Role of miR-203 in estrogen receptor-mediated signaling in the rat uterus and endometrial carcinoma.

The role of microRNAs (miRNA) in estrogen receptor (ER) signaling in the uterus and in endometrial cancer is not well understood. We therefore analyzed miRNA expression in uterine samples from a standard 3-day uterotrophic assay using young female adult rats to identify E2-regulated miRNAs. Microarray analysis identified 47 E2 down-regulated miRNAs including miR-30a, and 25 E2up-regulated miRNAs including miR-672, miR-203, and miR-146b. The strongly E2-upregulated miR-203 was selected for further analysis. miR-203 was deleted in the rat endometrial adenocarcinoma cell line, RUCA-I, using CRISPR/CAS9. Five clones devoid of miR-203 expression were generated. Proliferation was reduced and G2-arrest was observed in all miR-203 deficient RUCA-I clones. Transfection with a miR-203-3p mimic partially rescues this effect. Comparison of mRNA expression in three miR-203 knockout clones to wild type RUCA-I cells reveals 566 miR-203-upregulated and 592 miR-203-downregulated genes. 43 of the genes that are upregulated by miR-203 knockout in vitro are downregulated in the uterus by E2. Of these Acer2, Zbtb20, Ptn, Rcbtb2, Mum1l1, Hmgn3, and Nfat5 possess one or more seed sequence matches in their 3'-UTR that are predicted to be targets of miR-203. These data demonstrate the importance of E2 regulated miRNAs in general, and miR-203 in particular, for E2 regulated gene expression and physiological processes including proliferation and cell migration, in the uterus as well as in the etiology of endometrial carcinomas.

Progesterone, as well as 17ß-estradiol, is important for regulating AHR battery homoeostasis in the rat uterus.

Several studies indicate that the aryl hydrocarbon receptor (AHR), which plays an important role in mediating the toxicity of many industrial chemicals, plays an important role in the physiology of female reproductive tract organs. This makes it likely that the AHR and additional components of the AHR signalling pathway are under the control of female sex steroids. In a previous study, we could already demonstrate the regulation of many members of the AHR battery by 17ß-estradiol (E2) in the uterus of rats. In this study, we addressed the potential role of progesterone (P4) in this context. In a comparative approach using ovariectomized rats which were treated for 3 days with either vehicle control, E2, progesterone (P4) or the combination of both hormones in addition to sham-operated animals, we could demonstrate that in addition to E2, P4 is also an important factor in regulating AHR signalling in the rat uterus. P4 has effects similar to E2 on uterine Ahr, Arnt and Arnt2 mRNA levels, resulting in a downregulation of these genes, while the E2-mediated downregulation of key AHR response genes Cyp1a1, Gsta2 and Ugt1 is completely antagonized by P4. As with E2, P4 leads to an increase in uterine AHR levels, especially in the endometrial epithelium despite the decrease in corresponding mRNA levels. This indicates a complex gene-specific regulatory network involving E2, P4 and possibly AHR itself to maintain all components of the AHR signalling cascade at the required levels during all stages of the oestrous cycle and pregnancy.

Erythroidine alkaloids: a novel class of phytoestrogens.

