June 2016
Volume 57, Issue 7
Open Access
Cornea  |   June 2016
Assessment of DNA Damage and Cell Senescence in Corneal Epithelial Cells Exposed to Airborne Particulate Matter (PM2.5) Collected in Guangzhou, China
Author Affiliations & Notes
  • Zi-Xun Gao
    Institute of Ophthalmology School of Medicine, Jinan University, Guangzhou, China
    Key Laboratory for Regenerative Medicine, Ministry of Education, Jinan University, Guangzhou, China
  • Xi-Ling Song
    Department of Public Health and Preventive Medicine, Jinan University, Guangzhou, China
  • Shan-Shan Li
    Department of Clinical Medicine, School of Medicine, Jinan University, Guangzhou, China
  • Xiao-Rong Lai
    Department of Clinical Medicine, School of Medicine, Jinan University, Guangzhou, China
  • Yu-Lan Yang
    Department of Clinical Medicine, School of Medicine, Jinan University, Guangzhou, China
  • Guang Yang
    Department of Public Health and Preventive Medicine, Jinan University, Guangzhou, China
  • Zhi-Jie Li
    Institute of Ophthalmology School of Medicine, Jinan University, Guangzhou, China
    Key Laboratory for Regenerative Medicine, Ministry of Education, Jinan University, Guangzhou, China
  • Yu-Hong Cui
    Department of Histology and Embryology, Guangzhou Medical University, Guangzhou, China
  • Hong-Wei Pan
    Institute of Ophthalmology School of Medicine, Jinan University, Guangzhou, China
    Key Laboratory for Regenerative Medicine, Ministry of Education, Jinan University, Guangzhou, China
    Department of Clinical Medicine, School of Medicine, Jinan University, Guangzhou, China
  • Correspondence: Hong-Wei Pan, Institute of Ophthalmology, School of Medicine, Jinan University, 601 West Huangpu Avenue, Guangzhou 510632, China; panhongwei@hotmail.com
Investigative Ophthalmology & Visual Science June 2016, Vol.57, 3093-3102. doi:10.1167/iovs.15-18839
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      Zi-Xun Gao, Xi-Ling Song, Shan-Shan Li, Xiao-Rong Lai, Yu-Lan Yang, Guang Yang, Zhi-Jie Li, Yu-Hong Cui, Hong-Wei Pan; Assessment of DNA Damage and Cell Senescence in Corneal Epithelial Cells Exposed to Airborne Particulate Matter (PM2.5) Collected in Guangzhou, China. Invest. Ophthalmol. Vis. Sci. 2016;57(7):3093-3102. doi: 10.1167/iovs.15-18839.

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

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Abstract

Purpose: To assess the genotoxic effect of airborne particulate matter on corneal epithelial cells and investigate the role of reactive oxygen species (ROS) formation in this process.

Methods: Immortalized human corneal epithelial cells (HCECs) and primary bovine corneal epithelial cells were exposed to airborne particulate matter collected from Guangzhou for 24 hours. The cell viability and toxicity were measured by the CCK-8 test and lactate dehydrogenase (LDH) release, respectively. The DNA breaks and DNA repair were examined by alkaline comet assay and by immunofluorescence staining of the phosphorylated histone variant H2AX (γH2AX), respectively. Reactive oxygen species production was assessed by the fluorescent probe, CM-H2DCFDA. Cell senescence was evaluated with senescence-associated β-Galactosidase staining, and cell ultrastructure was observed with transmission electron microscopy.

Results: Exposure to PM2.5 at the concentration of 20 μg/mL to 200 μg/mL decreased cell viability and increased LDH release. Remarkably increased DNA double-stand breaks, increased expression of DNA repair-related protein γH2AX, elevated ROS formation, and altered cell ultrastructure were observed in HCECs after treatment with PM2.5. The genotoxic effect of PM2.5 was attenuated by the ROS inhibitor N-acetyl-l-cysteine (NAC).

Conclusions: Particulate matter 2.5 could induce DNA damage and cell senescence in corneal epithelial cells, probably by promoting ROS formation. Thus, whether long-term exposure of PM2.5 might be related to potential risk of abnormality in corneal epithelium renewal and regeneration should be further investigated.

