Estrogen Agonist Activity Research Articles

In Vitro Detection of Estrogen Activity in Plastic Products Using a Sensitive Bioassay: Failure to Acknowledge Limitations

Yang et al. (2011) used the in vitro E-SCREEN assay to infer that health risks from “estrogenic” plastics can be eliminated by using their proprietary materials, processes, and products to manufacture plastics. An in vitro cell proliferation assay such as the E-SCREEN is a sensitive indicator of in vitro estrogen agonist activity and potential estrogenic activity in vivo (e.g., in the rat uterotrophic assay). However, in vitro properties may not manifest in in vivo activity, and neither demonstrates a health risk. Without definitive evidence that in vivo activity leads to adverse health effects, the results of Yang et al. are unconvincing and fail to support changing current manufacturing processes for plastics. The value of in vitro and in vivo estrogenic assays for predicting adverse health effects is largely untested but would need to account for actual exposure levels, metabolism, distribution, excretion, and the affinity of parent compounds and metabolites for estrogen receptor binding and transcriptional activation relative to and in competition with physiological levels of potent endogenous hormones. The combined effects of these exposures would also need to be assessed in the context of dietary (e.g., milk, cheeses, vegetables, meats, and other foodstuffs) and environmental estrogens. An excellent in vitro/in vivo study of combined effects (Charles et al. 2007) showed that while relatively high levels of a putative synthetic estrogen mixture increased the estrogenic action of common dietary phytoestrogens, low levels were without effect. Thus, sensitive in vitro detection may not portend estrogenic effects amid the endogenous and dietary hormonal milieu. Yang et al. (2011) made inferences about the safety of plastic food packages, but it is unfortunate that they did not use an extraction method that was approved by the U.S. Food and Drug Administration (FDA 2007). This would have improved the reliability and applicability of their results. Although food typically contacts only the inside surface of containers, Yang et al. extracted materials from 4-mm squares of cut plastic, exposing the inside, outside, and cut surfaces to the extraction medium. Substances may leach into food from the exposed surface of a plastic container but do not typically migrate through the plastic layer (Franz and Welle 2009); thus Yang et al.’s extraction method differs from FDA-approved methods and the way foods normally contact containers. Experimental error was not reported, making comparison of these results with standard methods impossible. In the study by Yang et al. (2011), irradiation methods for simulating “stress” were not well characterized, but they appear to have involved all surfaces of the plastic squares. However, even clear plastics can filter ultraviolet (UV) rays, reducing the potential irradiation of inside container surfaces. Similarly, colorants were added to the extraction mixture; however, during the production of plastics, colorants are embedded and tightly linked. The extent to which these procedures may have confounded the data cannot be known, but the resulting tested extracts may be substantially different from residues that could enter food from plastic containers. Yang et al. (2011) indicated that without increasing production costs, they can identify and/or have developed monomers, additives, and processing agents that lack estrogenic activity. This conclusion appears to derive from data for resins P1, P2, P3, P4, P19, and maybe P18 in their Table 3. In the text the authors noted six MCF-7 assays, but it is unclear whether a single assay was conducted for each of the six stressor and extraction combinations (microwave, UV, autoclave, saline, and ethanol) or whether the whole series was completed six times. Regardless, the authors provided no estimate of assay variance, making it difficult to differentiate real differences from experimental error. In addition, the relative safety of these new agents, particularly antiandrogenic potential, has yet to be resolved. In conclusion, Yang et al. (2011) provided interesting observations but failed to acknowledge the significant limitations of their observations to human health risk assessment. They relied on a very limited in vitro screen to model a very complex system, and those reviewing the study should be aware of the limitations of the approach and the interpretation of such data.

