For Peer Review Effects Of Light-Emitting Diode Radiations On Human Retinal Pigment Epithelium Cells In Vitro Journal: Photochemistry and Photobiology Manuscript ID: Draft Wiley - Manuscript type: Research Article Date Submitted by the Author: n/a Complete List of Authors: Chamorro, Eva; Universidad Complutense de Madrid, Neuro-Computing and Neuro-Robotics Research Group Bonnin-Arias, Cristina; Universidad Complutense de Madrid, Neuro- Computing and Neuro-Robotics Research Group Pérez-Carrasco, Maria; Universidad Complutense de Madrid, Optometry and Vision Muñoz-Luna, Javier; Universidad Complutense de Madrid, Optics Vazquez-Molini, Daniel; Universidad Complutense de Madrid, Optics Sánchez-Ramos, Celia; Universidad Complutense de Madrid, Neuro- Computing and Neuro-Robotics Research Group Keywords: LED lighting, Light damage, Retinal Pigment Epithelium Photochemistry and Photobiology For Peer Review 1 Effects Of Light Emitting Diodes Radiations On Human Retinal 1 Pigment Epithelial Cells In Vitro 2 3 Eva Chamorro*1, Cristina Bonnin-Arias1, María Jesús Pérez-Carrasco2, Javier Muñoz 4 de Luna3, Daniel Vázquez3, Celia Sánchez-Ramos1,2 5 1. Neuro-Computing and Neuro-Robotics Research Group, Universidad Complutense de 6 Madrid, 2.Optometry and Vision Department, Escuela Universitaria de Óptica, 3.Optics 7 Department, Escuela Universitaria de Óptica. 8 *Corresponding author e-mail: eva.chamorro@opt.ucm.es (Eva Chamorro) 9 10 Page 1 of 18 Photochemistry and Photobiology For Peer Review 2 ABSTRACT 11 To investigate the effect of LED lighting on RPE cells, HRPEpiC cells were exposed to 3 12 light-darkness cycles (12hours/12hours) with blue light (468nm), green light (525nm), red 13 light (616nm). Cellular viability of HRPEpic cells was evaluated by labeling all nuclei with 14 DAPI, ROS production was determined by the H2DCFDA staining and fluorescence 15 microscopy, Mitochondrial membrane potential was quantified by the TMRM staining and 16 fluorescence microscopy, DNA damage was determined by the activation of H2AX histone 17 and apoptosis was evaluated by the activation of caspases-3, -7. The clearly show that LED 18 lighting radiation decrease 75-99% cellular viability and increase 66-89% cellular apoptosis, as 19 well as, increase the production of ROS and cause DNA damage. This study indicates that 3 20 light-darkness cycles (12hours/12hours) exposure to LED lighting affect to in vitro RPE cells. 21 22 Page 2 of 18Photochemistry and Photobiology For Peer Review 3 INTRODUCTION 23 Visible light spectrum can be absorbed by biologic chromophores in RPE cells, causing 24 cellular dysfunction and even death of cells (6). The blue region of the spectrum (400-500 nm) 25 although it is out of the UV-A range (1) has relatively high energy and can penetrate through 26 tissues to cells and their organelles. Cell culture studied revealed that blue light directly 27 induces the production of reactive oxygen species (ROS) in RPE mitochondria and leads to 28 apoptosis, potentially triggered by ROS damage mitochondrial DNA (10). 29 Human observers are exposed to a limited numbers of natural and artificial lights. Light 30 pollution is increasing exponentially in industrialized countries, with more sophisticated light 31 sources, with specific spectra and high intensities. LEDs, or Light-Emitting Diodes, were 32 developed as an energy-efficient option to traditional light bulbs. In the coming years, the light 33 output of LEDs will continue to increase, enabling to progressively suppress the least efficient 34 light sources and replace them by Light-Emitting Diodes (LED). By the first of September 35 2016, no more incandescent lights will be available in Europe for domestic lighting, and 36 inorganic or organic LEDs could become the next generation light sources (3). 37 The potential risks of these new light sources need to be explored. Due to specific spectral and 38 energetic characteristics of white LEDs as compared to other domestic light sources, some 39 concerns have been raised regarding their safety for human health and particularly potential 40 harmful risks for the eye (3). 41 The purpose of the study was to study the effects of LED lighting on RPE cells. Outcome 42 measures included cell viability, oxidative stress, mitochondrial membrane potential, DNA 43 damage and apoptosis. 