Environmental Estrogenic Chemicals Research Articles

Estrogen-induced reactive oxygen species, through epigenetic reprogramming, causes increased growth in breast cancer cells

Despite the progress made in cancer diagnosis and treatment, breast cancer remains the second leading cause of cancer-related death among the women. Exposure to elevated levels of endogenous estrogen or environmental estrogenic chemicals is an important risk factor for breast cancer. Estrogen metabolites and ROS generated during estrogen metabolism are known to play a critical role in estrogen carcinogenesis. However, the molecular mechanisms through which estrogen-induced ROS regulate gene expression is not clear. Epigenetic changes of DNA methylation and histone modifications are known to regulate genes expression. Therefore, the objective of this study was to evaluate whether estrogen-induced ROS, through aberrant expression of epigenetic regulatory genes and epigenetic reprogramming, causes growth of breast cancer cells. Estrogen responsive MCF-7 and T47D human breast cancer cells were exposed to natural estrogen 17 beta-estradiol (E2) and synthetic estrogen Diethylstilbestrol (DES) both alone and in combination with antioxidant N-acetyl cysteine. Effects of NAC-mediated scavenging of estrogen-induced ROS on cell growth, gene expression, and histone modifications were measured. The result of MTT and cell cycle analysis revealed significant abrogation of E2 and DES-induced growth by scavenging ROS through NAC. E2 and DES caused significant changes in expression of epigenetic regulatory genes for DNA methylation and histone modifications as well as changes in both gene activating and repressive marks in the Histone H3. NAC restored the expression of epigenetic regulatory genes and changes in histone marks. Novel findings of this study suggest that estrogen can induce growth of breast cancer cells through ROS-dependent regulation of epigenetic regulatory genes and epigenetic reprogramming of histone marks.

Repeated exposure to 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP) accelerates ligand-independent activation of estrogen receptors in long-term estradiol-deprived MCF-7 cells

It was previously identified that there may be an active metabolite of bisphenol A (BPA), 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP). An in vitro system was developed to detect MBP toxicity to the Michigan Cancer Foundation-7 (MCF-7) cells that had been repeatedly exposed to a low dose of the metabolite. MBP profoundly activated estrogen receptor (ER)-dependent transcription as a ligand, with an EC50 of 2.8 nM. Women are continuously exposed to numerous estrogenic environmental chemicals; but their susceptibility to these chemicals may be significantly altered after menopause. Long-term estrogen-deprived (LTED) cells, which display ligand-independent ER activation, are a postmenopausal breast cancer model derived from MCF-7 cells. In this study, we investigated the estrogenic effects of MBP on LTED cells in a repeated exposure in vitro model. The results suggest that i) nanomolar levels of MBP reciprocally disrupt the balanced expression of ERα and ERβ proteins, leading to the dominant expression of ERβ, ii) MBP stimulates ERs-mediated transcription without acting as an ERβ ligand, and iii) MBP utilizes mitogen-activated protein kinase and phosphatidylinositol-3 kinase signaling to evoke its estrogenic action. Moreover, the repeated exposure strategy was effective for detecting low-dose estrogenic-like effects caused by MBP in LTED cells.

Abstract P1-07-10: Estrogen-stimulated DNA damage in mammary epithelium varies among strains of rodents and women

