RESUMO
Estrogens and progestins are widely used in combination in human medicine and both are present in aquatic environment. Despite the joint exposure of aquatic wildlife to estrogens and progestins, very little information is available on their combined effects. In the present study we investigated the effect of ethinylestradiol (EE2) and Levonorgestrel (LNG), alone and in mixtures, on the expression of the brain specific ER-regulated cyp19a1b gene. For that purpose, recently established zebrafish-derived tools were used: (i) an in vitro transient reporter gene assay in a human glial cell line (U251-MG) co-transfected with zebrafish estrogen receptors (zfERs) and the luciferase gene under the control of the zebrafish cyp19a1b gene promoter and (ii) an in vivo bioassay using a transgenic zebrafish expressing GFP under the control of the zebrafish cyp19a1b gene promoter (cyp19a1b-GFP). Concentration-response relationships for single chemicals were modeled and used to design the mixture experiments following a ray design. The results from mixture experiments were analyzed to predict joint effects according to concentration addition and statistical approaches were used to characterize the potential interactions between the components of the mixtures (synergism/antagonism). We confirmed that some progestins could elicit estrogenic effects in fish brain. In mixtures, EE2 and LNG exerted additive estrogenic effects both in vitro and in vivo, suggesting that some environmental progestin could exert effects that will add to those of environmental (xeno-)estrogens. Moreover, our zebrafish specific assays are valuable tools that could be used in risk assessment for both single chemicals and their mixtures.
Assuntos
Aromatase/genética , Encéfalo/efeitos dos fármacos , Estrogênios/farmacologia , Etinilestradiol/farmacologia , Levanogestrel/farmacologia , Progestinas/farmacologia , Proteínas de Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Bioensaio , Encéfalo/metabolismo , Linhagem Celular , Interações Medicamentosas , Embrião não Mamífero , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/genética , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Luciferases/genética , Luciferases/metabolismo , Peixe-ZebraRESUMO
This study was investigated to evaluate the effect of intra-hippocampal injection of the nandrolone on spatial learning task in rats. The drug or vehicle was manually injected into the hippocampus with a 10-µl Hamilton syringe attached via polyethylene tubing to 27-gauge stainless-steel injection cannula. After 6 days of recovery, learning behaviors were evaluated using an 8-arm radial maze. The results showed that intra-hippocampal injection of nandrolone can impair trained spatial learning at a dose of 5 µl. We also observed a dense cytoplasm and nucleus in CA1 neurons as well as signs of necrosis. Nandrolone can impair the time required to reach the baited arm as well as the frequency of successful arm entries. At the 10 µl dose of nandrolone, neural hypertrophy and increased dentate gyrus volume were also observed.
Assuntos
Anabolizantes/toxicidade , Aprendizagem em Labirinto/efeitos dos fármacos , Memória/efeitos dos fármacos , Nandrolona/toxicidade , Anabolizantes/administração & dosagem , Animais , Giro Denteado/efeitos dos fármacos , Giro Denteado/patologia , Hipocampo/metabolismo , Hipertrofia , Injeções , Masculino , Nandrolona/administração & dosagem , Neurônios/efeitos dos fármacos , Neurônios/patologia , Ratos , Ratos WistarRESUMO
PURPOSE: To compare demographics, severity, and activity of thyroid eye disease (TED) in patients with hyperthyroidism (Hr-TED) vs primary hypothyroidism (Ho-TED). PATIENTS AND METHODS: In a cross-sectional study, demographics, complete eye examination, severity score (NOSPECS, total hundred eye score), clinical activity score, and Rundle grading were recorded for patients with TED and different thyroid disorders referred from an endocrinology clinic from 2003 to 2006. RESULTS: TED was clinically found in 303 patients (303/851, 35.6%). The majority of them (280/303, 92.4%) had Graves' hyperthyroidism and 23 (23/303, 7.5%) had primary hypothyroidism. Mean age, gender, mean severity score, mean activity score, Rundle grade, unilateral presentation of TED, smoking habit, mean duration of eye disease, and mean interval time of thyroid to TED were not significantly different between the two groups (0.06
Assuntos
Oftalmopatia de Graves/fisiopatologia , Hipertireoidismo/fisiopatologia , Hipotireoidismo/fisiopatologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Adulto JovemRESUMO
