Zinc oxide nanoparticles inhibit Ca2+-ATPase expression in human lens epithelial cells under UVB irradiation

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Zinc oxide nanoparticles inhibit Ca2+-ATPase expression in human lens epithelial cells under UVB irradiation

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Daoguang Wang a,1, Dadong Guo b,1, Hongsheng Bi a,b,⇑, Qiuxin Wu a,b, Qingmei Tian a,b, Yuxiang Du b a

Affiliated Eye Hospital of Shandong University of Traditional Chinese Medicine, No. 48#, Yingxiongshan Road, Jinan 250002, China Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Therapy of Ocular Diseases of Shandong Province, Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Therapy of Ocular Diseases in Universities of Shandong, Eye Institute of Shandong University of Traditional Chinese Medicine, No. 48#, Yingxiongshan Road, Jinan 250002, China b

a r t i c l e

i n f o

Article history: Received 27 January 2013 Accepted 12 September 2013 Available online xxxx Keywords: Zinc oxide Nanoparticle Ultraviolet B Calcium homeostasis Plasma membrane Ca2+-ATPase Human lens epithelial cell

a b s t r a c t Epidemiological and experimental studies have revealed that lens epithelial cells exposed to ultraviolet B (UVB) light could be induced apoptosis, and lens epithelial cell apoptosis can initiate cataractogenesis. Posterior capsular opacification (PCO), the most frequent complication after cataract surgery, is induced by the proliferation, differentiation, migration of lens epithelial cells. Thus, inhibiting the proliferation of lens epithelial cells could reduce the occurrence of PCO. It is reported that zinc oxide (ZnO) nanoparticles have great potential for the application of biomedical field including cancer treatment. In the present study, we investigated the cytotoxic effect of ZnO nanoparticles on human lens epithelial cell (HLEC) viability. In addition, changes in cell nuclei, apoptosis, reactive oxygen species and intracellular calcium ion levels were also investigated after cells treated with ZnO nanoparticles in the presence and absence of UVB irradiation. Meanwhile, the expression of plasma membrane calcium ATPase 1 (PMCA1) was also determined at gene and protein levels. The results indicate that ZnO nanoparticles and UVB irradiation have synergistic inhibitory effect on HLEC proliferation in a concentration-dependent manner. ZnO nanoparticles can increase the intracellular calcium ion level, disrupt the intracellular calcium homeostasis, and decrease the expression level of PMCA1. UVB irradiation can strengthen the effect of reduced expression of PMCA1, suggesting that both UVB irradiation and ZnO nanoparticles could exert inhibitory effect on HLECs via calcium-mediated signaling pathway. ZnO nanoparticles have great potential for the treatment of PCO under UVB irradiation. Ó 2013 Published by Elsevier Ltd.

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1. Introduction

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Higher energy wavelengths of ultraviolet light from sunlight including Ultraviolet B (UVB, 290–320 nm) are known to cause oxidative stress and ocular injury (Black et al., 2011; Kolozsvari et al., 2002; Podskochy, 2004). An increasing number of evidence indicate that chronic exposure of the eye to UVB irradiation may injure the lens, a process that causes aberrant epithelial cell growth and differentiation, as well as cell death via necrosis and apoptosis (Estil et al., 1997; Rogers et al., 2004), thereby forming cortical and posterior subcapsular cataract in humans and animals (Bochow et al., 1989; McCarty and Taylor, 1996; Michael et al., 2000; Wickert et al., 1999). At present, the most effective treatment of cataract is the surgical removal of the opacified lens and

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⇑ Corresponding author. Address: Eye Institute of Shandong University of Traditional Chinese Medicine, No. 48#, Yingxiongshan Road, Jinan 250002, China. Tel./fax: +86 531 82861167. E-mail address: [email protected] (H. Bi). 1 These authors contributed to the work equally and should be regarded as co-first authors.

intraocular lens implantation. However, posterior capsular opacification (PCO), which involved in the lens epithelial cell proliferation and migration, is still one of the most common postoperative morbidities. Thus, inhibiting lens epithelial cell proliferation could efficiently reduce the occurrence of PCO after cataract surgery. Currently, the PCO is usually treated with YAG laser capsulotomy. However, this procedure can cause several complications including intraocular lens optic damage, postoperative intraocular pressure elevation and retinal detachment. Thus, there is an urgent need to prevent PCO. Calcium ions (Ca2+) can enter the cell through Ca2+ channels. Meanwhile, calcium ions can also be elevated via sarcoplasmic/ endoplasmic reticulum via Ca2+-release transporters. Thus, to keep the intracellular calcium homeostasis, components are needed which can delicate regulate the intracellular calcium ion level. The crucial components in regulating calcium homeostasis in cells include the Na+/Ca2+ exchanger and plasma membrane calcium ATPase (PMCA) transporters, by which the intracellular calcium ions could be removed to maintain calcium homeostasis. For the regulation of intracellular sarcoplasmic calcium stores, it also

0887-2333/$ - see front matter Ó 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.tiv.2013.09.015

