Experimental Researches On The Ocular Bio-safety Of Novel Artificial Light-emitting Diodes(LED)

Part Ⅰ Effect of white LED light with different correlated color temperatures (CCT) on oxidative stress and DNA injury of human lens epithelial cells in culturePurposeOxidative stress on the lens epithelial cells (HLECs) is the most important factor in cataract formation. Cumulative light-exposure from widely used light-emitting diodes (LED) may pose a potential oxidative threat to the lens epithelium, due to the high-energy blue light component in the whitelight emission from diodes. In the interest of perfecting biosafety standards for LED domestic lighting, this study analyzed the photobiological effect of white LED light with different correlated color temperatures (CCT) on cell viability, reactive oxygen species (ROS), DNA injury, cell cycle and apotosis in cultured hLECs.MethodsThe HLECs were cultured and cumulatively exposed to multichromatic white LED light with CCT of 2954,5624, and 7378K. LED exposure system (provided by Hongyan electrical technology) functioned in a light cycle of 16 hours-on/8 hours-off. Cell viability was measured by Cell Counting Kit-8 (CCK-8) assay. DNA damage was determined by alkaline comet assay. Intracellular ROS generation, cell cycle, and apoptosis were quantified by flow cytometry. PARP-1, p21 and p53 mRNA and protein expression were quantified by western blotting and RT-qPCR.ResultsCompared with normal control group, after 2-cycle (day) exposure under 7378K LED lighting of 15001ux, we observed a significant decrease of cell viability, but not found in 2954K and 5624K group. The greatest reduction in cell viability (68.26%) occurred after 3-cycle exposure under 7378K LED lighting of 25001ux; The greatest ROS generation was also observed in cells treated by 7378K radiation; Alkaline comet assay showed 2954K LED light caused no significant damage to DNA, and greater damage was observed for the 5624K and 7378K irradiation-treated cells, which were more arrested in the G2/M phase in cell cycle analysis; A significant increase in apoptosis was observed in the 7378K group, exposed to illuminance levels of 1500 and 25001ux, which was accompanied by significant up-regulation of PARP-1, p53 and p21 wafl/cipl.ConclusionsCompared with 2954 and 5624K LED light, LED light having a CCT of 7378K caused overproduction of intracellular ROS, decreased cell viability, severe DNA damage, which triggered G2/M arrest, cell apoptosis. These results indicate that white LED with a high CCT could cause significant photobiological damage to HLECs.Part Ⅱ Illumination from light-emitting diodes (LED) disrupts pathological cytokines expression and activates relevant signal pathways in primary human retinal pigment epithelial cellsPurposeAge-related macular degeneration (AMD) is the leading cause of blindness in the aged people. The latest systemic review of epidemiological investigations revealed that excessive sunlight (including blue light) exposure increases the risk of AMD. With the drastically increasing use of high-energy light-emitting diodes (LED) light in our domestic environment nowadays, it is supposed to pose a potential oxidative threat to ocular health. Retinal pigment epithelium (RPE) is the major ocular source of pathological cytokines, which regulate local inflammation and angiogenesis. We hypothesized that high-energy LED light might disrupt the pathological cytokine expression of RPE, contributing to the pathogenesis of AMD. We hypothesized that high-energy LED light might disrupt the pathological cytokine expression of retinal pigment epithelial cells (RPECs), contributing to the pathogenesis of AMD.MethodsPrimary human RPECs were isolated from eyecups of normal eye donors and seeded into plate wells for growing to confluence. Two widely used multichromatic white LED with correlated color temperatures (CCT) of 2954K and 7378K were used in this experiment. The confluent primary RPE cells were under white LEDs light exposure until 24 hours (a light cycle of 2 hours-on/20 min-off). VEGF-A, IL-6, IL-8 and MCP-1 proteins and mRNAs were measured using an ELISA kit and RT-qPCR, respectively. Activation of mitogen-activated protein kinases (MAPKs), Akt, Janus kinase (JAK) and