RESUMO
Pyrethroid pesticides are frequently used for household insect control of insects and in agriculture and livestock. Flumethrin is a pyrethroid that is used against ectoparasites in many animals. The goal of this study was to evaluate the cytotoxic, apoptotic, genotoxic, and estrogenic effects of flumethrin on the mammalian breast cancer cell line (MCF-7). Compared with control groups, a dose-dependent decrease was observed in cell viability at concentrations of 100 µM and higher. The cytotoxic and apoptotic effects detected by LDH assay and AO/EtBr staining increased significantly at a concentration of 1000 µM. The expression of BCL2, which is an anti-apoptotic gene, significantly decreased, whereas BAX, TP53, and P21 expression significantly increased. The results of a comet assay indicated that flumethrin significantly changed tail length, tail % DNA, tail moment, and Olive tail moment in concentrations above 1 and 10 µM. In addition, a 0.1 µM concentration of flumethrin affected ERα receptor mediated cell proliferation and increased transcription of estrogen-responsive pS2 (TFF1) and progesterone receptor (PGR) genes. As a result, flumethrin-induced apoptosis and cytotoxicity at a high concentration, while induced genotoxicity even at lower concentrations. Flumethrin is an endocrine disrupting insecticide with estrogenic effects at very low concentrations.
Assuntos
Neoplasias da Mama/genética , Disruptores Endócrinos/toxicidade , Estrogênios/toxicidade , Mutagênicos/toxicidade , Piretrinas/toxicidade , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/genética , Dano ao DNA , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Proteínas Proto-Oncogênicas c-bcl-2/genética , Fator Trefoil-1/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
Estrogen has an important role in regulating glucose homeostasis, and existing evidence indicates that it might be involved in the development of hyperglycemia in pregnancy. It mediates its effect through estrogen receptors including the nuclear receptor ERß encoded by ESR2. The association between the ESR2 polymorphism rs1256031 and GDM susceptibility has not been investigated yet. This study aimed to evaluate the relationship between rs1256031 and GDM risk in Chinese population. A total of 241 GDM patients and 139 healthy pregnant women were recruited for this study. The rs1256031 genotype was examined by time-of-flight mass spectrometry and the association between rs1256031 and GDM susceptibility was assessed by binary logistic regression in three different genetic models. The polymorphism rs1256031 was not associated with GDM susceptibility in additive [OR (95% CI) = 0.871 (0.453,1.675); P = 0.680], dominant [OR (95% CI) = 0.908 (0.495,1.665); P = 0.755] or recessive [OR (95% CI) = 0.912 (0.591,1.408); P = 0.677] models after adjusting for confounding factors. We observed no association between the polymorphism rs1256031 in the ESR2 gene and GDM susceptibility in Chinese pregnant women.
Assuntos
Diabetes Gestacional , Receptor beta de Estrogênio/genética , Adulto , Povo Asiático/genética , China/epidemiologia , Diabetes Gestacional/diagnóstico , Diabetes Gestacional/etnologia , Diabetes Gestacional/genética , Estrogênios/metabolismo , Feminino , Predisposição Genética para Doença , Humanos , Hiperglicemia/metabolismo , Polimorfismo de Nucleotídeo Único , GravidezRESUMO
BACKGROUND: Non-functioning pituitary adenomas (NFPAs) are common pituitary tumors, and surgery is generally the only treatment option. Few attempts have been made to explore target molecules for the development of NFPA pharmacological treatments. METHOD: We quantitatively assessed the expression profiles of estrogen receptor (ER) transcripts and proteins in NFPA samples, using reverse transcription-digital polymerase chain reaction (RT-dPCR) and immunohistochemistry, and further investigated the correlations between the expression levels of ER and those of downstream responsive genes. All patients had undergone surgery at the same high-volume hospital. A total of 20 patients with NFPAs were included. All patients were new-onset, and none were diagnosed with intratumoral hemorrhages or cysts. RESULTS: NFPA samples exhibited a bimodal ESR1 expression pattern and were categorized into significantly different high- and low-ESR1 expression level groups (P < 0.05). In contrast, expression levels of ESR1 variants and ESR2 could barely be detected. Similar results were obtained through the immunohistochemical staining of NFPAs, using well-validated antibodies against ERs. The expression levels of ESR1 positively correlated with those of GREB1, an estrogen-responsive gene [correlation coefficient (r) = 0.623, P = 0.003]. CONCLUSIONS: ESR1 expression levels in NFPAs exhibited a bimodal pattern and were positively correlated with GREB1 expression levels. The accurate assessment of ER expression levels may further advance future NFPA-related research.
