- Email: [email protected]
Airborne particulate matter (PM2.5) triggers autophagy in human corneal epithelial cell line
Environmental Pollution 227 (2017) 314e322
Contents lists available at ScienceDirect
Environmental Pollution journal homepage: www.elsevier.com/locate/envpol
Airborne particulate matter (PM2.5) triggers autophagy in human corneal epithelial cell line* Qiuli Fu a, b, 1, Danni Lyu a, b, 1, Lifang Zhang a, b, Zhenwei Qin a, b, Qiaomei Tang a, b, Houfa Yin a, b, Xiaoming Lou c, Zhijian Chen c, **, Ke Yao a, b, * a
Eye Center of the 2nd Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, China Zhejiang Provincial Key Lab of Ophthalmology, Hangzhou, Zhejiang Province, China Department of Environmental and Occupational Health, Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou, Zhejiang Province, China
b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 December 2016 Received in revised form 24 April 2017 Accepted 26 April 2017
Purpose: To investigate particulate matter (PM2.5)-induced damage to human corneal epithelial cells (HCECs) and to determine the underlying mechanisms. Methods: HCECs were exposed to PM2.5 at a series of concentrations for various periods. Cell viability was measured by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell proliferation was evaluated via 5-ethynyl-2’-deoxyuridine (EdU) analysis, while autophagy was determined by immunofluorescence and Western blot. Results: PM2.5-induced cell damage of HCECs occurred in a time- and dose-dependent manner. Decreased cell viability and proliferation as well as increased apoptosis were observed in HCECs after PM2.5 exposure for 24 h. Autophagy in HCECs was slightly inhibited in the early stage (before 4 h) of exposure but significantly activated in the late stage (after 24 h), as evidenced by a decrease in the former and increase in the latter of the expression of the autophagy-associated markers LC3B, ATG5, and BECN1. Interestingly, rapamycin, an autophagy activator, attenuated early-stage but aggravated late-stage PM2.5induced cell damage, suggesting that the role of autophagy in HCECs may change over time during PM2.5 exposure. In addition, in the early stage, the expression of LC3B and ATG5 increased in cells co-treated with rapamycin and PM2.5 compared to rapamycin-only or PM2.5-only treated cells, suggesting that autophagy may benefit cell viability after PM2.5 exposure. Conclusions: The results indicate the potential role of autophagy in the treatment of PM2.5-induced ocular corneal diseases and provide direct evidence for the cytotoxicity, possibly involving an autophagic process, of PM2.5 in HCECs. © 2017 Elsevier Ltd. All rights reserved.
Keywords: PM2.5 Autophagy Human corneal epithelial cells
1. Introduction China, like many other industrialized countries, has suffered severe air pollution problems in recent years, which are set to continue for a long time to come. Although the adverse health
*
This paper has been recommended for acceptance by David Carpenter. * Corresponding author. Eye Center of the 2nd Affiliated Hospital, Medical College of Zhejiang University, Hangzhou 310009, Zhejiang Province, China. ** Corresponding author. Department of Environmental and Occupational Health, Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051, Zhejiang Province, China. E-mail addresses: [email protected] (K. Yao), [email protected] (Z. Chen). 1 Q.F. and D.L. contributed equally to this work. http://dx.doi.org/10.1016/j.envpol.2017.04.078 0269-7491/© 2017 Elsevier Ltd. All rights reserved.
effects of such pollution may be associated with its atmospheric nature, increasing evidence suggests that particulate matter (PM), especially that less than 2.5 mm in diameter (referred to as PM2.5), plays a major role in cardiovascular and chronic cardiopulmonary morbidity and mortality (Guo et al., 2009; Ma et al., 2011; Wu et al., 2010; Yang et al., 2012). The eye is one of the few organs of the human body that are constantly exposed to the external environment. The cornea, which is comprised of five layers: epithelium, Bowman's layer, stroma, Descemet's membrane, and endothelium, represents the outermost structure of the eye and is particularly vulnerable to air pollution. Recent studies have shown that both water and organic extracts of indoor dust have adverse impacts on the human cornea (Xiang et al., 2016a). Although most people spend less than 10% of their
Q. Fu et al. / Environmental Pollution 227 (2017) 314e322
day outdoors (Seaton et al., 1999), the atmospheric environment is undeniably important for human ocular health, especially for those who spend long periods in the open such as children and outdoor workers. It is estimated that the cornea of an adult engaged in light activity is exposed to approximately 34.2 mg of PM2.5 (100 mg/m3) per hour (see Supplementary Fig. 1 for the detailed calculation method). Individuals living in areas with high concentrations of pollutants frequently complain of ocular symptoms and signs, such as watering, burning, itchiness, and redness (Camara and Lagunzad, 2011). Clinical studies have shown that certain air pollutants increase the chances of outpatient visits for nonspecific conjunctivitis and that PM2.5 may exacerbate the development of allergic conjunctivitis (Chang et al., 2012; Mimura et al., 2014). Pathological studies of the ocular surface have revealed that exposure to ambient levels of air pollution could affect the density of conjunctival goblet cells (Berra et al., 2015; Torricelli et al., 2011, 2014). In previous studies on indoor dust-induced toxicity in human corneal epithelial cells (HCECs), it was suggested that dustinduced oxidative stress plays a key role in the cytotoxicity of primary HCECs, in which heavy metals and PAHs are of significant importance (Xiang et al., 2016a, b). Another study provided evidence of cytotoxicity and inflammatory and oxidative stress responses in human corneal and conjunctival epithelial cells incubated with diesel exhaust particles (DEPs) (Tau et al., 2013). However, it is unclear whether PM2.5 exerts similar effects and which molecular mechanisms are involved in ocular diseases caused by long-term exposure to a high degree of environmental pollution. Autophagy is a predominant cellular process involved in the delivery of cytoplasmic substrates to lysosomes for degradation. Its role as a self-destructive or cytoprotective process depends on various conditions (De Meyer et al., 2015; Gottlieb et al., 2009; Killian, 2012; Lopez-Alonso et al., 2013; Sun et al., 2012). Accumulated evidence indicates that autophagy plays crucial roles in a range of physiological and pathological processes, such as development, aging, aging-related diseases, cellular homeostasis, and cancer (Lapierre et al., 2015; Parzych and Klionsky, 2014). Some studies have shown that autophagy is significant in maintaining corneal homeostasis and transparency, as well as in degradation of invading microorganisms (Karnati et al., 2016; Wang et al., 2013). Recent research has revealed that autophagy is involved in PM2.5induced cardiovascular and respiratory diseases and that autophagy inhibitors are able to eliminate or alleviate PM2.5-induced symptoms (Chen et al., 2016; Deng et al., 2013, 2014). Based on these findings, we hypothesized that autophagy may also be critical in PM-induced ocular diseases. The aim of this study was to explore the effect of PM2.5 on HCECs in vitro. The results demonstrate that PM2.5 exposure significantly inhibits cell viability and proliferation in HCECs, and that activation of autophagy is altered in a time-dependent manner following PM2.5 exposure. The present work provides the first evidence that alteration of autophagy activity in HCECs may be one of the mechanisms underlying PM2.5-induced corneal diseases such as keratitis. 2. Materials and methods 2.1. PM2.5 collection In total, six samples were collected with 24 h’ sampling time each (from 9:00 a.m. local time each day to 9:00 a.m. the next day), on the 15th of every month from December 2015 to May 2016. The sampling site was set in the yard of the Center for Disease Control and Prevention of Zhejiang Province (ZJCDC), Hangzhou, Zhejiang, China, at a height equal to the respiratory zone, namely 1.5 m above
315
ground. Atmospheric PM samples were collected on 90-mm quartz fiber filters (QZ47DMCAN, MTL, America) using three mid-volume samplers (MVS6, LECKEL, Germany) at a constant flow rate of 38.3 L/min. The samples were weighted by an automated filterweighing system (WZZ-02, Weizhizhao, Hangzhou, China) before and after sampling to determine the mass of PM. All filters were immediately transported to the laboratory in a filter holder and stored at 20 C until analysis. Weather information, including temperature, relative humidity, wind speed, precipitation, atmospheric pressure, and sunshine hours for every sampling month, is listed in Supplementary Table 1. The PM samples were prepared as described in a previous study (Zhou et al., 2015). Briefly, the quartz filters containing the PM samples were soaked in 75% ethanol (dissolved in Mili-Q water) and sonicated for 30 min. The PM suspensions thereby obtained were dried by lyophilization and re-suspended in sterilized water to achieve a final concentration of 2 mg/mL. Finally, aliquots of the PM suspensions were stored at 80 C for later experiments. 2.2. Quality assurance/quality control (QA/QC) Quality control acceptance criteria were taken into account during atmospheric PM2.5 sampling and analysis of chemical components. For the former, three samplers were deployed simultaneously to collect the atmospheric PM2.5 samples. Surrogate recoveries, limits of detection (LOD), and relative standard deviations (RSD) of chemical components analysis are listed in Supplementary Table 2. None of the recoveries in the present study were outside the surrogate recovery. When a concentration was below the LOD, the value was assumed to be equal to LOD (upper bound) for calculation purposes. One test of procedural blank was carried out for every 20 samples. 