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Endocrinology Vol. 146, No. 12 5144-5150
Copyright © 2005 by The Endocrine Society

Estrogenic Activities in Rodent Estrogen-Free Diets

Paolo Ciana1, Andrea Brena1, Paolo Sparaciari, Elena Bonetti, Diego Di Lorenzo and Adriana Maggi

Department of Pharmacological Sciences (P.C., A.B., P.S., A.M.), Center of Excellence on Neurodegenerative Diseases, University of Milan, 20133 Milan, Italy; Third Laboratory/Biotechnology (E.B., D.D.L.), Civic Hospital of Brescia, University of Brescia, 25123 Brescia, Italy

Address all correspondence and requests for reprints to: Adriana Maggi, Center of Excellence on Neurodegenerative Diseases and Department of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy. E-mail: adriana.maggi{at}unimi.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Diets lacking soy and {alpha}-{alpha} derivatives that are considered to be estrogen-free by standard bioassays (uterotrophic assay and vaginal opening) have been revealed to contain considerable amounts of compounds able to transcriptionally activate the estrogen receptors (ERs) and stimulate luciferase expression in several organs of the ERE-Luc reporter mouse. By molecular imaging, we show that ER activation is present in nonreproductive organs to an extent similar to that observed with the administration of 17ß-estradiol, and it is not influenced by orchiectomy or treatment with an aromatase inhibitor. This, together with the use of a completely synthetic diet, proves that the activation of ERs observed is due to estrogenic compounds present in commercial diets and that it is not a secondary event determined by food consumption and metabolism. The pervasiveness of estrogenic compounds in nature poses the question of how relevant and necessary is the daily ingestion of natural compounds active through the ERs for the maintenance of a correct metabolism in both male and female mammals.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE FEMALE SEX hormone 17ß-estradiol modulates target cell activity by binding two receptor isotypes: estrogen receptors (ERs) {alpha} and ß (1, 2, 3). It is well known that these receptors are hormone-regulated transcription factors that recognize and bind specific DNA sequences [estrogen-responsive elements (EREs)] in the promoter of target genes to modulate transcription (4, 5, 6). For quite a long time, it was assumed that the physiological role of these receptors was circumscribed to the control of female reproductive functions. This assumption, however, is now challenged by a series of findings suggesting a much deeper involvement of ERs in mammalian physiology and metabolism. First, ER{alpha} and ERß expression is not limited to tissues responsible for reproductive functions because ERs are present in most cells in animals of both sexes (7, 8, 9, 10). Second, several dietary components, such as phytoestrogens, have strong estrogenic activity, suggesting that the ERs may be continuously challenged in both male and female organisms (11, 12, 13). Third, ER intracellular activities go far beyond the transcriptional control of ERE genes because ER may also interact with intranuclear or intracytoplasmic signaling proteins, such as activator protein-1 (14), nuclear factor {kappa}B (15, 16), signal transducer and activator of transcription-3 (17), G proteins (18), phosphatidylinositol-3OH kinase (19), and regulate their signal transduction potential. Thus, the exact contribution of ERs to mammalian physiology needs to be reassessed, particularly with regard to the activity of these receptors in nonreproductive organs.

To facilitate the study of ER action in reproductive and nonreproductive organs, we recently engineered the genome of a mouse model with a transgene containing an exogenous reporter gene, luciferase, driven by an ERE-thymidine kinase promoter. The presence of insulators ensures the ubiquitous expression of the reporter gene (20). This mouse model, named ERE-Luc, has proven to be a valuable tool to study ER transcriptional actions mediated by EREs (21, 22) in vitro and in vivo by biochemical and imaging methodologies (23, 24, 25).

