title: Retinal pigmented epithelial cells cytotoxicity and apoptosis through activation of the mitochondrial intrinsic pathway: role of indocyanine green, brilliant blue and implications for chromovitrectomy.
abstract: OBJECTIVE
  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.
authors.affiliation: Departamento de Oftalmologia, Instituto da Visão (IPEPO), Universidade Federal de São Paulo, São Paulo, SP, Brazil. penhaepm@yahoo.com.br
authors.forename: Fernando M
authors.initials: FM
authors.lastname: Penha
authors.forename: Marianne
authors.initials: M
authors.lastname: Pons
authors.forename: Elaine Fiod
authors.initials: EF
authors.lastname: Costa
authors.forename: Nilana Meza Tenório
authors.initials: NM
authors.lastname: Barros
authors.forename: Eduardo B
authors.initials: EB
authors.lastname: Rodrigues
authors.forename: Emmerson Badaró
authors.initials: EB
authors.lastname: Cardoso
authors.forename: Eduardo
authors.initials: E
authors.lastname: Dib
authors.forename: Mauricio
authors.initials: M
authors.lastname: Maia
authors.forename: Maria E
authors.initials: ME
authors.lastname: Marin-Castaño
authors.forename: Michel Eid
authors.initials: ME
authors.lastname: Farah
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D001557:Benzenesulfonates / Q000633:toxicity; D053453:Caspase 9 / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004396:Coloring Agents / Q000633:toxicity*; D045304:Cytochromes c / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007208:Indocyanine Green / Q000633:toxicity; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D001557:Benzenesulfonates; D004396:Coloring Agents; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D045304:Cytochromes c; C414357:Methyl Blue; D053453:Caspase 9; C006796:brilliant blue; D007208:Indocyanine Green