RESUMO
Organophosphorus chlorpyrifos (CPF) is currently considered an endocrine disruptor (ED), as it can imitate hormone actions both in vitro and in vivo. We recently reported that CPF induces migration and invasion in 2D cultures and changes the expression of key molecular markers involved in epithelial mesenchymal transition in MCF-7 and MDA-MB-231 cell lines. In this study, we investigated whether CPF could behave as a predisposing factor for tumors to become more metastatic and aggressive using 3D culture models. In MCF-7 cells, 0.05 µM CPF induced an increase in the number and size of mammospheres via estrogen receptor alpha (ERα) and c-SRC. Furthermore, 0.05 µM CPF increased the area of spheroids generated from MCF-7 cells, induced invasion using both Matrigel® and type 1 collagen matrices, and increased cell migration capacity via ERα in this 3D model. In turn, 50 µM CPF increased cell migration capacity and invasion using type 1 collagen matrix. In monolayers, CPF increased the phosphorylation and membrane translocation of c-SRC at both concentrations assayed. CPF at 0.05 µM boosted p-AKT, p-GSK-3ß and p-P38. While p-AKT rose in a ERα-dependent way, p-GSK-3ß was dependent on ERα- and c-SRC, and p-P38 was only dependent on c-SRC. On the other hand, the increase in p-AKT and p-P38 induced by 50 µM CPF was dependent on the c-SRC pathway. We also observed that 0.05 µM CPF increased IGF-1R and IRS-1 expression and that 50 µM CPF induced IGF-1Rß phosphorylation. In the MDA-MB-231 cell line, 0.05 and 50 µM CPF increased p-c-SRC. Finally, p-AKT and p-GSK-3ß were also induced by CPF at 0.05 and 50 µM, and an increase in p-P38 was observed at 50 µM. Taken together, these data provide support for the notion that CPF may represent a risk factor for breast cancer development and progression.
Assuntos
Neoplasias da Mama , Clorpirifos , Disruptores Endócrinos , Linhagem Celular Tumoral , Proliferação de Células , Clorpirifos/toxicidade , Disruptores Endócrinos/toxicidade , Feminino , Glicogênio Sintase Quinase 3 beta , Humanos , Fenótipo , FosforilaçãoRESUMO
Chlorpyrifos (CPF) is one of the most frequently used pesticide in extensive agriculture around the world and can be incorporated by humans and animals with possible consequences on health. The effects of this pesticide on carcinogenesis are not clear and there is no consensus concerning the risks of this compound. In previous work, we demonstrated that CPF induces proliferation of breast cancer cells both in vivo and in vitro. In this work we investigate whether CPF promotes the epithelial-mesenchymal transition (EMT) in breast cancer cells. Herein, we demonstrate that 50 µM CFP induces invasion in MCF-7 and MDA-MB-231 cells. In addition, 0.05 and 50 µM CPF increases migration in both cell lines. In MCF-7 cells, 0.05 and 50 µM CPF increase the metalloprotease MMP2 expression and decrease E-Cadherin and ß-Catenin expression diminishing their membrane location. Furthermore, 50 µM CPF induces Vimentin expression and Slug nuclear translocation in MCF-7 cells. 0.05 and 50 µM CPF increase MMP2 gelatinolytic activity and expression, decrease ß-Catenin expression and increase Vimentin expression in MDA-MB-231 cells. Inhibition of the oncoprotein c-Src reverses all the effects induced by CPF in MDA-MB-231 but not in MCF-7 indicating that c-Src is a kinase with a crucial role in the cells which grow in an estrogen-independent way. In MCF-7 cells both c-Src and estrogen receptor alpha must be blocked to completly inhibit the CPF-mediated effects. Our results show for the first time that the exposure to subthreshold concentrations of CPF promotes the modulation of EMT-molecular markers and pathways. These results, together with the ubiquitous distribution of the pesticide CPF, make it of utmost importance to take measures to minimize the risk of exposure to this compound.