Estrogen agonist/antagonist properties of dibenzyl phthalate (DBzP) based on in vitro and in vivo assays

The most commonly used phthalates have been banned or restricted for use as plasticizers in toys in some countries because of their endocrine-disrupting properties. Dibenzyl phthalate (DBzP) has been proposed as a possible alternative for the banned/restricted phthalates. In this study, the estrogen agonist/antagonist properties of DBzP were predicted by molecular docking and confirmed by yeast estrogen screen (YES) and immature mouse uterotrophic assays. The YES assay results showed a dose-dependent increase in DBzP estrogen agonist activity from 10 −6 to 10 −4 M, and at concentrations from 1.95 × 10 −6 M to higher, DBzP significantly inhibited the agonist activity of 10 −9 M 17β-estradiol (E 2), inhibiting 10 −9 M E 2 by 74.5% at its maximum effectiveness. The in vivo estrogen agonist/antagonist activities of DBzP were demonstrated in immature mouse uterotrophic assays. The antagonist activity of DBzP inhibited E 2-induced uterine growth promoted at 40 and 400 μg/kg bw (body weight) ( P < 0.05). In addition, we also analyzed the estrogen agonist/antagonist potentials of benzyl butyl phthalate (BBP) by YES, and found both were weaker than those of DBzP, suggesting DBzP would be more toxic than BBP and should not be used as an alternative plasticizer.

Potential Hormonal Activity in Surface and Ground Water in Natural Gas Drilling-Dense Areas.

The recent dramatic rise in the use of hydraulic fracturing for natural gas raises concerns that chemicals used in this process may contaminate surface and ground water. As demand increases for alternative energy sources, well proliferation has increased and is moving into locations within crucial water basins that supply fresh water for millions. Hydraulic fracturing involves injecting millions of gallons of water and hundreds of chemicals into the ground under high pressure. A portion will return to the surface, and is typically disposed of by injecting it back into the ground, spreading it over large areas, or storing it in large evaporation pits that may not adequately contain it. In 2005, the process of hydraulic fracturing was added as an exemption to the Safe Drinking Water Act, reducing the ability to regulate and study the impacts of this process on the environment. At the same time, there has been an increase of areas reporting contamination of local water supplies.Preliminary research has shown that dozens of the chemicals used in this process have endocrine disrupting activity, creating the potential for widespread contamination of water supplies with mixtures of these chemicals. The goal of this preliminary study is to compare the hormonal activity in surface and ground water as well as sediment samples collected in drilling-dense region and drilling-sparse areas of Colorado. Water will be tested for estrogen, androgen, thyroid, and progesterone receptor agonist and antagonist activity using MCF-7 and HepG-2 cell lines that have been transfected with hormone responsive reporter genes. A baseline of hormonal activity will be established from samples taken from drilling-sparse areas. Once we have that in place, we can more accurately determine if samples taken from drilling-dense areas show altered hormonal activity. Samples of surface and ground water will also be taken periodically from sites with new well production. This will provide a timeline to ascertain whether or not the water composition changes substantially throughout the entire drilling process.The implications of this are important as drilling extends into areas where potential drinking water contamination could be an even larger problem. The Marcellus Shale is one such area, covering a large portion of many northeastern states. This area is of high concern, as it includes the Delaware River Basin, supplying water to the entire greater New York City area. It has been suggested that citizens living near drilling of this type have experienced significant changes to their surface and drinking water. However, as there is currently no legislation controlling this process, there has been little to no scientific research done in this area. This study looks to gain preliminary information on the subject to better direct future research in this area. (poster)

Emerging data on the efficacy and safety of fulvestrant, a unique antiestrogen therapy for advanced breast cancer

