Effects of trypan blue on cell viability and gene expression in human retinal pigment epithelial cells

Abstract

Aim: To evaluate the effects of trypan blue on cell viability and gene expression in human retinal pigment epithelial (RPE) cells.

Methods: Three concentrations (0.06 mg/ml, 0.6 mg/ml, and 4 mg/ml) of trypan blue were applied to human ARPE19 cells for 1 minute. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RPE cells were sampled daily for 6 consecutive days to assess the effects of trypan blue on cell viability. The effects of trypan blue on the expression of apoptosis related and cell cycle arrest gene expressions including c-fos, c-jun, p53, and p21 were performed using reverse transcription-polymerase chain reaction and immunostaining.

Results: The MTT assay showed a concentration dependent suppression effect of trypan blue on cell viability, with higher reduction in the 0.6 mg/ml and 4 mg/ml trypan blue treated groups. No significant change in the expression of c-fos and c-jun was found with all three concentrations of trypan blue. An increase in p53 expression was found in the 4 mg/ml trypan blue treated group at 10-30 minutes after trypan blue application. Immunostaining showed a mild, albeit insignificant, increase of p53 expression in the RPE cells. No significant increase in p21 expression was observed in the 0.06 mg/ml trypan blue treated group but there were significant increases in p21 expression in both the 0.6 mg/ml (p = 0.032) and the 4 mg/ml (p = 0.025) treated groups.

Conclusions: Trypan blue may lead to toxicity on cultured RPE cells as indicated by the reduction in cell viability and changes in the expression of apoptosis related and cell cycle arrest genes at higher concentrations. The application of 0.06 mg/ml trypan blue for 1 minute appeared to have no significant effect on cultured RPE.

Figure 1

(A) MTT assay showing the proliferation of ARPE19 cells after the application of three concentrations of trypan blue (Tb) compared with control. Greater reduction in cell viability was found with higher concentration of trypan blue. (B) Trypan blue toxicity was expressed as the ratio of viable cells against control. There was a concentration dependent reduction in cell viability caused by trypan blue. Reduction in the percentage of viable cells was most marked in the first 2 days after application of trypan blue.

Figure 2

PCR signal intensities of c-fos, c-jun, and p53 in ARPE19 cells treated with different concentrations of trypan blue: (A) control; (B) 0.06 mg/ml; (C) 0.6 mg/ml, and (D) 4 mg/ml. Samples were collected at 0, 10, 20, 30, 40, 50, 90, and 120 minutes. The intensities were compared against that of the GAPDH.

Figure 3

Quantification of PCR signal intensities of (A) c-fos, (B) c-jun, and (C) p53 in ARPE19 after the application of trypan blue (Tb) compared with control. No significant increase in expression of c-fos or c-jun was found. For the 4 mg/ml trypan blue treated group, there was an increase in the expression of p53 from 10–30 minutes following trypan blue application.

Figure 4

Immunofluorescence of (A) p53 and (B) p21. No significant difference in the number of cells stained positive for p53 was observed for various concentrations of trypan blue. Significant increase in the number of p21 positively stained cells was observed in 0.6 mg/ml and 4 mg/ml trypan blue treated groups.