RESUMO
No abstract provided.
Assuntos
Pneumonia Aspirativa , Acidente Vascular Cerebral , Humanos , Malásia , Saúde Bucal , Fatores de RiscoRESUMO
Concern that some chemicals in our environment may affect human health by disrupting normal endocrine function has prompted research on interactions of environmental contaminants with steroid hormone receptors. We compared the activity of 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), an estrogenic metabolite of the organochlorine pesticide methoxychlor, at estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta). Human hepatoma cells (HepG2) were transiently transfected with either human or rat ERalpha or ERbeta plus an estrogen-responsive, complement 3-luciferase construct containing a complement 3 gene promoter sequence linked to a luciferase reporter gene. After transfection, cells were treated with various concentrations of HPTE in the presence (for detecting antagonism) or absence (for detecting agonism) of 17beta-estradiol. HPTE was a potent ERalpha agonist in HepG2 cells, with EC50 values of approximately 5 x 10(-8) and 10(-8) M for human and rat ERalpha, respectively. In contrast, HPTE had minimal agonist activity with either human or rat ERbeta and almost completely abolished 17beta-estradiol-induced ERbeta-mediated activity. Moreover, HPTE behaved as an ERalpha agonist and an ERbeta antagonist with other estrogen-responsive promoters (ERE-MMTV and vtERE) in HepG2 and HeLa cells. This study demonstrates the complexity involved in determining the mechanism of action of endocrine-active chemicals that may act as agonists or antagonists through one or more hormone receptors.
Assuntos
Fenóis/farmacologia , Receptores de Estrogênio/efeitos dos fármacos , Animais , Carcinoma Hepatocelular , Antagonistas de Estrogênios/metabolismo , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio , Receptor beta de Estrogênio , Expressão Gênica , Células HeLa , Humanos , Neoplasias Hepáticas , Metoxicloro/metabolismo , Fenóis/metabolismo , Ratos , Receptores de Estrogênio/agonistas , Receptores de Estrogênio/antagonistas & inibidores , Receptores de Estrogênio/genética , Transfecção , Células Tumorais CultivadasRESUMO
The estrogenic activity of dieldrin, toxaphene, and an equimolar mixture of both compounds (dieldrin/toxaphene) was investigated in the 21-day-old B6C3F1 mouse uterus, MCF-7 human breast cancer cells, and in yeast-based reporter gene assays. Treatment of the animals with 17beta-estradiol (E2) (0.0053 kg/day x3) resulted in a 3.1-, 4.8-, and 7.8-fold increase in uterine wet weight, peroxidase activity, and progesterone receptor binding, respectively. In contrast, treatment with 2.5, 15 and 60 micromol/kg (x3) doses of toxaphene, dieldrin, or dieldrin/toxaphene (equimolar) did not significantly induce a dose-dependent increase in any of the E2-induced responses. The organochlorine pesticides alone and the binary mixture did not bind to the mouse uterine estrogen receptor (ER) in a competitive binding assay using [3H]E2 as the radioligand. In parallel studies, estrogenic activities were determined in MCF-7 cells by using a cell proliferation assay and by determining induction of chloramphenicol acetyl transferase (CAT) activity in MCF-7 cells transiently transfected with plasmids containing estrogen-responsive 5'-promoter regions from the rat creatine kinase B and human cathepsin D genes. E2 caused a 24-fold increase in CAT activity in MCF-7 cells transiently transfected with creatine kinase B and a 3.8-fold increase in cells transiently transfected with the human cathepsin D construct. Treatment of MCF-7 cells with dieldrin, toxaphene, or an equimolar mixture of dieldrin plus toxaphene (10(-8)-10(-5) M) did not significantly induce cell proliferation or CAT activity in the transient transfection experiment with both plasmids. The relative competitive binding of the organochlorine pesticides was determined by incubating MCF-7 cells with 10(-9) M [3H]E2 in the presence or absence of 2 x 10(-7) M unlabeled E2 (to determine nonspecific binding), toxaphene (10(-5) M), dieldrin (10(-5) M), and equimolar concentrations of the dieldrin plus toxaphene mixture (10(-5) M). The binding observed for [3H]E2 in the whole cell extracts was displaced by unlabeled E2, whereas the organochlorine pesticides and binary mixture exhibited minimal to nondetectable competitive binding activity. E2 caused a 5000-fold induction of beta-galactosidase (beta-gal) activity in yeast transformed with the human ER and a double estrogen responsive element upstream of the beta-gal reporter gene. Treatment with 10(-6)-10(-4) M chlordane, dieldrin, toxaphene, or an equimolar mixture of dieldrin/toxaphene did not induce activity, whereas 10(-4) M endosulfan caused a 2000-fold increase in beta-gal activity. Diethylstilbestrol caused a 20-fold increase in activity in yeast transformed with the mouse ER and a single estrogen responsive element upstream of the beta-gal reporter gene. Dieldrin, chlordane, toxaphene, and endosulfan induced a 1.5- to 4-fold increase in activity at a concentration of 2.5 x 10(-5) M. Synergistic transactivation was not observed for any equimolar binary mixture of the pesticides at concentrations of either 2.5 x 10(-5) M or 2.5 x 10(-4) M. The results of this study demonstrate that for several estrogen-responsive assays in the mouse uterus, MCF-7 human breast cancer cells, and yeast-based reporter gene assays, the activities of both dieldrin and toxaphene were minimal, and no synergistic interactions were observed with a binary mixture of the two compounds.
