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Abstract

Nuclear Receptors are ligand-activated transcription factors that translate information about the lipid environment into specific genetic programs, a property that renders them good candidates to be mediators of rapid adaptation changes of a species. Lipid-based morphogens, endocrine hormones, fatty acids and xenobiotics might act through this class of transcription factors making them regulators able to fine-tune physiological processes. Here we review the basic concepts and current knowledge on the process whereby small molecules act through nuclear receptors and contribute to transgenerational changes. Several molecules shown to cause transgenerational changes like phthalates, BPA, nicotine, tributylin bind and activate nuclear receptors like ERs, androgen receptors, glucocorticoid receptors or PPARγ. A specific subset of observations involving nuclear receptors has focused on the effects of environmental stress or maternal behaviour on the development of transgenerational traits. While these effects do not involve environmental ligands, they change the expression levels of Estrogen and glucocorticoid receptors of the second generation and consequently initiate an altered genetic program in the second generation. In this review we summarize the available literature about the role of nuclear receptors in transgenerational inheritance. Copyright © 2015. Published by Elsevier Ltd.

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... As for many other EDCs, interactions with nuclear sex hormone receptors and their ligand-dependent transcription factor activity (so-called genomic signaling mechanism) have been implicated as the principal molecular mechanism responsible for endocrine disrupting activity of MXC and VIN (Manikkam et al., 2014;Ozgyin et al., 2015;van Ravenzwaay et al., 2013). Although MXC is a weak estrogen receptor (ER) agonist, its hydroxylated metabolites, such as HPTE (2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane), are potent ERa agonists and weak antagonists to both ERß and androgen receptor (AR) (Gaido et al., 2000). ...
... Although MXC and VIN are being intensively investigated as prototypical EDCs in order to understand their mechanisms of endocrine disruption, epigenetic regulation and transgenerational inheritance (Manikkam et al., 2014;Ozgyin et al., 2015;Skinner, 2014), and even though rapid alterations of cellular signaling are being increasingly recognized as an important mechanism contributing to the adverse effects of EDCs (Trevino et al., 2015;Watson et al., 2014;Wong and Walker, 2013), there is only limited information about effects of MXC and VIN on rapid intercellular and intracellular signaling events, such as GJIC or mitogen-activated protein kinases (MAPKs). ...
Article
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Methoxychlor and vinclozolin are well-recognized endocrine disrupting chemicals known to alter epigenetic regulations and transgenerational inheritance; however, non-endocrine disruption endpoints are also important. Thus, we determined the effects of methoxychlor and vinclozolin on the dysregulation of gap junctional intercellular communication (GJIC) and activation of mitogen-activated protein kinases (MAPKs) in WB-F344 rat liver epithelial cells. Both chemicals induced a rapid dysregulation of GJIC at non-cytotoxic doses, with 30 min EC50 values for GJIC inhibition being 10 μM for methoxychlor and 126 μM for vinclozolin. Methoxychlor inhibited GJIC for at least 24 h, while vinclozolin effects were transient and GJIC recovered after 4 h. Vinclozolin induced rapid hyperphosphorylation and internalization of gap junction protein connexin43, and both chemicals also activated MAPK ERK1/2 and p38. Effects on GJIC were not prevented by MEK1/2 inhibitor, but by an inhibitor of phosphatidylcholine-specific ph
... Likewise, maternal exposures to chemicals have been associated with altered hepatic metabolism and steatosis in offspring. Exposure throughout gestation and perinatal development to BPA may further exacerbate the nonalcoholic steatohepatitislike phenotype in male rats that were fed a high-fat diet post-weaning; in particular, BPA worsened the accumulation of lipids in hepatocytes as well as liver inflammation and oxidative stress fibrosis [600]. Liver function markers were unimpaired in BPA-exposed rats kept on a standard diet; however, these animals showed effects suggestive of subtle alterations of liver programming, such as moderately increased steatosis and altered expression of insulin signaling elements. ...
... Developmental exposure to BPA has also been shown to cause weight gain in offspring in some animal models and human studies (reviewed in [340]). BPA exposure throughout gestation and perinatal development exacerbates a nonalcoholic steatohepatitis-like phenotype in male rats that were fed a high-fat diet post-weaning; in particular, BPA worsened the accumulation of lipids in hepatocytes as well as liver inflammation and oxidative stress fibrosis [600]. ...
Article
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The recent epidemics of metabolic diseases, obesity, type 2 diabetes(T2D), liver lipid disorders and metabolic syndrome have largely been attributed to genetic background and changes in diet, exercise and aging. However, there is now considerable evidence that other environmental factors may contribute to the rapid increase in the incidence of these metabolic diseases. This review will examine changes to the incidence of obesity, T2D and non-alcoholic fatty liver disease (NAFLD), the contribution of genetics to these disorders and describe the role of the endocrine system in these metabolic disorders. It will then specifically focus on the role of endocrine disrupting chemicals (EDCs) in the etiology of obesity, T2D and NAFLD while finally integrating the information on EDCs on multiple metabolic disorders that could lead to metabolic syndrome. We will specifically examine evidence linking EDC exposures during critical periods of development with metabolic diseases that manifest later in life and across generations.
... In short, several language-related areas of the brain are affected by intrauterine nutrients, the prolongation of breastfeeding, improved child feeding practices, and delayed puberty, but also, by the extent of group size and the new social relationships, in a synergic and explosive cooperation. More than genetics-also-, language evolution may be a comprehensible example of epigenetics-given that the trait is now heritable for subsequent generations, perhaps through altered histone or DNA methylation patterns at nuclear receptors (Ozgyin et al., 2015), in a heritable manner, as epigenetics has been defined (Dressler, 2008), and tested for some maternal behaviors in laboratory animals (Champagne, 2008)-, being love a crucial factor: In families with higher levels of marital love when children were 12 months of age, mothers and fathers used a more diverse vocabulary with their 24 month-old children (Pancsofar et al., 2008), as presumably later, and also thousands of years ago. ...
Article
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Language is a specifically human mental function, although some neurobiological adaptations associated with communication can be found in other primates, in other mammalian orders, and even in other kinds of animals (evolutionary trend). Exposure to language is necessary for its acquisition (culture), there are specific alleles of some genes for human language (gene), and the brain circuits for language are mainly lateralized towards the left hemisphere (brain lateralization). However, some data suggest that the crucial factor for human verbal language, which originates in childhood in both, ontogeny and phylogeny, must be of motivational nature, and have at least the same importance as other genetic, brain or cultural factors. So, this article proposes that language was promoted by the building of love, and that it was maintained by hominid women as proto-language during some hundreds of thousands years through the maternal-filial interaction, until the first permanent settlements of the current human species, between 40,000 - 10,000 years ago. Also, that it was then when the woman transmitted speech to the man (it is further suggested that this may have been the Original Sin of the biblical Genesis), signalizing this transmission with the beginning of the symbolic thought, thus promoting the first artistic displays, like sculptures, painting or music, which were associated with the expansion of love and speech to the relationship between the sexes, with the consequent diversification of languages, mainly in the last 10,000 to 5,000 years. Love caused and causes human speech in both, phylogeny and ontogeny.
... Up to now, in the context of obesogenic EDCs and epigenetic modifications, the general picture of the interplays between DNA methylation, histone modifications, and noncoding RNA is still uncompleted, with most studies being focused on DNA methylation. Early-life EDCs exposure can alter epigenetic programming of obesity by activating or inhibiting nuclear receptors and other transcription factors, which in turn recruit chromatin-modifying complexes, such as methyl-and acetyltransferases, which regulate the expression of the target genes by directly altering epigenetic marks [130]. As described before, the principal research of obesogenic EDCs has been focused on PPARγ, considered the main target of obesogens. ...
Article
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The incidence of obesity has dramatically increased over the last decades. Recently, there has been a growing interest in the possible association between the pandemics of obesity and some endocrine-disrupting chemicals (EDCs), termed “obesogens”. These are a heterogeneous group of exogenous compounds that can interfere in the endocrine regulation of energy metabolism and adipose tissue structure. Oral intake, inhalation, and dermal absorption represent the major sources of human exposure to these EDCs. Recently, epigenetic changes such as the methylation of cytosine residues on DNA, post-translational modification of histones, and microRNA expression have been considered to act as an intermediary between deleterious effects of EDCs and obesity development in susceptible individuals. Specifically, EDCs exposure during early-life development can detrimentally affect individuals via inducing epigenetic modifications that can permanently change the epigenome in the germline, enabling changes to be transmitted to the next generations and predisposing them to a multitude of diseases. The purpose of this review is to analyze the epigenetic alterations putatively induced by chemical exposures and their ability to interfere with the control of energy metabolism and adipose tissue regulation, resulting in imbalances in the control of body weight, which can lead to obesity.
... The in vitro assays that provide data on the selected endocrine mechanisms are outlined in level 2 of the framework. The responses of nuclear receptors play important roles in TA [7,8]. Nuclear receptors like estrogen receptors (ERs) or androgen receptors (ARs) initiate the endocrine disruptor response. ...
... Groundbreaking work has shown the existence of transgenerational effects of some EDCs (Nilsson & Skinner, 2015), including DEHP (Doyle et al., 2013;Chen et al., 2015a,b;Quinnies et al., 2015). The mechanisms that mediate these effects are not fully understood (Ozgyin et al., 2015;Xin et al., 2015); it is likely that animal models, such as the phthalate model, in addition to gaining insight about the effects of EDCs, will also serve to understand the function of the epigenome in health and disease. ...
Article
Endocrine disruptors (ED) are environmental pollutants that mimic endogenous hormonal signals. Exposure to EDs during fetal and early life is a public health concern because these are periods characterized by high cellular plasticity that influence the physiology and development of disease later in life. Phthalates are plasticizers used in the industry to manufacture polyvinyl chloride products and several consumer products. Di(2-ethylhexyl) phthalate (DEHP) is one of the most produced plasticizers; it is ubiquitously found contaminating the environment, and has been shown to be an ED. Human exposure to phthalates starts during fetal development and continues after birth through contact of the newborn with the environment and contaminated food sources. We used a rat model in which pregnant dams are gavaged with DEHP from gestational day 14 until birth to study the long-term effects of DEHP. This window of fetal exposure results in decreased circulating testosterone and aldosterone levels in adult male offspring and estradiol in the female. The observed steroid changes are likely of an epigenetic origin as DEHP is rapidly cleared after birth. Here, we review the long-term effects of fetal exposure to DEHP with a focus on the molecular and epigenetic changes, including DNA methylation, which may mediate long-term endocrine dysfunction.
... However, BPA has been defined as a SERM, which means that BPA can act through several other pathways and can subsequently induce different effects in various cells and tissues (Nagel et al. 2001). Further, BPA has been shown to activate several other receptors with the potential to affect epigenetic mechanisms, such as the thyroid hormone and androgen receptors (Delfosse et al. 2014;Ozgyin et al. 2015). ...