In the last 2 yr, we have fully characterized the ERE-luc model, which appears to report reliably on the activity of liganded and unliganded ERs induced by endogenous (20, 26) and exogenous (21, 26, 27) stimuli. However, in the course of the various experiments, we observed a significant variability in the levels of luciferase in unstimulated animals that were fed estrogen-free diets. This observation prompted us to better evaluate the contribution of diets to ER transcriptional activity and to compare diets and feeding scheduling to establish a proper protocol to investigate the effects of estrogenic compounds of biological or chemical origin in the absence of major dietary interferences. Here we show that diets considered estrogen free, devoid of derivatives of vegetables known to produce phytoestrogens (such as {alpha}-{alpha} or soy), indeed contain estrogenic activities sufficient to induce a state of ER transcriptional activity compatible with the activity of endogenous molecules signaling through ERs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Diets
The characteristics of the five diets used in the present study are represented in Table 1Go. Three of these diets [Piccioni 48 (Piccioni, Gessate, Italy), Mucedola 4RF21TC (Mucedola, Settino Milanese, Italy), Harlan Teklad Global Maintenance 2014 (Harlan Italy, S. Pietro Al Natisone, Italy)] are commercially available, and two (Mucedola purified and synthetic) were custom made. Piccioni 48 is a complete and regular rodent diet with soy, {alpha}-{alpha}, and fish derivatives, therefore containing compounds known to be estrogenic. Mucedola purified is a casein-based diet without {alpha}-{alpha} or soy derivatives. Mucedola 4RF21TC is certified as estrogen free and accurately tested for the detection of estrogenic activities. In this diet, the percentage of phytoestrogens is certified to be less than 4 ppb (parts per billion) according to international standards (U.S. Food and Drug Administration National Center for Toxicological Research Standard No. 2, September 5, 1973). Teklad Global Maintenance Diet 2014 is considered to be one of the best diets for assessing chemicals for estrogenic activity (28). The last diet, Mucedola synthetic, is purely synthetic and manufactured under technical specifications by Mucedola (Settino, Milanese, Italy). This diet contains products and byproducts of cereals and sugars, free amino acids, fat, and vitamins. All of these diets are in the form of cylindrical pellets.


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  		TABLE 1. Rodent diets: comparative analysis

 
The animals involved in this study were housed in plastic cages with clear, hardwood chips bedding, fed ad libitum, and provided with filtered water. The animal room was maintained within a temperature range of 22–25 C and relative humidity of 50% ± 10%. There was a cycle of 12 h light, 12 h dark (lights on, 0700–1900 h).

Luciferase enzymatic assay
Tissue extracts of mice killed after 15 d of feeding with complete diet (Piccioni 48) and top-certificate diet (Mucedola 4RF21TC) were prepared as previously described (20). Briefly, tissues are homogenized in 200 µl of 100 mM KPO4 lysis buffer (pH 7.8 containing 1 mM dithiothreitol, 4 mM EGTA, 4 mM EDTA, and 0.7 mM phenylmethylsulfonyl fluoride), three cycles of freezing-thawing, and 30 min of minifuge centrifugation (Eppendorf, Hamburg, Germany) at maximum speed. Supernatants containing luciferase were collected, and protein concentrations were determined by Bradford assay. Luciferase enzymatic activity in tissue extracts was measured by a commercial kit (Luciferase assay system, Promega, Madison, WI) according to the instructions of the supplier. The light intensity was measured with a luminometer (Lumat LB 9501/16, Berthold, Wildbad, Germany) in 10-sec time periods and expressed as relative light units over 10 sec/µg protein.

Bioluminescence reporter imaging
Mice fed with each different diet were visualized with a Night Owl imaging unit (Berthold Technologies, Bad Wildbad, Germany) consisting of a Peltier cooled charge-coupled device slow-scan camera equipped with a 25 mmf/0.95 lens. The images were generated by a Night Owl LB981 image processor and transferred via video cable to a peripheral component interconnect (PCI) frame grabber using WinLight software (Berthold Technologies). For the detection of bioluminescence due to luciferase in presence of the substrate luciferin, mice were anesthetized and received ip injection of an aqueous solution of luciferin (beetle luciferin potassium salt; Promega, 25 mg/kg) 20 min before bioluminescence quantification. Mice were placed in the light-tight chamber, and a gray-scale image of the animals were first taken with dimmed light. For colocalization of the bioluminescent photon emission on the animal body, gray-scale and pseudocolor images were merged using WinLight32 (Berthold Technologies) imaging software. Luminescence measurements are expressed as the integration of the average brightness/pixel unit.

