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Hormone replacement therapy is a viable option to protect bone from postmenopausal osteoporosis. Systemically elevated estrogen levels, however, are disadvantageous because of the risk of harmful side effects in other organs. The rationale of the study presented here is to target a key enzyme in estradiol (E2) and testosterone (T) metabolism to increase E2 levels in an organ-specific manner, thereby avoiding the disadvantages of systemically increased E2 levels. The 17ß-hydroxysteroid dehydrogenase (17β-HSD2), which is e.g. expressed in bone, catalyzes the oxidation of E2 and T into estrone (E1) and androstenedione. We postulate that inhibiting 17β-HSD2 should lead to elevated E2 and T levels in organs expressing the enzyme. Therefore, we can use the benefits of E2 directly, or those of T following aromatization into E2, in the bone without affecting systemic levels. We tested for the first time, the novel and potent 17β-HSD2 inhibitor, compound 24 (C24), to explore the therapeutic potential of a 17β-HSD2 inhibition in an ovariectomy (ovx)-induced rat model of bone loss. We tested the inhibitor alone and, together with low dose estrogen supplementation to model estrogen levels in the postmenopausal situation. Female mature Wistar-Hannover rats were treated for 8 weeks with doses of 2, 10, 50 mg C24 per kg body weight per day alone or in the presence of estradiol benzoate (E2B) supplementation to alleviate ovx-induced bone loss. Ovx placebo and sham operated animals served as negative and positive controls. The experiment was evaluated regarding aspects of efficacy and safety: Bone was analyzed to evaluate bone protective effects, and uterus for potential, unwanted E2-mediated side effects. We observed a good bioavailability of C24 as very high plasma concentrations were measured, up to a group mean of 15,412 nM for the ovx C24-high group. Histomorphometrical analyses and in vivo &ex vivo μCT revealed significant bone protective effects for the lowest inhibitor concentration used. Irrespective of the plasma concentration, no proliferative effects in the uterus could be observed. These results support our approach of intracellular targeting key enzymes of E2 and T metabolism to increase E2 and T levels in an organ specific manner., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Selective androgen receptor modulators comprise compounds that bind as ligands to the androgen receptor and possess tissue-selective activities. Ideally, they show agonistic properties in anabolic target tissues, while inducing antagonistic or only weak agonistic effects in reproductive organs. Due to their myoanabolic effects, selective androgen receptor modulators are included in the list of prohibited substances and methods of the World Anti-Doping Agency. In the current investigation, the androgenic potential of RAD-140, GSK-2881078 and GLPG0492 was comparably investigated in two different in vitro bioassays. In the yeast androgen screen, the androgenic effects were lower than in the reporter gene assay in prostate carcinoma cells (e.g. for GSK-2881078, the EC 50 values were 4.44 × 10 -6 M in the yeast screen and 3.99 × 10 -9 M in the prostate cells respectively). For future investigations, it is of importance whether the yeast androgen screen, which has been proven to detect androgenic compounds in urine, can detect an abuse of the selective androgen receptor modulators. Molecular modeling of the binding to the androgen receptor ligand binding domain suggests slight differences in the binding modes of RAD-140, GSK-2881078 and GLPG0492. In conclusion, androgenic activity of the three non-steroidal compounds in the two different in vitro test systems confirmed the results of the in silico modeling of the androgen receptor binding., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Supplements with estrogenic activities are intensively investigated as potential alternatives for the treatment of menopausal symptoms. These investigations include studies on their safety regarding potential breast cancer risks. Therefore, the aim of this study was to assess whether or not a standardized hops (Humulus lupulus) extract, containing 0.42% of the estrogenic flavanone, 8-prenylnaringenin, would stimulate growth of methyl-nitrosourea (MNU) induced mammary cancer in ovariectomized (OVX) Sprague-Dawley (SD) rats or would impact on the proliferative activity within the normal mammary gland of Wistar rats. To induce tumorigenesis SD-rats received an intraperitoneal injection of 50mg/kg body weight of MNU on postnatal days PND 50 and 52. 28days later animals were OVX or were SHAM operated (positive control) and randomly allocated and maintained for 140days on either a phytoestrogen-free placebo diet (SHAM and negative control) or on the hops fortified diet. For the investigations in the normal mammary gland young adult Wistar rats were bilaterally OVX and randomly allocated to a control group fed to a phytoestrogen-free diet, or to a diet supplemented either with E 2 -benzoate or the hops extract. As a major result, the tumor incidence was 15% (3 tumors totally) in OVX controls, whereas it was 85% (39 tumors totally) in SHAM operated positive controls. No tumors were detectable in the hops group. In addition, no estrogenic activity of the hops extract was detectable in uterus and liver of these animals. In investigations on the normal mammary gland, no impact of hops extract on the expression of estrogen dependent proliferation markers or of progesterone receptor became apparent. In conclusion, the lack of growth stimulation of MNU-induced breast cancer in OVX SD-rats and the lack of stimulation proliferative events in the normal mammary gland of OVX Wistar rats by standardized hops extracts provides an important piece of evidence regarding the safety of these extracts in the management of menopausal symptoms., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Tendons and ligaments are crucial structures inside the musculoskeletal system. Still many issues in the treatment of tendon diseases and injuries have yet not been resolved sufficiently. In particular, the role of estrogen-like compound (ELC) in tendon biology has received until now little attention in modern research, despite ELC being a well-studied and important factor in the physiology of other parts of the musculoskeletal system. In this review we attempt to summarize the available information on this topic and to determine many open questions in this field., (Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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 17β-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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