Article
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Background: Bisphenol A (BPA) is an endocrine-disrupting chemical that may contribute to development of obesity and metabolic disorders. Humans are constantly exposed to low concentrations of BPA, and studies support that the developmental period is particularly sensitive. Objectives: The aim was to investigate the effects of low-dose developmental BPA exposure on metabolic parameters in male and female Fischer 344 (F344) rat offspring. Methods: Pregnant F344 rats were exposed to BPA via their drinking water, corresponding to 0.5 μg/kg BW/d (BPA0.5; n =21) or 50 μg/kg BW/d (BPA50; n =16), from gestational day (GD) 3.5 until postnatal day (PND) 22, and controls were given vehicle ( n =26). Body weight (BW), adipose tissue, liver (weight, histology, and gene expression), heart weight, and lipid profile were investigated in the 5-wk-old offspring. Results: Males and females exhibited differential susceptibility to the different doses of BPA. Developmental BPA exposure increased plasma triglyceride levels (0.81±0.10 mmol/L compared with 0.57±0.03 mmol/L, females BPA50 p =0.04; 0.81±0.05 mmol/L compared with 0.61±0.04 mmol/L, males BPA0.5 p =0.005) in F344 rat offspring compared with controls. BPA exposure also increased adipocyte cell density by 122% in inguinal white adipose tissue (iWAT) of female offspring exposed to BPA0.5 compared with controls (68.2±4.4 number of adipocytes/HPF compared with 55.9±1.5 number of adipocytes/HPF; p =0.03) and by 123% in BPA0.5 females compared with BPA50 animals (68.2±4.4 number of adipocytes/high power field (HPF) compared with 55.3±2.9 number of adipocytes/HPF; p =0.04). In iWAT of male offspring, adipocyte cell density was increased by 129% in BPA50-exposed animals compared with BPA0.5-exposed animals (69.9±5.1 number of adipocytes/HPF compared with 54.0±3.4 number of adipocytes/HPF; p =0.03). Furthermore, the expression of genes involved in lipid and adipocyte homeostasis was significantly different between exposed animals and controls depending on the tissue, dose, and sex. Conclusions: Developmental exposure to 0.5 μg/kg BW/d of BPA, which is 8-10 times lower than the current preliminary EFSA (European Food Safety Authority) tolerable daily intake (TDI) of 4 μg/kg BW/d and is within the range of environmentally relevant levels, was associated with sex-specific differences in the expression of genes in adipose tissue plasma triglyceride levels in males and adipocyte cell density in females when F344 rat offspring of dams exposed to BPA at 0.5 μg/kg BW/d were compared with the offspring of unexposed controls. https://doi.org/10.1289/EHP505.
... However, BPA has been defined as a SERM, which means that BPA can act through several other pathways and can subsequently induce different effects in various cells and tissues (Nagel et al. 2001). Further, BPA has been shown to activate several other receptors with the potential to affect epigenetic mechanisms, such as the thyroid hormone and androgen receptors (Delfosse et al. 2014;Ozgyin et al. 2015). ...
Article
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Background: Bisphenol A (BPA) is a component of polycarbonate plastics to which humans are regularly exposed at low levels, and an endocrine disruptor with effects on several hormonal systems. Bone is a sensitive hormone target tissue, and we have recently shown that in utero and lactational exposure to 25µg BPA/kg BW/day alters femoral geometry in rat offspring. Objective: To investigate bone effects in rat offspring after developmental exposure to a BPA dose in the range of human daily exposure (0.1-1.5µg/kg BW/day) as well as a dose to corroborate previous findings. Methods: Pregnant Fischer 344 rats were exposed to BPA via drinking water corresponding to 0.5µg/kg BW/day: [0.5], (n=21) or 50µg/kg BW/day: [50], (n = 16) from gestational day 3.5 until postnatal day 22, while controls were given only vehicle (n = 25). The offspring was sacrificed at 5 weeks of age. Bone effects were analyzed using peripheral quantitative computed tomography (pQCT), the 3-point bending test, plasma markers of bone turnover, and gene expression in cortical bone and bone marrow. Results: Compared to controls, male offspring developmentally exposed to BPA had shorter femurs. pQCT analysis revealed effects in the [0.5] group, but not in the [50] group; BPA reduced both trabecular area (-3.9%, p < 0.01) and total cross sectional area (-4.1%, p < 0.01) of femurs in the [0.5] group, whereas no effects were seen on bone density. Conversely, bone length and size were not affected in female offspring. However, the procollagen type I N-terminal propeptide (P1NP), a peptide formed during type 1 collagen synthesis, was increased in plasma (42%: p < 0.01) in female offspring exposed to [0.5] of BPA, although collagen gene expression was not increased in bone. The biomechanical properties of the bones were not altered in either sex. Bone marrow mRNA expression was only affected in male offspring. Conclusions: Developmental low-dose exposure to BPA resulted in sex-specific bone effects in rat offspring. A dose approximately eight times lower than the current temporary EFSA human tolerable daily intake of 4µg/kg BW/day, reduced bone length and size in male rat offspring. Long-term studies are needed to clarify whether the increased plasma levels of P1NP in female offspring reflect development of fibrosis.
... In neurons, there are thousands of molecules that quickly convert to second signaling molecules (SWARM #2) which slowly invade the whole cell like a cloud over minutes of time when strong molecular contact is made by external signaling molecules called ligands locking into the external membrane's receptors (Bacskai et al., 1993). Some extracellular signals like steroid hormones can even penetrate the neuronal membrane and lock onto receptors located deep in the cell nucleus triggering a similar molecular turnover cloud (Ozgyin, Erd} os, Bojcsuk, & Balint, 2015). The neuron is constantly producing and recycling receptors and redistributing them on its membrane. ...
Article
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This essay aims to outline a scientific approach to the investigation of consciousness emphasizing achievements and promise of hardcore bottom-up biology. We propose to contemplate what would be the minimal requirements of consciousness in the simplest of life forms. We show that, starting from the molecular nuts and bolts of such life forms, it is the extreme multitudinousness of the moving material components forming consciousness, and their organized swarming, that appears outstanding. This is in stark contrast with the impression obtained from introspection that consciousness is a single, unconstrained, immaterial stream.
... In general terms, evidences demonstrated that the obesogenic effects of potential EDCs are mediated by their ability to bind NRs. These receptors directly recruit methyl and acetyltransferase, thus altering epigenetic marks regulating gene expression (80). In this way, EDCs can modify chromatin states or the levels of DNA or histone methyltransferases (81). ...
Article
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Exposure to potential Endocrine Disrupting Chemicals (EDCs) pose a documented risk to both wildlife and human health. Many studies so far described declining sperm counts, genital malformations, early puberty onset, highlighting the negative impact on reproduction caused by the exposure to many anthropogenic chemicals. In the last years, increasing evidence suggested that these compounds, other than altering reproduction, affect metabolism and induce the onset of obesity and metabolic disorders. According to the “environmental obesogens” hypothesis, evidence exists that exposure to potential EDCs during critical periods when adipocytes are differentiating, and organs are developing, can induce diseases that manifest later in the life. This review summarizes the effects occurring at the hepatic level in different animal models, describing morphological alterations and changes of molecular pathways elicited by the toxicant exposure. Results currently available demonstrated that these chemicals impair normal metabolic processes via interaction with members of the nuclear receptor superfamily, including steroid hormone receptors, thyroid hormone receptors, retinoid X receptors, peroxisome proliferator–activated receptors, liver X receptors, and farnesoid X receptors. In addition, novel results revealed that EDC exposure can either affect circadian rhythms as well as up-regulate the expression of signals belonging to the endocannabinoid system, in both cases leading to a remarkable increase of lipid accumulation. These results warrant further research and increase the interest toward the identification of new mechanisms for EDC metabolic alterations. The last part of this review article condenses recent evidences on the ability of potential EDCs to cause “transgenerational effects” by a single prenatal or early life exposure. On this regard, there is compelling evidence that epigenetic modifications link developmental environmental insults to adult disease susceptibility. This review will contribute to summarize the mechanisms underlying the insurgence of EDC-induced metabolic alterations as well as to build integrated strategies for their better management. In fact, despite the large number of results obtained so far, there is still a great demand for the development of frameworks that can integrate mechanistic and toxicological/epidemiological observations. This would increase legal and governmental institution awareness on this critical environmental issue responsible for negative consequences in both wild species and human health.
... Dysregulation of these processes can have an impact on metabolic balance, leading to obesity [18,19] and related pathologies, such as type 2 diabetes [20,21], hypertriglyceridemia [22], and nonalcoholic fatty liver disease (NAFLD) [23]. New findings increasingly support the concept that nuclear receptors are involved in transgenerational adaptive changes through epigenetic mechanisms, which reflect the impact of adverse environments in early life [24]. Peroxisome proliferator-activated receptor α (Ppara) is a key member of the PPAR family of nuclear receptors, and is highly expressed in tissues with active fatty acid catabolism [25,26], especially in the liver. ...
Article
Objective: Diabetes exerts adverse effects on the initiation or progression of diabetes and metabolic syndrome in the next generation. In past studies, limited attention has been given to the fathers' role in shaping the metabolic landscape of offspring. Our study was designed to investigate how paternal hyperglycemia exerts an intergenerational effect in mammals as well as the underlying mechanisms. Methods: Hyperglycemia was introduced in male rats by intraperitoneally injected streptozotocin and these males were bred with healthy females to generate offspring. The metabolic profiles of the progeny were assessed; DNA methylation profiles and gene expression were investigated. Mutagenesis constructs of the Ppara promoter region, and a luciferase reporter assay were used to determine transcription factor binding sites (TFBSs) and the effects of hypermethylation on Ppara transcription. Results: Paternal hyperglycemia induced increased liver weight, and plasma TC, TG, LDL, accumulation of triglycerides in the liver. We discovered that CpG 13 in the amplified promoter region (-852 to -601) of Ppara was hypermethylated in adult offspring liver and expression of Ppara, Acox1, Cpt-1α, and Cd36 was down regulated. Hypermethylation of CpG site 13 in the Ppara promoter inhibited the gene transcription, probably through abrogation of SP1 binding. The same epigenetic alteration was discovered in the fetus (E16.5) liver of hyperglycemic father's progeny. Conclusions: Paternal hyperglycemia may induce epigenetic modification of Ppara in offspring's liver, probably through interaction with SP1 binding, causing impaired lipid metabolism. Our investigation may have implications for the understanding of father-offspring interactions with the potential to account for metabolic syndromes.
... Dysregulation of these processes can have an impact on metabolic balance, leading to obesity [18,19] and related pathologies, such as type 2 diabetes [20,21], hypertriglyceridemia [22], and nonalcoholic fatty liver disease (NAFLD) [23]. New findings increasingly support the concept that nuclear receptors are involved in transgenerational adaptive changes through epigenetic mechanisms, which reflect the impact of adverse environments in early life [24]. Peroxisome proliferator-activated receptor α (Ppara) is a key member of the PPAR family of nuclear receptors, and is highly expressed in tissues with active fatty acid catabolism [25,26], especially in the liver. ...
... ERs, particularly estrogen receptor beta (ESR2), are expressed in many developing tissues, indicating multiple important functions of estrogen signaling during development (reviewed in [23]). Experimental evidence shows that exposure to environmental toxicants such as bisphenol A (BPA) affects developmental processes, at least partially, through binding to estrogen receptors [24]. BPA also modulates meiosis by affecting chromosomal synapsing, DSBs repair or changing the expression of meiotic genes that rely on the ER function. ...
Article
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Chlordecone (CD) is an insecticide that was used in the French West Indies for several years to control the banana root borer pest. Given its nonsignificant degradation, it persists in the environment. CD is a carcinogenic compound with reproductive and developmental toxicity and is a recognized endocrine-disrupting chemical. In this study, we examined the effects of CD on female reproductive system of mice with the focus on epigenetic features in ovary. Our data show that gestational exposure to low dose of CD affects meiotic double-strand breaks repair in female embryos. In adult mice derived from CD-treated pregnant females, we observed delayed puberty, decreased number of primordial and increased number of atretic follicles. Gene expression analysis revealed that Rcbtb2 and Rbpms genes were not expressed in embryonic gonads. Estrogen signaling- and oocyte maturation-associated genes were downregulated in adult ovaries. The morphological changes were associated with altered epigenetic features: increased H2Aub and increased H3K27me3 and decreased H4ac and H3K4me3 in embryonic oocytes. The DNA damage-associated, γH2AX marks were detected in the follicles of treated but not control adult ovaries. We also found reduced H3K4me3 and H4ac in fully grown oocytes of the treated ovaries. The ChIP-seq analysis of H3K4me3 in adult ovaries showed that target genes of ZFP57 and TRIM28, which regulate pluripotency and imprinting, were significantly enriched in altered regions. Our study clearly demonstrates that gestational exposure to a low dose of CD impairs the function of female reproductive system and the changes are associated with altered epigenetic features. Electronic supplementary material The online version of this article (10.1186/s13072-019-0276-7) contains supplementary material, which is available to authorized users.
