Competitive Estrogen Receptor Research Articles

Safety effect of fermented oyster extract on the endocrine disruptor assay in vitro and in vivo

Oyster (Crassostrea gigas) is a marine bivalve mollusk widely distributed in coastal areas, and have been long widely used in industrial resources. Several studies demonstrated that fermented oyster (FO) extract attribute to bone health, but whether administration of FO play as an endocrine disruptor has not been studied. Therefore, in the present study, we investigated the effect of FO on the endocrine system in vitro and in vivo. As the results of the competitive estrogen receptor (ER) and androgen receptor (AR) binding affinities, FO was not combined with ER-α, ER-β, and AR. However, 17β-estradiol and testosterone, used as positive control, were interacted with ER and AR, respectively. Meanwhile, oral administration of 100 mg/kg and 200 mg/kg of FO doesn’t have any harmful effect on the body weight, androgen-dependent sex accessory organs, estrogen-dependent-sex accessory organs, kidney, and liver in immature rats. In addition, FO supplementation has no effect on the serum levels of luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone, and 17β-estradiol. However, the relative weight of androgen- and estrogen-dependent organs were significantly increased by subcutaneously injection of 4.0 mg/kg of testosterone propionate (TP) and by orally administration of 1.0 μg of 17α-ethynyl estradiol (EE) in immature male and female rats, respectively. Furthermore, TP and EE administration markedly decreased the serum LH and FSH levels, which are similar those of mature Sprague-Dawley (SD) rat. Furthermore, the testosterone and 17β-estradiol levels were significantly enhanced in TP and EE-treated immature rats. Taken together, our findings showed that FO does not interact with ER and AR, suggesting consequentially FO does not play as a ligand for ER and AR. Furthermore, oral administration of FO did not act as an endocrine disruptor including androgenic activity, estrogenic activity, and abnormal levels of sex hormone, indicating FO may ensure the safety on endocrine system to develop dietary supplement for bone health.

Down-regulation of hTERT and Cyclin D1 transcription via PI3K/Akt and TGF-β pathways in MCF-7 Cancer cells with PX-866 and Raloxifene

Human telomerase reverse transcriptase (hTERT) is the catalytic and limiting component of telomerase and also a transcription factor. It is critical to the integrity of the ends of linear chromosomes and to the regulation, extent and rate of cell cycle progression in multicellular eukaryotes. The level of hTERT expression is essential to a wide range of bodily functions and to avoidance of disease conditions, such as cancer, that are mediated in part by aberrant level and regulation of cell cycle proliferation. Value of a gene in regulation depends on its ability to both receive input from multiple sources and transmit signals to multiple effectors. The expression of hTERT and the progression of the cell cycle have been shown to be regulated by an extensive network of gene products and signaling pathways, including the PI3K/Akt and TGF-β pathways. The PI3K inhibitor PX-866 and the competitive estrogen receptor ligand raloxifene have been shown to modify progression of those pathways and, in combination, to decrease proliferation of estrogen receptor positive (ER+) MCF-7 breast cancer cells. We found that combinations of modulators of those pathways decreased not only hTERT transcription but also transcription of additional essential cell cycle regulators such as Cyclin D1. By evaluating known expression profile signatures for TGF-β pathway diversions, we confirmed additional genes such as heparin-binding epidermal growth factor-like growth factor (HB EGF) by which those pathways and their perturbations may also modify cell cycle progression.

Effect of tamoxifen on fatty degeneration and atrophy of rotator cuff muscles in chronic rotator cuff tear: An animal model study.

Fatty degeneration of the rotator cuff muscles is an irreversible change resulting from chronic rotator cuff tear and is associated with poor clinical outcomes following rotator cuff repair. We evaluated the effect of Tamoxifen, a competitive estrogen receptor inhibitor, on fatty degeneration using a mouse model for chronic rotator cuff tear. Sixteen adult mice were divided into two diet groups (Tamoxifen vs. Regular) and subjected to surgical creation of a large rotator cuff tear and suprascapular nerve transection in their left shoulder with the right shoulder serving as a control. The rotator cuff muscles were harvested at 16 weeks and subjected to histology and RT-PCR for adipogenic and myogenic markers. Histology showed substantially decreased atrophy and endomysial inflammation in Tamoxifen group, but no significant differences in the amount of intramuscular adipocytes and lipid droplets compared to the Regular group. With RT-PCR, the operated shoulders showed significant upregulation of myogenin and PPAR-γ, and downregulation of myostatin compared to the nonsurgical shoulder. No significant differences of gene expression were found between the two diet groups. Our study demonstrated that tamoxifen diet leads to decreased muscle atrophy and inflammatory changes following chronic rotator cuff tear, but has no apparent effect on adipogenesis.