Abstract Background: Estrogens play an important role in breast development, but prolonged exposures and higher levels have been associated with increased risk of breast cancer. Recently, estrogens and environmental estrogenic chemicals have been shown to promote the formation of estrogen receptor-mediated co-transcriptional R-loops that lead to DNA double strand breaks in breast cancer cell lines. However, the DNA damage mediated by the estrogen receptors in the breast epithelium may vary among individuals. Experimental Design: Strains of mice and rats were used to investigate the genetic basis for variation in estrogen-induced DNA damage. The BALB/c and C57BL/6 strains of mice were selected as they differ in susceptibility to mammary tumors (Majhi et al., 2021). Similarly, the ACI and BN strains of rats differ in their susceptibility to mammary tumors induced by estrogen exposure. The mice and rats were treated with 17β-estradiol (E2) for up to 28 days, then tissues were analyzed for γH2AX foci to assess the presence of DNA double strand breaks. Normal breast tissues from average-risk and high-risk patients were maintained in culture (patient-derived explant cultures or PDEs) and exposed to either control media or supplemented with 10nM E2, then analyzed for γH2AX foci. Results: Proliferative responses were similar in both BALB/c and C57BL/6 mouse strains. However, DNA damage and R loops were significantly higher in the mammary epithelium of BALB/c mice whereas only background levels were detected in C57BL/6 mice. The highest level of γH2AX foci was found colocalized in nuclei of cells that were positive for estrogen receptor alpha (ERα+). The E2-stimulated γH2AX in BALB/c mice was not associated with differences in estrogen receptor (Esr1) level or transcriptional responses in target genes Pgr, Areg or expression of Apobec3. Similarly, DNA damage was increased significantly in the mammary epithelium of mammary tumor-susceptible ACI rats treated with E2, but not in the tumor-resistant BN strain. The effect of E2 was also examined in humans using breast explant cultures. Levels of E2-induced γH2AX were significantly higher in tissues from donors undergoing prophylactic mastectomy due to familial risk compared to tissues from average risk women undergoing reduction mammoplasty. Among average risk donors, significant increases in γH2AX by E2 treatment was observed in only 1 of five donors. Conclusions: These data demonstrate that E2-induced DNA damage varies among strains of rodents and women. The levels of E2-stimulated DNA damage can provide a valuable diagnostic tool to identify subsets of individuals for whom estrogen exposure may be pathogenic in breast epithelial cells due to impaired DNA double strand break repair due to inherited risk alleles or epigenetic alterations. Citation Format: Karen A. Dunphy, Aman Sharma, Prabin Majhi, Sallie S Schneider, Giovanna M Crisi, Grace Makari-Judson, D Joseph Jerry. Estrogen-stimulated DNA damage in mammary epithelium varies among strains of rodents and women [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-07-10.

Asymmetric development of the male mouse mammary gland and its response to a prenatal or postnatal estrogen challenge

The CD-1 mouse mammary gland is sexually dimorphic, with males lacking nipples. Recent studies have revealed that the underlying epithelium in the male mammary gland is sensitive to estrogenic environmental chemicals. In ongoing investigations, we observed asymmetric morphology in the left and right male mouse mammary glands. Here, we quantified these asymmetries in the embryonic, prepubertal, pubertal and adult male mammary gland. We found that the right gland was typically larger with more branching points compared to the left gland. We next evaluated the response of the left and right glands to 17α-ethinyl estradiol (EE2) after perinatal or peripubertal exposures. We found that the right gland was more responsive to EE2 than the left at both periods of exposure. These results reveal novel aspects of male mammary gland biology and suggest that future studies should control for laterality in the evaluation of hazards associated with exposures to estrogenic chemicals.

Inhibitor of Differentiation-3 and Estrogenic Endocrine Disruptors: Implications for Susceptibility to Obesity and Metabolic Disorders.

The rising global incidence of obesity cannot be fully explained within the context of traditional risk factors such as an unhealthy diet, physical inactivity, aging, or genetics. Adipose tissue is an endocrine as well as a metabolic organ that may be susceptible to disruption by environmental estrogenic chemicals. Since some of the endocrine disruptors are lipophilic chemicals with long half-lives, they tend to bioaccumulate in the adipose tissue of exposed populations. Elevated exposure to these chemicals may predispose susceptible individuals to weight gain by increasing the number and size of fat cells. Genetic studies have demonstrated that the transcriptional regulator inhibitor of differentiation-3 (ID3) promotes high fat diet-induced obesity in vivo. We have shown previously that PCB153 and natural estrogen 17β-estradiol increase ID3 expression. Based on our findings, we postulate that ID3 is a molecular target of estrogenic endocrine disruptors (EEDs) in the adipose tissue and a better understanding of this relationship may help to explain how EEDs can lead to the transcriptional programming of deviant fat cells. This review will discuss the current understanding of ID3 in excess fat accumulation and the potential for EEDs to influence susceptibility to obesity or metabolic disorders via ID3 signaling.