... In males, aquaporin mRNA level in Oct-Nov were higher than in May-June. Since aquaporin channels are involved in the hydration of the seminal fluid in the acquisition of sperm motility (Boj et al., 2015), we could hypothesize that in spent testis, the aquaporin mRNA is not immediately translated into protein and could be stored until the next reproductive season as occurs in the gonad of several teleost species (Kagawa et al., 2011;Ozgyin et al., 2015;Sun et al., 2010b;Tingaud-Sequeira et al., 2010). ...
Presentation
In the frame of BioMedaqu Project, the University of Rome Tor Vergata, the Politecnica delle Marche and the Italian Association of Aquaculturists organize a training course on the most adequate procedures for monitoring of fish skeletal anomalies. BioMedaqu Project (https:// www.gigabiomedaqu.uliege.be/cms/c_4430156/en/portailgigabiomedaqu) is a Marie Sklodowska‐Curie Innovative Training Network (MCSAITN) with the aim of creating an innovative expertise combining research in skeletal biology of aquaculture fish species with that in biomedical models and humans.
... In males, aquaporin mRNA level in Oct-Nov were higher than in May-June. Since aquaporin channels are involved in the hydration of the seminal fluid in the acquisition of sperm motility (Boj et al., 2015), we could hypothesize that in spent testis, the aquaporin mRNA is not immediately translated into protein and could be stored until the next reproductive season as occurs in the gonad of several teleost species (Kagawa et al., 2011;Ozgyin et al., 2015;Sun et al., 2010b;Tingaud-Sequeira et al., 2010). ...
Article
The Atlantic Bluefin Tuna (ABFT, Thunnus thynnus) is one of the most intensely exploited fisheries resources in the world. In spite of the years of studies on ABFT, basic aspects of its reproductive biology remain uncertain. To gain insight regarding the seasonal changes of the reproductive characteristics of the eastern stock of ABFT, blood and tissue samples were collected from mature specimens caught in the Mediterranean basin during the reproductive (May-June) and non-reproductive season (Oct-Nov). Histological analysis of the gonads of May-June samples indicated that there were females which were actively spawning (contained post-ovulatory follicles) and females that were not actively spawning that had previtellogenic and fully vitellogenic oocytes. In males, testis were at early or late stage of spermatogenesis during the reproductive season. In Oct-Nov, ovaries contained mostly previtellogenic oocytes as well as β and α atretic follicles while the testis predominantly contained spermatogonia and few cysts with spermatocytes and spermatozoa. Gonadosomatic index (GSI) in females was highest among the actively spawning individuals while in males GSI was higher in early and late spermatogenic individuals compared to those that were spent. Plasma sex steroids levels varied with the reproductive season. In females, estradiol (E2), was higher in May-June while testosterone (T) and progesterone (P) did not vary. In males, E2 and T were higher in May-June while P levels were similar at the two sampling points. Circulating follicle stimulating hormone (FSH) was higher in Oct-Nov than in May-June both in males and females. Vitellogenin (VTG) was detected in plasma from both males and females during the reproductive season with levels in females significantly higher than in males. VTG was undetected in Oct-Nov samples. Since choriogenesis is an important event during follicle growth, the expression of three genes involved in vitelline envelope formation and hardening was measured and results showed significantly higher levels in ovaries in fish caught in May-June with respect to those sampled in Oct-Nov. In addition, a set of genes encoding for ion channels that are responsible for oocyte hydration and buoyancy, as well as sperm viability, were characterized at the two time points, and these were found to be more highly expressed in females during the reproductive season. Finally, the expression level of three mRNAs encoding for different lipid-binding proteins was analyzed with significantly higher levels detected in males, suggesting sex-specific expression. Our findings provide additional information on the reproductive biology of ABFT, particularly on biomarkers for the assessment of the state of maturation of the gonad, highlighting gender-specific signals and seasonal differences.
... For example, nuclear receptors, such as steroid receptors, can directly bind hormone-response elements present in the DNA upon activation by single or multiple ligands. Furthermore, they are able to recruit chromatin-modifying complexes including methyltransferases and acetyltransferases, which directly alter epigenetic marks involved in the regulation of target genes (Ozgyin et al., 2015). Therefore, EDCs can change the local chromatin state as well as modulate the expression of DNA or histone methyltransferases by activating or inhibiting nuclear receptors and other transcription factors (Rissman and Adli, 2014). ...
Article
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Metabolic chronic diseases, also named noncommunicable diseases (NCDs), are considered multifactorial pathologies, which are dramatically increased during the last decades. Noncommunicable diseases such as cardiovascular diseases, obesity, diabetes mellitus, cancers, and chronic respiratory diseases markedly increase morbidity, mortality, and socioeconomic costs. Moreover, NCDs induce several and complex clinical manifestations that lead to a gradual deterioration of health status and quality of life of affected individuals. Multiple factors are involved in the development and progression of these diseases such as sedentary behavior, smoking, pollution, and unhealthy diet. Indeed, nutrition has a pivotal role in maintaining health, and dietary imbalances represent major determinants favoring chronic diseases through metabolic homeostasis alterations. In particular, it appears that specific nutrients and adequate nutrition are important in all periods of life, but they are essential during specific times in early life such as prenatal and postnatal phases. Indeed, epidemiologic and experimental studies report the deleterious effects of an incorrect nutrition on health status several decades later in life. During the last decade, a growing interest on the possible role of epigenetic mechanisms as link between nutritional imbalances and NCDs development has been observed. Finally, because of the pivotal role of the hormones in fat, carbohydrate, and protein metabolism regulation throughout life, it is expected that any hormonal modification of these processes can imbalance metabolism and fat storage. Therefore, a particular interest to several chemicals able to act as endocrine disruptors has been recently developed. In this review, we will provide an overview and discuss the epigenetic role of some specific nutrients and chemicals in the modulation of physiological and pathological mechanisms.
... The nuclear receptors ER, AR and PPARy were suggested to be involved in triggering transgenerational inheritance via affecting DNA methylation; reviewed in [170]. For example, vincozolin causes transgenerational inheritance via AR signaling [152]. ...
Article
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Genetic studies traditionally focus on DNA as the molecule that passes information on from parents to their offspring. Changes in the DNA code alter heritable information and can more or less severely affect the progeny’s phenotype. While the idea that information can be inherited between generations independently of the DNA’s nucleotide sequence is not new, the outcome of recent studies provides a mechanistic foundation for the concept. In this review, we attempt to summarize our current knowledge about the transgenerational inheritance of environmentally induced epigenetic changes. We focus primarily on studies using mice but refer to other species to illustrate salient points. Some studies support the notion that there is a somatic component within the phenomenon of epigenetic inheritance. However, here, we will mostly focus on gamete-based processes and the primary molecular mechanisms that are thought to contribute to epigenetic inheritance: DNA methylation, histone modifications, and non-coding RNAs. Most of the rodent studies published in the literature suggest that transgenerational epigenetic inheritance through gametes can be modulated by environmental factors. Modification and redistribution of chromatin proteins in gametes is one of the major routes for transmitting epigenetic information from parents to the offspring. Our recent studies provide additional specific cues for this concept and help better understand environmental exposure influences fitness and fidelity in the germline. In summary, environmental cues can induce parental alterations and affect the phenotypes of offspring through gametic epigenetic inheritance. Consequently, epigenetic factors and their heritability should be considered during disease risk assessment.
... Another EDC target with important consequences for epigenetic regulation is the Estrogen Receptor (ERα) [76,87]. ERα is a transcription factor whose activation triggers estrogen-responsive elements present on the promoter region of the histone-lysine N-methyltransferase enzyme EZH2, key player in gene silencing. ...
Article
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Endocrine-disrupting chemicals (EDCs) are exogenous substances able to mimic or to interfere with the endocrine system, thus altering key biological processes such as organ development, reproduction, immunity, metabolism and behavior. High concentrations of EDCs are found in several everyday products including plastic bottles and food containers and they could be easily absorbed by dietary intake. In recent years, considerable interest has been raised regarding the biological effects of EDCs, particularly Bisphenol A (BPA) and phthalates, on human pregnancy and fetal development. Several evidence obtained on in vitro and animal models as well as by epidemiologic and population studies strongly indicated that endocrine disruptors could negatively impact fetal and placental health by interfering with the embryonic developing epigenome, thus establishing disease paths into adulthood. Moreover, EDCs could cause and/or contribute to the onset of severe gestational conditions as Preeclampsia (PE), Fetal Growth Restriction (FGR) and gestational diabetes in pregnancy, as well as obesity, diabetes and cardiovascular complications in reproductive age. Therefore, despite contrasting data being present in the literature, endocrine disruptors must be considered as a therapeutic target. Future actions aimed at reducing or eliminating EDC exposure during the perinatal period are mandatory to guarantee pregnancy success and preserve fetal and adult health.
... A variety of environmental chemicals may act similarly to, or in opposition of, vertebrate sex steroids by altering nuclear receptor signalling and gene transactivation (TA) (Ozgyin et al., 2015;Shang et al., 2002). When ligands bind to the nuclear receptors within cells, the ligand-receptor complex undergoes a conformational change that leads to receptor activation and a transcriptional response (Le Maire et al., 2010). ...
Article
We describe the characterisation and validation of an androgen receptor (AR) transactivation assay for detection of AR agonists and antagonists using a stably transfected human prostate cancer cell line. This 22Rv1/mouse mammary tumour virus glucocorticoid knock-out cell line based AR transactivation assay was validated by criteria in Organisation for Economic Cooperation and Development Guidance Document 34 to determine if the assay performed equally well to the AR EcoScreen Assay included in Test Guideline for AR Transactivation (OECD TG 458). There was no Glucocorticoid Receptor (GR) crosstalk, and no changes in the AR DNA sequence in cells after the successful knock out of GR. Subsequently, the concordance of classifications of the 22 test chemicals was 100% in all laboratories. The AR agonistic and antagonistic inter-laboratory coefficients of variation based on log[10% effect for 10 nM DHT, PC10] and log[inhibitory response of 800 pM DHT by at 30%, IC30] from comprehensive tests were 2.75% and 2.44%, respectively. The AR agonist/antagonist test chemical classifications were consistent across AR EcoScreen ARTA assay data for 82/89%, and the balanced accuracy, sensitivity, and specificity were 83/90%, 88/100% and 78/80%, respectively. This assay was successfully validated and was approved for inclusion in TG 458 in 2020.
... In recent decades, humans have been increasingly treated with synthetic hormone drugs and exposed to many environmental substances that act as EDCs (Diamanti- Kandarakis et al., 2009). Most of these substances affect molecular signaling through the superfamily of nuclear receptors, which act as DNA-binding TFs with powerful capabilities of modifying the epigenetic landscape and gene expression programs (Ozgyin et al., 2015). Numerous studies support the hypothesis that alterations in endocrine systems influence the epigenetic information of the germline which may lead to neurodevelopmental and behavioral abnormalities in subsequent generations. ...
