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
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
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
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
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 amino acid sequence of rainbow trout estrogen receptor (rtER) is highly conserved in the C domain but presents few similarities in the A/B and E domains with human estrogen receptor alpha (hER) [NR3A1]. A previous study has shown that rtER and hER have differential functional activities in yeast Saccharomyces cerevisiae. To determine the domain(s) responsible for these differences, chimeric human/rainbow trout estrogen receptors were constructed. The A/B, C/D or E/F regions of rtER were replaced by corresponding regions of hER and expressed in yeast cells. Ligand-binding and transcription activation abilities of these hybrid receptors were compared with those of wild-type rtER or hER. Surprisingly, our data revealed that the human C/D domains play an important role in the magnitude of transactivation of ER. Two other chimeric ERs carrying either a C or D domain of hER showed that the C domain was responsible for this effect whereas the D domain did not affect hybrid receptor activities. Moreover, a chimeric hER carrying the C domain of rtER showed maximal transcriptional activity similar to that observed with rtER. Gel shift assays showed that, whereas rtER and hER present a similar binding affinity to an estrogen response element (ERE) element, the rtER C domain is responsible for a weaker DNA binding stability compared to those of hER. In addition, the human C domain allows approximately 2 times faster association of ER to an ERE. Utilization of reporter genes containing one or three EREs confirms that rtER requires protein-protein interactions for its stabilization on DNA and that the C domain is involved in this stabilization. Moreover, AF-1 may be implicated in this synergistic effect of EREs. Interestingly, although E domains of these two receptors are much less conserved, replacement of this domain in rtER by its human counterpart resulted in higher estradiol sensitivity but no increase in the magnitude of transactivation. Data from the chimeric receptors, rtER(hC) and hER(rtC), demonstrated that rtER AF-1 and AF-2 activation domains activated transcription in the presence of estradiol similar to both AF-1 and AF-2 hER. This implies that these domains, which show poor sequence homology, may interact with similar basal transcription factors.
Assuntos
Oncorhynchus mykiss/genética , Receptores de Estrogênio/genética , Sequência de Aminoácidos , Animais , Ligação Competitiva , DNA Recombinante/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Dados de Sequência Molecular , Mutação , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Estrogênio/química , Receptores de Estrogênio/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Elementos de Resposta , Saccharomyces cerevisiae/genética , Ativação TranscricionalRESUMO
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 shown that charged amino acids near C530 of the human estrogen receptor (ER) are involved in receptor discrimination between estrogen and antiestrogen. We now examine the role of charged residues, adjacent to the three other cysteines (381, 417, and 447) in the hormone-binding domain of the human ER, in the hormone-binding, DNA-binding, and transcription activation abilities of the receptor. Mutation of the one charged amino acid nearest to C381 gave a mutant receptor (E380Q) requiring two to three times less estradiol (E2) than wild type (WT) ER to achieve maximal activity and having activity in the absence of added estrogen that was 6-fold higher than that of WT receptor. The enhanced ability of this mutant to bind to estrogen response element DNA in the absence and presence of estrogen may, at least in part, explain its elevated, seemingly constitutive trans-activation activity and its increased sensitivity to estrogen. While more sensitive to E2, this E380Q mutant was less sensitive than WT ER to antiestrogen for suppression of transcriptional activity. Mutation of all three charged residues nearest to C381 (the triple mutant D374N, E380Q, and E385Q) resulted in a greatly reduced potency of the receptor in trans-activation with no change in estrogen-binding affinity. When K449 (near C447), highly conserved among steroid receptors, was mutated to Q, 400-fold more E2 was required for maximal reporter gene trans-activation due to an unstable, temperature-sensitive hormone-receptor complex. In contrast, the mutant K416Q (near C417) was unaltered in E2-binding or receptor transcriptional activity. These studies reveal a region in the N-terminal portion of the hormone-binding domain (ca. amino acids 374-385) where alterations in charged residues result in either increases or decreases in receptor transcriptional activity with no change in receptor affinity for hormone. Our findings suggest that this region may be important in DNA binding and protein-protein interactions that modulate transcriptional activity of the ER. In addition, the region near C447, which is well conserved among steroid receptors, appears to be important in maintaining the receptor in a conformation that is stable at physiological (37 C) temperatures. To our knowledge, this is the first report of an ER (E380Q) with a sensitivity to E2 for trans-activation greater than that of WT receptor and having high trans-activation activity in the absence of added hormone.
