RESUMO
Polybrominated Diphenyl Ethers (PBDEs) are a major class of brominated flame retardants, and their widespread use has led them to be considered contaminants with emerging concern. PBDEs have been detected in the indoor air, house dust, food, and all environmental compartments. The congener BDE-47 (2,2',4,4'-tetrabromodiphenyl ether) is the most prevalent, and hepatotoxicity, neurotoxicity, immunological changes, endocrine disruption, and genotoxic potential have been related to its exposure. Although the BDE-47 molecular toxicity pathway is directly related to intrinsic apoptotic cell death, the role of autophagy in BDE-47 toxicity remains unclear. In this context, three-dimensional cell culture has emerged as a good strategy for the replacement of animals in toxicological testing. Here, we used HepaRG spheroids cultured in alginate microcapsules to investigate the role of autophagy in BDE-47-mediated hepatotoxicity. We developed mature and functional HepaRG spheroids by culturing them in alginate microcapsules. Histological analysis revealed that HepaRG spheroids formed an extracellular matrix and stored glycogen. No apoptotic and/or necrotic cores were observed. BDE-47 showed concentration- and time-dependent cytotoxicity in HepaRG spheroids. In the early exposure period, BDE-47 initially disrupted mitochondrial activity and increased the formation of acid compartments that promoted the increase in autophagic activity; however, this autophagy was blocked, and long-term exposure to BDE-47 promoted efficient apoptotic cell death through autophagy blockade, as evidenced by an increased number of fragmented/condensed nuclei. Therefore, for the first time, we demonstrated BDE-47 toxicity and its cell pathway induces cell death using a three-dimensional liver cell culture, the HepaRG cell line.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Retardadores de Chama , Animais , Éteres Difenil Halogenados/toxicidade , Cápsulas , Autofagia , Retardadores de Chama/toxicidadeRESUMO
ABSTRACT Objective To investigate if ICI 182,780 (fulvestrant), a selective estrogen receptor alpha/beta (ERα/ERβ) antagonist, and G-1, a selective G-protein-coupled receptor (GPER) agonist, can potentially induce autophagy in breast cancer cell lines MCF-7 and SKBr3, and how G-1 affects cell viability. Methods Cell viability in MCF-7 and SKBr3 cells was assessed by the MTT assay. To investigate the autophagy flux, MCF-7 cells were transfected with GFP-LC3, a marker of autophagosomes, and analyzed by real-time fluorescence microscopy. MCF-7 and SKBr3 cells were incubated with acridine orange for staining of acidic vesicular organelles and analyzed by flow cytometry as an indicator of autophagy. Results Regarding cell viability in MCF-7 cells, ICI 182,780 and rapamycin, after 48 hours, led to decreased cell proliferation whereas G-1 did not change viability over the same period. The data showed that neither ICI 182,780 nor G-1 led to increased GFP-LC3 puncta in MCF-7 cells over the 4-hour observation period. The cytometry assay showed that ICI 182,780 led to a higher number of acidic vesicular organelles in MCF-7 cells. G-1, in turn, did not have this effect in any of the cell lines. In contrast, ICI 182,780 and G-1 did not decrease cell viability of SKBr3 cells or induce formation of acidic vesicular organelles, which corresponds to the final step of the autophagy process in this cell line. Conclusion The effect of ICI 182,780 on increasing acidic vesicular organelles in estrogen receptor-positive breast cancer cells appears to be associated with its inhibitory effect on estrogen receptors, and GPER does notseem to be involved. Understanding these mechanisms may guide further investigations of these receptors' involvement in cellular processes of breast cancer resistance.
RESUMO Objetivo Avaliar o efeito dos compostos ICI 182,780 (fulvestranto), um antagonista seletivo dos receptores de estrógeno alfa/beta (REα/REβ), e do G-1, um agonista seletivo de receptores de estrógeno acoplados a proteínas-G (GPER), na possível indução de autofagia em linhagens de câncer de mama MCF-7 e SKBr3, bem como o efeito de G-1 na viabilidade celular. Métodos A viabilidade celular de células MCF-7 e SKBr3 foi avaliada pelo ensaio com MTT. Para investigar a indução da autofagia, células MCF-7 foram transfectadas com GFP-LC3, um marcador de autofagossomos, e analisadas por microscopia de fluorescência em tempo real. As células MCF-7 e SKBr3 foram incubadas com o indicador de compartimentos ácidos laranja de acridina e analisadas por citometria de fluxo como indicativo para autofagia. Resultados Em células MCF-7, o ICI 182,780 e rapamicina após 48 horas levaram à diminuição da viabilidade celular, enquanto o G-1 não alterou a viabilidade no mesmo período de tratamento. Nem o ICI 182,780 e nem o G-1 induziram aumento na pontuação de GFP-LC3 em células MCF-7 até 4 horas. Já os ensaios de citometria de fluxo demonstraram que ICI 182,780 levou ao aumento de compartimentos ácidos em células MCF-7. O G-1 não aumentou estes parâmetros em ambas as linhagens. Por outro lado, ICI 182,780 e G-1 não induziram à redução da viabilidade em células SKBr3 e nem à formação de compartimentos ácidos, como etapa final do processo autofágico. Conclusão O aumento de compartimentos ácidos pelo ICI 182,780 em células de câncer de mama positivas para receptores de estrógeno parece estar associado com seu efeito inibidor de receptores de estrógeno, mas sem o envolvimento de GPER. A compreensão desses mecanismos pode direcionar estudos sobre o envolvimento dos receptores nos processos celulares de resistência do câncer de mama.