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Investigations into the etiology of autism spectrum disorders have been largely confined to two realms: variations in DNA sequence and somatic developmental exposures. Here we suggest a third route—disruption of the germline epigenome induced by exogenous toxicants during a parent’s gamete development. Similar to cases of germline mutation, these molecular perturbations may produce dysregulated transcription of brain-related genes during fetal and early development, resulting in abnormal neurobehavioral phenotypes in offspring. Many types of exposures may have these impacts, and here we discuss examples of anesthetic gases, tobacco components, synthetic steroids, and valproic acid. Alterations in parental germline could help explain some unsolved phenomena of autism, including increased prevalence, missing heritability, skewed sex ratio, and heterogeneity of neurobiology and behavior.
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The aim of this study is to assess the androgen receptor (AR) agonistic/antagonistic effects on various chemicals, which are used in household products including cleaning agents and wetted tissues by in vitro OECD test guideline No. 458 (using AR-EcoScreen™ cell line) and the me-too test method (using 22Rv1cell line), which was adopted as OECD project No. 4.99. All chemicals were not determined as AR agonists. However α-dodecyl-ω-hydroxypoly (oxyethylene) and 3-iodo-2-propynyl butylcarbamate have shown a weak AR antagonistic effects with IC50 values of 2.18 ± 0.12 and 4.26 ± 0.17 μg/ml via binding affinity to AR in only 22Rv1/mouse mammary tumor virus using AR transcriptional activation assay, because of their different cytotoxicity on each applied cell line. This report firstly provides information about agonistic/antagonistic effects against human AR of various chemicals including surfactants and biocides by OECD in vitro stably transfected transcriptional activation assays. However, further in vivo and human model studies are needed to confirm their adverse effects.
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Obesity is among the most common and costly chronic disorders worldwide. Estimates suggest that in the United States obesity affects one-third of adults, accounts for up to one-third of total mortality, is concentrated among lower income groups, and increasingly affects children as well as adults. A lack of effective options for long-term weight reduction magnifies the enormity of this problem; individuals who successfully complete behavioral and dietary weight-loss programs eventually regain most of the lost weight. We included evidence from basic science, clinical, and epidemiological literature to assess current knowledge regarding mechanisms underlying excess body-fat accumulation, the biological defense of excess fat mass, and the tendency for lost weight to be regained. A major area of emphasis is the science of energy homeostasis, the biological process that maintains weight stability by actively matching energy intake to energy expenditure over time. Growing evidence suggests that obesity is a disorder of the energy homeostasis system, rather than simply arising from the passive accumulation of excess weight. We need to elucidate the mechanisms underlying this “upward setting” or “resetting” of the defended level of body-fat mass, whether inherited or acquired. The ongoing study of how genetic, developmental, and environmental forces affect the energy homeostasis system will help us better understand these mechanisms and are therefore a major focus of this statement. The scientific goal is to elucidate obesity pathogenesis so as to better inform treatment, public policy, advocacy, and awareness of obesity in ways that ultimately diminish its public health and economic consequences.
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There is now considerable evidence indicating the potential for endocrine disrupting chemicals to alter the epigenome and for subsets of these epigenomic changes or “epimutations” to be heritably transmitted to offspring in subsequent generations. While there have been many studies indicating how exposure to endocrine disrupting chemicals can disrupt various organs associated with the body’s endocrine systems, there is relatively limited information regarding the relative susceptibility of different specific organs, tissues, or cell types to endocrine disrupting chemical-induced epimutagenesis. Here we review available information about different organs, tissues, cell types, and/or cell lines which have been shown to be susceptible to specific endocrine disrupting chemical-induced epimutations. In addition, we discuss possible mechanisms that may be involved, or impacted by this tissue- or cell type-specific, differential susceptibility to different endocrine disrupting chemicals. Finally, we summarize available information indicating that certain periods of development display elevated susceptibility to endocrine disrupting chemical exposure and we describe how this may affect the extent to which germline epimutations can be transmitted inter- or transgenerationally. We conclude that cell type-specific differential susceptibility to endocrine disrupting chemical-induced epimutagenesis is likely to directly impact the extent to, or manner in, which endocrine disrupting chemical exposure initially induces epigenetic changes to DNA methylation and/or histone modifications, and how these endocrine disrupting chemical-induced epimutations can then subsequently impact gene expression, potentially leading to the development of heritable disease states.
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Sentence: Thyroid hormone-clearing type 3 deiodinase is located in spermatogonia, where it may serve as a critical modulator of the thyroid hormone exposure of the male germ line and its epigenetic information, with implications for neurodevelopmental and endocrine disorders in subsequent generations.
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Recent research supports a role for exposure to endocrine-disrupting chemicals (EDCs) in the global obesity epidemic. Obesogenic EDCs have the potential to inappropriately stimulate adipogenesis and fat storage, influence metabolism and energy balance and increase susceptibility to obesity. Developmental exposure to obesogenic EDCs is proposed to interfere with epigenetic programming of gene regulation, partly by activation of nuclear receptors, thereby influencing the risk of obesity later in life. The goal of this minireview is to briefly describe the epigenetic mechanisms underlying developmental plasticity and to evaluate the evidence of a mechanistic link between altered epigenetic gene regulation by early life EDC exposure and latent onset of obesity. We summarize the results of recent in vitro, in vivo, and transgenerational studies, which clearly show that the obesogenic effects of EDCs such as tributyltin, brominated diphenyl ether 47, and polycyclic aromatic hydrocarbons are mediated by the activation and associated altered methylation of peroxisome proliferator-activated receptor-γ, the master regulator of adipogenesis, or its target genes. Importantly, studies are emerging that assess the effects of EDCs on the interplay between DNA methylation and histone modifications in altered chromatin structure. These types of studies coupled with genome-wide rather than gene-specific analyses are needed to improve mechanistic understanding of epigenetic changes by EDC exposure. Current advances in the field of epigenomics have led to the first potential epigenetic markers for obesity that can be detected at birth, providing an important basis to determine the effects of developmental exposure to obesogenic EDCs in humans.
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Increasing evidence has associated the exposure of endocrine-disrupting chemicals (EDCs) with the cardiovascular (CV) system. This exposure is particularly problematic in a sensitive window of development, pregnancy. Pregnancy exposome can affect the overall health of the pregnancy by dramatic changes in vascular physiology and endocrine activity, increasing maternal susceptibility. Moreover, fetoplacental vascular function is generally altered, increasing the risk of developing pregnancy complications (including cardiovascular diseases, CVD) and predisposing the foetus to adverse health risks later in life. Thus, our review summarizes the existing literature on exposures to EDCs during pregnancy and adverse maternal health outcomes, focusing on the human placenta, vein, and umbilical artery associated with pregnancy complications. The purpose of this review is to highlight the role of fetoplacental vasculature as a model for the study of human cardiovascular endocrine disruption. Therefore, we emphasize that the placenta, together with the umbilical arteries and veins, allows a better characterization of the pregnant woman's exposome. Consequently, it contributes to the protection of the mother and foetus against CV disorders in life.
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Transgenerational effects of environmental toxins require either a chromosomal or epigenetic alteration in the germ line. Transient exposure of a gestating female rat during the period of gonadal sex determination to the endocrine disruptors vinclozolin (an antiandrogenic compound) or methoxychlor (an estrogenic compound) induced an adult phenotype in the F1 generation of decreased spermatogenic capacity (cell number and viability) and increased incidence of male infertility. These effects were transferred through the male germ line to nearly all males of all subsequent generations examined (that is, F1 to F4). The effects on reproduction correlate with altered DNA methylation patterns in the germ line. The ability of an environmental factor (for example, endocrine disruptor) to reprogram the germ line and to promote a transgenerational disease state has significant implications for evolutionary biology and disease etiology.
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Recently, we have suggested that down-regulation of homeostatic mesenchymal peroxisome proliferator-activated receptor γ signaling after in utero nicotine exposure might contribute to asthma. Here, we have exploited an in vivo rat model of asthma to determine if the effects of perinatal nicotine exposure on offspring pulmonary function and mesenchymal markers of airway contractility in both tracheal and lung parenchymal tissue are sex specific, and whether the protection afforded by the peroxisome proliferator-activated receptor g agonist, rosiglitazone (RGZ), against the perinatal nicotine-induced effect on offspring lung is also sex specific. Pregnant rat dams received placebo, nicotine, or nicotine plus RGZ daily from Embryonic Day 6 until Postnatal Day 21, at which time lung resistance, compliance, tracheal contractility, and the expression of structural and functional mesenchymal markers of pulmonary contractility were determined.Comparedwith control animals, perinatal nicotine exposure caused a significant increase in airway resistance and a decrease in airway compliance after a methacholine challenge in both male and female offspring, with more pronounced changes in the males. In contrast to this, the effects of perinatal nicotine exposure on acetylcholine-induced tracheal constriction, along with the expression of its mesenchymal markers, were observed exclusively in the male offspring. Concomitant treatment with RGZ normalized the nicotine-induced alterations in pulmonary function in both sexes, as well as the male-specific effects on acetylcholine-induced tracheal constriction, along with the affected mesenchymal markers. These data suggest that perinatal nicotine exposure causes sex-specific perinatal cigarette smoke exposure-induced asthma, providing a powerful phenotypic model for unequivocally determining the underlying nature of the cell molecular mechanism for this disease.
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Considerable variation is inherent both within and between comparative physiological data sets. Known sources for such variation include diet, gender, time of day and season of experiment, among many other factors, but a meta-analysis of physiological studies shows that surprisingly few studies report controlling for these factors. In fact, less than 3% of comparative physiological papers mention epigenetics. However, our understanding of epigenetic influences on physiological processes is growing rapidly, and it is highly likely that epigenetic phenomena are an additional 'hidden' source of variation, particularly in wild-caught specimens. Recent studies have shown epigenetic inheritance of commonly studied traits such as metabolic rate (water fleas Daphnia magna; emu, Dromaius novaellandiae), hypoxic tolerance, cardiac performance (zebrafish, Danio rerio), as well as numerous morphological effects. The ecological and evolutionary significance of such epigenetic inheritance is discussed in a comparative physiological context. Finally, against this context of epigenetic inheritance of phenotype, this essay also provides a number of caveats and warnings regarding the interpretation of transgenerational phenotype modification as a true epigenetic phenomenon. Parental effects, sperm storage, multiple paternity and direct gamete exposure can all be confounding factors. Epigenetic inheritance may best be studied in animal models that can be maintained in the laboratory over multiple generations, to yield parental stock that themselves are free of epigenetic effects from the historical experiences of their parents.
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Estrogen receptor alpha (ERα) activation functions AF-1 and AF-2 classically mediate gene transcription in response to estradiol (E2). A fraction of ERα is targeted to plasma membrane and elicits membrane-initiated steroid signaling (MISS), but the physiological roles of MISS in vivo are poorly understood. We therefore generated a mouse with a point mutation of the palmitoylation site of ERα (C451A-ERα) to obtain membrane-specific loss of function of ERα. The abrogation of membrane localization of ERα in vivo was confirmed in primary hepatocytes, and it resulted in female infertility with abnormal ovaries lacking corpora lutea and increase in luteinizing hormone levels. In contrast, E2 action in the uterus was preserved in C451A-ERα mice and endometrial epithelial proliferation was similar to wild type. However, E2 vascular actions such as rapid dilatation, acceleration of endothelial repair, and endothelial NO synthase phosphorylation were abrogated in C451A-ERα mice. A complementary mutant mouse lacking the transactivation function AF-2 of ERα (ERα-AF2(0)) provided selective loss of function of nuclear ERα actions. In ERα-AF2(0), the acceleration of endothelial repair in response to estrogen-dendrimer conjugate, which is a membrane-selective ER ligand, was unaltered, demonstrating integrity of MISS actions. In genome-wide analysis of uterine gene expression, the vast majority of E2-dependent gene regulation was abrogated in ERα-AF2(0), whereas in C451A-ERα it was nearly fully preserved, indicating that membrane-to-nuclear receptor cross-talk in vivo is modest in the uterus. Thus, this work genetically segregated membrane versus nuclear actions of a steroid hormone receptor and demonstrated their in vivo tissue-specific roles.