Assuntos
Receptores de Estrogênio/química , Sequências Reguladoras de Ácido Nucleico , Ativação Transcricional , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Células CHO , Cricetinae , DNA/metabolismo , Estrogênios/metabolismo , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação Puntual , Ligação Proteica , Receptores de Estrogênio/genética , Receptores de Estrogênio/fisiologia , Proteínas Recombinantes de Fusão/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/metabolismo , TemperaturaRESUMO
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
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.
Assuntos
Receptores de Estrogênio/química , Receptores de Estrogênio/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Estradiol/metabolismo , Estradiol/farmacologia , Evolução Molecular , Células HeLa , Humanos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Receptores de Estrogênio/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Transcrição Gênica , Ativação Transcricional , Leveduras/genéticaRESUMO
The estrogen-binding region of the cDNA for chicken ER reveals a mRNA of 3.5 kilobases (kb) in rainbow trout liver. The level of this messenger, which is very low in the liver of naive male animals, can be increased by estrogen stimulation. With this chicken probe, we have isolated a clone from a lambda gt10 trout liver cDNA library. The partial cDNA sequence, which encompasses most of the coding region, shows two domains of striking amino acid homology with human, avian, and Xenopus estrogen receptors (ERs) (DNA binding region: 90%, Hormone binding region: 60%). With this specific probe rainbow trout ER, we detected another messenger (4.5 kb) that is less expressed than the 3.5 kb messenger. The kinetics of stimulation of the two messengers is compared with the kinetics of accumulation of vitellogenin mRNA after E2 administration. This report constitutes the first identification of ER mRNA from a fish.
Assuntos
Estradiol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/metabolismo , RNA Mensageiro/análise , Receptores de Estrogênio/genética , Salmonidae/metabolismo , Truta/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Galinhas , Sondas de DNA , Feminino , Masculino , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , RNA Mensageiro/biossíntese , Homologia de Sequência do Ácido Nucleico , Truta/genética , Vitelogeninas/genéticaRESUMO
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
A characteristic of all estrogen receptors (ER) cloned from fish to date is the lack of the first 37-42 N-terminal amino acids specific to the A domain. Here we report the isolation and characterization from trout ovary of a full-length complementary DNA (cDNA) clone encoding an N-terminal variant form of the rainbow trout ER (rtER). Sequence analysis of open reading frame of this cDNA predicts a 622-amino acid protein. The C-terminal region of this protein, from amino acid position 45 to the end, was very similar to the previously reported rtER (referred to as the short form, or rtER(S)). In contrast, this novel rtER cDNA (referred to as the long form, or rtER(L)) contains an additional in-frame ATG initiator codon that adds 45 residues to the N-terminal region of the protein. This new N-terminal region may represent the A domain of ER found in tetrapod species. The first 227 bp of this new cDNA were similar to the 3'-end intronic sequence of the rtER gene intron 1. These data together with S1 nuclease, primer extension, and RT-PCR experiments demonstrate that the rtER(L) represents a second isoform of rtER that arises from an alternative promoter within the first intron of the gene. Transcripts encoding both rtER forms were expressed in the liver. In vitro translation of the rtER(L) cDNA produced 2 proteins with molecular masses of 71 and 65 kDa, whereas rtER(S) cDNA produced 1 65-kDa protein. Interestingly, Western blot analysis with a specific antibody against the C-terminal region of rtER revealed 2 receptor forms of 65 and 71 kDa in trout liver nuclear extracts, in agreement with the presence of the 2 distinct classes of rtER messenger RNA in this tissue. Functional analysis of both rtER isoforms revealed that although rtER(S) consistently exhibited a basal (estrogen-independent) trans-activation activity that could be further increased in the presence of estrogens, the novel isoform rtER(L) is characterized by a strict estrogen-dependent transcriptional activity. These data suggest that the additional 45 residues at the N-terminal region of rtER(L) clearly modify the hormone-independent trans-activation function of the receptor.
