Current Eye Research, 2015; 40(8): 792–799 ! Informa Healthcare USA, Inc. ISSN: 0271-3683 print / 1460-2202 online DOI: 10.3109/02713683.2014.958171
ORIGINAL ARTICLE
In Vitro Model for Predicting the Protective Effect of Ultraviolet-Blocking Contact Lens in Human Corneal Epithelial Cells
Curr Eye Res Downloaded from informahealthcare.com by Nyu Medical Center on 07/25/15 For personal use only.
Antonio Abengo´zar-Vela1,2, Cristina Arroyo1, Roberto Reinoso1,2, Amalia Enrı´quez-de-Salamanca1,2, Alfredo Corell1,2 and Marı´a Jesu´s Gonza´lez-Garcı´a1,2 1
Instituto de Oftalmobiologı´a Aplicada (IOBA), Universidad de Valladolid, Valladolid, Spain and 2 CIBER-BBN, Valladolid, Spain
ABSTRACT Purpose: To develop an in vitro method to determine the protective effect of UV-blocking contact lenses (CLs) in human corneal epithelial (HCE) cells exposed to UV-B radiation. Materials and Methods: SV-40-transformed HCE cells were covered with non-UV-blocking CL, UV-blocking CL or not covered, and exposed to UV-B radiation. As control, HCE cells were covered with both types of CLs or not covered, but not exposed to UV-B radiation. Cell viability at 24, 48 and 72 h, after UV-B exposure and removing CLs, was determined by alamarBlueÕ assay. Percentage of live, dead and apoptotic cells was also assessed by flow cytometry after 24 h of UV-B exposure. Intracellular reactive oxygen species (ROS) production after 1 h of exposure was assessed using the dye H2DCF-DA. Results: Cell viability significantly decreased, apoptotic cells and intracellular ROS production significantly increased when UVB-exposed cells were covered with non-UV-blocking CL or not covered compared to non-irradiated cells. When cells were covered with UV-blocking CL, cell viability significantly increased and apoptotic cells and intracellular ROS production did not increase compared to exposed cells. Conclusions: UV-B radiation induces cell death by apoptosis, increases ROS production and decreases viable cells. UV-blocking CL is able to avoid these effects increasing cell viability and protecting HCE cells from apoptosis and ROS production induced by UV-B radiation. This in vitro model is an alternative to in vivo methods to determine the protective effect of UV-blocking ophthalmic biomaterials because it is a quicker, cheaper and reliable model that avoids the use of animals. Keywords: Apoptosis, contact lens, corneal epithelial cells, oxidative stress, ultraviolet light
INTRODUCTION
radiation that reaches the earth is mainly UV-A and -B. Even though the UV-B radiation represents less than 1% of the total radiation reaching the earth’s surface, it is a radiation of high energy level and it has the greatest potential ocular damage.2 The eye is one of the organs more susceptible to UV light damage. The human cornea has a high absorption coefficient for UV-B radiation,3 acting as UV-B filter that absorbs approximately up to 92% of
The ultraviolet radiation (UV) is a part of the electromagnetic spectrum that covers the wavelength range from 100 to 400 nm, and is further divided into the following bands: UV-vacuum from 100 to 200 nm; UV-C from 200 to 280 nm; UV-B from 280 to 315; and UV-A from 315 to 400 nm.1 UV-C radiation is fully absorbed in the upper atmosphere. Thus, the UV
Received 27 June 2014; revised 12 August 2014; accepted 18 August 2014; published online 6 October 2014 Correspondence: Marı´a Jesu´s Gonza´lez-Garcı´a, Instituto de Oftalmobiologı´a Aplicada (IOBA), Universidad de Valladolid, Paseo de Bele´n 17, Valladolid 47011, Spain. Tel: +34 983184756. E-mail: [email protected]
792
Curr Eye Res Downloaded from informahealthcare.com by Nyu Medical Center on 07/25/15 For personal use only.