Air pollution has emerged as one of the most important issues in public health worldwide. Particulate matter with diameter of 2.5 μm or less (PM2.5) has been used as an important indicator for air quality monitoring. Extensive evidence has indicated the positive correlation between airborne particulate matter and mortality and morbidity. Epidemiologic and clinical studies also found that air pollution was associated with increased chances of outpatient visits for nonspecific conjunctivitis, and chronic exposure to air pollution occasionally causes obvious discomfort, such as burning, irritation, itching, tearing, and foreign body sensation.1 Numerous studies have addressed the biological effect of air pollution on ocular surface.26 One study has shown that exposure to air pollution reduces tear film stability and influences tear film osmolarity.3 Another study has demonstrated that air pollutants cause inflammation response in corneal and conjunctival epithelial cells and alter the mucin expression.2 
The ability of self-renewal of corneal epithelial cells (CECs) is critical for the integrity of ocular surface defense system. Previous studies have shown that PM2.5 can induce DNA damage in many cell types.710 Therefore, it is an important issue to evaluate whether PM2.5 exposure can induce DNA damage and cell senescence in CECs and in turn impair the ability of corneal epithelium repair and regeneration. 
In the present study, we placed emphasis on the genotoxic effect of PM2.5 exposure on CECs. We examined the DNA damage, ultrastructural change, and cell senescence in a model of human CECs (HCECs) exposed to PM2.5 obtained from Guangzhou, the third largest city in China, which experiences high PM level. To determine the possible reactive oxygen species (ROS) mechanisms in the PM2.5 action, we used N-acetyl-l-cysteine (NAC) to change the antioxidant levels in response to incense PM2.5 treatment. 
Materials
Dulbecco's modified Eagle's medium (DMEM)/F12 medium, fetal bovine serum (FBS), 0.25%Trypsin-EDTA, recombinant human epidermal growth factor (hEGF), insulin-transferrin-selenium supplement, and ROS detection reagents CM-H2DCFDA and Alexa Fluor 488-labeled donkey anti-rabbit IgG antibody were purchased from Invitrogen-Gibco (Carlsbad, CA, USA); 6-, 24-, and 96-well culture plates as well as cell culture flasks were from Corning (Corning, NY, USA). Comet Assay kit was from Trevigen, Inc. (Gaithersburg, MD, USA). Lactate dehydrogenase (LDH) Cytotoxicity Assay Kit was purchased from Pierce Biotechnology (Rockford, IL, USA); rabbit anti-phospho-Histone γH2AX (Ser139) primary antibody was from Millipore (Darmstadt, Germany); Cell Counting Kit-8 (CCK-8), Senescence β-Galactosidase Staining Kit, and NAC were from Bayotime Biotechnology Co. Ltd (Beijing, China). All reagent grade chemicals were from Sigma-Aldrich Corp. (St. Louis, MO, USA) unless otherwise indicated. 
Collection of Airborne Particulate Matter
The PM2.5 samples were collected in July 2014 by the South China Institute of Environmental Sciences, which is located in urban Guangzhou (23°07′N, 113°21′E). There was no obvious industrial pollution source near the monitoring station. Sampling was performed using Partisol air sampler (Model 2000H; Rupprecht & Patashnick Co., Inc., Albany, NY, USA), which separated, by means of a cyclone, particles with aerodynamic diameters below 2.5 μm using a vacuum pump (Gast, Benton Harbor, MI, USA) to draw air at a rate of 16.7 L/min. The air stream was connected to a 47-mm quartz filter (Whatman; Maidstone, Kent, UK). The sampling duration for each sample was 24 hours, starting at 9:30 AM each day. The collected filter samples were stored in a freezer at −4°C to prevent possible volatilization of particles. The PM2.5 samples were extracted from sampled filter strips by immersing them in deionized water and then sonicating them for 60 minutes in a water-bath sonicator. Particle suspensions were dried in a vacuum frozen desiccator (48 hours) (Heto Lab Equipment, Copenhagen, Denmark), weighed and stored at −20°C, and the resulting pellets were resuspended in sterile water (3 mg/mL) just before use. 
Cell Culture and Treatment
Immortalized HCECs, originally established and characterized by Liu et al,11 were used in this study. Human corneal epithelial cells were cultured in DMEM/F12 medium supplemented with 10% FBS, recombinant hEGF (10 ng/mL), Insulin-Transferrin-Selenium Supplement, and penicillin (100 U/mL) and streptomycin (100 μg/mL) in a 5% CO2 atmosphere at 37°C. Primary bovine CECs (BCECs) were collected from domestic adult cattle corneas and the eyes were obtained from the local slaughterhouse. The harvested corneas were placed epithelial side down in a 12-well culture plates and immersed with 1.2 U/mL dispase II at 37°C for 1 hour. The loosened epithelial cells were scraped gently using a cell scraper. The suspended BCECs were washed, seeded onto the cell culture dishes, and incubated at 37°C in a humidified atmosphere of 5% CO2. The BCECs were cultured in the same culture medium as HCECs and the cells at the third passage were used for the experiments. 
For experiments, cells were seeded at a concentration of 8 × 104cells/well in 6-well plates, 2 × 104 cells in 24-well plates, or 5 × 103 cells in 96-well plates, and treated with PM2.5 at the concentration indicated. The cellular responses were examined after exposure and the results compared with those of untreated cells (control). Cells were preincubated for 1 hour with antioxidants, NAC (1 mg/mL) before exposure to PM2.5 to investigate the role of ROS. 
Cell Count Kit-8 Assay
A CCK-8 (Beyotime) was used in this experiment to quantitatively evaluate cell viability. The HCECs were seeded in 96-well plates at 5×103 cells per well and then treated with PM2.5. Then 10 μL of CCK-8 solution were added to each well of the plate, which was then placed for 2 hours in an incubator (37°C and 5% CO2). The absorbance was measured at 450 nm using a microplate reader (680; Bio-Rad, Hercules, CA, USA). 
Lactate Dehydrogenase Release
To determine cellular toxicity of PM2.5, the level of lactate dehydrogenase (LDH) released from HCECs was measured. After exposure to PM2.5, cell-free supernatant aliquots were collected from each experimental sample, and LDH in the culture supernatants was measured using the Pierce LDH Cytotoxicity Assay Kit (cat. 88953; Thermo Scientific, Rockford, IL, USA,). All samples were assayed in triplicate for LDH content by a microplate reader (Bio-Rad 680) at wave length of 490 nm. According to the manufacturer's instructions, the cytotoxicity was calculated by subtracting the LDH activity of the spontaneous LDH release control (untreated) from the PM2.5-treated sample LDH activity, dividing by the total LDH activity ([maximum LDH release control activity] – [spontaneous LDH release control activity]), and multiplying by 100. 
Assessment of ROS Formation
The fluorescent dye, H2DCFDA, diffuses through cell membranes and is deacetylated by intracellular esterases to nonfluorescent 2′,7′-dichlorodihydrofluorescein. In the presence of ROS, dichlorodihydrofluorescein (DCFH) is oxidized to the highly fluorescent 2′,7′-dichlorodifluorescein (DCF). Briefly, HCECs were plated in 24-well plate at a density of 2 × 104 cells per well and cultured for 24 hours to allow cell adhesion. Then the culture medium was replaced with fresh medium contained various concentrations of PM2.5 for 24 hours. After treatment, the cells were washed with PBS solution twice and incubated with CM-H2DCFDA (5 μM in PBS) at 37°C for 30 minutes in the dark. The images were captured using a fluorescence microscope (Olympus IX51; Tokyo, Japan). 
Comet Assay
Human CECs and BCECs were plated on 24-well plate and exposed to PM2.5 and, after 24 hours of exposure, cells were collected by trypsinization. DNA damage has been evaluated by alkaline single-cell gel electrophoresis (Comet assay) with CometAssay kit (cat.4250-050-K; Trevigen) according to the recommended procedure. Briefly, cells were mixed in 1% low fusion point agarose and then placed on a slide. The sample was treated with lysis solution for 30 minutes, then rinsed in a 200 mM NaOH, 200 mM EDTA unwinding solution, and subjected to 21V alkaline electrophoresis for 30 minutes; SYBR Gold solution was added to each sample and analyzed under fluorescence microscopy (Olympus IX51). The length of tail, the percentage of DNA in the tail, and the tail moment (TM, measure of tail length × percent of DNA in the tail) of each comet were measured using an image analysis program CASP (Version1.2.3; available at http://casp.sourceforge.net). 
Immunofluorescence Staining
Human CECs and BCECs were washed in PBS and fixed with 4% paraformaldehyde in phosphate buffer for 30 minutes at 4°C and permeabilized with 0.5% Triton X-100 solution for 15 minutes. All the following procedures were performed at room temperature. After three washes with PBS, the cells were incubated in 1% BSA in PBS for 30 minutes, to block nonspecific binding. Afterward, the cells were incubated for 2 hours with rabbit anti-γH2AX (Ser139) at optimal dilutions in PBS containing 1% BSA to detect phosphorylated γH2AX at Ser 139. After they were washed with PBS-BSA (three times, 5 minutes each), the cells were incubated with the corresponding secondary antibodies for 1 hour; 4′,6-diamidino-2-phenylindole (DAPI) was used to counterstain the nuclei. Images were captured using a microscope (Olympus IX51). 
Senescence-Associated β-Galactosidase Assay (SA-β-gal)
Human CECs and BCECs were seeded on coverslips in 24-well culture plates for 24 hours. After exposure to PM2.5 at the indicated concentration for 24 hours, the activity of SA-β-gal was determined using the senescence-associated β-Galactosidase Staining Kit (Beyotime) according to the manufacturer's instructions. Briefly, cells were fixed with 2% paraformaldehyde at room temperature and, after 15 minutes, the cells were washed three times with PBS. Cells were incubated with the staining solution at 37°C overnight. The level of SA-β-gal was obtained by capturing the image with a digital camera (Olympus IX51). 
Transmission Electronic Microscopy
For transmission electron microscopy, HCECs were grown on glass slides to subconfluence and treated at appropriate concentrations of PM2.5 for 24 hours. Subconfluent cells were collected by trypsinization and fixed by 2.5% glutaraldehyde at 4°C for 2 hours, then washed with 0.1M PBS and postfixed in 1% osmium tetroxide. After dehydration in 50%, 70%, and 90% ethanol and 90% and 100% acetone sequentially, cells were subsequently embedded in Epon812 medium, and examined in a transmission electron microscope (XL-30ESEM; Philips, The Netherlands). 
Statistical Analysis
The quantitative and statistical analysis of immunofluorescence staining and SA-β-gal staining were analyzed by Image-pro plus (Version 6.0; Media Cybernetics, Inc., Silver Spring, MD, USA). This software is capable of providing the positive target distribution area and the integrated option density of the staining image. The data were analyzed to determine the mean optical density (MOD), which represented the concentration of the stain as measured per positive pixels. 
All data were presented as the mean ± SD of at least three independent experiments. Statistical analyses were performed using Student's t-test or, when multiple comparisons were made, 1-way ANOVA with post hoc Tukey's test using SPSS (SPSS 11.5; IBM SPSS Statistics, IBM Corporation, Chicago, IL). Values of P < 0.05 were considered statistically significant. 
Results
Cell Viability and Plasma Membrane Damage
The viability of HCECs was assayed with CCK-8 methods after treatment with PM2.5 As shown in Figure 1, cell viability was decreased to 95.04% ± 2.99%, 86.63% ± 2.96%, 82.62% ± 3.85%, and 47.04% ± 3.37% of the control after exposed to 20 μg/mL, 50 μg/mL, 100 μg/mL, and 200 μg/mL PM2.5, respectively. These results indicated that PM2.5 decreased HCECs viability dose-dependently. 
Figure 1
 