Please cite this article in press as: Wang, D., et al. Zinc oxide nanoparticles inhibit Ca2+-ATPase expression in human lens epithelial cells under UVB irradiation. Toxicol. in Vitro (2013), http:// dx.doi.org/10.1016/j.tiv.2013.09.015

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could be achieved by sarcoplasmic/endoplasmic reticular calcium ATPase (SERCA). In human lens, cellular calcium homeostasis is acquired by a delicate balance of calcium ions among passive inward movement from the extracellular milieu through membrane channels (Cooper et al., 1986), extrusion by PMCA (Hightower and Kinsey, 1980) and internal sequestration by SERCA (Duncan et al., 1993). There are lens epithelial cells and fiber cells in human lens. Studies have substantiated that no calcium pump is located in the fiber cells (Marian et al., 2005). Thus, the regulation of intracellular calcium homeostasis mainly depends on the Ca2+-ATPase pumps found in lens epithelial cells. PMCA has four isoforms (i.e., PMCA1-4), and is a high affinity Ca2+ pump. PMCA can use the energy from ATP hydrolysis to drive Ca2+ out of the cell against its electrochemical gradient (Brini, 2009). Therefore, PMCA plays an important role involved in the regulation of intracellular calcium homeostasis. It has been proved that zinc oxide (ZnO) nanoparticles could exert cytotoxic effect on both broncho-alveolar lavage cells and white blood cells in rats via interfering with zinc ion homeostasis (Kao et al., 2012), liver cells (Sharma et al., 2011, 2012), and human bronchial epithelial cells (Heng et al., 2010). Our previous studies have also revealed that ZnO nanoparticles can induce rat retinal ganglion cell death via reactive oxygen species pathway and Caspase pathways (Guo et al., 2013a). In addition, ZnO nanoparticles could induce apoptosis in human dermal fibroblasts via p53 and p38 pathways (Meyer et al., 2011). Recent researches have proposed that nanoparticles can modulate intracellular calcium levels and may play a role in calcium homeostasis (Young et al., 2009a,b; Guo et al., 2013b). This ability which regulates calcium homeostasis is believed to have important physiologic and pathologic implications. However, it is still unknown for the effect of ZnO nanoparticles on human lens epithelial cells (HLECs). In the meantime, the possible mechanism involved in the regulation of calcium homeostasis via PMCA has also not been addressed. Thus, in the present study, we explored the cytotoxic effects of ZnO nanoparticles on HLECs in the presence and absence of UVB irradiation through cell viability assay, 40 ,6-diamidino-2-phenylindole (DAPI) staining, hydroxyl radical assay kits, flow cytometry, quantitative real-time PCR and western blotting analysis, respectively. Meanwhile, the real-time cell electronic sensing (RT-CES) assay was also applied to explore the dynamic process and binding behavior between cells and ZnO nanoparticles with or without UVB irradiation. We found that ZnO nanoparticles could inhibit the proliferation of HLECs, decrease PMCA1 expression at gene and protein levels, increase the intracellular calcium ion level and further disrupt the intracellular calcium homeostasis, finally cause cell death. These results suggest that cytotoxic effect of ZnO nanoparticles under UVB irradiation on HLEC B-3 cells may be involved in calcium-mediated signaling pathway.

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2. Materials and methods

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2.1. ZnO nanoparticles

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The ZnO nanoparticles (>99.0% purity) capped with aminopolysiloxane were purchased from Jiangsu Changtai Nanometer Material Co., Ltd. and were characterized by a scanning electron microscope (ZEISS EVO, Germany). The particle size distribution and zeta potential of ZnO nanoparticles dissolved in 1640 medium were determined using a Malvern Zetasizer (Malvern Instruments, Britain) with specialized software (Zetasizer Nano ZS). For every experiment, ZnO nanoparticle suspensions were mixed vigorously, sonicated for 20 min on the ice prior to experiment, and then immediately applied to the related assay to minimize agglomeration.

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2.2. UV irradiation

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For UV irradiation, UVB (k = 310 nm) was provided by a UVB lamp (Nanjing Huaqiang Electronic Co., Ltd., China). The UVB dose was determined by a double channels UVB illuminometer (Photoelectric Instrument Factory of Beijing Normal University, China). The total exposure dose of UVB was 40 mJ/cm2 and the average intensity was 0.2 mW/cm2 at the working plane. In the present study, the effect of different concentrations of ZnO nanoparticles on HLECs was investigated under UVB irradiation for 200 s.

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2.3. Cell culture and preparation of ZnO nanoparticle solution

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A human lens epithelial cell line (HLEC B-3, purchased from ATCC) was maintained in RPMI-1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (Sigma, USA), 100 U/ mL penicillin (Sigma, USA) and 100 lg/mL streptomycin (Sigma, USA) and grown at 37 °C in a 5% CO2 humidified environment. Cell numbers were determined using an automated cell counter (TC10, Bio-rad, USA). For the preparation of ZnO nanoparticle suspensions, ZnO nanoparticles were dissolved in RPMI-1640 and were sonicated on the ice for 20 min prior to use.