In Vitro Model to Test UV-Blocking CLs 793 UV-B at 300 nm.4 In addition, corneal epithelium absorbs a substantial amount of the UV-B radiation that reaches the cornea, protecting the deeper corneal layers and eye structures from UV-B radiation.3,5 The result of this light absorption is the damage to the corneal epithelium by UV-B radiation. One of the most common acute effect of UV-B irradiation on the cornea is photokeratitis,6,7 characterized by both apoptosis (programmed cell death) and inflammation in corneal epithelium.8,9 Not only does acute UV exposure produce adverse ocular effects; but also chronic UV irradiation is one of the most important risk factor for ocular surface disorders. It is involved in solar keratopathy,10 pterygium,11,12 and ocular melanoma in conjunctiva,10,13 even in cataract formation14,15 and retina degeneration.16 Currently, silicone hydrogel (SiH) contact lenses (CLs) have quickly become the most popular type of soft CLs prescribed for daily wear representing 14.1% of daily disposable lens fits and 48.8% of reusable lens fits.17 In addition, today’s advanced techniques have allowed some manufacturers to incorporate different UV absorbing monomers into their SiHCLs. The different classes of UV-blocking CLs are labeled as Class I and Class II, according to the America National Standards Institute (ANSI). Class I UV filter blocks 90% of UV-A and 99% of UV-B, and Class II UV filter blocks 70% UV-A and 95% of UV-B, with 380 nm as upper limit of UV-B.18 Thus, there is a need to evaluate and compare the performance of these UV-blocking CLs. Some in vitro and in vivo studies have evidenced that UV-absorbing ophthalmic biomaterials have protective effects on cornea, lens and retina.19–23 However, only in vivo experiments have tested the protective effect of UV-blocking CLs on corneal cells.4,20,23–25 The aim of this study was to develop a suitable in vitro method as alternative to in vivo models to examine the protective effect of UV-blocking ophthalmic biomaterials, mainly CLs, when human corneal epithelial cells were exposed to UV-B radiation. Cellular viability, apoptosis and oxidative stress were analyzed.
MATERIALS AND METHODS Cell Line and Culture Medium SV-40-transformed human corneal epithelial (HCE) cells, a gift from Dr. Arto Urtti (University of Helsinki, Finland), were cultured in Dulbecco’s modified Eagle’s medium/nutrient mixed F12 (DMEM/F-12) L-glutamine supplemented with 15% fetal bovine serum (FBS), 10 ng/mL human epidermal growth factor (EGF), 0.5% dimethyl sulfoxide (DMSO), !
2015 Informa Healthcare USA, Inc.
0.1 mg/mL cholera toxin, 5 mg/mL insulin, 100 U/mL penicillin and 0.1 mg/mL streptomycin (all reagents from Sigma, St. Louis, MO), as previously described.26 The cells were maintained at 37 C with a 5% CO2 humidified atmosphere. When culture reached confluence, cells were harvested by trypsin and seeded into white 24-well plates with clear bottom (Perkin Elmer, Waltham, MA, USA) at a density of 60,000 cells per well; except for cell viability assay where cell density was 35,000 cells per well. Cells from passage 50 to 61 were used in this study.