Responsiveness of four gender-specific genes, figla, foxl2, scp3 and sox9a to 17α-ethinylestradiol in adult rare minnow Gobiocypris rarus

Proteins encoded by figla, foxl2, scp3 and sox9a play important roles in gonad differentiation and reproduction. In the present study, we aimed to determine the responsiveness of figla, foxl2, scp3 and sox9a to 17α-ethinylestradiol (EE2) in the gonads of adult Gobiocypris rarus. Full-length cDNAs of figla, scp3 and sox9a were cloned and characterized by RT-PCR and RACE methods. Expression patterns in adult tissues were investigated. Results indicated that figla was predominantly expressed in adult ovaries and scp3 was restrictively expressed in the male testes and sox9a was principally expressed in the brains of both genders and the testes of males. Gene expression profiles of figla, foxl2, scp3 and sox9a were analyzed in the gonads of adult G. rarus exposed to EE2 at 1, 5, 25, and 125ng/L for 3 and 6days. Three-day EE2 treatment at 1–125ng/L all caused a significant increase of figla transcript in testes and foxl2 transcript in ovaries. However, six-day EE2 exposure at 1–125ng/L repressed figla and scp3 transcript in testes and foxl2 transcript in ovaries. The present study indicates that the testicular transcripts of figla and scp3 in males and the ovarian foxl2 transcript in females have high responsiveness to EE2 and they can be used as sensitive molecular biomarkers for early warning to monitor the environmental estrogenic chemicals in fresh water environment. The present study also suggests that the effective EE2 dosage for feminization in male G. rarus might be at least 25ng/L.

Chronic Oxidative Stress Increases Growth and Tumorigenic Potential of MCF-7 Breast Cancer Cells

Accumulating evidence suggests that exposures to elevated levels of either endogenous estrogen or environmental estrogenic chemicals are associated with breast cancer development and progression. These natural or synthetic estrogens are known to produce reactive oxygen species (ROS) and increased ROS has been implicated in both cellular apoptosis and carcinogenesis. Though there are several studies on direct involvement of ROS in cellular apoptosis using short-term exposure model, there is no experimental evidence to directly implicate chronic exposure to ROS in increased growth and tumorigenicity of breast cancer cells. Therefore, the objective of this study was to evaluate the effects of chronic oxidative stress on growth, survival and tumorigenic potential of MCF-7 breast cancer cells. MCF-7 cells were exposed to exogenous hydrogen peroxide (H2O2) as a source of ROS at doses of 25 µM and 250 µM for acute (24 hours) and chronic period (3 months) and their effects on cell growth/survival and tumorigenic potential were evaluated. The results of cell count, MTT and cell cycle analysis showed that while acute exposure inhibits the growth of MCF-7 cells in a dose-dependent manner, the chronic exposure to H2O2-induced ROS leads to increased cell growth and survival of MCF-7 cells. This was further confirmed by gene expression analysis of cell cycle and cell survival related genes. Significant increase in number of soft agar colonies, up-regulation of pro-metastatic genes VEGF, WNT1 and CD44, whereas down-regulation of anti-metastatic gene E-Cadherin in H2O2 treated MCF-7 cells observed in this study further suggests that persistent exposure to oxidative stress increases tumorigenic and metastatic potential of MCF-7 cells. Since many chemotherapeutic drugs are known to induce their cytotoxicity by increasing ROS levels, the results of this study are also highly significant in understanding the mechanism for adaptation to ROS-induced toxicity leading to acquired chemotherapeutic resistance in breast cancer cells.

Effect of endogenous and exogenous hormones on testicular cancer: the epidemiological evidence

Testicular cancer is the most common type of malignancy in men aged 15-40 years. Although its incidence has increased over the past 40 years in most countries, the reasons for this rise are unclear. It has been suggested that a relative excess of endogenous estrogens during prenatal life and/or later exposures to various occupational and environmental estrogenic chemicals such as organochlorine compounds may play a causal role in the etiology of testicular cancer, but the issue is still open to further research. The purpose for this review is to summarize the epidemiologic literature about hormonal factors, endogenous hormones and environmental xenoestrogens, and testicular carcinogenesis. Future studies need to (a) consider the possible synergistic effect of exposure to environmental xenoestrogens and sex hormones, (b) focus on the most vulnerable life stages of exposure to endocrine disruptors and testicular cancer risk, (c) assess the possible additive role of androgen secretion occurring during puberty in tumor progression, and (d) consider more systematically gene-environment interactions.

