RESUMO
Inhalation is the main route of nanoparticles (NP) exposure during manufacturing. Although many mechanisms of toxicity have been described, the interaction of NP with relevant pneumocytes organelles is not widely understood. Considering that the physicochemical properties of NP influence their toxicological responses, the objective of this study was to evaluate whether exposure to different NP, crystalline Fe3O4 NP and amorphous SiO2 NP could alter pneumocytes organelles in alveolar epithelial cells. To achieve this goal, cell viability, ultrastructural changes, lysosomal damage, mitochondrial membrane potential (MMP), lipid droplets (LD) formation and cytokines production were evaluated by MTT, electron microscopy, lysotracker red staining, JC-1, Oil Red staining and Milliplex® assay respectively. Both NP were observed within lamellar bodies (LB), lysosomes, and cytoplasm causing morphological changes. Exposure to SiO2 NP at 6 h induced lysosomal activation, but not Fe3O4 NP. MMP decreased and LD increased at the highest concentrations after both NP exposure. Pro-inflammatory cytokines were released only after SiO2 NP exposure at 48 h. These results indicate that SiO2 NP have a greater impact than Fe3O4 NP on organelles responsible for energy, secretion, degradation and metabolism in pneumocytes leading to the development of respiratory disorders or the exacerbation of preexisting conditions. Therefore, the established biocompatibility for amorphous NP has to be reconsidered.
Assuntos
Células Epiteliais Alveolares/efeitos dos fármacos , Óxido Ferroso-Férrico/toxicidade , Nanopartículas/toxicidade , Dióxido de Silício/toxicidade , Células A549 , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Humanos , Gotículas Lipídicas/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacosRESUMO
Alveolar epithelia play an essential role in maintaining the integrity and homeostasis of lungs, in which alveolar epithelial type II cells (AECII) are a cell type with stem cell potential for epithelial injury repair and regeneration. However, mechanisms behind the physiological and pathological roles of alveolar epithelia in human lungs remain largely unknown, partially owing to the difficulty of isolation and culture of primary human AECII cells. In the present study, we aimed to characterize alveolar epithelia generated from A549 lung adenocarcinoma cells that were cultured in an air-liquid interface (ALI) state. Morphological analysis demonstrated that A549 cells could reconstitute epithelial layers in ALI cultures as evaluated by histochemistry staining and electronic microscopy. Immunofluorescent staining further revealed an expression of alveolar epithelial type I cell (AECI) markers aquaporin-5 protein (AQP-5), and AECII cell marker surfactant protein C (SPC) in subpopulations of ALI cultured cells. Importantly, molecular analysis further revealed the expression of AQP-5, SPC, thyroid transcription factor-1, zonula occludens-1 and Mucin 5B in A549 ALI cultures as determined by both immunoblotting and quantitative RT-PCR assay. These results suggest that the ALI culture of A549 cells can partially mimic the property of alveolar epithelia, which may be a feasible and alternative model for investigating roles and mechanisms of alveolar epithelia in vitro.
Assuntos
Humanos , Meios de Cultivo Condicionados , Técnicas de Cultura de Células/métodos , Células Epiteliais Alveolares/fisiologia , Células A549/fisiologia , Valores de Referência , Fatores de Tempo , Microscopia Eletrônica de Varredura , Immunoblotting , Contagem de Células , Reprodutibilidade dos Testes , Análise de Variância , Proteína C Associada a Surfactante Pulmonar/análise , Aquaporina 5/análise , Mucina-5B/análise , Reação em Cadeia da Polimerase em Tempo Real , Proteína da Zônula de Oclusão-1/análise , Fator Nuclear 1 de Tireoide/análiseRESUMO
Alveolar epithelia play an essential role in maintaining the integrity and homeostasis of lungs, in which alveolar epithelial type II cells (AECII) are a cell type with stem cell potential for epithelial injury repair and regeneration. However, mechanisms behind the physiological and pathological roles of alveolar epithelia in human lungs remain largely unknown, partially owing to the difficulty of isolation and culture of primary human AECII cells. In the present study, we aimed to characterize alveolar epithelia generated from A549 lung adenocarcinoma cells that were cultured in an air-liquid interface (ALI) state. Morphological analysis demonstrated that A549 cells could reconstitute epithelial layers in ALI cultures as evaluated by histochemistry staining and electronic microscopy. Immunofluorescent staining further revealed an expression of alveolar epithelial type I cell (AECI) markers aquaporin-5 protein (AQP-5), and AECII cell marker surfactant protein C (SPC) in subpopulations of ALI cultured cells. Importantly, molecular analysis further revealed the expression of AQP-5, SPC, thyroid transcription factor-1, zonula occludens-1 and Mucin 5B in A549 ALI cultures as determined by both immunoblotting and quantitative RT-PCR assay. These results suggest that the ALI culture of A549 cells can partially mimic the property of alveolar epithelia, which may be a feasible and alternative model for investigating roles and mechanisms of alveolar epithelia in vitro.