Introduction: Fulvestrant is an antiestrogen therapy with a unique mechanism of action. Unlike the selective estrogen receptor modulator tamoxifen, fulvestrant has no known estrogen agonist activity and is considered a pure antiestrogen. Its primary mechanism of action is thought to result from downregulation of the estrogen receptor (ER). Considerable data have demonstrated the efficacy of fulvestrant in postmenopausal women with ER-positive advanced breast cancer, both in the first-line setting and following disease progression on tamoxifen or aromatase inhibitors. Recent studies report improved benefit with alternative dosing strategies. At all administration schedules, fulvestrant has an excellent safety profile with no significant adverse effects.Areas covered: This article provides a review of the mechanism of action of fulvestrant and the preclinical and clinical data evaluating its use as a form of endocrine therapy. The reader will gain insight into the pharmacologic properties of the drug and its role in the treatment of advanced hormone receptor-positive breast cancer in postmenopausal women.Expert opinion: Based on data demonstrating the efficacy of fulvestrant, including prolonged clinical benefit in many patients, this well-tolerated antiestrogen is an important therapy for breast cancer. The optimal position of fulvestrant in the sequence of endocrine therapies for postmenopausal women and its role in combination regimens are not yet resolved.

The possible role of estrogen and selective estrogen receptor modulators in a rat model of Parkinson's disease

AimThe aim of the present study was to assess and compare the effect of 17β-estradiol and two different selective estrogen receptor modulators (SERMs), tamoxifen and raloxifene, as well as a selective estrogen receptor alpha agonist, propyl-pyrazole-triol (PPT) and a selective estrogen receptor beta agonist, diarylpropionitrile (DPN), on behavioral and biochemical alterations in 6-hydroxydopamine (6-OHDA)-induced nigral dopaminergic cell death in rats. Main methods80 female Wister rats were used. Animals were divided into eight equal groups: Group I; Sham operated, Group II; subjected to ovariectomy (OVX), Group III; OVX rats received striatal injection of 6-OHDA, Groups IV–VIII; OVX rats received striatal injection of 6-OHDA and were injected daily with 17β-estradiol, tamoxifen, raloxifene, PPT and DPN respectively for 5days before 6-OHDA and continued for further 2weeks. Key findingsResults showed that striatal injection of 6-OHDA produced significant behavioral alteration suggestive of PD, together with significant decrease in striatal dopamine, homovanillic acid (HVA) and 3,4-dihydroxyphenyl acetic acid (DOPAC) concentrations. 6-OHDA-induced nigral dopaminergic cell death was characterized by oxidative stress, evidenced by significant decrease in striatal glutathione peroxidase activity, as well as apoptosis, evidenced by significant increase in nigral caspase-3 activity. Treatment with 17β-estradiol, raloxifene, PPT, but neither tamoxifen nor DPN, resulted in significant amelioration of the behavioral and biochemical alterations induced by 6-OHDA. SignificanceThese findings suggest that estrogen and some SERMs having estrogenic agonist activity in the brain, like raloxifene, might exert beneficial effect in PD.

Selektive Estrogenrezeptor-Modulatoren (SERMs)

Selective estrogen receptor modulators (SERMs) have organ specific estrogen and estrogen-antagonistic effects. The two most commonly used SERMs are Tamoxifen and Raloxifen, which have antiestrogenic effects in the mammary gland and (unfortunately also) in the hypothalamus. From a prophylactic view, both substances decrease the development of mammary cancer (which is due to their estrogen-antagonistic properties) and of osteoporosis (which is due to their estrogen-agonistic effects). Because of their estrogen-antagonistic effect in hypothalamus the vegetative climacteric symptoms are increased. Their estrogen-agonistic actions in uterus are clinically undesired. Phytoestrogens (Isoflavone)-containing soy- and red clover-products have in the daily recommended doses minor, if any positive but probably also no adverse effects. Cimicifuga racemosa extracts have no estrogenic effects but they ameliorate climacteric vegetative complaints due to neurotransmitter-like activities.