Cell viability of HCECs after PM2.5 exposure assayed with CCK-8. Human CECs were incubated for 24 hours with medium containing various concentrations of PM2.5: 20 μg/mL, 50 μg/mL, 100 μg/mL, and 200 μg/mL. The cell viability was determined using CCK-8 according to the manufacturer's instructions. The results were expressed as mean ± SD of three independent experiments. **P < 0.01 versus control.
Figure 1
 
Cell viability of HCECs after PM2.5 exposure assayed with CCK-8. Human CECs were incubated for 24 hours with medium containing various concentrations of PM2.5: 20 μg/mL, 50 μg/mL, 100 μg/mL, and 200 μg/mL. The cell viability was determined using CCK-8 according to the manufacturer's instructions. The results were expressed as mean ± SD of three independent experiments. **P < 0.01 versus control.
Lactate dehydrogenase is a stable cytoplasmic enzyme in cells and is rapidly released into culture medium on damage of the plasma membrane of cells. We observed a significant increase of cytotoxicity to 17.86% ± 4.44% and 25.96% ± 6.86% in HCECs after exposure to PM2.5 at the concentration of 100 μg/mL and 200 μg/mL compared with the control group (12.89% ± 2.48%). However, when cells were preincubated with NAC (1 mg/mL), cytotoxicity was significantly reduced to 16.24% ± 3.02%, as shown in Figure 2. This suggested that oxidative stress was responsible for the cytotoxicity of PM2.5. 
Figure 2
 
Lactate dehydrogenase cytotoxicity assay for HCECs after PM2.5 exposure. Human CECs cultured in a 96-well plate were incubated for 24 hours with the medium containing various concentrations of PM2.5 as indicated with or without NAC (1 mg/mL). The results were presented as the percent cytotoxicity of the maximum LDH release control according to the manufacturer's instructions. Data were expressed as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 versus control; ##P < 0.01 versus 200 μg/mL PM2.5 treated group.
Figure 2
 