Several methods have been developed to evaluate and quantify the effects of Endocrine disruptor chemicals (EDC). Nevertheless, most of these methods are time-consuming or not enough sensitive to detect EDC at the environmental range. To link the biological effect of tested EDC to natural protein secretion, we have developed a new screening method based on the secretion of the cytokine CXCL12 (or SDF-1, Stroma-cell Derived Factor 1), which plays a capital role in cell survival and migration. We have demonstrated that CXCL12 secretion is regulated by estrogenic compounds in a dose-dependent way in ER-positive breast cancer cell lines (MCF-7 and T47D). By combining cell culture and ELISA test, we used this up-regulation of CXCL12 secretion to test several major environmental contaminants. Our results showed that 17ß-estradiol (from 10(-11) M), 17α-ethynylestradiol (from 10(-12) M), genistein (from 10(-8) M) and bisphenol A (from 10(-6) M) dose-regulate CXCL12 secretion in T47D. In contrast, antiestrogens, raloxifen and 4-hydroxytamoxifen, had no effect on the CXCL12 secretion, but were able to inhibit E2 effect. Moreover, we used cell proliferation assays to evaluate the effect of these different compounds on the growth of T47D cells. We found strong correlation (P = 0.7) between proliferation and CXCL12 secretion. However CXCL12 secretion was as sensitive as cell proliferation assays but appeared more rapid. Thus, this bioassay named CXCL-test (for Checking Xeno-estrogen activity by CXCL12 secretion in breast cancer cell Lines) constitutes a fast and sensitive method for the detection of estrogenic compounds allowing in 14 h to achieve a detection limit of 10(-11) M of E2 (2.7 ng/L).
Assuntos
Quimiocina CXCL12/metabolismo , Monitoramento Ambiental/métodos , Poluentes Ambientais/toxicidade , Moduladores de Receptor Estrogênico/toxicidade , Estrogênios/toxicidade , Compostos Benzidrílicos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Estradiol/toxicidade , Etinilestradiol/toxicidade , Feminino , Genisteína/toxicidade , Humanos , Fenóis/toxicidade , Cloridrato de Raloxifeno/toxicidade , Tamoxifeno/análogos & derivados , Tamoxifeno/toxicidade , Fatores de TempoRESUMO
Early expression of estrogen receptors (esr) and their role in regulating early expression of cyp19a1b encoding brain aromatase were examined in the brain of zebrafish. Using in toto hybridization and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), a significant increase in the expression of esr1, esr2a, and esr2b was observed between 24 and 48 hours postfertilization (hpf). In toto hybridization demonstrated that esr2a and esr2b, but not esr1, are found in the hypothalamus. Using real-time RT-PCR, an increase in cyp19a1b mRNAs occurs between 24 and 48 hpf, indicating that expression of cyp19a1b is temporally correlated with that of esr. This increase is blocked by the pure anti-estrogen ICI182,780. Furthermore, E2 treatment of cyp19a1b-GFP (green fluorescent protein) transgenic embryos results in appearance of GFP expression in the brain as early as 25 hpf. These results indicate that basal expression of cyp19a1b expression in the brain of developing zebrafish most likely relies upon expression of esr that are fully functional before 25 hpf.
Assuntos
Aromatase/metabolismo , Encéfalo , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Receptores de Estrogênio/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Aromatase/genética , Encéfalo/embriologia , Encéfalo/enzimologia , Embrião não Mamífero/anatomia & histologia , Embrião não Mamífero/enzimologia , Estradiol/análogos & derivados , Estradiol/metabolismo , Antagonistas de Estrogênios/metabolismo , Fulvestranto , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Estrogênio/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
To investigate the prevalence of smoking, asthma and asthma-related symptoms in relation to age, sex and smoking behaviour in Urmia, we carried out a cross-sectional study in 2004 using the International Union Against Tuberculosis and Lung Disease questionnaire, which includes some questions on smoking. We surveyed 2987 adults aged 20-44 years. Prevalence of wheeze, breathlessness with wheezing, asthma attack, use of asthma medication and nasal allergy were 4.8%, 3.3%, 1.2%, 1.1%, and 16.0% respectively. Current smokers comprised 16.4% of participants. Prevalence of asthma symptoms was significantly greater in smokers than non-smokers. Rates for asthma diagnosis and asthma medication were lower than the European Community Respiratory Health Survey median.