Assuntos
Adenoma/patologia , Biomarcadores Tumorais/metabolismo , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Hipofisárias/patologia , Adenoma/genética , Adenoma/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/genética , Feminino , Seguimentos , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Hipofisárias/genética , Neoplasias Hipofisárias/metabolismo , Prognóstico , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
To assess the effect of exposure to persistent organic pollutants (POPs) on the estrogen receptor (ER) signaling pathway in Baikal seals (Pusa sibirica), we investigated the molecular characterizations and functions of two Baikal seal ER (bsER) isoforms, bsERα and bsERß. The bsERα and bsERß cDNA clones isolated have an open reading frame of 595 and 530 amino acid residues, respectively. The tissue distribution analyses of bsER mRNAs showed that bsERα transcripts were primarily found in the ovary and uterus, and bsERß in the muscle in wild Baikal seals. The immunofluorescence staining assay showed that 17ß-estradiol (E2) treatment promoted the nuclear translocation of in vitro-expressed bsERα. Transient transfection of bsERα in U2OS cells enhanced the transcription of pS2, an ER target gene of E2. We then measured bsER-mediated transactivation potencies of POPs in an in vitro reporter gene assay system, in which a bsERα or bsERß expression vector was transfected into COS-1â¯cells. For comparison, transactivation potencies of POPs on mouse ERs (mERα and mERß) were also evaluated in the same manner. Results showed significant dose-dependent responses of bsERs and mERs when treated with p,p'-dichlorodiphenyltrichloroethane (p,p'-DDT), and p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE). bsERs and mERs showed no response when exposed to polychlorinated biphenyls (PCBs) or 2,3,7,8-tetrachlorodibenzo-p-dioxin. Comparison of the dose-response curves of DDTs across species (bsERs vs. mERs) showed that bsERα had a response similar to mERα, but bsERß was less sensitive than mERß. Comparing the lowest observable effective concentrations of p,p'-DDT (2.8⯵M) and p,p'-DDE (10⯵M) for in vitro bsERα-mediated transactivation with their hepatic concentrations in wild Baikal seals indicated that some individuals accumulated these compounds at levels comparable to the effective concentrations, suggesting the potential disruption of the bsERα signaling pathway in the wild population by these compounds. Co-transfection experiments with bsER and the aryl hydrocarbon receptor (AHR) suggested that high accumulation of estrogenic compounds exerts a synergistic effect with dioxin-like congeners on ER signaling through AHR activation in the wild seal population.
Assuntos
Dioxinas e Compostos Semelhantes a Dioxinas/toxicidade , Poluentes Ambientais/toxicidade , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/genética , Ativação Transcricional/efeitos dos fármacos , Animais , Dioxinas e Compostos Semelhantes a Dioxinas/metabolismo , Poluentes Ambientais/metabolismo , Feminino , Camundongos , RNA Mensageiro/metabolismo , Focas VerdadeirasRESUMO
The presence of veterinary drug residues in foods and the environment could potentially cause adverse effects on humans and wildlife. Several veterinary drugs were reported to exhibit endocrine disrupting effects via binding affinities to sexual hormone receptors such as estrogen and androgen receptors. Therefore, we confirmed the human estrogen receptor (ER) agonistic/antagonistic effects of 135 chemicals that were used as veterinary drugs in Korea by the official Organization for Economic Cooperation and Development (OECD) in vitro ER transcriptional activation (TA) assay using the VM7Luc4E2 cell line. In the case of ER agonist screening, 7 veterinary drugs (cefuroxime, cymiazole, trenbolone, zeranol, phoxim, altrenogest and nandrolone) were determined to be ER agonists. In addition, only zeranol was found to exhibit weak ER antagonistic activity. These 7 veterinary drugs, which were determined as ER agonists and/or antagonists by an OECD in vitro assay, were also found to have binding affinity to ERs. These results indicate that various veterinary drugs possess potential (anti-)estrogenic effects. However, further study is needed to determine the precise endocrine-disrupting effects of these compounds.