Contact Lenses Two types of CLs were tested in this study: non-UVblocking SiHCL (Lotrafilcon B; AirOptixÕ ; CIBA Vision, Duluth, GA); and class I UV-blocking SiHCL (Senofilcon A; AcuvueÕ OasysÕ ; Johnson & Johnson Vision Care, Limerick, Ireland). Technical details of CLs studied are summarized in Table 1. UV spectra for both CLs have been previously described,18 showing that UV-B transmittance for Lotrafilcon-B and Senofilcon-A CLs was 62.89% and 0.03% of UV-B radiation, respectively, being UV-A transmittance for Lotrafilcon-B and Senofilcon-A CLs 80.71% and 18.35%, respectively.18
Exposure of Cells to UV-B Radiation HCE cells were seeded and growth until preconfluence. Then, medium was removed, and phenol redfree Roswell Park Memorial Institute (RPMI) 1640 Medium (Sigma, St. Louis, MO) was added to each well. Prior to sink CLs in the well, CLs were gently soaked for 10 s with RPMI in order to avoid decreasing viability of HCE cells due to CLs borate-buffered packaging solution.27 Then, CLs were totally immersed in culture medium and placed on top of cell monolayer with concave surface facing upwards, covering more than 94.5% of the well surface area (well diameter 14.8 mm). Cells in the presence/ absence of CLs were then exposed to UV-B light generated from 6 W UV lamps (Vilber Lourmat, Marne-la-Valle´e, France) with peak intensity at 312 nm (range 280–380 nm; UV-C cut-off filter TABLE 1 Nominal parameters of CLs used in this study. Material Brand Company Base curve (mm) Diameter (mm) Central thickness (mm) Power (D) Water content (%) Dk: oxygen permeability UV-Blocking
Lotrafilcon B Õ
AirOptix CIBA Vision 8.6 14.2 0.08 –1.00 33 110 No
Senofilcon A AcuvueÕ OasysÕ Johnson & Johnson 8.8 14.0 0.07 –1.00 38 103 Class I
794 A. Abengo´zar-Vela et al. by cells to resorufin, a fluorescence compound. This conversion occurs intracellularly by mitochondrial, microsomal and cytosolic oxidoreductases.28 AlamarBlueÕ can distinguish metabolically active cells because non-viable cells have lower innate metabolic activity and thus generate a proportionally lower signal than healthy cells.
Curr Eye Res Downloaded from informahealthcare.com by Nyu Medical Center on 07/25/15 For personal use only.
Cell Apoptosis
FIGURE 1 The emission spectrum of the UV-B lamp used for this in vitro model.
included; Figure 1) for 5 min and 30 s. The lamp was placed on a stand where it emits uniform irradiance of 680 mW/cm2 at 15 cm, according to the manufacturer instruction. UV-B radiant exposure used was 200 mJ/ cm2; calculated with the following formula: H = Et. Here H is the radiant exposure (J/cm2), E is the irradiance (W/cm2) and t is the exposure time (s). Control cells were not irradiated. After irradiation, all CLs were removed, phenol red-free RPMI was replaced with the supplemented DMEM/F12 culture medium used for cell growth, and cells were incubated at 37 C for specific times according to the experiment carried out. Experiments were repeated three times and were always performed with new CLs.
Cell Viability Cell viability was assessed by alamarBlueÕ test (Life Technologies, Carlsbad, CA) following the manufacturer‘s instructions with some modifications. Briefly, after UV-B exposure and removing all CLs and phenol red-free RPMI, cells were maintained for 24 h of recovery in supplemented DMEM/F12 cell culture medium. Then, medium was discarded and attached cells were washed with phosphate buffered saline (PBS; Life Technologies), and 1 mL of alamarBlueÕ solution (10 % v/v in supplemented culture medium) was added at 24, 48 and 72 h of culture. Cells were incubated with alamarBlueÕ for 4 h at 37 C. Finally, alamarBlueÕ solution from each sample was collected and fluorescence at 560 nmex/590 nmem was measured by UV/Vis spectrophotometry (SpectraMaxÕ M5, Molecular Devices, Sunnyvale, CA). Samples were assayed in duplicates. AlamarBlueÕ , or resazurin, is a non-toxic and nonfluorescent compound that is reduced continuously
Percentages of live, early and late apoptotic, and dead cells were assessed by flow cytometry. After UV-B exposure and 24 h of recovery in supplemented DMEM/F12 culture medium, cell supernatants were collected and centrifuged, and well-attached cells were trypsinized and collected. Subsequently, detached cells from wells and cells from supernatants were pooled. After that, cell suspensions were washed with 2 mL of Cell Wash Solution (BD Biosciences, San Jose, CA), centrifuged at 500 g for 5 min, and stained with an annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) commercial kit (BeckmanCoulter, Fullerton, CA) as described previously.29 The flow cytometry analysis was performed with a Cytomics FC 500 Cytometer (Beckman-Coulter) using 488 nm excitation with an argon-ion laser for FITC and PI. The data collected were analyzed using the Cytomics CXP software program (BeckmanCoulter). At least 15,000 HCE cells were analyzed for each condition. Controls included cross-reactivity of the fluorescence signals of each channel. Flow cytometry results were expressed as means ± SEM.
