In recent years several studies demonstrated the presence of estrogen receptors in mammalian tissues and significantly improved our understanding of their ability to control biological processes in reproductive as well as non reproductive organs. Considering the manifold mechanisms and organs that are involved in estrogen action and the implication of estrogens in human female physiology, innovative approaches are required to shed light on the widespread activities of estrogen receptors in woman physiology. This is particularly relevant for the definition of novel, more efficacious hormonal replacement therapies or for the evaluation of the risk associated with the exposure to endocrine disruptors. The advent of genetic engineering and the development and exploitation of in vivo imaging techniques are providing new means for pre-clinical studies. The generation of the ERE-Luc mouse, a reporter animal developed for in vivo studies of the estrogen receptor activity, allows assessing the state of activity of the ER signaling pathway in all target tissues and organs at the same time, under physiological stimuli or as a consequence of a pharmacological treatment. This review summarizes the main steps in the generation and appraisal of the estrogen receptor reporter mouse ERE-Luc, designed for in vivo molecular imaging studies, and describes examples demonstrating the applicability of the ERE-Luc model to drug development and to the study of the in vivo effects of endogenous, dietary and environmental estrogens.
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â-ºReporter systems are novel tools to monitor complex biological events.
â-º ERE-Luc mouse model allows a systemic view of the transcriptional effects of estrogens.
â-º ERE-Luc mouse is a powerful tool for drug development.
â-º ERE-Luc mouse model is critical to study action and tissue specificity of phytoestrogens.
Keywords: Reporter mouse; Estrogen Receptor; SERM; Drug development; Phytoestrogens
In the recent years many reports have revealed the complexity of ER physiological activities and the multiplicity of estrogen targets. For this reason is critical to define whether and to what extent new ER ligands, intended for pharmacological purposes, are activating or repressing ERs in any given tissue. To this aim, cells transfected with reporter genes have been extensively employed to characterize the activity of new ER ligands. However, the application of reporter gene technology to animal engineering offers now the opportunity to take advantage of reporter tools to investigate a specific biochemical process in the whole animal, thus extending the analysis of ERs activity in the spatio-temporal dimension. In order to follow the transcription processes in a physiological context, several reporter animals have been generated; nevertheless, only recently the advances in reporter gene technology and in transgenesis allowed the generation of powerful tools designed to follow more and more complex biological events in the context of living beings.
Following this line of thoughts, the ERE-Luc model was generated by integrating into the mouse genome a luciferase reporter gene whose expression is under the control of transcriptionally activated ERs. In this transgenic animal any stimulus that induce receptor activation results in a proportional increase in luciferase synthesis; thus, the reporter expression represents a biosensor that measures the state of receptor activity. This unique feature makes possible to assess the activation state of the ER signaling pathway in all target tissues and organs concurrently, allowing the investigation of the consequences of physiological stimuli as well as of pharmacological treatments. For this reason the ERE-Luc model can be considered as a prototype of reporter mouse for SERMs development , to identify endocrine disrupter activities , to study cross-talk among membrane receptor and ERs and to discover novel estrogen target tissues . Above all, the ERE-Luc emerged as an invaluable tool to unveil the physiology of ER activation, thus shedding light onto a novel mechanism that is paving the way to more selective therapies to treat the menopause-associated disorders .
2. Reporter mice - a new way to look at drug action
The notable advances in molecular genetics achieved in the past decade as well as the possibility to manipulate cells in order to investigate drug activity using reporter genes led to significant improvements in drug development strategies. Nowadays, eukaryotic cells transfected with reporter genes (genes that encode an easily detectable protein) are extensively used to study cis-regulatory sequences or trans-acting factors that modulate the transcriptional activity of specific promoters.
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The application of reporter gene technology to animal genomes allows studying the activity of the selected promoter within a living organism, thus providing, with the aid of imaging technology, the opportunity to observe and quantify in real time the dynamics of transcription of simple or complex promoters.