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2.4. In vitro RT-CES cytotoxicity assay for HLECs

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The RT-CES assay was performed using a RT-CES analyzer (ACEA Biosciences (Hangzhou) Inc., China) according to the literature (Guo et al., 2013b). Initially, 1  104 cells were seeded into each well containing 200 lL cell culture medium used for the 16 sensor device and incubated overnight, then the medium was replaced with ZnO nanoparticle suspensions and treated with or without UVB irradiation, followed by incubation at 37 °C for at least 72 h in an incubator contained 5% CO2. Controls were cultivated under the same condition either without UVB radiation or without ZnO nanoparticles. The relevant experiments were repeated three times independently.

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2.5. Viability assay of cells treated with ZnO nanoparticles in the presence and absence of UVB irradiation

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The HLECs were seeded on the coverslip in 6-well plate (105 cells/well), then treated with different concentrations (i.e., 0, 2.5, 5.0, 10 lg/mL) of ZnO nanoparticles in the presence and absence of UVB irradiation, further all cells were cultured in an incubator with 5% CO2 at 37 °C for 24 h for optical microscopy assay. After treatment, the cells were observed by an optical microscope (Olympus IX51, Japan). Every experiment was repeated at least three times independently.

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2.6. DAPI staining

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To monitor the effect of ZnO nanoparticles on HLECs nuclei, the DAPI nuclear staining was carried out. The HLECs were seeded in a 6-well plate at a density of 5  104 cells per well and grown overnight, then incubated with different concentrations (i.e., 0, 2.5, 5.0, 10 lg/mL) in 1.5 mL volume of ZnO nanoparticles in the presence and absence of UVB irradiation and further cultured for additional 6 h, subsequently cells were washed with phosphate buffered saline (PBS, pH 7.4). After fixation in 4% polymerisatum for 15 min, the fixed cells were washed with PBS and were stained with 1 lg/mL of DAPI solution (Sigma, USA) for 30 min, then stained cells were examined using an inverted fluorescence microscope (Olympus IX71, Japan) and the typical photographs were captured.

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2.7. Analysis on externalization of phosphatidylserine (Annexin V/PI staining) HLECs were seeded in a 6-well plate at a density of 4  105 cells/ well and grown overnight, then cells were treated with different concentrations (i.e., 0, 2.5, 5.0, 10.0 lg/mL) of ZnO nanoparticles (final volume: 1.5 ml) in the presence and absence of UVB irradiation, further all cells were cultured for additional 24 h. After treatment, both floating and adherent cells were collected, and then cells were washed with PBS twice, followed by centrifugation at 600 g for 5 min. After that, the cells were resuspended in Annexin V-FITC binding buffer (Beyotime, China) at a density of 1  106 cells/mL. Further, 0.1 mL (1  105 cells) of the cell suspension was transferred to a 2 mL eppendorf plastic tube, where 5 lL of FITC-conjugated Annexin V (Beyotime, China) and 10 lL of PI (50 lg/mL) were added, then cells were blended gently and incubated in a dark room for 10 min, finally flow cytometric analysis was performed immediately after staining. Data acquisition and analysis were done by a flow cytometer (Accuri C6, USA). Cells in the early stages of apoptosis were Annexin V-positive and PI-negative, whereas cells in the late stages of apoptosis were positive for both Annexin V and PI. All assays were repeated at least three times independently.

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2.8. Hydroxyl radical level determination

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Fenton reaction is the most common chemical reaction to produce hydroxyl radicals (OH). During this reaction, the amount of hydrogen peroxide (H2O2) is proportional to the amount of OH, and in which, ferrous sulfate (FeSO4) is a source of ferrous ions. When given an electron acceptor, Griess, it can form the red substance, which is proportional to the number of OH. To determine the changes in the level of hydroxyl radicals in the medium after treatment with ZnO nanoparticles in the presence and absence of UVB irradiation, we determined the alterations in hydroxyl radical levels. Briefly, cells were treated with different concentrations of ZnO nanoparticles (0, 2.5, 5.0 and 10.0 lg/mL, respectively) in the presence and absence of UVB irradiation for 200 s, further cultured for an additional 6 h, then all cells were collected. The supernatant, obtained by centrifugation at 2000 g for 10 min, was used to determine the level of hydroxyl radicals. The determination was in accordance with the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, China) and every experiment was performed at least three times.

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2.9. Quantitative real-time PCR

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First, HLECs were seeded in a 6-well plate at a density of 4  10 cells/well and grown overnight, then the medium was replaced with different concentrations (i.e., 0, 2.5, 5.0, 10.0 lg/mL) of ZnO nanoparticle suspensions with or without UVB irradiation (final volume: 1.5 mL), further all cells were cultured for additional 2 h, then total RNA was extracted with Trizol reagent (Aidlab, China). After determination of total RNA using a micro-spectrophotometer (K5600, Beijing Kaiao Technology Development Co., Ltd.), cDNA was generated using a commercial kit (Aidlab, China) according to the manufacturer’s instructions. Quantitative real-time PCR was performed with SYBR Green Master Mix (Aidlab, China) using a Stratagene Mx3000P sequence detection system (Agilent Technologies, USA). The GAPDH was used as a positive control and a negative control without template RNA was also included. The primer sequences (forward/reverse) used in the reaction were as follows: Ca2+-ATPase (PMCA1), forward primer: 50 -CTCCCGCACCATGATGAAGAACA-30 , reverse primer: 50 -TCCCCAGACCAGCTCCCCAACAC-30 ; GAPDH, forward primer: 50 -GCGGGGCTCTCCAGAACATCAT-30 , and reverse primer: 50 -CCAGCCCCAGCGTCAAAGGTG-30 . The PCR program was 2+