SM6Met, a phytoestrogenic extract of Cyclopia subternata indigenous to the Western Cape province of South Africa, displays estrogenic attributes with potential for breast cancer chemoprevention. In this study, we report that SM6Met, in the presence of estradiol, induces a significant cell cycle G0/G1 phase arrest similar to the selective estrogen receptor modulator, tamoxifen. Furthermore, as a proof of concept, in the N-Methyl-N-nitrosourea induced rat mammary gland carcinogenesis model, SM6Met increases tumor latency by 7days and median tumor free survival by 42 days, while decreasing palpable tumor frequency by 32%, tumor mass by 40%, and tumor volume by 53%. Therefore, the current study provides proof of concept that SM6Met has definite potential as a chemopreventative agent against the development and progression of breast cancer., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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), 8‑prenylnaringenin (8PN) and 6‑(1,1‑dimethylally) naringenin (6DMAN), we investigated uterus and vagina derived from a three‑day 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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In the hippocampus, synaptic proteins, as studied so far, have been shown to be upregulated by 17β-estradiol, while inhibition of local estradiol synthesis consistently downregulates them. As an exception to this rule, we have previously shown that synaptopodin, an actin-associated postsynaptic protein, is downregulated in response to estradiol in dissociated cultured hippocampal neurons. In this study, we show, unexpectedly, that synaptopodin is downregulated in the hippocampus of aromatase knock-out mice and that inhibition of neuronal estradiol synthesis using the aromatase inhibitor letrozole also downregulates synaptopodin in these cultures. Moreover, the effects of estradiol and letrozole are additive, suggesting a subtle balance between available ligand and receptor. Using selective estrogen receptor agonists and antagonists, we consequently studied the effects of estrogen receptor subtypes on synaptopodin expression in our hippocampal cultures. We found that estradiol-induced downregulation of synaptopodin is mediated by estrogen receptor β. Estrogen receptor β in turn, is upregulated in response to intracellular estradiol ablation following inhibition of estradiol synthesis by letrozole in dissociated hippocampal cultures, as well as in the hippocampus of the aromatase knock-out mouse. Thus, it appears that both the application of estradiol, via binding to estrogen receptor β, and letrozole, via upregulation of estrogen receptor β, eventually result in a downregulation of synaptopodin. Our data show that the synaptic plasticity caused by estradiol is subject to a subtle balance of the levels of estrogen receptor subtypes regulated by the available ligands. In addition, both seem to be part of a homeostatic feedback mechanism., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The reproductive transition of women through peri- to postmenopause is characterized by changes in steroid hormone levels due to the cessation of the ovarian function. Beside several complaints associated with these hormonal changes, the deterioration of the trabecular bone micro-architecture and the loss of skeletal mass can cause osteoporosis. At this life stage, women often have a reproductive history of one to several pregnancies. The ovariectomized skeletally mature rat (>10 months old) is one of the most commonly used animal models for postmenopausal osteoporosis research. Despite the fact that mammals can undergo up to several reproductive cycles (primi-/pluriparous), nulliparous animals are often used and the question whether changes in the hormonal milieu subsequently affect the skeleton and influence the outcome of intervention studies is often neglected in study designs. Therefore, the aim of the present study was to compare the estrogen responsiveness of nulliparous and pluriparous rats. For this purpose, one year old virgin or retired breeder Lewis rats were either sham operated or ovariectomized, whereas half of the ovariectomized animals received subcutaneous 17β-estradiol pellets eight weeks after surgery. After another four weeks, the effects on the uterus were determined by expression analysis of estrogen-dependently regulated steroid receptor genes and well-established marker genes. Moreover, trabecular bone parameters in the tibia were analyzed by micro-computed tomography (μCT). Parity-dependency in estrogen responsiveness was observed with respect to the achieved serum E2 levels in response to similar E2 treatment. This led to differences both on the uterus wet weight and on the expression level of uterine target genes. In addition, a reversal of the ovariectomy-induced changes of the bone architecture after 17β-estradiol substitution was only observed among the nulliparous. In conclusion, the observations of this study support parity-dependent differences in the responses to estrogenic compounds in the uterus and the bone of rats. These results indicate that the parity-status has an impact on the outcome of studies aiming at the investigation of estrogenic effects of compounds potentially used in hormone replacement and thus, this should be taken into consideration for further studies and particularly for the discussion of data obtained with the preclinical ovariectomized rat animal model., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The reactive stromal phenotype is an important factor for prostate cancer progression and may be a new target for treatment and prevention. A new high efficiency preclinical protocol, the EPI bioassay, reflects the interaction of endocrine, paracrine and immune, (EPI) factors on induced androgen metabolism in human prostate reactive stroma. The bioassay is based on co-culturing human primary prostate stromal cells and LAPC-4 prostatic adenocarcinoma cells in a downscaled format of 96-well-plates for testing multiple doses of multiple target compounds. Metabolism of dehydroepiandrosterone (DHEA) with or without TGFβ1-induced stimulation (D+T) of the reactive stroma phenotype was assessed by increased testosterone in the media and PSA production of the epithelial prostate cancer cells. Using the non-metabolizable androgen R1881, effects from direct androgen action were distinguished from stromal androgen production from DHEA. Stromal cell androgenic bioactivity was confirmed using conditioned media from D+T-treated stromal cell monocultures in an androgen-inducible AR screening assay. We further showed that both agonists to estrogen receptor (ER), DPN (ERβ) and PPT (ERα), as well as estrogenic natural compounds including soy isoflavones attenuated D+T-induced PSA production. Studies with the pure ER agonists showed that activating either ERα or ERβ could inhibit both D+T-mediated and R1881-mediated PSA production with the D+T effect being more pronounced. In conclusion, natural compounds with estrogenic activity and pure ER agonists are very potent inhibitors of stromal conversion of DHEA to androgenic metabolites. More studies are needed to characterize the mechanisms involved in estrogenic modulation of the endocrine-immune-paracrine balance of the prostate microenvironment., (Published by Elsevier Ltd.)