Lactate dehydrogenase cytotoxicity assay for HCECs after PM2.5 exposure. Human CECs cultured in a 96-well plate were incubated for 24 hours with the medium containing various concentrations of PM2.5 as indicated with or without NAC (1 mg/mL). The results were presented as the percent cytotoxicity of the maximum LDH release control according to the manufacturer's instructions. Data were expressed as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 versus control; ##P < 0.01 versus 200 μg/mL PM2.5 treated group.
DNA Damage
The alkaline comet assay was used to evaluate genotoxic effects of PM2.5 on HCECs. Figure 3A shows the representative images of comet tails in each experimental group. Quantitative analysis indicated that PM2.5 caused significant DNA damage in HCECs in a dose-dependent manner, and this effect was attenuated by pretreatment with NAC. For the control, 100 μg/mL, 200 μg/mL, and 200 μg/mL plus NAC (1 mg/mL) group, the tail length was 18 ± 3.57 μm, 41 ± 7.80 μm, 85.08 ± 10.84 μm, and 59.83 ± 5.67 μm, respectively, the percentage DNA content in tail was 8.56% ± 3.67%, 17.18% ± 5.55%, 25.18% ± 8.26%, and 18.39% ± 6.20%, respectively, and the comet TM was 6.81 ± 1.14, 22.62 ± 6.48, 103.53 ± 9.52, and 20.19 ± 2.33, respectively, as shown in Figure 3B. 
Figure 3
 
Comet assay for HCECs after PM2.5 exposure. Human CECs cultured in a 24-well plate were incubated for 24 hours with medium containing various concentrations of PM2.5 with or without NAC (1 mg/mL), respectively. Comet assay was performed with a kit according to the manufacturer's instructions. The images were obtained using a fluorescence microscope (×400). (A) Representative images of comet tails from each experimental group: 0 μg/mL, 100 μg/mL, 200 μg/mL, and 200 μg/mL with NAC (1 mg/mL). (B) Quantitative analysis of the tail length, percent DNA in comet tails, and comet tails moment. Data were expressed as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 versus control; #P < 0.05, ##P < 0.01 versus 200 μg/mL PM2.5 treated group.
Figure 3
 
Comet assay for HCECs after PM2.5 exposure. Human CECs cultured in a 24-well plate were incubated for 24 hours with medium containing various concentrations of PM2.5 with or without NAC (1 mg/mL), respectively. Comet assay was performed with a kit according to the manufacturer's instructions. The images were obtained using a fluorescence microscope (×400). (A) Representative images of comet tails from each experimental group: 0 μg/mL, 100 μg/mL, 200 μg/mL, and 200 μg/mL with NAC (1 mg/mL). (B) Quantitative analysis of the tail length, percent DNA in comet tails, and comet tails moment. Data were expressed as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 versus control; #P < 0.05, ##P < 0.01 versus 200 μg/mL PM2.5 treated group.
Foci formation of γH2AX (phosphorylation at Ser139 of histone variant H2AX) has been used as a sensitive biomarker for DNA damage. In this study, we used an immunofluorescence method to detect the formation of γH2AX foci. The results showed γH2AX foci exhibited a dose-dependent increase in HCECs after exposure to PM2.5 for 24 hours, as shown in Figure 4. The MOD with γH2AX foci in the control group was 0.0317 ± 0.0032, whereas it increased to 0.0392 ± 0.0015 and 0.0529 ± 0.0025 in the 50-μg/mL and 200-μg/mL PM2.5 group, respectively. Preincubation with NAC for 1 hour before the PM2.5 exposure resulted in a significantly smaller MOD (0.0484 ± 0.0025) of γH2AX foci-positive cells compared with PM2.5-treated cells without NAC preincubation. 
Figure 4
 
Immunofluorescence staining for γH2AX (Ser139) in HCECs after PM2.5 exposure. Human CECs cultured in a 24-well plate were incubated for 24 hours with medium containing PM2.5 at the concentration of 0 μg/mL, 50 μg/mL, 200 μg/mL, and 200 μg/mL PM2.5 with NAC (1 mg/mL), respectively. (A) The cells were immunostained with anti-γH2AX (Ser139) antibody (green, top), and the nuclei were counterstained with DAPI (blue, bottom). Images were taken at the magnification of ×200. (B) Quantitative analysis of the fluorescent MOD. The data represent mean ± SD of five measurements. **P < 0.01 versus control; #P < 0.05 versus the 200 μg/mL PM2.5 treated group.
Figure 4
 
Immunofluorescence staining for γH2AX (Ser139) in HCECs after PM2.5 exposure. Human CECs cultured in a 24-well plate were incubated for 24 hours with medium containing PM2.5 at the concentration of 0 μg/mL, 50 μg/mL, 200 μg/mL, and 200 μg/mL PM2.5 with NAC (1 mg/mL), respectively. (A) The cells were immunostained with anti-γH2AX (Ser139) antibody (green, top), and the nuclei were counterstained with DAPI (blue, bottom). Images were taken at the magnification of ×200. (B) Quantitative analysis of the fluorescent MOD. The data represent mean ± SD of five measurements. **P < 0.01 versus control; #P < 0.05 versus the 200 μg/mL PM2.5 treated group.
Taken together, our results showed PM2.5 could induce remarkable DNA damage in HCECs and suppression of ROS formation might alleviate the DNA damage. 
To confirm our results in an immortalized CEC line, we repeated the main experiments in primary BCECs. The results of alkaline comet assay in BCECs showed PM2.5 caused obvious DNA damage in a dose-dependent manner. The results of γH2AX immunostaining in BCECs showed exposure to PM2.5 induced a dose-dependent increase in γH2AX foci formation. Both DNA damage and γH2AX foci formation induced by PM2.5 were attenuated by pretreatment with NAC, as shown in the online Supplementary Materials (Supplementary Figs. S1, S2). 
Cell Senescence
With senescence-associated β-galactosidase assay, we investigated PM2.5-induced cell senescence in HCECs. As shown in Figure 5, PM2.5 induced dose-dependently increase of cell senescence in HCECs. The MOD of senescent cells was 0.042 ± 0.041 in the control group, whereas it increased to 0.089 ± 0.017, 0.166 ± 0.042, 0.216 ± 0.022, and 0.273 ± 0.028 in the 20 μg/mL, 50 μg/mL, 100 μg/mL, and 200 μg/mL groups, respectively. N-acetyl-l-cysteine pretreatment decreased the MOD of staining cells to 0.124 ± 0.042 in the 200 μg/mL PM2.5 treatment group. This result indicated PM2.5 could induce HCEC senescence, probably via ROS formation. 
Figure 5
 