Assuntos
Dieldrin/farmacologia , Receptores de Estrogênio/análise , Toxafeno/farmacologia , Útero/efeitos dos fármacos , Animais , Ligação Competitiva , Neoplasias da Mama/metabolismo , Divisão Celular/efeitos dos fármacos , Dieldrin/administração & dosagem , Sinergismo Farmacológico , Estradiol/farmacologia , Feminino , Humanos , Camundongos , Peroxidase/metabolismo , Progesterona/metabolismo , Ratos , Toxafeno/administração & dosagem , Transfecção , Células Tumorais Cultivadas , Útero/metabolismoRESUMO
The ability of bisphenol A (BPA) to affect human estrogen receptor (ER) binding, expression of progesterone receptor (PR) mRNA and protein, and cell proliferation has been measured in the human endometrial cell line, ECC-1. Although less potent than 17beta-estradiol, BPA was able to bind to the human uterine ER. BPA also induced both mRNA and protein to levels similar to E2. BPA-mediated PR mRNA induction was antagonized by ICI, suggesting an ER-mediated pathway. Finally, E2 produced a 2-fold increase in cell number, while BPA showed no difference compared with vehicle control. The increase by E2 was inhibited by treatment with the either ICI 182,780 (ICI) or BPA, suggesting similar binding sites. Although ER binding is similar, E2 affected both proliferation and PR expression, while BPA only affected PR gene expression. The results of this study provide evidence that two ER agonists can act differentially in vitro to affect the expression of genes involved in regulating cellular growth and development, though the human risk potential remains to be determined.
Assuntos
Carcinoma/metabolismo , Neoplasias do Endométrio/metabolismo , Estrogênios não Esteroides/farmacologia , Fenóis/farmacologia , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/biossíntese , Compostos Benzidrílicos , Ligação Competitiva , Carcinoma/patologia , Divisão Celular/efeitos dos fármacos , Neoplasias do Endométrio/patologia , Estradiol/farmacologia , Estrogênios não Esteroides/metabolismo , Feminino , Humanos , Fenóis/metabolismo , RNA Mensageiro/biossíntese , Células Tumorais CultivadasRESUMO
We investigated the interaction of bisphenol A (BPA, an estrogenic environmental contaminant used in the manufacture of plastics) with the estrogen receptor alpha (ERalpha) transfected into the human HepG2 hepatoma cell line and expanded the study in vivo to examine the effect of BPA on the immature rat uterus. Bisphenol A was 26-fold less potent in activating ER-WT and was a partial agonist with the ERalpha compared to E2. The use of ERalpha mutants in which the AF1 or AF2 regions were inactivated has permitted the classification of ER ligands into mechanistically distinct groups. The pattern of activity of BPA with the ERalpha mutants differed from the activity observed with weak estrogens (estrone and estriol), partial ERalpha agonists (raloxifene or 4-OH-tamoxifen), or a pure antagonist (ICI 182, 780). Intact immature female Sprague-Dawley rats were exposed to BPA alone or with E2 for 3 days. Unlike E2, BPA had no effect on uterine weight; however, like E2, both peroxidase activity and PR levels were elevated, though not to the level induced by E2. Following simultaneous administration, BPA antagonized the E2 stimulatory effects on both peroxidase activity and PR levels but did not inhibit E2-induced increases of uterine weight. These results demonstrate that BPA is not merely a weak estrogen mimic but exhibits a distinct mechanism of action at the ERalpha.