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Asthma is a major public health hazard world-wide. Its transgenerational inheritance has been inferred from epidemiologic studies. More recently, using nicotine as a proxy for maternal smoking, we have demonstrated that an asthma-like phenotype can be inherited by rat offspring for up to two generations, i.e., multigenerationally, after the initial intrauterine exposure. We hypothesized that asthma transmission to offspring following perinatal nicotine exposure is not restricted up to F2 generation, but it also extends to subsequent generations. To test this hypothesis, using a well-established rat model of nicotine exposure-induced childhood asthma, we determined if perinatal nicotine exposure of F0 gestating dams would transmit asthma transgenerationally to F3 offspring. We now extend our findings to third generation offspring, including abnormal pulmonary function, particularly as it relates to the occurrence in the upper airway exclusively in males, and to its effects on molecular functional markers (fibronectin and PPARγ), previously shown to be consistent with the asthma phenotype, herein expressed in fibroblasts isolated from the lung. These data, for the first time, demonstrate the transgenerational transmission of the asthma phenotype to F3 offspring following perinatal nicotine exposure of F0 dams.
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The role of gene expression of the estrogen receptor-a form (ERa) in the regulation of female reproductive behavior was investigated in estrogen receptor knockout (ERKO) mice, deficient specifically for the ERa, but not the ERb, gene. Estrogen- or estrogen- plus progesterone- treated gonadectomized ERKO mice did not show any lordosis re- sponse. Detailed behavioral analysis revealed that ERKO females were also deficient in sexual behavioral interactions preceding the lordosis response. They were extremely rejective toward attempted mounts by stud male mice, which could not show any intromissions. During resident-intruder aggression tests, gonadally intact ERKO females were more aggressive toward female intruder mice than wild- type (WT) mice. Gonadectomy did not influence the levels of aggres- sive behavior, and their genotype differences when mice were tested both before and after gonadectomy. However, when mice were tested after gonadectomy for the first time, very few ERKO mice showed aggression. In contrast to aggression, male-type sexual behavior shown by resident mice toward female intruder mice during aggres- sion tests was not different between ERKO and WT mice and was completely abolished after gonadectomy of the resident mice. Finally, it was also found that ERKO females showed greatly reduced levels of parental behavior toward newborn pups placed in their home cage. These changes in parental behavior were not influenced by gonad- ectomy. ERKO females retrieved significantly fewer numbers of pups with longer latencies compared with wild-type (WT) or heterozygous (HZ) littermates when they were tested as gonadally intact or 20 - 65 days after gonadectomy. In addition, during parental behavior tests, a significantly higher percentage of ERKO mice exhibited infanticide compared with WT and HZ mice, which rarely showed infanticide. Taken together, these findings suggest that ERa gene expression plays a key role in female mice, not only for sexual behavior but also for other interrelated behaviors, such as parental and aggressive behaviors. In addition, persistence of genotype differences in parental and aggressive behavior after gonadectomy indicates that ERa acti- vation during neural developmental processes may also be involved in the regulation of these behaviors. (Endocrinology 139: 5070 -5081, 1998)
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The polymorphic nature of sequences which act as retinoic acid response elements (RAREs and RXREs) in transactivation assays in mammalian cells, suggests that elements consisting of a direct repetition of a half site motif, separated by 1 to 5 base pairs (DR1 to DR5), are targets for retinoic acid (RA) signalling. In a previous report we showed that in yeast cells, heterodimers of the retinoic acid receptors RARalpha and RXRalpha were required for efficient transcription of a reporter gene containing a DR5 element [Heery et al., (1993); Proc. Natl. Acad. Sci. USA, 90: 4281 - 4285]. Here we report that DR1 to DR5 elements containing a direct repeat of the 5'-AGGTCA-3' motif, and an inverted repeat of the same sequence with no spacer (IRO), behave as RAREs in yeast cells coexpressing RARalpha and RXRalpha, albeit with different efficacies. Heterodimer activity was strongest on a DR5 reporter gene, and the strength of activation of the reporter series (DR5 > DR1 > DR3 > DR2 = IRO = DR4) correlated with the ability of the heterodimer to bind to the corresponding sequences in vitro. Significantly, a reporter containing a DR1 element was selectively and efficiently activated in yeast cells expressing only RXRalpha. This activity was dependent on the induction by 9-cis retinoic acid of an activation function (AF-2) located in the RXRalpha ligand binding domain. In addition, a strong synergistic activity of RXRalpha was observed on a reporter containing the putative RXR element (RXRE) from the rat CRBPII gene promoter. Thus, RXRalpha can function independently as a transcription factor, in the absence of RARs or other heteromeric partners. Similarly, homodimers of RARalpha selectively stimulated the transcription of a DR5 reporter in a ligand-dependent manner, but less efficiently than RARalpha/RXRalpha heterodimers.
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Bisphenol A (BPA) is an industrial compound and a well known endocrine-disrupting chemical with estrogenic activity. The widespread exposure of individuals to BPA is suspected to affect a variety of physiological functions, including reproduction, development, and metabolism. Here we report that the mechanisms by which BPA and two congeners, bisphenol AF and bisphenol C (BPC), bind to and activate estrogen receptors (ER) α and β differ from that used by 17β-estradiol. We show that bisphenols act as partial agonists of ERs by activating the N-terminal activation function 1 regardless of their effect on the C-terminal activation function 2, which ranges from weak agonism (with BPA) to antagonism (with BPC). Crystallographic analysis of the interaction between bisphenols and ERs reveals two discrete binding modes, reflecting the different activities of compounds on ERs. BPA and 17β-estradiol bind to ERs in a similar fashion, whereas, with a phenol ring pointing toward the activation helix H12, the orientation of BPC accounts for the marked antagonist character of this compound. Based on structural data, we developed a protocol for in silico evaluation of the interaction between bisphenols and ERs or other members of the nuclear hormone receptor family, such as estrogen-related receptor γ and androgen receptor, which are two known main targets of bisphenols. Overall, this study provides a wealth of tools and information that could be used for the development of BPA substitutes devoid of nuclear hormone receptor-mediated activity and more generally for environmental risk assessment.
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Variations in maternal care affect the development of individual differences in neuroendocrine responses to stress in rats. As adults, the offspring of mothers that exhibited more licking and grooming of pups during the first 10 days of life showed reduced plasma adrenocorticotropic hormone and corticosterone responses to acute stress, increased hippocampal glucocorticoid receptor messenger RNA expression, enhanced glucocorticoid feedback sensitivity, and decreased levels of hypothalamic corticotropin-releasing hormone messenger RNA. Each measure was significantly correlated with the frequency of maternal licking and grooming (all r's > −0.6). These findings suggest that maternal behavior serves to “program” hypothalamic-pituitary-adrenal responses to stress in the offspring.
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Background: We have previously shown that exposure to tributyltin (TBT) modulates critical steps of adipogenesis through RXR/PPARγ and that prenatal TBT exposure predisposes multipotent mesenchymal stem cells (MSCs) to become adipocytes by epigenetic imprinting into the memory of the MSC compartment. Objective: We tested whether the effects of prenatal TBT exposure were heritable in F2 and F3 generations. Methods: We exposed C57BL/6J female mice (F0) to DMSO vehicle, the pharmaceutical obesogen rosiglitazone (ROSI), or TBT (5.42, 54.2, or 542 nM) throughout pregnancy via the drinking water. F1 offspring were bred to yield F2, and F2 mice were bred to produce F3. F1 animals were exposed in utero and F2 mice were potentially exposed as germ cells in the F1, but F3 animals were never exposed to the chemicals. We analyzed the effects of these exposures on fat depot weights, adipocyte number, adipocyte size, MSC programming, hepatic lipid accumulation, and hepatic gene expression in all three generations. Discussion: Prenatal TBT exposure increased most white adipose tissue (WAT) depot weights, adipocyte size, and adipocyte number, and reprogrammed MSCs toward the adipocyte lineage at the expense of bone in all three generations. Prenatal TBT exposure led to hepatic lipid accumulation and up-regulated hepatic expression of genes involved in lipid storage/transport, lipogenesis, and lipolysis in all three subsequent generations. Conclusions: Prenatal TBT exposure produced transgenerational effects on fat depots and induced a phenotype resembling nonalcoholic fatty liver disease through at least the F3 generation. These results show that early-life obesogen exposure can have lasting effects.
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Regulation of nuclear receptor gene expression involves dynamic and coordinated interactions with histone acetyl transferase (HAT) and deacetylase complexes. The estrogen receptor (ERα) contains two transactivation domains regulating ligand-independent and -dependent gene transcription (AF-1 and AF-2 (activation functions 1 and 2)). ERα-regulated gene expression involves interactions with cointegrators (e.g.p300/CBP, P/CAF) that have the capacity to modify core histone acetyl groups. Here we show that the ERα is acetylated in vivo.p300, but not P/CAF, selectively and directly acetylated the ERα at lysine residues within the ERα hinge/ligand binding domain. Substitution of these residues with charged or polar residues dramatically enhanced ERα hormone sensitivity without affecting induction by MAPK signaling, suggesting that direct ERα acetylation normally suppresses ligand sensitivity. These ERα lysine residues also regulated transcriptional activation by histone deacetylase inhibitors and p300. The conservation of the ERα acetylation motif in a phylogenetic subset of nuclear receptors suggests that direct acetylation of nuclear receptors may contribute to additional signaling pathways involved in metabolism and development.
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The medial preoptic area has been shown to be intricately involved in many behaviors, including locomotion, sexual behavior, maternal care, and aggression. The gene encoding estrogen receptor-α (ERα) protein is expressed in preoptic area neurons, and a very dense immunoreactive field of ERα is found in the preoptic region. ERα knockout animals show deficits in maternal care and sexual behavior and fail to exhibit increases in these behaviors in response to systemic estradiol treatment. In the present study, we used viral-vector mediated RNA interference to silence ERα expression specifically in the preoptic area of female mice and measured a variety of behaviors, including social and sexual aggression, maternal care, and arousal activity. Suppression of ERα in the preoptic area almost completely abolished maternal care, significantly increasing the latency to pup retrieval and significantly reducing the time the moms spent nursing and licking the pups. Strikingly, maternal aggression toward a male intruder was not different between control and preoptic ERα-silenced mice, demonstrating the remarkably specific role of ERα in these neurons. Reduction of ERα expression in preoptic neurons significantly decreased sexual behavior in female mice and increased aggression toward both sexual partners and male intruders in a seminatural environment. Estrogen-dependent increases in arousal, measured by home cage activity, were not mediated by ERα expression in the preoptic neurons we targeted, as ERα-suppressed mice had increases similar to control mice. Thus, we have established that a specific gene in a specific group of neurons is required for a crucially important natural behavior.
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Prenatal exposure to maternal stress can have lifelong implications for psychological function, such as behavioral problems and even the development of mental illness. Previous research suggests that this is due to transgenerational epigenetic programming of genes operating in the hypothalamic-pituitary-adrenal axis, such as the glucocorticoid receptor (GR). However, it is not known whether intrauterine exposure to maternal stress affects the epigenetic state of these genes beyond infancy. Here, we analyze the methylation status of the GR gene in mothers and their children, at 10-19 years after birth. We combine these data with a retrospective evaluation of maternal exposure to intimate partner violence (IPV). Methylation of the mother's GR gene was not affected by IPV. For the first time, we show that methylation status of the GR gene of adolescent children is influenced by their mother's experience of IPV during pregnancy. As these sustained epigenetic modifications are established in utero, we consider this to be a plausible mechanism by which prenatal stress may program adult psychosocial function.