Experimental animals
The ERE-Luc are transgenic mice engineered to express ubiquitously a transgene where the firefly luciferase gene is under the control of an estrogen-responsive promoter (20). The ubiquitous expression of the reporter and the short half-life of the luciferase allow monitoring spatiotemporally the transcriptional activity of the ER by biochemical or optical imaging technologies. In the present study, we used heterozygous males of 3 months of age obtained by mating male homozygous ERE-LUC mice with female C57BL/6 wild-type mice. Heterozygous mice were identified by PCR analysis as previously described (20). The aromatase inhibitor Arimidex (kindly provided by Dr. Alan Wakeling, AstraZeneca, Macclesfield, UK) was given sc at 1700 h for three consecutive days to a group of four animals fed ad libitum Mucedola 4RF21TC; luciferase activity was monitored at 0900 h in the mornings of d 0 and 3.

To assess the effect of endogenous sex hormones, groups of four animals were orchiectomized and at d 15 fed ad libitum Mucedola 4RF21TC. Photon emission was monitored at 1700 h and at 0900 h of the consecutive day.

All animal experiments described in the present study were carried out in accordance with European guidelines for humane animal care and use of experimental animals, approved by the Italian Ministry of Research and University, and controlled by the panel of experts of the Department of Pharmacological Sciences, University of Milan.

Statistic analysis
Statistical analysis was performed with ANOVA followed by the Scheffé test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The ERE-Luc reporter mice reveal ERs activity in mice fed with putative estrogen-free diets
Experiments were carried out in adult (3-month-old) male ERE-Luc mice. First, we compared the effects of a regular diet (Piccioni 48) and an estrogen-free diet (Mucedola 4RF21TC) on the state of ER transcriptional activation by measuring luciferase activity in a series of tissue extracts by enzymatic assay. Mice were fed ad libitum for 15 d with either the Piccioni 48 or Mucedola 4RF21TC diet. As shown in Fig. 1Go, when we compared the former with the latter group of animals, luciferase activity was significantly higher in mice fed the complete Piccioni 48 diet than in those fed Mucedola 4RF21TC; the dietary effect was particularly visible in brain, spleen, liver, kidney, prostate, and lung. Similar results were obtained with Mucedola purified, as demonstrated by bioluminescence analysis. As shown in Fig. 2Go (Fig. 2AGo, white bars), mice fed Mucedola purified had high photon emission and, therefore, high ER state of activity in several parts of the body: in the chest, reflecting ER activity in the liver; in the abdomen; in bones of the limbs; and tail. Conversely, very low bioluminescence was found in mice fed the Mucedola 4RF21TC diet (Fig. 2BGo, light gray bars). However, with the use of different batches of Mucedola 4RF21TC diet, the level of bioluminescence could change considerably (Fig. 2CGo, dark gray bars). This variability of effects was not dependent on the chow supplier because it was also observed with diets of similar certified quality purchased by other suppliers (Fig. 2DGo, black bars). Furthermore, the state of ER activation seen with the certified diets was not reconcilable with a sensitization or up-regulation of the ERs due to a prolonged treatment with estrogen-free diets, because ER activation was not observed in mice maintained with regular diet and then switched to estrogen-free a few days before the measurement of luciferase content (not shown).



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  		FIG. 1. High ER transcriptional activity in mice fed the regular diet. Adult 3-month-old ERE-luc male mice were fed ad libitum the regular diet (Piccioni 48) or estrogen-free diet (Mucedola 4RF21TC) for 15 d. Then, animals were euthanized, and different tissue samples were assayed for luciferase activity. On the y-axis is represented the ratio of luciferase activity calculated in mice fed the complete vs. estrogen-free diet. A total of six animals were analyzed for each diet in two separate experiments. Bars represent the mean ± SEM of the two experiments.

 


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  		FIG. 2. Differential activation of ER in ERE-luc mice fed the three putative estrogen-free diets. Adult male mice were fed ad libitum for 2 wk the three different putative estrogen-free diets and were visualized by a charge-coupled device. Panels show optical imaging of ERE-Luc representative male mice fed the following: Mucedola purified (A); Mucedola 4RF21TC, batch 1 (B); Mucedola 4RF21TC, batch 2 (C); and Harlan Teklad 2014 (D). Bars show the quantification of photon emission from three different body regions (chest, abdomen, and bone) in mice fed the Mucedola purified (white bars); Mucedola 4RF21TC, batch 1 (light gray bars); Mucedola 4RF21TC, batch 2 (dark gray bars); and Harlan Teklad 2014 (black bars) diets. Bioluminescence is expressed as integration of the average brightness/pixel unit (cts/s) and reported as mean ± SEM of values obtained from four animals for each diet. *, P < 0.05; **, P < 0.01 vs. 4RF21TC diet, batch 1.