Assuntos
Estradiol/farmacologia , Estrogênios não Esteroides/farmacologia , Fenóis/farmacologia , Receptores de Estrogênio/efeitos dos fármacos , Animais , Compostos Benzidrílicos , Carcinoma Hepatocelular , Estradiol/metabolismo , Receptor alfa de Estrogênio , Feminino , Humanos , Neoplasias Hepáticas , Mutagênese , Tamanho do Órgão/efeitos dos fármacos , Peroxidase/metabolismo , Fenóis/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Estrogênio/genética , Receptores de Estrogênio/fisiologia , Receptores de Progesterona/metabolismo , Transfecção , Células Tumorais Cultivadas , Útero/anatomia & histologia , Útero/efeitos dos fármacos , Útero/metabolismoRESUMO
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a potent modulator of epithelial cell growth and differentiation, has been shown to induce transforming growth factor (TGF)-alpha in cultures of human keratinocytes and in the human keratinocyte cell line, SCC-12F. In this report, we investigated the mechanisms by which TCDD alters TGF-alpha expression. In addition, we studied the actions of TCDD on TGF-beta 1 and TGF-beta 2 expression. Treatment of SCC-12F cells with TCDD resulted in an increase in TGF-alpha and a reduction in TGF-beta 2 mRNA levels while mRNA levels for TGF-beta 1 were unchanged. Changes in TGF-alpha and TGF-beta 2 expression were maximal by 24 h. No change in the rate of transcription of TGF-alpha was detected following treatment with TCDD as determined by nuclear run-off analysis. TCDD treatment resulted in a stabilization of TGF-alpha mRNA as judged by an approximately 2-fold higher level of TGF-alpha mRNA in treated versus control cells in the presence of actinomycin D. In contrast to TGF-alpha, the rate of transcription of TGF-beta 2 was significantly reduced following TCDD treatment. These findings demonstrate that the induction of TGF-alpha expression in SCC-12F cells by TCDD occurs post-transcriptionally, primarily by mRNA stabilization, while TGF-beta 2 expression is reduced due to a decrease in the rate of TGF-beta 2 gene transcription.
Assuntos
Queratinócitos/metabolismo , Dibenzodioxinas Policloradas/farmacologia , Processamento Pós-Transcricional do RNA , Transcrição Gênica , Fator de Crescimento Transformador alfa/biossíntese , Fator de Crescimento Transformador beta/biossíntese , Northern Blotting , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/genética , Expressão Gênica/efeitos dos fármacos , Gliceraldeído-3-Fosfato Desidrogenases/biossíntese , Gliceraldeído-3-Fosfato Desidrogenases/genética , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Cinética , Plasmídeos , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , Transcrição Gênica/efeitos dos fármacos , Fator de Crescimento Transformador alfa/genética , Fator de Crescimento Transformador beta/genéticaRESUMO
In the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related chemicals, the Ah receptor nuclear translocator (Arnt) forms a heterodimeric complex with the ligand-bound Ah receptor, leading to recognition of dioxin-responsive elements within the enhancer of the CYP1A1 gene and transcription activation by an unknown mechanism. To understand the role of Arnt in transcription activation by the Ah receptor-Arnt heterodimer, we performed a deletion analysis of Arnt to locate domains that are directly involved in transcription activation. We showed that the C-terminal 34 amino acids of Arnt encode a transcription activation domain (TAD) that functions independently of other sequences in the Ah receptor complex when attached to the heterologous Gal4 DNA binding domain. Deletion of the C-terminal acidic-rich 14 amino acids completely abolishes activity. Sequences important in Arnt TAD function were independent of the glutamine-rich region which is an important structural feature in the TAD of other transcription factors. The strength of the Arnt TAD when compared with the strong TAD from the herpes simplex virus VP16 protein was cell-type specific. Both the Arnt and VP16 TAD were equally strong in COS-1 cells, but the Arnt TAD had weak activity in an Arnt-deficient mouse hepatoma cell line and was not needed for restoration of CYP1A1 activation. These results imply that for CYP1A1 activation the Ah receptor provides the dominant activation function for the heterodimer in hepatoma cells. The potential of the Arnt TAD to contribute to activation by the Ah receptor complex is likely determined by availability or activity of cell-specific factors with which the TAD interacts.