Screening for estrogenic and antiestrogenic activities of plants growing in Egypt and Thailand

Background:There is a growing demand for the discovery of new phytoestrogens to be used as a safe and effective hormonal replacement therapy.Materials and Methods:The methanol extracts of 40 plants from the Egyptian and Thailand folk medicines were screened for their estrogen agonist and antagonist activities. The estrogenic and antiestrogenic effects of the tested extracts were carried out using the yeast two-hybrid assay system expressing ERα and ERβ. In addition, all the extracts were subjected to a naringinase treatment and retested for their estrogenic activity.Results:The methanol extracts of Derris reticulata and Dracaena lourieri showed the most potent estrogenic activity on both estrogen-receptor subtypes, while, the methanol extracts of Butea monosperma, Erythrina fusca, and Dalbergia candenatensis revealed significant estrogenic activity on ERβ only. Nigella sativa, Sophora japonica, Artabotrys harmandii, and Clitorea hanceana showed estrogenic effect only after naringinase treatment. The most potent antiestrogenic effect was revealed by Aframomum melegueta, Dalbergia candenatensis, Dracena loureiri, and Mansonia gagei.

Tetra-methoxystilbene modulates ductal growth of the developing murine mammary gland

Extensive data suggest that estradiol contributes to the development of breast cancer by acting as a mitogen and exerting direct genotoxic effects after enzymatic conversion to 4-hydroxyestradiol (4-OHE2) via cytochrome P450 1B1 (CYP1B1). The mammary gland, ovary, and uterus all express CYP1B1. Overexpression of this enzyme has been associated with an increased risk of breast cancer and blockade might reduce this carcinogenic effect. For this reason, we conducted systematic in vitro and in vivo studies of a CYP1B1 inhibitor, TMS (2,3',4,5'-tetramethoxystilbene). We found that TMS blocked the enzymatic conversion of radiolabeled estradiol to both 2-hydroxyestradiol (2-OHE2) and 4-OHE2, but did not inhibit Cyp1b1 message formation. In vivo studies using mass spectrometry showed that TMS inhibited formation of 2-OHE2 and 4-OHE2 and the resulting estrogen-DNA adducts. To examine its biologic actions in vivo, we investigated whether TMS could block the hyperplastic changes that occur in the developing breast of aromatase-transfected mice. We found that TMS induced a significant reduction of ductal structures in mice less than 6 months in age. In older mice, no reduction in breast morphology occurred. These latter studies uncovered unexpected estrogen agonistic actions of TMS at high doses, including a paradoxical stimulation of breast ductal structures and the endometrium. These studies suggest that the enzyme inhibitory properties of TMS, as well as the effects on developing breast, could implicate a role for TMS in breast cancer prevention, but only in low doses and on developing breast.

Synthetic analogs of daidzein, having more potent osteoblast stimulating effect

A series of didzein derivatives were synthesized and assessed for stimulation of osteoblast differentiation using primary cultures of rat calvarial osteoblasts. Data suggested that three synthetic analogs, 1c, 3a and 3c were several folds more potent than daidzein in stimulating differentiation and mineralization of osteoblasts. Further, these three compounds did not show any estrogen agonistic activity, however had mild estrogen antagonistic effect. Out of the three compounds, 3c was found to maximally increase the mineralization of bone marrow osteoprogenitor cells. Compound 3c also robustly increased the mRNA levels of osteogenic genes including bone morphogenetic protein-2 and osteocalcin in osteoblasts. Unlike daidzein, 3c did not inhibit osteoclastogenesis. Collectively, we demonstrate osteogenic activity of daidzein analogs at significantly lower concentrations than daidzein.

ChemInform Abstract: Structure-Activity Relationship of Antiestrogens. Phenolic Analogues of 2,3-Diaryl-2H-1-benzopyrans.