Erythrina poeppigiana is a medicinal plant which is widely used in Asia, Latin America, and Africa in traditional remedies for gynecological complications and maladies. In continuation of studies for the discovery of novel phytoestrogens, four erythroidine alkaloids, namely α-erythroidine, ß-erythroidine, and their oxo-derivatives 8-oxo-α-erythroidine and 8-oxo-ß-erythroidine, were isolated and structurally characterized from the methanolic extract of the stem bark of E. poeppigiana. Due to the high amounts of erythroidines in the extract and considering the widespread utilization of Erythrina preparations in traditional medicine, the exploration of their estrogenic properties was performed. The estrogenicity of the isolated erythroidines was assayed in various estrogen receptor-(ER)-dependent test systems, including receptor binding affinity, cell culture based ER-dependent reporter gene assays, and gene expression studies in cultured cells using reverse transcription polymerase chain reaction techniques. α-Erythroidine and ß-erythroidine showed binding affinity values for ERα of 0.015 ± 0.010% and 0.005 ± 0.010%, respectively, whereas only ß-erythroidine bound to ERß (0.006 ± 0.010%). In reporter gene assays, both erythroidines exhibited a significant dose-dependent estrogenic stimulation of ER-dependent reporter gene activity in osteosarcoma cells detectable already at 10 nM. Results were confirmed in the MVLN cells, a bioluminescent variant of MCF-7 breast cancer cells. Further, α-erythroidine and ß-erythroidine both induced the enhanced expression of the specific ERα-dependent genes trefoil factor-1 and serum/glucocorticoid regulated kinase 3 in MCF-7 cells, confirming estrogenicity. Additionally, using molecular docking simulations, a potential mode of binding on ERα, is proposed, supporting the experimental evidences. This is the first time that an estrogenic profile is reported for erythroidine alkaloids, potentially a new class of phytoestrogens.

Hop extracts and hop substances in treatment of menopausal complaints.

Hop extract is a long used medicinal product and, regarding hormonal activities, in 1999 a number of prenylflavanones have been identified as its major constituents with 8-prenylnaringenin (8-PN) being the main active estrogenic compound. There have been several in vivo studies performed that demonstrate the potential of hop extract and the single compound 8-PN to alleviate climacteric symptoms like osteoporosis, vasomotoric complaints, and sexual motivation. On the other hand, only a few clinical studies have been performed so far, and these mainly focused on menopausal discomforts, especially hot flushes, yielding rather inconclusive results. Despite preferentially activating estrogen receptor α, 8-PN is only slightly uterotrophic, but it also elucidates estrogenic effects on the mammary gland. In conclusion, although hop extract and especially 8-PN are promising candidates as a relief for climacteric symptoms, data on the safety and efficacy is still scarce.

Effect of 17ß-estradiol and flavonoids on the regulation of expression of newly identified oestrogen responsive genes in a rat raphe nuclei-derived cell line.

Due to the health risks attributed to perimenopausal hormone therapy, phytoestrogens such as flavonoids are receiving widespread attention to help alleviate menopausal symptoms, including hormone-driven mood disorders. Based on our previous reporter gene study regarding their transactivational activity in raphe nuclei cells from a brain region involved in regulation of mood disturbances, we herein study their effects on the regulation of expression of 17ß-estradiol (E2)-regulated genes. DNA microarray was used to globally assess E2-induced gene expression in RNDA cells, a rat raphe nuclei-derived cellular model expressing oestrogen receptor ß. Out of 212 regulated genes, six were selected for verification and as endpoints for the effect of flavonoids on the regulation of mRNA expression in proliferating as well as differentiating RNDA cells. Under proliferative conditions, E2 up-regulated mRNA expression of Cml-5, Sox-18 and Krt-19. Similar effects were observed in response to 8-prenylnaringenin (8-PN), genistein (GEN), daidzein (DAI) and equol (EQ). In line with E2, mRNA expression of Nefm and Zdhhc-2 was down-regulated following 8-PN, GEN, DAI, EQ and naringenin treatment. No regulation was observed on Slc6a4 mRNA expression in response to E2 or the flavonoids in proliferating RNDA cells. When cells were shifted to conditions promoting differentiation, changes in cell morphology, in mRNA expression levels and in responsiveness towards E2 and the tested flavonoids were noticed. These expression studies additionally highlighted some of the genes as markers for RNDA cellular differentiation. RNDA cells should prove useful to elucidate molecular and cellular mechanisms of exogenous oestrogen receptor ligands with neural cell populations.

The social status of the male Nile tilapia (Oreochromis niloticus) influences testis structure and gene expression.

