November 2010
Volume 51, Issue 11
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Retinal Cell Biology  |   November 2010
Comparative Effects of Six Intraocular Vital Dyes on Retinal Pigment Epithelial Cells
Author Affiliations & Notes
  • María-Celia Morales
    From the Instituto Clínico-Quirúrgico de Oftalmología, Vizcaya, Spain; and
  • Vanesa Freire
    From the Instituto Clínico-Quirúrgico de Oftalmología, Vizcaya, Spain; and
  • Aintzane Asumendi
    University of the Basque Country, Leioa, Vizcaya, Spain.
  • Javier Araiz
    From the Instituto Clínico-Quirúrgico de Oftalmología, Vizcaya, Spain; and
  • Itxaso Herrera
    From the Instituto Clínico-Quirúrgico de Oftalmología, Vizcaya, Spain; and
  • Gonzalo Castiella
    From the Instituto Clínico-Quirúrgico de Oftalmología, Vizcaya, Spain; and
  • Iñigo Corcóstegui
    From the Instituto Clínico-Quirúrgico de Oftalmología, Vizcaya, Spain; and
  • Gonzalo Corcóstegui
    From the Instituto Clínico-Quirúrgico de Oftalmología, Vizcaya, Spain; and
  • Corresponding author: María-Celia Morales, ICQO, c/Virgen de Begoña 34, E-48006 Bilbao, Vizcaya, Spain; morales@icqo.org
Investigative Ophthalmology & Visual Science November 2010, Vol.51, 6018-6029. doi:10.1167/iovs.09-4916
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      María-Celia Morales, Vanesa Freire, Aintzane Asumendi, Javier Araiz, Itxaso Herrera, Gonzalo Castiella, Iñigo Corcóstegui, Gonzalo Corcóstegui; Comparative Effects of Six Intraocular Vital Dyes on Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(11):6018-6029. doi: 10.1167/iovs.09-4916.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To evaluate and compare the effects of the following dyes on human pigmented epithelial cells: indocyanine green (ICG), infracyanine green (IfCG), trypan blue (TB), bromophenol blue (BrB), patent blue (PB), and Brilliant Blue G (BBG).

Methods.: ARPE-19 cells cultured in vitro were exposed to these dyes, and acute and chronic toxicity were evaluated. Cell viability was measured by colorimetry (MTT assay), morphology was observed by phase-contrast microscopy, membrane permeability (CMP) was evaluated by flow cytometry with propidium iodide (PI), and mitochondrial membrane potential (ΔΨm) was measured with 3,3′-dihexyloxacarbocyanine (DiOC6(3)).

Results.: Each of the studied dyes exhibited toxicity after acute exposure at surgical doses. The presence of light often reduced cell viability, especially when measured 3 hours after incubation in the case of ICG, TB, BrB, and BBG. Morphologic changes were induced by ICG, IfCG, and BBG. Both CMP and ΔΨm were altered after exposure to surgical doses of ICG, TB, PB, and a fourfold surgical dose of BrB. Chronic exposure to residual amounts of some dyes was associated with reduced proliferation and even cell death.

Conclusions.: It appears to be prudent to use the lowest possible dose of each dye, to minimize the risk of toxic effects. This precaution may be particularly important in the case of BrB, which should not be used in excess of 0.5%. In addition, abundant irrigation of the vitreous cavity after surgery to completely remove traces of dye may be of crucial importance, particularly in the case of ICG, in minimizing chronic toxicity.

