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Sex plays a role in the incidence and outcome of neurological illnesses, also influencing the response to treatments. Despite sexual differentiation of the brain has been extensively investigated, the study of sex differences in microglia, the brain's resident immune cells, has been largely neglected until recently. To fulfill this gap, our laboratory developed several tools, including cellular and animal models, which bolstered in-depth studies on sexual differentiation of microglia and its impact on brain physiology, as well as on the onset and progression of neurological disorders. Here, we summarize the current status of knowledge on the sex-dependent function of microglia, and report recent evidence linking these cells to the sexual bias in the susceptibility to neurological brain diseases., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Epidemiological and clinical studies have largely demonstrated major differences in the prevalence of metabolic disorders in males and females, but the biological cause of these dissimilarities remain to be elucidated. Mammals are characterized by a major change in reproductive strategies and it is conceivable that these changes subjected females to a significant evolutionary pressure that perfected the coupling between energy metabolism and reproduction., Scope of Review: This review will address the plausibility that female liver functions diverged significantly from males given the role of liver in the control of metabolism. Indeed, it is well known that the liver is sexually dimorphic, and this might be relevant to explain the lower susceptibility to hepatic diseases and liver-derived metabolic disturbances (such as the cardiovascular diseases) characteristic of females during their fertile period. Furthermore, estrogens and the hepatic ERα play a significant role in liver sexual-specific functions and in the control of metabolic functions., Conclusions: A better grasp of the role of male and female sex steroids in the liver of the two sexes may therefore represent an important element to conceive novel treatments aimed at preventing metabolic diseases particularly in ageing women or limiting undesired side effect in the treatment of gender dysphoria., (Copyright © 2018. Published by Elsevier GmbH.)

Sex has a role in the incidence and outcome of neurological illnesses, also influencing the response to treatments. Neuroinflammation is involved in the onset and progression of several neurological diseases, and the fact that estrogens have anti-inflammatory activity suggests that these hormones may be a determinant in the sex-dependent manifestation of brain pathologies. We describe significant differences in the transcriptome of adult male and female microglia, possibly originating from perinatal exposure to sex steroids. Microglia isolated from adult brains maintain the sex-specific features when put in culture or transplanted in the brain of the opposite sex. Female microglia are neuroprotective because they restrict the damage caused by acute focal cerebral ischemia. This study therefore provides insight into a distinct perspective on the mechanisms underscoring a sexual bias in the susceptibility to brain diseases., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Recent studies have demonstrated that in mice, the estrogen receptor alpha (ERα) is expressed in the liver and has a direct effect on the regulation of the hepatic genes relevant for energy metabolism and drug metabolism. The sex-related differential expression of the hepatic ERα raises the questions as to whether this receptor is responsible for the sexual differences observed in the physiopathology of the liver.

Introduction: In Alzheimer's disease (AD), pathologic amyloid-beta (Aβ) is synaptotoxic and impairs neuronal function at the microscale, influencing brain networks at the macroscale before Aβ deposition. The latter can be detected noninvasively, in vivo, using resting-state functional MRI (rsfMRI), a technique used to assess brain functional connectivity (FC)., Methods: RsfMRI was performed longitudinally in TG2576 and PDAPP mice, starting before Aβ deposition to determine the earliest FC changes. Additionally, the role of pathologic Aβ on early FC alterations was investigated by treating TG2576 mice with the 3D6 anti-Aβ-antibody., Results: Both transgenic models showed hypersynchronized FC before Aβ deposition and hyposynchronized FC at later stages. Early anti-Aβ treatment in TG2576 mice prevented hypersynchronous FC and the associated synaptic impairments and excitatory/inhibitory disbalances., Discussion: Hypersynchrony of FC may be used as a new noninvasive read out of early AD and can be recovered by anti-Aβ treatment, encouraging preventive treatment strategies in familial AD., (Copyright © 2016 The Alzheimer's Association. Published by Elsevier Inc. All rights reserved.)