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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 ERβ. 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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Knockout of the Cyp-19 gene (aromatase) renders mice to have insufficient endogenous estrogen production and contributes to the development of symptoms related the metabolic syndrome, including excess adiposity and insulin resistance. This study comparatively assessed the estrogen responsiveness in animal models of genetical versus surgical (ovariectomy) origin of estrogen deficiency. Evaluation of physiological parameters and gene expression pattern in response to estrogens revealed differences in estrogen responsiveness between aromatase deficient and castrated or intact wild-type mice. ArKO mice had a significantly higher bodyweight than matched ovariectomized wild-type mice. The weight of the completely regressed uterus following ovariectomy was higher than the uterine weight of ArKO mice. Further, alterations in metabolic parameters like increased serum leptin levels and decreased plasma glucose levels in genetically deficient mice became apparent. Finally, expression pattern of estrogen responsive genes differed in the two experimental models of estrogen deficiency. Both, in uterine and adipose tissues the regulation of expression of some genes either was inversed of regulation or considerably differed in the magnitude of the response in the two models. Our studies demonstrate that the cause of estrogen deficiency significantly impacts on estrogen responsiveness and may be of relevance for investigations on aspects of estrogen deficiency and metabolic and/or menopausal symptoms., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Many flavonoids, a major group of phenolic plant-derived secondary metabolites, are known to possess estrogen-like bioactivities. However, little is known about their estrogenic properties in the central nervous system due to the lack of suitable cellular models expressing sufficient amounts of functional estrogen receptor beta (ERbeta). To overcome this deficit, we have created a cellular model, which is serotonergic in origin, to study properties of estrogenic substances by stably transducing RN46A-B14 cells derived from raphe nuclei region of the rat brain with a lentiviral vector encoding a human ERbeta. We clearly showed that the transgenic human ERbeta is a spontaneously expressed and a functional receptor. We have further assessed the estrogenicity of three different isoflavones and four different naringenin-type flavanones in this cell line utilizing a luciferase reporter gene assay. Genistein (GEN), Daidzein (DAI), Equol (EQ), Naringenin (NAR) and 8-prenylnaringenin (8-PN) showed strong estrogenic activity in a concentration-dependent manner as compared to 7-(O-prenyl)naringenin-4'-acetate (7-O-PN) which was only slightly estrogenic and 6-(1,1-dimethylallyl)naringenin (6-DMAN) that neither showed estrogenic nor anti-estrogenic activity in our model. All observed effects could be antagonized by the anti-estrogen fulvestrant. Moreover, co-treatment of cells with 17beta-estradiol (E2) and either GEN or DAI showed a slight additive effect as compared to EQ. On the other hand, 8-PN in addition to 7-O-PN, but not NAR and 6-DMAN, were able to slightly antagonize the responses triggered by E2. Our newly established cellular model may prove to be a useful tool in explicating basic physiological properties of ERbeta in the brain and may help unravel molecular and cellular mechanisms involved in serotonergic mood regulation by estrogen or potential plant-derived secondary metabolites., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Eight isoflavones derivatives, with isoprenyl and/or 7-methoxy substitution, isolated from Erythrina poeppigiana (Fabaceae) have been investigated for their estrogenic properties in receptor subtype-specific reporter gene assays. First we focused on their estrogen receptor alpha and beta (ERalpha and ERbeta) selectivity, second we addressed structure-activity relationships, using bone-derived human osteosarcoma cell line (U2OS cells) stably expressing ERalpha or transiently expressing ERbeta. Our results show that a substitution at position 3' together with a 7-methoxy substitution on the genistein skeleton is associated with a statistically significant activation of the ERalpha- and ERbeta-dependent reporter gene expression in U2OS cells starting from 0.1nM. Particularly, the 7-methoxy-3'-isoprenyl (1) and the 7-methoxy-3'-(3-methyl-2-hydroxybuten-3-yl) (3) derivatives of genistein induces an ERalpha- and ERbeta-coupled luciferase activity at a concentration ten times lower than that of genistein, for which a statistically significant effect was observable at 1nM. On the other hand, isoprenyl substitution at position 6 of the A ring, compound 5, seems to have very little impact on the genistein ability to induce ER-coupled luciferase activity in U2OS cells, while a double prenylation at positions 8 and 3', compound 7, is associated with an almost complete loss of function on the reporter gene activation in U2OS-ERalpha, but in ERbeta expressing system the effectiveness remains on a statistically significant level, demonstrating an "exclusive ERbeta-selectivity" in U2OS human osteosarcoma cells, and therefore 7 can be considered as an isotype-selective ER ligand. Finally all the tested isoflavones derivatives appear to exhibit a slightly pronounced ERbeta preference, depending upon the position and the nature of the substituent moiety on the isoflavone skeleton. The estrogen-like effect of these prenylated isoflavone derivatives could be inhibited by the pure ER antagonist ICI 182 780, indicating that these effects were primarily mediated through ERs., (Copyright 2010 Elsevier Ltd. All rights reserved.)

A variety of plant derived substances, so-called phytoestrogens (PEs), although structurally not related to steroids, produce effects similar to the mammalian estradiol. However, little is known so far about the structural requirements which determine PE activities. Taking into consideration that prenylation reactions are relatively common in plant secondary metabolism, the activity of a set of three PE derivatives of genistein and naringenin, namely genistein, 8-prenylgenistein (8PG), 6-(1,1-dimethylallyl)genistein (6DMAG), naringenin, 8-prenylnaringenin (8PN) and 6-(1,1-dimethylallyl)naringenin (6DMAN) was compared regarding structure-estrogenicity relationships in three functionally different estrogen receptor assays. Strong estrogenic activities were recorded for 6DMAN and 8PN in all assays used, while the parent compound naringenin showed only very weak estrogenicity. In contrast, in the case of genistein derivatives, only genistein itself exhibited estrogenic activity in a yeast based assay. In MVLN breast cancer cells, a bioluminescent MCF-7-derived cell line, the estrogenic activity of all three genistein derivatives was similar. Studying alkaline phosphatase activity in Ishikawa endometrial cancer cells as an estrogenic response marker revealed a similar pattern of estrogenicity of the genistein derivatives compared to the yeast based assay although a slight estrogenic effect of 6DMAG and 8PG was apparent. In summary, this study demonstrates that prenylation often found in plant secondary metabolism differentially modifies estrogenic properties of PEs depending on the basic structure of the respective PE., (Copyright 2009 Elsevier Ltd. All rights reserved.)