One of the most important innovations in vitreoretinal surgery over the past 10 years has been the introduction of vital dyes to improve the visualization of preretinal tissues and membranes. 1 4 Staining these structures facilitates the peeling of the internal limiting membrane (ILM) of the retina during vitrectomy and reduces surgical risks. 5 9  
In 2000, indocyanine green (ICG) became the first dye to be used to stain the ILM, and it is currently the most commonly used surgical dye in ophthalmology. Nevertheless, despite its routine use, it has generated controversial discussion over the past decade. ICG has been found to be associated with the risk of retinal damage, 10 13 atrophy of the retinal pigment epithelium (RPE), 14 damage to the photoreceptors and RPE cells, 15,16 lower visual function outcome, 17 22 loss of epiretinal cellular integrity, 23 and cellular toxicity, 24 29 among other harmful effects. Recently, several groups have reported that ICG may persist in the ocular cavity, even 6 weeks after its application in surgery. 30 32 Therefore, the need for the investigation of alternative dyes for vitrectomy has become apparent. Since then, several alternative stains have been introduced into vitreoretinal surgery and their number is constantly growing. 33 37 However, it is not clear which dye would be ideal in terms of reduced toxicity, higher affinity, and minimal residual permanence. 
Infracyanine Green (IfCG) is a dye with a chemical formula and pharmacologic properties similar to those of ICG. However, IfCG is synthesized without sodium iodine, which seems to represent a clear advantage, because it is believed that iodine damages the cornea and retina. 38 40 On the other hand, iodide-free IfCG is not water soluble and has to be dissolved in a 5% glucose solvent. 41 43  
Trypan blue (TB) is used in microscopy for staining dead tissues. In ophthalmology, TB has preferential affinity for the epiretinal membrane (ERM). 44 48 Although it may not enable ILM visualization as well as ICG, it does exhibit better biocompatibility. 49 52  
Bromophenol blue (BrB) is an acid-base indicator that has recently been proposed as a promising alternative biostain for vitrectomy, because it stains the ERM and ILM and has induced no damage in either in vitro or in vivo studies. 35,53,54  
Patent blue (PB) exhibits moderate affinity for the ERM and low affinity for the ILM. 54 Little is known about the possible side effects of PB. Some toxic effects have been reported, but the data are conflicting. 55 57  
Brilliant blue G (BBG), or Coomassie blue, is commonly used for protein determination and gel electrophoresis. 58 60 In 2006, this dye was reported to stain the ILM and to have no significant in vivo toxicity. 61 64  
The purpose of the present study was to compare the in vitro effect on human RPE cells of a panel of the dyes most commonly used in vitrectomy. The effects of several concentrations of the dyes were examined, and the same experimental conditions were used for each of the six dyes, thus facilitating a more precise comparison of the specific effects of each dye. 
Methods
Cell Culture
The well-characterized human retinal pigment epithelial cell line ARPE-19 65 was purchased from the American Type Culture Collection (Manassas, VA). The cells were maintained in a 1:1 mixture of Dulbecco's modified Eagle's medium (BE12-709F; Lonza Ibérica, Barcelona, Spain) and Ham's F-12 (FG-0815; Biochrom AG, Berlin, Germany), supplemented with 10% fetal bovine serum (FBS; Lonza Ibérica), 2 mM l-glutamine (BE17-605E, Lonza Ibérica), and 1% penicillin-streptomycin (DE17-602E; Lonza Ibérica) in a humidified 5% CO2 atmosphere at 37°C. The cells were detached with 0.5% trypsin-0.2% ethylene-diamine-tetraacetic acid (EDTA; T4174; Sigma-Aldrich, St. Louis, MO) and subcultivated (1:3–1:4) twice a week. 
First, we studied the proliferation characteristics of ARPE-19 cells to determine the appropriate plating density for each type of assay. For acute toxicity assays, the number of cells per well must be enough to allow detection (i.e., above the limit of detection of the assay method) in the event that cell death occurs. In contrast, for chronic toxicity assays, the cells should be seeded at a density that allows them to proliferate without experiencing contact inhibition forces due to confluence. To this end, we characterized the proliferation of cells seeded at a variety of initial concentrations (cells/well) in 96-well plates (data not shown). On the basis of these studies, we found that it was optimal to seed 104 cells/well for acute toxicity assays (subconfluent) and 3000 cells/well for chronic toxicity assays, since these densities allow the assays to be performed at least 72 hours later under nonconfluent conditions. 
Dye Preparation
Surgical concentrations of each of the six dyes were prepared based on previously published data. However, to characterize the dose dependency of each dye, we also examined the effects of a higher and a lower concentration of each dye. ICG 51,66 (Pulsion Medical Systems, Munich, Germany) and IfCG 42,43 (Laboratories SERB, Paris, France) were used at 2% (20 mg/mL), 0.5% (5 mg/mL), and 0.05% (0.5 mg/mL). TB, 51,66 BrB 35 (Sigma-Aldrich), and PB 55,57 (Fluka, Buchs, Switzerland) were diluted at 1% (10 mg/mL), 0.25% (2 mg/mL), and 0.025% (0,2 mg/mL). Finally, BBG 67 (Sigma-Aldrich) was prepared at 0.2% (2 mg/mL), 0.05% (0.5 mg/mL), and 0.005% (0.05 mg/mL). ICG powder was reconstituted immediately before use, first in phosphate-buffered saline (PBS), and subsequent dilutions were made with a 1:1 mixture of DMEM-F12. IfCG was diluted in a dextrose monohydrate vehicle provided by the manufacturer, and serial dilutions were also made with the same vehicle, because IfCG, being hydrophobic is insoluble in other aqueous-based vehicles, such as PBS. We assayed the cytotoxicity of this vehicle in ARPE-19 cells, and it was found to have none (data not shown). All dilutions of TB, BrB, PB, and BBG were prepared in a DMEM-F12 mixture. 
Each of the six dyes was diluted 1:500, 1:1000, and 1:2000 with respect to the normal surgical concentration, to evaluate the toxicity associated with the chronic presence of residual amounts of dye after surgery. 
Acute Toxicity Evaluation
To evaluate the acute toxicity associated with vital dyes, we reproduced, as much as possible, the conditions used during vitreoretinal surgery. Thus, the ARPE-19 cells were seeded at 104 cells/well in 96-well plates and left to attach to the substrate until the next day. Then, the cells were exposed to 50 μL of each of the just-described solutions for 3 minutes. Afterward, the dyes were removed, and the cells were washed three times with DMEM-F12 and reincubated with fresh culture medium. Cell viability was measured 1.5, 3, and 24 hours later. All experiments were performed in quadruplicate and repeated three times. Results are expressed as the mean percentage ± SD of viable cells with respect to the control cells incubated without dye. 
Since the cytotoxicity of dyes may be photosensitive, we also performed these assays in the presence of a maximum-power 35-W halogen xenon light with 520 lumens. This light source was placed at a distance of 10 mm from the cell cultures. 
Chronic Toxicity Evaluation
Residual dye can persist in the vitreous cavity after surgery if washing is not thoroughly performed. To emulate this condition, we evaluated the chronic toxicity of high dilutions of dye. Cells were seeded at 3000 cells/well in 96-well plates and left to attach until the next day. Then, they were incubated with 200 μL of a dye dilution, which was 1:500, 1:1000, or 1:2000 of the standard surgical concentration of each dye for 0, 24, 48, and 72 hours. Cell viability was subsequently evaluated. All experiments were performed in quadruplicate and repeated three times. The results are expressed as the mean proliferation rate ± SD of viable cells with respect to the control cells at the start of incubation (t = 0 hours). 
Cell Viability Assay
Cell viability was assessed by using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT; Sigma-Aldrich). MTT is reduced by mitochondrial and cytosolic dehydrogenases in living cells to a purple formazan dye that is spectrophotometrically detectable. After exposure to the dye, the cells were incubated at 37°C with 0.5 mg/mL MTT in culture medium. After 3 hours, the MTT solution was removed, and 100 μL/well dimethylsulfoxide was added. Optical densities were determined at 540 nm with a microplate reader (ELx800 Microplate Reader; BioTek Instruments, Winooski, VT). 
IfCG can interfere with the measurement of formazan. This artifact was reduced by measuring the optical density immediately after MTT was added. The results reflected the effect of IfCG before the formazan reaction took place in the cells. These results were subtracted from the optical densities measured after 3 hours' incubation with MTT. 
Light Microscopy
Phase-contrast micrographs were taken to illustrate changes in cell morphology after acute dye exposure. Cells were seeded at 2 × 105 cells/well into 12-well plates and allowed to adhere overnight. Then, the ells were exposed to 250 μL dye solution for 3 minutes, at a final concentration per well equivalent to surgical concentration. After 1.5 hours, cell morphology was observed with a phase-contrast microscope (Eclipse TS 100; Nikon, Tokyo, Japan) at a final magnification of ×100, and images were captured (ProgRes CapturePro, ver. 2.6; Imaging Planet, Goleta, CA). 
Cell Membrane Permeability and Mitochondrial Membrane Potential
Changes in cell membrane permeability (CMP) were evaluated by propidium iodide (PI) incorporation, whereas alterations to mitochondrial membrane potential (ΔΨm) were measured by 3,3′-dihexyloxacarbocyanine iodide (DiOC6(3)) labeling (Invitrogen-Molecular Probes, Eugene, OR). PI stains nuclear DNA and cytoplasmic RNA, but penetrates only the cells that have a permeable cell membrane, which is a manifestation cells as they die. In contrast, ΔΨm decreases before mitochondrial death, and DiOC6(3) is a ΔΨm-sensitive dye. 
At 1.5, 3, and 24 hours after exposure to the surgical concentration of dye in the presence and absence of a halogen xenon light, the cells were incubated with 100 nM DiOC6(3) in culture medium for 20 minutes at 37°C. In this case, we also seeded 2 × 105 cells/well in 12-well plates and exposed them to 250-μL dye solutions to induce cytotoxicity. The cells were then washed and resuspended in fresh medium with 5 μg/mL PI. Fluorescence emission of at least 10,000 viable cells was analyzed by flow cytometry (Epics Elite ESP; Beckman Coulter Corp., Brea, CA). 
Results
Acute Toxicity
Ninety minutes after a 3-minute exposure to the different dyes, the cells showed signs of toxicity. Cell viability, as measured by the MTT assay, was significantly reduced with all dyes in comparison with that of the control cells, which had not been exposed to the dyes (Fig. 1). The degree of reduction of cell viability depended on the dye used (Fig. 1, Table 1), but significant differences were not observed between the different concentrations used, except in the case of BrB, which, at 1%, induced substantial toxicity (22.26% viability; Fig. 1). 
Figure 1.
 
