binding. Both genistein and daidzein were found to have little effect on glutamate receptor binding, while the binding of [³H]muscimol to GABA_A receptors was markedly inhibited. However, 17β-estradiol did not affect GABA_A receptor binding suggesting that the toxic effect of genistein and daidzein could be due to their inhibition of the GABA_A receptor resulting in further enhancement of excitation by glutamate and leading to cellular damage. Ying Jin, Heng Wu contributed equally to this article.     

Involvement of D-Asp in P450 aromatase activity and estrogen receptors in boar testis

Summary. Mammalian testis contains D-aspartic acid (D-Asp), which enhances testosterone production. D-Asp, on other hand, also stimulates 17β-estradiol synthesis in the ovary of some lower vertebrates. We studied boar testis in order to determine if D-Asp intervenes in 17β-estradiol synthesis in the testis of those mammals which produce significant amounts of estrogens as well as testosterone. The boar testis contains D-Asp (40 ± 3.6 nmol/g tissue) which, according to immunohistological techniques, is localized mainly in Leydig cells, and, to a lesser extent, in sustentacular (Sertoli), peritubular and some germ cells. The enzyme P450aromatase is present in Leydig cells and few germ cells. In vitro experiments showed that the addition of D-Asp to testicular tissue extracts induced a significant increase of aromatase activity, as evaluated by testosterone conversion into 17β-estradiol. The enzyme’s K_m was not affected by D-Asp (about 25 nM in both control and D-Asp added tests). On the basis of these results we suggest that, as in the ovary, D-Asp is involved in the local control of aromatase activity of boar testis and, therefore, it intervenes in the 17β-estradiol production. In the testis, the D-Asp targets are presumably the Leydig cells, which having also a nuclear estrogen receptor are, in turn, one of the putative targets of the 17β-estradiol that they produce (autocrine effect).     

NMR analysis reveals 17β-estradiol induced conformational change in ERβ ligand binding domain expressed in <Emphasis Type="Italic">E. coli</Emphasis>

Nuclear magnetic resonance (NMR) spectroscopy is a useful biophysical technique to study the ligand–protein interaction. In this report, we have used bacterially produced ERβ and its domains for studying the functional analysis of ligand–protein interaction. Briefly, ERβ and its transactivation domain (TAD) and ligand binding domain (LBD) were subcloned and overexpressed using a prokaryotic expression system. The recombinant proteins were purified using Ni⁺²-IDA affinity chromatography and analyzed by NMR. Purified ERβ and TAD show similar conformation in the absence or presence of 17β-estradiol. However, LBD shows altered conformation in the presence of 17β-estradiol. These findings suggest that ERβ produced in bacteria exhibits a conformation such that its LBD remains masked and consequently it binds less to 17β-estradiol. Such study may help to develop the therapeutic approaches for controlling the estradiol-mediated gene expression in hormone dependent diseases.     

Estrogen-like properties of brominated analogs of bisphenol A in the MCF-7 human breast cancer cell line

Tetrabromobisphenol A (TeBBPA) is a four-meta-brominated variant of bisphenol A (BPA) and is one of the most commonly used brominated flame retardants worldwide. We compared the estrogenic potency of TeBBPA, BPA and the brominated analogs mono- (MBBPA), di- (DBBPA), and tribromobisphenol A (TrBBPA) in the estrogen-dependent human breast cancer cell line MCF-7. All of the compounds competed with 17β-estradiol for binding to the estrogen receptor, although the affinity of the test chemicals to the estrogen receptor was much lower than that of 17β-estradiol. TrBBPA and TeBBPA showed a considerably lower access to the estrogen receptors within intact MCF-7 cells incubated in 100% serum compared to incubation in serum-free medium, indicating a strong binding to serum proteins. BPA, MBBPA, and DBBPA showed only a slightly reduced access to the receptors. All of the test compounds induced proliferation in MCF-7 cells, the potential decreasing with increasing number of bromo-substitutions. TeBBPA did not induce maximal cell growth, indicating cytotoxic effects at high concentrations. BPA and the brominated analogs, except TeBBPA, induced progesterone receptor and pS2 to the same extent as 17β-estradiol, although at much higher concentrations. Our studies demonstrate that compared to 17β-estradiol, BPA and the brominated analogs have much lower estrogenic potencies for all of the endpoints tested, TeBBPA being the least estrogenic compound. This revised version was published online in July 2006 with corrections to the Cover Date.     