Dietary intake of 17α-ethinylestradiol promotes leukocytes infiltration in the gonad of the hermaphrodite gilthead seabream

A wide variety of chemicals discharged from industrial and municipal sources have been reported to disrupt the endocrine system of animals, which may be exposed via the food chain and contaminated water. 17α-Ethinylestradiol (EE2), a drug used in oral contraceptives and hormone replacement therapy, has a widespread presence in the aquatic environment. Current knowledge on the sensitivity of marine fish to estrogenic environmental chemicals is limited. We report here the effects of dietary intake of EE2 on gilthead seabream, a marine hermaphrodite teleost, focusing on the immune events that take place in the gonad. When seabream males were fed with 5, 50, 125 and 200μg EE2/g food for 7, 14, 21 and 28days an infiltration of acidophilic granulocytes and B lymphocytes occurred in the testis as the same time that spermatogenesis is disrupted. Moreover, the dietary intake of EE2 promoted a dose-dependent up-regulation of the expression of genes coding for cytokines, chemokines and adhesion molecules correlated with a leukocyte infiltration.

Permanent Oviduct Posteriorization after Neonatal Exposure to the Phytoestrogen Genistein

Background: Preimplantation embryo loss during oviduct transit has been observed in adult mice after a 5-day neonatal exposure to the phytoestrogen genistein (Gen; 50 mg/kg/day).Objective: We investigated the mechanisms underlying the contribution of the oviduct to infertility.Methods: Female mice were treated on postnatal days 1–5 with corn oil or Gen (50 mg/kg/day). We compared morphology, gene expression, and protein expression in different regions of the reproductive tracts of Gen-treated mice with those of control littermates at several time points.Results: Neonatal Gen treatment resulted in substantial changes in expression of genes that modulate neonatal oviduct morphogenesis, including Hoxa (homeobox A cluster), Wnt (wingless-related MMTV integration site), and hedgehog signaling genes. An estrogen receptor antagonist blocked these effects, indicating that they were induced by the estrogenic activity of Gen. Oviducts of adults treated neonatally with Gen had abnormal morphology and were stably “posteriorized,” as indicated by altered Hoxa gene patterning during the time of treatment and dramatic, permanent up-regulation of homeobox genes (e.g., Pitx1, Six1) normally expressed only in the cervix and vagina.Conclusions: Neonatal exposure to estrogenic environmental chemicals permanently disrupts oviduct morphogenesis and adult gene expression patterns, and these changes likely contribute to the infertility phenotype.

Exposure to Environmental Estrogenic Chemicals and Breast Cancer

The human population is now exposed on a daily basis to a multitude of environmental pollutant chemicals that would not have been present a century ago, and many of these chemicals have been detected in the human breast. The fatty nature of human breast tissue makes it a particular target for lipophilic as well as hydrophilic pollutant chemicals, which may enter the human body through oral, respiratory, or dermal routes. These chemicals possess a range of endocrine-disrupting properties and genotoxic activity, but from a breast cancer perspective the greatest concern has centered around their ability to mimic or interfere with the action of estrogen. The breast is an endocrine target organ and exposure to estrogen is a known risk factor for breast cancer.

Redox sensitive Pyk2 as a target for therapeutics in breast cancer

Breast cancer progression is dependent on the formation of new blood vessels that not only help the tumor by supplying additional nutrients, but also allow cancer cells to spread from the breast to distant sites in the body. Several studies suggest a positive correlation between new vessel formation and estrogens. Estrogenic environmental chemicals such as PCBs have been shown to increase the expression of factors known to promote vessel formation in breast tumors. These studies highlight a growing concern that women exposed to estrogenic environmental compounds may be more susceptible to either aggressive metastatic tumors or a high recurrence of breast cancer. Our concept offers a fundamental new understanding of the way the environment contributes to breast cancer progression. This review will be focused on a highly novel Pyk2 signaling complex as a target for therapy of estrogen dependent breast tumor angiogenesis. A better understanding of the role of Pyk2 signaling in estrogen dependent tumor vascularization may lead to the development of a new therapy against aggressive breast cancer using small molecule inhibitors of Pyk2.