Senescence of HCECs after PM2.5 exposure determined with SA-β-gal staining. Human CECs cultured in a 24-well plate were incubated for 24 hours with medium containing various concentrations of PM2.5: 0 μg/mL, 20 μg/mL, 50 μg/mL, 100 μg/mL, 200 μg/mL, and 200 μg/mL with 1 mg/mL NAC, respectively. (A) Representative images (×200) of senescent cells determined with SA-β-gal staining. (B) Quantitative analysis of the MOD of staining cells. Data were expressed as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 versus control; ##P < 0.01 versus 200 μg/mL PM2.5 treated group.
Figure 5
 
Senescence of HCECs after PM2.5 exposure determined with SA-β-gal staining. Human CECs cultured in a 24-well plate were incubated for 24 hours with medium containing various concentrations of PM2.5: 0 μg/mL, 20 μg/mL, 50 μg/mL, 100 μg/mL, 200 μg/mL, and 200 μg/mL with 1 mg/mL NAC, respectively. (A) Representative images (×200) of senescent cells determined with SA-β-gal staining. (B) Quantitative analysis of the MOD of staining cells. Data were expressed as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 versus control; ##P < 0.01 versus 200 μg/mL PM2.5 treated group.
The results of senescence-associated β-galactosidase assay in BCECs also showed cell senescence was increased by PM2.5 exposure and NAC pretreatment reduced cell senescence, as shown in the online supplement materials (Supplementary Fig. S3). 
Cellular Morphology and Ultrastructure
We observed the morphologic changes of the HCECs after exposure to PM2.5 with an optical microscope and transmission electron microscopy. As shown in Figure 6, PM2.5 treatment induced irregular cellular shape, remarkable cytoplasmic shrinkage, and decreased cell adhesion. Pretreatment with NAC alleviated the morphologic changes to a certain extent. 
Figure 6
 
Morphology of HCECs after PM2.5 exposure. Human CECs cultured in a 6-well plate were incubated for 24 hours with medium containing PM2.5 at the concentration of 0 μg/mL, 20 μg/mL, 50 μg/mL, 100 μg/mL, 200 μg/mL or 200 μg/mL with NAC (1 mg/mL), respectively. The morphology was observed using an optical microscope (×200). It was shown that the PM2.5 treatment induced irregular cellular shape, remarkable cytoplasmic shrinkage, and decreased cell adhesion. The NAC treatment attenuated the morphology change induced by PM2.5 incubation.
Figure 6
 
Morphology of HCECs after PM2.5 exposure. Human CECs cultured in a 6-well plate were incubated for 24 hours with medium containing PM2.5 at the concentration of 0 μg/mL, 20 μg/mL, 50 μg/mL, 100 μg/mL, 200 μg/mL or 200 μg/mL with NAC (1 mg/mL), respectively. The morphology was observed using an optical microscope (×200). It was shown that the PM2.5 treatment induced irregular cellular shape, remarkable cytoplasmic shrinkage, and decreased cell adhesion. The NAC treatment attenuated the morphology change induced by PM2.5 incubation.
With transmission electron microscopy, we observed the uptake and subcellular location of PM2.5 particles in the HCECs, as well as the pathologic changes in cellular ultrastructure. Chromatin condensation and margination along the inner nuclear membrane, cytoplasmic condensation, and membrane blebbing could be observed (Figs. 7A, 7B). Uptake of particulate matter particles and appearance of cellular vesicles could be seen in some cells (Figs. 7C, 7D). 
Figure 7
 
Transmission electron microscopy observation of HCECs after PM2.5 exposure. Human CECs were incubated for 24 hours with medium containing PM2.5 (200 μg/mL). Chromatin condensation and margination along the inner nuclear membrane, cytoplasmic condensation, and membrane blebbing could be observed (A, B). Uptake of particulate matter particles and appearance of cellular vesicles could be seen in some cells (C, D). N, nucleus; V, vesicle; arrows, PM2.5 aggregates. Transmission electron microscopy scale bars: (A) 2 μm, (B) 2 μm, (C) 2 μm, (D) 1 μm. Images were taken at the magnification of 9k, 15k, 9k, and 24k, respectively.
Figure 7
 
Transmission electron microscopy observation of HCECs after PM2.5 exposure. Human CECs were incubated for 24 hours with medium containing PM2.5 (200 μg/mL). Chromatin condensation and margination along the inner nuclear membrane, cytoplasmic condensation, and membrane blebbing could be observed (A, B). Uptake of particulate matter particles and appearance of cellular vesicles could be seen in some cells (C, D). N, nucleus; V, vesicle; arrows, PM2.5 aggregates. Transmission electron microscopy scale bars: (A) 2 μm, (B) 2 μm, (C) 2 μm, (D) 1 μm. Images were taken at the magnification of 9k, 15k, 9k, and 24k, respectively.
Reactive Oxygen Species Formation
To determine whether ROS formation may be one of the mechanisms causing cytotoxicity of PM2.5, we detected ROS in HCECs with a ROS fluorescent probe CM-H2DCFDA. After exposure to PM2.5 for 24 hours, the ROS was increased in a dose-dependent manner in each group. The fluorescent MOD was increased from 0.0082 ± 0.0026 in the control group to 0.0128 ± 0.0025, 0.0201 ± 0.0027, and 0.0244 ± 0.0032 in the 20 μg/mL, 50 μg/mL, and 100 μg/mL PM2.5 groups, respectively. Pretreatment with NAC (1 mg/mL) could inhibit the increase of ROS induced by PM2.5, with a MOD of 0.0171 ± 0.0010, as shown in Figure 8
Figure 8
 