Assessment of the viability of ARPE-19 cells exposed for 3 minutes to different concentrations of six vital dyes (MTT assays). Viability was measured 1.5, 3, and 24 hours after treatment. The results are expressed as the mean percentage ± SD with respect to the control (n = 3 for each case). *Statistically significant differences with respect to control (P ≤ 0.01) and †between concentrations (P ≤ 0.01).
Figure 1.
 
Assessment of the viability of ARPE-19 cells exposed for 3 minutes to different concentrations of six vital dyes (MTT assays). Viability was measured 1.5, 3, and 24 hours after treatment. The results are expressed as the mean percentage ± SD with respect to the control (n = 3 for each case). *Statistically significant differences with respect to control (P ≤ 0.01) and †between concentrations (P ≤ 0.01).
Table 1.
 
Acute Toxicity of Drugs, Measured According to Cell Viability
Table 1.
 
Acute Toxicity of Drugs, Measured According to Cell Viability
Incubation Time (h)
1.5 3 24
Light Light Light
ICG
    0.05% 68.60 ± 26.76 48.68 ± 6.97 93.10 ± 19.45 55.19 ± 11.72 98.56 ± 28.39 99.55 ± 21.40
    0.5% 53.87 ± 23.73 67.94 ± 27.14 78.20 ± 8.63 51.70 ± 18.64 96.28 ± 31.81 87.68 ± 32.89
    2.0% 47.36 ± 15.58 66.52 ± 19.88 80.25 ± 12.90 45.25 ± 19.73 87.26 ± 31.17 96.20 ± 43.64
IfCG
    0.05% 58.58 ± 9.60 54.56 ± 19.77 78.34 ± 7.97 53.29 ± 14.47 77.62 ± 15.52 91.88 ± 20.95
    0.5% 76.30 ± 30.05 102.70 ± 56.73 101.66 ± 21.07 75.51 ± 19.15 84.75 ± 25.15 118.35 ± 26.60
    2.0% 59.82 ± 21.80 71.35 ± 15.73 85.02 ± 35.52 69.34 ± 11.13 63.25 ± 11.10 119.95 ± 12.08
TB
    0.025% 61.77 ± 17.06 65.75 ± 11.54 78.63 ± 15.47 52.56 ± 10.84 82.84 ± 17.20 64.01 ± 8.39
    0.25% 56.62 ± 13.61 60.63 ± 6.00 74.49 ± 8.78 43.28 ± 9.34 80.73 ± 15.76 73.65 ± 14.73
    1.0% 64.38 ± 18.26 58.16 ± 7.76 69.47 ± 17.12 31.70 ± 9.51 72.68 ± 29.68 64.20 ± 23.48
BrB
    0.025% 65.74 ± 18.84 54.49 ± 6.57 82.83 ± 16.41 49.01 ± 13.64 75.42 ± 15.18 72.79 ± 10.45
    0.25% 64.44 ± 19.01 47.48 ± 16.48 88.36 ± 11.67 45.90 ± 11.60 70.26 ± 15.28 75.25 ± 15.26
    1.0% 22.26 ± 10.06 16.32 ± 3.11 31.68 ± 12.46 17.47 ± 8.63 28.17 ± 4.93 33.16 ± 17.83
PB
    0.025% 59.16 ± 19.18 60.47 ± 2.19 92.52 ± 20.94 64.14 ± 19.91 76.81 ± 12.27 80.25 ± 8.39
    0.25% 67.00 ± 16.47 84.21 ± 16.18 86.14 ± 16.92 72.62 ± 23.18 83.88 ± 20.06 89.17 ± 14.73
    1.0% 73.99 ± 18.94 75.70 ± 17.13 78.83 ± 16.96 69.20 ± 24.33 85.85 ± 17.21 86.80 ± 23.48
BBG
    0.005% 59.64 ± 27.37 48.92 ± 15.00 68.06 ± 10.32 43.93 ± 10.76 67.28 ± 6.73 67.19 ± 8.55
    0.05% 55.29 ± 17.69 59.85 ± 11.31 75.11 ± 17.70 47.35 ± 19.14 64.13 ± 6.95 61.04 ± 7.85
    0.2% 46.51 ± 13.38 40.84 ± 12.15 63.94 ± 14.80 36.71 ± 9.48 50.41 ± 6.50 47.03 ± 10.48
Cell viability increased to almost control levels after a 3-hour incubation and was sustained during incubations of up to 24 hours with ICG, IfCG, and PB (Fig. 1, Table 1). Cells treated with TB also experienced increased viability after 3 hours versus 1.5 hours. However, the differences were not statistically significant until 24 hours after dye exposure (82.84%, 80.73%, and 72.68% viability associated with dye concentrations of 0.025%, 0.25%, and 1%, respectively). 
Cells exposed to BrB and BBG did not recover their viability over time. In both cases, a slight transitory recovery of viability was apparent, but by 24 hours, viability levels were similar to those at 1.5 hours. In the case of BBG, a dose of 0.2% reduced viability significantly more than the other two concentrations when the cells were assayed 24 hours later (Fig. 1). 
Influence of Light on Acute Toxicity
Acute toxicity assays were also performed in the presence of light, to simulate intraocular surgery conditions. Significant differences in cell viability were detected when the cells were exposed to the dyes (ICG, IfCG, TB, BrB, and BBG) in the presence of light for 3 minutes and assayed 3 hours later (Fig. 2), but curiously, not after other incubation times (Table 1). In fact, 1.5 and 24 hours after dye exposure, cell viability was found to be similar for the cells incubated in the presence or absence of light at all the concentrations tested (Table 1). Finally, the presence of light had no synergistic effect with the PB dye at any concentration or with surgical and higher concentrations of IfCG 3 hours after exposure to the dye (Fig. 2). 
Figure 2.
 