 
It is important to underline that the degree of stimulation of liver ERs with selected batches of the top-certificate diet was as high as the stimulation induced by treatment with a concentration of estradiol (50 µg/kg) sufficient to induce overphysiological circulating levels of estradiol with respect to what was measured in female mice (Fig. 3Go, A and B). Yet, irrespective of the batch of estrogen-free diet used, we were unable to observe any change in the uterus weight when the diets were given to ovariectomized mice (data not shown). This is in line with what has been reported by previous authors (29, 30).



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  		FIG. 3. Luciferase expression in ERE-luc mice treated with exogenous 17ß-estradiol. A, Optical imaging of the bioluminescence of a representative ERE-luc male mice before (left) and upon (right) treatment with 50 µg/kg of 17ß-estradiol sc. B, Quantification of photon emission from three body regions of the animal (chest, abdomen, and bone) expressed as the integration of the average brightness/pixel unit (cts/s). Each bar represents the mean ± SEM of values from five animals. **, P < 0.01 vs. controls.

 
ER activity correlate with food assumption
To demonstrate that the luciferase activity also observed with estrogen-free diets was due to dietary estrogenic compounds, we investigated whether 1) luciferase activity correlated with food assumption and 2) the use of a synthetic diet resulted in blockade of luciferase synthesis.

We quantified photon emission of animals fed with Mucedola 4RF21TC diet in the evening and morning of the first two experimental days. In the second experimental day, after the morning measurement, animals were divided into two subgroups: the first was fed only during the daytime and the second only at night for three consecutive days; then photon emission was measured again in an evening and in a morning session. As shown in Fig. 4Go, in the initial group of animals fed the Mucedola 4RF21TC diet ad libitum, luciferase activity in the morning was significantly higher than in the evening. This is compatible with a diet effect because it is well known that mice, as nocturnal animals, eat preferably during the night. In addition, in the two experimental subgroups, animals fed at night showed elevated luciferase levels in the morning; vice versa animals fed only during the day had luciferase activity higher in the evening. Considering that rodents do not consume as much food during the daytime as they do at night, the lower level of luciferase found in the evening in the animals fed during the day led us to conclude that the so-called estrogen-free diet actually contained estrogenic compounds.



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  		FIG. 4. Mice fed at night have higher luciferase activity than mice fed at daytime. A, Bioluminescence analysis was done on four male ERE-luc mice fed ad libitum the Mucedola 4RF21TC diet the first day in the evening (at 1800 h) and in the morning of the second day (at 0900 h) (upper panels). Then, animals were divided into two subgroups: one fed at night (bottom left panels), the other one at daytime (bottom right panels). Mice were visualized as before, the fourth day in the evening (at 1800 h) or the fifth day in the morning (at 0900 h). B, Quantification of photon emission from the liver (chest) of the four mice fed ad libitum (left), the two of them fed at night (center), or the other two fed at daytime (right) the Mucedola 4RF21TC diet. For each group, white bars and black bars represent bioluminescence acquired respectively in the evening or in the morning. Each bar represents the mean ± SEM of values from four animals. The experiment was repeated two times. **, P < 0.01; *, P < 0.05 the corresponding evening group; {Delta}{Delta}, P < 0.01 vs. the group fed during daytime, morning detection.