44 Page 3 of 18 Photochemistry and Photobiology For Peer Review 4 MATERIALS AND METHODS 45 Cell culture of human RPE: The human retinal pigment epithelial cell line HRPEpiC 46 (ScienceCell Research Laboratories, USA), was grown in a low-serum epithelial cell culture 47 medium (ScienceCell Research Laboratories, USA). After primary cultures became confluent, the 48 cells were detached from the culture dish with the use of the Trypsin/EDTA solution (Sigma-49 Aldrich, USA). Cells were plated on 96well, black clear Imaging Plate (Becton, Dickinson and 50 Company, USA) with Poly-L-Lysine (Sigma-Aldrich, USA) Coating (density = 5000cells/well). 51 The cells were incubated in a humidified atmosphere of 5% CO2 and 95% air at 37ºC, and the 52 culture medium was changed every 24 h. 53 Light exposure: Illumination was produced by a LED-based system. Cells plated on imaging 54 plate were exposed to 3 light-darkness cycles (12hours/12hours) with blue light (468nm), green 55 light (525nm), red light (616nm) or white light in well chambers (light intensity was 5mW/cm2). 56 Although this value is not very frecuent in daylive situations it can be found in several cases, 57 beside we have select this value in order to compare with other studies about this subject (9, 11) 58 This value imply 34.150 lux for an incandescent lightsource or 33.446 lx for a D65 (skylight) 59 lightsource. It is similar to the horizontal irradiance for example for a lying person looking up for 60 clear sky day when the sun is around 37,5º(12) or a person at 20 cm of a 100w incandescent 61 lamp(2) 62 Control group consisted of RPE cells kept in the dark. Figure 1 shows a schematic diagram of the 63 LED lighting irradiation system and spectral irradiance of LED lighting. 64
65 Cell viability: Cell nuclei were labeled by incubating the cells with the nuclear stain 4′6-66 Page 4 of 18Photochemistry and Photobiology For Peer Review 5 diamidine-2-phenylindole dihydrochloride, DAPI, (Sigma-Aldrich, USA) for 1 hour. The viable 67 cells were counted under a BD Pathway 855 fluorescence microscope (Becton, Dickinson and 68 Company, USA) and analysis of the image data was performed using Attovision software (Becton, 69 Dickinson and Company, USA). 70 Measurement of intracellular ROS production: Oxydative stress was measure by using the 71 dye (5-(and-6)chloromethyl-2’,7’-dichlorodihydrofluorescein diactate acetyl ester H2DCFDA 72 (Invitrogen, Germany) at a final concentration of 1:1000 for 30 min at 37 C in the dark. Excess 73 dye was removed by washing in PBS. Fluorescence intensity was measured in a BD Pathway 855 74 Bioimager (Becton, Dickinson and Company, USA) using an excitation band pass filter at 492-75 495nm and an emission cutoff filter at 517-527nm. 76 Measurement of mitochondrial membrane potential (MMA): Mitochondrial damage was 77 assessed by using the dye Tetramethylrhodamine, methyl ester, TMRM (Invitrogen, Germany) at a 78 final concentration of 1:1000 for 30 min at 37 C in the dark. Excess dye was removed by washing 79 in PBS. Fluorescence intensity was measured in a BD Pathway 855 Bioimager (Becton, Dickinson 80 and Company, USA) using an excitation band pass filter at 549 nm and an emission cutoff filter at 81 572 nm. 82 Immunocytochemical Detection of Histone H2AX and Activated Caspase-3 and -7: 83 DNA damage and apoptosis was evaluated by immunocytochemistry, evaluating the activation of 84 histone H2AX and caspases-3 and -7. At designated time period, cells were washed with 85 phosphate-buffered saline, (PBS, Sigma-Aldrich, USA) and fixed with 4% paraformaldehyde 86 (Sigma-Aldrich, USA) for 1 hour. Cells were suspended in 0.3% Triton X-100-PBS (Sigma-87 Aldrich, USA) in a 3% Bovine Serum Albumin, BSA (Sigma-Aldrich, USA) 1% (w/v) in PBS for 88 30 min to suppress. The cells were then incubated in 2.5% PBS+BSA containing either a 89 Page 5 of 18 Photochemistry and Photobiology For Peer Review 6 combination of 1:400 diluted antiphospho-histone H2AX (Abcam, UK) and 1:400 anticaspase-3 90 rabbit antibody (Cell Signaling Technology, USA). The cells were then incubated for 1 hour and 91 washed twice with PBS, and resuspended in 1:400 diluted goat antimouse Alexa Fluor 633 92 conjugated (Invitrogen, Germany) and 1:400 diluted goat antirabbit Alexa Fluor 488 (Invitrogen, 93 Germany) for 30 min at room temperature in the