Phenolic analogues of 2-[4-(2-piperidinoethoxy)phenyl]-3-phenyl-2H-1-benzopyran (1), a novel antiestrogen, were synthesized and evaluated for their structure-activity relationship. Incorporation of OH at position 7 was found to improve receptor affinity of the benzopyran while having no effect on its action as an antagonist. Similar substitution of 2-phenyl as well potentiated receptor affinity as well as antagonist activity of the prototype. The monophenol 19 and the diphenol 25 were thus found to be good receptor ligands, devoid of estrogen agonist activity and associated with marked antiestrogenic activity of comparable order. Both caused nearly complete inhibition of the estradiol stimulated uterine growth in rats as well as mice and were thus found to be better antiestrogens than tamoxifen, trioxifen, and LY-117018. A binding-site model for estrogen receptor rationalizing the structure-activity relationship of benzopyrans in relation to that of the triarylethylene and the triarylpropenone antiestrogens has been discussed.

Estrogenic activity of a naringinase-treated extract of Sophora japonica cultivated in Egypt

The naringinase-treated methanol extract of Sophora japonica L. (Fabaceae) seeds showed potent estrogen agonist activity. Through bioassay-guided isolation of the main active constituents from the naringinase-treated methanol extract of S. japonica, the aglycones genistein and kaempferol were found to be the main phytoestrogens in the naringinase-treated extract. In addition, kaempferol was nearly equipotent to genistein as an estrogen agonist. Concerning the compounds isolated from the untreated methanol extract, sophoricoside showed weak estrogenic activity on ERβ only.

Influence of estradiol and triiodothyronine on breast cancer cell lines proliferation and expression of estrogen and thyroid hormone receptors

To better understand the estrogen (E2) agonist action of triiodothyronine (T3) the effects of these hormones on ER negative MDA-MB-231 breast cancer cells were compared with those on S30, a clone of MDA-MB-231 stably transfected with ERalpha cDNA, in terms of proliferation and modulation of hormone receptors. Growth experiments showed that MDA-MB-231 was not modulated by any hormone or tamoxifen (TAM). Treatment with E2, 10(-8)M or 10(-9)M had little effect on S30 proliferation. T3 at 10(-8)M significantly inhibited proliferation. This effect was not reverted by TAM. Treatments with 10(-8)M concentration of E2 or T3 reduced ERalpha gene expression in S30, an effect partially blocked by association with TAM, with no effect on TR expression. These results suggest that, in S30, 10(-8)M T3 has a similar action to E2 relative to ERalpha gene modulation. Such results emphasize the need of determining T3 levels, before the introduction of antiestrogenic forms of treatment in breast cancer patients.

Fulvestrant treatment is associated with cholesterol plasma level reduction in hormone receptor-positive metastatic breast cancer patients

Background: Fulvestrant is a pure anti-estrogen hormonal agent formally lacking any estrogen-agonist activity. We analyze the effect(s) of fulvestrant treatment on estrogen target systems in hormone-sensitive advanced breast cancer patients. Methods: Fifty-one patients (median age 65 [range 48 - 82] years) were enrolled. All patients received previous hormonal treatments, with 90.2% receiving ≥2 courses. Last hormonal treatment was exemestane, letrozole, anastrozole and other in 30-10-7-4/51 patients respectively. Median withdrawal time was 18 days (range 3–1456). Complete fasting lipid blood profile and coagulation indices were assessed before fulvestrant administration, every 3 months and at discontinuation time. Endometrial mucosa thickness was evaluated before fulvestrant administration and at end-study time. Results: Patients received a median of 5 fulvestrant injections (range 3-19). We observed a partial response in one patient, disease stability in 21 and disease progression in 29 patients with a clinical benefit of 43.2% and a median time to progression of 5 [range 3-20] months. Total cholesterol levels significantly decreased during treatment (219.8 ± 45.3 vs 201.4 ± 42.1 mg/dl; p = 0.0054) together with LDL-cholesterol (129.7 ± 41.39 vs 112.3 ± 37.1 mg/dl; p = 0.018). HDL-cholesterol and triglycerides did not show significant changes. Reduction of total and LDL-cholesterol was independent from last hormonal treatment or treatment duration. All coagulation indices and mean endometrial mucosa thickness value did not vary. Conclusions: we observed a lipid lowering effect of fulvestrant possibly related to an influence on lipid metabolism by a mechanism in which a role could be played by progesterone receptor.