 
Endogenous testosterone does not contribute to ER activation
To finally rule out the possibility that the observed activation of ERs was not due to a direct action of the substances present in the diets, but to circulating testosterone, luciferase activity was detected in the ERE-Luc mice before and 15 d after gonadectomy (Fig. 5AGo). In accordance with the previous experiments, photon emission was detected in the evening and in the morning of the consecutive day. Orchiectomy did not significantly change the effect of diet on ER activation observed in intact animals. This ruled out the presence in the diets of components able to increase the activity of the enzymes converting circulating testosterone into 17ß-estradiol or other estrogenic metabolites [e.g. 5-{alpha}-androstane-3ß and 17ß-diol]. To further prove a lack of contribution by testosterone in the observed ER activation, ERE-Luc mice received sc injections for three consecutive days of the aromatase inhibitor Arimidex (0.5 mg/kg). The state of ER activation in these animals was monitored in the morning, before and after treatment (Fig. 5BGo). Although this treatment was able to block the female estrous cycle in a parallel experiment (data not shown), we did not observe any significant change in photon emission in the male mice. This again points to the presence of estrogenic compounds in the diet.



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  		FIG. 5. Dietary ER activation is not influenced by endogenous production of 17ß-estradiol or estrogenic metabolites. A, Photon emission from mice fed ad libitum the Mucedola 4RF21TC diet was quantified in the evening and the consecutive morning, before orchiectomy, and 15 d after. For each group, white bars and black bars represent bioluminescence acquired in the evening and in the morning, respectively. Each bar represents the mean ± SEM of values from four animals. The experiment was repeated twice. **, P < 0.01 vs. each corresponding evening group. B, Animals received injections for three consecutive days of 0.5 mg/kg of Arimidex sc. Photon emission of these mice fed ad libitum Mucedola 4RF21TC was quantified in the morning before and after treatment. The experiment was repeated twice on a total of eight animals. Each bar represents the mean ± SEM of values of the two experiments.

 
Long-term exposure to a synthetic diet deprived of estrogenic compounds results in unliganded activation of ERs
Next, we tested the effects of administration of a purely synthetic diet. Mice were, therefore, fed ad libitum the Mucedola synthetic diet composed of chemicals known to be devoid of estrogenic activities. As expected, with 1 wk of treatment, the consistent luciferase activity detected in the chest was completely abolished. No significant change was observed in the other organs where the reporter activity continued to be low (Fig. 6Go). Then, we maintained the mice with the synthetic diet for the following 2 wk. After the second week of treatment, the levels of luciferase started to augment in the abdomen and head; at the third week, the levels were significantly increased in the abdomen, bone, and head. We believe that the luciferase changes observed with the synthetic diet are not attributable to a regulation of ER number because, in a dose-dependence study, these animals responded to 17ß-estradiol, as did mice maintained with the regular, so-called, estrogen-free diet. We suggest that the prolonged deficiency of alimentary estrogens resulted in the induction of endogenous compounds able to transcriptionally activate ERs. These compounds appeared to differentially affect the various organs: in mice deprived of dietary estrogens for a long time, luciferase activity was significantly increased in bone, abdomen, and head but not in the chest, where we failed to see any increase of luciferase activity in all of the animals studied.



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  		FIG. 6. Feeding mice the synthetic diet for 1 wk abolishes ER activation in the chest. Four ERE-luc male mice were maintained with a Mucedola 4RF21TC diet and then fed ad libitum a Mucedola synthetic diet deprived of estrogenic activity. A, Bioluminescence was acquired just before (baseline) or at 1, 2, or 3 wk of feeding with synthetic diet. B, Bars represent the quantification of photon emission from particular body regions (chest, abdomen, head, and bone) of the animals at the different time points. White bars (baseline), light gray bars (first week), dark gray bars (second week), and black bars (third week) represent the mean ± SEM of values from the four animals. The experiment was repeated with superimposable results. *, P < 0.05; **, P <0.01 vs. baseline.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study reveals a pervasive presence of compounds active on ERs in rodent aliments and points to the usefulness of reporter animals to monitor the presence of estrogenic compounds or endocrine disrupters in the alimentary chain.