dark. After three washing steps, the fluorescence 94 of the samples was measured in the Pathway 855 automated fluorescence microscope (Becton, 95 Dickinson and Company, USA) using an excitation band pass filter at 632 nm and an emission 96 cutoff filter at 647 nm for caspase -3,-7 detection. For histone H2AX detection, an excitation band 97 pass filter at 488 nm and an emission cutoff filter at 594 nm were used. 98 Statistical analysis: Each experiment was repeated three times. The values were given as 99 mean±SD. Data were analyzed using an unpaired two-tailed t-test by Statgraphics version 100 Centurion XVI.I (USA). A p value less than 0.05 was considered statistically significant. 101 RESULTS 102 Cell viability 103 Nonirradiated RPE cells grew well but irradiation inhibited the growth of RPE cells. The 104 difference in cell number of RPE cells irradiated by blue, green or white LED lighting and not 105 irradiated was statistically very significant (p<0.01). Maximum damage was observed in cells 106 exposed to blue LED lighting. In the experiments, 99%, 88% and 75% of the cells irradiated 107 became nonviable after blue, green or white light. Red light caused a slight decrease of number of 108 RPE cells. However, the difference in cell number of RPE cells irradiated by red light and not 109 irradiated was statistically nonsignificant (Figures 3A and 4A). 110 111 Page 6 of 18Photochemistry and Photobiology For Peer Review 7 Measurement of intracellular ROS production 112 Low level production of reactive oxygen species was observed in RPE cells maintained in the 113 dark. However, a significant increase in the level of ROS was observed after 3 light-darkness 114 cycles (12hours/12hours) with blue light, green light or red light. Non increase of cellular 115 cytoplasm fluorescence was detected in cells exposed to white LED lighting in comparison with 116 non irradiated cells (Figures 2A, 3B and 4B). 117 Measurement of mitochondrial membrane potential 118 After 3 light-darkness cycles of irradiation, no significant effect on mitochondrial membrane 119 potential was detectable compared to control cells for any of the different LED lighting (Figures 120 2B, 3C and 4C). 121 Effects of light on DNA damage of RPE 122 Significant DNA damage was observed for light exposed RPE cells. The fluorescence microscopic 123 data for all irradiated RPE cells show the increased degradation of nucleic acids in comparison 124 with the control cells. Maximum damage was showed to cells exposed to blue LED lighting 125 (Figures 2C, 3D and 4D). 126 Detection of apoptosis 127 Percentage of apoptotic cells was increased on light exposed RPE cells in comparison with RPE 128 cells maintained in the dark. The death of non irradiated RPE cells reached a frequency of 3.7%. 129 However, cell death was 86%, 84%, 66% and 89% for blue, green, red and white irradiated RPE, 130 respectively (Figures 2D, 3E and 4E). 131
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134 DISCUSSION 135 Epidemiological studies suggest an association between visible-light exposure and increased 136 risk of advanced age-related macular degeneration (AMD). Visible light can affect the retina 137 and RPE by photochemical, thermal and mechanical mechanism.(13). Experimental evidence 138 has demonstrated that the retina and RPE are much more sensitive to blue light damage than 139 red or green light (7, 8, 5). The most researches have been focused on evaluate the response of 140 the retina to light from conventional lighting sources as halogen or fluorescent. 141 It has been speculated that LED lighting radiation may cause ocular damage (3), however, the 142 potential risks of these new light sources has not been explored. In this study, we have 143 demonstrated that LED lighting is able to damage RPE cells. The results of this study clearly 144 show that LED lighting radiation decrease 75-99% cellular viability and increase 66-89% 145 cellular apoptosis, as well as, increase the production of ROS and cause DNA damage. 146 The present results are consistent with previous reports that suggest that visible light of 147 conventional light sources could be ( por ser algo más dilomatico?) able to cause cell damage. 