A recent clinical study with a two-year application of the extract ERr 731 from Rheum rhaponticum demonstrated its efficacy and potentially suggested it safety regarding unwanted endometrial side effects. The aim of the present study is to provide experimental proof for the latter observation in a preclinical experimental animal model by assessing dose-dependent effects of ERr 731 - either alone or in combination with estradiol (E2) - on growth and proliferation in the uterus of ovariectomized (ovx) rats. ERr 731 was given in a dose corresponding to human therapeutic application and additionally in three pharmacologically relevant doses. In addition to uterine wet weight, this study examines the effects of ERr 731 on the uterine mRNA expression of the proliferation marker Ki67, proliferating cell nuclear antigen (PCNA), insulin-like growth factor-1 (IGF-1), type 1 IGF receptor (IGF-1R), the two estrogen receptor (ER) subtypes alpha and beta (ERalpha and ERbeta) and the estrogen-responsive gene complement C3 (C3). ERr 731 did neither stimulate an uterotrophic response in the uterotrophic assay with ovx rats nor stimulate or modulate the expression of genes associated with proliferation. In combination with E2, ERr 731 reduced the E2-induced uterine growth stimulation. These observations were further substantiated by the expression pattern of genes related to proliferation control, in view of the fact that the E2-induced elevation of Ki67 mRNA and PCNA protein levels in the uterus were counteracted by simultaneous treatment of the animals with ERr 731. In conclusion, the experimental findings presented here provide further evidence for the safety of ERr 731 towards unwanted uterine and endometrial proliferative events in response to ERr 731 and support observations from recent clinical trials.

Isoflavones (ISOs) are bioactive food ingredients of the traditional East Asian diet and currently discussed as alternatives to classical hormone replacement therapies and for reducing the prevalence of hormone-dependent cancers. Although there are many studies on ISOs, not much is known about their long-term effects. Therefore, we performed an animal experiment analyzing the effects of three different diets: a phytoestrogen-free diet, a diet supplemented with genistein (700 microg/g diet) and an ISO-high diet (232 microg daidzein and 240 microg genistein/g) at two distinct time points, juvenile (21 days) and adult (97 days). Exposure started prior to mating of the parents and throughout the life of the offspring. We observed a stronger increase of uterine wet weights in juvenile offspring with genistein exposure (1018+/-350 mg/kg BW) than with ISO-high diet (497+/-133 mg/kg BW). Whereas the expression of proliferation related genes (PCNA; Ki67; IGF-1; IGF-1R), analyzed by real-time-qPCR and Western blot, were significantly down-regulated in juvenile animals exposed to genistein. Additionally, genistein exposure led to estrogenic responses, observed upon increase of complement C3 and decrease of estrogen receptors gene expressions, while the exposure to ISO-high diet did not show these effects. In conclusion, both the time point on which phytoestrogen exposure starts together with the composition of the ingested phytoestrogen containing diet are of great importance for the biological response of the offspring.

The special extract ERr 731 from the roots of Rheum rhaponticum is the major constituent of Phytoestrol N which is used for the treatment of climacteric symptoms in menopausal women. However, the molecular mode of action of ERr 731 was unknown. For the first time, ERr 731 and its aglycones trans-rhapontigenin and desoxyrhapontigenin were investigated with regard to the activation of the estrogen receptor-alpha or estrogen receptor-beta (ERalpha, ERbeta). The related hydroxystilbenes cis-rhapontigenin, resveratrol and piceatannol were studied as comparators. As controls, 17beta-estradiol or the selective ERalpha-(propylpyrazoltriol) or ERbeta-agonists (diarylpropionitril) were used. Neither in ERalpha-expressing yeast cells, in the ERalpha-responsive Ishikawa cells, nor in human endometrial HEC-1B cells transiently transfected with the ERalpha an activation of ERalpha by ERr 731 or the other single compounds was detected. Furthermore, an antiestrogenic effect was not observed. In contrast in human endometrial HEC-1B cells transiently transfected with the ERbeta, 100 ng/ml ERr 731 and the single compounds significantly induced the ERbeta-coupled luciferase activity in a range comparable to 10(-8)M 17beta-estradiol. All effects were abolished with the pure ER antagonist ICI 182780, indicating an ER-specific effect. The ERbeta agonistic activity by ERr 731 could be of importance for its clinical use, as central functions relevant to climacteric complaints are proposed to be mediated via ERbeta activation.

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.

The knowledge about safety of phytoestrogens on proliferative endpoints in the endometrium is rather limited, particularly when low amounts of estrogens are present like in postmenopausal women. Therefore, we now studied how genistein (GEN) exposure affects proliferative endpoints in the endometrium in estrogenized animals. We investigated the effects of GEN (10 mg/(kgday) BW) on uterine proliferation and on general uterine response markers in intact female rats and ovariectomized (OVX) female rats co-treated with different doses of estradiol (E2; 1 or 4 microg/(kgday) BW). In parallel we investigated generalized hepatic effects of GEN in this co-stimulatory protocol. In agreement to our previous results, GEN treatment of OVX animals for 3 days results in a faint stimulation of the uterine wet weight. In intact animals and in OVX animals co-treated with E2 no effects of GEN on uterine wet weight were detectable. GEN treatment did not affect the uterine epithelial height in intact animals but resulted in a decrease of the protein and mRNA expression of the proliferation marker PCNA. In OVX animals co-treated with E2, GEN antagonized the E2 stimulated increase of the uterine epithelial height and epithelial PCNA expression. Besides PCNA, GEN effects on the uterine mRNA expression of IGF-1, IGF-1R, Complement C3, estrogen receptor-alpha (ERalpha) and -beta (ERbeta), as well as progesterone receptor were investigated in intact and OVX co-treated animals. Overall there was a tendency in all combinatorial groups that GEN counteracts E2 function in uterine tissue. Surprisingly, while investigating estrogenic response markers in liver, we observed very strong effects of GEN on hepatic marker gene expression. GEN significantly down-regulated CaBP9K and IGFBP1 mRNA levels in intact animals. In OVX animals hepatic CABP9K and IGFBP1 mRNA levels were not affected by E2 treatment. GEN treatment, even in combination with E2, decreased the hepatic CaBP9K expression below the levels observed in untreated animals. Interestingly co-treatment of OVX rats with low dose E2 and GEN resulted in a significant increase of IGFBP1 mRNA expression. Summarising our results we conclude that (1) GEN treatment in the presence of E2 is safe regarding proliferative responses in the endometrium of adult animals; (2) the observation of differences of the GEN activity in intact and OVX/E2 substituted animals can be taken as a hint that GEN may interact mechanistically with progestins which has to be proven in detail in future investigations and (3) the detection of strong effects of the phytoestrogen GEN on hepatic gene expression may point to the need of future investigations to rule out the possibility of adverse responses in this organ.

