Retinal Pigmented Epithelial Cells Cytotoxicity and Apoptosis through Activation of the Mitochondrial Intrinsic Pathway: Role of Indocyanine Green, Brilliant Blue and Implications for Chromovitrectomy

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.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)National Institutes of Health CenterResearch to Prevent BlindnessDepartment of Defense (DOD)Universidade Federal de São Paulo, Inst Visao IPEPO, Dept Oftalmol, São Paulo, BrazilUniv Miami, Bascom Palmer Eye Inst, Miami, FL USAUniversidade Federal de São Paulo, Inst Visao IPEPO, Dept Oftalmol, São Paulo, BrazilNational Institutes of Health Center: P30EY014801Department of Defense (DOD): W81XWH-09-1-0675Web of Scienc

In Vivo, in Vitro Toxicity and in Vitro Angiogenic Inhibition of Sunitinib Malate

Purpose: To evaluate the in vivo and in vitro toxicity of sunitinib malate, a multikinase inhibitor molecule.Design: Experimental, Prospective, Controlled.Methods: Human retinal pigment epithelial (ARPE-19) and human umbilical vein endothelialcells (HUVECS) were used in a culture toxicity test and exposed to different concentrations of sunitinib malate for 18 hours. the HUVECs also were cultured to evaluate the angiogenesis inhibitory effect of sunitinib malate. Fundus photography and angiographic, electrophysiologic, and histopathologic evaluations with light and electron microscopy were performed in two groups of five rabbits each that received different intravitreal concentrations of the drug. Each rabbit received 0.1 ml of sunitinib malate in the right eye (one group with 12.5 mg/ml, the other group with 25 mg/ml); all animals received 0.1 ml of physiologic saline solution in the left eye. After sacrifice, the eyes were enucleated and fixed with modified Karnovsky solution.Results: No toxicity related to sunitinib malate was observed using an in vitro model with the 12.5 and 25 mg/ml solutions in HUVEC and ARPE cell cultures. No toxicity was observed in the in vivo model with 12.5 mg/ml, but light microscopy showed that the 25 mg/ml solution damaged the photoreceptors layer. No functional changes in the electroretinogram were observed in any group.Conclusions: Sunitinib malate 12.5 mg/ml caused no toxicity in in vivo and in vitro models, but the 25 mg/ml concentration caused retinal changes suggesting toxicity in the in vivo model. Further research with the drug is needed in models of ocular neovascularization.São Paulo Fed Univ UNIFESP, Dept Ophthalmol, Vitreoretinal Dis Sect, São Paulo, BrazilVis Inst IPEPO, Dept Ophthalmol, Ocular Pharmacol Sect, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, São Paulo, BrazilUniversidade Federal de São Paulo, Ctr Electron Microscopy, São Paulo, BrazilSão Paulo Fed Univ UNIFESP, Dept Ophthalmol, Vitreoretinal Dis Sect, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, São Paulo, BrazilUniversidade Federal de São Paulo, Ctr Electron Microscopy, São Paulo, BrazilWeb of Scienc