Assuntos
Asma/epidemiologia , Fumar/epidemiologia , Saúde da População Urbana/estatística & dados numéricos , Adulto , Distribuição por Idade , Asma/diagnóstico , Asma/tratamento farmacológico , Asma/etiologia , Distribuição de Qui-Quadrado , Estudos Transversais , Dispneia/epidemiologia , Feminino , Inquéritos Epidemiológicos , Humanos , Irã (Geográfico)/epidemiologia , Masculino , Vigilância da População , Prevalência , Hipersensibilidade Respiratória/epidemiologia , Sons Respiratórios/etiologia , Distribuição por Sexo , Fumar/efeitos adversosRESUMO
In contrast to other vertebrates, in which the adult brain shows limited adult neurogenesis, teleost fishes exhibit an unparalleled capacity to generate new neurons as adults, suggesting that their brains present a highly permissive environment for the maintenance and proliferation of adult progenitors. Here, we examine the hypothesis that one of the factors permitting establishment of this favourable environment is estradiol. Indeed, recent data showed that radial glial cells strongly expressed one of two aromatase duplicated genes. Aromatase is the estrogen-synthesizing enzyme and this observation is of great interest, given that radial glial cells are progenitor cells capable of generating new neurons. Given the well-documented roles of estrogens on cell fate, and notably on cell proliferation, these data suggest that estradiol could be involved in maintaining and/or activating these progenitors. Examination of recent data in birds and mammals suggests that the situation in fish could well be an exaggeration of a more general mechanism implicating estrogens in neurogenesis. Indeed, there is accumulating evidence that estrogens are involved in embryonic, adult or reparative neurogenesis in other vertebrates, notably in mammals.
Assuntos
Encéfalo/citologia , Estrogênios/biossíntese , Peixes/anatomia & histologia , Células-Tronco/metabolismo , Animais , Evolução Biológica , Proliferação de Células , Neurônios/fisiologiaRESUMO
To investigate the prevalence of smoking, asthma and asthma-related symptoms in relation to age, sex and smoking behaviour in Urmia, we carried out a cross-sectional study in 2004 using the International Union Against Tuberculosis and Lung Disease questionnaire, which includes some questions on smoking. We surveyed 2987 adults aged 20-44 years. Prevalence of wheeze, breathlessness with wheezing, asthma attack, use of asthma medication and nasal allergy were 4.8%, 3.3%, 1.2%, 1.1%, and 16.0% respectively. Current smokers comprised 16.4% of participants. Prevalence of asthma symptoms was significantly greater in smokers than non-smokers. Rates for asthma diagnosis and asthma medication were lower than the European Community Respiratory Health Survey median
Assuntos
Fumar , Prevalência , Estudos Transversais , Inquéritos e Questionários , Distribuição por Idade , Distribuição por Sexo , Sinais e Sintomas Respiratórios , AsmaRESUMO
AIM: To investigate the effect of vitamin E on oxidative stress status in the small intestine of diabetic rats. METHODS: Twenty-four male Wistar rats were randomly divided into three groups: Control (C), non-treated diabetic (NTD) and vitamin E-treated diabetic (V(E)TD) groups. The increases in lipid peroxidation, protein oxidation and superoxide dismutase (SOD) in these three groups was compared after 6 wk. RESULTS: There was no significant difference in catalase activity between NTD and control rats. Compared to NTD rats, the treatment with vitamin E significantly decreased lipid peroxidation and protein oxidation, and also increased catalase activity and SOD. CONCLUSION: The results revealed the occurrence of oxidative stress in the small intestine of diabetic rats. Vitamin E, as an antioxidant, attenuates lipid peroxidation and protein oxidation, and increases antioxidant defense mechanism.