Assessment of the viability of ARPE-19 cells exposed for 3 minutes to different concentrations of six dyes in the presence or absence of illumination and incubated for a further 3 hours (MTT assays). Solid bars: results without illumination; hashed bars: results with illumination. The results are expressed as the mean percentage ± SD with respect to the control. In each case, n = 3; *statistically significant differences between results in the presence or absence of illumination (P ≤ 0.01).
Figure 2.
 
Assessment of the viability of ARPE-19 cells exposed for 3 minutes to different concentrations of six dyes in the presence or absence of illumination and incubated for a further 3 hours (MTT assays). Solid bars: results without illumination; hashed bars: results with illumination. The results are expressed as the mean percentage ± SD with respect to the control. In each case, n = 3; *statistically significant differences between results in the presence or absence of illumination (P ≤ 0.01).
Alterations in Cell Morphology
ARPE-19 cells typically grow in stable monolayers as flattened cells, exhibiting a hexagonal shape, epithelial-like morphology, and functional polarity. These cells, like their in vivo counterparts, form tight junctions with transepithelial resistance. However, 1.5 hours after exposure to surgical doses of some dyes, the cells exhibited an altered morphology (Fig. 3). We assayed only surgical concentrations of the dyes, because we had not observed significant differences between the three concentrations used in acute toxicity assays, with the exception of BrB. 
Figure 3.
 
Micrographs of monolayers of ARPE-19 cells at the end of 1.5 hours' incubation after exposure to various dyes at concentrations typically used for surgery. Gross morphologic differences between cultures exposed to dyes and control cultures are apparent. The fields in these images are representative of the whole culture. Magnification, ×100.
Figure 3.
 
Micrographs of monolayers of ARPE-19 cells at the end of 1.5 hours' incubation after exposure to various dyes at concentrations typically used for surgery. Gross morphologic differences between cultures exposed to dyes and control cultures are apparent. The fields in these images are representative of the whole culture. Magnification, ×100.
In Figure 3, we can see that ∼50% of the ARPE-19 cells assumed a more rounded shape after exposure to ICG. Cell monolayers exposed to IfCG retained a green film, even after they were rinsed with PBS. The only way to remove this film was by rinsing with the vehicle provided by the manufacturer. These cells presented alterations in their shape and organization. It appeared that exposure to IfCG altered the previous epithelial-like morphology, with cell groups forming a palisade, indicative of cell contact alterations. 
In contrast, TB, BrB, and PB seemed to cause no observable changes in the morphology of the ARPE19 cells. Cell density appeared to be lower after exposure to TB and PB than in control cells, indicative of reduced proliferation. However, the cells that were incubated with a higher concentration (1%) of BrB showed significant morphologic changes. Thus, cells shrank, acquired a more rounded shape, detached from the plates, and exhibited a significant decrease in cell survival. 
Finally, BBG-treated cells appeared to undergo mild morphologic changes, but their shape was not as evidently rounded as that of ICG-treated cells. However, some groups of cells were reminiscent of the palisade formed by the IfCG-treated cells. 
CMP and ΔΨm
We next studied CMP by labeling with PI and the state of the ΔΨm by assessing the incorporation of the ΔΨm-sensitive dye DiOC6(3), with a view toward examining the nature of the toxicity associated with some of the dyes. 
As shown in Table 2, IfCG, BrB, and BBG did not induce significant changes in CMP or in ΔΨm. IfCG appeared to decrease both parameters, but the differences were not statistically significant with respect to the corresponding control. ICG did not appear to affect CMP or ΔΨm 1.5 hours after dye exposure. In contrast, 24 hours after dye exposure in the presence of light, CMP had increased by twofold with respect to control cells; ΔΨm also increased, but not to the same extent (Fig. 4). 
Figure 4.
 
Effect of dye exposure on CMP and ΔΨm. Membrane permeability was measured by labeling with PI 1.5 and 24 hours after exposure to surgical doses of ICG, TB, and PB and 1% BrB (with illumination). The corresponding ΔΨm values were measured by labeling with DiOC6(3) 24 hours after exposure to the dye. Representative histograms of three experiments are shown. The solid curves represent control cells and empty curves represent treated cells. *Statistically significant differences (P ≤ 0.01) with respect to control.
Figure 4.
 
Effect of dye exposure on CMP and ΔΨm. Membrane permeability was measured by labeling with PI 1.5 and 24 hours after exposure to surgical doses of ICG, TB, and PB and 1% BrB (with illumination). The corresponding ΔΨm values were measured by labeling with DiOC6(3) 24 hours after exposure to the dye. Representative histograms of three experiments are shown. The solid curves represent control cells and empty curves represent treated cells. *Statistically significant differences (P ≤ 0.01) with respect to control.
Table 2.
 
CMP and ΔΨm Status after Acute Exposure to Dyes at Normal Surgical Concentrations
Table 2.
 