Assuntos
Movimento Celular/efeitos dos fármacos , Clorpirifos/toxicidade , Disruptores Endócrinos/toxicidade , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Praguicidas/toxicidade , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteína Tirosina Quinase CSK/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Relação Dose-Resposta a Droga , Transição Epitelial-Mesenquimal/genética , Receptor alfa de Estrogênio/genética , Feminino , Humanos , Células MCF-7 , Metaloproteinase 2 da Matriz/genética , Transdução de SinaisRESUMO
Hexachlorobenzene (HCB) is an organochlorine pollutant widely distributed in the environment around the entire world. Previous reports from our group and others have demonstrated that this compound is as an endocrine disruptor. We have also reported that HCB presents a co-carcinogenic effect in N-Nitroso-N-methyl-urea-induced mammary tumours in rats. In this work, we studied the effects of HCB on cell cycle progression and cell cycle regulating protein expression in the estrogen-sensitive breast cancer cell line, MCF-7. Here, we show that HCB alters cell cycle in a concentration-dependent way. The lowest assessed concentration (0.005µM) promotes the cell cycle progression, enhances cyclin D1 expression, and reduces the nuclear localization of p27 accompanied by an increased interaction between p27 and c-Src kinase. On the other hand, 5µM HCB delays the cell cycle progression and promotes the formation of the cyclin E-CDK2-p27 protein complex. Our results show that HCB stimulates cell proliferation through cell cycle modulation and c-Src involvement in MCF-7 cells. Here, we report for the first time that differential mechanisms of action of HCB on mammary cell cycle progression are triggered at different concentrations of this pollutant.
Assuntos
Ciclo Celular/efeitos dos fármacos , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Hexaclorobenzeno/toxicidade , Proteínas Oncogênicas/metabolismo , Quinases da Família src/metabolismo , Proteína Tirosina Quinase CSK , Divisão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ciclina D1/genética , Ciclina D1/metabolismo , Ciclina E/genética , Quinase 2 Dependente de Ciclina/genética , Inibidor de Quinase Dependente de Ciclina p27/genética , Humanos , Células MCF-7 , Proteínas Oncogênicas/genética , Fosforilação , Quinases da Família src/genéticaRESUMO
3,5,3'-triiodothyroacetic acid (TRIAC) has been used to suppress pituitary TSH secretion with reported attenuation of extrapituitary effects. We investigated whether equivalent doses of T (3) and TRIAC preventing the induction of goiter by methimazole (MMI) had a different or similar impact on peripheral tissues, such as liver and bone. In particular, we compared the effects of both compounds on the activity of the hepatic thyroid hormone-responsive enzymes, malic enzyme and L-glicerol-3-P dehydrogenase; bone mineral density and biochemical parameters of bone turnover, such as bone alkaline phosphatase (b-ALP) and the carboxy-terminal telopeptide region of type I collagen (beta-CTX); and the activity of thyroid ornithine decarboxylase (ODC). We also compared the effects of T (3) and TRIAC on the involution of MMI-induced goiter. Our results showed that TRIAC was more effective than T (3) to reduce MMI-induced goiter in a short-term goiter involution assay. TRIAC increased hepatic enzymes activity and beta-CTX levels, a parameter of bone resorption, more than T (3). However, bone mineral density was not altered by either treatment. Both compounds even reduced ODC activity at doses that were not effective at the pituitary level. These results demonstrate increased TRIAC hepatic and antigoitrogenic activity compared to T (3). TRIAC induces an imbalance in bone remodeling without affecting bone mineral density. Further studies are required to clarify this point.
Assuntos
Osso e Ossos/patologia , Bócio/prevenção & controle , Fígado/patologia , Tri-Iodotironina/análogos & derivados , Tri-Iodotironina/uso terapêutico , Animais , Densidade Óssea/efeitos dos fármacos , Osso e Ossos/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Fígado/efeitos dos fármacos , Ratos , Ratos Wistar , Tireotropina/sangueRESUMO
Hexachlorobenzene (HCB) is a polyhalogenated aromatic hydrocarbon widely distributed in the environment. In animal testing, HCB has been shown to be a reproductive toxin. Previous investigations of the effects of HCB on ovarian function have yielded equivocal results. Thus, the effects of chronic administration of HCB (1 g kg(-1) body wt.) on the ovary and pituitary hormone levels, hepatic and uterine oestradiol receptors, ovarian histopathological changes and oestrus cycle characteristics were investigated in spontaneously cycling rats. Our data demonstrate that HCB treatment, under the conditions of the present study, reduced circulating levels of oestradiol and prolactin without differences in serum concentrations of progesterone. Follicle-stimulating hormone serum levels were elevated. Hexachlorobenzene treatment resulted in irregularity of cycles, characterized mainly as prolonged periods of oestrus with a reduced number of ova recovered. In addition, HCB administration resulted in significantly decreased uterine nuclear oestrogen receptor levels. Histopathological examination revealed degenerative changes of the ovarian follicles and germinal epithelium and increased numbers of atresic follicles.