Assuntos
Antioxidantes/farmacologia , Diabetes Mellitus Experimental/fisiopatologia , Intestino Delgado/fisiopatologia , Estresse Oxidativo/efeitos dos fármacos , Vitamina E/farmacologia , Animais , Catalase/metabolismo , Intestino Delgado/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Distribuição Aleatória , Ratos , Ratos Wistar , Estreptozocina , Superóxido Dismutase/metabolismoRESUMO
The present study was designed to evaluate the effect of Vitamin E (Vit. E) on diabetes-induced changes in small intestine, lipid peroxidation and plasma antioxidant capacity in rats. Twenty-four rats were divided into three groups (n=8), namely control, non-treated diabetic (NTD) and Vit. E-treated diabetic (VETD) groups. The VETD group received 300 mg of Vit. E daily in drinking water. After 6 weeks, the length and weight of small intestine, villus height, crypt depth and muscular layer thickness showed a significant increase in the NTD group compared to the control group. In the VETD group, these parameters did not show any significant difference compared to the control group. The level of malondialdehyde (MDA) in the red blood cells showed a signficant increase in the NTD group, but not in the VETD group, compared to the control group. The plasma antioxidant capacity showed a significant increase in VETD compared to the NTD group. These findings indicate that Vit. E significantly improved small intestinal changes in diabetic rats and that these effects could be mediated at least in part by enhanced plasma antioxidant capacity and reduced lipid peroxidation (AU)
Se estudia en el presente trabajo el efecto de la vitamina E sobre los cambios debidos a la diabetes en la estructura intestinal, peroxidación lipídica y capacidad antioxidante del plasma en la rata. Se utilizaron 24 animales, divididos en tres grupos (n=8): control, diabéticos no tratados (NTD) y diabéticos tratados con Vit. E (VETD). El grupo VETD recibió 300 mg de Vit. E diariamente en el agua de bebida. Tras 6 semanas, se observó aumento significativo en el grupo NTD de la longitud y peso del intestino delgado, espesor de la capa muscular, altura de las vellosidades y profundidad de las criptas respecto del grupo control. En cambio, en el grupo VETD, esos parámetros no se modificaron significativamente respecto de los controles. De modo similar, el nivel de malondialdehido (MDA) en los eritrocitos aumentó significativamente respecto del control en el grupo NTD y no en el VETD. La capacidad antioxidante del plasma mostró un significativo incremento en el grupo VETD respecto del control y NTD. Estos hechos indican que la vitamina E mejora las alteraciones intestinales debidas a la diabetes y que los efectos podrían estar parcialmente mediados por la disminución de la peroxidación lipídica y el aumento de la capacidad antioxidante del plasma (AU)
Assuntos
Animais , Ratos , Vitamina E/farmacocinética , Peroxidação de Lipídeos , Estresse Oxidativo , Complicações do Diabetes/tratamento farmacológico , Estudos de Casos e Controles , Diabetes Mellitus Experimental/fisiopatologia , Intestino Delgado/fisiopatologia , Malondialdeído/sangue , Antioxidantes/farmacocinética , Substâncias Protetoras/farmacocinética , Modelos Animais de DoençasRESUMO
Age-related cataract is an ever-increasing health problem among the elderly population worldwide. In recent years, there has been speculation that the levels of micronutrients in ocular tissue may play a role in the pathogenesis of cataract, especially zinc, since it is found in high concentrations in the eye tissues, including the aqueous humor. 44 subjects diagnosed with cataract were chosen for study and matched with 21 healthy controls to determine the concentration of zinc in aqueous humor. The findings revealed that a significantly higher concentration of zinc was present in the aqueous humor of our study population compared to controls (p < 0.001). These findings, in agreement with several previous studies, amplify the need for further investigation to determine if these higher levels are in actuality a consequence of the disease or a factor in the formation of cataract.