CMP and ΔΨm Status after Acute Exposure to Dyes at Normal Surgical Concentrations
AFI Incubation Time (h)
1.5 3 24
Light Light Light
ICG
    IP 0.90 ± 0.03 1.29 ± 0.34 1.10 ± 0.13 1.00 ± 0.12 0.81 ± 0.13 2.17 ± 1.70*
    DiOC6(3) 0.83 ± 0.01 1.04 ± 0.29 0.93 ± 0.04 1.06 ± 0.05 0.70 ± 0.03 1.54 ± 0.65
IfCG
    IP 0.71 ± 0.35 0.90 ± 0.31 0.67 ± 0.21 0.59 ± 0.01 0.88 ± 0.27 1.03 ± 0.01
    DiOC6(3) 0.72 ± 0.38 0.81 ± 0.32 0.73 ± 0.26 0.53 ± 0.17 0.72 ± 0.20 1.15 ± 0.20
TB
    IP 1.18 ± 0.04 1.53 ± 0.59* 0.64 ± 0.06 1.05 ± 0.47 0.90 ± 0.16 0.80 ± 0.13
    DiOC6(3) 1.20 ± 0.09 1.40 ± 0.37 0.85 ± 0.05 0.77 ± 0.04 0.79 ± 0.02* 0.63 ± 0.19*
1%BrB
    IP 3.02 ± 0.16* 2.83 ± 0.16* 1.20 ± 0.11 1.11 ± 0.14 1.86 ± 0.28* 1.36 ± 0.34
    DiOC6(3) 0.98 ± 0.01 0.92 ± 0.08 0.96 ± 0.08 0.80 ± 0.15 1.16 ± 0.11 1.14 ± 0.28
BrB
    IP 0.97 ± 0.08 0.90 ± 0.13 0.79 ± 0.12 0.65 ± 0.05 0.62 ± 0.05 0.75 ± 0.11
    DiOC6(3) 0.89 ± 0.10 0.81 ± 0.09 0.81 ± 0.18 0.80 ± 0.20 0.64 ± 0.09 0.84 ± 0.16
PB
    IP 1.58 ± 0.16 1.35 ± 0.59 1.14 ± 0.16 1.41 ± 0.05 1.47 ± 0.39 2.13 ± 0.56*
    DiOC6(3) 1.21 ± 0.13 1.12 ± 0.35 0.97 ± 0.12 0.94 ± 0.20 1.05 ± 0.19 1.57 ± 0.23
BBG
    IP 1.12 ± 0.42 0.96 ± 0.16 0.95 ± 0.14 1.09 ± 0.01 0.82 ± 0.27 0.75 ± 0.54
    DiOC6(3) 0.99 ± 0.19 0.95 ± 0.03 0.96 ± 0.05 0.96 ± 0.09 0.77 ± 0.08 0.83 ± 0.40
In the TB-exposed cells, we observed a discreet increase in CMP at 1.5 hours, especially in the presence of light; but 24 hours later, the values were similar to those of control cells. However, at this time, ΔΨm decreased significantly in the presence and absence of light (Fig. 4, Table 2). 
We also evaluated the effects of a higher concentration of BrB, since we had observed significant effects of this higher dose in acute toxicity assays. At 1.5 hours after exposure to 1% BrB in the presence or absence of light, the cells exhibited a threefold increase in CMP (Fig. 4, Table 2). We observed two subpopulations of cells with different fluorescence intensity, but by 24 hours after dye exposure, there was only one (Fig. 4), consistent with the decrease in CMP (1.86- and 1.36-fold) and with the ΔΨm value's being similar to that found in control cells. 
PB treatment produced an increase in CMP (1.58- and 1.35-fold in the absence and presence of light, respectively), as early as 1.5 hours after exposure. CMP increased to 2.13-fold 24 hours after PB exposure in the presence of light, but not in its absence. ΔΨm was also observed to increase, but to a dissimilar degree (Fig. 4, Table 2). 
Chronic Toxicity
Finally, the cells were chronically exposed to lower concentrations of dyes to simulate the effect of dye persistence in the vitreous cavity after surgery. Prolonged exposure to dilute ICG produced significant toxicity as early as 24 hours' incubation (Fig. 5). The 1:2000 dilution produced a just significant decrease (72.29%) in the cell proliferation rate (Fig. 5), but the 1:500 and 1:1000 dilutions induced massive cell death at 48 and 72 hours of incubation (Fig. 5). 
Figure 5.
 
Assessment of proliferation of ARPE-19 cells after prolonged exposure to high dilutions of six dyes (MTT assay). The results are expressed as the mean proliferation rate ± SD of viable cells with respect to control culture cells at t = 0 hours. In each case, n = 3; *statistically significant differences (P ≤ 0.01) with respect to control.
Figure 5.
 