Nuclear factor (NF)-κB signal pathways after LED illumination were evaluated by western blotting analysis. The level of reactive oxygen species (ROS) using chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate (CM-H2DCFDA). Inhibitors of relevant signal pathways and anti-oxidants were added to the primary RPECs before LED illumination to evaluate their biological functions.ResultsBy the improved enzymatic methods, we successfully isolated the primary RPECs with high growth activity from the donor eye cup, seeding these primary cells with appropriate density to remain the epithelial phenotype of RPECs. And these cultured primary RPECs exhibited significantly higher secretory levels than immortalized ARPE-19 cell lines. We found that 7378K light, but not 2954K upregulated the VEGF-A, IL-6, IL-8 and downregulated MCP-1 proteins and mRNAs levels in a time-dependent manner. In parallel, initial activation of MAPKs and NF-κB signal pathways were also observed after 7378K light exposure. Mechanistically, antioxidants for eliminating ROS and targeted inhibitors of MAPKs and NF-kB significantly blocked 7378K light-induced changes of specific cytokines, respectively.ConclusionsWhite LED light with higher CCT disrupted the expression of VEGF-A, IL-6, IL-8 and MCP-1 via MAPKs and NF-kB pathways in primary human RPECs. Supplementation of Vitamin C and NAC in primary RPECs eliminated the ROS, suppressed the signal pathways activation and attenuated the changes of these pathological cytokines.Part Ⅲ Retinal injury and genome-wide expression pattern induced by white LED light with different correlated color temperatures (CCTs) in micePurposeNowadays the widely used white light-emitting diodes (LED) deliver higher levels of blue light than do conventional domestic light sources. The high intensity of blue component is the main source of concern about the health risks of LED with respect to their light-toxicity to the retina. This study analyzed the retinal injury and genome-wide expression changes induced by white LED light with different correlated color temperatures (CCT) in a mouse model.MethodsWhite LED light with CCT of 2954,5624, and 7378K were used for exposure experiments on 10-week-old Babl/c mice. Illuminance of 250,500,1000,30001ux light stimulated the mouse for 7 days,14 days and 28 days (light cycle of 12 hours-on/12 hours-off), respectively. Hematoxylin and eosin (H&E) staining was used to observe the retinal morphology, TdT-mediated dUTP Nick end labeling (TUNEL) method detecting the cell apoptosis in outer nuclear layer (ONL). Retinal genome-wide expression patterns of control group,2954K group and 7378K group were analyzed by Affymetrix GeneChip (Mouse Genome 4302.0) to describe the overall difference of gene expression in mouse retina.ResultsH&E staining revealed that LED radiation damage mainly induced the declining number of ONL nuclei in mice.7378K white LED light with 2501ux for 28-day exposure resulted in significantly reduction of ONL nuclei. Only until increasing the light intensity to 30001ux,2954K white LED light leaded to a significant reduction in the number of ONL in mice; By the illuminance of 30001ux,5624K and 7378K group appeared a significant increase of TUNEL positive apoptotic nuclei. However, under the illuminance of 10001ux, no increased apoptotic nuclei were observed; Genome-wide expression analysis showed that compared with control group,121 up-regulated genes, 458 down-regulated genes were found in 7378K group,59 up-regulated genes, only 4 down-regulated genes found in 2954K group; GO and KEGG enrichment analysis revealed that differentially expressed genes of 7378K group participated in the 341 Term/GO,16 related Pathway (no apoptosis signaling pathways found); differentially expressed genes of 2954K group participated in 12 Term/G0,7 related Pathway.ConclusionsWhite LED light induced mouse retinal injury was present in a CCT-dependent manner. White LED light with higher CCT is more likely to cause retinal injury in mice, significantly reducing the number of ONL nuclei, however apoptosis pathway may not be the only mechanism. Mouse retina expression profile chip analysis further supported the different influence on mouse retina by two white LED light with different CCT.