Assuntos
Humor Aquoso/química , Catarata/metabolismo , Zinco/análise , Envelhecimento/fisiologia , Estudos de Casos e Controles , Catarata/etiologia , Feminino , Nível de Saúde , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
The present study was designed to evaluate the effect of Vitamin E (Vit. E) on diabetes-induced changes in small intestine, lipid peroxidation and plasma antioxidant capacity in rats. Twenty-four rats were divided into three groups (n=8), namely control, non-treated diabetic (NTD) and Vit. E-treated diabetic (VETD) groups. The VETD group received 300 mg of Vit. E daily in drinking water. After 6 weeks, the length and weight of small intestine, villus height, crypt depth and muscular layer thickness showed a significant increase in the NTD group compared to the control group. In the VETD group, these parameters did not show any significant difference compared to the control group. The level of malondialdehyde (MDA) in the red blood cells showed a significant increase in the NTD group, but not in the VETD group, compared to the control group. The plasma antioxidant capacity showed a significant increase in VETD compared to the NTD group. These findings indicate that Vit. E significantly improved small intestinal changes in diabetic rats and that these effects could be mediated at least in part by enhanced plasma antioxidant capacity and reduced lipid peroxidation.
Assuntos
Antioxidantes/farmacologia , Complicações do Diabetes/tratamento farmacológico , Complicações do Diabetes/metabolismo , Intestino Delgado/metabolismo , Vitamina E/farmacologia , Animais , Antioxidantes/metabolismo , Complicações do Diabetes/patologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Radicais Livres/metabolismo , Intestino Delgado/patologia , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Ratos , Ratos Wistar , Vitamina E/sangueRESUMO
We have previously cloned and characterized three estrogen receptors (ER) in the zebrafish (zfERalpha, zfERbeta1 and zfERbeta2). We have also shown that they are functional in vitro and exhibit a distinct expression pattern, although partially overlapping, in the brain of zebrafish. In this paper, we have shown that the hepatic expression of these zfER genes responds differently to estradiol (E2). In fact, a 48-h direct exposure of zebrafish to E2 resulted in a strong stimulation of zfERalpha gene expression while zfERbeta1 gene expression was markedly reduced and zfERbeta2 remained virtually unchanged. To establish the potential implication of each zfER in the E2 upregulation of the zfERalpha gene, the promoter region of this gene was isolated and characterized. Transfection experiments with promoter-luciferase reporter constructs together with different zfER expression vectors were carried out in different cell contexts. The data showed that in vivo E2 upregulation of the zfERalpha gene requires ERalpha itself and a conserved transcription unit sequence including at least an imperfect estrogen-responsive element (ERE) and an AP-1/ERE half site at the proximal transcription initiation site. Interestingly, although in the presence of E2 zfERalpha was the most potent at inducing the expression of its own gene, the effect of E2 mediated by zfERbeta2 represented 50% of the zfERalpha activity. In contrast, zfERbeta1 was unable to upregulate the zfERalpha gene whereas this receptor form was able to tightly bind E2 and activate a reporter plasmid containing a consensus ERE. Altogether, these results indicated that the two ERbeta forms recently characterized in teleost fish could have partially distinct and not redundant functions.
Assuntos
Estradiol/metabolismo , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Sequência de Bases , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/genética , Regulação da Expressão Gênica , Humanos , Fígado/fisiologia , Dados de Sequência Molecular , Mutação , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética , Alinhamento de Sequência , Sítio de Iniciação de Transcrição , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
In oviparous species, in addition to a full-length estrogen receptor alpha (ER alpha), another ER alpha isoform lacking the A domain and exhibiting a ligand-independent transactivation function has been consistently reported. Although both isoforms are expressed in the liver, their respective sites of expression in other potential target tissues are unknown. In contrast to the situation in Xenopus and chicken, the two isoforms of rainbow trout (Oncorhynchus mykiss) are generated from two classes of transcripts with different 5' untranslated sequences issued from the same gene by alternative splicing and promoter usage. The aim of this study was to take advantage of the unique organization of the rainbow trout ER alpha gene to investigate the tissue distribution of these two messenger species along the reproductive axis of female trout. The S1 nuclease assay and in situ hybridization were used, with probes specific for each of the transcripts. Reverse transcription polymerase chain reaction (RT-PCR) with primers specific for each of the isoforms also was performed. The data indicated that the full-length ER alpha is expressed in liver, brain, pituitary, and ovary, whereas expression of the isoform with the truncated A domain is restricted to the liver, demonstrating a tissue-specific expression of these two ER alpha isoforms. The presence of a short liver-specific isoform in oviparous species suggests its role in the development and/or maintenance of the unique function of the liver in the vitellogenesis process.