Lipoprotein synthesis is controlled by estrogens, but the exact mechanisms underpinning this regulation and the role of the hepatic estrogen receptor α (ERα) in cholesterol physiology are unclear. Utilizing a mouse model involving selective ablation of ERα in the liver, we demonstrate that hepatic ERα couples lipid metabolism to the reproductive cycle. We show that this receptor regulates the synthesis of cholesterol transport proteins, enzymes for lipoprotein remodeling, and receptors for cholesterol uptake. Additionally, ERα is indispensable during proestrus for the generation of high-density lipoproteins efficient in eliciting cholesterol efflux from macrophages. We propose that a specific interaction with liver X receptor α (LXRα) mediates the broad effects of ERα on the hepatic lipid metabolism., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Estrogen receptors (ER) are known to play an important regulatory role in mammary gland development as well as in its neoplastic transformation. Although several studies highlighted the contribution of ER signaling in the breast transformation, little is known about the dynamics of ER state of activity during carcinogenesis due to the lack of appropriate models for measuring the extent of receptor signaling in time, in the same animal. To this aim, we have developed a reporter mouse model for the non-invasive in vivo imaging of ER activity: the ERE-Luc reporter mouse. ERE-Luc is a transgenic mouse generated with a firefly luciferase (Luc) reporter gene driven by a minimal promoter containing an estrogen responsive element (ERE). This model allows to measure receptor signaling in longitudinal studies by bioluminescence imaging (BLI). Here, we have induced sporadic mammary cancers by treating systemically ERE-Luc reporter mice with DMBA (9,10-dimethyl 1,2-benzanthracene) and measured receptor signaling by in vivo imaging in individual animals from early stage until a clinically palpable tumor appeared in the mouse breast. We showed that DMBA administration induces an increase of bioluminescence in the whole abdominal area 6 h after treatment, the signal rapidly disappears. Several weeks later, strong bioluminescence is observed in the area corresponding to the mammary glands. In vivo and ex vivo imaging analysis demonstrated that this bioluminescent signal is localized in the breast area undergoing neoplastic transformation. We conclude that this non-invasive assay is a novel relevant tool to identify the activation of the ER signaling prior the morphological detection of the neoplastic transformation.

Context and Objective: During their reproductive years, women are generally protected from cardiovascular disease events by their estrogen-replete status. Our starting hypothesis was that lower estrogen levels after menopause are associated with macrophage activation profiles skewed toward proinflammatory phenotypes. Research Design and Setting: This was an in vitro and ex vivo study in human blood-derived macrophages., Subjects: We obtained blood from 12 healthy male donors for the in vitro study and from 5 premenopausal and 8 postmenopausal women for the ex vivo study., Outcome: We measured macrophage immunophenotypes in the resting state and after activation with M1-associated (lipopolysaccharide [LPS]/interferon-γ [IFN-γ]) or M2-associated (IL-4/IL-13) stimuli and expression of estrogen receptors (ERs) and other transcription factors., Results: Unpolarized macrophages expressed both ERα and ERβ, and ERα but not ERβ levels were decreased by M1 stimuli. LPS/IFN-γ also induced down-regulation of CD163 and CD206, markers of alternative activation, and increased cell-bound TNF-α and IL-10. These effects were prevented by 17β-estradiol treatment through impaired nuclear factor-κB liberation. In agreement with a role for 17β-estradiol in attenuating the inflammatory response, M1/M2 subpopulations in monocytes and unstimulated macrophages from premenopausal and postmenopausal donors were similar. In contrast, M2 activation appeared to be blunted in macrophages from postmenopausal women, leading to an increased M1/M2 response ratio., Conclusions: Estrogen treatment prevented LPS/IFN-γ action on human M2 macrophage markers and cytokine production, whereas menopausal estrogen loss was associated with an impaired response to alternative activation, suggesting that these mechanisms affect the cardiovascular risk profile in relation to menopausal status.

Estrogens play an important role in the regulation of energy homeostasis in female mammals and a reduced ovarian function, due to natural aging or surgery, is associated with body weight increase and fat redistribution. This disruption of energy homeostasis may constitute a trigger for several pathologies known to be associated with climacterium; however, so far, limited attention has been devoted to the ability of estrogen replacement therapies (ERT) to reinstate the balanced energy metabolism characteristic of cycling female mammals. The purpose of the present study was to compare the efficacy of selected ERTs in reversing the ovariectomy-induced gain in body weight. To this aim female ERE-Luc mice were ovariectomized and, after 3 weeks, treated per os for 21 days with: conjugated estrogens, two selective estrogen receptor modulators (bazedoxifene and raloxifene), and the combination of bazedoxifene plus conjugated estrogens (tissue-selective estrogen complex, TSEC). The study shows that the therapy based on TSEC was the most efficacious in reducing the body weight accrued by ovariectomy (OVX). In addition, by means of in vivo imaging, the TSEC treatment was shown to increase estrogen receptor (ER) transcriptional activity selectively in the arcuate nucleus, which is a key area for the control of energy homeostasis. Finally, quantitative analysis of the mRNAs encoding orexigenic and anorexigenic peptides indicated that following ERT with TSEC there was a significant change in Agrp, NPY, and Kiss-1 mRNA accumulation in the whole hypothalamus. Considering that prior studies showed that ERT with TSEC was able to mimic the rhythm of ER oscillatory activity during the reproductive cycle and that such fluctuations were relevant for energy metabolism, the present observations further point to the ER tetradian oscillation as an important component of the ER signaling necessary for the full hormone action and therefore for an efficacious ERT.