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set as follows: 95 °C for 3 min, followed by 45 cycles of a 95 °C denaturation for 25 s, 60 °C for 30 s and 72 °C extension for 30 s. A standard curve for each gene was generated from serial dilutions of PCR products to monitor amplification efficiency and to relatively quantify mRNA abundance. Each experiment was carried out three times and the DDct values were calculated by normalizing the gene expression levels to the expression of GAPDH. The relative expression level of each gene was expressed as a fold change.

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2.10. Western blot analysis

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First, HLECs were seeded in a 6-well plate at a density of 4  105 cells/well in 1.5 mL volume and grown overnight, then the medium was replaced with different concentrations (i.e., 0, 2.5, 5.0, 10.0 lg/mL) of ZnO nanoparticle suspensions with or without UVB irradiation (1.5 mL per well) and further cultured for 4 h. Subsequently, both floating and adherent cells were collected using 0.25% trypsin, followed by treatment using a cell-lysis buffer (Beyotime, Jiangsu, China), and centrifuged at 12,000g for 5 min. Supernatant was then collected from the lysates and the total protein concentration of each sample was measured using a bicinchoninic acid (BCA) method. The equal amounts of protein samples were loaded onto a 10% SDS gel and transferred to nitrocellulose membranes in an electrophoresis apparatus at 150 mA for 90 min. The membranes were incubated with primary antibodies: b-actin antibody (1:1000, Abcam, UK) and Ca2+-ATPase antibody (1:1000, Abcam, UK). After the membranes were washed, they were incubated with a goat anti-rabbit IgG horseradish peroxidase (HRP)-conjugated antibody (1:3000, Boster, China) as the secondary antibody in assay buffer. Finally, immunoblotting signals were visualized using the ECL-plus Kit (Chemicon, USA). Quantification of Ca2+-ATPase protein was performed by a Chemiluminescence imager (LAS-4000 mini, Fujifilm, Japan).

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2.11. Measurement of intracellular Ca2+ levels

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Changes in intracellular Ca2+ level were determined using flow cytometry with Fluo-3/AM probes after different treatment. In brief, HLE B-3 cells were seeded in a 12-well plate at a density of 4  105 cells per well and grown overnight, then the medium was replaced with different concentrations (i.e., 0, 2.5, 5.0, 10.0 lg/ mL) of ZnO nanoparticles (final volume: 1.5 mL). After treatment with or without UVB irradiation for 200 s, all cells were incubated at 37 °C in a 5% CO2 humidified incubator for 30 min. Further cells were loaded with 10 lmol/L Fluo-3 acetoxymethyl ester (Molecular Probes, USA) at 37 °C for 30 min, harvested and washed with PBS (pH 7.4). The maximal Fluo-3 fluorescence intensity (Fmax) was determined by adding 10 lmol/L Ionomycin (Beyotime Institute of Biotechnology, China). The minimal fluorescence (Fmin) was measured by quenching Fluo-3 fluorescence with the addition of 5 mmol/L of EGTA. Finally, the cells were gently resuspended in PBS at a concentration of approximately 1  105 cells/mL, and then analyzed by a flow cytometer (Accuri C6, USA) at the excitation of 488 nm and emission of 525 nm. The intracellular Ca2+ concentration was calculated using the following formula:

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2þ

½Ca free

F  F min ¼ Kd  F max  F

ð1Þ

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where Fmax is the maximal Fluo-3 fluorescence intensity and Fmin is the minimal fluorescence. F is the fluorescence at intermediate calcium levels. Kd represents the dissociation constant of Ca2+ bound to the dye. Herein, Kd is 400 nmol/L for Fluo-3 probe.

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2.12. Statistics analysis

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Data were expressed as mean ± SD (standard deviation) from at least three independent experiments. One-tailed unpaired

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Student’s t-test was used for significance testing and P < 0.05 is considered statistically significant.

nanoparticles (Fig. 2A), in which the electrode impedance became higher following the culture time. And this decrease affected by nanoparticles was also concentration- and time-dependent manners. Also, these results were consistent with that of our apoptotic assay. Furthermore, when ZnO nanoparticles were stimulated by the UVB irradiation for 200 s, we observed that the electrode impendence was extremely decreased, especially after culture for about 6 h. Meanwhile, we noted that massive cell death occurred in a short culture time after treatment (Fig. 2E–H). These observations demonstrated that the synergistic cytotoxic effect on HLECs occurred between ZnO nanoparticles and UVB irradiation, i.e., UVB irradiation could distinctly enhance the suppression ability of ZnO nanoparticles on HLEC proliferation.