Assuntos
Expressão Gênica , Oncorhynchus mykiss , Especificidade de Órgãos , Receptores de Estrogênio/genética , Animais , Autorradiografia , Receptor alfa de Estrogênio , Feminino , Hibridização In Situ , Fígado/química , Masculino , Ovário/química , Hipófise/química , Prosencéfalo/química , Isoformas de Proteínas/genética , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Endonucleases Específicas para DNA e RNA de Cadeia SimplesRESUMO
The transcriptional activity of ERalpha (or NR3A1) after binding of ligand is mediated through synergistic action between activation functions (AFs) AF-1 and AF-2 and the transcriptional machinery. This is functionally achieved by bridging coactivators such as CEBP binding protein/p300 and members of the p160 subfamily such as steroid receptor coactivator protein-1 (SRC-1). We previously identified a conserved potential alpha-helical structure within the AF-1 functional core, and by evaluating point mutants of human ERalpha (hERalpha) within this region, we show that in transfection experiments this structure is required for synergism between SRC-1 and hERalpha. We report that the transcriptional synergism between AF-1 mutants and SRC-1 was abolished in AF-1-sensitive cells such as HepG2, whereas it was reduced by 50% in CHO-K1 cells, which have a mixed context that is sensitive to both the AF-1 and AF-2 regions of hERalpha. Glutathione-S-transferase pulldown assays demonstrate that the AF-1 core is able and sufficient for the hERalpha N-terminal region to interact with SRC-1. Interestingly, an enhancement of this recruitment in the presence of the hERalpha ligand-binding domain was observed, which was found to be dependent on a direct interaction between the N-terminal B domain and the ligand-binding domain. Another functional consequence of this physical interaction, which is promoted by both partial and full agonists of hERalpha, was an increase in the phosphorylation state of the N-terminal domain. Binding of 4-hydroxytamoxifen (OHT) to the hERalpha C-terminal region induced a functional AF-1 conformation in vitro through this N- and C-terminal interaction. The involvement of an SRC-1-mediated pathway in transactivation mediated by hERalpha AF-1 was further substantiated by transfection experiments using the OHTresponsive human C3 promoter, which showed that OHT-induced hERalpha AF-1 activity was enhanced by SRC-1 and required the AF-1 alpha-helical structure. In conclusion, we demonstrate that the synergism between AF-1 and AF-2 is mediated in part by a cooperative recruitment of SRC-1 by both the AF-1 alpha-helical core and AF-2 regions and that it is stabilized by a direct interaction between the B and C-terminal domains. This interaction of SRC-1 with the AF-1 alpha-helical core is essential for both E2- and OHT-induced ERalpha activity.
Assuntos
Estradiol/análogos & derivados , Receptores de Estrogênio/química , Receptores de Estrogênio/metabolismo , Tamoxifeno/análogos & derivados , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Coenzima A/metabolismo , Cricetinae , RNA Helicases DEAD-box , Estradiol/metabolismo , Estradiol/farmacologia , Antagonistas de Estrogênios/metabolismo , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio , Histona Acetiltransferases , Humanos , Ligantes , Dados de Sequência Molecular , Mutação , Coativador 1 de Receptor Nuclear , Fosforilação , Alcamidas Poli-Insaturadas , Conformação Proteica , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , RNA Helicases/genética , RNA Helicases/metabolismo , Coelhos , Receptores de Estrogênio/genética , Tamoxifeno/metabolismo , Transativadores/química , Transativadores/genética , Fatores de Transcrição/genéticaRESUMO
Estrogenic potency of 4-n-nonylphenol diethoxylate, 4-n-nonylphenol (NP) and metabolites were tested using two bioassays: rainbow trout hepatocyte culture and recombinant yeast stably expressing rainbow trout estrogen receptor (rtER) and containing estrogen-dependent reporter genes. Since NP was the only compound active in both systems, its interaction with rtER was studied in more detail. Qualitative and quantitative differences were observed in the presence of 17beta-estradiol (E2) or NP when estrogen-dependent promoters containing one to three estrogen-responsive elements were used in yeast. Moreover, limited proteolysis of rtER after E2 or NP binding presented different patterns after SDS-PAGE analysis suggesting that NP induces a differential conformation of rtER compare to E2. This finding may have important implications with respect to the biological activity of NP. Thus, the effects of NP on the activation of an E2-dependent gene and on sexual differentiation were assessed on all-male trout embryos exposed to NP for 1 h per day for 10 days. Although in situ hybridization demonstrated that E2, and to a lesser extend NP, were able to increase rtER mRNA level in the liver of embryos, no indication of total or partial sexual reversion was observed (even in E2 treated fishes) when the gonads were examined 8 months after hatching.