Estimation of safety/risk profile of the phytoestrogens 8-prenylnaringenin, 6-(1.1-dimethylallyl)naringenin and naringenin in MCF-7 cells and the rat mammary gland

Evaluation of stimulation of proliferation in female reproductive organs by phytoestrogens of reproductive tract organs is important to estimate their safety/risk profile if used as an alternative to hormone therapy. To assess the proliferative capacity of the flavanones 8-Prenylnaringenin (8-PN), 6-(1.1-dimethylallyl)naringenin (6-DMAN) and Naringenin (Nar) on the mammary gland we compared their estrogenic properties to 17β-Estradiol (E2) in three independent assays. We performed competitive estrogen receptor (ER) ligand binding assays, MCF-7 proliferation studies and a three day exposure experiment in young adult ovariectomized female rats. 8-PN displayed a high relative binding affinity for both ERs with a preference for ERα (19.46%) compared to ERβ (6.5%). Among the tested flavanones 8-PN had the strongest mitotic effect on MCF-7 cells. In vivo 8-PN increased the number of terminal end buds in the mammary gland and the protein expression of the proliferation markers Ki-67 and PCNA in epithelial ductal cells and further led to an increase of Amphiregulin (Areg) mRNA expression, endpoints correlated to mammary gland proliferation. 6-DMAN also possesses a higher relative binding affinity for the ERα than for the ERβ and stimulated the cell proliferation of MCF-7 cells. It enhanced Ki-67 protein expression and β-Casein mRNA expression in vivo. Nar displayed the weakest estrogenic properties and was the only tested compound with a higher affinity for the ERβ than the ERα. It displayed proliferative effects on MCF-7 cells and slightly induced Ki-67 protein expression in vivo.

Abstract 625: Proliferative effects of the phytoestrogens 8-Prenylnaringenin, 6-(1.1-dimethylallyl)naringenin and Naringenin in MCF-7 cells and the rat mammary gland

Abstract Evaluation of stimulation of proliferation in female reproductive organs by phytoestrogens of reproductive tract organs is important to estimate their safety/risk profile if used as an alternative to hormone therapy. To assess the proliferative capacity of the flavanones 8-Prenylnaringenin (8-PN), 6-(1.1-dimethylallyl)naringenin (6-DMAN) and Naringenin (Nar) on the mammary gland we compared their estrogenic properties to 17β-Estradiol (E2) in three independent assays. We performed competitive estrogen receptor (ER) ligand binding assays, MCF-7 proliferation studies and a three day exposure experiment in young adult ovariectomized female rats. 8-PN displayed a high relative binding affinity for both ERs with a preference for ERα (19.46 %) compared to ERβ (6.5 %). Among the tested flavanones 8-PN had the strongest mitotic effect on MCF-7 cells. In vivo 8-PN increased the number of terminal end buds (TEB) in the mammary gland and the protein expression of the proliferation markers Ki-67 and PCNA in epithelial ductal cells and further led to an increase of Amphiregulin (Areg) mRNA expression, endpoints correlated to mammary gland proliferation. 6-DMAN also possesses a higher relative binding affinity for the ERα than for the ERβ (1.46 %, 0.84 %) and stimulated the cell proliferation of MCF-7 cells. It enhanced Ki-67 protein expression and β-Casein mRNA expression in vivo. Nar displayed the weakest estrogenic properties and was the only tested compound with a higher affinity for the ERβ than the ERα (0.5 %, 0.08 %). It displayed proliferative effects on MCF-7 cells and slightly induced Ki-67 protein expression in vivo. Citation Format: Janina Helle, Oliver Zierau, Kristin Kräker, Annekathrin Keiler, Günter Vollmer, JoEllen Welsh, Georg Kretzschmar. Proliferative effects of the phytoestrogens 8-Prenylnaringenin, 6-(1.1-dimethylallyl)naringenin and Naringenin in MCF-7 cells and the rat mammary gland. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 625. doi:10.1158/1538-7445.AM2014-625