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3.2. The RT-CES dynamic study for the inhibition of ZnO nanoparticles on HLECs in the presence of UV irradiation

3.3. The cellular viability

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RT-CES assay is a labeling-free assay which allows real-time, automatically and continually monitoring cellular status alterations during the whole process of the cell-chemical interaction. Thus, in this work we introduce the RT-CES assay to study the dynamic response of target HLECs exposed to ZnO nanoparticles with and without UVB irradiation. As shown in Fig. 2B–D, our observations demonstrated that after ZnO nanoparticles were injected into the cell system, the electrode impendence would be lower compared with that of negative system in the absence of

Initially, the cellular viability of HLECs was observed after treatment with ZnO nanoparticles in the presence and absence of UVB irradiation. The typical images were shown in Fig. 3. As it can be seen, the untreated cells were grown well (Fig. 3A), and the viability of HLECs treated with UVB irradiation alone for 200 s was similar to that of the untreated cells (Fig. 3E). Compared with those of untreated cells (Fig. 3A), the majority of HLECs still grew well even after an incubation period of 24 h with 2.5 lg/mL of ZnO nanoparticles with (Fig. 3F) or without UVB irradiation

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3. Results

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3.1. Characterization of ZnO nanoparticles

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The average diameter of ZnO nanoparticles was about 30 nm (Fig. 1A), and the histogram of the size distribution of ZnO nanoparticles was shown in Fig. 1B. For zeta potential analysis, the result indicates that ZnO nanoparticles dissolved in 1640 medium possessed 10.6 mV (Fig. 1C).

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Fig. 1. Typical image of ZnO nanoparticles captured by a scanning electron microscope ((A), bar = 100 nm). The size distribution (B) and zeta potential (C) of ZnO nanoparticles dissolved in 1640 medium were measured by a Malvern zetasizer.

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Fig. 2. Dynamic response of HLECs exposed to different concentrations of ZnO nanoparticles in the presence and absence of UVB irradiation for 200 s. Curve A, B, C and D were those treated with 0, 2.5, 5.0 and 10.0 lg/mL of ZnO nanoparticles in the absence of UV irradiation, respectively; Curve E, F, G and H were those treated with 0, 2.5, 5.0 and 10.0 lg/mL of ZnO nanoparticles in the presence of UVB irradiation, respectively. The comparison of decrease for cell activity after ZnO nanoparticletreatment was incubated for more than 72 h.

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(Fig. 3B). However, after ZnO nanoparticles increased to 5.0 lg/mL, the rate of cell death increased more apparent in the presence of UVB irradiation (Fig. 3G) than that without UVB irradiation (Fig. 3C), and their viability had a remarkable difference. When HLECs treated with ZnO nanoparticles (10.0 lg/mL) in the presence and absence of UVB irradiation, it was noted that both cases could inhibit the proliferation of most of HLECs (Fig. 3D and H). These results revealed that the functionalized ZnO nanoparticles could induce apoptosis and/or necrosis to inhibit cell growth when cells exposed to higher concentrations of ZnO nanoparticles, whereas it has little effect on target HLECs when exposed to lower doses of ZnO nanoparticles. Importantly, UVB irradiation plays an important role in enhancing ZnO nanoparticle-induced cell death.

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3.4. DAPI staining

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To investigate the effects of different concentrations of ZnO nanoparticles on the cellular morphology in the presence and

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absence of UVB irradiation, HLECs were incubated with ZnO nanoparticles for 6 h and were stained with DAPI solution. Compared with normal cells (Fig. 4A), the ZnO nanoparticle-treated HLECs displayed a concentration-dependent canonical apoptotic transformation, including cell shrinkage (Fig. 4B and C) and nuclear condensation (Fig. 4D). Additionally, the application of UVB irradiation can further enhance the cytotoxic effect of ZnO nanoparticles on HLECs, indicating UVB irradiation can play a role in enhancing ZnO nanoparticle-induced cytotoxic effect on HLECs.

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3.5. Annexin V/PI staining assay

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The use of the double staining of Annexin V-FITC and propidium iodide (PI) allows separation between live cell populations, cells entering early apoptosis and those in late-stage apoptosis/necrosis. For cells, necrotic cells could expose phosphatidylserine from the inner face of the plasma membrane to the cell surface and lose membrane function simultaneously soon after cell damage. Using a DNA binding dye such as PI with Annexin V, apoptotic cells could be identified and discriminated from necrotic cells. In our studies, the late apoptotic rate of HLECs rose from 1.4% to 5.3%, 14.8%, and 70.2% (Fig. 5A) after treatment with different concentrations (i.e., 0, 2.5, 5.0, 10.0 lg/mL) of ZnO nanoparticles for 24 h. However, the late apoptotic rate of HLECs rose from 7.5% to 19.9%, 46.1% and 75.8% (Fig. 5B) in the presence of UVB irradiation, respectively. These results also suggest that 24 h exposure to ZnO nanoparticles apparently increases the cell death in the presence of UVB irradiation. Meanwhile, the cytotoxicity and inhibitory effects of ZnO nanoparticles on target cells were in a concentration-dependent manner.