Assuntos
Fungicidas Industriais/toxicidade , Hexaclorobenzeno/toxicidade , Reprodução/efeitos dos fármacos , Animais , Estradiol/sangue , Estro/efeitos dos fármacos , Feminino , Gonadotropinas Hipofisárias/sangue , Gonadotropinas Hipofisárias/metabolismo , Fígado/efeitos dos fármacos , Tamanho do Órgão , Ovário/efeitos dos fármacos , Ovário/fisiologia , Ovário/ultraestrutura , Progesterona/sangue , Ratos , Ratos Wistar , Receptores de Estradiol/efeitos dos fármacos , Receptores de Estrogênio/efeitos dos fármacos , Útero/efeitos dos fármacosRESUMO
The effect of the in vivo administration of hexachlorobenzene (HCB) (100 mg/100 g bw) for 30 days, on the activities of brown adipose tissue (BAT) lipogenic enzymes, i.e. malic enzyme (ME), and glucose-6-phosphate dehydrogenase (G6PD) and the mitochondrial non lipogenic enzyme, L-glycerol-3-phosphate dehydrogenase (LG3PD), was studied in male Wistar rats, submitted to various neurohormonal manipulations. BAT ME, G6PD and LG3PD activities showed significant reductions in response to HCB treatment both in euthyroid and surgically thyroidectomized rats, showing that the effect does not depend on the presence of thyroid hormones. These results differ from those obtained for hepatic ME and G6PD activities, which increased in HCB intoxicated rats without alteration in LG3PD. HCB decreased BAT ME activity under BAT denervation. Administration of HCB resulted in time and dose-dependent decreases in the activity of BAT malic enzyme. The basal activity of ME was increased in hypothyroid rats, while that of LG3PD was reduced. A stimulatory effect of receptor-saturating doses of triiodothyronine (T3) (50 microg/100 g body weight) was observed on BAT ME and LG3PD activities in thyroidectomized rats, showing that the enzymes responded to thyroid hormone stimulation in a normal manner. The stimulatory effect of saturating doses of T3 on ME and LG3PD was reduced by HCB. The results presented herein unequivocally show that brown adipose tissue is a specific target in HCB-induced toxicity, which in turn involves severe alterations in the regulation of BAT lipogenesis.
Assuntos
Tecido Adiposo Marrom/enzimologia , Glicerolfosfato Desidrogenase/metabolismo , Hexaclorobenzeno/farmacologia , Lipídeos/biossíntese , NADP/biossíntese , Tecido Adiposo Marrom/efeitos dos fármacos , Animais , Peso Corporal/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Wistar , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/enzimologia , Tiroxina/sangue , Tri-Iodotironina/sangueRESUMO
Hexachlorobenzene (HCB) is a widespread environmental pollutant. Chronic exposure of laboratory animals to HCB triggers porphyria, induction of liver microsomal enzymes, low levels of T4 reproductive dysfunction's, liver and thyroid tumors. Previous findings from our laboratory have shown that HCB increased the activity of the liver thyroid-responsive enzymes: malic enzyme (ME), glucose-6-phosphate dehydrogenase (G6PD) without any change in the mytochondrial alpha-glycerophosphate dehydrogenase (alpha-GPD). In this study we have demonstrated that HCB treatment increased ME mRNA. We also have investigated if HCB affected: a) the thyroid hormone receptor (TR) concentration and binding affinity for its ligands, b) specifically the ME gene expression, or other thyroid hormone responsive enzymes were affected as well, c) Protein/DNA complex formed on the thyroid responsive element (TRE). Livers from female Wistar rats intoxicated with HCB (100 mg/100 g b.w.), for 9 and 15 days, were analyzed. Northern blot hybridization analysis, have demonstrated that ME mRNA levels increased 4 times and 2 times after 9 and 15 days intoxication respectively, without any alterations in the mRNA levels of other thyroid hormone responsive enzymes such as glyceraldheyde 3- phosphate dehydrogenase, phosphoenolpyruvatecarboxikinase and alpha-GPD. These results suggest that HCB affects specifically, ME gene expression. Hepatic T3 and T4 levels evaluated by RIA were not affected by HCB. Scatchard analyses showed that TR affinity and number of sites were not altered after 9 and 15 days of HCB treatment (control, Ka: 1.9 nM, Bmax 3.9 f/mol 100 micrograms DNA: HCD 9 days Ka: 2.1 nM, Bmax 4.5 fmol/100 micrograms DNA: HCB 15 days Ka 1.9 nM. Bmax 5.1 fmol/100 micrograms DNA intoxication, neither at 9 nor at 15 days. Electrophoresis mobility shift assay showed that HCB did not modify nuclear protein extract affinity for the TREs sequence. Our results suggest that TR itself was not directly involved in the induction of ME gene expression by HCB. Nevertheless TR could interact with other transcription factors in the overexpression of ME gene.