A key step in the generation of a new reporter animal model is the choice of the candidate reporter gene, that must satisfy several prerequisites in order to be used for molecular imaging studies: (i) they must be detectable by in vivo studies: their expression indeed must provide information about the location, the magnitude and the persistence of gene expression directly in vivo; (ii) their expression must reflect the expression of the gene endogenously driven by the promoter under study; (iii) the turnover rate of the reporter proteins must be short enough to allow rapid assessment of changes in the molecular process under investigation occurring in a short time.
The introduction of reporter genes into animal genomes has a huge impact on preclinical experimentation, thus providing the opportunity to appreciate physiological and pathological mechanisms by providing measurable endpoints useful for the assessment of drug efficacy in all tissues in vivo. In order to be exploited for drug development, a mouse model must satisfy several requirements that are indispensable for the proper evaluation of drug candidates, thus allowing: (i) rapid assessment of all the organs where a specific compound is active; (ii) evaluation of the response to the drug after repeated administration; (iii) measurement of the response in each tissue, dependent on the route of administration; (iv) evaluation of the minimal concentration of drug necessary to obtain the pharmacological response independently of its plasma levels; (v) identification of active metabolites and their proï¬le of action; and (vi) detection of sites of drug accumulation during chronic administration.
As result of the generation of novel tools to monitor more and more complex biological events in the context of living beings, granted by the recent advances in reporter gene technology and in transgenesis, several reporter animals have been generated to study transcription processes in a physiological context. Notwithstanding, if we take in consideration the abovementioned points, only few of the reporter mice generated up to now are applicable to drug development. Even the numerous mice generated for the specific investigation of the activity of ligands on specific receptors are not designed to have a ubiquitous expression of the reporter and to guarantee that their signalling is free from influences resulting from position effects . In addition, several of these mouse models have reporters unsuitable for in vivo imaging analysis or have a turnover rate that is too long to enable a dynamic assessment of drug action . Finally, other models rely on the activity of receptors missing functionally important domains . Even though these models can provide information on the capability of the receptor to recognize a ligand, still they are not appropriate to understand the physiological consequences of such an event, which is critical to evaluate the pharmacological potential of the investigated compound.
The ERE-Luc mouse is the first example of a reporter animal designed for in vivo studies for drug development offering the opportunity to exploit the reporter tools to the entire animal, and extending the analysis of ERs activation in the spatio-temporal dimension.
3. The generation of ERE-Luc Reporter mouse model
Estrogens control specific gene networks to modulate target cell activities by signaling through two nuclear receptors (estrogen receptors, i.e. ERs), ERalpha and ERbeta. Cell-based approaches developed in the last decade have provided major insights into the transcriptional regulation of ERs at the promoter of target genes, where the interplay between ERs and coactivators and corepressors provides the receptors with tissue specificity of action.
ER transcriptional activation is controlled by a multiplicity of mechanisms . In addition, growth factors stimulate unliganded ER to induce the transcription of selected target genes . Activated ERs may cross-talk with other signaling pathways by influencing the activity of other transcription factors like AP-1, STATs, NFkB, SP-1 or by interacting with components of different transduction pathways such as Src , MAPK and G-proteins . Given that the estrogen action involves a multiplicity of mechanisms and organs, and considered the relevance of estrogens in human female physiology, it is necessary to take advantage of innovative approaches to clarify the exact physiological relevance of these alternative mechanisms in each target tissue. Cell systems cannot offer views into the hormonal activity on an entire organism: the ideal model to investigate the physiology and pharmacology of estrogen and cognate receptor activity is a reporter animal system.
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The ERE-Luc model was designed to satisfy the following parameters: (i) the reporter gene is ubiquitously expressed and responsive to molecules activating ERs in all the mouse cells; (ii) the reporter is a protein not expressed by the mouse, easily measured by quantitative assays, a good antigen to facilitate its cellular localization by immunohistochemistry and visible by non-invasive imaging technologies.