2.3. Scanning electron microscopy (SEM) analysis of PM The morphology of PM was observed via SEM (Hitachi, Tokyo, Japan). The PM samples on quartz filters were coated with platinum and observed using an automatic mode. 2.4. Analysis of the size distribution and chemical components of the PM The size distribution of the PM suspended in pure water was analyzed using a Nano-Zetasizer (Malvern Instruments Ltd., Worcestershire, UK) based on the dynamic light-scattering measurement technique. Chemical analysis of the characteristics of the PM samples was performed immediately after collection. The organic (polycyclic aromatic hydrocarbons, PAHs), ion, and metal compositions of the samples were analyzed. In all, 17 PAHs (acenaphthene, acenaphthylene, anthracene, benz(a)anthracene, benzo[a]pyrene, benzo[b] fluorathene, benzo[ghi]perylene, benzo[k]fluoranthene, chrysene, decafluorobiphenyl, dibenz[a,h]anthracene, fluoranthene, fluorine, indeno[123-cd]pyrene, naphthalene, phenanthrene, and pyrene) were analyzed. The concentrations of ions, including nitrate (NO3), sulfate (SO42), ammonium (NH4þ), and chloride (Cl), were measured. The concentrations of 12 metals, including lead (Pb), aluminum (Al), manganese (Mn), arsenic (As), selenium (Se), antimony (Sb), chromium (Cr), nickel (Ni), cadmium (Cd), thallium (Tl), mercury (Hg), and beryllium (Be). 2.5. Cell culture and exposure to PM2.5 HCEC lines (PCS-700-010) were purchased from ATCC (VA, USA). The cells were cultured in DMEM/F12 (Gibco, CA, USA) with 15%
316
Q. Fu et al. / Environmental Pollution 227 (2017) 314e322
fetal bovine serum (FBS) (Gibco, CA, USA), passaged with 0.25% trypsin and 0.02% ethylene diamine tetra-acetic acid (EDTA) (Gibco, CA, USA) every three days. The HCECs were then seeded onto 6well, 24 well, 96-well plates or 10 cm dishes overnight to allow attachment before PM2.5 treatment. Subsequently, all the culture medium was replaced with fresh medium containing PM2.5 suspension, supplemented with 100 U/mL penicillin and 0.1 mg/mL streptomycin (Gibco, CA, USA). 2.6. Western blot HCECs were seeded onto 10 cm culture dishes at a density of 5 104 cells/mL with 10 mL of medium overnight before PM2.5 treatment. They were then exposed to PM2.5 (50 mg/mL) for 1, 2, 4, 6, 12, 24, 36, or 48 h. Rapamycin (an autophagy activator) and 3Methyladenine (3-MA, an autophagy inhibitor) were administered for autophagy regulation. After treatment, the cells were lysed with lysis buffer (Sangon, Shanghai, China) containing phenylmethanesulfonyl fluoride (PMSF) (Sangon, Shanghai, China), protease inhibitor (Sangon, Shanghai, China), and phosphatase inhibitors (Sangon, Shanghai, China). The protein concentration was determined using a spectrophotometer (iMark Microplate Absorbance Reader; Bio-Rad, CA, USA). Extracts from each sample were loaded onto gels, transferred onto membranes, and immunoblotted with primary antibodies including rabbit anti-LC3B (Sigma Aldrich, MO, USA; 1:1000), rabbit anti-BECN1 (CST, MA, USA; 1:1000), and rabbit anti-ATG5 (CST, MA, USA; 1:1000), followed by incubation with HRP-conjugated anti-rabbit IgG (CST, MA, USA; 1:5000). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal control. 2.7. 3 -(4,5-dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium bromide (MTT) assay HCECs were seeded at a density of 1 104 to 3 104 cells/mL (adjusted according to the culture time) onto 96-well plates with 200 mL of medium per well overnight before PM2.5 treatment. The next day, HCECs were treated with various concentrations of PM2.5 (0, 0.5, 1, 2, 5, 10, 25, 50, 75, and 100 mg/mL) for 24 h or 50 mg/mL of PM2.5 for 0, 0.5, 1, 2, 4, 6, 12, 24, or 48 h in 96-well plates. Before MTT (Sigma Aldrich, MO, USA) was added (final concentration of 350 mg/mL), the culture medium with PM2.5 was removed, and each well was washed three times with phosphate-buffered saline (PBS). After incubation in a humid atmosphere of 37 C with 5% CO2 for 4 h, the culture medium was removed and dimethyl sulfoxide (DMSO) (Sigma Aldrich, MO, USA) was added. Finally, the absorbance in each well was recorded at 490 nm using a spectrophotometer (iMark Microplate Absorbance Reader; Bio-Rad, CA, USA). 2.8. 5-Ethynyl-2’-deoxyuridine (EdU) analysis HCECs were seeded at a density of 1.5 104 cells/mL onto 24well plates with 1 mL of medium per well overnight before PM2.5 treatment. To examine the effect of PM2.5 on cell proliferation, the HCECs were first exposed to PM2.5 for 12, 24, 36, or 48 h. Then, half of the medium containing PM2.5 was replaced with fresh medium containing 20 mM of EdU (final concentration of 10 mM) and cultured with cells for 2 h. To detect cell proliferation, EdUlabeled cells were processed according to the manufacturer's protocol (Click-iT EdU Imaging Kit, ThermoFisher, MA, USA). Finally, images were captured using an Olympus IX71 microscope (Olympus, Tokyo, Japan), equipped with DP2-BSW software (Olympus, Tokyo, Japan). The percentage of EdUþ cells within the total number was then quantified.
2.9. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis of HCECs HCECs were seeded at a density of 3 104 cells/mL onto 24-well plates with 1 mL of medium per well overnight before PM2.5 treatment. To examine the PM-induced cell apoptosis in HCECs, the cells were first exposed to PM2.5 (50 mg/mL) for 24 h. Apoptosis was scored by TUNEL (Click-iT Plus TUNEL Assay, Life Technologies, CA, USA) analysis of the PM2.5-treated and fixed HCECs, according to the manufacturer's protocol. 2.10. Flow cytometric analysis HCECs were seeded at a density of 1.5 105 cells/mL onto 6-well plates with 2 mL of medium per well overnight before PM2.5 treatment. HCECs were exposed to 50 mg/mL of PM2.5 for 24 h. Apoptotic cells were then identified and quantified by Annexin VFITC and propidium iodide (PI) Apoptosis Detection Kit (Biyuntian, Beijing, China). The cells were harvested with trypsin-EDTA and washed twice with cold PBS. Cells were incubated in 195 mL of Annexin V-FITC binding buffer supplemented with 5 mL of Annexin V-FITC and 10 mL of PI at room temperature in the dark for 15 min. The apoptosis of cells was analyzed by a flow cytometer (FACSCanto II, BD, NJ, USA). 2.11. Immunofluorescence examinations HCECs were seeded at a density of 3 104 cells/mL onto 24-well plates with 1 mL of medium per well overnight before PM2.5 treatment. The HCECs were seeded on cleaned and autoclaved cover glass, followed by PM2.5 (50 mg/mL) exposure for 24 h. As a positive control, cells were incubated with rapamycin (1 mM) and serum deprivation for 4 h. Following exposure, the cells were washed with PBS and fixed with paraformaldehyde (4% in PBS). The cell samples were then permeabilized with TritonX-100 (Sigma Aldrich, MO, USA; 0.4% in PBS) for 15 min and incubated overnight with primary antibodies: rabbit anti-caspase-3 (CST, MA, USA; 1:100) or rabbit anti-LC3B (Sigma Aldrich, MO, USA; 1:100), followed by incubation with secondary antibodies (1:1000) labeled with Alexa Fluor 488 or 555 (Invitrogen, CA, USA). Nuclei were labeled with 4,6-diamido-2-phenylindole dihydrochloride (DAPI) (Sigma Aldrich, MO, USA; final concentration of 1 mg/mL). Images were captured using a Leica TCS SP8 confocal microscope (Leica, Wetzla, Germany) and processed with Image J software (National Institutes of Health, DC, USA). 2.12. Statistical analysis The results are presented as means ± standard deviation (SD). Data were analyzed using GraphPad Prism 6.0c (GraphPad Software, CA, USA). For all data, a one-way analysis of variance (ANOVA) was applied using SPSS 22.0.0.0 (IBM, NY, USA), followed by a post hoc multiple comparison using the least significant difference test when comparisons of more than two groups were required. 3. Results 3.1. Physical and chemical characteristics of PM2.5 The SEM analysis showed the morphology of PM particles in the quartz filters (Fig. 1A). Dynamic light-scattering measurements revealed PM with sizes ranging between 1106 nm and 3091 nm. The sizes of more than 91.3% of the particles were less than 2305 nm (Fig. 1B). Therefore, most of the PM used in the experiments was less than 2.5 mm in size.
Q. Fu et al. / Environmental Pollution 227 (2017) 314e322
317
Fig. 1. Physical characterizations of PM and cytotoxicity in HCECs. (A) SEM image of PM. (B) Particle size distribution analyzed by dynamic light scattering. (C and D) Cell viability analyzed by MTT assay. The cells were treated with various concentrations of PM2.5 for 24 h (C) or with 50 mg/mL of PM2.5 for different periods (D). The data are presented as means ± SD of at least three independent experiments. *p < 0.05 and **p < 0.01 vs. control. #p < 0.05 and ##p < 0.01. (E) Images of EdU-labeled cells detected by fluorescent microscope. The cells were treated with PM2.5 (50 mg/mL) for 12, 24, 36, or 48 h. Control, vehicle control. (F) Percentage of EdUþ cells. Ten fields (200 ) from each group were randomly chosen for statistical analysis. Control, vehicle control. The data are presented as means ± SD. **p < 0.01 vs. control. #p < 0.05.
The organic, ion, and metal composition of PM collected in October, November, and December 2015 and April 2016 was measured. Of the 17 PAHs measured, decalfluorobiphenyl (120.260 ± 146.447 ng/m3) was the most abundant (Supplementary 2 Table 3). Four ions (NO 3 , SO4 , NH4, and Cl ) were detected, with 3 concentrations of 13.97 ± 10.16 mg/m , 12.47 ± 7.07 mg/m3, 8.84 ± 5.77 mg/m3, and 1.76 ± 1.14 mg/m3 respectively (Supplementary Table 4). Among the 11 metals detected in the PM, Pb, Al, and Mn were the three most prominent, with concentrations of 78.36 ± 34.45 ng/m3, 56.33 ± 32.86 ng/m3, and 36.39 ± 13.89 ng/ m3 respectively (Supplementary Table 5).