Assessment of proliferation of ARPE-19 cells after prolonged exposure to high dilutions of six dyes (MTT assay). The results are expressed as the mean proliferation rate ± SD of viable cells with respect to control culture cells at t = 0 hours. In each case, n = 3; *statistically significant differences (P ≤ 0.01) with respect to control.
Exposure to the three dilutions of BrB and BBG resulted in a slightly lower proliferation rate after 72 hours of incubation (Fig. 5). Chronic exposure to TB was initially associated with a mild inhibition of proliferation, but 72 hours later, the cells had resumed a proliferation rate that was similar to that of the control cells (Fig. 5). 
Prolonged exposure to dilutions of 1:500, 1:1000, and 1:2000 of IfCG and PB did not produce any differences in cell proliferation with respect to control cell cultures (Fig. 5). 
Discussion
Since the introduction of ICG in vitrectomy, it has been widely accepted that vital dyes represent useful agents to improve visualization and peeling of the ILM. However, over the past few years, controversial evidence has accumulated indicating that these dyes may have harmful effects. The discrepancies among these reports are due principally to the fact that each study used different manufacturers, conditions, dilution procedures, exposure times, illumination, and cell models, among other differences. 
In this study, we ruled out these variables as possible causes for variations in results, by examining six vital dyes at the same time and under the same conditions. This experimental design allowed us to focus on the specific biological effects associated with each of the dyes and to determine which dye may be more appropriate in a given circumstance, on the basis of the characteristic effects and risks associated with each dye. The experimental conditions that we used mimic surgical conditions as closely as possible: We used short exposure times (3 minutes) and dye concentrations that are typically administered to patients during surgery. We also examined the effects of concentrations that were higher and lower than those routinely used in surgery, to determinate not only cell viability but also cell morphology and functional status. Finally, we assayed the effects of chronic exposure to highly diluted dyes to simulate the effect on RPE cells of residues of dye persisting in the vitreous cavity after surgery. 
Indocyanine Green
ICG was the first dye to be used for intraocular surgery and is currently the dye of choice, having been more extensively characterized than any other dye. However, it is also one of the most controversial dyes in use today, having yielded contradictory results, even under similar conditions. The present study corroborates those that have reported that partial toxic effects are associated with this dye. Thus, we observed a reduction in cell viability after the cells had been exposed to ICG, but then, surprisingly, the cells recovered viability. To resolve this paradox, it should be borne in mind that the MTT method, which is frequently used in in vitro studies to determine toxicity, measures alterations in mitochondrial enzymes that metabolize the MTT reagent. 50 Therefore, low viability according to the MTT method does not necessarily mean that cells are irreversibly on the road to cell death. Moreover, it has been demonstrated that a 3-minute exposure to 2.5 mg/mL ICG suppresses cell growth, but in the absence of signs of necrosis, 68 further supporting the idea that a low MTT value does not necessarily reflect an irreversible loss of cell viability. 
Some investigators have reported that the toxic effects of ICG are time and dose dependent. 68,69 However, our results indicate that the effects of ICG are not strongly dose dependent, at least at the physiological concentrations examined. We did find, however, that ICG's effects are photosensitive. Nevertheless, this photosensitivity, rather than indicating a loss of cell viability may be due to a delay in cell recuperation (due to a longer arrest of the cell cycle in the presence of light 23 ), since differences in viability are minimal by 24 hours after exposure. In keeping with this idea, we did not observe signs of cell death by phase-contrast microscopy. Rather, we saw a noticeable change in morphology, which some researchers have associated with an increase in the expression of apoptotic genes. 67,70  
To clarify the nature of the biological processes that underlie the toxicity associated with the dyes, we measured cell membrane permeability and mitochondrial function; however, the results were somewhat contradictory. Thus, shortly after ICG exposure, despite the fact that we had measured reduced viability according to the MTT method, both membrane permeability and mitochondrial function were essentially unchanged. However, 24 hours after dye exposure in the presence of light, we found that there was no mitochondrial dysfunction; rather, cell membrane permeability had increased to such an extent that if it continued, it would probably have led to the death of the cells within this culture, as has been reported elsewhere. 23,68 This interpretation, moreover, is consistent with our results from chronic toxicity assays. 
It has already been observed that small quantities of ICG may remain within the cytosol and in cell membranes after exposure, even after 6 weeks, despite extensive washing and that this residual dye may have harmful effects. 14,30 Our study also points to a potential risk associated with these residual amounts of ICG; however, at a 1:2000 dilution, we observed only a delay in cell proliferation. These controversial results support the findings by some, 43,69 but contrast with those reported by others, 50 thus contributing to the essentially controversial nature of this dye. 
Infracyanine Green
IfCG has been the object of only a few studies, despite the fact that its use was introduced shortly after ICG and that it has been recommended for reducing the risk of toxic effects due to the hypo-osmolarity of the ICG vehicle. 39 Even today, it is still considered that IfCG, due to the absence of iodine in its formulation, has reduced adverse effects. 71,72 Nevertheless, some groups have also reported that ICG and IfCG have similar effects. 43  
In the present study, we did not find evidence of significant acute toxicity associated with IfCG. It is possible that mitochondrial metabolism is altered initially, but it recuperates later (MTT assay observations; Fig. 1). Similarly, the effect of light on IfCG incubation seemed to be negligible. It is important to remember that IfCG can interfere in the MTT assay. For this reason, MTT assay results associated with IfCG should be readjusted by subtracting the baseline value that is obtained before the MTT reaction takes place. On the other hand, it is possible that the PBS used for the washings after exposure to IfCG can provoke flocculation of residual IfCG. It would be more appropriate to wash with a glucose solution, but that would not mimic the saline solutions used in the clinical context for infusion of the vitreous. 
Finally, it should be remembered that IfCG can be phagocytosed by RPE cells, remaining in the interior of these cells for long periods, with a risk of inducing chronic toxicity. 43 However, we did not find any signs of toxicity associated with low concentrations IfCG (Fig. 5). In contrast, we did observe changes in cell morphology and in intercellular unions (Fig. 3), which could be due to the accumulation of low concentrations of IfCG in the cell interior. These morphologic alterations may well be due to changes in the permeability of the plasma membrane, since there is less incorporation of PI than in control cells, particularly at shorter times after exposure. However, DiOC6(3) incorporation was also reduced, indicative of reduced mitochondrial membrane activity. Nevertheless, the differences with respect to control cultures and those treated with IfCG were not found to be statistically significant. 
Trypan Blue
The effects of TB have been well characterized, perhaps because it is the second most commonly used dye. However, it seems clear that its use should be restricted to the staining of the ERM, 36,69,71 since it does not stain as well as ICG. As with ICG, there is little consensus about its effects. 51,66,69 Thus, after TB exposure, we observed an initial statistically significant decrease in cell viability (Fig. 1), probably associated with reduced mitochondrial activity. However, viability recovered 24 hours later. Curiously enough, not all cells appeared to recover, indicating that the remaining percentage of cells (∼20%) may have undergone cell death, as reported by others. 23 This process seems to be aggravated by the presence of light, since TB's effects exhibit marked photosensitivity. This reduction in the number of cells with respect to the control culture was also apparent in the phase-contrast images. In keeping with these observations, we found that the plasma membrane permeability of a percentage of cells increased initially and was further enhanced in the presence of light (Table 2). Subsequently, mitochondrial function decreased significantly, possibly due to the percentage of cells that had undergone apoptosis. 
Regarding the chronic toxicity of TB, it has been reported that it induces arrest of the cell cycle at G0–G1 via increased expression of p21. 73 This effect was found to be more evident during the first 2 days and less significant from the sixth day on. 73 Our results showing growth delay due to chronic exposure to TB corroborate these findings. Indeed, we found growth delay to be statistically significant 24 hours after incubation, but not subsequently. 
Bromphenol Blue
BrB has not been used widely as a dye in ophthalmology. The few available studies have not revealed any toxic effects in vitro, 35 or in the retina or lens at low concentrations (0.2%). However, at this concentration, the dye does not stain very intensely. 53,54 In contrast, we found that concentrations ≤0.25% (concentrations used in the clinical setting) led to reduce RPE cell viability. Moreover, this reduction was enhanced significantly in the presence of light (Fig. 2), but surprisingly, was not accompanied by visible changes in culture morphology. This finding suggests that the observed effects of the BrB dye may be due to reduced MTT metabolism, consistent with our data regarding the status of the membranes (Table 2). 
The situation is radically different when higher concentrations of BrB were used to increase the intensity of the staining. Thus, after exposure to 1% BrB, massive cell death was found and there were no signs of recuperation according to microscopy and flow cytometry. The mean PI fluorescence intensity initially revealed two distinct populations; later, one of these subpopulations disappeared, suggesting a cellular explosion that is typical of necrosis. This subpopulation may represent the cells that had exhibited reduced viability. It is likely that these cells died by necrosis, since no changes were observed in DiOC6(3), a marker of apoptotic processes. For these reasons, we highlight the importance of careful management of concentrations of this dye in particular, since high concentrations may have significant toxic effects. 
As occurred with other dyes, small traces of BrB were found to delay the rhythm of cell growth. It would be interesting to evaluate whether BrB induces arrest of the cell cycle at G0–G1 via increased expression of p21. 
Patent Blue
The utility of the PB dye for applications in ophthalmology has only recently been discovered, and relatively little is known about its effects. For this reason, the present findings are of particular interest. 
We found that PB initially induced a statistically significant decrease in viability (but the cells subsequently recuperated), an apparent reduction in cell density and, surprisingly, a slight increase in cell membrane permeability. Taken together, these findings indicate that, after exposure to PB, a small percentage of cells may die by necrosis (indicated by increased PI staining); however, the extent of necrosis does not appear to significantly affect culture viability, since cell viability was not found to be significantly reduced 3 hours after exposure to PB. In addition, cell viability assays revealed that the presence of light exerted no significant effect on viability associated with PB. Nevertheless, the significant increase in plasma membrane permeability 24 hours after dye exposure in the presence of light (Fig. 4, Table 2) brings into question the photosensitivity of the effects of PB on RPE cells. 
Brilliant Blue G
Useful applications of BBG in the area of ophthalmology were discovered not long ago. 61 Since then, its security has been demonstrated in both in vitro and in vivo studies. 61 63 However, a decrease in cell viability, similar to that which we observed, with the same exposure time (3 minutes) and with similar concentrations, has been reported. 69 This reduction has been attributed to a cytostatic effect, 67 since it has been shown that BBG acts as a P2X7 receptor antagonist, leading to reduced cell growth. 74 This feature may contribute to a beneficial postoperative effect by reducing the formation of fibrous material. 
In other studies of BBG, the influence of light on BBG's effects was not examined. We found BBG to be mildly phototoxic and to induce variations in the morphologic aspect of cultures. However, the cells appeared to be healthy, and no significant changes were observed by flow cytometry (Table 2). Thus, our findings corroborate the reports that BBG does not lead to apoptosis or necrosis and provide further support for the idea that reduced cell viability, as measured in MTT assays, represents a delay in cell growth rather than the induction of death. 
In contrast to other studies of the chronic toxicity of BBG, we found only a slight reduction in growth rate associated with this dye. 69 The reduced effect could have occurred because BBG is more hydrosoluble than ICG and IfCG; it would thus penetrate less into the cells and be more easily washed away, leaving less residues after surgery. 
Results obtained in vitro cannot necessarily be extrapolated to in vivo clinical situations. Nevertheless, we consider that the in vitro finding that all dyes have the capacity to induced at least growth arrest, strongly suggests that caution should be exercised when using these dyes in the clinical setting. Residual amounts of dye can be retained in the vitreous cavity, even after its irrigation and this can lead to chronic cytotoxic effects, especially with low water-soluble dyes (i.e., hydrophobic dyes such as ICG). Thus, the vitreous cavity should be copiously and thoroughly irrigated after staining, to minimize the risk of chronic cytotoxicity. 
Finally, our results suggest that the dyes routinely used in ophthalmic surgery may exert toxic effects, not only by altering cell viability, but also by perturbing cell morphology and membrane status, since the dyes were found to lead to reduced or altered functional and metabolic capacity, with substantial consequences for cell behavior in vitro. In light of these studies, we consider it prudent to reduce the duration of exposure to these six dyes during surgery to the extent possible and thus minimize the risk of inducing toxic effects. 
Footnotes
 Disclosure: M.-C. Morales, None; V. Freire, None; A. Asumendi, None; J. Araiz, None; I. Herrera, None; G. Castiella, None; I. Corcóstegui, None; G. Corcóstegui, None
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Figure 1.
 