Optical imaging techniques are based on bioluminescence or fluorescence. The reporter genes used in bioluminescence imaging protocols such as luciferase generate photons proportionally to the level of luciferase expression in the presence of the appropriate substrate, i.e. luciferin.
The main limitation when bioluminescent reporters are used is that the light signal is absorbed and scattered in the tissue volume intersected by these low energy photons in the path from the emission site to the detector system. Red light is more efficiently transmitted through tissues than light with lower wavelengths, therefore efforts are being made to generate reporters that emit photons at wavelengths above 600Â nm. Luciferase activity from Photinus pyralis produces a broad spectrum peaking at 560Â nm with a wide fraction above 600Â nm, whereas wild-type green fluorescent protein (GFP) from Aequorea victoria, upon excitation, emits light with a peak at 509Â nm. In order to shift the emission closer to infrared wavelengths, recently GFP and luciferase mutant variants have been created . Nevertheless, GFP needs to be excited by an external light source. As consequence, the effect of tissue scattering and absorption is doubled with respect to bioluminescent probes luciferase enzymatic activity that results in the direct emission of photons, thus limiting the use of GFP to very small organisms. In addition, the luciferase gene offers a number of notable advantages: (i) encodes an insect enzyme not present in mammalians, thus ensuring the absence of background due to endogenous product; (ii) is easily quantitated by highly sensitive enzymatic assay; (iii) allows localization studies with specific, commercially available antibodies; (iv) allows optic imaging in live animals. Most importantly, luciferase has a very short turn-over rate and it does not accumulate, thus providing a dynamic view of the state of ERs activity. On this basis, the firefly luciferase gene was preferred as a reporter gene in the generation of this first animal model.
In the phase of selection of the estrogen-responsive promoter, the strategy of using strong promoters was discarded because of their high activity, which might have masked hormonal response or would have introduced new estrogen-independent elements for transcription. Therefore, it was pursued the idea to use a promoter ensuring a low basal state of reporter expression to emphasize the ubiquitous capacity of the reporter to be expressed in all tissues and the hormone-dependent transcription.
To this aim, using deletion mutants of the minimal promoter from the thymidine kinase (tk) gene from herpes simplex virus or the basal TATA box linked to a variable number of estrogen receptor-responsive elements (ERE), several different estrogen-inducible promoters were generated and tested in transient transfection assays in different cell lines. The combination of 2 palindromic EREs spaced 8Â bp apart located at 55Â bp from the constitutive tk promoter (âˆ’109 fragment) provided the required low basal transcription and the highest estrogen-induced reporter expression and was therefore selected .In order to bypass position effects and to ensure the ubiquitous expression of the transgene, we took advantage of the insulator strategy using insulators from the chicken genome : the Î²-globin hypersensitive site 4 (HS4) and the matrix attachment region (MAR) . A comparative analysis of luciferase expression in clones obtained in stable transfection assays carried out with pERE, pMAR and pHS4 plasmids showed that the presence of insulator sequences increased the number of luciferase expressing clones from 19 to 40-70%, thus enhancing the inducibility of luciferase expression by estrogen treatment. Subsequent studies demonstrated that the generated mouse has ubiquitous, regulated expression of the reporter
This model has a much broader potential than the classical preclinical investigation models, since it allows to: (i) observe in which organs or tissues a given compound is active, independently of previously acquired knowledge of the target tissue; (ii) define the effects of and changes in response after repeated administration of a given compound; (iii) determine the exact response of each tissue to the administration of a given compound independently of the route of administration; (iv) evaluate the minimal drug concentration needed to stimulate ERs independently of the drug's plasma levels; (v) discover the generation of active metabolites and their profile of action; (vi) verify potential sites of drug accumulation during chronic administration; and (vii) by combining pharmacological and toxicological studies, it can dramatically shorten the time needed to develop a given compound.
Taking advantage of transgenic mice, it is possible to combine pharmacological and toxicological studies, thus decreasing the number of animals necessary for drug development.