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Phosphorylation of estrogen receptor-α (ERα) is critical for its transcription factor activity and may determine its predictive and therapeutic value as a biomarker for ERα-positive breast cancers. Recent attention has turned to the poorly understood ERα hinge domain, as phosphorylation at serine 305 (Ser305) associates with poor clinical outcome and endocrine resistance. We show that phosphorylation of a neighboring hinge domain site, Ser294, analyzed by multiple reaction monitoring mass spectrometry of ERα immunoprecipitates from human breast cancer cells is robustly phosphorylated exclusively by ligand (estradiol and tamoxifen) activation of ERα and not by growth factor stimulation (EGF, insulin, heregulin-β). In a reciprocal fashion, Ser305 phosphorylation is induced by growth factors but not ligand activation of ERα. Phosphorylation at Ser294 and Ser305 is suppressed upon co-stimulation by EGF and ligand, respectively, unlike the N-terminal (AF-1) domain Ser118 and Ser167 sites of ERα where phosphorylation is enhanced by ligand and growth factor co-stimulation. Inhibition of cyclin-dependent kinases (CDK) by roscovitine or SNS-032 suppresses ligand-activated Ser294 phosphorylation without affecting Ser118 or Ser104/Ser106 phosphorylation. Likewise, cell-free studies using recombinant ERα and specific cyclin-CDK complexes suggest that Ser294 phosphorylation is primarily induced by the transcription-regulating and cell-cycle-independent kinase CDK7. Thus, CDK-dependent phosphorylation at Ser294 differentiates ligand-dependent from ligand-independent activation of Ser305 phosphorylation, showing that hinge domain phosphorylation patterns uniquely inform on the various ERα activation mechanisms thought to underlie the biologic and clinical diversity of hormone-dependent breast cancers.
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Morphoregulator genes like members of the Hox gene family regulate uterine development and are associated with endocrine-related processes such as endometrial proliferation and differentiation in the adult uterus. Exposure to neonatal endocrine disruptors could affect signaling events governed by Hox genes, altering the developmental trajectory of the uterus with lasting consequences. We investigated whether neonatal exposure to bisphenol A (BPA) alters Hoxa10 and Hoxa11 mRNA uterine expression shortly after treatment as well as in the adult. Moreover, we studied whether xenoestrogen exposure may affect the adult uterine response to hormonal stimuli. Newborn females received vehicle, 0.05 mg/kg.d BPA, 20 mg/kg*d BPA, or diethylstilbestrol (0.2 microg/kg*d) on postnatal d 1, 3, 5, and 7). At postnatal d 8, real time RT-PCR assays showed a decrease in Hoxa10 and Hoxa11 expression in all xenoestrogen-treated groups. To evaluate the long-term effects, we used adult ovariectomized rats with hormonal replacement. The subepithelial stroma in BPA- and diethylstilbestrol-treated animals showed an impaired proliferative response to steroid treatment associated with a silencing of Hoxa10 but not associated with changes in the methylation pattern of the Hoxa10 promoter. BPA animals showed that the Hoxa10 reduction was accompanied by an increased stromal expression of the silencing mediator for retinoic acid and thyroid hormone receptor. The spatial coexpression of steroid receptors Hoxa10 and silencing mediator for retinoic acid and thyroid hormone receptor was established using immunofluorescence. Our data indicate that postnatal BPA exposure affects the steroid hormone-responsiveness of uterine stroma in adulthood. Whether this impaired hormonal response is associated with effects on uterine receptivity and decidualization is currently under investigation.
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Estrogen signaling pathways, because of their central role in regulating the growth and survival of breast tumor cells, have been identified as suitable and efficient targets for cancer therapies. Agents blocking estrogen activity are already widely used clinically, and many new molecules have entered clinical trials, but intrinsic or acquired resistance to treatment limits their efficacy. The basic molecular studies underlying estrogen signaling have defined the critical role of estrogen receptors (ER) in many aspects of breast tumorigenesis. However, important knowledge gaps remain about the role of posttranslational modifications (PTM) of ER in initiation and progression of breast carcinogenesis. Whereas major attention has been focused on the phosphorylation of ER, many other PTM (such as acetylation, ubiquitination, sumoylation, methylation, and palmitoylation) have been identified as events modifying ER expression and stability, subcellular localization, and sensitivity to hormonal response. This article will provide an overview of the current and emerging knowledge on ER PTM, with a particular focus on their deregulation in breast cancer. We also discuss their clinical relevance and the functional relationship between PTM. A thorough understanding of the complete picture of these modifications in ER carcinogenesis might not only open new avenues for identifying new markers for prognosis or prediction of response to endocrine therapy but also could promote the development of novel therapeutic strategies.
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The occurrence of halogenated analogs of the xenoestrogen bisphenol A (BPA) has been recently demonstrated both in environmental and human samples. These analogs include brominated [e.g., tetrabromobisphenol A (TBBPA)] and chlorinated [e.g., tetrachlorobisphenol A (TCBPA)] bisphenols, which are both flame retardants. Because of their structural homology with BPA, such chemicals are candidate endocrine disruptors. However, their possible target(s) within the nuclear hormone receptor superfamily has remained unknown. We investigated whether BPA and its halogenated analogs could be ligands of estrogen receptors (ERs) and peroxisome proliferator-activated receptors (PPARs) and act as endocrine-disrupting chemicals. We studied the activity of compounds using reporter cell lines expressing ERs and PPARs. We measured the binding affinities to PPARγ by competitive binding assays with [3H]-rosiglitazone and investigated the impact of TBBPA and TCBPA on adipocyte differentiation using NIH3T3-L1 cells. Finally, we determined the binding mode of halogenated BPAs to PPARγ by X-ray crystallography. We observed that TBBPA and TCBPA are human, zebrafish, and Xenopus PPARγ ligands and determined the mechanism by which these chemicals bind to and activate PPARγ. We also found evidence that activation of ERα, ERβ, and PPARγ depends on the degree of halogenation in BPA analogs. We observed that the bulkier brominated BPA analogs, the greater their capability to activate PPARγ and the weaker their estrogenic potential. Our results strongly suggest that polyhalogenated bisphenols could function as obesogens by acting as agonists to disrupt physiological functions regulated by human or animal PPARγ.
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Nicotine exposure alters normal homeostatic pulmonary epithelial-mesenchymal paracrine signaling pathways, resulting in alveolar interstitial fibroblast (AIF)-to-myofibroblast (MYF) transdifferentiation. Though this has been described under in vitro conditions, it is not known if the same phenomenon also takes place in vivo. A well-established rodent model of lung damage following perinatal nicotine exposure was used. By probing for the well-established markers of fibroblast differentiation (parathyroid hormone-related protein [PTHrP], peroxisome proliferator-activated receptor gamma [PPARgamma], adipocyte differentiation-related protein, alpha-smooth muscle actin, and fibronectin) at the mRNA, protein, and tissue levels, the authors provide the first in vivo evidence for nicotine-induced AIF-to-MYF transdifferentiation. In addition, these data also provide the first evidence for nicotine-induced up-regulation of Wnt signaling, accompanying the down-regulation of PTHrP/PPARgamma signaling in vivo following nicotine exposure during pregnancy. These data provide an integrated mechanism for in utero nicotine-induced lung damage and how it could permanently alter the "developmental program" of the developing lung by disrupting critically important epithelial-mesenchymal interactions. More importantly, these data are likely to provide specific interventions to augment the pulmonary mesenchymal lipogenic pathway to ameliorate nicotine-induced in utero lung injury.
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Endocrine-disrupting chemicals (EDCs) represent a broad class of exogenous substances that cause adverse effects in the endocrine system by interfering with hormone biosynthesis, metabolism, or action. The molecular mechanisms of EDCs involve different pathways including interactions with nuclear hormone receptors (NHRs) which are primary targets of a large variety of environmental contaminants. Here, based on the crystal structures currently available in the Protein Data Bank, we review recent studies showing the many ways in which EDCs interact with NHRs and impact their signaling pathways. Like the estrogenic chemical diethylstilbestrol, some EDCs mimic the natural hormones through conserved protein-ligand contacts, while others, such as organotins, employ radically different binding mechanisms. Such structure-based knowledge, in addition to providing a better understanding of EDC activities, can be used to predict the endocrine-disrupting potential of environmental pollutants and may have applications in drug discovery.
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Fetal exposure to environmental estrogens may contribute to hypofertility and/or to testicular germ cell cancer. However, many of these xenoestrogens have only a weak affinity for the classical estrogen receptors (ERs,) which is 1,000-fold less potent than the affinity of 17beta-estradiol (E(2)). Thus, several mechanisms have been suggested to explain how they could affect male germ cell proliferation at low environmental relevant concentrations. In this study we aimed to explore the possible promoting effect of bisphenol A (BPA) on human testicular seminoma cells. BPA is a well-recognized estrogenic endocrine disruptor used as a monomer to manufacture poly carbonate plastic and released from resin-lined food or beverage cans or from dental sealants. BPA at very low concentrations (10(-9) to 10(-12) M) similar to those found in human fluids stimulated JKT-1 cell proliferation in vitro. BPA activated both cAMP-dependent protein kinase and cGMP-dependent protein kinase pathways and triggered a rapid (15 min) phosphorylation of the transcription factor cAMP response-element-binding protein (CREB) and the cell cycle regulator retinoblastoma protein (Rb). This nongenomic activation did not involve classical ERs because it could not be reversed by ICI 182780 (an ER antagonist) or reproduced either by E(2) or by diethylstilbestrol (a potent synthetic estrogen), which instead triggered a suppressive effect. This activation was reproduced only by E(2) coupled to bovine serum albumin (BSA), which is unable to enter the cell. As with E(2)-BSA, BPA promoted JKT-1 cell proliferation through a G-protein-coupled nonclassical membrane ER (GPCR) involving a Galpha(s) and a Galpha(i)/Galpha(q) subunit, as shown by the reversible effect observed by the corresponding inhibitors NF449 and pertussis toxin. This GPCR-mediated nongenomic action represents--in addition to the classical ER-mediated effect--a new basis for evaluating xenoestrogens such as BPA that, at low doses and with a high affinity for this GPCR, could interfere with the developmental programming of fetal germ cell proliferation and/or differentiation when they cross the placenta.
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Maternal care influences hypothalamic-pituitary-adrenal (HPA) function in the rat through epigenetic programming of glucocorticoid receptor expression. In humans, childhood abuse alters HPA stress responses and increases the risk of suicide. We examined epigenetic differences in a neuron-specific glucocorticoid receptor (NR3C1) promoter between postmortem hippocampus obtained from suicide victims with a history of childhood abuse and those from either suicide victims with no childhood abuse or controls. We found decreased levels of glucocorticoid receptor mRNA, as well as mRNA transcripts bearing the glucocorticoid receptor 1F splice variant and increased cytosine methylation of an NR3C1 promoter. Patch-methylated NR3C1 promoter constructs that mimicked the methylation state in samples from abused suicide victims showed decreased NGFI-A transcription factor binding and NGFI-A-inducible gene transcription. These findings translate previous results from rat to humans and suggest a common effect of parental care on the epigenetic regulation of hippocampal glucocorticoid receptor expression.