High estrogen concentrations in receiving river discharge from a concentrated livestock feedlot

Environmental estrogenic chemicals interrupt endocrine systems and generate reproductive abnormalities in wildlife, especially natural and synthetic estrogenic steroid hormones such as 17β-estradiol (E2), estrone (E1), estriol (E3), 17α-ethynylestradiol (EE2), and diethylstilbestrol (DES). Concentrated animal feedlot operations (CAFOs) are of particular concern since large amounts of naturally excreted estrogens are discharged into aquatic environments. This study investigated E2, E1, E3, EE2, and DES with high performance liquid chromatography/tandem mass (HPLC–MS/MS) analyses along Wulo Creek in southern Taiwan, near a concentrated livestock feedlot containing 1,030,000 broiler chickens, 934,000 laying hens, 85,000 pigs, and 1500 cattle. Sampling was performed from December 2008 to May 2009, in which 54 samples were collected. Experimental results indicate that concentrations of EE2 were lower than the limit of detection (LOD), and concentrations of DES were only detected twice. Concentrations ranged from 7.4 to 1267 ng/L for E1, from not detected (ND) to 313.6 ng/L for E2, and from ND to 210 ng/L for E3. E1 had the highest average mass fraction (72.2 ± 3.6%), which was significantly higher than E3 (16.2 ± 1.7%) and E2 (11.5 ± 2.6%). Additionally, the mean E2 equivalent quotient (EEQ) ranged from 17.3 to 137.9 ng-E2/L. Despite having a markedly lower concentration than E1, E2 more significantly contributed (52.4 ± 6.0%) EEQ than E1 (19.7 ± 3.5%). Moreover, the concentrations of E2, E1, and E3 upstream were significantly higher than concentrations downstream, suggesting a high attenuation effect and fast degradation in the study water. Most concentrations in winter season were higher than those of spring season due to the low dilution effect and low microbial activity in the winter season. Based on the results of this study, we recommend further treatment of the wastewater discharge from the feedlot.

Good Laboratory Practices and Safety Assessments: Another View

In a letter responding to an article by Myers et al. (2009), Becker et al. (2009) claimed that industry’s Good Laboratory Practices (GLP)-compliant studies are superior to traditional academic peer-review in predicting the risk of toxic agents. I have read almost 30,000 experimental, etiologic, and epidemiologic papers (most in part), and it is evident that industry GLP studies do not report the same risks of a chemical when published in peer-reviewed studies from academia. This may be explained by biases in industry experiments and epidemiology, especially in design, due to the financial interests of industry sponsors—some receiving billions of dollars in revenue per chemical each year. For pharmaceuticals, dozens of published reviews show a strong correlation between industry sponsorship and findings of safety; I know of four such strong correlations in studies of industrial chemical risks (Bekelman et al. 2003; Fagin and Lavelle 1999; Swaen and Meijers 1988; vom Saal and Hughes 2005). Becker et al. (2009) relied on a commentary by a former editor at the Nature research journals (Jennings 2006) to claim that peer-review gives inferior data compared with GLP studies. Actually, Jennings (2006) wrote about improving, not abandoning, peer review. He presented data showing that the long-term value of scientific papers in neuroscience (judged by experts) correlates with the quality of the journals in which they were published (based on impact factor). That is a cardinal finding because industry supports various journals and their scientific associations, but their GLP studies are rarely published in high-quality journals (again, based on my readings). Evidently, industry’s GLP data are not reliable enough to publish, while financial independence of authors and editors, as well as peer review, are markers of good quality data. Since the widespread experimental testing frauds at Industrial Bio-Test Laboratories (Schneider 1983) and Craven Laboratories (U.S. Environmental Protection Agency 1994), which generated the GLP reforms, industry has issued oceans of GLP-compliant studies for submission to regulatory agencies. Few are submitted for publication, but almost all (in my experience) are submitted to journals that publish many industry-sponsored studies. Critically, industry and their regulatory agencies took the opportunity proferred by the requirement to comply with GLP to exclude almost all academic high-quality, non-GLP studies from risk assessments of existing chemicals (and the toxicity of new agents are primarily evaluated by the parties who want to sell it). For existing chemicals, I have always found that the effective toxicity doses in regulatory (GLP) studies are higher than those in the peer-reviewed literature, for several end points. It is important for individuals who value the contributions that science makes to society (reliable data)—or those who are cautious about toxicity of low-dose and cocktail agents that may affect biochemical signals, especially during development—to continue lobbying public agencies to incorporate academia’s peer-reviewed studies and to use disclosure of financial interests to give appropriate credence to industry’s data in chemical risk assessments. I also call on independent academics to be less competitive and make their methods and data more freely available.