4. ERE-Luc mouse as a tool to study the dynamics of Estrogen Receptor activity
Among the huge number of studies on the molecular mechanism of estrogen receptor action, only very few have been focused on the characterization of the receptor activation in the spatio-temporal dimension. The use of molecular imaging technologies is smoothing the progress of these studies, offering the possibility to follow biological processes on a recurring basis in the same individual. Biochemical, immunohistochemical as well as pharmacological criteria have been applied to the ERE-Luc mouse to verify that the luciferase expression can be considered as a measure of the state of ER activity , thus establishing that, by measuring the photon emission originated from the luciferase/luciferin enzymatic reaction within the estrogen target tissue, the state of ER activation can be visualized and quantitated. Photons are externally detected and quantitated by a cool-charged coupled device camera (CCD-camera). Using a computer-assisted analysis is then possible to localize the photon emission with high sensitivity and with a resolution of 3-5Â mm by merging the luminescence detected with the CCD and a light picture of the animal . This non-invasive procedure can be applied several times to the same individual without its sacrifice; thus, it is possible to monitor the modulation in receptor activation in different organs during all the main physiological changes taking place in life, from embryonic state to adulthood.
The application of this technique allowed to challenge the well-established knowledge on estrogen action in the control of reproductive-related functions. By detecting the photon emission from a female ERE-Luc animal during estrous cycle we surprisingly observed a lack of correlation between the serum level of 17Î²-estradiol and the ER activation in tissues that are not directly involved in the reproductive function (e.g. bone, intestine, thymus, aorta, areas of the brain like cortex, hippocampus, etc.). The maximal receptor activity in these "non-reproductive tissues" has been detected at diestrus phase, when low levels of serum estrogens are present. This unexpected finding has been confirmed by post-mortem measurement in tissue extracts of the luciferase gene product activity coupled with the expression of the progesterone receptor, a natural estrogen target gene.
In order to define the dynamics of estrogen receptor activity as function of the estrogen synthesis in the ovaries, we evaluated the oscillation of ER activity in intact and ovariectomized (ovx) mice . To this aim, we measured daily luciferase-dependent photon emission in intact and ovx mice for 21 d. We observed that in the cycling mice (Fig. 1A) luciferase activity oscillated with time and the amplitude of the oscillation was different in each body area and the frequency of oscillation resulted to be tetradian (lasting 4 days, average in all tissues 4.4d) in most body areas. This result was predictable, considering that the length of estrous cycle in mice is 4-5 d; however, ER oscillations did not occur synchronously in all the body areas. In addition, quite unexpectedly ER activity fluctuated also after ablation of the ovaries with an oscillation period of about 4 d (Fig. 1B) with an amplitude lower than in intact mice and with altered phasing among organs: for instance, oscillation in the hepatic and genital areas was seldom in phase.
These results show that in intact mice ER activity oscillates with a frequency that is compatible with the estrous cycle, but the master regulator of ER oscillatory activity cannot be 17Î²-estradiol (E2), because the oscillations were asynchronous and persisted after ovariectomy. The asynchrony of ER activity in the different body areas of intact mice was verified by quantitative real-time PCR analysis of mRNAs encoded by endogenous ER target genes , thus confirming the reliability of luciferase as indicator of ER transcriptional activity. Therefore, luciferase can be used as a surrogate target gene in studies on the effect of selected HT.