148 Sparrow et al (11) analyzed human RPE cells irradiated with blue light (430nm, 8mW/cm2), 149 green light (550nm, 8mW/cm2) and white light (246 mW/cm2).The light was delivered from a 150 tungsten halogen source for 20 minutes and it was observed that illuminated RPE cells 151 remained viable. In another study, Godley et al (6) exposed confluent cultures of human 152 primary retinal epithelial cells to visible light (390-550nm at 2.8mW/cm2) of a metal halide 153 Page 8 of 18Photochemistry and Photobiology For Peer Review 9 lamp for 0-9 hours and analyzed cell viability and ROS production. Cells maintained in the 154 absence of blue light exposure showed no decrease in viability, no mitochondrial or nuclear 155 DNA damage and low level production of ROS; however blue light-irradiated cells showed an 156 increasing loss of viability (approximately 10%), time-dependent increase in the levels of ROS 157 and maximal mitochondrial DNA damage 3 hours after exposure with evidence of some repair 158 mechanism. 159 On the other hand, Chu et al (4) studied changes on viability of RPE as a result of blue and red 160 halogen light irradiation. Early passages of human RPE cells were exposed to blue light 161 (460nm, 0.4mW/cm2) and red light (640nm, 1mW/cm2) for 48 hours. Cell viability was not 162 significantly affected by blue-light irradiation or red-light irradiation at low doses. After that, 163 Youn et al (2009)(14) investigated light-induced retinal damage in human RPE cells exposed 164 to specific narrow wavebands of blue light obtained using interference filters and an arc lamp 165 system(400nm at an irradiance of 1.555 mW/cm2, 420nm at an irradiance of 1.466 mW/cm2 166 and 435.8nm at an irradiance of 1.351 mW/cm2) for 3-12 hours. Cells exposed to 400 nm 167 light showed decrease in cell viability, degradation of mitochondria and nucleic acids damage; 168 however, no alterations was observed for 420 and 435.8 nm light-exposed RPE cells. 169 It is relevant the researches carry out by Roehlecke et al (10) in which are evaluated the in 170 vitro response of RPE cells exposed to blue LED lighting. Cells were irradiated with 405nm 171 light at an output power of 0.3 mW/cm2 or 1 mW/cm2 for 3, 24 or 72 hours. The data shown a 172 significantly stimulated ROS production and a decrease of mitochondrial membrane potential 173 after 24 hours of exposure to blue light, but no apoptosis or viability changes was evidenced. 174 They used low doses of light for up to 72 hours without a repair time, in order to establish an 175 in vitro model system in which light irradiation induced mild stress without causing cell death. 176 Page 9 of 18 Photochemistry and Photobiology For Peer Review 10 It has been suggested that cells may adapt to the light-induced stress and therefore survive (10) 177 so in the present study we have exposed cells to 3 light-darkness cycles (12hours/12hours) 178 instead of continuous light. 179 In conclusion, 3 light-darkness cycles (12hours/12hours) exposure to LED lighting affect to 180 growth RPE cells, produce cellular stress increasing ROS levels accompanying of an 181 increasing of DNA damage and apoptotic cells. Future investigation will determine the 182 intensities and wavelengths of LED lighting which are lethal and nonlethal for ocular tissues, 183 as well as the effect of optical filters in RPE cells protection. This information will be 184 necessary in order to develop appropriate normative for this growing industry field. 185 ACKNOWLEDGMENTS: This work has been supported in part by Fundación Mapfre 186 (Spain) 187 REFERENCES 188 1. International Lighting Vocabulary. In Standard Cie S 017/E:2011 Ilv. (Edited By 189 Commission Internationale De L'eclairage (Cie)). 190 2. (2005) General Light Brochure. (Edited By Osram). 191 3. Behar-Cohen, F., C. Martinsons, F. Vienot, G. Zissis, A. Barlier-Salsi, J. P. Cesarini, O. 192 Enouf, M. Garcia, S. Picaud And D. Attia (2011) Light-Emitting Diodes (Led) For 193 Domestic Lighting: Any Risks For The Eye? Prog Retin Eye Res 30, 239-57. 194 4. Chu, R., X. Zheng, D. Chen And D. N. Hu (2006) Blue Light Irradiation Inhibits The 195 Production Of Hgf By Human Retinal Pigment Epithelium Cells In Vitro. Photochem 196 Photobiol 82, 1247-50. 197 Page 10 of 18Photochemistry and Photobiology For Peer Review 11 5. Dorey, C. K., F. C. Delori And K. Akeo (1990) Growth Of Cultured Rpe And Endothelial 198 Cells Is Inhibited By Blue Light But Not Green Or Red Light. Curr Eye Res 9, 549-59. 