The aim of this study was to characterize carbonic anhydrase II (CA2), as novel estrogen responsive gene, towards its usefulness to elucidate the molecular mechanisms of phytoestrogen action. Effects of estradiol-17beta (E2), and the phytoestrogens genistein (Gen), daidzein (Dai), as well as 8-prenylnaringenin (8PN) on CA2 mRNA expression were investigated in vivo in the uterus and liver of Wistar rats, and in vitro in Fe33 hepatoma cells. Relative amounts of mRNA levels of CA2 were measured by real-time RT-PCR. In vivo CA2 expression in uterus and liver is down-regulated by estrogen in time dependent manner with the most pronounced effect detectable 72 h after treatment. Treatment with Gen results in a slight down-regulation of CA2 expression in the uterus. In liver a response to Gen is detectable only after 7 h, where the expression of the gene is down-regulated to 60%. Treatment with Dai and 8PN for 72 h results in a slight down-regulation of CA2 in both tissues. In contrast in Fe 33 cells CA2 gene expression was up-regulated in response to the treatment with E2 for 7 h. In summary, we could demonstrate that the modulation of CA2 gene expression following treatment with E2 and Gen in rat uterus is comparable to the uterotrophic response of these substances, but with an inverted pattern. Remarkably, of all phytoestrogens 8PN exhibited the strongest uterotrophic response but only induced a very faint decrease of CA2 expression. In addition, we provide the first pieces of evidence that 8PN, like Gen and Dai, cannot be considered as a pure agonist. In conclusion, CA2 shows estrogen sensitivity not only in both tissues studied, but also in many others. Further, it exhibits a differential sensitivity thereby being capable to discriminate between different molecular qualities of phytoestrogens, like demonstrated for Gen and 8PN.

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.

8-prenylnaringenin (8-PN) and preparations containing 8-prenylnaringenin have been suggested for use in medicinal and cosmetic applications like hormone replacement or bust enhancement. However, the safety of application is still under considerable debate. Recently it has been shown that human liver microsomes are converting 8-prenylnaringenin to 12 metabolites, with (E)-8-(4''-hydroxyisopentenyl)naringenin (8-PN-OH) and (E)-8-(4''-oxoisopentenyl)naringenin (8-PN=O) being among the most abundant. Applying two independent in vitro test systems we demonstrate that these two metabolites of 8-prenylnaringenin are estrogenic in vitro. These results represent an important piece of information towards the discussion of safety of use of preparations containing 8-prenylnaringenin.

Xenoestrogens, phytoestrogens and synthetic estrogens, are able to bind to estrogen receptors, and to mimic estrogenic activities in a cell and tissue specific manner. For the characterization of environmental estrogens mainly mammary derived and yeast based models have been used. The aim of this study was therefore to assess selected natural and synthetic compounds in an endometrial derived model. We measured the relative estrogenic potency of phytoestrogens (genistein, daidzein, coumestrol, some naringenins), synthetic estrogens (bisphenol A, octylphenol, nonylphenol, o,p'-DDT), mycoestrogen (zearalanone) as well as extracts of Cimicifuga racemosa on alkaline phosphatase (AlkP) activity in the endometrial derived adenocarcinoma cell line Ishikawa. We used a modified multiwell plate in vitro bioassay based on the estrogen-specific and dose-dependent enhancement of AlkP activity in this cell line. Estradiol, which induced AlkP at levels as low as 10(-8)M, was used as positive control. Most of the compounds studied showed a clear dose-dependent estrogenic effect. Compared to the vehicle control (ethanol) all phyto- and mycoestrogens, stimulated the AlkP activity 2-4-fold at a concentration of 10(-6)M. The synthetic chemicals bisphenol A and nonylphenol showed an effect at 10(-6)M, octylphenol at 10(-5)M. Effects of o,p'-DTT could not be measured. ICI 182,780, a pure estrogen receptor antagonist, significantly inhibited these effects. The latter result demonstrated the estrogen receptor dependency of this process. In summary, most of the phytoestrogens and industrial chemicals tested, behaved as estrogen receptor agonists in terms of the stimulation of AlkP activity.

Despite the wide use of extracts from the rhizome of black cohosh (Cimicifuga racemosa) for the treatment of menopausal complaints, surprisingly little is known on their potential estrogenic properties, e.g. on estrogen dependent gene transcription. In addition, available informations on the effects on cell proliferation are contradictory. We therefore, tested for estrogenic and antiestrogenic effects of Cimicifuga racemosa extracts on proliferation of MCF-7 cells and on gene expression using ethanolic and iso-propanolic extracts of this medical plant. Estrogenic properties of plant extracts could neither be detected in proliferation assays, nor on gene expression using an estradiol-inducible yeast assay or the estrogen-inducible MVLN cells. In contrast, in all three experimental systems Cimicifuga racemosa antagonized estradiol induced activities. Estradiol induced stimulation of proliferation was inhibited by a dosage >1 microg/ml of extract concentration, gene expression was suppressed by doses of 100-1000 microg/ml of Cimicifuga racemosa extracts. From these results we conclude, that extracts from the rhizome of Cimicifuga racemosa contain compounds with antiestrogenic properties.