Assuntos
Mapeamento Cromossômico , Receptores de Hidrocarboneto Arílico/genética , Transativadores/química , Fatores de Transcrição/genética , Ativação Transcricional/genética , Sequência de Aminoácidos , Aminoácidos/química , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Células Cultivadas , Humanos , Neoplasias Hepáticas Experimentais/química , Camundongos , Dados de Sequência Molecular , Receptores de Hidrocarboneto Arílico/química , Proteínas Recombinantes de Fusão/química , Fatores de Transcrição/químicaRESUMO
There is a concern that chemicals in our environment are affecting human health by disrupting normal endocrine function. Much of the concern has focused on chemicals that can interact directly with steroid hormone receptors. We have used a yeast-based assay to assess chemical interactions with the estrogen, androgen, and progesterone receptors. The yeast transformants used in this study contained the human estrogen, androgen, or progesterone receptor along with the appropriate steroid responsive elements upstream of the beta-galactosidase reporter gene. Chemicals were added to yeast cultures in doses ranging from 10(-12) to 10(-4) M and following incubation, the yeasts were then lysed and assayed for beta-galactosidase activity. Diethylstilbesterol and 17-beta estradiol were most active in the estrogen receptor assay, followed by the phytoestrogen, coumestrol. p-Nonylphenol and bisphenol A were approximately 5000- and 15,000-fold less active, respectively, than estradiol. Methoxychlor, DDT and its metabolites, o,p'-DDD, and o,p'-DDE ranged in potency from 5 to 24 X 10(6) less potent than estradiol. Testosterone and dihydrotestosterone were most potent in the androgen receptor assay, followed by estradiol and progesterone. p,p'-DDE was approximately 10(6)-fold less potent than testosterone. None of the industrial chemicals tested interacted with the progesterone receptor. These data demonstrate the utility of using yeast-based receptor assays for detecting chemical interaction with steroid receptors and these assays should serve as a useful component of an in vitro-in vivo strategy to assess the effects of chemicals on endocrine function.
Assuntos
Glândulas Endócrinas/fisiopatologia , Poluentes Ambientais/toxicidade , Inseticidas/toxicidade , Receptores Androgênicos/efeitos dos fármacos , Receptores de Estrogênio/efeitos dos fármacos , Receptores de Progesterona/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , DDT/toxicidade , Relação Dose-Resposta a Droga , Flutamida/análogos & derivados , Flutamida/farmacologia , Hormônios/farmacologia , Humanos , Técnicas In Vitro , Metoxicloro/toxicidade , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/genética , Sensibilidade e Especificidade , Transcrição GênicaRESUMO
The estrogenic activity of 2',4',6'-trichloro-4-biphenylol (HO-PCB3), 2',3',4',5'-tetrachloro-4-biphenylol (HO-PCB4), and an equimolar mixture of both compounds (HO-PCB3/HO-PCB4) was investigated in the 21-day-old B6C3F1 mouse uterus, MCF-7 and MDA-MB-231 human breast cancer cells, HepG2 cells, and in a yeast-based reporter gene assay. Treatment of the animals with 17beta-estradiol (E2) (0.02 microg/kg/day x3) resulted in increased uterine wet weight, peroxidase activity and progesterone receptor binding. Treatment with 18, 73, 183 or 366 micromol/kg (x3) doses of HO-PCB3, HO-PCB4, or HO-PCB3/HO-PCB4 (equimolar) caused a dose-dependent increase in estrogenic activity; a maximal-induced response was not observed at any dose and the activity of the mixture was additive. Binding of E2, HO-PCB3, HO-PCB4, and HO-PCB3/HO-PCB4 to the mouse uterine estrogen receptor (ER) was determined in a competitive binding assay using [3H]E2 as the radioligand. The IC50 values were 1.1 x 10(-8), 3.4 x 10(-6), 9.9 x 10(-7), and 4.25 x 10(-6) m, respectively. HO-PCB3 and HO-PCB4 maximally induced MCF-7 cell proliferation, rat creatine kinase, and human complement C3 (C3-LUC) reporter gene activity at concentrations of 10(-5) to 10(-6) m, and these compounds were 10(3) to 10(4) less potent than E2. The HO-PCB3/HO-PCB4 mixture was active at the high concentration (10(-5) m) and was additive for these responses. HO-PCB3 and HO-PCB4 also exhibited estrogenic activity in human HepG2 cells cotransfected with C3-LUC and an ER expression plasmid, and the estrogenic activity of the HO-PCB mixture was additive. Similar results were obtained in yeast transformed with the human ER and a double estrogen responsive element upstream of the beta-gal reporter gene. The effects of variable ER expression on the potential synergistic interactions of HO-PCB3/HO-PCB4 were investigated in HepG2 cells cotransfected with C3-LUC (405 ng/well) and variable amounts of ER expression plasmid (270, 27, 2.7, or 0.27 ng/well). The results show that as ER levels decreased, the magnitude of the induction response by E2, HO-PCB3, HO-PCB4, and HO-PCB3/HO-PCB4 also decreased. However, the activities of the HO-PCB mixture were additive at high and low levels of ER. Similar results were obtained in MDA-MB-231 cells cotransfected with C3-LUC and variable amounts of ER expression plasmid. The results of this study demonstrate that for several estrogen-responsive assays in the mouse uterus; MCF-7, HepG2, and MDA-MBA-231 human cancer cells; and a yeast based-reporter gene assay, both HO-PCB3 and HO-PCB4 exhibited estrogenic activity. The estrogenic activity of an equimolar mixture of these compounds was additive at high and low levels of ER expression.