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The nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARgamma) has important roles in adipogenesis and immune response as well as roles in both lipid and carbohydrate metabolism. Although synthetic agonists for PPARgamma are widely used as insulin sensitizers, the identity of the natural ligand(s) for PPARgamma is still not clear. Suggested natural ligands include 15-deoxy-delta12,14-prostaglandin J2 and oxidized fatty acids such as 9-HODE and 13-HODE. Crystal structures of PPARgamma have revealed the mode of recognition for synthetic compounds. Here we report structures of PPARgamma bound to oxidized fatty acids that are likely to be natural ligands for this receptor. These structures reveal that the receptor can (i) simultaneously bind two fatty acids and (ii) couple covalently with conjugated oxo fatty acids. Thermal stability and gene expression analyses suggest that such covalent ligands are particularly effective activators of PPARgamma and thus may serve as potent and biologically relevant ligands.
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Background: Traumatic experiences in childhood are linked to adult depression and cardiovascular disease. Depression is twice as common in women than men, and depression after cardiovascular events is more common in women than men. However, sex differences in these relationships have not been comprehensively investigated using a nationally representative sample in which demographic factors related to these illnesses can be controlled. Method: Data come from the Part 2 sample of the U.S. National Comorbidity Survey, a nationally representative sample containing over 5000 adults. Relationships between childhood maltreatment (sexual abuse, physical abuse, neglect), adult depression (DSM-III-R), and cardiovascular disease were examined using multiple logistic regression models with a specific emphasis on the evaluation of sex differences. Results: Childhood maltreatment was associated with a significant increase in cardiovascular disease for women only and with a significant increase in lifetime depression for both genders. A history of childhood maltreatment removed the natural protection against cardiovascular disease for women and depression for men. Although depression and cardiovascular disease were correlated, depression did not contribute to the prediction of cardiovascular disease in women when controlling for history of childhood maltreatment. Conclusions: Gender is important in evaluating potential psychiatric and physical correlates of childhood maltreatment. Maltreatment is a potent risk factor for cardiovascular disease in women and for depression in both women and men. Effective clinical assessment should recognize the role of childhood abuse or neglect in adult health and disease. Research on the consequences of childhood maltreatment should focus on both psychiatric and physical outcomes.
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Bisphenol-A (BPA) is one of the highest-volume chemicals produced worldwide and the widespread exposure of individuals to BPA is suspected to affect a variety of physiological functions, including reproduction, development, and metabolism. Its estrogenic activity has been well documented in the last 15 years. In addition to estrogen receptors, BPA has been also shown to bind to and activate the estrogen-related receptor γ and pregnane X receptor and inhibit the androgen receptor. Halogenated BPAs were also shown to activate the peroxisome proliferator-activated receptor γ and inhibit thyroid hormone receptors. In this chapter, we review recent studies shedding light on the structural and molecular mechanisms by which BPA and its halogenated derivatives interfere with nuclear hormone receptor signaling. These data provide guidelines for the development of safer substitutes devoid of hormonal activity and may help environmental risk assessment.
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Estrogen receptor alpha (ERα) is a ligand-activated transcription factor. Upon estrogen stimulation, ERα recruits a number of coregulators, including both coactivators and corepressors, to the estrogen response elements, modulating gene activation or repression. Most coregulator complexes contain histone-modifying enzymes to control ERα target gene expression in an epigenetic manner. In addition to histones, these epigenetic modifiers can modify nonhistone proteins including ERα, thereby constituting another layer of transcriptional regulation. Here we show that SET and MYND domain containing 2 (SMYD2), a histone H3K4 and H3K36 methyltransferase, directly methylates ERα protein at lysine 266 (K266) both in vitro and in cells. In breast cancer MCF7 cells, SMYD2 attenuates the chromatin recruitment of ERα to prevent ERα target gene activation under an estrogen-depleted condition. Importantly, the SMYD2-mediated repression of ERα target gene expression is mediated by the methylation of ERα at K266 in the nucleus, but not the methylation of histone H3K4. Upon estrogen stimulation, ERα-K266 methylation is diminished, thereby enabling p300/cAMP response element-binding protein-binding protein to acetylate ERα at K266, which is known to promote ERα transactivation activity. Our study identifies a previously undescribed inhibitory methylation event on ERα. Our data suggest that the dynamic cross-talk between SMYD2-mediated ERα protein methylation and p300/cAMP response element-binding protein-binding protein-dependent ERα acetylation plays an important role in fine-tuning the functions of ERα at chromatin and the estrogen-induced gene expression profiles.
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Concern that some chemicals in our environment may affect human health by disrupting normal endocrine function has prompted research on interactions of environmental contaminants with steroid hormone receptors. We compared the activity of 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), an estrogenic metabolite of the organochlorine pesticide methoxychlor, at estrogen receptor α (ERα) and estrogen receptor β (ERβ). Human hepatoma cells (HepG2) were transiently transfected with either human or rat ERα or ERβ plus an estrogen-responsive, complement 3-luciferase construct containing a complement 3 gene promoter sequence linked to a luciferase reporter gene. After transfection, cells were treated with various concentrations of HPTE in the presence (for detecting antagonism) or absence (for detecting agonism) of 17β-estradiol. HPTE was a potent ERα agonist in HepG2 cells, with EC50 values of approximately 5 × 10⁻⁸ and 10⁻⁸m for human and rat ERα, respectively. In contrast, HPTE had minimal agonist activity with either human or rat ERβ and almost completely abolished 17β-estradiol-induced ERβ-mediated activity. Moreover, HPTE behaved as an ERα agonist and an ERβ antagonist with other estrogen-responsive promoters (ERE-MMTV and vtERE) in HepG2 and HeLa cells. This study demonstrates the complexity involved in determining the mechanism of action of endocrine-active chemicals that may act as agonists or antagonists through one or more hormone receptors.
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The nuclear receptor PPARγ/RXRα heterodimer regulates glucose and lipid homeostasis and is the target for the antidiabetic drugs GI262570 and the thiazolidinediones (TZDs). We report the crystal structures of the PPARγ and RXRα LBDs complexed to the RXR ligand 9-cis-retinoic acid (9cRA), the PPARγ agonist rosiglitazone or GI262570, and coactivator peptides. The PPARγ/RXRα heterodimer is asymmetric, with each LBD deviated ∼10° from the C2 symmetry, allowing the PPARγ AF-2 helix to interact with helices 7 and 10 of RXRα. The heterodimer interface is composed of conserved motifs in PPARγ and RXRα that form a coiled coil along helix 10 with additional charge interactions from helices 7 and 9. The structures provide a molecular understanding of the ability of RXR to heterodimerize with many nuclear receptors and of the permissive activation of the PPARγ/RXRα heterodimer by 9cRA.
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STEROID hormone receptors control gene expression through binding, as dimers, to short palindromic response elements located upstream of the genes they regulate1–3. An independent domain of ~70 amino acids directs this sequence-specific DNA binding and is highly conserved between different receptor proteins and related transcription factors4–6. This domain contains two zinc-binding Cys2–Cys2 sequence motifs, which loosely resemble the 'zinc-finger' motifs of TFIIIA7–10. Here we describe the structure of the DNA-binding domain from the oestrogen receptor, as determined by two-dimensional 1H NMR techniques. The two 'zinc-finger'-like motifs fold to form a single structural domain and are thus distinct from the independently folded units of the TFIIIA-type zinc fingers11–13. The structure consists of two helices perpendicular to each other. A zinc ion, coordinated by four conserved cysteines, holds the base of a loop at the N terminus of each helix. This novel structural domain seems to be a general structure for protein-DNA recognition.
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The present study investigated whether the medial preoptic area (MPOA) mediates estrogen stimulation of maternal behavior in the male as it does in the female. Previous studies have shown that lesions of the medial preoptic area prevent sensitization of maternal behavior in male rats and that in gonadectomized, hormonally primed males, systemically administered estradiol benzoate stimulates short-latency maternal behavior. These findings are similar to those found in females. In the present study adult males were gonadectomized and hormonally primed with subcutaneously implanted capsules of estradiol (Days 1–16) and progesterone (Days 3–15) and then were stereotaxically implanted bilaterally in the MPOA with implants containing 10% estradiol. Tests with young pups were started 48 h later and continued for 10 days (11 tests). Control groups were implanted in the MPOA with cholesterol or were injected subcutaneously with estradiol benzoate (100 μg/kg). Estradiol implanted males had shorter latencies for maternal behavior (retrieving, crouching, licking pups) than cholesterol implanted males, but their latencies were slightly longer than those of estradiol benzoate injected males. The medial preoptic area, therefore, mediates estrogen stimulation of maternal behavior in males as it does in females.
Article
Bisphenol A (BPA) is an estrogenic endocrine disruptor commonly used in manufacture of polycarbonate plastics and epoxy resins. Due to its ubiquitous presence in the environment, health concerns are increasing. Earlier studies from our group have shown that neonatal exposure of male rats to BPA affected spermatogenesis leading to impairment in fertility during adulthood. Further we also observed an altered gene expression of ERα and ERβ in adult testis upon BPA exposure. Based on these results, we hypothesized that apart from endocrine action, BPA might mediate perturbations in expression of ERs via epigenetic mechanism. The present study was undertaken to determine the effect of exposure of neonatal male rats to BPA on DNA methylation profile of estrogen receptor promoter region and on DNA methylation machinery. In order to test this hypothesis, neonatal male rats were subcutaneously injected with 2.4μg of BPA/day for the first five days of life, i.e., on postnatal days (PND) 1-5, while control group received vehicle (sesame oil). Animals were sacrificed during adulthood (PND-125) and testes were dissected out for analysis. Methylation pattern of promoter region of ERα and ERβ was analyzed in the testis by bisulfite sequencing and expression levels of DNA methyltransferases by quantitative RT-PCR and Western blotting respectively. Bisulfite sequencing revealed significant hypermethylation of ERα promoter to varying extents from 40% to 60%, and ERβ promoter region with varying extent from 20% to 65%. Approximately 2-fold increase in Dnmt3a and Dnmt3b expression at transcript and protein level was also observed. The experimental evidence demonstrated that the neonatal exposure of rats to BPA led to aberrant DNA methylation in testis, indicating methylation mediated epigenetic changes as one of the possible mechanisms of BPA induced adverse effects on spermatogenesis and fertility.
Article
Diethylstilbestrol (DES) and bisphenol-A (BPA) are estrogen-like endocrine-disrupting chemicals that induce persistent epigenetic changes in the developing uterus. However, DES exposure in utero is also associated with an increased risk of breast cancer in adult women. Similarly, fetal exposure to BPA induces neoplastic changes in mammary tissue of mice. We hypothesized that epigenetic alterations would precede the increased risk of breast neoplasia after in utero exposure to endocrine disruptors. Enhancer of Zeste Homolog 2 (EZH2) is a histone methyltransferase that has been linked to breast cancer risk and epigenetic regulation of tumorigenesis. We examined the effect of BPA and DES on EZH2 expression and function in MCF-7 cells and in mammary glands of mice exposed in utero. DES and BPA treatment approximated human exposure. EZH2 functional activity was assessed by measuring histone H3 trimethylation. Treatment of MCF-7 cells with DES or BPA led to a 3- and 2-fold increase in EZH2 mRNA expression, respectively (p < 0.05) as well as increased EZH2 protein expression. Mice exposed to DES in utero showed a >2-fold increase in EZH2 expression in adult mammary tissue compared with controls (p < 0.05). EZH2 protein was elevated in mammary tissue of mice exposed to DES or BPA. Histone H3 trimethylation was increased in MCF-7 cells treated with BPA or DES. Similarly, mice exposed to BPA or DES in utero showed increased mammary histone H3 trimethylation. Developmental programming of EZH2 is a novel mechanism by which in utero exposure to endocrine disruptors leads to epigenetic regulation of the mammary gland.