Gene expression profile of endothelial cells exposed to estrogenic environmental compounds: Implications to pulmonary vascular lesions

Aims The cardiovascular system is an important target of estrogenic compounds. Considering the recent studies that question previously reported cardio-protective effects of estrogen, there is a growing concern that estrogenic environmental compounds may contribute to the pathology of vascular lesion formation. Main methods Real-time quantitative PCR was used to monitor the expression of genes involved in vascularization. Using Bayesian network modeling, we determined a gene network that estrogenic chemicals modulate in human vascular endothelial cells. Key findings We showed that planar and coplanar polychlorinated biphenyls (PCBs) induce the expression of different genes compared to estradiol. Non-planar PCB congener 153 induced NOTCH3 which is a new finding as well as CCL2 and IL8 similar to what has been reported by other non-planar PCBs in endothelial cells. Our gene network indicated that experimental treatments signal a network containing TGF-β receptor and NOTCH3; molecules biologically relevant to signaling pulmonary vascular lesions. Significance We report in the present study that exposure of vascular endothelial cells to environmentally relevant concentrations of estrogenic PCBs induce gene networks implicated in the process of inflammation and adhesion. Our data suggest that PCBs can promote vascular lesion formation by activating gene networks involved in endothelial cell adhesion, cell growth, and pro-inflammatory molecules which were different from natural estrogen. Since inflammation and adhesion are a hallmark in the pathology of endothelial cell dysfunction, reconstructing gene networks provide insight into the potential mechanisms that may contribute to the vascular risks associated with estrogenic environmental chemicals.

Development of a rapid screening and surveillance for estrogenic chemicals in environment based on recombinant yEGFP yeast cell

A recombinant yEGFP gene yeast strain necessary for the routine screening of estrogen activity in the chemical product of the environment was described. Two plasmids, one containing human estrogen receptor (hER) cDNA fused to the GPD gene promoter and another yEGFP inserted under the control of ERE, were constructed. The use of hER cDNA and the yEGFP reporter in the yeast cell sensor resulted in estrogenic chemical product-dependent light emission of yEGFP without additions owing its advantages: a simple and reagent-free measurement of GFP, and a non-toxic protein characteristic. The time needed for the optimal induction of light emission was 4h. The maximal fold induction of 8.80 over uninduced levels at the concentration of 10−5M of bisphenol A and 0.1nM sensitivities for four different estrogenic chemicals tested were obtained. Five different chemicals which could not bind to hER did not cause an induction of yEGFP. This bioassay can be performed completely in 96-well plates. Thus, this test system can be used as a rapid screening system for the surveillance of estrogenic chemical products in the environment. The yEGFP assay is sensitive, reproducible, and cheap, which makes it highly suitable to be used as a high throughput system.

Female sexual behavior, estrous cycle and gene expression in sexually dimorphic brain regions after pre- and postnatal exposure to endocrine active UV filters

The developing female brain represents a potential target for estrogenic environmental chemicals because it depends on estrogen but is exposed to low endogenous estrogen levels, thus facilitating competition by exogenous estrogen receptor (ER) agonists. We investigated effects of two estrogenic UV filters, 4-methylbenzylidene camphor (4-MBC) and 3-benzylidene camphor (3-BC). 4-MBC has been detected in human milk, indicating potential exposure of fetus and infant. The two chemicals were administered in chow to rats of the parent generation before mating, during pregnancy and lactation, and to their offspring until adulthood. Female sexual behavior was recorded on videotape in adult female offspring on proestrus evening at the beginning of the dark phase. 4-MBC (7 and 24 mg/kg bw/day) and 3-BC (2.4 and 7 mg/kg bw/day) reduced proceptive behavior (jump and ear wiggling) and receptive behavior (lordosis quotient), and increased rejection behavior towards the male. Estrous cycles were not affected by 4-MBC but disturbed by 3-BC. mRNAs encoding for genes involved in female sexual behavior, ERα, ERβ, progesterone receptor (PR) and steroid receptor coactivator-1 (SRC-1), were measured by real-time RT-PCR in ventromedial hypothalamic nucleus (VMH) and medial preoptic area of adult male and female offspring (studied in diestrus) after pre- and postnatal exposure to 3-BC (0.24, 0.7, 2.4 and 7 mg/kg bw/day). Gene expression was affected in a sex- and region-specific manner. PR mRNA in female VMH was reduced to male levels at dose levels of 2.4 and 7 mg/kg bw/day 3-BC. Our data demonstrate that female sexual behavior represents a sensitive target of endocrine disrupters and point to an involvement of PR in VMH.