3.2. Time- and dose-dependent impacts of PM2.5 on cytotoxicity in HCECs Light microscopy images showed that HCECs became bigger and flatter after exposure to PM2.5 (50 mg/mL) for 24 h compared to unexposed cells (Supplementary Fig. 2). The results reveal that the HCECs were very sensitive to incubation with PM, even PM2.5 at a concentration as low as 0.5 mg/mL. A dose-dependent decrease in cell viability was observed when the HCECs were exposed to different doses of PM2.5 for 24 h (Fig. 1C). It was indicated that there was no significant difference of cell viability observed among
318
Q. Fu et al. / Environmental Pollution 227 (2017) 314e322
50, 75, 100 mg/mL conditions. Therefore, a concentration at 50 mg/ mL was selected for the subsequent study. In addition, a timedependent decrease in cell viability was also found when the HCECs were exposed to PM2.5 (50 mg/mL) for various periods. A significant difference in cell viability was observed after 4 h of PM2.5 exposure (Fig. 1D). 3.3. Effect of PM2.5 exposure on cell proliferation and apoptosis of HCECs The percentage of EdUþ cells was significantly decreased in the PM2.5-exposed cells (Fig. 1E), pointing to adverse effects of PM2.5 on normal HCECs turnover. More importantly, longer exposure periods (more than 12 h) resulted in decreased cell proliferation (Fig. 1F). Additionally, after exposure of 50 mg/mL of PM2.5 for 24 h, only a few TUNELþ and cleaved caspase-3þ cells were observed (Supplementary Figs. 3A and B). Meanwhile, the results of the Annexin V-FITC/PI apoptosis detection test revealed that the percentage of early apoptotic cells (Supplementary Fig. 3C, lower right quadrant, 6.73± 1.05%) and late apoptotic cells (Supplementary Fig. 3C, upper right quadrant, 10.03± 0.91%) of HCECs treated with 50 mg/mL of PM2.5 for 24 h increased compared to that in the control cells (Supplementary Fig. 3C, 3.27± 2.20% and 4.73± 0.29%, respectively). 3.4. Influence of PM2.5 exposure on autophagy in HCECs The results of the immunofluorescence study show that the percentage of cells containing LC3B puncta significantly increased after exposure to PM2.5 (50 mg/mL) for 24 h, with the number of cells close to those induced by rapamycin, compared to the unexposed group (Fig. 2A and B). Additionally, the LC3B puncta per cell were more abundant in the PM-exposed cells and rapamycininduced positive control, compared to the unexposed cells (Fig. 2C). Interestingly, the results of Western blot showed that the expression of LC3B II, the activated form of LC3B, decreased in the first 4 h (with no significant difference), then slowly returned to the baseline, but increased with longer PM2.5 exposure periods (24, 36, and 48 h) when compared to the unexposed cells (Fig. 3A and B). Similar results were observed in the expressions of BECN1 and ATG5, which decreased shortly after exposure to PM2.5 and increased thereafter (Fig. 3D and E). These observations, together with the results that the cell viability decreased sharply with longer exposure to PM2.5, especially after 12 h, indicate that autophagy may play different roles under various PM2.5 exposure periods. 3.5. Dual effects of autophagy on PM2.5-induced cell damage in the early and late stages As shown in Fig. 4A, rapamycin partially attenuated PM2.5induced cell damage in the early stage (before 4 h), but the reverse effect was observed in the late stage (after 24 h), in which rapamycin accelerated PM2.5-induced cell damage. The results of Western blot showed that autophagy was triggered in the PM2.5 and rapamycin co-treated cells and that the expressions of LC3B II and ATG5 were upregulated under this condition compared to the cells treated with PM2.5 or rapamycin alone (Fig. 4BeF). In addition, the elevated expressions of LC3B II and ATG5 could be partially abolished by 3-MA. These results indicate that the protective effect of rapamycin in the early stage may be due to the activation of autophagy, which was slightly inhibited in the early stage. It is also suggested that when autophagy is activated by PM2.5, rapamycin may aggravate its adverse effects by triggering autophagy activity in the late stage of PM2.5 exposure. Therefore, our results point to autophagy having a range of roles in HCECs, depending on the
exposure period to PM2.5. 4. Discussion In the present study, following PM2.5 exposure, the viability of HCECs decreased in a time- and dose-dependent manner. PM2.5 exposure had an accelerative effect on apoptosis in HCECs, and significant inhibition on proliferation was also revealed. More importantly, autophagy was slightly inhibited in the early stage but activated in HCECs in the late stage of PM2.5 exposure, which may contribute to the effect of rapamycin on PM2.5-induced cell damage at different stages. Although the relationship between PM and eye discomfort has been revealed in a recent epidemiological investigation (Mimura et al., 2014), the underlying pathological mechanisms are yet to be elucidated. Previous studies have shown that house dust induces cytotoxicity, oxidative stress, proinflammatory response, and mitochondrial dysfunction in primary HCECs (Xiang et al., 2016a, b). Evidence from a study of DEPs, one of the constituents of PM, also showed that oxidative stress caused by DEPs led to cytotoxicity and inflammatory responses in human conjunctiva cells (Tau et al., 2013). However, among the mechanisms investigated and involved in house dust- or DEPs-induced cell damage, autophagy has not been studied. To address this, we examined the effects of PM2.5 on HCECs and the role of autophagy as the underlying mechanism. Atmospheric DEPs exist as individual particles or chains of aggregates, with most in the invisible sub-micrometer range of 100 nm (Avino and Manigrasso, 2016), also known as ultrafine particles. However, in the present study, 81.1% of the PM collected and analyzed was in the range of 1484e2305 nm, suggesting that DEPs may not have been the main component of the PM2.5 used in the present study. Both DEPs and PM are complex mixtures of chemical and biological elements, such as PAHs and ions, which are not only adsorbed on the surface of PM but also deeply encrusted in its structure, as previously reported (Deng et al., 2013; Leung et al., 2014). However, the physical and chemical characteristics of DEPs and PM have been neglected in most clinical and epidemiological research (Guo et al., 2009; Ma et al., 2011; Tau et al., 2013; Wu et al., 2010; Yang et al., 2012). In addition, the results of toxicological studies into DEPs' and PM's toxicity vary according to the region where the study was conducted (Chen et al., 2016; Deng et al., 2013, 2014; Mimura et al., 2014; Tau et al., 2013; Zhou et al., 2015). A previous study on the possible influence of PM on the environment in the Yangtze Delta region of China, where Hangzhou is located, showed that the air in this region contained relatively considerable PM carrying high concentrations of ammonium, nitrate, and sulfate (Xu et al., 2002), which is consistent with the findings of the present study. These results indicate that the PM in Hangzhou may have significant impacts on human ocular health. PAHs are widely recognized environmental pollutants due to their carcinogenic and mutagenic properties that impact human health (Mishra et al., 2016). A previous study showed that concentrations of PAHs in PM2.5 from indoor dust at public places in Hangzhou accounted for 71.5% of total particulate PAHs, of which naphthalene was the most abundant (Lu et al., 2008). Therefore, PM2.5, rather than PM10, was chosen for the study of PM-induced human ocular health problems. Among all the PAHs found in PM2.5 in the present study, decalfluorobiphenyl was the most abundant, in comparison to naphthalene in a previous study (Lu et al., 2008), which may be due to their differences in origins. Another study also suggested that PAHs play an essential role in house dust-induced toxicity in primary HCECs (Xiang et al., 2016a). Additionally, Pb, Al, and Mn were found to be the most commonly found metals in the PM2.5 used in the present study, which is consistent with the adverse effect of metals
Q. Fu et al. / Environmental Pollution 227 (2017) 314e322
319
Fig. 2. PM2.5-induced autophagosome formation in HCECs through immunofluorescence examination. (A) Representative immunofluorescence images of the punctate staining of LC3B in HCECs treated with the vehicle control, PM2.5 (50 mg/mL) for 24 h, or rapamycin (1 mM) and serum deprivation for 4 h (B and C) Percentage of autophagic cells (B) and average puncta per autophagic cell (C). At least three fields (630 ) from each group were randomly chosen for analysis. Control, vehicle control. The data are presented as means ± SD. **p < 0.01 vs. control.
on human health as suggested by previous studies on house dust (Xiang et al., 2016a, b). The aforementioned components may play key roles in the cytotoxicity of PM2.5 in HCECs; however, the individual biological functions of each element remain to be determined in further experiments. In the PM2.5-exposed HCECs, we observed a time- and dosedependent decrease in cell viability. This occurred even when the HCECs were exposed to PM2.5 at concentrations as low as 0.5 mg/ mL, and the viability decreased significantly in accordance with a rise in the concentration of PM2.5. When compared to cytotoxicity induced in HCECs exposed to DEPs (Tau et al., 2013), PM2.5 exposure appeared to induce greater cytotoxicity, with even low doses of PM2.5 resulting in a marked decrease in viability, which may be due to the different diameters of particulate and chemical components attached. As mentioned above, the diameters of PM2.5 used in the present study are larger than those of DEPs; therefore, PM2.5 may carry more harmful chemicals. The distinctive characteristics of PM2.5 in the present study may as well attribute to the accelerative effect of PM2.5 on apoptosis in HCECs. This result is inconsistent with the findings on DEPs exposure in HCECs though (Tau et al., 2013), which may be due to the differences in size and
the harmful components attached to PM2.5 and DEPs. In airway epithelial cells, PM-induced oxidative stress triggers autophagy, which is essential for inflammation and mucus hyperproduction (Chen et al., 2016). Accordingly, we hypothesized that PM-induced oxidative stress in ocular surface epithelial cells would also trigger autophagy. A previous study of the cytotoxicity of DEPs in human conjunctival epithelial cells showed that DEPs induced oxidative stress and inflammatory responses, but it was unclear whether autophagy was involved (Tau et al., 2013). Although the essential role of autophagy in PM-induced injury in airway epithelium cells has been well documented in both cells and mouse models (Chen et al., 2016), its role in human ocular surface epithelium cells has remained elusive. To our knowledge, the present study provides the first evidence of the involvement of autophagy in the mechanism of PM2.5-induced cell damage in ocular surface epithelial cells. After 24 h of exposure to PM2.5, autophagy was markedly activated in the HCECs. The upregulation of autophagy may have been the main cause of the cell damage, as the latter deteriorated when the cells were co-treated with PM2.5 and rapamycin, an autophagy activator. These results are consistent with the previous findings in lung epithelial cells, in which PM-
320
Q. Fu et al. / Environmental Pollution 227 (2017) 314e322
Fig. 3. Time-dependent effect of PM2.5 exposure on autophagy in HCECs. (A) The expression levels of LC3B, ATG5, and BECN1 in the PM2.5-exposed HCECs detected by Western blot. The cells were exposed to PM2.5 (50 mg/mL) for different periods. GAPDH was used as the internal control. Control, vehicle control. Positive control (PC), treatment with rapamycin (1 mM) and serum deprivation for 4 h (B to E) Quantitative analysis of relative expression levels of LC3B II (B), LC3B II/LC3B I (C), ATG5 (D), and BECN1 (E). Control, vehicle control. The data are presented as means ± SD of at least three independent experiments. **p < 0.01 vs. control. #p < 0.05.
induced cell damage was reduced in transgenic autophagydeficient LC3/ and Becn1þ/ mice (Chen et al., 2016). Interestingly, in the present study, autophagy was slightly inhibited in the early stage of PM2.5 exposure in the HCECs, which has not been reported previously in other cells or tissues. Additionally, administration of rapamycin in the first six hours partially reversed the PM2.5-induced cell damage, suggesting that autophagy may act as a protective factor in the early stage of PM2.5 exposure in HCECs.