Article
Evidence is emerging that estrogen receptor alpha (ERalpha) is central to the rapid transduction of estrogen signaling to the downstream kinase cascades; however, the mechanisms underlying this nongenomic function are not fully understood. Here we report a paradigm of ERalpha regulation through arginine methylation by PRMT1, which transiently methylates arginine 260 within the ERalpha DNA-binding domain. This methylation event is required for mediating the extranuclear function of the receptor by triggering its interaction with the p85 subunit of PI3K and Src. Furthermore, we find that the focal adhesion kinase (FAK), a Src substrate involved in the migration process, is also recruited in this complex. Our data indicate that the methylation of ERalpha is a physiological process occurring in the cytoplasm of normal and malignant epithelial breast cells and that ERalpha is hypermethylated in a subset of breast cancers.
Article
Although rapid, membrane-activated estrogen receptor (ER) signaling is no longer controversial, the biological function of this nongenomic signaling is not fully characterized. We found that rapid signaling from membrane-associated ER regulates the histone methyltransferase enhancer of Zeste homolog 2 (EZH2). In response to both 17beta-estradiol (E2) and the xenoestrogen diethylstilbestrol, ER signaling via phosphatidylinositol 3-kinase/protein kinase B phosphorylates EZH2 at S21, reducing levels of trimethylation of lysine 27 on histone H3 in hormone-responsive cells. During windows of uterine development that are susceptible to developmental reprogramming, activation of this ER signaling pathway by diethylstilbestrol resulted in phosphorylation of EZH2 and reduced levels of trimethylation of lysine 27 on histone H3 in chromatin of the developing uterus. Furthermore, activation of nongenomic signaling reprogrammed the expression profile of estrogen-responsive genes in uterine myometrial cells, suggesting this as a potential mechanism for developmental reprogramming caused by early-life exposure to xenoestrogens. These data demonstrate that rapid ER signaling provides a direct linkage between xenoestrogen-induced nuclear hormone receptor signaling and modulation of the epigenetic machinery during tissue development.
Article
Bisphenol-A (BPA) is a nonsteroidal estrogen that is ubiquitous in the environment. The homeobox gene Hoxa10 controls uterine organogenesis, and its expression is affected by in utero BPA exposure. We hypothesized that an epigenetic mechanism underlies BPA-mediated alterations in Hoxa10 expression. We analyzed the expression pattern and methylation profile of Hoxa10 after in utero BPA exposure. Pregnant CD-1 mice were treated with BPA (5 mg/kg IP) or vehicle control on d 9-16 of pregnancy. Hoxa10 mRNA and protein expression were increased by 25% in the reproductive tract of mice exposed in utero. Bisulfite sequencing revealed that cytosine-guanine dinucleotide methylation was decreased from 67 to 14% in the promoter and from 71 to 3% in the intron of Hoxa10 after in utero BPA exposure. Decreased DNA methylation led to an increase in binding of ER-alpha to the Hoxa10 ERE both in vitro as and in vivo as determined by EMSA and chromatin immunoprecipitation, respectively. Diminished methylation of the ERE-containing promoter sequence resulted in an increase in ERE-driven gene expression in reporter assays. We identify altered methylation as a novel mechanism of BPA-induced altered developmental programming. Permanent epigenetic alteration of ERE sensitivity to estrogen may be a general mechanism through which endocrine disruptors exert their action.
Article
Estrogen receptor alpha (ERalpha) is a member of a large conserved superfamily of steroid hormone nuclear receptors which regulates many physiological pathways by acting as a ligand-dependent transcription factor. Evidence is emerging that estrogens also induce rapid signaling to the downstream kinase cascades; however the mechanisms underlying this nongenomic function remain poorly understood. We have recently shown that ERalpha is methylated specifically by the arginine methyltransferase PRMT1 at arginine 260 in the DNA-binding domain of the receptor. This methylation event is required for mediating the extra-nuclear function of the receptor which would thereby interact with Src/FAK and p85 and propagate the signal to downstream transduction cascades that orchestrate cell proliferation and survival. Of particular interest, a possible role of methylated ERalpha in mammary tumorigenesis is also evident by the fact that, as demonstrated by immunohistochemical studies on a cohort of breast cancer patients, ERalpha is methylated in normal epithelial breast cells and is hypermethylated in a subset of breast cancers. Hypermethylation of ERalpha in breast cancer might cause hyperactivation of cellular kinase signaling, notably of Akt, described as a selective survival advantage for primary tumor cells even in the presence of anti-estrogens. A detailed understanding of the molecular mechanisms that control estrogen signaling in breast cancer is a crucial step in identifying new effective therapies.
Article
Endocrine-disrupting chemicals (EDCs), among which is the antiandrogen vinclozolin (VCZ), have been reported to affect the male reproductive system. In this study, VCZ was administered to pregnant mice at the time of embryo sex determination, and its possible effects on the differentially methylated domains (DMDs) of two paternally (H19 and Gtl2) and three maternally (Peg1, Snrpn, and Peg3) imprinted genes were tested in the male offspring. The CpGs methylation status within the five gene DMDs was analyzed in the sperm, tail, liver, and skeletal muscle DNAs by pyrosequencing. In the sperm of controls, the percentages of methylated CpGs were close to the theoretical values of 100 and 0% in paternally or maternally imprinted genes respectively. VCZ decreased the percentages of methylated CpGs of H19 and Gtl2 (respective values 83.1 and 91.5%) and increased those of Peg1, Snrpn, and Peg3 (respective values 11.3, 18.3, and 11.2%). The effects of VCZ were transgenerational, but they disappeared gradually from F1 to F3. The mean sperm concentration of the VCZ-administered female offspring was only 56% of that of the controls in the F1 offspring, and it was back to normal values in the F2 and F3 offspring. In the somatic cells of controls, the percentages of methylated CpGs were close to the theoretical value of 50% and, surprisingly, VCZ altered the methylation of Peg3. We propose that the deleterious effects of VCZ on the male reproductive system are mediated by imprinting defects in the sperm. The reported effects of EDCs on human male spermatogenesis might be mediated by analogous imprinting alterations.
Article
Some phenols have been suspected to modulate the endocrine systems of wildlife and humans, but less is known about their interactions with different types of nuclear receptors. In this study, the ability of 2-tert-butylphenol, 2-isopropylphenol, 4-tert-octylphenol (4-t-OP), 2,4-dichlorophenol (2,4-DCP), 3,4-dichlorophenol (3,4-DCP), pentachlorophenol (PCP), bisphenols A (BPA), tetrabromobisphenol A (TBBPA), tetrachlorobisphenol A (TCBPA) and 4-phenylphenol to activate estrogen receptor (ER), androgen receptor (AR), progesterone receptor (PR) and estrogen-related receptor (ERR) were determined using a set of recombined yeast strains. It was found that 4-t-OP, 3,4-DCP, PCP, BPA, TBBPA, TCBPA and 4-phenylphenol were ERalpha agonists, while 4-t-OP, PCP and 4-phenylphenol showed ERalpha antagonistic activities. 2-tert-Butylphenol, 4-t-OP, 2-isopropylphenol, 2,4-DCP, 3,4-DCP, BPA, TCBPA and 4-phenylphenol were antagonists for AR, whereas none of the compounds studied were found to be an AR agonist. TCBPA, TBBPA and PCP were PR antagonists, and 2-tert-butylphenol, 3,4-DCP, 4-t-OP, 4-phenylphenol and 2-isopropylphenol were weak inhibitors on expression under control of the PR. None of the phenols were PR agonists. 2-tert-Butylphenol, 4-t-OP and PCP were ERRgamma inverse agonists, while 2,4-DCP, 3,4-DCP, PCP, BPA, TBBPA and TCBPA exhibited the ability to reverse the ERR inhibition induced by 4-hydroxytamoxifen. Based on the functional agonistic or antagonistic receptor-mediated effects, we further discussed the possible action mechanisms of these phenols as endocrine disrupting chemicals.
Article
Steroid hormones and steroid receptors (SRs) play a crucial role in spermatogenesis. Steroid receptor coregulators are the major determinants of SR functioning, and any alteration in their expression is known to be associated with impaired spermatogenesis. Since Bisphenol A (BPA) exposure leads to an impairment of spermatogenesis, we hypothesized that this effect could be associated with the altered expression of steroid receptors and their coregulators in the testes. The present study describes the effect of perinatal exposure of rats to BPA on the expression profile of testicular steroid receptor coregulators in the F(1) generation. These effects were further studied in the F(2) and F(3) generations to determine vertical transmission. Pregnant female rats (F(0)) were gavaged daily with BPA (1.2 and 2.4 microg/kg bw) (or vehicles for controls) from gestation day 12 through postnatal day (PND) 21 to obtain the F(1) and subsequent F(2) and F(3) generations. Immunohistochemical localization of steroid receptor coactivator-1 (SRC-1), G-receptor integrating protein-1 (GRIP-1), p300/CBP/cointegrator-associated protein (p/CIP) and nuclear corepressor (NCoR) was carried out in the testes of F(1), F(2) and F(3) generation adult rats. A significant reduction in the expression of SRC-1 and NCoR, with a parallel increase in the expression of p/CIP and GRIP-1, was observed in the testes of rats exposed perinatally to BPA. Surprisingly, a similar pattern was observed in the testes of F(2) and F(3) rats. Perinatal exposure of male rats to BPA leads to transgenerational perturbations in the expression profile of testicular steroid receptor coregulators.
Article
To verify whether anti-androgens cause transgenerational effects on spermatogenesis and DNA methylation in rats, gravid Crl:CD(SD) female rats (4 or 5/group, gestational day (GD) 0=day sperm detected) were intraperitoneally treated with anti-androgenic compounds, such as vinclozolin (100 mg/kg/day), procymidone (100 mg/kg/day), or flutamide (10 mg/kg/day), from GD 8 to GD 15. Testes were collected from F1 male pups at postnatal day (PND) 6 for DNA methylation analysis of the region (210 bp including 7 CpG sites) within the lysophospholipase gene by bisulfite DNA sequencing method. F0 and F1 males underwent the sperm analysis (count, motility and morphology), followed by DNA methylation analysis of the sperm. Remaining F1 males were cohabited with untreated-females to obtain F2 male pups for subsequent DNA methylation analysis of the testes at PND 6. These analyses showed no effects on spermatogenesis and fertility in F1 males of any treatment group. DNA methylation status in testes (F1 and F2 pups at PND 6) or sperms (F1 males at 13 weeks old) of the treatment groups were comparable to the control at all observation points, although DNA methylation rates in testes were slightly lower than those in sperm. In F0 males, no abnormalities in the spermatogenesis, fertility and DNA methylation status of sperm were observed. No transgenerational abnormalities of spermatogenesis and DNA methylation status caused by anti-androgenic compounds were observed.
Article
Perinatal exposure to diethylstilbestrol (DES) can have numerous adverse effects on the reproductive organs later in life, such as vaginal clear-cell adenocarcinoma. Epigenetic processes including DNA methylation may be involved in the mechanisms. We subcutaneously injected DES to neonatal C57BL/6 mice. At days 5, 14, and 30, expressions of DNA methyltransferases (Dnmts) Dnmt1, Dnmt3a, and Dnmt3b, and transcription factors Sp1 and Sp3 were examined. We also performed restriction landmark genomic scanning (RLGS) to detect aberrant DNA methylation. Real-time RT-PCR revealed that expressions of Dnmt1, Dnmt3b, and Sp3 were decreased at day 5 in DES-treated mice, and that those of Dnmt1, Dnmt3a, and Sp1 were also decreased at day 14. RLGS analysis revealed that 5 genomic loci were demethylated, and 5 other loci were methylated by DES treatment. Two loci were cloned, and differential DNA methylation was quantified. Our results indicated that DES altered the expression levels of Dnmts and DNA methylation.
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