The distinct regulation of ERs in reproductive and non-reproductive tissues may reflect a differential functionality of ligand-bound versus unliganded-activated receptors, thus inspiring the intriguing hypothesis that hormone-dependent ER functions were acquired only later in the phylogenetic evolution in relation of the development of reproductive functions. According to this view, before this acquisition, the ERs were orphan receptors modulating the response to membrane receptor signaling pathways in different organs. As demonstrated by our imaging studies, ER in liver is very active and may be activated by factors other than estrogens. Indeed, in the past we observed that liver ER was significantly activated after food ingestion even when the synthetic diet utilized to feed the mice was deprived of known estrogenic compounds . It took us several experiments to realize that, among all macronutrients, amino acids (aa) were responsible for a transcriptional activation of liver ER strictly associated and necessary for the proper progression of the estrous cycle . Furthermore, by genome-wide experiments we observed that regulation of energy metabolism by ERÎ± is tightly linked to reproductive functions as indicated by several findings: (i) in adult mice the expression of genes involved in fatty acid and cholesterol synthesis oscillated synchronously with the estrous cycle (possibly in function of a potential egg fertilization); (ii) in prepuberal female mice the expression of the two sets of genes had an opposite trend (the high cholesterol synthesis being probably due to the requirements of a growing organism); (iii) in the later stages of pregnancy, characterized by high circulating estrogen levels , there was a impressive decrease in the expression of all enzymes; and, most relevant, (iv) in the absence of a cycle due to age or surgical menopause, the expression of most of the genes in study lost the oscillatory pattern, and the mRNAs levels were generally significantly higher than at proestrus. It's noteworthy that a long-term dysregulation of the estrous cycle and tetradian ERÎ± oscillatory activity (e.g. ovariectomy, aging, alteration of IGF-1 signaling) resulted also in accumulation of fat deposits in liver. Experiments on liver Igf1-/- mice and liver specific ERÎ± KO mice showed that the amount of deposited lipid is inversely proportional to the synchrony between the estrous cycle and production of mRNAs for fatty acid/cholesterol synthesis. These results underline the importance of the tetradian activity of hepatic ERÎ± to poise the receptor to adjust to the different energy requirements associated to the reproductive stage. These findings are particularly relevant to understand the etiology of metabolic disorders associated with impaired ovarian functions (e.g. PCOS) or the cessation of the reproductive cycle , suggesting to revise current rationales in the treatment of the post-menopause, taken into due consideration the periodic nature of ER signaling for a more efficacious use of estrogens.
5. SERM classification exploiting the ERE-Luc mouse
The reported experiments raised doubt on whether the existing approaches in hormone administration are correctly mimicking the physiological activation of the receptors in terms of asynchronous activation in reproductive versus non-reproductive tissues. Using ERE-Luc mice, we demonstrated that as consequence of long-term stimulation with natural and synthetic ligands, ER-mediated transcriptional activity oscillates with pulses characterized by frequency and amplitude strictly associated with the type of estrogenic compound administered, its dosage, and the organ in study. We investigated the effect of long-term (21 d) hormone replacement on ER signaling by whole-body in vivo imaging. Estrogens and selective ER modulators (17Î²-estradiol (E2), conjugated estrogens (CE, Premarin), bazedoxifene (BZA), lasofoxifene (LAS), ospemifene (OSP), raloxifene (RAL), and tamoxifen (TAM) were administered daily at doses equivalent to those used in humans as calculated by the allometric approach. During this study, photon emission was measured once a day in selected body areas by means of a segmentation algorithm. ER activity was measured in cycling and ovariectomized mice as controls. The study demonstrated that ER-dependent transcriptional activity oscillated in time, and the strict association of frequency and amplitude of the transcription pulses with the target tissue and the estrogenic compound administered. In all of the studied body areas, each compound showed a different profile of activity. In the skeletal and genital areas of mice treated with E2, we observed an increment in luciferase activity over time of exposure; in contrast, photon emission in the hepatic area increased rapidly after E2 administration and decreased with time. LAS administration led to little to no change in hepatic and skeletal areas, whereas photon emission in genital area was higher than in controls just before the end of the treatment. In the OVX mice ER activity did not change noticeably in all anatomical areas during the treatments. The analysis of the photon emission evidenced a fluctuation over consecutive days of exposure in all mice studied, including the OVX. Further analysis of the bioluminescence profile in time showed that with some of the drugs, the oscillatory pattern had a fixed amplitude and frequency (e.g. LAS in the genital area). The comparison of the effects of the different treatments in each experimental group evidenced that such an oscillation was a characteristic response to the specific ligand in the various tissues examined.