CdCl2 salt is widely used in exposure oriented studies, while the biological exposure of Cadmium (Cd) occurs mostly through diet. Hence, we designed a in vivo imaging methodology with sexually immature male ERE-Luc reporter mice to test the estrogen-like (EL) effects of Cd as a natural component in wheat and flax bread based diets (containing 17.57 and 49.22 ug/kg Cd concentrations respectively) and CdCl2 per-oral dose of 1 ug/kg/bw/day. Total exposure of ingested and % bioaccumulation of Cd in selected organs were estimated as 547 ng (4.4%), 776 ng (0.3%) and 2131.8 ng (0.1%) corresponding to CdCl2, wheat and flax bread based diet treatments respectively. Cd from CdCl2 bioaccumulated more readily, despite the exposure of Cd is higher with bread based diets. Longitudinal in vivo imaging did not reveal significant changes in luciferase activity. White adipose tissue (WAT) and prostate were identified as novel target organs of Cd. Indeed, the rest of the observed EL effects, endogenous target gene expression and necropsy findings are not consistent to any particular organ or treatment. This implies that, the observed EL effects due to low doses of Cd (either from CdCl2 or dietary form) occur only as subtle changes at the molecular level, but inadequate to cause significant changes at the anatomo-pathological level during the 21 day exposure period. The study demonstrates the sensitivity of the methodology to assess EL effects of food embedded Cd and underlines the limitations of directly extrapolating the results of suspected chemicals in their pure form to dietary exposure scenarios.

Noninvasive, imaging-based methodologies provide for the first time the possibility to spatio-temporally investigate physiopathological events and long-term effects of drug administration of exposure to environmental and alimentary toxic compounds. Hence, this novel methodology could enable us to measure the dynamics of specific molecular pathways in live animals. In the last few years, animals, particularly mice, were genetically modified to respond to a specific stimulus with the production of proteins, named "reporters," that are easily detected and quantitated by in vivo and ex vivo imaging. These "reporter mice" are gradually being applied to the pharmaco-toxicological research. In the generation of a reporter mouse useful for pharmaco-toxicological studies several elements need to be considered in the selection of the reporter system: the half-life of proteins should be compatible with the necessities of the study to assess the onset and the termination of the stimulus of interest, in all tissues the response should be proportional to the given stimulus, and the imaging modalities requested for reporter measurements should be applicable to high-throughput screening. Bioluminescence-based imaging (BLI) in small animals has the advantage over other modalities that does not require too sophisticated equipment or specifically and highly trained personnel, and furthermore may be carried out at a relative rapidity and low cost; for these reasons several luciferases have been developed for in vivo imaging applications and used in the generation of reporter mice. We here describe a BLI-based reporter mouse created to respond to estrogenic stimuli, which has been applied to the study of female physiopathology as well as for the identification of the effects of selective drugs or toxic compounds present in the environment and in the alimentary chain.

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 introduction of genetic engineering and the development and application of in vivo imaging techniques offer new tools 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 activity state of the ER signaling pathway in all target tissues and organs at once, under physiological stimuli or as a result 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 suitability of the ERE-Luc model for drug development and for the investigation of the effects of endogenous, environmental, and dietary estrogens in vivo. This article is part of a Special Issue entitled 'Phytoestrogens'., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Although several lines of evidence have indicated that menopause is associated with increased susceptibility to neurological disorders, the mechanisms involved in this phenomenon remain to be elucidated. Because neuroinflammation is a common feature of a number of brain diseases, we hypothesized that the cessation of ovarian functions and the consequent decrease in estrogen receptor (ER)-mediated antiinflammatory activity may represent a trigger for postmenopausal brain dysfunctions. The aim of the present study was to investigate the effects of aging and surgical menopause on the activity of ER in neuroinflammation. The present study shows that ER genes are expressed in the hippocampus, but ER transcriptional activity decreases significantly beginning at 12 months of age in intact and ovariectomized mice. With ovariectomy, we observe an age-dependent accumulation of mRNA encoding inflammatory mediators (e.g. TNFα, IL1β, and macrophage inflammatory protein-2) and changes in the morphology of astroglia and microglia. In addition, we show that aging itself is coupled with an exaggerated response to acute inflammatory stimuli with a major accumulation of TNFα, IL1β, macrophage inflammatory protein-2, and macrophage chemoattractant protein-1 mRNA in response to lipopolysaccharide administration. The response to acute inflammatory stimuli appears to be differentially modulated by the duration of hormone deprivation in 12-month-old mice. Taken together, the present results show that aging is associated with decreased ER activity, despite continuous ER synthesis, and that age-dependent neuroinflammation is strongly influenced by hormone deprivation.

