RESUMO
PURPOSE: To investigate the in vitro effect of vital dyes on toxicity and apoptosis in a human retinal pigment epithelial cell line. METHODS: ARPE-19 cells were exposed to brilliant blue (BBG), Evans Blue (EB), bromophenol blue (BroB), indocyanine green (ICG), infracyanine green (IfCG), light green (LG), fast green (FG), indigo carmine (IC) and Congo red (CR). Balanced salt solution was used as the control. Five different concentrations and 2 exposure times were tested. Cell viability was determined by the MTS (1-solution methyl thiazolyl tetrazolium) assay and apoptosis by Bax expression on Western blot. RESULTS: All dyes significantly reduced cell viability after 3 min of exposure at all concentrations (p < 0.01), except for BBG that was safe at concentrations up to 0.25 mg/ml and CR up to 0.05 mg/ml, while LG was safe at all concentrations. Toxicity was higher after 30 min of exposure. Expression of Bax was upregulated after all dye exposures, except BBG; ICG had the highest Bax expression (p < 0.01). CONCLUSIONS: Overall the safest dye was BBG followed by LG, IfCG, FG, CR, IC, BroB, EB and ICG. ICG was toxic at all concentrations and exposure times tested. Moreover, BBG was the only dye that did not induce apoptosis in ARPE-19 cells.
Assuntos
Apoptose/efeitos dos fármacos , Corantes/toxicidade , Epitélio Pigmentado da Retina/efeitos dos fármacos , Western Blotting , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Fatores de Tempo , Vitrectomia , Proteína X Associada a bcl-2/metabolismoRESUMO
PURPOSE: To investigate the in vitro effect of four vital dyes on toxicity and apoptosis in a human retinal pigment epithelial (RPE) cell line. METHODS: ARPE-19 cells were exposed to brilliant blue (BriB), methyl blue (MetB), acid violet (AcV) and indocyanine green (ICG). Balanced salt solution was used as control. Five different concentrations of each dye (1, 0.5, 0.25, 0.05 and 0.005 mg/mL) and two exposure times (3 and 30 min) were tested. Cell viability was determined by cell count and MTS assay and cell toxicity by LDH assay. Real-time PCR and Western blotting were used to access the apoptosis process. RESULTS: ICG significantly reduced cell viability after 3 minutes of exposure at all concentrations (p<0.01). BriB was safe at concentrations up to 0.25 mg/mL and MetB at concentrations up to 0.5 mg/mL, while AcV was safe up to 0.05 mg/ml, after 3 minutes of exposure. Toxicity was higher, when the cells were treated for 30 minutes. Expression of Bax, cytochrome c and caspase-9 was upregulated at the mRNA and protein level after ICG exposure, while Bcl-2 was downregulated. AcV and MetB were similar to control. However, BriB resulted in upregulation of Bcl-2, an antiapoptotic protein. CONCLUSIONS: The safest dye used on RPE cells was MetB followed by BriB and AcV. ICG was toxic at all concentrations and exposure times tested. Moreover, ICG was the only dye that induced apoptosis in ARPE-19 cells. BriB significantly increased Bcl-2 protein levels, which might protect against the apoptosis process.