The possible adverse effects of the so-called environmental estrogens have raised considerable concern. Developmental, endocrine and reproductive disorders in wildlife animals have been linked to high exposure to persistent environmental chemicals with estrogen-like activity (xenoestrogens); yet, the potential impact of environmental estrogens on human health is currently under debate also due to lack of data. A battery of in vitro assays exist for identifying compounds with estrogenic activity, but only a few models are available to assess estrogenic potency in a multiparametric analysis. We have recently established the endometrial adenocarcinoma cell line RUCA-I; it enables us to compare estrogenic effects both in vitro and in vivo as these cells are estrogen responsive in vitro and grow estrogen sensitive tumors if inoculated in syngeneic animals in vivo. Here we report in vitro data concerning (a) the relative binding affinity of the selected synthetic chemicals Bisphenol A, nonylphenol, p-tert-octylphenol, and o,p-DDT to the estrogen receptor of RUCA-I cells and (b) the relative potency of these compounds in inducing increased production of complement C3, an endogenous estrogen-responsive gene. Competitive Scatchard analysis revealed that xenoestrogens bound with an at least 1000-fold lower affinity to the estrogen receptor of RUCA-I cells than estradiol itself, thereby exhibiting the following affinity ranking, estradiol>>>nonylphenol>bisphenol A approximately p-tert-octylphenol>o,p-DDT. Despite these low binding affinities, bisphenol A, nonylphenol and p-tert-octylphenol increased production of complement C3 in a dose dependent manner. Compared with estradiol, only 100-fold higher concentrations were needed for all the compounds to achieve similar levels of induction, except o,p-DDT which was by far less potent. Northern blot analyses demonstrated that the increased production of complement C3 was mediated by an increased transcription. In summary, cultured RUCA-I cells represent a valuable endometrial derived model system to assess the relative potencies and the molecular mode of action of environmental estrogens in vitro. Our results further show that no intimate correlation exists between the relative binding affinity and the biological response of these compounds. Therefore, data obtained from single-parametric analyses may result in misleading conclusions. On the other hand, the presented in vitro data will provide us with tools to study the activity of xenoestrogens in vivo and thus carry risk assessment one step further.

The function of the uterus is regulated by female sex steroids and it is, therefore, used as the classical target organ to detect estrogenic action. Uterine response to estrogens involves the activation of a large pattern of estrogen-sensitive genes. This fact offers the opportunity to analyze the estrogenic activity of xeno- and phytoestrogens, and the mechanisms of their molecular action by a correlation of the uterotropic activity and their ability to modulate the expression of estrogen-sensitive genes. We have analyzed the expression of androgen receptor (AR), progesterone receptor (PR), estrogen receptor (ER), clusterin (CLU), complement C3 (C3), and GAPDH mRNA in the rat uterus following oral administration of ethinylestradiol (EE), bisphenol A (BPA), o,p'-DDT (DDT), p-tert-octylphenol (OCT) and daidzein (DAI). A significant stimulation of the uterine wet weight could be observed after administration of all the substances. The activity of all analyzed compounds to stimulate uterine weight was low in comparison to EE. DDT has the highest activity to stimulate uterine weight whereas BPA and DAI turned out to be less potent. The analysis of gene expression revealed a very specific profile of molecular action in response to the different compounds which cannot be detected by judging the uterotropic response alone. A dose dependent analysis revealed that C3 mRNA is already modulated at doses where no uterotropic response was detectable. Although DAI and BPA were very weak stimulators of uterine growth, these substances were able to alter the expression of AR, ER and C3 very strongly. Based on these investigations the analyzed compounds can be subdivided into distinct classes: First, compounds which exhibit a similar gene expression fingerprint as EE (e.g. OCT); second, compounds exhibiting a significant uterotropic activity, but inducing a pattern of gene expression different from EE (e.g. DDT); and third, compounds like BPA and especially DAI which exhibit a very low uterotropic activity, but nevertheless modulate the expression of estrogen-sensitive genes. These findings strongly suggest that the fingerprint of uterine gene expression is a very sensitive tool to investigate estrogenicity of natural and synthetic compounds and offers the possibility to get information in regard to the molecular mechanisms involved in the action of the respective compounds.

In previous investigations it was shown that the synthetic estrogen diethylstilbestrol (DES) induces a rise of the intracellular calcium level ([Ca2+]i) in C6 rat glioma cells [P. Tas, H. Stopper, K. Koschel, D. Schiffmann, Influence of the carcinogenic oestrogen diethylstilboestrol on the intracellular calcium level in C6 rat glioma cells. Toxic. In vitro 5 (1991) 463-465] which is accompanied by changes of the arrangement of the cytoskeleton. In the present study, we compared the induction of these effects in COS (monkey kidney cells) lacking estrogen receptors (ER) with those in RUCA-I (rat endometrial carcinoma) cells containing ER. The [Ca2+]i in RUCA-I and COS cells following 17beta-estradiol (ES), genistein (GEN), daidzein (DZ) and coumestrol (CES) treatment was analyzed. A significant increase of [Ca2+]i induced by all compounds was observed in RUCA-I cells. No effects were detected in COS cells after ES and GEN treatment. The anti-estrogen ICI 182780 completely blocked the ES-and GEN-induced rise of [Ca2+]i. Dose and time dependencies of changes of calcium levels were analyzed and a biphasic response could be observed. The actin staining showed disintegrated stress fibers in RUCA-I cells. The degree of the observed effects correlates with the known estrogenicity of the applied compounds (DES > ES > GEN). It remains to be elucidated whether or not the effects observed are mediated by the "classic" genomic estrogen receptor pathway or by alternate nongenomic or receptor-independent pathways.