The effect of PM2.5 on ROS generation in HCECs. Human CECs cultured in a 24-well plate were incubated for 24 hours with medium containing PM2.5 at the concentrations of 0 μg/mL, 20 μg/mL, 50 μg/mL, 100 μg/mL, and 100 μg/mL with NAC (1 mg/mL), respectively. The intracellular ROS was detected with a fluorescent probe CM-H2DCFDA. (A) Representative images under fluorescent microscope (×200). (B) The fluorescent MOD of staining cells. Data were presented as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 versus control; #P < 0.05 versus 100 μg/mL PM2.5-treated group.
Figure 8
 
The effect of PM2.5 on ROS generation in HCECs. Human CECs cultured in a 24-well plate were incubated for 24 hours with medium containing PM2.5 at the concentrations of 0 μg/mL, 20 μg/mL, 50 μg/mL, 100 μg/mL, and 100 μg/mL with NAC (1 mg/mL), respectively. The intracellular ROS was detected with a fluorescent probe CM-H2DCFDA. (A) Representative images under fluorescent microscope (×200). (B) The fluorescent MOD of staining cells. Data were presented as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 versus control; #P < 0.05 versus 100 μg/mL PM2.5-treated group.
Discussion
Corneal epithelium, located at the outmost layer of the cornea, is vulnerable to various damage. It has been shown that many factors, such as ultraviolet radiation and preservative substance, impair corneal epithelium by inducing DNA damage.12,13 Particulate matter 2.5 has raised increasing concern for the potential human health impacts in recent years, but the influence of PM2.5 on the eye, especially on the ocular surface, has not been well understood. It has been found that CECs could engulf particular matter decades ago.14 However, there is no report regarding the genotoxic effect of PM2.5 on corneal epithelium. In this study, we found PM2.5 could induce DNA damage, activation of DNA repair response, as well as cell senescence. These results will provide a better understanding of the potential threat of air pollution to the eye. 
DNA damage refers to a variety of structurally abnormalities in the DNA, including single- and double-strand breaks, 8-hydroxydeoxyguanosine residues, and polycyclic aromatic hydrocarbon adducts. Generally, DNA damage can be recognized by enzymes and thus can be correctly repaired. The single-cell gel electrophoresis assay, also known as comet assay, is a sensitive technique for detection of DNA damage at the level of the individual cell and has been extensively applied in cell culture studies to observe the genotoxic effect of PM2.5.1517 In this study, in both the HCEC line and primary BCECs, we observed a high level of strand break after exposure to PM2.5 in a dose-dependent manner, and the level of DNA damage was decreased at the presence of ROS inhibitor. 
The cellular response to DNA damage, namely DNA damage response (DDR), may involve activation of a cell-cycle checkpoint, commencement of transcriptional programs, execution of DNA repair, or when the damage is severe, initiation of apoptosis. A variant of the H2A protein family, H2AX is an important regulator of DDR. It is phosphorylated at serine 139 by kinases such as ataxia telangiectasia mutated (ATM) and ATM-Rad3-related (ATR) within 1 to 3 minutes after DNA damage, known as γ-H2AX. The phosphorylated protein, γ-H2AX, is the first step in recruiting and localizing DNA repair proteins.18,19 The detection and visualization of γ-H2AX allow the assessment of DNA damage, related DNA damage proteins, and DNA repair.20 In this study, we used an immunofluorescence method to detect the DNA damage, and found the expression of γ-H2AX was remarkably increased in HCECs and BCECs exposed to PM2.5, suggesting the initiation of DNA repair in response to PM2.5 exposure. 
The relationship between DNA damage and aging has been investigated for decades. In 1967, Alexander21 proposed that DNA damage was the primary cause of aging. Thereafter, increasing experimental evidence supports the idea that oxidative DNA damage plays a key role in aging of various tissues. Recent studies suggested that DNA damage might inhibit self-renewal and induce senescence in tissue-specific stem cells.2224 Corneal epithelial stem cells (CESCs), also referred to as limbal stem cells, are stem cells located in the basal epithelial layer of the corneal limbus. Many studies have demonstrated that CESCs serve as the origin of CEC proliferation and thus is of great importance for corneal epithelium regeneration. Because there is no consensus to date regarding the definitive markers for CESCs, and identification and isolation of these cells are still challenging,25 we did not try to isolate primary CESCs in our study. We think that PM2.5 might exert a similar toxic effect on CESCs as CECs. With senescence-associated β-galactosidase assay methods, we detected PM2.5 could induce cell senescence in HCECs and BCECs. Senescent cells have bystander effects of spreading senescence toward their neighboring cells by secreting inflammatory cytokines and producing ROS, thus resulting in age-related diseases or promoting the process of aging.26,27 It has been demonstrated that the aging cornea undergoes structural alteration and is more susceptible to infection.28,29 Therefore, we infer that PM2.5 might result in deficiency of corneal epithelium regeneration and susceptibility to infection by inducing CESC senescence and aging. 
Reactive oxygen species are chemically reactive molecules containing oxygen, such as hydrogen peroxide, superoxide anion, and hydroxyl radical. Reactive oxygen species are produced as byproducts of normal cellular metabolism, and they have important roles in cell signaling and homeostasis.30,31 Reactive oxygen species can be dramatically increased under certain environmental or pathophysiological conditions, and thus result in an imbalance between ROS production and intracellular defense mechanisms, which is known as oxidative stress. Reactive oxygen species can damage cellular molecules and the cell structures (e.g., lipids, proteins, or DNA). A number of studies have shown that ROS formation is a critical mechanism by which particulate matter exerts it toxic effect in different cell types.3234 In agreement with the findings in other cell types, we found that PM2.5 could induce ROS production in HCECs, and suppression of ROS by NAC could alleviate the genotoxic effect of PM2.5. 
Particulate matter 2.5 is a complex mixture of organic and inorganic substances originating from a variety of sources, including power plants, industrial processes, and traffic, and the chemical and physical characteristics of PM2.5 vary with the location, weather, and time of year. Therefore, it is difficult to determine the contribution of each component in the PM2.5 to the DNA damaging effect and ROS formation. In this study, we collected PM2.5 samples from Guangzhou, China, a large city with severe traffic congestion and a high level of PM2.5. Even some investigators have found obvious visibility degradation due to light scattering and absorption by fine particles in the atmosphere in Guangzhou.35,36 Our results indicate that the PM2.5 sample collected in Guangzhou has evident DNA damaging effect probably by inducing ROS formation, suggesting its potential threat to the corneal defense system. However, to what extent such effects are influenced by the disparity in PM2.5 components collected from different sources or locations remains unclear. 
In summary, we found that PM2.5 could induce evident DNA damage and cell senescence in both immortalized and primary CECs, and this effect was partly abolished by ROS inhibitor. These results suggest that PM2.5-induced oxidative stress probably plays a key role in DNA damage in CECs, which may contribute to PM2.5-induced impairment of corneal epithelium. Yet, the in vivo effect of long-term exposure to PM2.5 on corneal epithelium should be further investigated to obtain an overall understanding of the toxic effect of PM2.5 and its relationship with potential risk of corneal lesions. 
Acknowledgments
Supported by the National Natural Science Foundation of China (81570814, 81300271, 91543132), Natural Science Foundation of Guangdong Province, China (2014A030313363), Scientific Research project of Guangzhou Municipal Education Department (1201410345), and Scientific Research project of Guangzhou Medical University (2012C02) 
Disclosure: Z.-X. Gao, None; X.-L. Song, None; S.-S. Li, None; X.-R. Lai, None; Y.-L. Yang, None; G. Yang, None; Z.-J. Li, None; Y.-H. Cui, None; H.-W. Pan, None 
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Figure 1
 