Dominant and territorial behaviour are known social phenomena in cichlids and social stress influences reproduction and growth. The gonadotropic hormones trigger spermatogenesis and subordinate males have typically lower levels of gonadotropins than dominant males. In this study, we compared testis morphology and gene expression of dominant and subordinate Nile tilapia males (d- and s-males) in socially stable communities. The d-males had the highest gonadosomatic index but they were not the largest animals in the majority of studied cases. Long-term d-males showed large groups of Leydig cells and hyperplasia of the tunica albuginea due to numerous cytochrome-P450-11ß-hydroxylase (Cyp11b) expressing myoid cells. Increased Cyp11b expression in d-males was reflected by elevated 11-ketotestosterone plasma values. However, immunofluorescence microscopy and expression analysis of selected genes revealed that most s-males conserved their capability for spermatogenesis and are, therefore, ready for reproduction when the social environment changes. Moreover, in s-males gene expression analysis by quantitative RT-PCR showed increased transcript levels for germ line-specific genes (vasa, sox2 and dmc1) and Sertoli-specific genes (amh, amhrII and dmrt1) whereas gene expression of key factors for steroid production (sf1 and cyp11b) were reduced. The Nile tilapia is a promising model to study social cues and gonadotropic signals on testis development in vertebrates.

Cadmium modulates expression of aryl hydrocarbon receptor-associated genes in rat uterus by interaction with the estrogen receptor.

Estrogen-like effects of the heavy metal cadmium have been reported in both in vitro and in vivo studies. Yet, the molecular mechanisms involved in the hormonal activity of cadmium ions have not been fully elucidated. There are extensive data on cross-talk between aryl hydrocarbon receptor (AhR) and estrogen receptor (ER). Recently, 17ß-estradiol (E(2)) was found to modulate the expression of AhR and AhR-regulated genes in rat uterus (Kretzschmar et al. in Mol Cell Endocrinol 321:253-257, 2010). Thus, we hypothesized that cadmium may also affect AhR signaling and examined whether cadmium or E(2) modulate AhR-associated genes via the ER in rat uterus. Ovariectomized Wistar rats received E(2) (0.5 mg/kg bw) or cadmium chloride (0.05 and 2 mg/kg bw i.p.) alone and in combination with the pure anti-estrogen ZK191703. We also co-treated a group with E(2) and cadmium 2 mg/kg bw to assess how they act in concert. Uterus wet weight, uterus epithelial height, complement C3 mRNA, and progesterone receptor (PR) protein expression served as estrogen response parameters, and expression of Mt1a mRNA was analyzed as a cadmium responsive gene. The expression of AhR protein and AhR-associated gene expression, i.e., Ahr, Arnt1, Arnt2, Cyp1a1, and Gsta2, were analyzed to examine effects on AhR-mediated signaling pathways in the uterus of all groups. Both, E(2) and cadmium induced C3 and PR expression, and this was antagonized by ZK191703. Mt1a expression was clearly induced by cadmium but slightly reduced by E(2) compared to controls. Uterine Ahr, Arnt1, Arnt2, and Cyp1a1 expression was modulated by E(2) via the ER since down-regulation by E(2) was reversed by anti-estrogen. Cadmium apparently also modulated Cyp1a1 expression via the ER. Furthermore, cadmium-induced AhR was antagonized by E(2,) and anti-estrogen-induced Gsta2 expression was antagonized by cadmium. Together our findings provide evidence for cross-talk of ER and AhR in the rat uterus.

Regulation of uterine AHR battery gene expression by 17ß-Estradiol is predominantly mediated by estrogen receptor α.