Assuntos
Fungicidas Industriais/intoxicação , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hexaclorobenzeno/intoxicação , Fígado/enzimologia , RNA Mensageiro/efeitos dos fármacos , Tiroxina/fisiologia , Tri-Iodotironina/fisiologia , Animais , Citosol/enzimologia , Feminino , Gliceraldeído-3-Fosfato Desidrogenases/efeitos dos fármacos , Glicerolfosfato Desidrogenase/efeitos dos fármacos , Fígado/efeitos dos fármacos , Mitocôndrias Hepáticas/enzimologia , Fosfoenolpiruvato Carboxilase/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores dos Hormônios Tireóideos/efeitos dos fármacos , Receptores dos Hormônios Tireóideos/metabolismo , Fatores de TempoRESUMO
The time and dose-dependent effects of the in vivo administration of hexachlorobenzene (HCB), on hepatic microsomal membrane functions, were studied in female Wistar rats. Administration of HCB (100 mg/100 g b.w.) resulted in time-dependent decreases in the activity of two membrane-bound enzymes: 5'nucleotidase and Na+/K+ ATPase. HCB was found to cause a significant rise in protein tyrosine kinase (PTK) activity during the early stages of intoxication (day 2), followed by a significant decrease at 10 days, returning to control levels after 20 days of treatment. A stimulatory effect of HCB on in vitro endogenous microsomal protein phosphorylation was observed from 2 days of intoxication up to 30 days of treatment, with an important stimulation of phosphorylation at 5 days. Administration of HCB (100 mg/100 g b.w.) for 10 days caused a 50% reduction in epidermal growth factor receptor (EGF-R) ligand binding. The effects of known specific inhibitors of protein phosphatases on endogenous protein phosphorylation were studied. HCB affected the labelling of several bands, as well as the 5'nucleotidase and PTK activities, in a dose-dependent manner. In conclusion, this study indicated that the in vivo administration of HCB results in a significant alteration of membrane function.
Assuntos
Hexaclorobenzeno/farmacologia , Membranas Intracelulares/efeitos dos fármacos , Microssomos Hepáticos/efeitos dos fármacos , Poluentes do Solo/farmacologia , 5'-Nucleotidase/metabolismo , Animais , Relação Dose-Resposta a Droga , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Feminino , Membranas Intracelulares/enzimologia , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/ultraestrutura , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Ratos , Ratos Wistar , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
El hexaclorobenceno (HCB) es un tóxico ampliamente distribuído en la biosfera. La exposición crónica de animales de laboratorio al HCB provoca disfunciones tiroideas. Previamente hemos demostrado que el HCB incrementa la actividad de enzimas hepáticas reguladas por hormonas tiroideas (HT) tales como: enzima málica (EM) y glucosa-6fosfato de dehidrogenasa (G6PD) sin alterar la actividad de la alpha-glicerol fosfato deshidrogenasa mitocondrial (alpha-GPD). En éste estudio hemos investigado si el HCB afectaba: a) la concentración del receptor de hormonas tiroideas (RT3) y su afinidad por el ligando, b) la expresión del gen de EM y de otras enzimas HT-dependientes, c) los complejos proteína/DNA formados sobre el elemento de respuesta a hormonas tiroideas (TRE). Se utilizaron hígados de ratas hembras Wistar intoxicadas con HCB (100 mg/100 g P.C.), por 9 y 15 días. El análisis de Scatchard mostró que ni la afinidad ni el número de sitios RT3 estaban alterados luego de 9 y 15 días de tratamiento con HCB (Control, Ka: 1,9 nM, Bmáx:3.9 fmol/100mug DNA; HCB9díasKa2.1nM, Bmáx4.5 fmol/100mug DNA; HCB15 días Ka 1.9nM, Bmáx5.1 fmol/100mug DNA). Tampoco los niveles de RNAm de TRbeta1 medidos por ensayos de protección