Assessment of the viability of ARPE-19 cells exposed for 3 minutes to different concentrations of six vital dyes (MTT assays). Viability was measured 1.5, 3, and 24 hours after treatment. The results are expressed as the mean percentage ± SD with respect to the control (n = 3 for each case). *Statistically significant differences with respect to control (P ≤ 0.01) and †between concentrations (P ≤ 0.01).
Figure 1.
 
Assessment of the viability of ARPE-19 cells exposed for 3 minutes to different concentrations of six vital dyes (MTT assays). Viability was measured 1.5, 3, and 24 hours after treatment. The results are expressed as the mean percentage ± SD with respect to the control (n = 3 for each case). *Statistically significant differences with respect to control (P ≤ 0.01) and †between concentrations (P ≤ 0.01).
Figure 2.
 
Assessment of the viability of ARPE-19 cells exposed for 3 minutes to different concentrations of six dyes in the presence or absence of illumination and incubated for a further 3 hours (MTT assays). Solid bars: results without illumination; hashed bars: results with illumination. The results are expressed as the mean percentage ± SD with respect to the control. In each case, n = 3; *statistically significant differences between results in the presence or absence of illumination (P ≤ 0.01).
Figure 2.
 
Assessment of the viability of ARPE-19 cells exposed for 3 minutes to different concentrations of six dyes in the presence or absence of illumination and incubated for a further 3 hours (MTT assays). Solid bars: results without illumination; hashed bars: results with illumination. The results are expressed as the mean percentage ± SD with respect to the control. In each case, n = 3; *statistically significant differences between results in the presence or absence of illumination (P ≤ 0.01).
Figure 3.
 
Micrographs of monolayers of ARPE-19 cells at the end of 1.5 hours' incubation after exposure to various dyes at concentrations typically used for surgery. Gross morphologic differences between cultures exposed to dyes and control cultures are apparent. The fields in these images are representative of the whole culture. Magnification, ×100.
Figure 3.
 
Micrographs of monolayers of ARPE-19 cells at the end of 1.5 hours' incubation after exposure to various dyes at concentrations typically used for surgery. Gross morphologic differences between cultures exposed to dyes and control cultures are apparent. The fields in these images are representative of the whole culture. Magnification, ×100.
Figure 4.
 
Effect of dye exposure on CMP and ΔΨm. Membrane permeability was measured by labeling with PI 1.5 and 24 hours after exposure to surgical doses of ICG, TB, and PB and 1% BrB (with illumination). The corresponding ΔΨm values were measured by labeling with DiOC6(3) 24 hours after exposure to the dye. Representative histograms of three experiments are shown. The solid curves represent control cells and empty curves represent treated cells. *Statistically significant differences (P ≤ 0.01) with respect to control.
Figure 4.
 
Effect of dye exposure on CMP and ΔΨm. Membrane permeability was measured by labeling with PI 1.5 and 24 hours after exposure to surgical doses of ICG, TB, and PB and 1% BrB (with illumination). The corresponding ΔΨm values were measured by labeling with DiOC6(3) 24 hours after exposure to the dye. Representative histograms of three experiments are shown. The solid curves represent control cells and empty curves represent treated cells. *Statistically significant differences (P ≤ 0.01) with respect to control.
Figure 5.
 
Assessment of proliferation of ARPE-19 cells after prolonged exposure to high dilutions of six dyes (MTT assay). The results are expressed as the mean proliferation rate ± SD of viable cells with respect to control culture cells at t = 0 hours. In each case, n = 3; *statistically significant differences (P ≤ 0.01) with respect to control.
Figure 5.
 
Assessment of proliferation of ARPE-19 cells after prolonged exposure to high dilutions of six dyes (MTT assay). The results are expressed as the mean proliferation rate ± SD of viable cells with respect to control culture cells at t = 0 hours. In each case, n = 3; *statistically significant differences (P ≤ 0.01) with respect to control.
Table 1.
 