The aryl hydrocarbon receptor (AHR) is known to mediate the cellular response to numerous xenobiotics including dioxin. Surprisingly AHR knockout mice provide evidence for the involvement of the AHR signalling cascade in estrogen regulated physiological functions of the female reproductive system. Several studies already aimed to investigate the impact of the AHR mediated xenobiotic response pathway on estrogen receptor (ER) signalling, whereas on contrary availability of data describing the effect of 17ß-Estradiol (E2) on the AHR signalling cascade is rather limited. In this study we observed an inhibitory effect of E2 treatment on uterine Ahr, Arnt, Arnt2, Ahrr, Cyp1a1, Ugt1 and Nfe2l2 gene expression in ovariectomized Wistar rats, whereas Cyp1b1, Nqo1 and Gsta2 displayed an increased transcription. The usage of the ER selective agonists, 16α-LE(2) (ERα selective) and 8ß-VE(2) (ERß selective), enabled us to distinguish between ER subtype specific responses. On mRNA level the observed changes in gene expression were mainly mediated by ERα except for the expression of Nqo1. In most cases the activation of ERß caused effects opposite to the ones observed following activation of ERα. Despite the significant changes in AHR mRNA levels immunohistochemical staining uterine tissue section did not reveal changes of the AHR protein level. Taken together our results validate, support and extend the hypothesis of uterine crosstalk between AHR and ER signalling pathways. Furthermore they give an insight into how the AHR and its related genes may participate in E2 dependent uterine physiological processes and provide another potential mechanism of action for xenoestrogens.

AHR agonistic effects of 6-PN contribute to potential beneficial effects of Hops extract.

Hops (Humulus lupulus) is used as an alternative to hormone replacement therapy due to the phytoestrogen, 8-prenylnaringenin (8-PN). To examine the potential risks/benefits of hops extract and its compounds (8-PN and 6-prenylnaringenin, 6-PN), we aimed to evaluate the estrogen receptor α (ERα) and aryl hydrocarbon receptor (AHR) signaling pathways in human endometrial cancer cells. Hops extract, 8-PN and 6-PN showed estrogenic activity. Hops extract and 6-PN activated both ERα and AHR pathways. 6-PN increased the expression of the tumor suppressor gene (AHRR), and that of genes involved in the estrogen metabolism (CYP1A1, CYP1B1). Although 6-PN might activate the detoxification and genotoxic pathways of estrogen metabolism, hops extract as a whole only modulated the genotoxic pathway by an up-regulation of CYP1B1 mRNA expression. These data demonstrate the relevant role of 6-PN contained in the hops extract as potential modulator of estrogen metabolism due to its ERα and AHR agonist activity.

Time dependency of uterine effects of naringenin type phytoestrogens in vivo.

Phytoestrogens exhibit significant estrogen agonistic/antagonistic properties in animals and humans. Naturally occurring flavonoids with a naringenin backbone like 8-prenylnaringenin (8-PN) and 6-(1,1-dimethylallyl)naringenin (6-DMAN) are considered to be some of the most potent phytochemicals activating nuclear receptors. 8-PN is a more potent estrogenic substance while 6-DMAN appears to have a higher antiandrogenic potency, however these are less well characterized compared to other phytoestrogens such as genistein. The aim of this study was to assess the estrogenic properties of 8-PN and 6-DMAN in an ovariectomized in vivo rat model. 8-PN and 6-DMAN were applied at concentrations of 15mg/kgBW. We assessed the uterotrophic response after 7h, 24h and 72h of treatment. In contrast to 8-PN, 6-DMAN did not alter uterine wet weight or the level of expression of proliferation markers at any time point. In contrast to the uterotrophic response, 6-DMAN stimulated uterine mRNA expression of estrogen responsive genes carrying an estrogen response element (ERE) in the ovariectomized rats, but to a lesser extent than E2 and 8-PN. In all treatment regimens, the mRNA expression of estrogen receptors alpha and beta mRNA was measured. In summary, we assessed the time dependent uterine responses and estrogenic activities of 6-DMAN and 8-PN. In contrast to 8-PN which mimicked the E2 induced responses on uterine wet weight and gene expression, 6-DMAN has no uterotrophic effect and only regulated the mRNA expression of genes carrying an ERE. Therefore, 6-DMAN is an exciting candidate molecule for future investigations and potentially a natural occurring selective estrogen receptor modulator.