Extranuclear Coactivator Signaling Confers Insensitivity to Tamoxifen

Tamoxifen is one of many standard therapeutic options currently available for estrogen receptor-alpha-positive breast cancer patients. Emerging data have suggested that levels of estrogen receptor coregulatory proteins play a significant role in acquiring resistance to antiestrogen action. It has been suggested that high levels of estrogen receptor coactivators and its mislocalization may enhance the estrogen agonist activity of tamoxifen and contribute to tamoxifen resistance. In an effort to understand the impact of nongenomic signaling and its contribution to hormone resistance in a whole-animal setting, we generated a transgenic mouse expressing a cytoplasmic version of proline-, glutamic acid-, and leucine-rich protein-1 (PELP1) mutant defective in its nuclear translocation (PELP1-cyto) and implanted these mice with tamoxifen pellets to assess its responsiveness. We show that mammary glands from these mice developed widespread hyperplasia with increased cell proliferation and enhanced activation of mitogen-activated protein kinase and AKT as early as 12 weeks of age. Treatment with tamoxifen did not inhibit this hyperplasia; instead, such treatment exaggerated hyperplasia with an enhanced degree of alteration, indicative of hypersensitivity to tamoxifen. Analysis of molecular markers in the transgenic mammary glands from the tamoxifen-treated transgenic mice showed higher levels of proliferation markers proliferating cell nuclear antigen and activated mitogen-activated protein kinase than in untreated PELP1-cyto cell-derived mice. We also found that nude mice with MCF-7/PELP1-cyto cell-derived tumor xenografts did not respond to tamoxifen. Using immunohistochemical analysis, we found that 43% of human breast tumor samples had high levels of cytoplasmic PELP1, which shows a positive correlation between tumor grade and proliferation. Patients whose tumors had high levels of cytoplasmic PELP1 exhibited a tendency to respond poorly to tamoxifen compared with patients whose tumors had low levels of cytoplasmic PELP1. These findings suggest that PELP1 localization could be used as a determinant of hormone sensitivity or vulnerability. The establishment of the PELP1-cyto transgenic mouse model is expected to facilitate the development of preclinical approaches for effective intervention of breast tumors using cytoplasmic coregulators and active nongenomic signaling.

Identification and Structure−Activity Relationships of Chromene-Derived Selective Estrogen Receptor Modulators for Treatment of Postmenopausal Symptoms

As part of a program aimed at the development of selective estrogen receptor modulators (SERMs), novel chromene scaffolds, benzopyranobenzoxapanes, were discovered. Many compounds showed binding affinity as low as 1.6-200 nM, displayed antagonist behaviors in the MCF-7 human breast adenocarcinoma cell line as well in Ishikawa cell line with IC(50) values in the range 0.2-360 nM. On the basis of the side chain substitution, various compounds demonstrated strong inhibitory activity in anti-uterotropic assay. Compound 7-(R) and its major metabolites 5-(R) and 6-(R) were evaluated in several in vivo models of estrogen action. Relative to a full estrogen agonist (ethynyl estradiol) and the SERM raloxifene, 7-(R) was found to be a potent SERM that behaved as antagonist in the uterus and exhibited estrogen agonistic activity on bone, plasma lipids, hot flush, and vagina. The overall pharmacokinetic profile and stability were significantly improved compared to those of the phase 2 development compound 9-(R).