Assuntos
Fígado/metabolismo , Ovário/metabolismo , Receptores de Estrogênio/genética , Truta/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar , Éxons , Feminino , Biblioteca Gênica , Dados de Sequência Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , Coelhos , Receptores de Estrogênio/química , Receptores de Estrogênio/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Reticulócitos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiaeRESUMO
We have cloned and sequenced the p53-encoding cDNA of rainbow trout (Salmo gairdneri). The encoded product contains the characteristics found in all p53 proteins: (i) the five highly conserved domains, (ii) an acidic N terminus, (iii) a hydrophilic C terminus, and (iv) a penultimate serine residue. Furthermore, we demonstrate that the rainbow trout p53 is able to specifically interact with the SV40 large T antigen.
Assuntos
Genes p53/genética , Truta/genética , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Animais , Antígenos Transformantes de Poliomavirus/metabolismo , Sequência de Bases , Evolução Biológica , Clonagem Molecular , Sondas de DNA/genética , Humanos , Dados de Sequência Molecular , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genéticaRESUMO
RT-PCR was undertaken on total RNA extracts from bone and scales of the rainbow trout, Oncorhynchus mykiss. The rainbow trout estrogen receptor (ER)-specific primers used amplified a single product of expected size from each tissue which, using Southern blotting, strongly hybridized with a 32P-labelled rtER probe under stringent conditions. These data provide the first in vivo evidence of ER mRNA in bone and scale tissues of rainbow trout and suggest that the effects of estrogen observed in this study (increased bone mineral and decreased scale mineral contents, respectively) may be mediated directly through ER.
Assuntos
Osso e Ossos/metabolismo , Cálcio/metabolismo , Estradiol/farmacologia , Oncorhynchus mykiss/metabolismo , Receptores de Estrogênio/biossíntese , Animais , Northern Blotting , Osso e Ossos/efeitos dos fármacos , Fosfatos/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro , Receptores de Estrogênio/genéticaRESUMO
A double immunocytochemical procedure, with two different chromogens, was used to compare the respective distributions of estrogen receptor-immunoreactive cells and gonadotrophin-releasing hormone-immunoreactive neurons on the same sections of the brains of adult male and female rainbow trout (Oncorhynchus mykiss). Estrogen receptor-immunoreactive cells were observed in the ventral and lateral telencephalon, the preoptic region, the mediobasal hypothalamus, and the ventromedial thalamic nucleus. Gonadotrophin-releasing hormone-immunoreactive perikarya were detected in the olfactory bulbs, the ventral telencephalon, the preoptic area, and the mediobasal hypothalamus. Double-staining studies showed that, although some estrogen receptor-positive cells were in close proximity to gonadotrophin-releasing hormone-immunoreactive perikarya, careful examination of 550 gonadotrophin-releasing hormone-positive cells from five adult females and two adult males failed to demonstrate any evidence that gonadotrophin-releasing hormone neurons coexpress estrogen receptor in the brain of the rainbow trout. The present study provides, for the first time in teleosts, morphological evidence that gonadotrophin-releasing hormone neurons do not represent major direct targets for estradiol, suggesting that the positive feedback effects of estradiol onto the gonadotrophin-releasing hormone system are likely to be conveyed via other cell populations.