Estrogen and environmental estrogenic chemicals exert developmental effects on rat hypothalamic neurons and glias

We investigated effects of 17β-estradiol ( E 2) and endocrine disrupters, nonylphenol (NP) and bisphenol-A (BPA), focusing on the neuronal development in cultures of fetal rat hypothalamic cells. We applied different concentrations of E 2, NP or BPA to the cultured hypothalamic cells and observed their effects on dendritic and synaptic development by immunocytochemistry using anti-microtubule associated protein-2 (MAP2) and anti-synapsin I antibodies, respectively. Administration of E 2 for 7 days affected MAP2-positive area as well as synapsin I-positive area. NP and BPA also influenced neuronal developments. The significant increase both in MAP2- and synapsin I-positive areas was observed at 10 and/or 100 nM of them, while 1 μM of them reduced the positive areas. Synaptic densities calculated from synapsin I-positive area/MAP2-positive area were not constant among different doses of three chemicals, but increased at 10 and/or 100 nM and decreased at 1 μM. Furthermore, immunostaining of NP-treated cells with the antibody against glial fibrillary acidic protein (GFAP) revealed that glial development was similarly influenced by NP. Therefore, the present results demonstrated that not only E 2 but also the environmental estrogenic chemicals, NP and BPA, affect development of fetal rat hypothalamic cells in vitro.

EXPRESSION OF THE ESTROGEN RECEPTOR ALPHA GENE IN THE ANAL FIN OF JAPANESE MEDAKA, ORYZIAS LATIPES, BY ENVIRONMENTAL CONCENTRATIONS OF BISPHENOL A

The anal fin in Japanese medaka, Oryzias latipes, is a typical sexual secondary character. In the present study, we focused on this organ and examined the effects of low doses of a natural estrogen, 17beta-estradiol (E(2)), and an environmental xenoestrogen, bisphenol A (BPA), in vivo by monitoring estrogen receptor (ER) alpha gene expression. Groups of adult male and female medaka were immersed in 10(-9) M E(2) or 10(-10) to 10(-8) M BPA and the levels of ERalpha gene transcripts in the anal fins were measured by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). One day of treatment with each concentration of BPA examined and 10(-9) M E(2) increased the levels of ERalpha mRNA in female anal fins by 3-fold as compared with controls. In the male specimens, neither 10(-9) M E(2) nor 10(-10) M BPA showed remarkable effects on the anal fins as compared with the results in females, but 10(-9) and 10(-8) M BPA increased the levels of ERalpha mRNA by 2.3- and 3.3-fold with 1 day of exposure, respectively. The present results showed that medaka anal fins may be a sensitive bio-indicator for screening of environmental estrogenic chemicals.

Modulation of Coactivator Recruitment by Cooperative Ligand Binding to Human Estrogen Receptor .ALPHA. and .BETA.

Estrogen receptor (ER) is a member of the nuclear receptor superfamily, and functions as a ligand-dependent transcription factor with roles in cell growth and differentiation. In addition to endogenous estrogen, 17beta-estradiol (E(2)) and artificial antagonists, many suspected environmental estrogenic chemicals are reported to bind to ER, with various affinities and transcriptional responses. ER is also an allosteric protein and shows a positive cooperative interaction with E(2). Cooperativity affects inter-subunit interaction, and while ligand-bound ER interacts with coactivators, antagonist-bound ER does not. We therefore hypothesized that ligand-binding characteristics influence coactivator recruitment to the ER dimer, and thereby affect transcriptional activity. We investigated the interaction between ER and human Steroid Receptor Coactivator-1 (SRC-1), in the presence of compounds exhibiting various Hill coefficients. In the case of both ER subtypes (ERalpha and ERbeta), the Hill coefficients of the compounds tested correlated with the affinity of the ER-ligand complex to SRC-1, with the exception of ERbeta-4-n-nonylphenol and ER-antagonist complexes. This is the first report to investigate the relationship between Hill coefficients of ligand binding and coactivator interaction with the ER-ligand complex. We also examined the proteolytic digestion of ER using trypsin, in the presence and absence of compounds with various Hill coefficients, to investigate ligand-dependent conformational changes in ER. We used not only agonists and antagonists but also compounds of weak biological activity (partial agonists). Our results shed light on the subtle modulation of transcriptional activation by chemical agents.