After the analysis of bioluminescence profiles generated in the studied areas, five independent parameters (peak number, peak amplitude, peak period, AUC, and potency), namely descriptors, were used to describe unique features of the drug effect. The selected descriptors were inserted in a clustering algorithm in order to define the extent to which the drugs act differently or similarly with each other. More importantly, by comparing the sets of parameters of drug-treated mice with those found in OVX or cycling animals, we were able to assess the effectiveness of each drug in replacing the endogenous hormones, thus representing the best replacement therapy.
By using all the descriptors that characterize the effect of SERMs on ER in the different ERE-Luc anatomical areas (genital, skeletal, hepatic, abdominal, and thymic), we created a single phenogram to classify the drugs according to their spatio-temporal activity. This analysis includes the features of drug action in time in different organs, and may be considered as a multivariate fingerprint of drug efficacy (Fig. 2).
An example showing the feasibility of this approach is provided by our experiment aimed to confirm the relevance of ER rhythmic oscillations on lipid accumulation . In details, we ovariectomized ERE-Luc mice of 2 months of age for 4-5 weeks prior initiating a 21-day treatment with vehicle, CE, BZA, TSEC (Tissue Selective Estrogen Compound) and RAL. The drugs were administered at a dosage previously shown to induce a proper oscillation of ER in liver and intestine. The measurement of luciferase activity in liver and intestine and the application of Fourier transform led us to measure the frequency of the oscillations induced by the treatments.
Next, we evaluated the synchrony of the peaks of ER activity in liver and intestine and evaluated the data obtained by agglomerative hierarchical clustering to identify the classes of compounds which better mimicked the ER oscillatory behavior characteristic of cycling mice. The results (Fig. 3A) clearly indicated that ovx changed significantly the oscillatory pattern of the receptor (as indicated by the Manhattan distance from the cycling mice) and that CE and RAL did not significantly improve the distance from the cycling controls: this indicated their inability to induce a physiological oscillation. Conversely, BZA and TSEC were found to cluster with cycling animals, thus indicating to be able to reinstate an oscillation more similar to cycling animals than to ovx. When we measured liver FFA content, we found that lipids were increased significantly in ovx mice treated with vehicle for 21 days (+ 84% vs. intact cycling mice); among the selected HRT only TSEC and BZA prevented liver FFA accumulation. When hormone replacement therapy was not reinstating the correct oscillatory pattern, with CE and RAL, lipid accumulation occurred (Fig. 3B).
Experience with Selective Estrogen Receptor Modulators (SERMs) has demonstrated the immense variability of the in vivo action of estrogenic molecules: the long-term treatments performed induced a state of activation of ER which was dependent on the tissue evaluated, the dosage utilized, and the time of treatment, challenging the ability to establish the parameters necessary to evaluate the extent of beneficial/negative effects associated with each estrogenic compound. We believe that the dynamics of ER transcriptional activity are very relevant from the physiological point of view and need to be reproduced faithfully in hormone replacement therapy. To maximize the efficacy of hormone replacement therapies, we should identify ligands that can closely mimic the temporal effects of endogenous hormones. Clustering compounds on the bases of their effects in space and time on ER transcriptional activity and assessing the extent to which the pharmacological treatment overcomes the effect of ovariectomy and mimics the activity of ER in cycling mice may open the way to the identification of an efficacious and safe treatment for the postmenopause. Furthermore, classifying alimentary and environmental endocrine disrupters and comparing their activity to well-studied compounds may facilitate the understanding of their real toxicity.