Assuntos
Bioensaio , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio , Receptor beta de Estrogênio , Estrogênios/farmacologia , Drogas Veterinárias/farmacologia , Criação de Animais Domésticos , Animais , Aquicultura , Linhagem Celular , Receptor alfa de Estrogênio/agonistas , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/agonistas , Receptor beta de Estrogênio/antagonistas & inibidores , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Peixes , Humanos , Gado , Organização para a Cooperação e Desenvolvimento Econômico , Ativação Transcricional , TransfecçãoRESUMO
Most satiety-inducing obesity therapeutics, despite modest efficacy, have safety concerns that underscore the need for effective peripherally acting drugs. An attractive therapeutic approach for obesity is to optimize/maximize energy expenditure by increasing energy-utilizing thermogenic brown adipose tissue. We used in vivo and in vitro models to determine the role of estrogen receptor ß (ER-ß) and its ligands on adipose biology. RNA sequencing and metabolomics were used to determine the mechanism of action of ER-ß and its ligands. Estrogen receptor ß (ER-ß) and its selective ligand reprogrammed preadipocytes and precursor stem cells into brown adipose tissue and increased mitochondrial respiration. An ER-ß-selective ligand increased markers of tricarboxylic acid-dependent and -independent energy biogenesis and oxygen consumption in mice without a concomitant increase in physical activity or food consumption, all culminating in significantly reduced weight gain and adiposity. The antiobesity effects of ER-ß ligand were not observed in ER-ß-knockout mice. Serum metabolite profiles of adult lean and juvenile mice were comparable, while that of adult obese mice was distinct, indicating a possible impact of obesity on age-dependent metabolism. This phenotype was partially reversed by ER-ß-selective ligand. These data highlight a new role for ER-ß in adipose biology and its potential to be a safer alternative peripheral therapeutic target for obesity.-Ponnusamy, S., Tran, Q. T., Harvey, I., Smallwood, H. S., Thiyagarajan, T., Banerjee, S., Johnson, D. L., Dalton, J. T., Sullivan, R. D., Miller, D. D., Bridges, D., Narayanan, R. Pharmacologic activation of estrogen receptor ß increases mitochondrial function, energy expenditure, and brown adipose tissue.
Assuntos
Tecido Adiposo Marrom/metabolismo , Metabolismo Energético/fisiologia , Receptor beta de Estrogênio/agonistas , Receptor beta de Estrogênio/metabolismo , Isoquinolinas/farmacologia , Mitocôndrias/fisiologia , Tecido Adiposo Branco/fisiologia , Animais , Biomarcadores , Dieta Hiperlipídica , Receptor beta de Estrogênio/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Resistência à Insulina , Masculino , Camundongos , Camundongos Knockout , Obesidade/sangue , Obesidade/metabolismoRESUMO
Estrogen Receptor-ß (ERß) has been implicated in many cancers. In prostate and breast cancer its function is controversial, but genetic studies implicate a role in cancer progression. Much of the confusion around ERß stems from antibodies that are inadequately validated, yet have become standard tools for deciphering its role. Using an ERß-inducible cell system we assessed commonly utilized ERß antibodies and show that one of the most commonly used antibodies, NCL-ER-BETA, is non-specific for ERß. Other antibodies have limited ERß specificity or are only specific in one experimental modality. ERß is commonly studied in MCF-7 (breast) and LNCaP (prostate) cancer cell lines, but we found no ERß expression in either, using validated antibodies and independent mass spectrometry-based approaches. Our findings question conclusions made about ERß using the NCL-ER-BETA antibody, or LNCaP and MCF-7 cell lines. We describe robust reagents, which detect ERß across multiple experimental approaches and in clinical samples.