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3.6. The alterations in hydroxyl radical levels

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Reactive oxygen species (ROS) can damage most cell components, including DNA, membranes and proteins. In particular, the hydroxyl radical is known as the most reactive species in ROS and is toxic for the cells. In the present study, we investigated the alterations in hydroxyl radical level in HLECs after treatment with different concentrations of ZnO nanoparticles in the presence and absence of UVB irradiation. The results indicate that untreated

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Fig. 3. The morphology of HLECs treated with ZnO nanoparticles in the absence and presence of UVB irradiation. (A) untreated cells (control); (B) cells treated with 2.5 lg/mL of ZnO nanoparticles; (C) cells treated with 5.0 lg/mL of ZnO nanoparticles; (D) cells treated with 10.0 lg/mL of ZnO nanoparticles; (E) cells treated with UVB irradiation for 200 s; (F) cells treated with 2.5 lg/mL of ZnO nanoparticles under UVB irradiation for 200 s; (G) cells treated with 5.0 lg/mL of ZnO nanoparticles under UVB irradiation for 200 s and (H) cells treated with 10.0 lg/mL of ZnO nanoparticles under UVB irradiation for 200 s. Bar = 20 lm.

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Fig. 4. Typical nuclear morphologic changes in HLECs. Cells treated with different concentrations of ZnO nanoparticles in the absence and presence of UVB irradiation for 200 s and further cultured for 6 h, then typical pictures were captured. (A) untreated cells (control); (B) cells treated with 2.5 lg/mL of ZnO nanoparticles; (C) cells treated with 5.0 lg/mL of ZnO nanoparticles; (D) cells treated with 10.0 lg/mL of ZnO nanoparticles; (E) cells after UVB irradiation for 200 s; (F) cells treated with 2.5 lg/mL of ZnO nanoparticles under UVB irradiation for 200 s; (G) cells treated with 5.0 lg/mL of ZnO nanoparticles under UVB irradiation for 200 s; and (H) cells treated with 10.0 lg/mL of ZnO nanoparticles under UVB irradiation for 200 s. Arrows indicate the changed cells with nuclear condensation. Strong fluorescent spots showed apoptotic nuclei. Bar = 20 lm.

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cells generated less hydroxyl radicals whereas the levels of hydroxyl radicals gradually increased with the increase of concentrations of ZnO nanoparticles exposed to HLECs. Remarkably, after UVB irradiation, the levels of hydroxyl radicals increased significantly in the presence of ZnO nanoparticles (Fig. 6), and it was in a concentration-dependent manner.

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3.7. Expression of Ca2+-ATPase

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The effect of different concentrations of ZnO nanoparticles on the PMCA1 expression was explored by quantitative real-time PCR and western blotting analysis in the presence and absence of UVB irradiation. The results indicate that after treatment with different concentration ZnO nanoparticles, the mRNA expression of Ca2+-ATPase in HLECs was decreased to 0.75-, 0.57-, and 0.41-fold compared with that in the untreated cells (Fig. 7A), whereas the mRNA expression level of PMCA1 was decreased significantly to 0.64-, 0.24-, and 0.09-fold compared with that in the presence of ZnO nanoparticles with UVB irradiation (Fig. 7B). In the meantime, the levels of Ca2+-ATPase protein in HLECs in the absence (Fig. 7C) and presence of UVB irradiation (Fig. 7D) demonstrate that with the increase of concentrations of ZnO nanoparticles incubated with HLECs, it was found that the level of Ca2+-ATPase protein was significantly decreased under the conditions with or without UVB irradiation.

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3.8. Intracellular Ca2+ concentrations

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As shown in Fig. 8, the intracellular Ca2+ levels rose from 92.59 ± 2.09 to 128.19 ± 7.19, 167.82 ± 4.81, 402.21 ± 6.23 nmol/L after HLECs exposure to different concentrations (0, 2.5, 5.0 and 10.0 lg/mL, respectively) of ZnO nanoparticles for 30 min. However, the levels of intracellular Ca2+ increased from 156.34 ± 4.59 to 173.88 ± 7.17, 289.02 ± 9.09, 488.36 ± 48.16 nmol/L after treatment with different concentrations of ZnO nanoparticles under UVB irradiation, respectively. These results demonstrate that the intracellular Ca2+ was elevated with the increase of ZnO nanoparticles, and UVB irradiation treatment could further elevate the levels of intracellular Ca2+.