The results of the present study are based on a cellular model using an HCEC cell line, which is stable but may lose some characteristics over time compared to primary cultures (Xiang et al., 2016a, b). Therefore, studies that use primary HCECs are also needed to further investigate PM2.5-induced cytotoxicity and its mechanisms. In addition, the cellular model used in the present study is based on a monolayer structure, which partially represents the natural stratified corneal epithelium in vivo. Therefore, a 3D
Q. Fu et al. / Environmental Pollution 227 (2017) 314e322
321
Fig. 4. Effect of autophagy on PM2.5-induced cytotoxicity. (A) Cell viability measured by MTT assay. The HCECs were treated with PM2.5 (50 mg/mL) for various periods, with or without pre-treatment of rapamycin (1 mM) and serum deprivation for 2 h. The data are presented as means ± SD of at least three independent experiments. *p < 0.05 and **p < 0.01. (B) The expression levels of LC3B, ATG5, and BECN1 in HCECs detected by Western blot. The cells were treated with the vehicle control (Group C); PM2.5 (50 mg/mL) for 4 h (Group P); rapamycin (1 mM) with serum deprivation for 4 h (Group R); co-treatment with PM2.5 (50 mg/mL) and rapamycin (1 mM) and serum deprivation for 4 h (Group R þ P); pretreatment with rapamycin (1 mM) and serum deprivation for 0.5 h, followed by the addition of PM2.5 (50 mg/mL) for 3.5 h (Groups ReP); 3-MA (5 mM) for 4 h (Group M); cotreatment with 3-MA (5 mM) and PM2.5 (50 mg/mL) for 4 h (Group M þ P); co-treatment with 3-MA (5 mM), rapamycin (1 mM) and serum deprivation for 4 h (Group M þ R); or co-treatment with 3-MA (5 mM), rapamycin (1 mM), PM2.5 (50 mg/mL) and serum deprivation for 4 h (Group M þ R þ P). GAPDH was used as the internal control. (CeF) Quantitative analysis of the relative expression levels of LC3B II (C), LC3B II/LC3B I (D), ATG5 (E), and BECN1 (F). The data are presented as means ± SD of at least three independent experiments. **p < 0.01 vs. control; #p < 0.05, ##p < 0.01 vs. Group P; yp < 0.05, yyp < 0.01 vs. Group R; zp < 0.05, zzp < 0.01 vs. Group R þ P; xxp < 0.01 vs. Group ReP; jjp < 0.05, jjjjp < 0.01 vs. Group M; ¶¶p < 0.01 vs. Group M þ P.
stratified corneal epithelium model is necessary in the future. As well, in the present study, a cellular model of PM exposure at rather high concentrations for short periods was used to simulate acute and strong exposure, which may be related to PM-induced acute keratitis. However, it is also of great significance to focus on lower concentrations and longer periods to simulate chronic mild exposure, which may contribute to dry eye, chronic inflammation, etc. Additionally, experiments in animal models are also needed to shed
light on the molecular mechanism of PM2.5-induced injury in HCECs. With this aim in mind, an appropriate method to simulate PM2.5-induced injury in animals is urgently needed. In summary, the present study provides the first evidence that PM2.5 exposures over certain concentrations and culture periods result in HCECs dysfunction, in which autophagy plays an essential role. The results indicate that regulation of the autophagic process may have potential value in the treatment of PM-induced ocular
322
Q. Fu et al. / Environmental Pollution 227 (2017) 314e322
surface diseases. Acknowledgements This work has been supported by National Natural Science Foundation of China (81371001, 81300641, 81570822, 81670833, 81502786), Project of National Clinical Key Discipline of Chinese Ministry of Health, Zhejiang Key Laboratory Fund of China (2011E10006), Zhejiang Province Key Research and Development Program (2015C03042), Program of Zhejiang Medical technology (2015KYA109), Science and Technology Program of Zhejiang (2014C03025), National Natural Science Foundation of Zhejiang Province (LQ14H260003), and Analysis Center of Agrobiology and Environmental Sciences & Institute of Agrobiology and Environmental Sciences, Zhejiang University. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.envpol.2017.04.078. References Avino, P., Manigrasso, M., 2016. Dynamic of submicrometer particles in urban environment. Environ. Sci. Pollut. Res. Int. Berra, M., Galperin, G., Dawidowski, L., Tau, J., Marquez, I., Berra, A., 2015. Impact of wildfire smoke in Buenos Aires, Argentina, on ocular surface. Arq. Bras. Oftalmol. 78, 110e114. Camara, J.G., Lagunzad, J.K., 2011. Ocular findings in volcanic fog induced conjunctivitis. Hawaii Med. J. 70, 262e265. Chang, C.J., Yang, H.H., Chang, C.A., Tsai, H.Y., 2012. Relationship between air pollution and outpatient visits for nonspecific conjunctivitis. Invest Ophthalmol. Vis. Sci. 53, 429e433. Chen, Z.H., Wu, Y.F., Wang, P.L., Wu, Y.P., Li, Z.Y., Zhao, Y., Zhou, J.S., Zhu, C., Cao, C., Mao, Y.Y., Xu, F., Wang, B.B., Cormier, S.A., Ying, S.M., Li, W., Shen, H.H., 2016. Autophagy is essential for ultrafine particle-induced inflammation and mucus hyperproduction in airway epithelium. Autophagy 12, 297e311. De Meyer, G.R., Grootaert, M.O., Michiels, C.F., Kurdi, A., Schrijvers, D.M., Martinet, W., 2015. Autophagy in vascular disease. Circ. Res. 116, 468e479. Deng, X., Zhang, F., Rui, W., Long, F., Wang, L., Feng, Z., Chen, D., Ding, W., 2013. PM2.5-induced oxidative stress triggers autophagy in human lung epithelial A549 cells. Toxicol Vitro 27, 1762e1770. Deng, X., Zhang, F., Wang, L., Rui, W., Long, F., Zhao, Y., Chen, D., Ding, W., 2014. Airborne fine particulate matter induces multiple cell death pathways in human lung epithelial cells. Apoptosis 19, 1099e1112. Gottlieb, R.A., Finley, K.D., Mentzer Jr., R.M., 2009. Cardioprotection requires taking out the trash. Basic Res. Cardiol. 104, 169e180. Guo, Y., Jia, Y., Pan, X., Liu, L., Wichmann, H.E., 2009. The association between fine particulate air pollution and hospital emergency room visits for cardiovascular diseases in Beijing, China. Sci. Total Environ. 407, 4826e4830. Karnati, R., Talla, V., Peterson, K., Laurie, G.W., 2016. Lacritin and other autophagy associated proteins in ocular surface health. Exp. Eye Res. 144, 4e13. Killian, M.S., 2012. Dual role of autophagy in HIV-1 replication and pathogenesis. AIDS Res. Ther. 9, 16. Lapierre, L.R., Kumsta, C., Sandri, M., Ballabio, A., Hansen, M., 2015. Transcriptional and epigenetic regulation of autophagy in aging. Autophagy 11, 867e880. Leung, P.Y., Wan, H.T., Billah, M.B., Cao, J.J., Ho, K.F., Wong, C.K., 2014. Chemical and
biological characterization of air particulate matter 2.5, collected from five cities in China. Environ. Pollut. 194, 188e195. Lopez-Alonso, I., Aguirre, A., Gonzalez-Lopez, A., Fernandez, A.F., AmadoRodriguez, L., Astudillo, A., Batalla-Solis, E., Albaiceta, G.M., 2013. Impairment of autophagy decreases ventilator-induced lung injury by blockade of the NFkappaB pathway. Am. J. Physiol. Lung Cell Mol. Physiol. 304, L844eL852. Lu, H., Zhu, L.Z., Chen, S.G., 2008. Pollution level, phase distribution and health risk of polycyclic aromatic hydrocarbons in indoor air at public places of Hangzhou, China. Environ. Pollut. 152, 569e575. Ma, Y., Chen, R., Pan, G., Xu, X., Song, W., Chen, B., Kan, H., 2011. Fine particulate air pollution and daily mortality in Shenyang, China. Sci. Total Environ. 409, 2473e2477. Mimura, T., Ichinose, T., Yamagami, S., Fujishima, H., Kamei, Y., Goto, M., Takada, S., Matsubara, M., 2014. Airborne particulate matter (PM2.5) and the prevalence of allergic conjunctivitis in Japan. Sci. Total Environ. 487, 493e499. Mishra, N., Ayoko, G.A., Morawska, L., 2016. Atmospheric polycyclic aromatic hydrocarbons in the urban environment: occurrence, toxicity and source apportionment. Environ. Pollut. 208, 110e117. Parzych, K.R., Klionsky, D.J., 2014. An overview of autophagy: morphology, mechanism, and regulation. Antioxid. Redox Signal 20, 460e473. Seaton, A., Soutar, A., Crawford, V., Elton, R., McNerlan, S., Cherrie, J., Watt, M., Agius, R., Stout, R., 1999. Particulate air pollution and the blood. Thorax 54, 1027e1032. Sun, Y., Li, C., Shu, Y., Ju, X., Zou, Z., Wang, H., Rao, S., Guo, F., Liu, H., Nan, W., Zhao, Y., Yan, Y., Tang, J., Zhao, C., Yang, P., Liu, K., Wang, S., Lu, H., Li, X., Tan, L., Gao, R., Song, J., Gao, X., Tian, X., Qin, Y., Xu, K.F., Li, D., Jin, N., Jiang, C., 2012. Inhibition of autophagy ameliorates acute lung injury caused by avian influenza A H5N1 infection. Sci. Signal 5, ra16. Tau, J., Novaes, P., Matsuda, M., Tasat, D.R., Saldiva, P.H., Berra, A., 2013. Diesel exhaust particles selectively induce both proinflammatory cytokines and mucin production in cornea and conjunctiva human cell lines. Invest Ophthalmol. Vis. Sci. 54, 4759e4765. Torricelli, A.A., Novaes, P., Matsuda, M., Alves, M.R., Monteiro, M.L., 2011. Ocular surface adverse effects of ambient levels of air pollution. Arq. Bras. Oftalmol. 74, 377e381. Torricelli, A.A., Matsuda, M., Novaes, P., Braga, A.L., Saldiva, P.H., Alves, M.R., Monteiro, M.L., 2014. Effects of ambient levels of traffic-derived air pollution on the ocular surface: analysis of symptoms, conjunctival goblet cell count and mucin 5AC gene expression. Environ. Res. 131, 59e63. Wang, N., Zimmerman, K., Raab, R.W., McKown, R.L., Hutnik, C.M., Talla, V., Tyler, M.F.t., Lee, J.K., Laurie, G.W., 2013. Lacritin rescues stressed epithelia via rapid forkhead box O3 (FOXO3)-associated autophagy that restores metabolism. J. Biol. Chem. 288, 18146e18161. Wu, S., Deng, F., Niu, J., Huang, Q., Liu, Y., Guo, X., 2010. Association of heart rate variability in taxi drivers with marked changes in particulate air pollution in Beijing in 2008. Environ. Health Perspect. 118, 87e91. Xiang, P., Liu, R.Y., Sun, H.J., Han, Y.H., He, R.W., Cui, X.Y., Ma, L.Q., 2016a. Molecular mechanisms of dust-induced toxicity in human corneal epithelial cells: water and organic extract of office and house dust. Environ. Int. 92e93, 348e356. Xiang, P., He, R.W., Han, Y.H., Sun, H.J., Cui, X.Y., Ma, L.Q., 2016b. Mechanisms of housedust-induced toxicity in primary human corneal epithelial cells: oxidative stress, proinflammatory response and mitochondrial dysfunction. Environ. Int. 89e90, 30e37. Xu, J., Bergin, M.H., Yu, X., Liu, G., Zhao, J., Carrico, C.M., Baumann, K., 2002. Measurement of aerosol chemical, physical and radiative properties in the Yangtze delta region of China. Atmos. Environ. 36, 161e173. Yang, C., Peng, X., Huang, W., Chen, R., Xu, Z., Chen, B., Kan, H., 2012. A timestratified case-crossover study of fine particulate matter air pollution and mortality in Guangzhou, China. Int. Arch. Occup. Environ. Health 85, 579e585. Zhou, Z., Liu, Y., Duan, F., Qin, M., Wu, F., Sheng, W., Yang, L., Liu, J., He, K., 2015. Transcriptomic analyses of the biological effects of airborne PM2.5 exposure on human bronchial epithelial cells. PLoS One 10, e0138267.