Acute Toxicity of Drugs, Measured According to Cell Viability
Table 1.
 
Acute Toxicity of Drugs, Measured According to Cell Viability
Incubation Time (h)
1.5 3 24
Light Light Light
ICG
    0.05% 68.60 ± 26.76 48.68 ± 6.97 93.10 ± 19.45 55.19 ± 11.72 98.56 ± 28.39 99.55 ± 21.40
    0.5% 53.87 ± 23.73 67.94 ± 27.14 78.20 ± 8.63 51.70 ± 18.64 96.28 ± 31.81 87.68 ± 32.89
    2.0% 47.36 ± 15.58 66.52 ± 19.88 80.25 ± 12.90 45.25 ± 19.73 87.26 ± 31.17 96.20 ± 43.64
IfCG
    0.05% 58.58 ± 9.60 54.56 ± 19.77 78.34 ± 7.97 53.29 ± 14.47 77.62 ± 15.52 91.88 ± 20.95
    0.5% 76.30 ± 30.05 102.70 ± 56.73 101.66 ± 21.07 75.51 ± 19.15 84.75 ± 25.15 118.35 ± 26.60
    2.0% 59.82 ± 21.80 71.35 ± 15.73 85.02 ± 35.52 69.34 ± 11.13 63.25 ± 11.10 119.95 ± 12.08
TB
    0.025% 61.77 ± 17.06 65.75 ± 11.54 78.63 ± 15.47 52.56 ± 10.84 82.84 ± 17.20 64.01 ± 8.39
    0.25% 56.62 ± 13.61 60.63 ± 6.00 74.49 ± 8.78 43.28 ± 9.34 80.73 ± 15.76 73.65 ± 14.73
    1.0% 64.38 ± 18.26 58.16 ± 7.76 69.47 ± 17.12 31.70 ± 9.51 72.68 ± 29.68 64.20 ± 23.48
BrB
    0.025% 65.74 ± 18.84 54.49 ± 6.57 82.83 ± 16.41 49.01 ± 13.64 75.42 ± 15.18 72.79 ± 10.45
    0.25% 64.44 ± 19.01 47.48 ± 16.48 88.36 ± 11.67 45.90 ± 11.60 70.26 ± 15.28 75.25 ± 15.26
    1.0% 22.26 ± 10.06 16.32 ± 3.11 31.68 ± 12.46 17.47 ± 8.63 28.17 ± 4.93 33.16 ± 17.83
PB
    0.025% 59.16 ± 19.18 60.47 ± 2.19 92.52 ± 20.94 64.14 ± 19.91 76.81 ± 12.27 80.25 ± 8.39
    0.25% 67.00 ± 16.47 84.21 ± 16.18 86.14 ± 16.92 72.62 ± 23.18 83.88 ± 20.06 89.17 ± 14.73
    1.0% 73.99 ± 18.94 75.70 ± 17.13 78.83 ± 16.96 69.20 ± 24.33 85.85 ± 17.21 86.80 ± 23.48
BBG
    0.005% 59.64 ± 27.37 48.92 ± 15.00 68.06 ± 10.32 43.93 ± 10.76 67.28 ± 6.73 67.19 ± 8.55
    0.05% 55.29 ± 17.69 59.85 ± 11.31 75.11 ± 17.70 47.35 ± 19.14 64.13 ± 6.95 61.04 ± 7.85
    0.2% 46.51 ± 13.38 40.84 ± 12.15 63.94 ± 14.80 36.71 ± 9.48 50.41 ± 6.50 47.03 ± 10.48
Table 2.
 
CMP and ΔΨm Status after Acute Exposure to Dyes at Normal Surgical Concentrations
Table 2.
 
CMP and ΔΨm Status after Acute Exposure to Dyes at Normal Surgical Concentrations
AFI Incubation Time (h)
1.5 3 24
Light Light Light
ICG
    IP 0.90 ± 0.03 1.29 ± 0.34 1.10 ± 0.13 1.00 ± 0.12 0.81 ± 0.13 2.17 ± 1.70*
    DiOC6(3) 0.83 ± 0.01 1.04 ± 0.29 0.93 ± 0.04 1.06 ± 0.05 0.70 ± 0.03 1.54 ± 0.65
IfCG
    IP 0.71 ± 0.35 0.90 ± 0.31 0.67 ± 0.21 0.59 ± 0.01 0.88 ± 0.27 1.03 ± 0.01
    DiOC6(3) 0.72 ± 0.38 0.81 ± 0.32 0.73 ± 0.26 0.53 ± 0.17 0.72 ± 0.20 1.15 ± 0.20
TB
    IP 1.18 ± 0.04 1.53 ± 0.59* 0.64 ± 0.06 1.05 ± 0.47 0.90 ± 0.16 0.80 ± 0.13
    DiOC6(3) 1.20 ± 0.09 1.40 ± 0.37 0.85 ± 0.05 0.77 ± 0.04 0.79 ± 0.02* 0.63 ± 0.19*
1%BrB
    IP 3.02 ± 0.16* 2.83 ± 0.16* 1.20 ± 0.11 1.11 ± 0.14 1.86 ± 0.28* 1.36 ± 0.34
    DiOC6(3) 0.98 ± 0.01 0.92 ± 0.08 0.96 ± 0.08 0.80 ± 0.15 1.16 ± 0.11 1.14 ± 0.28
BrB
    IP 0.97 ± 0.08 0.90 ± 0.13 0.79 ± 0.12 0.65 ± 0.05 0.62 ± 0.05 0.75 ± 0.11
    DiOC6(3) 0.89 ± 0.10 0.81 ± 0.09 0.81 ± 0.18 0.80 ± 0.20 0.64 ± 0.09 0.84 ± 0.16
PB
    IP 1.58 ± 0.16 1.35 ± 0.59 1.14 ± 0.16 1.41 ± 0.05 1.47 ± 0.39 2.13 ± 0.56*
    DiOC6(3) 1.21 ± 0.13 1.12 ± 0.35 0.97 ± 0.12 0.94 ± 0.20 1.05 ± 0.19 1.57 ± 0.23
BBG
    IP 1.12 ± 0.42 0.96 ± 0.16 0.95 ± 0.14 1.09 ± 0.01 0.82 ± 0.27 0.75 ± 0.54
    DiOC6(3) 0.99 ± 0.19 0.95 ± 0.03 0.96 ± 0.05 0.96 ± 0.09 0.77 ± 0.08 0.83 ± 0.40
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