Effects of the chemically synthesized flavanone 7-(O-prenyl)naringenin-4'-acetate on the estrogen signaling pathway in vivo and in vitro.

The flavanone naringenin is known to possess only weak estrogenic properties, but some of its derivatives such as 8-prenylnaringenin are potent phytoestrogens. The aim of this study was to further clarify structure-function relationships of flavanones regarding their estrogenic or antiestrogenic properties by characterizing the new chemically synthesized naringenin derivative 7-(O-prenyl)naringenin-4'-acetate (7-O-PN). A yeast based reporter gene assay and MVLN cells, a MCF-7-derived cell line that possesses a luciferase reporter gene under the control of a vitellogenin estrogen responsive element, were used to investigate estrogenic actions of 7-O-PN in vitro. Estradiol (E2) has been used as a positive control. Subsequently a 3-day rat uterotrophic assay was performed to test for estrogenic effects. In addition, mRNA expression of estrogen sensitive genes in the uteri of these rats was measured using real time rtPCR. While E2 leads to a strong dose dependent signal in the yeast based reporter gene assay and in MVLN cells, 7-O-PN shows mild E2 antagonistic properties at concentrations 10(-8) and 10(-7)M, E2 agonistic properties at 10(-6) and 10(-5)M in MVLN cells and no effects on the yeast based system. In contrast to E2 treatment, 7-O-PN treatment did not increase uterus wet weight compared to the negative control. These findings are supported by mRNA expression studies of proliferation markers. Additionally, mRNA expression studies of estrogen regulated genes revealed very strong antiestrogenic properties of 7-O-PN regarding regulation of complement C3 expression while some estrogenic effects could be observed on the expression of estrogen receptor beta, clusterin and possibly on progesterone receptor and vascular endothelial growth factor.

Effects of the aryl hydrocarbon receptor agonist 3-methylcholanthrene on the 17ß-estradiol regulated mRNA transcriptome of the rat uterus.

Polycyclic aromatic hydrocarbons (PAHs) are products of incomplete combustion of organic compounds, abundant in exhaust fumes and cigarette smoke. They act by binding to the aryl hydrocarbon receptor (AHR) which induces expression of phase 1 and phase 2 enzymes in the liver. PAH induced AHR activation may also lead to adverse effects by modulating other pathways, for example estrogen receptor (ER) signaling in the female reproductive tract. We have investigated the effects of the PAH 3-methylcholanthrene (3-MC) on 17ß-estradiol (E2) dependent signaling in the uterus of ovariectomized rats to characterize the cross talk between AHR and ER on an mRNA transcriptome wide scale. A standard three day uterotrophic assay was performed in young adult Lewis rats. Treatment induced effects were analyzed using histology, immunohistochemistry and gene expression analysis by microarray and qPCR. 3-MC shows broad E2 antagonistic effects on uterine mRNA transcription of the vast majority of E2 regulated genes, significantly altering prostaglandin biosynthesis, complement activation, coagulation pathways and other inflammatory response pathways. The regulation of ER expression in the uterus, but not the regulation of E2 metabolism in the liver, was identified as a potentially important factor in mediating this general antiestrogenic effect. The regulation of prostaglandin biosynthesis by E2 is important for inflammation-like events during pregnancy including the initiation of birth. Our results suggest that adverse effects of PAHs on prostaglandin related pathways are likely caused by the interference with E2 signaling, specifically by inhibiting the E2 mediated downregulation of PGF2α. Characterization of the generalized antagonistic effect of 3-MC on E2 dependent signaling in the rat uterus thus contributes to a better understanding of molecular mechanisms of the toxicity of PAHs in female reproductive organs.