Contents lists available at ScienceDirect
Environmental Pollution journal homepage: www.elsevier.com/locate/envpol
Airborne particulate matter (PM2.5) triggers autophagy in human corneal epithelial cell line* Qiuli Fu a, b, 1, Danni Lyu a, b, 1, Lifang Zhang a, b, Zhenwei Qin a, b, Qiaomei Tang a, b, Houfa Yin a, b, Xiaoming Lou c, Zhijian Chen c, **, Ke Yao a, b, * a
Eye Center of the 2nd Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, China Zhejiang Provincial Key Lab of Ophthalmology, Hangzhou, Zhejiang Province, China Department of Environmental and Occupational Health, Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou, Zhejiang Province, China
b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 December 2016 Received in revised form 24 April 2017 Accepted 26 April 2017
Purpose: To investigate particulate matter (PM2.5)-induced damage to human corneal epithelial cells (HCECs) and to determine the underlying mechanisms. Methods: HCECs were exposed to PM2.5 at a series of concentrations for various periods. Cell viability was measured by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell proliferation was evaluated via 5-ethynyl-2’-deoxyuridine (EdU) analysis, while autophagy was determined by immunofluorescence and Western blot. Results: PM2.5-induced cell damage of HCECs occurred in a time- and dose-dependent manner. Decreased cell viability and proliferation as well as increased apoptosis were observed in HCECs after PM2.5 exposure for 24 h. Autophagy in HCECs was slightly inhibited in the early stage (before 4 h) of exposure but significantly activated in the late stage (after 24 h), as evidenced by a decrease in the former and increase in the latter of the expression of the autophagy-associated markers LC3B, ATG5, and BECN1. Interestingly, rapamycin, an autophagy activator, attenuated early-stage but aggravated late-stage PM2.5induced cell damage, suggesting that the role of autophagy in HCECs may change over time during PM2.5 exposure. In addition, in the early stage, the expression of LC3B and ATG5 increased in cells co-treated with rapamycin and PM2.5 compared to rapamycin-only or PM2.5-only treated cells, suggesting that autophagy may benefit cell viability after PM2.5 exposure. Conclusions: The results indicate the potential role of autophagy in the treatment of PM2.5-induced ocular corneal diseases and provide direct evidence for the cytotoxicity, possibly involving an autophagic process, of PM2.5 in HCECs. © 2017 Elsevier Ltd. All rights reserved.
Keywords: PM2.5 Autophagy Human corneal epithelial cells
1. Introduction China, like many other industrialized countries, has suffered severe air pollution problems in recent years, which are set to continue for a long time to come. Although the adverse health
*
This paper has been recommended for acceptance by David Carpenter. * Corresponding author. Eye Center of the 2nd Affiliated Hospital, Medical College of Zhejiang University, Hangzhou 310009, Zhejiang Province, China. ** Corresponding author. Department of Environmental and Occupational Health, Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051, Zhejiang Province, China. E-mail addresses: [email protected] (K. Yao), [email protected] (Z. Chen). 1 Q.F. and D.L. contributed equally to this work. http://dx.doi.org/10.1016/j.envpol.2017.04.078 0269-7491/© 2017 Elsevier Ltd. All rights reserved.
effects of such pollution may be associated with its atmospheric nature, increasing evidence suggests that particulate matter (PM), especially that less than 2.5 mm in diameter (referred to as PM2.5), plays a major role in cardiovascular and chronic cardiopulmonary morbidity and mortality (Guo et al., 2009; Ma et al., 2011; Wu et al., 2010; Yang et al., 2012). The eye is one of the few organs of the human body that are constantly exposed to the external environment. The cornea, which is comprised of five layers: epithelium, Bowman's layer, stroma, Descemet's membrane, and endothelium, represents the outermost structure of the eye and is particularly vulnerable to air pollution. Recent studies have shown that both water and organic extracts of indoor dust have adverse impacts on the human cornea (Xiang et al., 2016a). Although most people spend less than 10% of their
Q. Fu et al. / Environmental Pollution 227 (2017) 314e322
day outdoors (Seaton et al., 1999), the atmospheric environment is undeniably important for human ocular health, especially for those who spend long periods in the open such as children and outdoor workers. It is estimated that the cornea of an adult engaged in light activity is exposed to approximately 34.2 mg of PM2.5 (100 mg/m3) per hour (see Supplementary Fig. 1 for the detailed calculation method). Individuals living in areas with high concentrations of pollutants frequently complain of ocular symptoms and signs, such as watering, burning, itchiness, and redness (Camara and Lagunzad, 2011). Clinical studies have shown that certain air pollutants increase the chances of outpatient visits for nonspecific conjunctivitis and that PM2.5 may exacerbate the development of allergic conjunctivitis (Chang et al., 2012; Mimura et al., 2014). Pathological studies of the ocular surface have revealed that exposure to ambient levels of air pollution could affect the density of conjunctival goblet cells (Berra et al., 2015; Torricelli et al., 2011, 2014). In previous studies on indoor dust-induced toxicity in human corneal epithelial cells (HCECs), it was suggested that dustinduced oxidative stress plays a key role in the cytotoxicity of primary HCECs, in which heavy metals and PAHs are of significant importance (Xiang et al., 2016a, b). Another study provided evidence of cytotoxicity and inflammatory and oxidative stress responses in human corneal and conjunctival epithelial cells incubated with diesel exhaust particles (DEPs) (Tau et al., 2013). However, it is unclear whether PM2.5 exerts similar effects and which molecular mechanisms are involved in ocular diseases caused by long-term exposure to a high degree of environmental pollution. Autophagy is a predominant cellular process involved in the delivery of cytoplasmic substrates to lysosomes for degradation. Its role as a self-destructive or cytoprotective process depends on various conditions (De Meyer et al., 2015; Gottlieb et al., 2009; Killian, 2012; Lopez-Alonso et al., 2013; Sun et al., 2012). Accumulated evidence indicates that autophagy plays crucial roles in a range of physiological and pathological processes, such as development, aging, aging-related diseases, cellular homeostasis, and cancer (Lapierre et al., 2015; Parzych and Klionsky, 2014). Some studies have shown that autophagy is significant in maintaining corneal homeostasis and transparency, as well as in degradation of invading microorganisms (Karnati et al., 2016; Wang et al., 2013). Recent research has revealed that autophagy is involved in PM2.5induced cardiovascular and respiratory diseases and that autophagy inhibitors are able to eliminate or alleviate PM2.5-induced symptoms (Chen et al., 2016; Deng et al., 2013, 2014). Based on these findings, we hypothesized that autophagy may also be critical in PM-induced ocular diseases. The aim of this study was to explore the effect of PM2.5 on HCECs in vitro. The results demonstrate that PM2.5 exposure significantly inhibits cell viability and proliferation in HCECs, and that activation of autophagy is altered in a time-dependent manner following PM2.5 exposure. The present work provides the first evidence that alteration of autophagy activity in HCECs may be one of the mechanisms underlying PM2.5-induced corneal diseases such as keratitis. 2. Materials and methods 2.1. PM2.5 collection In total, six samples were collected with 24 h’ sampling time each (from 9:00 a.m. local time each day to 9:00 a.m. the next day), on the 15th of every month from December 2015 to May 2016. The sampling site was set in the yard of the Center for Disease Control and Prevention of Zhejiang Province (ZJCDC), Hangzhou, Zhejiang, China, at a height equal to the respiratory zone, namely 1.5 m above
315
ground. Atmospheric PM samples were collected on 90-mm quartz fiber filters (QZ47DMCAN, MTL, America) using three mid-volume samplers (MVS6, LECKEL, Germany) at a constant flow rate of 38.3 L/min. The samples were weighted by an automated filterweighing system (WZZ-02, Weizhizhao, Hangzhou, China) before and after sampling to determine the mass of PM. All filters were immediately transported to the laboratory in a filter holder and stored at 20 C until analysis. Weather information, including temperature, relative humidity, wind speed, precipitation, atmospheric pressure, and sunshine hours for every sampling month, is listed in Supplementary Table 1. The PM samples were prepared as described in a previous study (Zhou et al., 2015). Briefly, the quartz filters containing the PM samples were soaked in 75% ethanol (dissolved in Mili-Q water) and sonicated for 30 min. The PM suspensions thereby obtained were dried by lyophilization and re-suspended in sterilized water to achieve a final concentration of 2 mg/mL. Finally, aliquots of the PM suspensions were stored at 80 C for later experiments. 2.2. Quality assurance/quality control (QA/QC) Quality control acceptance criteria were taken into account during atmospheric PM2.5 sampling and analysis of chemical components. For the former, three samplers were deployed simultaneously to collect the atmospheric PM2.5 samples. Surrogate recoveries, limits of detection (LOD), and relative standard deviations (RSD) of chemical components analysis are listed in Supplementary Table 2. None of the recoveries in the present study were outside the surrogate recovery. When a concentration was below the LOD, the value was assumed to be equal to LOD (upper bound) for calculation purposes. One test of procedural blank was carried out for every 20 samples. 