Over the past two decades, our understanding of estrogen receptor physiology in mammals widened considerably as we acquired a deeper appreciation of the roles of estrogen receptor alpha and beta (ERα and ERβ) in reproduction as well as in bone and metabolic homeostasis, depression, vascular disorders, neurodegenerative diseases and cancer. In addition, our insights on ER transcriptional functions in cells increased considerably with the demonstration that ER activity is not strictly dependent on ligand availability. Indeed, unliganded ERs may be transcriptionally active and post-translational modifications play a major role in this context. The finding that several intracellular transduction molecules may regulate ER transcriptional programs indicates that ERs may act as a hub where several molecular pathways converge: this allows to maintain ER transcriptional activity in tune with all cell functions. Likely, the biological relevant role of ER was favored by evolution as a mean of integration between reproductive and metabolic functions. We here review the post-translational modifications modulating ER transcriptional activity in the presence or in the absence of estrogens and underline their potential role for ER tissue-specific activities. In our opinion, a better comprehension of the variety of molecular events that control ER activity in reproductive and non-reproductive organs is the foundation for the design of safer and more efficacious hormone-based therapies, particularly for menopause. This article is part of a Special Issue entitled: Translating Nuclear receptors from health to disease., (Copyright © 2011 Elsevier B.V. All rights reserved.)

By the use of in vivo imaging, we investigated the dynamics of estrogen receptor (ER) activity in intact, ovariectomized, and hormone-replaced estrogen response element-luciferase reporter mice. The study revealed the existence of a long-paced, noncircadian oscillation of ER transcriptional activity. Among the ER-expressing organs, this oscillation was asynchronous and its amplitude and period were tissue dependent. Ovariectomy affected the amplitude but did not suppress ER oscillations, suggesting the presence of tissue endogenous oscillators. Long-term administration of raloxifene, bazedoxifene, combined estrogens alone or with basedoxifene to ovariectomized estrogen response element-luciferase mice showed that each treatment induced a distinct spatiotemporal profile of ER activity, demonstrating that the phasing of ER activity among tissues may be regulated by the chemical nature and the concentration of circulating estrogen. This points to the possibility of a hierarchical organization of the tissue-specific pacemakers. Conceivably, the rhythm of ER transcriptional activity translates locally into the activation of specific gene networks enabling ER to significantly change its physiological activity according to circulating estrogens. In reproductive and nonreproductive organs this hierarchical regulation may provide ER with the signaling plasticity necessary to drive the complex metabolic changes occurring at each female reproductive status. We propose that the tissue-specific oscillatory activity here described is an important component of ER signaling necessary for the full hormone action including the beneficial effects reported for nonreproductive organs. Thus, this mechanism needs to be taken in due consideration to develop novel, more efficacious, and safer hormone replacement therapies.

Throughout evolution, organisms have devised strategies to limit fertility in case of prolonged starvation. In mammals, the liver plays a central role in the orchestration of mechanisms allowing for the maintenance of energy homeostasis. We here demonstrate that dietary amino acids regulate the transcriptional activity of hepatic estrogen receptor alpha (ERα) through an mTOR-dependent mechanism. As a result of ERα activation, hepatic IGF-1 mRNA and blood IGF-1 are increased. Conversely, calorie restriction or selective ablation of ERα in the liver decrease blood IGF-1 to levels inadequate for the correct proliferation of the lumen epithelium in the uterus and the progression of the estrous cycle. We propose that the liver acts as critical mediator of energetic and reproductive functions responsible for the blockade of the estrous cycle in case of protein scarcity. Our findings may provide novel insights to understand the cause of selected forms of infertility and metabolic alterations in women after menopause., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Prostate cancer (PC) develops in response to an abnormal activation of androgen receptor induced by circulating androgens and, in its initial stages, is pharmacologically controlled by androgen blockade. However, androgen ablation therapy often allows androgen-independent PC development, generally characterized by increased invasiveness. We previously reported that 5alpha-androstane-3beta,17beta-diol (3beta-Adiol) inhibits the migration of PC cell lines via the estrogen receptor beta (ERbeta) activation. Here, by combining in vitro assays and in vivo imaging approaches, we analyzed the effects of 3beta-Adiol on PC proliferation, migration, invasiveness, and metastasis in cultured cells and in xenografts using luciferase-labeled PC3 (PC3-Luc) cells. We found that 3beta-Adiol not only inhibits PC3-Luc cell migratory properties, but also induces a broader anti-tumor phenotype by decreasing the proliferation rate, increasing cell adhesion, and reducing invasive capabilities in vitro. All these 3beta-Adiol activities are mediated by ERbeta and cannot be reproduced by the physiological estrogen, 17beta-estradiol, suggesting the existence of different pathways activated by the two ERbeta ligands in PC3-Luc cells. In vivo, continuous administration of 3beta-Adiol reduces growth of established tumors and counteracts metastasis formation when PC3-Luc cells are engrafted s.c. in nude mice or are orthotopically injected into the prostate. Since 3beta-Adiol has no androgenic activity, and cannot be converted to androgenic compounds, the effects here described entail a novel potential application of this agent against human PC.