a RNasa fueron afectados por HCB. Ensayos de Northern Blot han demostrado que los niveles de RNAm de EM se incrementaban 4 veces y 2 veces con respecto al control después de 9 y 15 días de intoxicación respectivamente, sin observarse alteraciones en los niveles de RNAm de otras enzimas cuya expresión es regulada por HT como gliceraldehído - 3 - fosfato deshidrogenasa (GAPDH) y fosfoenolpiruvatocarboxiquinasa (PEPCK) ni tampoco en la alpha-GPD mitocondrial. Ensayos de retardo en gel mostraron que el HCB no modificó la afinidad de las proteínas presentes en extractos nucleares por el TRE presente en el promotor de EM. Nuestros resultados sugieren que el RT3 no está involucrado en forma directa en la inducción de la expresión del gen de EM por HCB, sin embargo podría interaccionar con otros factores de transcripción en la sobreexpresión del gen de EM. (AU)
Assuntos
Ratos , Animais , Tri-Iodotironina/farmacologia , Tiroxina/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Malato Desidrogenase/genética , Fígado/enzimologia , Hexaclorobenzeno/toxicidade , Fungicidas Industriais/toxicidade , Receptores dos Hormônios Tireóideos/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , Fígado/efeitos dos fármacos , Receptores dos Hormônios Tireóideos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo , Sensibilidade e Especificidade , Northern Blotting , Transcrição Gênica , Ratos Wistar , Citosol/enzimologia , Mitocôndrias Hepáticas/enzimologia , Gliceraldeído-3-Fosfato Desidrogenases/efeitos dos fármacos , Fosfoenolpiruvato Carboxilase/efeitos dos fármacos , Glicerolfosfato Desidrogenase/efeitos dos fármacosRESUMO
El hexaclorobenceno (HCB) es un tóxico ampliamente distribuído en la biosfera. La exposición crónica de animales de laboratorio al HCB provoca disfunciones tiroideas. Previamente hemos demostrado que el HCB incrementa la actividad de enzimas hepáticas reguladas por hormonas tiroideas (HT) tales como: enzima málica (EM) y glucosa-6fosfato de dehidrogenasa (G6PD) sin alterar la actividad de la alpha-glicerol fosfato deshidrogenasa mitocondrial (alpha-GPD). En éste estudio hemos investigado si el HCB afectaba: a) la concentración del receptor de hormonas tiroideas (RT3) y su afinidad por el ligando, b) la expresión del gen de EM y de otras enzimas HT-dependientes, c) los complejos proteína/DNA formados sobre el elemento de respuesta a hormonas tiroideas (TRE). Se utilizaron hígados de ratas hembras Wistar intoxicadas con HCB (100 mg/100 g P.C.), por 9 y 15 días. El análisis de Scatchard mostró que ni la afinidad ni el número de sitios RT3 estaban alterados luego de 9 y 15 días de tratamiento con HCB (Control, Ka: 1,9 nM, Bmáx:3.9 fmol/100mug DNA; HCB9díasKa2.1nM, Bmáx4.5 fmol/100mug DNA; HCB15 días Ka 1.9nM, Bmáx5.1 fmol/100mug DNA). Tampoco los niveles de RNAm de TRbeta1 medidos por ensayos de protección a RNasa fueron afectados por HCB. Ensayos de Northern Blot han demostrado que los niveles de RNAm de EM se incrementaban 4 veces y 2 veces con respecto al control después de 9 y 15 días de intoxicación respectivamente, sin observarse alteraciones en los niveles de RNAm de otras enzimas cuya expresión es regulada por HT como gliceraldehído - 3 - fosfato deshidrogenasa (GAPDH) y fosfoenolpiruvatocarboxiquinasa (PEPCK) ni tampoco en la alpha-GPD mitocondrial. Ensayos de retardo en gel mostraron que el HCB no modificó la afinidad de las proteínas presentes en extractos nucleares por el TRE presente en el promotor de EM. Nuestros resultados sugieren que el RT3 no está involucrado en forma directa en la inducción de la expresión del gen de EM por HCB, sin embargo podría interaccionar con otros factores de transcripción en la sobreexpresión del gen de EM.