2.3. Scanning electron microscopy (SEM) analysis of PM The morphology of PM was observed via SEM (Hitachi, Tokyo, Japan). The PM samples on quartz filters were coated with platinum and observed using an automatic mode. 2.4. Analysis of the size distribution and chemical components of the PM The size distribution of the PM suspended in pure water was analyzed using a Nano-Zetasizer (Malvern Instruments Ltd., Worcestershire, UK) based on the dynamic light-scattering measurement technique. Chemical analysis of the characteristics of the PM samples was performed immediately after collection. The organic (polycyclic aromatic hydrocarbons, PAHs), ion, and metal compositions of the samples were analyzed. In all, 17 PAHs (acenaphthene, acenaphthylene, anthracene, benz(a)anthracene, benzo[a]pyrene, benzo[b] fluorathene, benzo[ghi]perylene, benzo[k]fluoranthene, chrysene, decafluorobiphenyl, dibenz[a,h]anthracene, fluoranthene, fluorine, indeno[123-cd]pyrene, naphthalene, phenanthrene, and pyrene) were analyzed. The concentrations of ions, including nitrate (NO3), sulfate (SO42), ammonium (NH4þ), and chloride (Cl), were measured. The concentrations of 12 metals, including lead (Pb), aluminum (Al), manganese (Mn), arsenic (As), selenium (Se), antimony (Sb), chromium (Cr), nickel (Ni), cadmium (Cd), thallium (Tl), mercury (Hg), and beryllium (Be). 2.5. Cell culture and exposure to PM2.5 HCEC lines (PCS-700-010) were purchased from ATCC (VA, USA). The cells were cultured in DMEM/F12 (Gibco, CA, USA) with 15%
316
Q. Fu et al. / Environmental Pollution 227 (2017) 314e322
fetal bovine serum (FBS) (Gibco, CA, USA), passaged with 0.25% trypsin and 0.02% ethylene diamine tetra-acetic acid (EDTA) (Gibco, CA, USA) every three days. The HCECs were then seeded onto 6well, 24 well, 96-well plates or 10 cm dishes overnight to allow attachment before PM2.5 treatment. Subsequently, all the culture medium was replaced with fresh medium containing PM2.5 suspension, supplemented with 100 U/mL penicillin and 0.1 mg/mL streptomycin (Gibco, CA, USA). 2.6. Western blot HCECs were seeded onto 10 cm culture dishes at a density of 5 104 cells/mL with 10 mL of medium overnight before PM2.5 treatment. They were then exposed to PM2.5 (50 mg/mL) for 1, 2, 4, 6, 12, 24, 36, or 48 h. Rapamycin (an autophagy activator) and 3Methyladenine (3-MA, an autophagy inhibitor) were administered for autophagy regulation. After treatment, the cells were lysed with lysis buffer (Sangon, Shanghai, China) containing phenylmethanesulfonyl fluoride (PMSF) (Sangon, Shanghai, China), protease inhibitor (Sangon, Shanghai, China), and phosphatase inhibitors (Sangon, Shanghai, China). The protein concentration was determined using a spectrophotometer (iMark Microplate Absorbance Reader; Bio-Rad, CA, USA). Extracts from each sample were loaded onto gels, transferred onto membranes, and immunoblotted with primary antibodies including rabbit anti-LC3B (Sigma Aldrich, MO, USA; 1:1000), rabbit anti-BECN1 (CST, MA, USA; 1:1000), and rabbit anti-ATG5 (CST, MA, USA; 1:1000), followed by incubation with HRP-conjugated anti-rabbit IgG (CST, MA, USA; 1:5000). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal control. 2.7. 3 -(4,5-dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium bromide (MTT) assay HCECs were seeded at a density of 1 104 to 3 104 cells/mL (adjusted according to the culture time) onto 96-well plates with 200 mL of medium per well overnight before PM2.5 treatment. The next day, HCECs were treated with various concentrations of PM2.5 (0, 0.5, 1, 2, 5, 10, 25, 50, 75, and 100 mg/mL) for 24 h or 50 mg/mL of PM2.5 for 0, 0.5, 1, 2, 4, 6, 12, 24, or 48 h in 96-well plates. Before MTT (Sigma Aldrich, MO, USA) was added (final concentration of 350 mg/mL), the culture medium with PM2.5 was removed, and each well was washed three times with phosphate-buffered saline (PBS). After incubation in a humid atmosphere of 37 C with 5% CO2 for 4 h, the culture medium was removed and dimethyl sulfoxide (DMSO) (Sigma Aldrich, MO, USA) was added. Finally, the absorbance in each well was recorded at 490 nm using a spectrophotometer (iMark Microplate Absorbance Reader; Bio-Rad, CA, USA). 2.8. 5-Ethynyl-2’-deoxyuridine (EdU) analysis HCECs were seeded at a density of 1.5 104 cells/mL onto 24well plates with 1 mL of medium per well overnight before PM2.5 treatment. To examine the effect of PM2.5 on cell proliferation, the HCECs were first exposed to PM2.5 for 12, 24, 36, or 48 h. Then, half of the medium containing PM2.5 was replaced with fresh medium containing 20 mM of EdU (final concentration of 10 mM) and cultured with cells for 2 h. To detect cell proliferation, EdUlabeled cells were processed according to the manufacturer's protocol (Click-iT EdU Imaging Kit, ThermoFisher, MA, USA). Finally, images were captured using an Olympus IX71 microscope (Olympus, Tokyo, Japan), equipped with DP2-BSW software (Olympus, Tokyo, Japan). The percentage of EdUþ cells within the total number was then quantified.
2.9. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis of HCECs HCECs were seeded at a density of 3 104 cells/mL onto 24-well plates with 1 mL of medium per well overnight before PM2.5 treatment. To examine the PM-induced cell apoptosis in HCECs, the cells were first exposed to PM2.5 (50 mg/mL) for 24 h. Apoptosis was scored by TUNEL (Click-iT Plus TUNEL Assay, Life Technologies, CA, USA) analysis of the PM2.5-treated and fixed HCECs, according to the manufacturer's protocol. 2.10. Flow cytometric analysis HCECs were seeded at a density of 1.5 105 cells/mL onto 6-well plates with 2 mL of medium per well overnight before PM2.5 treatment. HCECs were exposed to 50 mg/mL of PM2.5 for 24 h. Apoptotic cells were then identified and quantified by Annexin VFITC and propidium iodide (PI) Apoptosis Detection Kit (Biyuntian, Beijing, China). The cells were harvested with trypsin-EDTA and washed twice with cold PBS. Cells were incubated in 195 mL of Annexin V-FITC binding buffer supplemented with 5 mL of Annexin V-FITC and 10 mL of PI at room temperature in the dark for 15 min. The apoptosis of cells was analyzed by a flow cytometer (FACSCanto II, BD, NJ, USA). 2.11. Immunofluorescence examinations HCECs were seeded at a density of 3 104 cells/mL onto 24-well plates with 1 mL of medium per well overnight before PM2.5 treatment. The HCECs were seeded on cleaned and autoclaved cover glass, followed by PM2.5 (50 mg/mL) exposure for 24 h. As a positive control, cells were incubated with rapamycin (1 mM) and serum deprivation for 4 h. Following exposure, the cells were washed with PBS and fixed with paraformaldehyde (4% in PBS). The cell samples were then permeabilized with TritonX-100 (Sigma Aldrich, MO, USA; 0.4% in PBS) for 15 min and incubated overnight with primary antibodies: rabbit anti-caspase-3 (CST, MA, USA; 1:100) or rabbit anti-LC3B (Sigma Aldrich, MO, USA; 1:100), followed by incubation with secondary antibodies (1:1000) labeled with Alexa Fluor 488 or 555 (Invitrogen, CA, USA). Nuclei were labeled with 4,6-diamido-2-phenylindole dihydrochloride (DAPI) (Sigma Aldrich, MO, USA; final concentration of 1 mg/mL). Images were captured using a Leica TCS SP8 confocal microscope (Leica, Wetzla, Germany) and processed with Image J software (National Institutes of Health, DC, USA). 2.12. Statistical analysis The results are presented as means ± standard deviation (SD). Data were analyzed using GraphPad Prism 6.0c (GraphPad Software, CA, USA). For all data, a one-way analysis of variance (ANOVA) was applied using SPSS 22.0.0.0 (IBM, NY, USA), followed by a post hoc multiple comparison using the least significant difference test when comparisons of more than two groups were required. 3. Results 3.1. Physical and chemical characteristics of PM2.5 The SEM analysis showed the morphology of PM particles in the quartz filters (Fig. 1A). Dynamic light-scattering measurements revealed PM with sizes ranging between 1106 nm and 3091 nm. The sizes of more than 91.3% of the particles were less than 2305 nm (Fig. 1B). Therefore, most of the PM used in the experiments was less than 2.5 mm in size.
Q. Fu et al. / Environmental Pollution 227 (2017) 314e322
317
Fig. 1. Physical characterizations of PM and cytotoxicity in HCECs. (A) SEM image of PM. (B) Particle size distribution analyzed by dynamic light scattering. (C and D) Cell viability analyzed by MTT assay. The cells were treated with various concentrations of PM2.5 for 24 h (C) or with 50 mg/mL of PM2.5 for different periods (D). The data are presented as means ± SD of at least three independent experiments. *p < 0.05 and **p < 0.01 vs. control. #p < 0.05 and ##p < 0.01. (E) Images of EdU-labeled cells detected by fluorescent microscope. The cells were treated with PM2.5 (50 mg/mL) for 12, 24, 36, or 48 h. Control, vehicle control. (F) Percentage of EdUþ cells. Ten fields (200 ) from each group were randomly chosen for statistical analysis. Control, vehicle control. The data are presented as means ± SD. **p < 0.01 vs. control. #p < 0.05.
The organic, ion, and metal composition of PM collected in October, November, and December 2015 and April 2016 was measured. Of the 17 PAHs measured, decalfluorobiphenyl (120.260 ± 146.447 ng/m3) was the most abundant (Supplementary 2 Table 3). Four ions (NO 3 , SO4 , NH4, and Cl ) were detected, with 3 concentrations of 13.97 ± 10.16 mg/m , 12.47 ± 7.07 mg/m3, 8.84 ± 5.77 mg/m3, and 1.76 ± 1.14 mg/m3 respectively (Supplementary Table 4). Among the 11 metals detected in the PM, Pb, Al, and Mn were the three most prominent, with concentrations of 78.36 ± 34.45 ng/m3, 56.33 ± 32.86 ng/m3, and 36.39 ± 13.89 ng/ m3 respectively (Supplementary Table 5).