The effects of long-term raloxifene and estradiol treatments on bone in a patient with congenital aromatase deficiency

Introduction In adult aromatase-deficient men, estrogen treatment has always resulted in a rapid skeletal maturation with epiphyseal closure and improved BMD. Raloxifene is a SERM with proven estrogen agonist action on bone that leads to an improvement in BMD and a reduction in bone turnover. The present study reports the effects of raloxifene and transdermal estradiol treatment, respectively, on epiphyseal closure and BMD in an aromatase-deficient man, over a 24-month follow-up, with the aim of obtaining further insight into the role of estrogens in the male skeletal homeostasis. Materials and methods A 25-year-old Caucasian man with aromatase deficiency, a bone age of 15.3 years, unfused epiphyses and an impaired BMD was initially administered raloxifene (60 mg/day per os) for 12 months, while transdermal estradiol (25 μg twice weekly) was administered for the subsequent 12 months. During the follow-up, the effects of the two treatments on epiphyseal closure, BMD and bone turnover markers were investigated. An iliac crest bone biopsy was performed only before and after the raloxifene treatment, but it was not repeated after transdermal estradiol treatment. Results No changes in bone age were observed after raloxifene therapy, whereas a complete epiphyseal closure was achieved with transdermal estradiol treatment. Compared with baseline values, raloxifene treatment led to improved BMD both at the ultradistal forearm and 33% radius; the transdermal estradiol treatment resulted in a further slight increase in BMD at the 33% radius, but not at the ultradistal forearm. The baseline bone biopsy showed elevated bone remodelling in trabecular bone, while the second biopsy following raloxifene treatment revealed a decrease in remodelling. Discussion This study shows that the management of aromatase deficiency in the male cannot consider raloxifene as a first choice treatment, but should be still based on estrogen replacement treatment since in this patient the completion of bone maturation has only been obtained once estradiol substitution was performed. The present case also demonstrates that raloxifene is able to improve BMD in aromatase-deficient men.

In vivo evaluation of substituted 3-phenyl,7-methoxy-benzopyrans as modified estrogens

AbstractSubstituted 3-phenyl,7-methoxy-benzopyran derivatives and vitamin D 3 (cholecalciferol, 1) were evaluated for their estrogen agonistic and antagonistic activities in immature female Sprague-Dawley rat model. The benzopyran derivatives 17 and 18, which were made as hybrids of estrogen and vitamin D 3 (pseudo vitamin D 3 analogs), showed significant estrogen agonistic activity (up to 48%) and weak estrogen antagonistic activity (up to 6%) at 10 mg/kg, whereas vitamin D 3 showed significant estrogen agonistic (82%) and antagonistic activities (39%) at 10 mg/kg.Graphical abstractSubstituted 3-phenyl, 7-methoxy-benzopyran derivavtives and vitamin-D3 (Cholecalciferol, 1) wereevaluated for their estrogen agonistic and antagonistic activities in mature female Sprague-Dawley ratmodel. The benzopyran derivatives, which were made as hybrids of estrogen and vitamin-D3 (pseudovitamin-D3 analogs), showed significant estrogen agonistic activity (upto 48%) and weak estrogenantagonistic activity (upto 6%) at 10 mg kg -1 . Whereas, vitamin-D3 showed significant estrogenagonistic (82%) and antagonistic activities (39%) at 10 mg kg -1 .[IMAGE]

Effects of Egb 761 on bone mineral density, bone microstructure, and osteoblast function: Possible roles of quercetin and kaempferol

The effects of standardized and concentrated extract of Ginkgo biloba, Egb 761, were studied on estrogen deficiency-induced bone loss in ovariectomized (OVx) rats rendered osteopenic. Upon osteopenia development, Egb 761 was administered at a dose of 100mgkg(?1)day(?1) by oral gavage to OVx rats whereas control group received vehicle. Following 5 weeks of treatment, the OVx+Egb 761 group (n=12) of rats exhibited significantly higher whole body BMD and lower bone turnover markers (serum alkaline phosphatase and osteocalcin) than OVx rats that were given vehicle (n=12). BMD levels in excised bones were also found to be higher in both trabecular (most robustly in lumbar vertebrae) and cortical bones of OVx+Egb 761 compared with OVx+vehicle group. Egb 761 did not exhibit estrogen agonistic activity at the uterine level. Microcomputed tomography demonstrated that OVx+Egb 761 group had better bone microarchitectural parameters compared with OVx+vehicle group. Moreover, OVx+Egb 761 group had higher femoral mRNA levels of osterix, type I collagen and osteocalcin compared with OVx+vehicle group. Determination of levels of three flavonoids of Egb 761, that are known to have bone conserving property, in serum and bone marrow suggests that kaempferol and quercetin, and not rutin, likely mediate the beneficial actions observed with Egb 761 treatment. These results show for the first time that oral administration of Egb 761 restores bone mass in aged OVx rats.

