Potencial de diferenciação de células-tronco mesenquimais tecido adiposo humanas expostas ao 2,3,7,8-tetraclorodibenzo-p-dioxina (tcdd) e bisfenol a (bpa)
Data
2018-01-18
Autores
Nunes, Helga Caputo [UNESP]
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
Células tronco mesenquimais (CTMs) possuem papel relevante na manutenção da homeostase e reparação em casos de lesão tecidual através da renovação do estoque celular. Tais células são implicadas na medicina regenerativa e terapia celular como fontes promissoras, sendo o tecido adiposo uma rica e relevante fonte dessas células. Nesse sentido, as células tronco mesenquimais derivadas de tecido adiposo (CTMs- TA) surgem como alternativa segura em estudos pré-clínicos e clínicos. As chamadas “Boas Práticas em Cultura Celular” (do inglês Good Manufacturing Practices - GMPs) tem sido utilizadas como uma nova abordagem para obtenção de controle de qualidade para fins terapêuticos. Neste contexto, o controle toxicológico dessas células surgem como uma questão relevante. Considera-se que os disruptores endócrinos mimetizam a ação de certos hormônios endógenos. Dessa forma, o BPA é conhecido por mimetizar o receptor de estrógeno e também causar alterações no processo de adipogênese. Já o TCDD possui afinidade com o receptor aril hidrocarboneto (AhR), assim como interfere através das vias de sinalização de estrógenos e andrógenos. Desta maneira, o presente estudo teve como objetivo geral testar o potencial de diferenciação das CTMs- TA humanas e de rato na presença dos compostos BPA e TCDD. Os objetivos específicos foram: a quantificação de proteínas relacionadas ao fenótipo de células- tronco, bem como de receptores específicos de ligação desses compostos, testes para verificação do possível perfil apoptótico e genotóxico dessas substâncias e quantificação do potencial de diferenciação adipogênica e osteogênica dessas células quando expostas. Primeiramente, CTMs-TA advindas de cultura primária humana ou de rato, foram obtidas, expandidas e caracterizadas. Assim, essas células foram expostas à 1uM e 10uM de BPA e 10nM de TCDD durante 7 dias. Análises por citometria de fluxo, ensaios de MTT, Western Blot para as proteínas ABCG2, BAK, SOX2, AhRe ERβ, ensaio de Apoptose/necrose, ensaio de genotoxicidade e quantificação espectrofotométrica do potencial de diferenciação das CTMs-TA foram realizados e analisados. A análise do MTT revelou que o BPA (1uM) para CTMs humanas alterou a atividade metabólica no sentido anti-proliferativo, já para as células de rato, tanto o BPA (10uM) quanto o TCDD (10nM) apresentaram caráter indutor de proliferação. Nas células humanas, o TCDD aumentou a concentração das proteínas ABCG2 e AhR. Para BAK, SOX2 e ERβ, nenhuma diferença foi observada entre exposições ou grupos analisados Ambos compostos testados induziram apoptose nas células de rato e mostraram-se genotóxicos após a análise do ensaio do cometa. Para a quantificação da diferenciação adipogênica, a concentração de 1uM de BPA foi capaz de induzir maior diferenciação, o que não ocorreu com nenhuma das concentrações dos compostos quanto à diferenciação osteogênica. No presente trabalho foram analisadas tanto CTMs humanas quanto de ratos, e, nesse sentido, foi possível verificar acentuada diferença entre os comportamentos celulares de cada espécie e verificar a existências de poucos trabalhos sobre a presente temática. Sendo assim, com os presentes achados, é possível concluir que o BPA na concentração de 1uM apresentou atividade tóxica para hASCs fortalecendo os achados de que doses baixas têm maior ação prejudicial que doses mais elevadas. Além de enfatizar a ação do TCDD via receptor AhR nas CTMs, os nossos resultados mostram que o TCDD elevou da expressão da proteína ABCG2 nessas células, o que indica que este composto pode alterar o fenótipo de células-tronco bem como a expressão de receptores do tipo bomba de influxo e efluxo de xenobióticos.