3.2. Time- and dose-dependent impacts of PM2.5 on cytotoxicity in HCECs Light microscopy images showed that HCECs became bigger and flatter after exposure to PM2.5 (50 mg/mL) for 24 h compared to unexposed cells (Supplementary Fig. 2). The results reveal that the HCECs were very sensitive to incubation with PM, even PM2.5 at a concentration as low as 0.5 mg/mL. A dose-dependent decrease in cell viability was observed when the HCECs were exposed to different doses of PM2.5 for 24 h (Fig. 1C). It was indicated that there was no significant difference of cell viability observed among
318
Q. Fu et al. / Environmental Pollution 227 (2017) 314e322
50, 75, 100 mg/mL conditions. Therefore, a concentration at 50 mg/ mL was selected for the subsequent study. In addition, a timedependent decrease in cell viability was also found when the HCECs were exposed to PM2.5 (50 mg/mL) for various periods. A significant difference in cell viability was observed after 4 h of PM2.5 exposure (Fig. 1D). 3.3. Effect of PM2.5 exposure on cell proliferation and apoptosis of HCECs The percentage of EdUþ cells was significantly decreased in the PM2.5-exposed cells (Fig. 1E), pointing to adverse effects of PM2.5 on normal HCECs turnover. More importantly, longer exposure periods (more than 12 h) resulted in decreased cell proliferation (Fig. 1F). Additionally, after exposure of 50 mg/mL of PM2.5 for 24 h, only a few TUNELþ and cleaved caspase-3þ cells were observed (Supplementary Figs. 3A and B). Meanwhile, the results of the Annexin V-FITC/PI apoptosis detection test revealed that the percentage of early apoptotic cells (Supplementary Fig. 3C, lower right quadrant, 6.73± 1.05%) and late apoptotic cells (Supplementary Fig. 3C, upper right quadrant, 10.03± 0.91%) of HCECs treated with 50 mg/mL of PM2.5 for 24 h increased compared to that in the control cells (Supplementary Fig. 3C, 3.27± 2.20% and 4.73± 0.29%, respectively). 3.4. Influence of PM2.5 exposure on autophagy in HCECs The results of the immunofluorescence study show that the percentage of cells containing LC3B puncta significantly increased after exposure to PM2.5 (50 mg/mL) for 24 h, with the number of cells close to those induced by rapamycin, compared to the unexposed group (Fig. 2A and B). Additionally, the LC3B puncta per cell were more abundant in the PM-exposed cells and rapamycininduced positive control, compared to the unexposed cells (Fig. 2C). Interestingly, the results of Western blot showed that the expression of LC3B II, the activated form of LC3B, decreased in the first 4 h (with no significant difference), then slowly returned to the baseline, but increased with longer PM2.5 exposure periods (24, 36, and 48 h) when compared to the unexposed cells (Fig. 3A and B). Similar results were observed in the expressions of BECN1 and ATG5, which decreased shortly after exposure to PM2.5 and increased thereafter (Fig. 3D and E). These observations, together with the results that the cell viability decreased sharply with longer exposure to PM2.5, especially after 12 h, indicate that autophagy may play different roles under various PM2.5 exposure periods. 3.5. Dual effects of autophagy on PM2.5-induced cell damage in the early and late stages As shown in Fig. 4A, rapamycin partially attenuated PM2.5induced cell damage in the early stage (before 4 h), but the reverse effect was observed in the late stage (after 24 h), in which rapamycin accelerated PM2.5-induced cell damage. The results of Western blot showed that autophagy was triggered in the PM2.5 and rapamycin co-treated cells and that the expressions of LC3B II and ATG5 were upregulated under this condition compared to the cells treated with PM2.5 or rapamycin alone (Fig. 4BeF). In addition, the elevated expressions of LC3B II and ATG5 could be partially abolished by 3-MA. These results indicate that the protective effect of rapamycin in the early stage may be due to the activation of autophagy, which was slightly inhibited in the early stage. It is also suggested that when autophagy is activated by PM2.5, rapamycin may aggravate its adverse effects by triggering autophagy activity in the late stage of PM2.5 exposure. Therefore, our results point to autophagy having a range of roles in HCECs, depending on the
exposure period to PM2.5. 4. Discussion In the present study, following PM2.5 exposure, the viability of HCECs decreased in a time- and dose-dependent manner. PM2.5 exposure had an accelerative effect on apoptosis in HCECs, and significant inhibition on proliferation was also revealed. More importantly, autophagy was slightly inhibited in the early stage but activated in HCECs in the late stage of PM2.5 exposure, which may contribute to the effect of rapamycin on PM2.5-induced cell damage at different stages. Although the relationship between PM and eye discomfort has been revealed in a recent epidemiological investigation (Mimura et al., 2014), the underlying pathological mechanisms are yet to be elucidated. Previous studies have shown that house dust induces cytotoxicity, oxidative stress, proinflammatory response, and mitochondrial dysfunction in primary HCECs (Xiang et al., 2016a, b). Evidence from a study of DEPs, one of the constituents of PM, also showed that oxidative stress caused by DEPs led to cytotoxicity and inflammatory responses in human conjunctiva cells (Tau et al., 2013). However, among the mechanisms investigated and involved in house dust- or DEPs-induced cell damage, autophagy has not been studied. To address this, we examined the effects of PM2.5 on HCECs and the role of autophagy as the underlying mechanism. Atmospheric DEPs exist as individual particles or chains of aggregates, with most in the invisible sub-micrometer range of 100 nm (Avino and Manigrasso, 2016), also known as ultrafine particles. However, in the present study, 81.1% of the PM collected and analyzed was in the range of 1484e2305 nm, suggesting that DEPs may not have been the main component of the PM2.5 used in the present study. Both DEPs and PM are complex mixtures of chemical and biological elements, such as PAHs and ions, which are not only adsorbed on the surface of PM but also deeply encrusted in its structure, as previously reported (Deng et al., 2013; Leung et al., 2014). However, the physical and chemical characteristics of DEPs and PM have been neglected in most clinical and epidemiological research (Guo et al., 2009; Ma et al., 2011; Tau et al., 2013; Wu et al., 2010; Yang et al., 2012). In addition, the results of toxicological studies into DEPs' and PM's toxicity vary according to the region where the study was conducted (Chen et al., 2016; Deng et al., 2013, 2014; Mimura et al., 2014; Tau et al., 2013; Zhou et al., 2015). A previous study on the possible influence of PM on the environment in the Yangtze Delta region of China, where Hangzhou is located, showed that the air in this region contained relatively considerable PM carrying high concentrations of ammonium, nitrate, and sulfate (Xu et al., 2002), which is consistent with the findings of the present study. These results indicate that the PM in Hangzhou may have significant impacts on human ocular health. PAHs are widely recognized environmental pollutants due to their carcinogenic and mutagenic properties that impact human health (Mishra et al., 2016). A previous study showed that concentrations of PAHs in PM2.5 from indoor dust at public places in Hangzhou accounted for 71.5% of total particulate PAHs, of which naphthalene was the most abundant (Lu et al., 2008). Therefore, PM2.5, rather than PM10, was chosen for the study of PM-induced human ocular health problems. Among all the PAHs found in PM2.5 in the present study, decalfluorobiphenyl was the most abundant, in comparison to naphthalene in a previous study (Lu et al., 2008), which may be due to their differences in origins. Another study also suggested that PAHs play an essential role in house dust-induced toxicity in primary HCECs (Xiang et al., 2016a). Additionally, Pb, Al, and Mn were found to be the most commonly found metals in the PM2.5 used in the present study, which is consistent with the adverse effect of metals
Q. Fu et al. / Environmental Pollution 227 (2017) 314e322
319
Fig. 2. PM2.5-induced autophagosome formation in HCECs through immunofluorescence examination. (A) Representative immunofluorescence images of the punctate staining of LC3B in HCECs treated with the vehicle control, PM2.5 (50 mg/mL) for 24 h, or rapamycin (1 mM) and serum deprivation for 4 h (B and C) Percentage of autophagic cells (B) and average puncta per autophagic cell (C). At least three fields (630 ) from each group were randomly chosen for analysis. Control, vehicle control. The data are presented as means ± SD. **p < 0.01 vs. control.
on human health as suggested by previous studies on house dust (Xiang et al., 2016a, b). The aforementioned components may play key roles in the cytotoxicity of PM2.5 in HCECs; however, the individual biological functions of each element remain to be determined in further experiments. In the PM2.5-exposed HCECs, we observed a time- and dosedependent decrease in cell viability. This occurred even when the HCECs were exposed to PM2.5 at concentrations as low as 0.5 mg/ mL, and the viability decreased significantly in accordance with a rise in the concentration of PM2.5. When compared to cytotoxicity induced in HCECs exposed to DEPs (Tau et al., 2013), PM2.5 exposure appeared to induce greater cytotoxicity, with even low doses of PM2.5 resulting in a marked decrease in viability, which may be due to the different diameters of particulate and chemical components attached. As mentioned above, the diameters of PM2.5 used in the present study are larger than those of DEPs; therefore, PM2.5 may carry more harmful chemicals. The distinctive characteristics of PM2.5 in the present study may as well attribute to the accelerative effect of PM2.5 on apoptosis in HCECs. This result is inconsistent with the findings on DEPs exposure in HCECs though (Tau et al., 2013), which may be due to the differences in size and
the harmful components attached to PM2.5 and DEPs. In airway epithelial cells, PM-induced oxidative stress triggers autophagy, which is essential for inflammation and mucus hyperproduction (Chen et al., 2016). Accordingly, we hypothesized that PM-induced oxidative stress in ocular surface epithelial cells would also trigger autophagy. A previous study of the cytotoxicity of DEPs in human conjunctival epithelial cells showed that DEPs induced oxidative stress and inflammatory responses, but it was unclear whether autophagy was involved (Tau et al., 2013). Although the essential role of autophagy in PM-induced injury in airway epithelium cells has been well documented in both cells and mouse models (Chen et al., 2016), its role in human ocular surface epithelium cells has remained elusive. To our knowledge, the present study provides the first evidence of the involvement of autophagy in the mechanism of PM2.5-induced cell damage in ocular surface epithelial cells. After 24 h of exposure to PM2.5, autophagy was markedly activated in the HCECs. The upregulation of autophagy may have been the main cause of the cell damage, as the latter deteriorated when the cells were co-treated with PM2.5 and rapamycin, an autophagy activator. These results are consistent with the previous findings in lung epithelial cells, in which PM-
320
Q. Fu et al. / Environmental Pollution 227 (2017) 314e322
Fig. 3. Time-dependent effect of PM2.5 exposure on autophagy in HCECs. (A) The expression levels of LC3B, ATG5, and BECN1 in the PM2.5-exposed HCECs detected by Western blot. The cells were exposed to PM2.5 (50 mg/mL) for different periods. GAPDH was used as the internal control. Control, vehicle control. Positive control (PC), treatment with rapamycin (1 mM) and serum deprivation for 4 h (B to E) Quantitative analysis of relative expression levels of LC3B II (B), LC3B II/LC3B I (C), ATG5 (D), and BECN1 (E). Control, vehicle control. The data are presented as means ± SD of at least three independent experiments. **p < 0.01 vs. control. #p < 0.05.
induced cell damage was reduced in transgenic autophagydeficient LC3/ and Becn1þ/ mice (Chen et al., 2016). Interestingly, in the present study, autophagy was slightly inhibited in the early stage of PM2.5 exposure in the HCECs, which has not been reported previously in other cells or tissues. Additionally, administration of rapamycin in the first six hours partially reversed the PM2.5-induced cell damage, suggesting that autophagy may act as a protective factor in the early stage of PM2.5 exposure in HCECs.