Estrogen stimulation of ovarian surface epithelial cell proliferation

Summary Ovarian cancer is the leading cause of gynecological cancer mortality, and 85–90% of this malignancy originates from the ovarian surface epithelium (OSE). The etiology of ovarian epithelial cancer is unknown but a role for estrogens has been suspected. However, the effect of estrogens on OSE cell proliferation remains to be determined. Using the rabbit model, our studies have demonstrated that 17β-estradiol stimulates OSE cell proliferation and the formation of a papillary ovarian surface morphology similar to that seen in human ovarian serous neoplasms of low malignant potential. Immunohistochemical staining of ovarian tissue sections with an antibody to the estrogen receptor α demonstrates its expression in both OSE cells and stromal interstitial cells. In primary ovarian cell cultures, the proliferative response of the epithelial cells to 17β-estradiol depends on the expression of the estrogen receptor α in the epithelial cells. However, when the epithelial cells are grown together with ovarian stromal cells, their proliferative response to this hormone is greatly enhanced, suggesting the involvement of stromal-epithelial interactions. These studies suggest a role for estrogens and the estrogen receptor α in OSE growth.     

Relaxin and β-estradiol modulate targeted matrix degradation in specific synovial joint fibrocartilages: progesterone prevents matrix loss

Relaxin, a 6-kDa polypeptide hormone, is a potent mediator of matrix turnover and contributes to the loss of collagen and glycosaminoglycans (GAGs) from reproductive tissues, including the fibrocartilaginous pubic symphysis of several species. This effect is often potentiated by β-estradiol. We postulated that relaxin and β-estradiol might similarly contribute to the enhanced degradation of matrices in fibrocartilaginous tissues from synovial joints, which may help explain the preponderance of diseases of specific fibrocartilaginous joints in women of reproductive age. The objective of this study was to compare the in vivo effects of relaxin, β-estradiol, and progesterone alone or in various combinations on GAG and collagen content of the rabbit temporomandibular joint (TMJ) disc fibrocartilage, knee meniscus fibrocartilage, knee articular cartilage, and the pubic symphysis. Sham-operated or ovariectomized female rabbits were administered β-estradiol (20 ng/kg body weight), progesterone (5 mg/kg), or saline intramuscularly. This was repeated 2 days later and followed by subcutaneous implantation of osmotic pumps containing relaxin (23.3 μg/kg) or saline. Tissues were retrieved 4 days later and analyzed for GAG and collagen. Serum relaxin levels were assayed using enzyme-linked immunosorbent assay. Relaxin administration resulted in a 30-fold significant (p < 0.0001) increase in median levels (range, approximately 38 to 58 pg/ml) of systemic relaxin. β-estradiol, relaxin, or β-estradiol + relaxin caused a significant loss of GAGs and collagen from the pubic symphysis and TMJ disc and of collagen from articular cartilage but not from the knee meniscus. Progesterone prevented relaxin- or β-estradiol-mediated loss of these molecules. The loss of GAGs and collagen caused by β-estradiol, relaxin, or β-estradiol + relaxin varied between tissues and was most prominent in pubic symphysis and TMJ disc fibrocartilages. The findings suggest that this targeted modulation of matrix loss by hormones may contribute selectively to degeneration of specific synovial joints.     

Effects of steroid hormones in fetal bovine serum on plating and cloning of human cells in vitro

Summary Fetal bovine sera from each of three different commercial sources were tested for their ability to support cloning of human fibroblastoid cells in vitro. Cloning efficiencies varied according to serum source. Serum (10 samples) from company A did not support growth, while sera (10 samples) from companies B and C provided adequate to excellent conditions for cloning and growth. Cells from neonatal foreskin or embryonic lung responded to each serum similarly. Bovine serum albumin type H7 from company C supported cell growth in media without serum. Sera containing 1.0 ng per ml or more of progesterone inhibited growth, whereas sera containing less than 1.0 ng per ml supported cloning and growth. In the low progesterone sera, the concentration of 17-β-estradiol exceeded 100 pg per ml. Growth supporting sera could be made non-supportive by adding 0.1 μg per ml of progesterone. The addition to non-supportive sera of 0.1 μg per ml of 17-β-estradiol or hydrocortisone made these sera supportive of cell growth. Addition of estrogen or hydrocortisone to a culture medium that inhibits growth, with subsequent reversal of the inhibitory effect, implies that these hormones competitively regulate growth of responsive cells in vitro. Supported in part by NIH-NCI-EC2074.