Cell viability of HCECs after PM2.5 exposure assayed with CCK-8. Human CECs were incubated for 24 hours with medium containing various concentrations of PM2.5: 20 μg/mL, 50 μg/mL, 100 μg/mL, and 200 μg/mL. The cell viability was determined using CCK-8 according to the manufacturer's instructions. The results were expressed as mean ± SD of three independent experiments. **P < 0.01 versus control.
Figure 1
 
Cell viability of HCECs after PM2.5 exposure assayed with CCK-8. Human CECs were incubated for 24 hours with medium containing various concentrations of PM2.5: 20 μg/mL, 50 μg/mL, 100 μg/mL, and 200 μg/mL. The cell viability was determined using CCK-8 according to the manufacturer's instructions. The results were expressed as mean ± SD of three independent experiments. **P < 0.01 versus control.
Figure 2
 
Lactate dehydrogenase cytotoxicity assay for HCECs after PM2.5 exposure. Human CECs cultured in a 96-well plate were incubated for 24 hours with the medium containing various concentrations of PM2.5 as indicated with or without NAC (1 mg/mL). The results were presented as the percent cytotoxicity of the maximum LDH release control according to the manufacturer's instructions. Data were expressed as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 versus control; ##P < 0.01 versus 200 μg/mL PM2.5 treated group.
Figure 2
 
Lactate dehydrogenase cytotoxicity assay for HCECs after PM2.5 exposure. Human CECs cultured in a 96-well plate were incubated for 24 hours with the medium containing various concentrations of PM2.5 as indicated with or without NAC (1 mg/mL). The results were presented as the percent cytotoxicity of the maximum LDH release control according to the manufacturer's instructions. Data were expressed as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 versus control; ##P < 0.01 versus 200 μg/mL PM2.5 treated group.
Figure 3
 
Comet assay for HCECs after PM2.5 exposure. Human CECs cultured in a 24-well plate were incubated for 24 hours with medium containing various concentrations of PM2.5 with or without NAC (1 mg/mL), respectively. Comet assay was performed with a kit according to the manufacturer's instructions. The images were obtained using a fluorescence microscope (×400). (A) Representative images of comet tails from each experimental group: 0 μg/mL, 100 μg/mL, 200 μg/mL, and 200 μg/mL with NAC (1 mg/mL). (B) Quantitative analysis of the tail length, percent DNA in comet tails, and comet tails moment. Data were expressed as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 versus control; #P < 0.05, ##P < 0.01 versus 200 μg/mL PM2.5 treated group.
Figure 3
 
Comet assay for HCECs after PM2.5 exposure. Human CECs cultured in a 24-well plate were incubated for 24 hours with medium containing various concentrations of PM2.5 with or without NAC (1 mg/mL), respectively. Comet assay was performed with a kit according to the manufacturer's instructions. The images were obtained using a fluorescence microscope (×400). (A) Representative images of comet tails from each experimental group: 0 μg/mL, 100 μg/mL, 200 μg/mL, and 200 μg/mL with NAC (1 mg/mL). (B) Quantitative analysis of the tail length, percent DNA in comet tails, and comet tails moment. Data were expressed as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 versus control; #P < 0.05, ##P < 0.01 versus 200 μg/mL PM2.5 treated group.
Figure 4
 
Immunofluorescence staining for γH2AX (Ser139) in HCECs after PM2.5 exposure. Human CECs cultured in a 24-well plate were incubated for 24 hours with medium containing PM2.5 at the concentration of 0 μg/mL, 50 μg/mL, 200 μg/mL, and 200 μg/mL PM2.5 with NAC (1 mg/mL), respectively. (A) The cells were immunostained with anti-γH2AX (Ser139) antibody (green, top), and the nuclei were counterstained with DAPI (blue, bottom). Images were taken at the magnification of ×200. (B) Quantitative analysis of the fluorescent MOD. The data represent mean ± SD of five measurements. **P < 0.01 versus control; #P < 0.05 versus the 200 μg/mL PM2.5 treated group.
Figure 4
 