Assuntos
Hormônio Liberador de Gonadotropina/metabolismo , Neurônios/metabolismo , Oncorhynchus mykiss/metabolismo , Receptores de Estrogênio/biossíntese , Animais , Autorradiografia , Encéfalo/citologia , Química Encefálica/fisiologia , Feminino , Imuno-Histoquímica , Hibridização In Situ , MasculinoRESUMO
In order to perform later studies on the transcriptional regulation of hormone-dependent genes in fish liver, we firstly examined the potential of trout liver nuclear extracts in a cell-free transcription system. As reporter genes, we used DNA sequences without G (G-free cassettes) under the control of three promoters derived from the 5' flanking sequence of the Xenopus vitellogenin B1 gene; two of them were responsive to the oestrogen receptor (ER) through oestrogen responsive elements (ERE). Maximal transcriptional activity was obtained within a range of 40-130 micrograms protein per extract depending on the extract preparation. Transcription was maximal in reactions carried out at 25 degrees C. Similar transcriptional activities for the three promoters were observed when transcription was performed in extracts from untreated male trout. In contrast, we observed a 4.5- to 6-fold increase in the transcription with ERE-containing promoters in comparison with that with the minimal promoter bearing only a TATA box when extracts from oestradiol-treated male trout were used. This effect was correlated with the increase in the nuclear ER concentration induced by in vivo hormonal treatment. This enhanced transcription was specifically inhibited by the addition of a 25- to 100-fold excess of ERE oligonucleotide competitor. These data demonstrated, therefore, that transcription was ERE-dependent in this system and suggest strongly that it was mediated by the trout ER. Addition of oestradiol or the anti-oestrogens hydroxytamoxifen or ICI 164384 had no effect on the transcriptional activity of the two ERE-containing promoters, indicating that transcription was hormone-independent in trout liver nuclear extracts.
Assuntos
Estradiol/farmacologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Oncorhynchus mykiss/genética , Transcrição Gênica/efeitos dos fármacos , Animais , Sequência de Bases , Ligação Competitiva , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Sistema Livre de Células , Primers do DNA/genética , Técnicas In Vitro , Ligantes , Masculino , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Oncorhynchus mykiss/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Receptores de Estrogênio/genética , XenopusRESUMO
Rainbow trout hepatocyte primary culture was used to test the influence of some xenobiotics on the expression of two genes implicated in reproduction, those for the estrogen implicated in reproduction, those for the estrogen receptor (ER) and vitellogenin (Vg). We showed that chlordecone, nonylphenol, a polychlorobiphenol (PCB) mixture (Aroclor 1245) and lindane were able to induce ER and Vg mRNA accumulation. Antiestrogens, 4-hydroxytamoxifen and ICI 164,384, prevented the effects of the xenobiotics, indicating that the induction of gene expression is mediated by the ER. Among these four xenobiotics, only chlordecone and nonylphenol were able to displace the binding of [3H]estradiol to ER-enriched COS-1 extracts, and to activate an estrogen-dependent reporter gene (ERE-TK-CAT) cotransfected with an expression vector containing ER cDNA. The results suggest that chlordecone and nonylphenol are direct inducers of rainbow trout ER and Vg gene expression, whereas PCBs and lindane act through their hepatic metabolites. Moreover, pentachlorophenol acts as an antagonist of the induction by estradiol of rainbow trout ER and Vg gene expression.
Assuntos
Expressão Gênica/efeitos dos fármacos , Fígado/metabolismo , Receptores de Estrogênio/biossíntese , Vitelogeninas/biossíntese , Xenobióticos/farmacologia , Actinas/biossíntese , Animais , Células Cultivadas , Cloranfenicol O-Acetiltransferase/biossíntese , Clordecona/farmacologia , Relação Dose-Resposta a Droga , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Hexaclorocicloexano/farmacologia , Cinética , Fígado/efeitos dos fármacos , Neoplasias Hepáticas Experimentais , Oncorhynchus mykiss , Fenóis/farmacologia , Bifenilos Policlorados/farmacologia , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Receptores de Estrogênio/metabolismo , Proteínas Recombinantes/biossíntese , Timidina Quinase/biossíntese , Transfecção , Células Tumorais CultivadasRESUMO
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.