Assessment of the proliferative capacity of the flavanones 8-prenylnaringenin, 6-(1.1-dimethylallyl)naringenin and naringenin in MCF-7 cells and the rat mammary gland

8-Prenylnaringenin (8-PN) and naringenin (Nar) are phytoestrogens found in food items and nutritional supplements, while 6-(1.1-dimethylallyl)naringenin (6-DMAN) is a component of an African plant. Besides their assumed beneficial effects they may promote mammary and endometrial cancer. We therefore assessed their proliferative and estrogenic potential on the mammary gland in vitro and in vivo. In competitive estrogen receptor (ER) ligand binding assays 8-PN displayed a high relative binding affinity for both ERs with a preference for ERα and had the strongest mitotic effect on MCF-7 cells among the test substances. In a three day exposure in young adult ovariectomized female rats 15mg/kg 8-PN had the highest capacity to increase the number of terminal end buds (TEB) in the mammary gland and stimulated expression of proliferation markers in epithelial ductal cells, followed by 6-DMAN and Nar, but overall their capacity to stimulate proliferation was weak in comparison to 17β-Estradiol (E2).

Tamoxifen-Independent Recombination in the RIP-CreER Mouse

BackgroundThe inducible Cre-lox system is a valuable tool to study gene function in a spatial and time restricted fashion in mouse models. This strategy relies on the limited background activity of the modified Cre recombinase (CreER) in the absence of its inducer, the competitive estrogen receptor ligand, tamoxifen. The RIP-CreER mouse (Tg (Ins2-cre/Esr1) 1Dam) is among the few available β-cell specific CreER mouse lines and thus it has been often used to manipulate gene expression in the insulin-producing cells of the endocrine pancreas.Principal FindingsHere, we report the detection of tamoxifen-independent Cre activity as early as 2 months of age in RIP-CreER mice crossed with three distinct reporter strains.SignificanceEvidence of Cre-mediated recombination of floxed alleles even in the absence of tamoxifen administration should warrant cautious use of this mouse for the study of pancreatic β-cells.

Screening of chemicals with anti-estrogenic activity using in vitro and in vivo vitellogenin induction responses in zebrafish ( Danio rerio)

Growing concern over possible adverse effects of endocrine-disrupting chemicals (EDCs) has driven the development of associated screening methods. The use of the vitellogenin (VTG) induction response in cultured teleost hepatocytes has been suggested as an in vitro screening assay for EDCs. However, current data do not sufficiently support this assay in the routine screening of chemicals. This study established and validated the use of primary cultured hepatocytes from zebrafish to screen chemicals for anti-estrogenic activities. Here we measured the transcript levels of selected hepatic estrogen-response genes, including vtg1, vtg2 and erα. Two model anti-estrogens, letrozole (LET), an aromatase inhibitor, and tamoxifen (TAM), a competitive estrogen receptor, were selected as representative chemicals. Additionally, comparisons between in vitro and in vivo assays were performed. As expected, there were concentration-dependent decreases for all three genes in the liver of female zebrafish exposed to LET in vivo for 72 h. Similar responses were observed in males. As for in vitro testing, no discernable alterations in the gene transcripts were found in hepatocytes from males or females. In the case of TAM, exposure for 72 h caused transcriptional reduction of hepatic estrogen-response genes in females in vivo and in vitro. In males, low concentrations of TAM resulted in increased expression of genes, while the expression decreased slightly at higher concentrations. Since these observations were in agreement with the pharmaceutical properties of two tested chemicals, the primary hepatocyte culture could be a promising tool for screening suspected EDCs.