Assuntos
Anticorpos Antineoplásicos/farmacologia , Receptor beta de Estrogênio/imunologia , Mama/efeitos dos fármacos , Mama/metabolismo , Linhagem Celular Tumoral , Doxiciclina/farmacologia , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Indicadores e Reagentes , Masculino , Peptídeos , Próstata/efeitos dos fármacos , Próstata/metabolismo , Reprodutibilidade dos TestesRESUMO
The need for development and validation of in vitro hormone receptor transactivation assays as important alternative tools to study interactions with sex hormone receptors is outlined by international organisations, as such assays should be included in the OECD conceptual framework for the testing and assessment of endocrine active chemicals. Therefore as part of the European Union (EU)-sponsored 6th framework project ReProTect, the validation study with MELN cells, MCF-7 cells (ER+, estrogen receptor positive) which were stably transfected with the estrogen responsive gene ERE-betaGlob-Luc-SVNeo was set up. Standard operating procedures including a prescreen assay for unknown chemicals, an ER-agonist assay and an ER-antagonist assay were developed at the Flemish Institute for Technological Research, Belgium, and successfully transferred to Bayer Schering Pharma AG, Germany. Test results were obtained for 16 chemicals, and it was demonstrated that the MELN assay is transferable, robust and reproducible which allowed to rank chemical compounds according to their strong to weak affinity for the estrogen-alpha receptor, or identify negative chemicals within the test range up to 10(-5)M. Besides the screening for agonism, we demonstrated the suitability of MELN cells to test for antagonistic activity, which is of added value compared to current validated assays. As the MELN assay successfully passed the first modules of the ECVAM validation procedure, it now should be considered for further steps including the definition of a prediction model and application domain to get it accepted as an alternative screening assay, contributing to the 3R's with a reduction of animal experiments.
Assuntos
Alternativas aos Testes com Animais , Bioensaio/métodos , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor beta de Estrogênio/antagonistas & inibidores , Bioensaio/normas , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Antagonistas de Estrogênios/química , Receptor alfa de Estrogênio/agonistas , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/agonistas , Receptor beta de Estrogênio/genética , Humanos , Luciferases/genética , Ligação Proteica , Reprodutibilidade dos Testes , TransfecçãoRESUMO
Two 10-mer DNA probes, or one 20-mer DNA probe, respectively, hybridize with a 21-mer target DNA to form a vacancy or bulge opposite the target nucleotide. The former double-DNA-probe method and the latter bulge form method are applicable to the detection of single-nucleotide polymorphisms (SNPs). A small fluorescent dye enters into the vacancy or bulge and binds with a target nucleotide via a hydrogen bonding interaction, which causes fluorescence quenching. The interaction between fluorescent dye and the target nucleotide is confirmed by measuring the melting temperature and fluorescence spectra. The fluorescent dye, ADMND (2-amino-5,7-dimethyl-1,8-naphthyridine), is found to selectively bind with C over A or G. The methods proposed here are economic, convenient, and effective for the fluorescence detection of SNPs. Finally, the double-DNA-probe method and bulge form method are successfully applied to the detection of C/G and C/A mutations in the estrogen receptor 2 gene and progesterone receptor gene using ADMND.
Assuntos
Corantes Fluorescentes/química , Naftiridinas/química , Polimorfismo de Nucleotídeo Único , Citosina , Sondas de DNA , Receptor beta de Estrogênio/genética , Fluorescência , Mutação , Receptores de Progesterona/genética , Temperatura de TransiçãoRESUMO
Bone mineral density (BMD), an important risk factor for osteoporosis, is a complex trait likely affected by multiple genes. The linkage and/or association of 13 polymorphic loci of seven candidate genes (estrogen receptor alpha [ERalpha] and beta [ERbeta], calcium-sensing receptor, vitamin D receptor, collagen type 1alpha1, low-density lipoprotein [LDL] receptor-related protein 5 [LRPS], and transforming growth factor beta1) were evaluated in 177 southern Chinese pedigrees of 674 subjects, with each pedigree identified through a proband having a BMD Z score of -1.28 or less at the hip or spine. A suggestive linkage was detected between the IVS1-351A/G polymorphism of ERalpha and spine BMD, and between the 1082G/A, 1730G/A, and D14S1026 polymorphisms of ERbeta and BMD at both spine and hip. The quantitative transmission disequilibrium test (QTDT) detected total family association between 1730G/A of ERbeta and BMD at spine and hip; between D14S1026 of ERbeta and hip BMD; and between the 266A/G and 2220C/T polymorphisms of LRP5 and hip BMD. Similar total family associations were detected when only the females were analyzed. In addition, the IVS1-397T/C polymorphism of ERalpha was associated with spine BMD, and the 266A/G and 2220C/T polymorphisms of LRP5 were associated with femoral neck BMD in the females. A within-family association was detected with the IVS1-397T/C polymorphism of ERalpha, and the 266A/G and 2220C/T polymorphisms of LRP5 in the females. The effect of each polymorphism on BMD variance ranged from 1% to 4%. In conclusion, ERalpha, ERbeta and LRP5 are important candidate genes determining BMD variation, especially in females.