199 6. Godley, B. F., F. A. Shamsi, F. Q. Liang, S. G. Jarrett, S. Davies And M. Boulton (2005) 200 Blue Light Induces Mitochondrial Dna Damage And Free Radical Production In 201 Epithelial Cells. J Biol Chem 280, 21061-6. 202 7. Ham, W. T., H. A. Mueller, J. J. Ruffolo And A. M. Clarke (1979) Sensitivity Of The 203 Retina To Radiation-Damage As A Function Of Wavelength. Photochemistry And 204 Photobiology 29, 735-743. 205 8. Ham, W. T., H. A. Mueller And D. H. Sliney (1976) Retinal Sensitivity To Damage From 206 Short Wavelength Light. Nature 260, 153-155. 207 9. Hui, S., L. Yi And Q. L. Fengling (2009) Effects Of Light Exposure And Use Of Intraocular 208 Lens On Retinal Pigment Epithelial Cells In Vitro. Photochemistry And Photobiology 209 85, 966-969. 210 10. Roehlecke, C., A. Schaller, L. Knels And R. H. Funk (2009) The Influence Of Sublethal 211 Blue Light Exposure On Human Rpe Cells. Mol Vis 15, 1929-38. 212 11. Sparrow, J. R., A. S. Miller And J. Zhou (2004) Blue Light-Absorbing Intraocular Lens 213 And Retinal Pigment Epithelium Protection In Vitro. J Cataract Refract Surg 30, 873-214 8. 215 12. Vazquez, D. And E. Bernabeu (1997) Quantitative Estimation Of Clear Sky Light In 216 Madrid. Energy Build. 26, 331-336. 217 13. Wu, J., S. Seregard And P. V. Algvere (2006) Photochemical Damage Of The Retina. Surv 218 Ophthalmol 51, 461-81. 219 14. Youn, H. Y., B. R. Chou, A. P. Cullen And J. G. Sivak (2009) Effects Of 400 Nm, 420 220 Nm, And 435.8 Nm Radiations On Cultured Human Retinal Pigment Epithelial Cells. J 221 Photochem Photobiol B 95, 64-70. 222 223 224 Page 11 of 18 Photochemistry and Photobiology For Peer Review 12 FIGURE CAPTIONS 225 Figure 1. Schematic diagram of the LED lighting irradiation system and spectral irradiance of 226 the different LED lighting sources: blue, green, red and white light 227 Figure 2. Representative images of effects of LED lighting on human retinal pigment 228 epithelial cells in vitro. HRPEpiC cells were exposed to blue, green, red and white LED 229 lighting (irradiated cells) or maintained in the dark (control) for 3 light-darkness cycles 230 (12hours/12hours). A. Cellular viability of HRPEpic cells determined by labeling all nuclei 231 with DAPI. B. ROS production determined by the H2DCFDA staining and fluorescence 232 microscopy; an increase of fluorescence in cells indicates oxidative stress. C. Mitochondrial 233 membrane potential determined by the TMRM staining and fluorescence microscopy. 234 Reduction or absence of fluorescence indicates decrease of MMP. D. DNA damage 235 determined by the activation of H2AX histone. E. Apoptosis determined by the activation of 236 caspases-3,-7. The white arrows indicate apoptotic cells. 237 Figure 3. Effects of monochromatic LED lighting on human retinal pigment epithelial cells in 238 vitro. HRPEpiC cells were exposed to blue, green and red LED lighting (irradiated cells) or 239 maintained in the dark (control) for 3 light-darkness cycles (12hours/12hours). The graph 240 displays mean fluorescence intensity radios of irradiated cells versus unirradiated controls. 241 Bars represent mean ± SD from n=3-5 experiments. The asterisk (*) indicates significant 242 differences as compared to controls (p<0.05, t-student test). A) Cellular viability of HRPEpic 243 cells determined by labeling all nuclei with DAPI. B) ROS production determined by the 244 H2DCFDA staining and fluorescence microscopy. C) Mitochondrial membrane potential 245 determined by the TMRM staining and fluorescence microscopy. D) DNA damage determined 246 Page 12 of 18Photochemistry and Photobiology For Peer Review 13 by the activation of H2AX histone. E) Apoptosis determined by the activation of caspases-3, -247 7 is observed as a pink coloration around DAPI stained cells. 248 Figure 4. Effects of white LED lighting on human retinal pigment epithelial cells in vitro. 