High-Content Screening and Mechanism-Based Evaluation of Estrogenic Botanical Extracts

Symptoms associated with menopause can greatly affect the quality of life for women. Botanical dietary supplements have been viewed by the public as safe and effective despite a lack of evidence indicating a urgent necessity to standardize these supplements chemically and biologically. Seventeen plants were evaluated for estrogenic biological activity using standard assays: competitive estrogen receptor (ER) binding assay for both alpha and beta subtypes, transient transfection of the estrogen response element luciferase plasmid into MCF-7 cells expressing either ER alpha or ER beta, and the Ishikawa alkaline phosphatase induction assay for both estrogenic and antiestrogenic activities. Based on the combination of data pooled from these assays, the following was determined: a) a high rate of false positive activity for the competitive binding assays, b) some extracts had estrogenic activity despite a lack of ability to bind the ER, c) one extract exhibited selective estrogen receptor modulator (SERM) activity, and d) several extracts show additive/synergistic activity. Taken together, these data indicate a need to reprioritize the order in which the bioassays are performed for maximal efficiency of programs involving bioassay-guided fractionation. In addition, possible explanations for the conflicts in the literature over the estrogenicity of Cimicifuga racemosa (black cohosh) are suggested.

Modeling binding equilibrium in a competitive estrogen receptor binding assay

Although the free concentration is more significant in the environmental chemistry and toxicology of receptor-mediated toxicants, few studies have been conducted to use it as a dose-metric. The relative binding affinity of three model endocrine disrupting compounds, diethylstilbestrol (DES), ethynylestradiol (EE2), and bisphenol A (BPA), were evaluated using a competitive ELISA with human estrogen receptor α. After measuring the available receptors and the dissociation constant for 17β-estradiol, binding inhibition curves using the free concentration as the dose-metric were obtained by assuming species equilibrium in the ELISA system and compared with apparent inhibition curves generated using the nominal concentration as the dose-metric. Because ligand binding to estrogen receptors may reduce its free concentration in the assay system, the differences between the two curves for free and nominal concentrations are more significant for more strongly binding ligands. The ratio of a compound’s nominal concentration causing 50% inhibition (IC50) to the IC50 of DES, the positive control, was strongly affected by specific assay conditions, while that estimated by modeling free concentration is independent of receptor concentration, indicating that the free concentration is a better dose-metric for a competitive binding assay.

Review of the Potential Environmental and Human Health–Related Hazards and Risks from Long-Term Exposure to p-tert-Octylphenol

ABSTRACT Para-tert-octylphenol (OP) is an important chemical intermediate mainly used for production of phenolic resins and lacquers. OP has weak estrogen-like activity in vitro, with induction of vitellogenin in fish hepatocytes and competitive estrogen receptor binding and estrogen-responsive gene expression in mammalian systems. Induction of vitellogenin in fish plasma and liver, in vivo, has also been measured and weak estrogen-like activity in rodent screening assays occurs but at much lower potency than predicted by the in vitro activity. Conventional aquatic wildlife toxicity testing shows effects on survival, growth and development, and reproduction occurring at concentrations lower than the estrogen-like responses predicted from screening assays. Studies in rodents have provided evidence of metabolic saturation at high doses of OP, and a multigeneration reproductive study confirms the lack of estrogen-like activity below this metabolic saturation as well as the lack of reproductive toxicity. This article reviews the weight of evidence for the aquatic and mammalian data and compares the results of the definitive assays to those of the screening tests. The results show that the estrogen-like activity in screening tests is not predictive of results from definitive testing, and that risk assessment decisions should be made using conventional toxicity endpoints.

Posttranscriptional regulation of albumin gene expression in Xenopus liver: evidence for an estrogen receptor-dependent mechanism.

We have previously shown that estrogen administration to male Xenopus laevis results in the posttranscriptional suppression of serum albumin mRNA concurrent with the transcriptional activation of the genes for the yolk protein precursor vitellogenin. To determine whether the posttranscriptional regulation of albumin gene expression is mediated through a mechanism involving the high affinity estrogen receptor protein or through a receptor-independent mechanism involving a middle affinity cytoplasmic estrogen-binding protein we examined the effects of the competitive estrogen receptor antagonist 4-hydroxytamoxifen. Administration of 4-hydroxytamoxifen 24 h before estradiol completely blocked both the suppression of albumin mRNA and the transcriptional activation of the vitellogenin genes. Albumin gene transcription remained constitutive under all treatment regimens. Competitive binding experiments demonstrated that 4-hydroxytamoxifen has an affinity for the estrogen receptor similar to that of estradiol. However, 4-hydroxytamoxifen displays little or no interaction with the middle affinity cytoplasmic estrogen-binding protein. These data indicate that the estrogen receptor occupies a key role in the posttranscriptional regulation of albumin mRNA.