6. ERE-Luc and phytoestrogens
Human beings, as consequence of their individual diet, are distinctively exposed to phytoestrogens, chemicals produced by plants mimicking estrogens by binding to estrogen receptors (ERs) with high affinity. The phytoestrogen-ER complex may interact with other nuclear factors and co-activators and regulate the transcription of selected genes . The isoflavone genistein is the most abundant of these compounds, and is commonly found in edible plants like legumes . The intake of a soy-nuts serving or a single capsule of a soy-extract containing 64Â mg of total isoflavones , induce a peak of plasma levels to a concentration considered to have the capability to perturb endocrine signaling. Since ER expression is not restricted only to reproductive organs, phytoestrogens might influence a number of physiological functions and also cause collateral effects in non reproductive organs. Studies in adult rodents have linked phytoestrogen intake with altered bone development , obesity , alteration of thymic functions , myelotoxicity and alterations in the sexually dimorphic behavior .
Notwithstanding, the clinical community has a less than negative position towards phytoestrogens: it is frequently reported that soy or isoflavones may foster beneficial effects against age-related pathologies. Several effects have been associated to diets containing estrogenic compounds, and the complexity of ER physiological activities is well known. For this reason is mandatory to consider a more systemic approach to the study of the effects of protracted exposure to phytoestrogen-rich diets. The availability of the ERE-Luc reporter mouse provides a suitable tool to study the effects of acute or chronic exposure to specific diets on ER transcriptional activity.
Recently, we investigated the effect of short-term and long-term consumption of isoflavones as pure compounds or as part of a specific diet on ER activity . ERE-Luc animals were allowed to drink soymilk (or water for controls) ad libitum for 20Â days. During this period of time luciferase activity was assessed daily by BLI. Luciferase activity clearly increased at the beginning of the treatment in the chest area of the soymilk group and remained significantly higher than in controls for the total duration of the experiment. The extent of photon emission was of the same order of magnitude (800Â cts/s) as the acute treatment with E2 (800-1200Â cts/s). No major changes were observed in thymus, limb, and genital area. Interestingly, the treatment affected photon emission in abdomen area, where the effect slightly increased over time and was highest around day 15. These data clearly evidence that in definite organs such as the liver, the continuous exposure to estrogens enclosed in soymilk maintains high levels of ER activity. Then, we studied the effect of chronic treatment with pure genistein using two doses 1 and 5Â mg/kg/day of the compound. Genistein treatment did not induce changes in ER activity in abdomen. Oscillations of photon emission were observed in this organ in both controls and treated mice: the origin of the changes related to the prolonged treatment with gavage is unknown.
The main finding of this study is that factors different from phytoestrogens (the food matrix) contribute to the global estrogenicity of soymilk. In order to explain the findings, we hypothesized three possible mechanisms : i.) differential receptor regulation: the administration of free isoflavones is likely to cause a rapid increase of their plasmatic concentrations, with simultaneous induction of a peak of ER activity that, recurring for some times, leads to desensitization of the receptor to the ligand or to its decreased transcriptional capacity. On the other hand, the continuous consumption of low concentration of phytoestrogens with soymilk may cause a permanent status of ER activation without ER down-regulation. Preliminary analysis did not show a clear decrease in ER protein in liver after long-term treatment with genistein, however it is conceivable that, in spite of the presence of ERs, the activity of the receptor could be blunted at the level of its interactions with co-regulators. ii.) Differential bioavailability: the simultaneous administration of genistein and daidzein failed to increase ER activity in both acute and chronic studies. A possible reason could be that daidzein or its metabolites modulate liver catabolic enzymes leading to a higher catabolism/excretion of the administered compounds . It is conceivable that the presence of the soy matrix, by limiting the kinetics of absorption, distribution, metabolism and excretion of isoflavones, could optimize/synchronize their permanence in blood or in other tissues with the availability of ER to be transcriptionally activated. iii.) The presence of estrogenic compounds other than isoflavones in soymilk: evidences suggests that soy proteins may have a direct effect on human liver contributing to the changes in LDL receptor activity (reviewed in Sirtori et al. ). interestingly, hepatic LDL receptor is still widely accepted as an estrogen-target gene . Hence, we may speculate that dietary proteins regulate estrogen receptor activity, in a similar way to what was observed for other nuclear factors with other macro-nutrients such as fatty acids or glucose .