Assuntos
Fenóis/toxicidade , Receptores de Estrogênio/efeitos dos fármacos , Diferenciação Sexual/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Animais , Feminino , Masculino , Oncorhynchus mykiss , Conformação Proteica , Receptores de Estrogênio/químicaRESUMO
Three in-vitro bioassays were used to compare the oestrogenic potency of chemicals used as growth promoter in beef cattle in certain non-European Union countries (17beta-oestradiol, alpha-zearalanol, testosterone, trenbolone, trenbolone acetate, melengestrol acetate) or found as food contaminant such as the mycotoxin zearalenone and some of their metabolites (17alpha-oestradiol, oestrone, 17alpha-epitestosterone, 19-nortestosterone, androstendione, zearalanone, alpha-zearalanol, beta-zearalanol, alpha-zearalenol, beta-zearalenol). The strong oestrogens 17alpha-ethinyl oestradiol and diethylstilboestrol were used as standards. The first bioassay was based on the activation of a reporter gene by oestrogens in recombinant yeast expressing human or rainbow trout oestrogen receptor. In the second bioassay, the vitellogenin gene induction of rainbow trout hepatocyte cultures was used as a biomarker for the exposure to oestrogens. The third bioassay was based on the alkaline phosphatase gene induction by oestrogens in the human endometrial Ishikawa cell line. The assessment of oestrogenic potency of these chemicals clearly demonstrates the strong oestrogenicity of the mycotoxin zearalenone and its metabolites and particularly alpha-zearalenol which was as potent as ethinyl oestradiol and diethylstilboestrol in the human endometrial Ishikawa cell line.
Assuntos
Estrogênios/farmacologia , Substâncias de Crescimento/farmacologia , Fosfatase Alcalina/genética , Animais , Bovinos , Células Cultivadas , Citocromos c1/genética , DNA Recombinante , Dietilestilbestrol/farmacologia , Relação Dose-Resposta a Droga , Estradiol/farmacologia , Etinilestradiol/farmacologia , Expressão Gênica/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Acetato de Melengestrol/farmacologia , Oncorhynchus mykiss/genética , Receptores de Estrogênio/genética , Proteínas Recombinantes de Fusão , Saccharomyces cerevisiae , Testosterona/farmacologia , Acetato de Trembolona/análogos & derivados , Acetato de Trembolona/farmacologia , Vitelogeninas/genética , Zeranol/farmacologia , beta-Galactosidase/análise , beta-Galactosidase/genéticaRESUMO
Here, we describe a rapid, convenient, and quantitative beta-galactosidase assay in liquid culture of recombinant yeast that expresses the estrogen receptor. This assay allows large-scale screening of chemicals (more than 600 samples/day) for the evaluation of their direct estrogenic potency and accurate determination of their EC50 with minimal manipulations. The assay, which is based on digestion of the yeast cell wall by lyticase (zymolase), a beta-glucanase isolated from Arthrobacter luteus, followed by a hypoosmotic shock lysis, is performed completely in 96-well plates. This protocol for using recombinant yeast with the two-hybrid technology significantly advances recombinant yeast manipulation.