Using a mouse model engineered to measure estrogen receptor (ER) transcriptional activity in living organisms, 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 were administered daily at doses equivalent to those used in humans as calculated by the allometric approach. As controls, ER activity was measured also in cycling and ovariectomized mice. The study demonstrated that ER-dependent transcriptional activity oscillated in time, and the frequency and amplitude of the transcription pulses was strictly associated with the target tissue and the estrogenic compound administered. Our results indicate that the spatiotemporal activity of selective ER modulators is predictive of their structure, demonstrating that the analysis of the effect of estrogenic compounds on a single surrogate marker of ER transcriptional activity is sufficient to classify families of compounds structurally and functionally related. For more than one century, the measure of drug structure-activity relationships has been based on mathematical equations describing the interaction of the drug with its biological receptor. The understanding of the multiplicity of biological responses induced by the drug-receptor interaction demonstrated the limits of current approach and the necessity to develop novel concepts for the quantitative analysis of drug action. Here, a systematic study of spatiotemporal effects is proposed as a measure of drug efficacy for the classification of pharmacologically active compounds. The application of this methodology is expected to simplify the identification of families of molecules functionally correlated and to speed up the process of drug discovery.

Reporter mice associated to molecular imaging represent a major asset for the study of the spatio-temporal effects of drugs in living animals. The field is still relatively young and so far the number of animals genetically modified to express a given reporter gene ubiquitously and under the control of specific drugs is still limited. For a reporter animal the indispensable elements for the application to drug research and development are (i) the short life of the reporter enabling to have a clear view of the onset as well as the termination of drug effects, (ii) the generalized, drug-dependent activation of the reporter, and (iii) imaging modality suitable for high-throughput analysis. Because of its relative cheapness and ease to perform, in addition to all the above considerations, bioluminescence-based imaging is now regarded as the best imaging technology to be applied to the field of drug research. We show here the application of reporter mouse systems for drug screening in living animals in order to compare drug potency on target and specificity of action.

Accumulating evidence shows that estrogens are protective factors in inflammatory lung diseases and are involved in the gender-related incidence of these pathologies. The aim of this study was to identify which estrogen receptor (ER), ER-alpha and/or ER beta, mediates hormone antiinflammatory effects in lung and how gender or aging modify this effect. Acute lung inflammation in wild type, ER alpha or ER beta knockout animals was induced by pleural injection of carrageenan; female mice were used and sham operated, ovariectomized, or ovariectomized and treated with 17beta-estradiol (E(2)) before carrageenan. Our data show that ER alpha, and not ER beta, mediates E(2)-induced reduction of the inflammatory response. By real-time PCR and immunohistochemistry assays, we demonstrate ER alpha expression in the resident and infiltrated inflammatory cells of the lung, in which ER beta could not be detected. In these cells, E(2)-mediated reduction in the expression of inflammatory mediators was also due to ER alpha. In parallel, we observed that female mice were more prone to inflammation as compared with males, suggesting a gender-related difference in lung susceptibility to inflammatory stimuli, whereas the effect of E(2) was similar in the two sexes. Interestingly, aging results in a strong increase in the inflammatory response in both sexes and in the disruption E(2)/ER alpha signaling pathway. In conclusion, our data reveal that E(2) is able to regulate lung inflammation in a gender-unrelated, age-restricted manner. The specific involvement of ER alpha in hormone action opens new ways to identify drug targets that limit the inflammatory component of lung pathologies.

Suppuration of the preputial gland in mice occurs as a septic complication of fight wounds around the external genitalia. Currently reported bacterial isolates from these lesions are limited to Staphylococcus aureus, Pasteurella pneumotropica, and Klebsiella oxytoca. In the context of a pilot experiment aimed at defining the aging phenotype of estrogen receptor beta knockout (BERKO) mice, 2 male mice (1 of the BERKO line and the other from the age- and sex-matched wild-type control group) were discovered at necropsy to have preputial gland lesions. In both cases, histopathologic examination confirmed severe suppuration and abscesses of the preputial glands associated with systemic reactive (secondary) amyloidosis. Both Gram staining and Bacillus Calmette-Guérin immunohistochemistry highlighted the presence of numerous bacillary to rod-shaped bacteria within the preputial lesions. Subsequent PCR analysis coupled with denaturing gradient gel electrophoresis identified Corynebacterium mastitidis in the preputial gland abscesses. This organism is isolated infrequently from the milk of sheep with subclinical mastitis and was identified as part of the normal microflora of the human ocular surface. No information regarding the epidemiology and pathogenesis of C. mastitidis infection in laboratory animals is currently available, and to our knowledge this report is the first description of C. mastitidis infection in mice.

During the past decade the remarkable progress in molecular genetics and the possibility to engineer cells to express genes reporting on the activity of specific promoters has produced major changes in biological research. The description and validation of reporter mice for non-invasive assessment of biological and biochemical processes in living subjects and the results obtained with the models reporting on the activity of estrogen and peroxisome proliferator receptors clearly showed that such technologies have the potential to enhance our understanding of disease and drug activity. Although reporter-gene technology is in its infancy, reporter animals already represent a valuable tool for biomedical investigation. The present chapter aims at critically illustrating the methodology to be applied when dealing with reporter systems and in vivo imaging.