Immunofluorescence staining for γH2AX (Ser139) in HCECs after PM2.5 exposure. Human CECs cultured in a 24-well plate were incubated for 24 hours with medium containing PM2.5 at the concentration of 0 μg/mL, 50 μg/mL, 200 μg/mL, and 200 μg/mL PM2.5 with NAC (1 mg/mL), respectively. (A) The cells were immunostained with anti-γH2AX (Ser139) antibody (green, top), and the nuclei were counterstained with DAPI (blue, bottom). Images were taken at the magnification of ×200. (B) Quantitative analysis of the fluorescent MOD. The data represent mean ± SD of five measurements. **P < 0.01 versus control; #P < 0.05 versus the 200 μg/mL PM2.5 treated group.
Figure 5
 
Senescence of HCECs after PM2.5 exposure determined with SA-β-gal staining. Human CECs cultured in a 24-well plate were incubated for 24 hours with medium containing various concentrations of PM2.5: 0 μg/mL, 20 μg/mL, 50 μg/mL, 100 μg/mL, 200 μg/mL, and 200 μg/mL with 1 mg/mL NAC, respectively. (A) Representative images (×200) of senescent cells determined with SA-β-gal staining. (B) Quantitative analysis of the MOD of staining cells. Data were expressed as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 versus control; ##P < 0.01 versus 200 μg/mL PM2.5 treated group.
Figure 5
 
Senescence of HCECs after PM2.5 exposure determined with SA-β-gal staining. Human CECs cultured in a 24-well plate were incubated for 24 hours with medium containing various concentrations of PM2.5: 0 μg/mL, 20 μg/mL, 50 μg/mL, 100 μg/mL, 200 μg/mL, and 200 μg/mL with 1 mg/mL NAC, respectively. (A) Representative images (×200) of senescent cells determined with SA-β-gal staining. (B) Quantitative analysis of the MOD of staining cells. Data were expressed as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 versus control; ##P < 0.01 versus 200 μg/mL PM2.5 treated group.
Figure 6
 
Morphology of HCECs after PM2.5 exposure. Human CECs cultured in a 6-well plate were incubated for 24 hours with medium containing PM2.5 at the concentration of 0 μg/mL, 20 μg/mL, 50 μg/mL, 100 μg/mL, 200 μg/mL or 200 μg/mL with NAC (1 mg/mL), respectively. The morphology was observed using an optical microscope (×200). It was shown that the PM2.5 treatment induced irregular cellular shape, remarkable cytoplasmic shrinkage, and decreased cell adhesion. The NAC treatment attenuated the morphology change induced by PM2.5 incubation.
Figure 6
 
Morphology of HCECs after PM2.5 exposure. Human CECs cultured in a 6-well plate were incubated for 24 hours with medium containing PM2.5 at the concentration of 0 μg/mL, 20 μg/mL, 50 μg/mL, 100 μg/mL, 200 μg/mL or 200 μg/mL with NAC (1 mg/mL), respectively. The morphology was observed using an optical microscope (×200). It was shown that the PM2.5 treatment induced irregular cellular shape, remarkable cytoplasmic shrinkage, and decreased cell adhesion. The NAC treatment attenuated the morphology change induced by PM2.5 incubation.
Figure 7
 
Transmission electron microscopy observation of HCECs after PM2.5 exposure. Human CECs were incubated for 24 hours with medium containing PM2.5 (200 μg/mL). Chromatin condensation and margination along the inner nuclear membrane, cytoplasmic condensation, and membrane blebbing could be observed (A, B). Uptake of particulate matter particles and appearance of cellular vesicles could be seen in some cells (C, D). N, nucleus; V, vesicle; arrows, PM2.5 aggregates. Transmission electron microscopy scale bars: (A) 2 μm, (B) 2 μm, (C) 2 μm, (D) 1 μm. Images were taken at the magnification of 9k, 15k, 9k, and 24k, respectively.
Figure 7
 
Transmission electron microscopy observation of HCECs after PM2.5 exposure. Human CECs were incubated for 24 hours with medium containing PM2.5 (200 μg/mL). Chromatin condensation and margination along the inner nuclear membrane, cytoplasmic condensation, and membrane blebbing could be observed (A, B). Uptake of particulate matter particles and appearance of cellular vesicles could be seen in some cells (C, D). N, nucleus; V, vesicle; arrows, PM2.5 aggregates. Transmission electron microscopy scale bars: (A) 2 μm, (B) 2 μm, (C) 2 μm, (D) 1 μm. Images were taken at the magnification of 9k, 15k, 9k, and 24k, respectively.
Figure 8
 
The effect of PM2.5 on ROS generation in HCECs. Human CECs cultured in a 24-well plate were incubated for 24 hours with medium containing PM2.5 at the concentrations of 0 μg/mL, 20 μg/mL, 50 μg/mL, 100 μg/mL, and 100 μg/mL with NAC (1 mg/mL), respectively. The intracellular ROS was detected with a fluorescent probe CM-H2DCFDA. (A) Representative images under fluorescent microscope (×200). (B) The fluorescent MOD of staining cells. Data were presented as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 versus control; #P < 0.05 versus 100 μg/mL PM2.5-treated group.
Figure 8
 
The effect of PM2.5 on ROS generation in HCECs. Human CECs cultured in a 24-well plate were incubated for 24 hours with medium containing PM2.5 at the concentrations of 0 μg/mL, 20 μg/mL, 50 μg/mL, 100 μg/mL, and 100 μg/mL with NAC (1 mg/mL), respectively. The intracellular ROS was detected with a fluorescent probe CM-H2DCFDA. (A) Representative images under fluorescent microscope (×200). (B) The fluorescent MOD of staining cells. Data were presented as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 versus control; #P < 0.05 versus 100 μg/mL PM2.5-treated group.
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