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2+

4. Discussion

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To date, more and more nanomaterials have been applied in cancer therapy and drug delivery including ZnO nanoparticles (Zhang et al., 2011; Hackenberg et al., 2012). PCO is a common complication after cataract surgery, and HLEC proliferation and migration are the cause of PCO. Using lens epithelial cell line (HLE B-3) which derived from human lens epithelium, we explored the possibility and potential for the application of ZnO nanoparticles in eye disease treatment. First, we investigated the effects of ZnO nanoparticles on HLECs in the presence and absence of UVB irradiation. We explored the changes in morphology, apoptosis, ROS levels, Ca2+-ATPase expression at gene and protein levels and intracellular calcium ions. Our results demonstrated that cell viability decreased significantly when exposed to higher concentrations of ZnO nanoparticles, whereas little impact on target cells at lower concentrations, indicating ZnO nanoparticles exert cytotoxic effect on HLECs in a concentration-dependent manner. Meanwhile, longer exposure time to ZnO nanoparticles with UVB irradiation could also inhibit the growth of HLECs. In addition, it is found that drug-sensitive and drug-resistant leukemia cell lines exhibit different sensitivity when exposed to ZnO nanoparticles (Guo et al., 2008) and this is also consistent with the report (Lanone et al., 2009), indicating the cytotoxicity of nanomaterials possesses specific selection in cell lines. Real-time cell electronic sensing (RT-CES) assay could provide dynamic information to identify the interaction between target cells and reagents. The basic principle of the RT-CES system is to monitor the changes in electrode impedance induced by the interaction between testing cells and electrodes, where the presence of the cells will lead to an increase in the electrode impedance. The more cells attached to the sensor, the higher the impedance that could be monitored with RT-CES. The RT-CES array has been proven to be a valuable and reliable way for real-time monitoring of dynamic changes induced by cell-chemical interaction (Joko Q2 et al., 2013; Kim et al., 2012a,b). Apoptosis is associated with a distinct set of biochemical and physical changes involving the cytoplasm, nucleus and plasma membrane. Early in apoptosis, cells round up, lose contact with their neighbors and shrink. In the cytoplasm, the endoplasmic reticulum dilates and the cisternae

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Fig. 5. The apoptosis was measured by flow cytometry after Annexin V/PI staining either for cells treated with ZnO nanoparticles alone (A) or treated with ZnO nanoparticles under UVB irradiation for 200 s (B). (a) untreated cells; (b) cells treated with 2.5 lg/mL of ZnO nanoparticles; (c) cells treated with 5.0 lg/mL of ZnO nanoparticles; and (d) cells treated with 10.0 lg/mL of ZnO nanoparticles. Data were expressed as mean ± SD.

Please cite this article in press as: Wang, D., et al. Zinc oxide nanoparticles inhibit Ca2+-ATPase expression in human lens epithelial cells under UVB irradiation. Toxicol. in Vitro (2013), http:// dx.doi.org/10.1016/j.tiv.2013.09.015

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Fig. 6. Changes in hydroxyl radicals generated from HLECs exposed to ZnO nanoparticles with or without UV irradiation for 200 s. Data were obtained from three independent experiments and results were represented as mean ± SD. NPs, nanoparticles;  p<0.05; and  p < 0.01.

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swell to form vesicles and vacuoles. In the nucleus, chromatin condenses and aggregates into dense compact masses (Lawen, 2003). In the present study, the DAPI staining analysis revealed that after treatment of ZnO nanoparticles in the presence and absence of UVB irradiation, the cell nuclei were changed significantly, including the nucleus turned rippled or creased, or nuclear condensation (Fig. 4), suggesting that ZnO nanoparticles could influence the morphology of cell nuclei and further disrupt their function. The evidence was collected showing an important role of the endoplasmic reticulum in the maintenance of intracellular calcium homeostasis, protein synthesis, posttranslational modification, and proper folding of proteins as well as their sorting and trafficking. Many stimuli can cause stress resulting in apoptosis (Rao et al., 2004) through the unfolded protein response and Ca2+ signaling mechanisms. Our findings demonstrate that the cells exposed to ZnO nanoparticles could lead to the marked decrease of HLEC

proliferative capacity using RT-CES assay (Fig. 2) as well as the increase of hydroxyl radical levels (Fig. 6), and it was in a concentration-dependent manner. These results were also consistent with that of Annexin V/PI staining determination (Fig. 5). ROS generation has been reported in response to inhaled particles, fibers, and nanomaterials (Mossman, 2003; Lin et al., 2006a,b). Studies showed that ROS were involved in the mechanism by which nanoparticles induce opening of plasma membrane Ca2+ transporters in response to calcium release from the endoplasmic reticulum by thapsigargin (Stone et al., 2000). The molecular basis of UVB damage has been linked to the generation of ROS, although the exact basis of this damage is still unknown (Heck et al., 2003). PMCA is a sensitive target of various ROS free, such as radical-induced oxidative damage and inactivation in rat brain synaptic plasma membranes or purified from erythrocyte membranes (Zaidi et al., 2009; Lushington et al., 2005). The abnormality of intracellular calcium level has major effects on cellular metabolism, signal transduction, and gene expression (Huang et al., 2010). To address the possible mechanism of ZnO nanoparticle-induced imbalance of calcium homeostasis in HLECs, we further explored the expression levels of PMCA1 before and after cells exposed to different concentrations of ZnO nanoparticles with or without UVB irradiation for both mRNA and protein levels. In our study, b-actin was used as an internal control for protein expression and GAPDH was used as an internal control for mRNA, because the results for both mRNA and protein expression were consistent. The results (Fig. 7) demonstrate that with the increase of ZnO nanoparticles exposed to target HLECs, the lower levels of PMCA1 were observed and in a concentration-dependent manner, UVB irradiation could further inhibit the expression of PMCA1 at gene and protein levels. After exposure to ZnO nanoparticles under UVB irradiation, HLECs can generate excessive ROS, further excessive ROS can attack the cell membrane; destruct the membrane integrity, leading to the high permeability of cell membrane. The high permeability of cell membrane will attribute to the influx of calcium ions from extracellular environment. Meanwhile, the excessive ROS within cells could also enhance the calcium ion release from internal stores, leading to the elevated intracellular calcium level. In addition, excessive ROS can also damage the activity of PMCA,