249 HRPEpiC cells were exposed to white LED lighting (irradiated cells) or maintained in the dark 250 (control) for 3 light-darkness cycles (12hours/12hours). The graph displays mean fluorescence 251 intensity radios of irradiated cells versus unirradiated controls. Bars represent mean ± SD from 252 n=3-5 experiments. The asterisk (*) indicates significant differences as compared to controls 253 (p<0.05, t-student test). A) Cellular viability of HRPEpic cells determined by labeling all 254 nuclei with DAPI. B) ROS production determined by the H2DCFDA staining and fluorescence 255 microscopy. C) Mitochondrial membrane potential determined by the TMRM staining and 256 fluorescence microscopy. D) DNA damage determined by the activation of H2AX histone. E) 257 Apoptosis determined by the activation of caspases-3, -7 is observed as a pink coloration 258 around DAPI stained cells. 259 Page 13 of 18 Photochemistry and Photobiology For Peer Review Table 1. Cell viability, ROS production, mitochondrial membrane potential, DNA damage and apoptosis of cultured RPE irradiated with blue, green, red and white LED lighting. Values indicate fluorescence intensity, mean ± SD. Control Blue light Green light Red light White light Viability (FU) 855±403 10±2* 99±114* 339±1 217±108* ROS (FU) 593±78 737±19* 855±30* 1004±49* 656±26* MMA (FU) 634±19 620±39 823±30 780±128 770±18 DNA damage (FU) 131±41 2537±589* 2258±738* 1920±286* 2697±493* Apoptosis (%) 3.7±0.02 86.1±0.03* 83.9±0.05* 65.5±0.07* 88.8±0.02* *p<0.05 compared to the control Page 14 of 18Photochemistry and Photobiology For Peer Review Schematic diagram of the LED lighting irradiation system and spectral irradiance of the different LED lighting sources: blue, green, red and white light 73x96mm (300 x 300 DPI) Page 15 of 18 Photochemistry and Photobiology For Peer Review Representative images of effects of LED lighting on human retinal pigment epithelial cells in vitro. HRPEpiC cells were exposed to blue, green, red and white LED lighting (irradiated cells) or maintained in the dark (control) for 3 light-darkness cycles (12hours/12hours). A. Cellular viability of HRPEpic cells determined by labeling all nuclei with DAPI. B. ROS production determined by the H2DCFDA staining and fluorescence microscopy; an increase of fluorescence in cells indicates oxidative stress. C. Mitochondrial membrane potential determined by the TMRM staining and fluorescence microscopy. Reduction or absence of fluorescence indicates decrease of MMP. D. DNA damage determined by the activation of H2AX histone. E. Apoptosis determined by the activation of caspases-3,-7. The white arrows indicate apoptotic cells. 379x329mm (96 x 96 DPI) Page 16 of 18Photochemistry and Photobiology For Peer Review Effects of monochromatic LED lighting on human retinal pigment epithelial cells in vitro. HRPEpiC cells were exposed to blue, green and red LED lighting (irradiated cells) or maintained in the dark (control) for 3 light- darkness cycles (12hours/12hours). The graph displays mean fluorescence intensity radios of irradiated cells versus unirradiated controls. Bars represent mean ± SD from n=3-5 experiments. The asterisk (*) indicates significant differences as compared to controls (p<0.05, t-student test). A) Cellular viability of HRPEpic cells determined by labeling all nuclei with DAPI. B) ROS production determined by the H2DCFDA staining and fluorescence microscopy. C) Mitochondrial membrane potential determined by the TMRM staining and fluorescence microscopy. D) DNA damage determined by the activation of H2AX histone. E) Apoptosis determined by the activation of caspases-3, -7 is observed as a pink coloration around DAPI stained cells. 117x300mm (150 x 150 DPI) Page 17 of 18 Photochemistry and Photobiology For Peer Review Effects of white LED lighting on human retinal pigment epithelial cells in vitro. HRPEpiC cells were exposed to white LED lighting (irradiated cells) or maintained in the dark (control) for 3 light-darkness cycles (12hours/12hours). The graph displays mean fluorescence intensity radios of irradiated cells versus unirradiated controls. Bars represent mean ± SD from n=3-5 experiments. The asterisk (*) indicates significant differences as compared to controls (p<0.05, t-student test). A) Cellular viability of HRPEpic cells determined by labeling all nuclei with DAPI. B) ROS production determined by the H2DCFDA staining and fluorescence microscopy. C) Mitochondrial membrane potential determined by the TMRM staining and fluorescence microscopy. D) DNA damage determined by the activation of H2AX histone. E) Apoptosis determined by the activation of caspases-3, -7 is observed as a pink coloration around DAPI stained cells. 103x297mm (150 x 150 DPI) Page 18 of 18Photochemistry and Photobiology