Mesenchymal stem cells (CTMs) plays a relevant role in the maintenance of homeostasis and repair in cases of tissue injury through cell stock renovation. Such cells are implicated in regenerative medicine and cell therapy as promising sources, been the adipose tissue a rich and relevant source. In this sense, mesenchymal stem cells derived from adipose tissue (ASCs) appears to be a safe alternative in preclinical and clinical studies. The so- called Good Manufacturing Practices (GMPs) have been used as a new approach to obtaining quality control for therapeutic purposes. In this context, the toxicological control approach becomes a relevant issue. Endocrine disrupting chemicals are thought to mimic the action of certain endogenous hormones. Thus, BPA is known to mimic the estrogen receptor and also cause changes in the process of adipogenesis. TCDD, on the other hand, has affinity with the aryl hydrocarbon receptor (AhR), as well as interfering with the estrogen and androgen signaling pathways. In this way, the present study had the rationale to test the differentiation potencial of human and rat MSCs in the presence of BPA and TCDD. The specific objectives were: quantification of proteins related to the stem cell phenotype, as well as specific binding receptors of these compounds, verification of apoptotic and genotoxic profile of these substances and also the quantification of the potential of adipogenic and osteogenic differentiation of these cells when exposed. First, ASCs from human or rat primary culture were obtained, expanded and characterized. Thus, these cells were exposed to 1uM and 10uM BPA and 10nM TCDD for 7 days. Flow cytometric assays, MTT assays, Western Blot for ABCG2, BAK, SOX2, AhR and ERβ assays, Apoptosis/ necrosis assay, genotoxicity assay, and spectrophotometric quantification of ASCs differentiation potential were performed and analyzed. MTT analysis revealed that BPA (1uM) for human ASCs altered the metabolic activity in an anti-proliferative sense, whereas for both rat cells, both BPA (10uM) and TCDD (10nM) had a proliferation-inducing behaviour. In human cells, TCDD increased the concentration of ABCG2 and AhR proteins. For BAK, SOX2 and ERβ, no difference was observed between exposures or groups analyzed. Both compounds tested induced apoptosis in rat cells and were genotoxic after comet assay analysis. For the quantification of adipogenic differentiation, the concentration of 1uM of BPA was able to induce greater differentiation, which did not occur with any of the concentrations of the compounds regarding the osteogenic differentiation. In the present study, was investigated both human and rat mesenchymal stem cells, accordingly, we observed marked difference between the cellular behaviors from diferente species and few studies about the current moment. Thus, with these findings, we conclude that BPA in the concentration of 1uM showed toxic activity to hASCs strengthening the findings that low doses has more harmful action than higher doses. In addition to emphasizing the action of TCDD via the AhR receptor on the MSCs, our results show that TCDD increased expression of ABCG2 protein in these cells, suggesting that this compound may alter stem cells phenotype as well as expression of pump-like receptors of influx and efflux of xenobiotics.
Mesenchymal stem cells (CTMs) plays a relevant role in the maintenance of homeostasis and repair in cases of tissue injury through cell stock renovation. Such cells are implicated in regenerative medicine and cell therapy as promising sources, been the adipose tissue a rich and relevant source. In this sense, mesenchymal stem cells derived from adipose tissue (ASCs) appears to be a safe alternative in preclinical and clinical studies. The so- called Good Manufacturing Practices (GMPs) have been used as a new approach to obtaining quality control for therapeutic purposes. In this context, the toxicological control approach becomes a relevant issue. Endocrine disrupting chemicals are thought to mimic the action of certain endogenous hormones. Thus, BPA is known to mimic the estrogen receptor and also cause changes in the process of adipogenesis. TCDD, on the other hand, has affinity with the aryl hydrocarbon receptor (AhR), as well as interfering with the estrogen and androgen signaling pathways. In this way, the present study had the rationale to test the differentiation potencial of human and rat MSCs in the presence of BPA and TCDD. The specific objectives were: quantification of proteins related to the stem cell phenotype, as well as specific binding receptors of these compounds, verification of apoptotic and genotoxic profile of these substances and also the quantification of the potential of adipogenic and osteogenic differentiation of these cells when exposed. First, ASCs from human or rat primary culture were obtained, expanded and characterized. Thus, these cells were exposed to 1uM and 10uM BPA and 10nM TCDD for 7 days. Flow cytometric assays, MTT assays, Western Blot for ABCG2, BAK, SOX2, AhR and ERβ assays, Apoptosis/ necrosis assay, genotoxicity assay, and spectrophotometric quantification of ASCs differentiation potential were performed and analyzed. MTT analysis revealed that BPA (1uM) for human ASCs altered the metabolic activity in an anti-proliferative sense, whereas for both rat cells, both BPA (10uM) and TCDD (10nM) had a proliferation-inducing behaviour. In human cells, TCDD increased the concentration of ABCG2 and AhR proteins. For BAK, SOX2 and ERβ, no difference was observed between exposures or groups analyzed. Both compounds tested induced apoptosis in rat cells and were genotoxic after comet assay analysis. For the quantification of adipogenic differentiation, the concentration of 1uM of BPA was able to induce greater differentiation, which did not occur with any of the concentrations of the compounds regarding the osteogenic differentiation. In the present study, was investigated both human and rat mesenchymal stem cells, accordingly, we observed marked difference between the cellular behaviors from diferente species and few studies about the current moment. Thus, with these findings, we conclude that BPA in the concentration of 1uM showed toxic activity to hASCs strengthening the findings that low doses has more harmful action than higher doses. In addition to emphasizing the action of TCDD via the AhR receptor on the MSCs, our results show that TCDD increased expression of ABCG2 protein in these cells, suggesting that this compound may alter stem cells phenotype as well as expression of pump-like receptors of influx and efflux of xenobiotics.
Descrição
Palavras-chave
célula-tronco mesenquimal derivada de tecido adiposo, disruptores endócrinos, bisfenol A, TCDD, Medicina Regenerativa