Estrogens are believed to play a crucial role in growth regulation and differentiation of the normal endometrial tissue as well as in the carcinogenesis of the endometrium. Therefore, the influence of estrogens and antiestrogens on gene expression in the estrogen receptor-positive rat endometrial adenocarcinoma cell line RUCA-I was investigated. Differentially expressed genes were detected by differential display PCR of RNA of untreated, estradiol-treated and antiestrogen-treated RUCA-I cells. By means of the PCR technique, 14 differentially expressed fragments could be detected. Three of these 14 differentially expressed fragments were confirmed by Northern blotting. The steady state mRNA levels of the three gene fragments named AH41, AH42 and AH44 were downregulated by the antiestrogen ICI 164384. Further characterization revealed that the fragment AH41 is not expressed in stromal cells but in the human and rodent epithelial cell lines, BG-1 and RUCA-II. A comparison of the cDNA sequence of fragment AH41 with the EMBL database showed no high homology to known genes. Therefore, fragment AH41 has to be regarded as a fragment of a novel, estradiol-sensitive gene.

The recently established, estrogen receptor positive rat endometrial adenocarcinoma cell line RUCA-I was tested for estrogen responsiveness in vivo and in vitro. In vivo, 10(6) RUCA-I cells were injected subcutaneously into intact, ovariectomized, or ovariectomized, estradiol-substituted syngenic DA/Han rats. All animals developed well differentiated endometrial adenocarcinoma, that had metastasized to peripheral lymph nodes and into the lung. Ovariectomy reduced tumor and lymph node weight, as well as number of lung metastases significantly compared to controls. In another series of experiments, treatment with the pure anti-estrogen ZK 119010 basically gave the same results as seen in ovariectomized animals, whereas tamoxifen treatment had no effect on metastasis of RUCA-I cells. These findings clearly demonstrate the estrogen dependency of growth and metastasis of RUCA-I cells in vivo. In vitro, we assessed the estrogenic and anti-estrogenic potency of various anti-estrogens, thereby investigating their effects on the expression of components of the complement C3 complex as an estradiol-induced protein and on the expression of fibronectin as an estrogen-repressed protein. Evaluating the relative anti-estrogenic potency of these anti-estrogens we found that ICI 164384 and ICI 182780 behaved as complete antagonists in vitro. Tamoxifen, like estradiol, stimulated complement C3 production and repressed fibronectin expression and has to be regarded as an agonist in this particular in vitro system. ZK 119010 if given alone had no significant influence on the biosynthesis of complement C3 and of fibronectin if compared to the unstimulated control. In addition, another estrogen dependent parameter was identified. Estrogen and anti-estrogen treatment affected glycosylation of complement C3 components. After estradiol treatment predominantly the higher glycosylated epitope of complement C3 became detectable, which could be transformed into the low molecular weight epitope by treatment with hyaluronidase. Finally, we compared the anti-proliferative effects of ICI 164384 and of tamoxifen in vitro. Both anti-estrogens slightly inhibited the growth of RUCA-I rat endometrial adenocarcinoma cells. In conclusion, RUCA-I cells represent a powerful, endometrial derived experimental model to test the agonistic and antagonistic properties of anti-estrogens on growth and metastasis in vivo and on gene expression in vitro. The effects of the tested anti-estrogens on gene expression of RUCA-I cells were found to be useful in predicting their effectiveness on tumor growth in vivo.

We recently established and characterized two rat endometrial adenocarcinoma cell lines which we called RUCA-I and RUCA-II. Despite high estrogen receptor levels neither cell line responded to estradiol in conventional cell culture conditions on plastic and in the presence of charcoal stripped fetal calf serum. We further demonstrated that culturing of these cells on a reconstituted basement membrane induced the estrogen responsiveness for both proliferation and gene expression. Particularly, the expression of components of the complement C3 system, which represent major estradiol inducible proteins in the rat uterus in vivo, were found to be under the control of estrogens and antiestrogens. In this paper the search for estrogen repressed proteins is reported. For this purpose secretory proteins of RUCA-I cells were metabolically labelled with 35S-methionine and tested for the presence of estrogen-repressed, antiestrogen-inducible protein species. Analyzing cell culture supernatants of RUCA-I cells by polyacrylamide gel electrophoresis under reducing conditions a protein with an apparent size of approx. 250-270 kDa became conspicuous. The formation and secretion of this protein was suppressed by estradiol and induced by the antiestrogen ICI 164384. Gel electrophoresis performed under non-reducing conditions and hyaluronidase digestion showed that this estrogen-repressed protein represents a dimeric glycoprotein. By immunoprecipitation this glycoprotein was identified as fibronectin. Investigations of steady state mRNA levels of fibronectin by rtPCR suggested a post-transcriptional regulation of this molecule by estradiol. This is the first report on repression of components of the extracellular matrix by estradiol and induction by the complete antiestrogen ICI 164384. The consequences of this finding in regard to growth and invasion of endometrial tumors are discussed.

We recently described the establishment and the characterization of two rat endometrial adenocarcinoma cell lines which we called RUCA-I and RUCA-II. Despite fairly high estrogen receptor levels neither cell line responded to estradiol in conventional cell culture conditions on plastic and in the presence of serum. A limited hormonal response to the antiestrogen tamoxifen was detectable in RUCA-I but not in RUCA-II cells. To advance our cell culture conditions we plated RUCA-I cells on a layer of reconstituted basement membrane (Harbor Matrix) in the presence of a serum-free defined medium. These cell culture conditions induced hormone responsiveness of RUCA-I cells and permitted a stimulation of proliferation by estradiol. Further, two estradiol-induced secretory proteins with an apparent molecular weight of 115 kD and 60 kD could be identified by SDS-gelelectrophoresis if analyzed under reducing conditions. These proteins migrated as a single band in a non-reducing electrophoresis gel and were identified as components of the complement C3 system. Additionally, our results suggest that the effects of extracellular matrix and hormones on the expression of these proteins are additive. We conclude that processes of functional differentiation are most likely to occur in this in vitro model, particularly since the expression of components of the complement C3 system was under estrogenic control. Complement C3 proteins represent major estradiol-inducible secretory protein of the immature rat uterus in vivo. Culturing RUCA-I cells on top of a layer of reconstituted basement membrane provides a novel tool to study the importance of the extracellular environment on the hormone-induced gene expression in endometrial carcinogenesis in vitro.