Evaluation of estrogenic activity of parabens and their chlorinated derivatives by using the yeast two‐hybrid assay and the enzyme‐linked immunosorbent assay

We assessed the estrogen agonist activities of 21 parabens and their chlorinated derivatives by using yeast two-hybrid assays incorporating either the human or medaka (Oryzias latipes) estrogen receptor alpha (hERalpha and medERalpha, respectively), and by using hERalpha competitive enzyme-linked immunosorbent assay (ER-ELISA). In the two-hybrid assay with hERalpha, five parabens and three chlorinated derivatives exhibited estrogenic activity, and their relative activity (17beta-estradiol [E2] = 1) ranged from 2.0 x 10(-5) to 2.0 x 10(-4), with the highest activity observed in i-butylparaben. In the medERalpha assay, six parabens and six chlorinated derivatives exhibited estrogenic activity and their relative activity ranged from 2.7 x 10(-5) to 3.5 x 10(-3), with the highest activity observed in benzylparaben, its monochlorinated derivative, i-butylparaben, and n-butylparaben. Although medERalpha demonstrated an activity to E2 that was three times lower than that demonstrated by hERalpha, medERalpha has a higher sensitivity to parabens than hERa (1.3-8.9 times). Five parabens and two chlorinated derivatives exhibited a binding affinity to ERa in the ER-ELISA; of the parabens, i-butylparaben exhibited the strongest binding affinity. The yeast two-hybrid assay and the ER-ELISA also revealed that many of the assayed chlorinated parabens were much weaker than the parent compound. In addition, the results mainly showed that parabens with a bulk substituent (e.g., i-butyl and benzyl groups) had a higher activity than those with a sterically small substituent. It is considered that derivatization masks the apparent estrogenic activity of parabens, but the resulting chlorinated compounds may represent a potential hazard and therefore other toxicity tests should be performed to determine the toxicity of the chlorinated derivatives.

Gene expression changes during the development of estrogen-independent and antiestrogen-resistant growth in breast cancer cell culture models

We have established estrogen-independent and antiestrogen-resistant cell lines from hormone-dependent MCF-7 breast cancer cells by long-term culture in the absence of estrogen, or in the presence of antiestrogen toremifene, respectively. By using a cDNA microarray we compared gene expression profiles among estrogen-independent, antiestrogen-resistant and long-term estrogen-treated MCF-7 cells. We also determined how the expression of the differentially expressed genes has developed during the long-term culture of the cell lines. Of the screened 1176 cancer-related genes, FOSL1, TIMP1, L1CAM, GDF15, and MYBL2 were found to be differentially expressed between the cell lines. A change in FOSL1 and TIMP1 expression could be attributed to the development of antiestrogen resistance, whereas induced L1CAM expression was implicated in the development of estrogen-independent growth of the cells. Estrogen regulated genes GDF15 and L1CAM became regulated by toremifene in the later passage number of toremifene-resistant cells, which might be an indication of the developed estrogen-agonistic activity of toremifene in these cells. Our findings suggest a pattern where the hormone-responsive cancer cells, which survive E2 deprivation and/or antiestrogen treatment, first acquire necessary changes in gene expression for transition to maximal growth in the new hormonal environment. Then, after prolonged treatment with antiestrogen, the antiestrogen-resistant cells may eventually generate an E2-agonistic response to antiestrogen, probably acquiring additional growth advantage.