In the search for xenoestrogens within food additives, we have analyzed the Joint FAO-WHO expert committee database, containing 1500 compounds, using an integrated in silico and in vitro approach. This analysis identified 31 potential estrogen receptor alpha ligands that were reduced to 13 upon applying a stringent filter based on ligand volume and binding mode. Among the 13 potential xenoestrogens, four were already known to exhibit an estrogenic activity, and the other nine were assayed in vitro, determining the binding affinity to the receptor and biological effects. Propyl gallate was found to act as an antagonist, and 4-hexylresorcinol was found to act as a potent transactivator; both ligands were active at nanomolar concentrations, as predicted by the in silico analysis. Some caution should be issued for the use of propyl gallate and 4-hexylresorcinol as food additives.

Endocrine disrupters (EDs) are environment and food contaminants known to alter metabolic functions of mammals by interfering with specific endocrine pathways. Many EDs act on steroid hormone target cells by interacting with intracellular receptors (IRs) like estrogen receptors, androgen receptors, and thyroid hormone receptors; other receptors may be engaged. IRs are ligand-operated transcription factors acting in concert with general or cell-specific coregulators. The newly acquired awareness on the panoply of IR functions has increased the concern on potential, unsought, harmful effects of EDs on human health and has questioned the capability of currently available methodologies to identify and study EDs in the environment and in the food chain. Indeed, current in vivo and in vitro methodologies restrict the analysis to very specific organs or cell systems, with obvious limitations in predicting the systemic metabolic consequences of ED exposure. The emphasis recently laid by Regulatory Authorities, including European Center for the Validation of Alternative Methods, on the generation of in vitro model systems for toxicological analyses discouraged the development of models suitable to envision the whole spectrum of ED body actions required when studying compounds acting through IRs. Molecular imaging now provides the opportunity to quantify ED effects in living organisms enabling, for the first time, to acquire a full comprehension of the systemic effects of acute and prolonged exposure to EDs, solving the issue of the potential harm due to repeated low-dose exposure. The systems here reviewed are of unquestionable toxicological relevance and need to be taken into consideration to improve the methodology currently available and in use.

The widespread use of diets containing estrogenic compounds raises questions on how relevant the presence of phytoestrogens may be, in order to allow a correct development of the reproductive ability and sexual maturity in humans and animals. The isoflavone genistein is the most estrogenically active molecule present in soy. Here we show that genistein, through an estrogen receptor (ER)-mediated action, modulates gene expression in the whole body of male mice in a dose- and time-dependent manner, at all ages. By luciferase bioassays, we show that genistein-induced ER activation is present in reproductive and nonreproductive organs of the transgenic mice Estrogen Responsive Element (ERE)-tK-LUC, although to an extent that is lower than what observed with the administration of estradiol. Peak activity was registered at genistein doses of 500-5000 microg/kg, at 12 h from the administration by gavage. In the liver, ER-alpha and ER-beta messenger RNAs and two target genes, CYP17 and the progesterone receptor, were modulated by genistein. CYP17 and PR time-dependent induction was similar to that of luciferase. ER-alpha protein level followed an opposite regulation by genistein and estradiol. Genistein passed from the lactating mother to the suckling offspring at levels sufficient to activate gene expression in reproductive and nonreproductive tissues of the pups, with maximal upregulation at 16-24 h. We also followed responsiveness to genistein in the testis, from early development to adult age. Testis are well responsive to genistein as well as to estradiol already at day 14.5 of fetal development, as determined by exposing organotypic cultures from mouse fetus testis. Ovaries were not responsive under the same conditions. Activation of luciferase correlates with an activation of cell proliferation in testis, but not in the ovaries. Prolonged exposure (15 days) to genistein also decreases prostate weight like estradiol. In conclusion, our results show that genistein affects reproductive and nonreproductive organs of male mice in a dose- and time-dependent manner, at all developmental ages.

Isoflavones are important dietary compounds that are consumed with the daily diet and elicit important biological actions. Here we report on the ability of genistein to partially accumulate in body depots of male mice, be released following fasting, and modulate the actions of estradiol and environmental estrogens in reproductive and nonreproductive target organs of estrogen-reporter mice (ERE-tK-luciferase). After the consumption of 50 mg/kg/day for 3 days, genistein accumulates in body compartments where it remains at functionally active levels for at least 15 days. Following 48 h of fasting, its concentration increased in serum from 99 +/- 13 to 163 +/- 17 nM. These levels are sufficient to exert an estrogenic effect in the testis and liver, as revealed by a twofold increase in luciferase gene expression. beta-Benzene-hexachloride (betaBHC) given at the concentration of 100 mg/kg/day for 3 days also accumulates in the body and is released by fasting, reaching serum levels of 176 +/- 33 nM, upregulating the luciferase gene in the liver and inhibiting its expression in the testis. When genistein was given in combination with betaBHC at doses sufficient to induce accumulation of both in body depots, the genistein mobilized by fasting reversed the action of the mobilized betaBHC in the testis. Acute administration of nutritional doses of genistein inhibited the action of estradiol and reversed the antiestrogenic action of o,p'-DDT: 1,1,1,-trichloro-2(p-chlorophenyl)-2-(o-chlorophenyl)ethane in the liver and the antiestrogenic action of betaBHC in the testis. Genistein had an additive effect with the ER agonist p,p'-DDT: 1,1,1,-trichloro-2,2-bis(p-chlorophenyl)ethane in the liver. The observed effects may be relevant to a protective action of phytoestrogens against estrogen receptor-interacting pollutants as well as the dietary modulation of estradiol action.