We were previously investigating the expression of the extracellular matrix glycoprotein tenascin in normal and malignant endometrial tissues of humans and rodents. These studies suggested that the expression of tenascin was induced by proliferating epithelia (normal and particularly malignant) and was downregulated with their differentiation. The aim of this study was to investigate the hormone dependency of tenascin expression in (a) the transplantable EnDA endometrial tumor model with or without estrogen deprivation (ovariectomy) of the animals, (b) DMBA-induced rat mammary tumors with or without a hormonal treatment of the animals [ovariectomy, antiestrogen (tamoxifen) or antiprogestin (ZK 98299) treatment] and (c) in the rat prostate of untreated or androgen deprived animals (orchiectomy, flutamide-, casodex- or cyproterone acetate (CPA)-treatment). 1. Estrogen withdrawal by ovariectomy did not affect tenascin expression in transplantable EnDA endometrial adenocarcinoma, meaning the entire extracellular space of the stromal mesenchyme was decorated by tenascin immunoreactivity. 2. In untreated DMBA-induced rat mammary tumors almost the entire extracellular space of the stroma was stained by tenascin immunoreactivity. Ovariectomy and antiestrogen treatment did not affect tenascin expression. In contrast, antiprogestin treatment induced terminal differentiation of mammary tumor cells and in parallel downregulated tenascin expression. 3. In the normal rat prostate no tenascin was detectable by immunocytochemistry. However, following androgen deprivation we found tenascin expression in the stroma of the prostate. The most prominent expression was observable after CPA-treatment, possibly due to its progestagenic potency. In conclusion, the hormones and antihormones tested show no direct effect on the stromal expression of tenascin. However, proliferative activity and a low degree of differentiation of the epithelium induces tenascin expression, whereas epithelial differentiation apparently shuts down tenascin expression. Preliminary in vitro studies suggest that paracrine acting growth factors trigger the hormonal regulation of tenascin expression.

The catecholestrogens, namely 2-hydroxyestradiol (2-OH-E2) and 4-hydroxyestradiol (4-OH-E2) are important, naturally occurring metabolites of E2. Here we studied their role on estrogen dependent processes. Using the MCF-7 cell line as a model system we analyzed the potency of 2- and 4-OH-E2 on the synthesis of the 160 kDa secreted protein and on the transcription of the pS2 mRNA. Both processes are known to be E2 inducible and are mediated by the estrogen receptor. Control incubations using E2 and antiestrogens were performed to validate the assay procedure and to enable us to comparatively study the effects of the catecholestrogens. Stimulating MCF-7 cells for 2 days with 10(-8) M 2- or 4-OH-E2 resulted in an induction of the synthesis of the 160 kDa protein and in an increase in pS2 mRNA. Following hormonal stimulation with 2- or 4-OH-E2 [35S]methionine labeling of MCF-7 cells increased the level of newly synthesized and secreted 160 kDa protein 54 and 88% compared with the inductive potency of E2 (100%). The pS2 mRNA in MCF-7 cells was increased by a 2 day treatment with 10(-8) M 2- or 4-OH-E2 by 48 and 79%, respectively, compared to E2. Therefore, we conclude that the estrogen receptor is transcriptionally active in MCF-7 cells upon binding of catecholestrogens. The estrogen receptor in vivo may be active if the intracellular concentration of catecholestrogens generated is sufficient to allow occupation of the receptor. The possible action of these hormones in vivo is discussed.

Catecholestrogens are important metabolites of estradiol and estrone in the human. Considerable interest has focused on the catecholestrogens 2-hydroxy- and 4-hydroxyestradiol since they bind to the estrogen receptor with an affinity in the range of estradiol. Using the MCF-7 cell line, we analysed the capacity of purified catecholestrogens to transform the estrogen receptor into its high affinity nuclear binding form and to affect receptor-dependent processes such as proliferation and expression of the progesterone receptor (PR). Incubations with 2-hydroxy- and 4-hydroxyestradiol at 10(-8) M for 1 h resulted in tight nuclear binding of the estrogen receptor. During treatment of the cells with catecholestrogens we obtained a marked increase in proliferation rate of 36 and 76% for 2-hydroxy- and 4-hydroxyestradiol, respectively, relative to the inductive effect of estradiol (100%). The PR level, was slightly increased by treatment with 2-hydroxyestradiol (10%), whereas treatment with 4-hydroxyestradiol increased the PR level at 28%, compared to estradiol (100%). From these results we conclude that the 2- and 4-hydroxylated derivatives of estradiol are active hormones and are able to initiate estrogen receptor mediated processes in MCF-7 cells.

The binding affinity and relative estrogenic potency of 2-bromo-, 4-bromo-, 2-methyl- and 4-methylestradiol was evaluated in MCF-7 breast cancer cells. The relative binding affinities compared to estradiol were 47% for 2-methyl-, 25% for 4-methyl-, 37% for 4-bromo- and 17% for 2-bromoestradiol. However, both 2- and 4-methyl- as well as 2- and 4-bromoestradiol were able (a) to translocate the cytosolic estrogen receptor into the nucleus and (b) to induce the progesterone receptor in a concentration dependent manner. Finally, all ring-A substituted estrogens used in this study induced the pS2 mRNA as demonstrated by Northern-blotting. From these findings we conclude that 2-bromo-, 4-bromo-, 2-methyl- and 4-methylestradiol are agonistic ligands for the estrogen receptor in MCF-7 breast cancer cells.