The results of the present study are based on a cellular model using an HCEC cell line, which is stable but may lose some characteristics over time compared to primary cultures (Xiang et al., 2016a, b). Therefore, studies that use primary HCECs are also needed to further investigate PM2.5-induced cytotoxicity and its mechanisms. In addition, the cellular model used in the present study is based on a monolayer structure, which partially represents the natural stratified corneal epithelium in vivo. Therefore, a 3D
Q. Fu et al. / Environmental Pollution 227 (2017) 314e322
321
Fig. 4. Effect of autophagy on PM2.5-induced cytotoxicity. (A) Cell viability measured by MTT assay. The HCECs were treated with PM2.5 (50 mg/mL) for various periods, with or without pre-treatment of rapamycin (1 mM) and serum deprivation for 2 h. The data are presented as means ± SD of at least three independent experiments. *p < 0.05 and **p < 0.01. (B) The expression levels of LC3B, ATG5, and BECN1 in HCECs detected by Western blot. The cells were treated with the vehicle control (Group C); PM2.5 (50 mg/mL) for 4 h (Group P); rapamycin (1 mM) with serum deprivation for 4 h (Group R); co-treatment with PM2.5 (50 mg/mL) and rapamycin (1 mM) and serum deprivation for 4 h (Group R þ P); pretreatment with rapamycin (1 mM) and serum deprivation for 0.5 h, followed by the addition of PM2.5 (50 mg/mL) for 3.5 h (Groups ReP); 3-MA (5 mM) for 4 h (Group M); cotreatment with 3-MA (5 mM) and PM2.5 (50 mg/mL) for 4 h (Group M þ P); co-treatment with 3-MA (5 mM), rapamycin (1 mM) and serum deprivation for 4 h (Group M þ R); or co-treatment with 3-MA (5 mM), rapamycin (1 mM), PM2.5 (50 mg/mL) and serum deprivation for 4 h (Group M þ R þ P). GAPDH was used as the internal control. (CeF) Quantitative analysis of the relative expression levels of LC3B II (C), LC3B II/LC3B I (D), ATG5 (E), and BECN1 (F). The data are presented as means ± SD of at least three independent experiments. **p < 0.01 vs. control; #p < 0.05, ##p < 0.01 vs. Group P; yp < 0.05, yyp < 0.01 vs. Group R; zp < 0.05, zzp < 0.01 vs. Group R þ P; xxp < 0.01 vs. Group ReP; jjp < 0.05, jjjjp < 0.01 vs. Group M; ¶¶p < 0.01 vs. Group M þ P.
stratified corneal epithelium model is necessary in the future. As well, in the present study, a cellular model of PM exposure at rather high concentrations for short periods was used to simulate acute and strong exposure, which may be related to PM-induced acute keratitis. However, it is also of great significance to focus on lower concentrations and longer periods to simulate chronic mild exposure, which may contribute to dry eye, chronic inflammation, etc. Additionally, experiments in animal models are also needed to shed
light on the molecular mechanism of PM2.5-induced injury in HCECs. With this aim in mind, an appropriate method to simulate PM2.5-induced injury in animals is urgently needed. In summary, the present study provides the first evidence that PM2.5 exposures over certain concentrations and culture periods result in HCECs dysfunction, in which autophagy plays an essential role. The results indicate that regulation of the autophagic process may have potential value in the treatment of PM-induced ocular
322
Q. Fu et al. / Environmental Pollution 227 (2017) 314e322
surface diseases. Acknowledgements This work has been supported by National Natural Science Foundation of China (81371001, 81300641, 81570822, 81670833, 81502786), Project of National Clinical Key Discipline of Chinese Ministry of Health, Zhejiang Key Laboratory Fund of China (2011E10006), Zhejiang Province Key Research and Development Program (2015C03042), Program of Zhejiang Medical technology (2015KYA109), Science and Technology Program of Zhejiang (2014C03025), National Natural Science Foundation of Zhejiang Province (LQ14H260003), and Analysis Center of Agrobiology and Environmental Sciences & Institute of Agrobiology and Environmental Sciences, Zhejiang University. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.envpol.2017.04.078. References Avino, P., Manigrasso, M., 2016. Dynamic of submicrometer particles in urban environment. Environ. Sci. Pollut. Res. Int. Berra, M., Galperin, G., Dawidowski, L., Tau, J., Marquez, I., Berra, A., 2015. Impact of wildfire smoke in Buenos Aires, Argentina, on ocular surface. Arq. Bras. Oftalmol. 78, 110e114. Camara, J.G., Lagunzad, J.K., 2011. Ocular findings in volcanic fog induced conjunctivitis. Hawaii Med. J. 70, 262e265. Chang, C.J., Yang, H.H., Chang, C.A., Tsai, H.Y., 2012. Relationship between air pollution and outpatient visits for nonspecific conjunctivitis. Invest Ophthalmol. Vis. Sci. 53, 429e433. Chen, Z.H., Wu, Y.F., Wang, P.L., Wu, Y.P., Li, Z.Y., Zhao, Y., Zhou, J.S., Zhu, C., Cao, C., Mao, Y.Y., Xu, F., Wang, B.B., Cormier, S.A., Ying, S.M., Li, W., Shen, H.H., 2016. Autophagy is essential for ultrafine particle-induced inflammation and mucus hyperproduction in airway epithelium. Autophagy 12, 297e311. De Meyer, G.R., Grootaert, M.O., Michiels, C.F., Kurdi, A., Schrijvers, D.M., Martinet, W., 2015. Autophagy in vascular disease. Circ. Res. 116, 468e479. Deng, X., Zhang, F., Rui, W., Long, F., Wang, L., Feng, Z., Chen, D., Ding, W., 2013. PM2.5-induced oxidative stress triggers autophagy in human lung epithelial A549 cells. Toxicol Vitro 27, 1762e1770. Deng, X., Zhang, F., Wang, L., Rui, W., Long, F., Zhao, Y., Chen, D., Ding, W., 2014. Airborne fine particulate matter induces multiple cell death pathways in human lung epithelial cells. Apoptosis 19, 1099e1112. Gottlieb, R.A., Finley, K.D., Mentzer Jr., R.M., 2009. Cardioprotection requires taking out the trash. Basic Res. Cardiol. 104, 169e180. Guo, Y., Jia, Y., Pan, X., Liu, L., Wichmann, H.E., 2009. The association between fine particulate air pollution and hospital emergency room visits for cardiovascular diseases in Beijing, China. Sci. Total Environ. 407, 4826e4830. Karnati, R., Talla, V., Peterson, K., Laurie, G.W., 2016. Lacritin and other autophagy associated proteins in ocular surface health. Exp. Eye Res. 144, 4e13. Killian, M.S., 2012. Dual role of autophagy in HIV-1 replication and pathogenesis. AIDS Res. Ther. 9, 16. Lapierre, L.R., Kumsta, C., Sandri, M., Ballabio, A., Hansen, M., 2015. Transcriptional and epigenetic regulation of autophagy in aging. Autophagy 11, 867e880. Leung, P.Y., Wan, H.T., Billah, M.B., Cao, J.J., Ho, K.F., Wong, C.K., 2014. Chemical and
biological characterization of air particulate matter 2.5, collected from five cities in China. Environ. Pollut. 194, 188e195. Lopez-Alonso, I., Aguirre, A., Gonzalez-Lopez, A., Fernandez, A.F., AmadoRodriguez, L., Astudillo, A., Batalla-Solis, E., Albaiceta, G.M., 2013. Impairment of autophagy decreases ventilator-induced lung injury by blockade of the NFkappaB pathway. Am. J. Physiol. Lung Cell Mol. Physiol. 304, L844eL852. Lu, H., Zhu, L.Z., Chen, S.G., 2008. Pollution level, phase distribution and health risk of polycyclic aromatic hydrocarbons in indoor air at public places of Hangzhou, China. Environ. Pollut. 152, 569e575. Ma, Y., Chen, R., Pan, G., Xu, X., Song, W., Chen, B., Kan, H., 2011. Fine particulate air pollution and daily mortality in Shenyang, China. Sci. Total Environ. 409, 2473e2477. Mimura, T., Ichinose, T., Yamagami, S., Fujishima, H., Kamei, Y., Goto, M., Takada, S., Matsubara, M., 2014. Airborne particulate matter (PM2.5) and the prevalence of allergic conjunctivitis in Japan. Sci. Total Environ. 487, 493e499. Mishra, N., Ayoko, G.A., Morawska, L., 2016. Atmospheric polycyclic aromatic hydrocarbons in the urban environment: occurrence, toxicity and source apportionment. Environ. Pollut. 208, 110e117. Parzych, K.R., Klionsky, D.J., 2014. An overview of autophagy: morphology, mechanism, and regulation. Antioxid. Redox Signal 20, 460e473. Seaton, A., Soutar, A., Crawford, V., Elton, R., McNerlan, S., Cherrie, J., Watt, M., Agius, R., Stout, R., 1999. Particulate air pollution and the blood. Thorax 54, 1027e1032. Sun, Y., Li, C., Shu, Y., Ju, X., Zou, Z., Wang, H., Rao, S., Guo, F., Liu, H., Nan, W., Zhao, Y., Yan, Y., Tang, J., Zhao, C., Yang, P., Liu, K., Wang, S., Lu, H., Li, X., Tan, L., Gao, R., Song, J., Gao, X., Tian, X., Qin, Y., Xu, K.F., Li, D., Jin, N., Jiang, C., 2012. Inhibition of autophagy ameliorates acute lung injury caused by avian influenza A H5N1 infection. Sci. Signal 5, ra16. Tau, J., Novaes, P., Matsuda, M., Tasat, D.R., Saldiva, P.H., Berra, A., 2013. Diesel exhaust particles selectively induce both proinflammatory cytokines and mucin production in cornea and conjunctiva human cell lines. Invest Ophthalmol. Vis. Sci. 54, 4759e4765. Torricelli, A.A., Novaes, P., Matsuda, M., Alves, M.R., Monteiro, M.L., 2011. Ocular surface adverse effects of ambient levels of air pollution. Arq. Bras. Oftalmol. 74, 377e381. Torricelli, A.A., Matsuda, M., Novaes, P., Braga, A.L., Saldiva, P.H., Alves, M.R., Monteiro, M.L., 2014. Effects of ambient levels of traffic-derived air pollution on the ocular surface: analysis of symptoms, conjunctival goblet cell count and mucin 5AC gene expression. Environ. Res. 131, 59e63. Wang, N., Zimmerman, K., Raab, R.W., McKown, R.L., Hutnik, C.M., Talla, V., Tyler, M.F.t., Lee, J.K., Laurie, G.W., 2013. Lacritin rescues stressed epithelia via rapid forkhead box O3 (FOXO3)-associated autophagy that restores metabolism. J. Biol. Chem. 288, 18146e18161. Wu, S., Deng, F., Niu, J., Huang, Q., Liu, Y., Guo, X., 2010. Association of heart rate variability in taxi drivers with marked changes in particulate air pollution in Beijing in 2008. Environ. Health Perspect. 118, 87e91. Xiang, P., Liu, R.Y., Sun, H.J., Han, Y.H., He, R.W., Cui, X.Y., Ma, L.Q., 2016a. Molecular mechanisms of dust-induced toxicity in human corneal epithelial cells: water and organic extract of office and house dust. Environ. Int. 92e93, 348e356. Xiang, P., He, R.W., Han, Y.H., Sun, H.J., Cui, X.Y., Ma, L.Q., 2016b. Mechanisms of housedust-induced toxicity in primary human corneal epithelial cells: oxidative stress, proinflammatory response and mitochondrial dysfunction. Environ. Int. 89e90, 30e37. Xu, J., Bergin, M.H., Yu, X., Liu, G., Zhao, J., Carrico, C.M., Baumann, K., 2002. Measurement of aerosol chemical, physical and radiative properties in the Yangtze delta region of China. Atmos. Environ. 36, 161e173. Yang, C., Peng, X., Huang, W., Chen, R., Xu, Z., Chen, B., Kan, H., 2012. A timestratified case-crossover study of fine particulate matter air pollution and mortality in Guangzhou, China. Int. Arch. Occup. Environ. Health 85, 579e585. Zhou, Z., Liu, Y., Duan, F., Qin, M., Wu, F., Sheng, W., Yang, L., Liu, J., He, K., 2015. Transcriptomic analyses of the biological effects of airborne PM2.5 exposure on human bronchial epithelial cells. PLoS One 10, e0138267.