A standardized Humulus lupulus (L.) ethanol extract partially prevents ovariectomy-induced bone loss in the rat without induction of adverse effects in the uterus.

BACKGROUND: Hops (Humulus lupulus (L.)) dietary supplements are of interest as herbal remedies to alleviate menopausal symptoms, such as hot flushes, depression and anxiety. So far, the evidence regarding estrogenic and related properties of hops preparations has been considered insufficient for a market authorization for menopausal indications. PURPOSE: The study aims to investigate a chemically standardized hops extract regarding its safety in the uterus, as wells as its efficacy to prevent bone loss in the ovariectomized rat model. STUDY DESIGN/METHODS: Female Wistar rats were ovariectomized and divided into a control group receiving phytoestrogen-free diet, a group treated with E2benzoate (0.93 mg/kg body weight/d) and a group treated with the standardized hops extract (60 mg/kg body weight/d) for 8 weeks. Micro-computed tomography of the tibiae and vertebrae, as wells as histological changes in the uterus and tibia were analyzed. RESULTS: Neither uterotrophic nor proliferative effects were observed in the endometrium in response to the oral 8-week administration of the hops extract. However, site-dependent skeletal effects were observed. The hops extract significantly decreased the number of osteoclasts in the tibial metaphysis and prevented reduction of the trabecular thickness that resulted from estradiol depletion. In contrast, the hops extract did not prevent the ovariectomy-induced micro-architectural changes in the lumbar vertebra. Certain parameters (e.g. thickness and number of trabeculae) were even found to be below the values determined in the ovariectomized control group. CONCLUSION: Taken together, the results provide evidence for the safety of the standardized hops extract and point to a weak bone type-specific, protective effect on bone loss following estradiol depletion.

Cross-Talk in the Female Rat Mammary Gland: Influence of Aryl Hydrocarbon Receptor on Estrogen Receptor Signaling.

BACKGROUND: Cross-talk between the aryl hydrocarbon receptor (AHR) and the estrogen receptor (ER) plays a major role in signaling processes in female reproductive organs. OBJECTIVES: We investigated the influence of the AHR ligand 3-methylcholanthrene (3-MC) on ER-mediated signaling in mammary gland tissue of ovariectomized (ovx) rats. METHODS: After 14 days of hormonal decline, ovx rats were treated for 3 days with 4 µg/kg 17ß-estradiol (E2), 15 mg/kg 8-prenylnaringenin (8-PN), 15 mg/kg 3-MC, or a combination of these compounds (E2 + 3-MC, 8-PN + 3-MC). Whole-mount preparations of the mammary gland were used to count terminal end buds (TEBs). Protein expression studies (immunohistochemistry, immunofluorescence), a cDNA microarray, pathway analyses, and quantitative real-time polymerase chain reaction (qPCR) were performed to evaluate the interaction between AHR- and ER-mediated signaling pathways. RESULTS: E2 treatment increased the number of TEBs and the levels of Ki-67 protein and progesterone receptor (PR); this treatment also changed the expression of 325 genes by more than 1.5-fold. Although 3-MC treatment alone had marginal impact on gene or protein expression, when rats were co-treated with 3-MC and E2, 3-MC strongly inhibited E2-induced TEB development, protein synthesis, and the expression of nearly half of E2-induced genes. This inhibitory effect of 3-MC was partially mirrored when 8-PN was used as an ER ligand. The anti-estrogenicity of ligand-activated AHR was at least partly due to decreased protein levels of ERα in ductal epithelial cells. CONCLUSION: Our data show transcriptome-wide anti-estrogenic properties of ligand-activated AHR on ER-mediated processes in the mammary gland, thereby contributing an explanation for the chemopreventive and endocrine-disrupting potential of AHR ligands. CITATION: Helle J, Bader MI, Keiler AM, Zierau O, Vollmer G, Chittur SV, Tenniswood M, Kretzschmar G. 2016. Cross-talk in the female rat mammary gland: influence of aryl hydrocarbon receptor on estrogen receptor signaling. Environ Health Perspect 124:601-610; http://dx.doi.org/10.1289/ehp.1509680.