Due to the increasing production of molecules endowed with endocrine-disruptor activity, the development of standardizable protocols to test for hormonally active compounds has been a major goal for regulatory agencies worldwide. Several researchers have analyzed the diets commonly used for rodent maintenance to identify those that did not interfere with investigation of endocrine disruptors or other compounds active through ERs. These studies were based on HPLC identification of known estrogenic compounds (such as phytoestrogens) and analysis of the effects of the diet on vaginal opening or uterus weight (29, 30, 31). Using these conventional methodologies, estrogenic substances were not identified in diets such as Teklad 2014, which were, as a consequence, recommended when testing for the effects of mixtures of compounds with putative estrogenic action (28). As a result of the availability of a transgenic mouse model with ubiquitous expression of a reporter gene driven by activated ERs, we were able to reveal the presence of estrogenic activities in diets currently believed to be estrogen free. Our data show that these activities could not be ascribed to a dietary-induced metabolic production of 17ß-estradiol or estrogenic metabolites but to direct effects of estrogenic compounds on ER signaling. These compounds are not present in trace amounts but at concentrations sufficient to induce a state of ER transcriptional activation compatible with endogenous hormonal activities in nonreproductive organs. Interestingly, these so-called estrogen-free diets were able to induce a significant state of transcriptional activity in organs other than those implicated with reproductive function but, in agreement with previous studies, failed to modify the uterus weight when used with ovariectomized female mice. This is not surprising, given the current knowledge of the mechanism of estrogen and ER action; it is well known that ER ligands, once localized within the hydrophobic pocket of ER hormone-binding domain, induce differential conformational changes that enable interaction with specific coregulators and other macromolecules of the transcription machinery (32). Depending on the ligand and on the cell targeted, ER effects on gene transcription have been shown to be extremely variable (33, 34, 35). Thus it is likely that diets ineffective on ER in uterus may be very estrogenic in other organs, as shown here.

The present study further proves the ERE-Luc model as suitable for testing the effects of endogenous as well as exogenous estrogenic compounds and provides a protocol to be used for in vivo testing of the estrogenic activity of xenobiotics and the discovery of novel sources of estrogenic compounds.

The difficulty in finding a diet totally deprived of estrogenic activity imposes further consideration because, in our view, it proves how widespread are estrogenic compounds in nature. This is partially expected because it is known that phytoestrogens are present in several plant derivatives, and endocrine disrupters may be easily found as contaminants in the plant and animal extracts used to feed rodents. On the other hand, the finding that dietary compounds may also have an extensive activity on the endogenous ERs in male mice poses the question of how relevant and necessary is the daily ingestion of xenobiotics active through the ERs for the maintenance of a balanced metabolism. The finding that long-term treatment with a diet completely deprived of estrogenic compounds triggers the production of endogenous molecules that activate ERs provides further support to the theory that ERs are involved in tissue metabolism and homeostasis. We do not know which endogenous compound is responsible of ER activation after exposure to a diet deprived of estrogens, but we could speculate that both ligand-dependent and ligand-independent mechanisms could be implicated in the activation of ERs. It is well known that organs other than ovaries can synthesize estrogens. On the other hand, it is well known that unliganded ERs may be transcriptionally activated by intracellular kinases transducing the signaling of membrane receptors such as neurotransmitters (36) or growth factors (21, 37, 38, 39, 40). We have recently shown that, at least in cycling female mice, unliganded activation of ERs is typically found in nonreproductive organs, whereas in reproductive organs and liver, ER activity appears to be more under the control of 17ß-estradiol. On the basis of these findings, it could be speculated that unliganded ER activation is unrelated to ER specialized functions, such as the control of reproduction, but is related to more general metabolic effects. A view supported by the finding that in animals fed the synthetic diet long term, the ER is activated in bone and abdomen but not in chest, where most of luciferase bioluminescence is due to ER activity in liver.

The present study leads to the speculation that perhaps a minimal state of activity of these receptors is a physiological necessity for most mammalian tissues in animals of both sexes and that a correct supply of exogenous estrogens might be a necessity.


   Acknowledgments
 
We thank M. Rebecchi and C. Meda for technical assistance and S. Oldoni for animal care. This work was supported by the European Community Programs EDERA (EC-QLK4-CT-2002-02221), Network of Excellence EMIL, CASCADE, and DIMI and Italian Grants COFIN (nos. 2002058785) and FIRB (nos. RBNE0157EH and RBNE01PASK) and the Italian Association for Cancer Research.


   Footnotes
 
First Published Online September 8, 2005

1 P.C. and A.B. contributed equally to this study. Back

Abbreviations: ER, Estrogen receptor; ERE, estrogen-responsive element.

Received June 1, 2005.

Accepted for publication September 1, 2005.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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