There is a growing interest in peroxisome proliferator-activated receptors (PPARs) as major players in the regulation of lipid and carbohydrate metabolism. Drugs targeting PPARs were in fact shown to have major relevance for the treatment of diseases associated with aging, such as arteriosclerosis and diabetes. However, a variety of toxic effects associated with PPAR ligand administration has been documented, including hepatocarcinogenesis, which may severely limit its therapeutic use. A better comprehension of the multiplicity of PPAR physiological functions is therefore mandatory for the development of novel, safer drugs. We here describe the generation of a novel transgenic mouse for the detection of the generalized activities of PPARs, the PPAR responsive element-Luc reporter mouse. In this model luciferase expression is under the control of a PPAR-inducible promoter in all target organs. By optical imaging and ex vivo analysis, we were able to demonstrate the remarkable gender specificity of the PPAR transcriptional activity in liver. In fact, in the liver of female PPAR responsive element-Luc, the PPAR reporter transgene is more than one order of magnitude less expressed, thus leading to the conclusion that the signaling in females is much less activated than in males. Diet or hormonal manipulations as demonstrated here by treatments with high-fat diet or gonad removal and hormone replacement do not influence this low activation. The extent of the gender difference in PPAR transcriptional activity and the ineffectiveness of hormone treatments or diet to significantly elevate liver PPAR activity in females led us to hypothesize that gender-specific epigenetic events occurring during development may affect PPAR signaling in the liver. This study sets the ground for understanding the differential susceptibility of the two genders to metabolic disorders; furthermore, the model generated provides a novel opportunity for the molecular characterization of PPAR activity in pathophysiological conditions.

It has been previously demonstrated that 17beta-estradiol (E(2)) inhibits the response of microglia, the resident brain macrophages, to acute injuries in specific brain regions. We here show that the effect of E(2) in acute brain inflammation is widespread and that the hormone reduces the expression of inflammatory mediators, such as monocyte chemoattractant protein-1, macrophage inflammatory protein-2, and TNF-alpha, induced by lipopolysaccharide, demonstrating that microglia are a direct target of estrogen action in brain. Using the APP23 mice, an animal model of Alzheimer's disease reproducing chronic neuroinflammation, we demonstrate that ovary ablation increases microglia activation at beta-amyloid (Abeta) deposits and facilitates the progression of these cells toward a highly reactive state. Long-term administration of E(2) reverts the effects of ovariectomy and decreases microglia reactivity compared with control animals. In this animal model, these events do not correlate with a reduced number of Abeta deposits. Finally, we show that E(2) inhibits Abeta-induced expression of scavenger receptor-A in macrophage cells, providing a mechanism for the effect of E(2) on Abeta signaling observed in the APP23 mice. Altogether, our observations reveal a substantial involvement of endogenous estrogen in neuroinflammatory processes and provide novel mechanisms for hormone action in the brain.

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 17beta-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.

Prostate cancer growth depends, in its earlier stages, on androgens and is usually pharmacologically modulated with androgen blockade. However, androgen-ablation therapy may generate androgen-independent prostate cancer, often characterized by an increased invasiveness. We have found that the 5alpha-reduced testosterone derivative, dihydrotestosterone (the most potent natural androgen) inhibits cell migration with an androgen receptor-independent mechanism. We have shown that the dihydrotestosterone metabolite 5alpha-androstane-3beta,17beta-diol (3beta-Adiol), a steroid which does not bind androgen receptors, but efficiently binds the estrogen receptor beta (ERbeta), exerts a potent inhibition of prostate cancer cell migration through the activation of the ERbeta signaling. Very surprisingly, estradiol is not active, suggesting the existence of different pathways for ERbeta activation in prostate cancer cells. Moreover, 3beta-Adiol, through ERbeta, induces the expression of E-cadherin, a protein known to be capable of blocking metastasis formation in breast and prostate cancer cells. The inhibitory effects of 3beta-Adiol on prostate cancer cell migration is counteracted by short interfering RNA against E-cadherin. Altogether, the data showed that (a) circulating testosterone may act with estrogenic effects downstream in the catabolic process present in the prostate, and (b) that the estrogenic effect of testosterone derivatives (ERbeta-dependent) results in the inhibition of cell migration, although it is apparently different from that linked to estradiol on the same receptor and may be protective against prostate cancer invasion and metastasis. These results also shed some light on clinical observations suggesting that alterations in genes coding for 3beta-hydroxysteroid dehydrogenases (the enzymes responsible for 3beta-Adiol formation) are strongly correlated with hereditary prostate cancer.