No estrogen-like effects of an isopropanolic extract of Rhizoma Cimicifugae racemosae on uterus and vena cava of rats after 17 day treatment.

The effects of black cohosh extracts (Rhizoma Cimicifugae racemosae) on primary estrogen target organs, like mammary gland and endometrium are better described then those on other estrogen-sensitive systems e.g. the vasculature. We therefore treated ovariectomized DA/Han rats for 17 days with an isopropanolic Cimicifuga racemosa rhizoma extract (iCR) alone and in combination with the pure antiestrogen fulvestrant. As control groups vehicle, estradiol, fulvestrant, and estradiol fulvestrant cotreatment were used. Effects of all substances were investigated by vena cava and uterine gene expression analysis using real-time-PCR. Uterus wet weight was increased after estradiol treatment compared to the negative controls but none of the other treatments including the treatment with iCR had a uterotrophic effect. While estradiol-induced changes in uterine gene expression were mainly analogous to those detectable in shorter term experiments, iCR showed no or slightly antiestrogenic effects on gene expression in the uterus. This is mirrored in the vena cava where iCR had a very minor impact on the expression of the genes analyzed. While C. racemosa is effectively used for treatment of peri- and post-menopausal symptoms for a long time its mechanism of action remains unresolved. Contrary to earlier suggestions C. racemosa does not seem to act as an estrogen agonist, but possibly as a weak antiestrogen.

Assessment of the effects of naringenin-type flavanones in uterus and vagina.

The potential utilization of plant secondary metabolites possessing estrogenic properties as alternatives to the classical hormone replacement therapy (HRT) for the relief of postmenopausal complaints asks for an evaluation regarding the safety in reproductive organs. In order to contribute to the estimation of the safety profile of the flavanones naringenin (Nar), 8­prenylnaringenin (8PN) and 6­(1,1­dimethylally) naringenin (6DMAN), we investigated uterus and vagina derived from a three­day uterotrophic assay in rats. Also, we investigated the metabolite profile resulting from the incubation of the three substances with liver microsomes. While no metabolites were detectable for naringenin, hydroxylation products were observed for 8PN and 6DMAN after incubation with human as well as rat liver microsomes. The parent compound naringenin did not evoke any estrogenic responses in the investigated parameters. A significant increase of the uterine wet weight, uterine epithelial thickness and proliferating vaginal cells was observed in response to 8PN, questioning the safety of 8PN if applied in the human situation. In contrast, no estrogenic effects on the reproductive organs were observed for 6DMAN in the conducted study, rendering it the compound with a more promising safety profile, therefore justifying further investigations into its efficacy to alleviate postmenopausal discomforts.

Alpinumisoflavone and abyssinone V 4'-methylether derived from Erythrina lysistemon (Fabaceae) promote HDL-cholesterol synthesis and prevent cholesterol gallstone formation in ovariectomized rats.