Assuntos
Estrogênios/farmacologia , Expressão Gênica , Receptores de Estrogênio/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , beta-Galactosidase/análise , Animais , Permeabilidade da Membrana Celular , Clorofórmio/farmacologia , Estradiol/farmacologia , Estrona/farmacologia , Etinilestradiol/farmacologia , Glucana Endo-1,3-beta-D-Glucosidase/farmacologia , Humanos , Complexos Multienzimáticos/farmacologia , Oncorhynchus mykiss/genética , Peptídeo Hidrolases/farmacologia , Proteínas Recombinantes de Fusão , Saccharomyces cerevisiae/enzimologia , Dodecilsulfato de Sódio/farmacologia , Transfecção , beta-Galactosidase/genéticaRESUMO
This study aims to characterise Prolactin receptor (PRLR) in rainbow trout for which no information is available despite the availability of Salmonid PRL preparations. By screening a freshwater rainbow trout intestine cDNA library with a probe corresponding to the extracellular domain (ECD) of tilapia PRLR, we have cloned a 2.5 kb insert coding for the PRLR. The mature protein of 614 amino acid residues is similar to PRLR isolated in tilapia and also the long form of mammalian PRLR. Analysis of PRLR gene expression in osmoregulatory organs revealed the presence of a unique transcript, thus confirming the involvement of this hormone in the control of osmoregulation in this fish species. By using surface plasmon resonance (SPR) technology, kinetic measurement of interaction between trout PRL and its receptor ECD was studied. This approach allowed us to demonstrate the formation of a transient, unstable homodimeric complex. This unstability could explain the inability to perform binding experiments using homologous PRL. In contrast, heterologous lactogenic ligands were able to interact through a more stable complex. Whether these characteristics of PRL-receptor interaction in rainbow trout are different to what occurs in tilapia where a homologous radioreceptor assay was developed would require further studies.
Assuntos
Oncorhynchus mykiss/metabolismo , Prolactina/metabolismo , Receptores da Prolactina/metabolismo , Animais , Sequência de Bases , Dimerização , Cinética , Dados de Sequência Molecular , Oncorhynchus mykiss/genética , Ligação Proteica , Estrutura Terciária de Proteína , RNA/metabolismo , Receptores da Prolactina/genética , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Ressonância de Plasmônio de Superfície , Distribuição Tecidual , Equilíbrio HidroeletrolíticoRESUMO
Transcriptional activation by the estrogen receptor (NR3A1, or ER) requires specific ligand-inducible activation functions located in the amino (AF-1) and the carboxyl (AF-2 and AF-2a) regions of the protein. Although several detailed reports of ER structure and function describe mechanisms whereby AF-2 activates transcription, less precise data exist for AF-1. We recently reported that the rainbow trout and human estrogen receptors (rtERs and hERs, respectively), two evolutionary distant proteins, exhibit comparable AF-1 activities while sharing only 20% homology in their N-terminal region. These data suggested that the basic mechanisms whereby AF-1 and the ER N-terminal region activate transactivation might be evolutionary conserved. Therefore, a comparative approach between rtER and hER could provide more detailed information on AF-1 function. Transactivation analysis of truncated receptors and Gal4DBD (DNA binding domain of the Gal4 factor) fusion proteins in Saccharomyces cerevisiae defined a minimal region of 11 amino acids, located at the beginning of the B domain, necessary for AF-1 activity in rtER. Hydrophobic cluster analysis (HCA) indicated the presence of a potential alpha-helix within this minimal region that is conserved during evolution. Both rtER and hER sequences corresponding to this potential alpha-helical structure were able to induce transcription when fused to the Gal4DBD, indicating that this region can transactivate in an autonomous manner. Furthermore, point mutations in this 11-amino acid region of the receptors markedly reduced their transcriptional activity either within the context of a whole ER or a Gal4DBD fusion protein. Data were confirmed in mammalian cells and, interestingly, ERs with an inverted alpha-helix were as active as their corresponding wild-type proteins, indicating a conserved role in AF-1 for these structures. Moreover, using two naturally occurring rtER N-terminal variants possessing or not the A domain (rtER(L) and rtER(S), respectively), together with A domain-truncated hER and chimeric rtER/hER receptors, we demonstrated that the A domain of the ER plays an inhibitory role in ligand-independent activity of the receptor. In vitro and in vivo protein-protein interaction assays using both rtER and hER demonstrated that this repression is likely to be mediated by a ligand-sensitive direct interaction between the A domain and the C-terminal region of the ER.