Fig. 7. Quantitative real-time PCR analysis of Ca2+-ATPase mRNA expression (a and b) and western blotting analysis of Ca2+-ATPase protein level (c and d) relevant to b-actin in HLECs treated with different concentrations of ZnO nanoparticles in the presence and absence of UVB irradiation. The graph showed the changes in the gene expression level of Ca2+-ATPase after exposure to ZnO nanoparticles (i., e., 0, 2.5, 5.0, 10.0 lg/mL) in the absence (a and c) and presence (b and d) of UVB irradiation for 200 s. Data were expressed as mean ± SD. ( P < 0.05,  P < 0.01, ANOVA and the Dunnett post hoc test). NPs, nanoparticles.

Please cite this article in press as: Wang, D., et al. Zinc oxide nanoparticles inhibit Ca2+-ATPase expression in human lens epithelial cells under UVB irradiation. Toxicol. in Vitro (2013), http:// dx.doi.org/10.1016/j.tiv.2013.09.015

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Fig. 8. Intracellular calcium ion levels were determined by Fluo-3/AM probes using flow cytometry after treatment with different concentrations of ZnO nanoparticles in the presence and absence of UVB irradiation. Cells were exposed to different concentrations of ZnO nanoparticles in the presence and absence of UVB irradiation, and cultured for additional 30 min, then all cells were collected. After treatment with Fluo-3/AM according to the manufacturer’s instructions, the intracellular calcium ion levels were measured using a flow cytometer. NPs, nanoparticles.

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destruct the regulatory role of PMCA which transport the excessive intracellular Ca2+ out of the cytoplasm, and thus disrupt the intracellular calcium homeostasis. It is reported that exposure of hippocampal neurons to exogenous hydrogen peroxide (H2O2) and glutamate resulted in PMCA degradation and inactivation (Kip and Strehler, 2007; Pottorf et al., 2006), indicating that PMCA can not only be a Ca2+ transporting protein the sole function of which is to maintain Ca2+ levels, but also be a very precisely-controlled regulator of important Ca2+-dependent signal transduction pathways that promote cell survival and/or cell death. Thus, our findings substantiate the importance of calcium-mediated signaling pathway involved in the regulation of PMCA in HLECs death induced by ZnO nanoparticles with UVB irradiation. As we know, disrupted intracellular calcium homeostasis can be detrimental to target cells. Thus, the intracellular Ca2+ concentration would maintain a normal level to keep normal physiological function of cells. It was reported that the Ca2+ level in human lens epithelial cell was about 96 ± 20 nmol/L (Riach et al., 1995) and UVB irradiation could apparently increase intracellular Ca2+ level (Ding and Wang, 2003). In our study, the intracellular Ca2+

Scheme 1. Principle of inhibitory effect of ZnO nanoparticles on HLECs under UVB irradiation.

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concentration was about 92.59 ± 2.09 nmol/L in untreated cells, and our study also shows an elevation of intracellular Ca2+ occurred after treatment with ZnO nanoparticles in the presence of UVB irradiation (Fig. 8), further the elevated intracellular calcium ion level can disrupt the intracellular calcium homeostasis in HLECs, and finally the disrupted calcium homeostasis leads to the cell death through calcium-dependent signaling pathway. As shown in Scheme 1, we presume that after treatment with ZnO nanoparticles in the presence of UVB irradiation, HLECs could produce excessive ROS within cells. The excessive ROS can damage cell membrane and enhance its permeability, further leads to the outer calcium ions into cytoplasm. The elevated intracellular calcium ions can disrupt the intracellular calcium homeostasis. Moreover, the excessive intracellular ROS and disrupted calcium homeostasis could initiate the PMCA-mediated calcium signaling pathway, finally cause cell death.

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In summary, we have explored the effect of different concentrations of ZnO nanoparticles on HLECs in the presence and absence of UVB irradiation using RT-CES, flow cytometry, real-time quantitative PCR and western blotting techniques. Results indicate that ZnO nanoparticles could inhibit HLEC proliferation in a concentrationdependent manner, and UVB irradiation can enhance the inhibitory effect mediated by ZnO nanoparticles. Both ZnO nanoparticles and UVB irradiation could generate excessive reactive oxygen species in HLECs, reduce the expression of PMCA1 at gene and protein levels. Meanwhile, ZnO nanoparticles and UVB irradiation can increase the intracellular calcium ion levels, disrupt the intracellular calcium homeostasis and finally cause HLEC death. Our investigations indicate that ZnO nanoparticles and UVB irradiation have synergistic cytotoxic effect on HLECs, suggesting that UVB irradiation and ZnO nanoparticles could exert inhibitory effect on HLECs via calcium-mediated signaling pathway. ZnO nanoparticles have great potential for the application in PCO treatment under UVB irradiation.

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Conflict of Interest

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The authors declare that they have no competing interests.

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Acknowledgment

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This work was supported by the Natural Science Foundation of Shandong Province (ZR2010HM032).

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