During the past decade remarkable progress in molecular genetics and the possibility of manipulating cells so that the expression of genes can directly 'report' on drug activity has produced major changes in drug development strategies. The recent description and pharmacological validation of reporter mice for in vivo analysis of hormone receptor activity opens new horizons for drug discovery. These novel animal models, in association with in vivo imaging technologies, provide a global view of the target tissues of drug action following acute and repeated drug treatment, thus enabling the prediction of potential side-effects in the early phase of preclinical studies. It is anticipated that further improvements of transgene architecture will lead to models that combine pharmacokinetic, pharmacodynamic and toxicological studies in a single step, which should provide a tremendous saving in time and, paradoxically, the number of animals to be sacrificed in the development of novel pharmacologically active molecules.

Background: Previous reports from our group have shown that 17beta-estradiol reduces the synthesis and activity of inducible nitric oxide synthase (iNOS) in rat aortic smooth muscle cells (SMC) in response to inflammatory mediators. In this study, we investigated the effect of 17beta-estradiol on iNOS function in aortic SMC from streptozotocin-diabetic rats., Methods and Results: Comparative analysis of NO release and of iNOS mRNA and protein content after 24-hour stimulation with a cytokine mixture revealed milder iNOS activation in diabetic than in control SMC. Furthermore, 17beta-estradiol dose-dependently blocked iNOS synthesis and activity in control but not in diabetic SMC. The defective estrogen response in diabetic SMC at 24 hours could not be attributed to reduced expression of estrogen receptors (ER). In fact, mRNA and protein levels of ERalpha and, to a greater extent, of ERbeta, were increased in diabetic compared with nondiabetic SMC. Cytokines decreased ERalpha and ERbeta expression in both groups. However, 17beta-estradiol dose-dependently restored the expression of ERalpha but further downregulated that of ERbeta, indicating a differential regulation of ER isoforms., Conclusions: Estrogenic control of iNOS was impaired in diabetic SMC. This was associated with a larger increase of ERbeta than of ERalpha protein, whereas 17beta-estradiol regulated the two isoforms in an opposite fashion. Thus, modifications in the estrogen modulation of iNOS and in the expression pattern of ER may be involved in diabetic vascular dysfunction.

Several clinical studies have shown that bone loss may be attributed to osteoclast recruitment induced by mediators of inflammation. In different experimental paradigms we have recently demonstrated that estrogen exhibits antiinflammatory activity by preventing the induction of inducible nitric oxide synthase (iNOS) and other components of the inflammatory reaction. To verify whether this could explain the estrogen-dependent blockade of osteoporosis, we investigated the effect of ovariectomy in mice in which iNOS activity had been blunted by genetic or pharmacological manipulation. The consequences of iNOS blockade were evaluated initially on bone formation and resorption by histomorphometric analysis. The proximal tibiae of mice with iNOS genotypes revealed that 32 d after ovariectomy bone volume and bone formation rate were significantly decreased, and osteoclast surface was increased. Conversely, in iNOS knockout (iNOSKO) and wild-type (WT) mice treated with a specific inhibitor of iNOS, N-iminoethyl-L-lysine, ovariectomy did not result in bone depletion. In WT mice, ovariectomy also affected bone formation, as shown by a decreased mineral apposition rate. Also in this case, iNOS inactivation prevented the effect of ovariectomy. Immunocytochemical analysis showed that after ovariectomy iNOS protein accumulates in chondrocytes, and a significant increase in nitrotyrosine and poly(ADP-ribose) synthetase staining was observed in the femur metaphyses. The increase in nitrotyrosine and poly(ADP-ribose) synthetase formation induced by ovariectomy was significantly reduced in sections from iNOSKO mice. These data indicate that in WT mice the observed induction of iNOS has functional relevance, because it leads to overproduction of nitric oxide and accumulation of highly reactive molecules, triggering a local inflammatory reaction. These inflammatory foci attract cytokines, well known actors in the mechanism of osteoclastogenesis. In iNOSKO mice the measurements of IL-1 beta, IL-6, and TNFalpha plasma levels showed that ovariectomy fails to elicit the increase observed in WT animals and suggests that iNOS plays a primary role in the protective effects of estrogens. To further support this hypothesis, we show that estradiol-dependent activation of estrogen receptor-alpha blocks phorbol 12-acetate 13-myristate-induced transcription of iNOS promoter in transfected cells, thus demonstrating that the promoter of iNOS is under estrogen negative control. Our findings point to a key role of iNOS in mediating the negative effects of estrogen depletion on bones and provide a novel mechanistic explanation for the effects of menopause in osteoporosis and possibly also in other diseases in which the inflammatory component is elevated.