The main benefit of using reporter animals is that they provide measurable endpoints useful to evaluate the effects of natural and synthetic estrogens in all tissues of living animals. This radically changes the study of ER physiological activities as well as preclinical experimentation thus providing the opportunity to unravel physiological and pathological mechanisms and to assess and compare the effects of selected treatment in healthy animals as well as in models of specific diseases.
The studies described in this review show the power of reporter mouse technology that allows: i.) global view of the tissues affected by the compound of interest for the recognition of the organs potentially affected; ii.) unequivocal assessment of dosage and timing necessary to elicit the activity of the receptor; iii.) longitudinal studies in a single individual during repeated exposure, unravels the sites of compound accumulation and activity, or the dynamics of the response of target to the treatment.
The presented results further demonstrate the applicability of the ERE-Luc model to the study of the in vivo effects of endogenous, dietary and environmental estrogens. ERs are expressed in most tissues in mammals and estrogens have unpredictable tissue-specific actions on ERs: therefore, the ERE-Luc mouse model represents the most appropriate model to obtain a realistic, systemic view of the transcriptional effects of endogenous or exogenous estrogens. The relevance of the ERE-Luc mouse as a model to identify novel selective ER modulators (SERMs) and to profile their activity is self-evident; furthermore, the use of the ERE-Luc model revealed the widespread action and tissue specificity of endocrine disrupters and xenobiotics such as phytoestrogens.
The work discussed in this article was supported by Pfizer Pharmaceutical Co. and the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement nÂ° 278850 (INMiND).
Figure 1. Profile of photon emission in time in cycling and ovx ERE-Luc mice. Photon emission was measured daily at 1500 h in 6-month-oldERE-Luc mice cycling or ovariectomized 3 wk before the initiation of the study. Each animal group was constituted of nine mice. The figure represents daily photon emission (plotted as photons per second per square centimeter) in head, genital, and hepatic areas of a single mouse representative of the pattern of ER activity in intact (A) and ovx(B) female mice. From Della Torre, et al. 2011 .
Figure 2. Phenetics of drug action. Each was normalized on the average calculated on intact cycling (cyc) females (considered equal to 100). A matrix was built for each anatomical area: each column contains one descriptor, and each cell contains the descriptor averages for each experimental group. Agglomerative hierarchical clustering was computed with a Manhattan metric and a complete linkage method with an R code available online (Agglomerative Nesting version 1.0.2, Office for Research Development and Education; http://www.wessa.net/). In the dendrogram, distances between branch lengths represents the distance of the menopause model (OVX) vs. the physiology model (cyc); hormone replacement efficacy is measured by its ability to mimic ER activity in the cycling mice. A and B, SERM classification (hierarchical clustering) in the genital area (A) and in skeletal area (B); (C), multidimensional imaging descriptors from all anatomical areas measured (genital, skeletal, hepatic, abdominal, and thymic) are clustered as above; dendrogram branches group families of structurally related compounds. From Rando, et al. 2010 .
Figure 3. Reinstatement of ER tetradian cycle by appropriate hormone replacement therapy prevents the effect of ovariectomy. (A) Dendrogram analysis of the efficacy of selected HRT. The distances between branch lengths represents the distance of the physiology model (CYC) vs. the surgical menopause model (VEH). Hormone replacement efficacy is measured by its ability to mimic ER activity in the cycling mice. (B) Free fatty acids (FFA) content of hepatic tissue harvested from intact cycling mice (CYC), and from ovx mice treated for 21 days with vehicle (VEH) and selected HRT. CE: conjugated estrogen; BZA: Bazedoxifene; TSEC: tissue selective estrogen complex; RAL: Raloxifene. Data are shown as mean Â± s.e.m. n=6-8 mice per treatment. Statistics were calculated by two-tailed t-test. *, pVal<0.05 for comparison with OVX. #, pVal<0.05 for comparison with VEH. Livers of cycling mice were collected at metestrus and the experiment was repeated twice. From Villa, et. al 2012 .