OBJECTIVES: Erythrina lysistemon was found to improve lipid profile in ovariectomized rats. Alpinumisoflavone (AIF) and abyssinone V 4'-methylether (AME) derived from this plant induced analogous effects on lipid profile and decreased atherogenic risks. To highlight the molecular mechanism of action of these natural products, we evaluated their effects on the expression of some estrogen-sensitive genes associated with cholesterol synthesis (Esr1 and Apoa1) and cholesterol clearance (Ldlr, Scarb1 and Cyp7a1). METHODS: Ovariectomized rats were subcutaneously treated for three consecutive days with either compound at the daily dose of 0.1, 1 and 10 mg/kg body weight (BW). Animals were sacrificed thereafter and their liver was collected. The mRNA of genes of interest was analysed by quantitative real-time polymerase chain reaction. KEY FINDINGS: Both compounds downregulated the mRNA expression of Esr1, a gene associated with cholesterogenesis and cholesterol gallstone formation. AME leaned the Apoa1/Scarb1 balance in favour of Apoa1, an effect promoting high-density lipoprotein (HDL)-cholesterol formation. It also upregulated the mRNA expression of Ldlr at 1 mg/kg/BW per day (25%) and 10 mg/kg/BW per day (133.17%), an effect favouring the clearance of low-density lipoprotein (LDL)-cholesterol. Both compounds may also promote the conversion of cholesterol into bile acids as they upregulated Cyp7a1 mRNA expression. CONCLUSION: AIF and AME atheroprotective effects may result from their ability to upregulate mechanisms promoting HDL-cholesterol and bile acid formation.

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 15 mg/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 17ß-Estradiol (E2).

Long-term effects of the rhapontic rhubarb extract ERr 731® on estrogen-regulated targets in the uterus and on the bone in ovariectomized rats.

The efficacy of ERr 731(®), a commercially available extract isolated from Rheum rhaponticum, in terms of menopausal complaints like hot flushes, depression, anxiety and vaginal dryness has been proven in a two-year clinical study. Further a recent preclinical study excluded unwanted side effects on the endometrium by showing a lack of stimulation of proliferation marker genes by ERr 731(®) or its constituents in the 3-day uterotrophic assay. The present study aimed at further substantiating the safety of ERr 731(®) in terms of endometrial hyperplasia and at the same time test for potential estrogenic effects in the bone. Therefore, ovariectomized (ovx) rats were treated in a dietary long-term administration for 90 days. Hence, the modulation of proliferation in the uterus was investigated by examining the effects on the mRNA expression of proliferation marker genes (Mki67, Pcna), on the estrogen-responsive gene C3 and on the estrogen receptors ERα and ERß. We additionally performed densitometry analysis of the proximal tibia metaphysis using peripheral computed tomography (pQCT) and quantified bone homeostasis markers in the serum to examine potential effects on the bone. In this study design, neither an uterotrophic response nor a modulation of proliferation marker genes on mRNA level has been observed as response to the long-term application of the rhapontic extract. Furthermore, no impact of the two administered ERr 731(®) doses on the E2 deprivation-induced bone loss has been evident at the end of the study. In conclusion, the observations from previous trials regarding the endometrial safety of ERr 731(®) have been supported by our experimental findings that exclude a stimulatory activity on proliferation in the uterus in a long-term administration in the young adult rat but no effect on the bone mineral density could be observed.

Age dependency of estrogen responsiveness in the uterus and adipose tissue of aromatase-knockout (ArKO) mice.

Aging is often associated with weight gain caused by metabolic changes including an increase of body fat. In this study we assessed the impact of age on estrogen responsiveness in the uterus and adipose tissue (AT) in aromatase-knockout (ArKO) mice. ArKO mice at the age of three or twelve months respectively were treated s.c. with vehicle, E(2) (10 µg/kg BW/d) or genistein (15 mg/kg BW/d) for three days. In the ArKO mouse model we were able to demonstrate that estrogen treatment resulted in an age specific response pattern both on a physiological and molecular level. Assessment of basal gene expression levels revealed significant age dependent differences only for elevated Esr1 levels in the uterus and leptin levels in infrarenal fat as well as lower levels of Pparg in the gonadal fat tissue. Investigating age dependency of estrogen responsiveness we were able to show that the E(2) and genistein resulted in age related pattern of regulation of expression of Esr1 and Lep in infrarenal and gonadal AT as well as the uterine expression of Pgr, Ltf and Pparg. In conclusion, evidence is provided that aging has an impact on the effectiveness of estrogen regulated processes in uterus and AT of ArKO mice. It remains to be elucidated whether or not this is associated with weight gain caused by an increase in body fat mass.