Reactive Oxygen Species Production UV-induced intracellular reactive oxygen species (ROS) production was determined using 20 ,70 -dihydrodichlorofluorescein diacetate (H2DCFDA; Sigma) probe. Non-fluorescent H2DCF-DA passively diffuses into cells, where intracellular esterases cleave it to the polar non-fluorescent compound H2DCF. Then, H2DCF is oxidized to highly fluorescent DCF by intracellular ROS. Cells were seeded in 24 well-plates in supplemented DMEM/F12 medium. When cells reached preconfluence, medium was discarded, serum-free DMEM/F12 medium was added and cells were maintained in it for 24 h. After that, medium was removed and cells were loaded with 10 mM H2DCF-DA solution for 30 min. Then, medium was replaced with phenol red-free RPMI, CLs were immersed in wells with the concave side facing up, and cells were exposed to 200 mJ/cm2 UV-B radiation. Control cells were placed under the UV lamp, but were not irradiated. CLs were then removed and cells were cultured for 1 h at 37 C. Finally, fluorescence was measured by UV/Vis spectrophotometry at 498 nmex/522 nmem.30 After fluorescence was read, medium was removed and well plates Current Eye Research
In Vitro Model to Test UV-Blocking CLs 795 with attached cells were stored at 80 C before determining the protein content of the cell lysates by using the bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Rockford, IL), according to manufacturer’s instructions. Samples were performed in duplicates and measurements were from three independent experiments. Data were expressed as mean fluorescence in arbitrary units (a.u.) normalized to total amount of protein (mg) per well.
Curr Eye Res Downloaded from informahealthcare.com by Nyu Medical Center on 07/25/15 For personal use only.
Statistical Analysis Statistical differences for cell viability assay between each CL group at each time point tested were assessed by two-way repeated-measures analyses of variance (ANOVA) followed by the Bonferroni post-hoc test, and differences between time points were assessed by one-way ANOVA followed by the Bonferroni post-hoc test. Cell apoptosis and ROS production data were analyzed using Student’s unpaired t-test for comparing UVB-unexposed cells with exposed cells and statistical differences between groups were assessed using one-way ANOVA followed by the Bonferroni post-hoc test. p Values below 0.05 were considered significant.
RESULTS Effect of Contact Lens with UV Filter on Cell Viability Viability of HCE cells over time after exposure to UV-B radiation in presence of either UV-blocking CL or non-UV-blocking CL is shown in Figure 2. Any of
FIGURE 2 Effect of UV-blocking contact lens (CL) on UVB-exposed HCE cells viability at 24, 48 and 72 h after UV irradiation. Data are expressed as fluorescence intensity at 590 nm in arbitrary units (a.u.). Each bar represents the mean ± SEM from three independent experiments. *Denotes significantly different compared to corresponding UVB-unexposed cell group: *p50.05, ***p50.001. +Denotes significantly different comparing non-UV-blocking CL group versus UV-blocking CL group for UV-B exposed cells: +++ p50.001. #Denotes significantly different compared to 24 h data: #p50.05, ##p50.01, ###p50.001. !
2015 Informa Healthcare USA, Inc.
the two types of SiHCLs altered the viability of UV-unexposed control cells at any time point measured. When HCE cells in the absence of CL or in the presence of the non-UVB-blocking CL were irradiated with 200 mJ/cm2, their viability dramatically decreased after 24, 48 and 72 h of recovery, compared to UV-unexposed cells (p50.001). In contrast, when cells were covered with UV-blocking CL and exposed to UV-B radiation, viability increased over time with a significant difference at 48 h (p50.05), compared to its control. Cell viability values for exposed cells covered with UV-blocking CL were significantly higher than those found for exposed cells covered with non-UV-blocking CL at all time points (p50.001).
Effect of Contact Lens with UV Filter on Apoptosis To further confirm the protective effect of a UV-blocking filter in the CL against UV-B exposure, apoptosis assay by flow cytometry was carried out in HCE cells after 24 h of UV exposure (Figure 3). Percentages of live, early and late apoptotic, and dead cells were 81.95 ± 3.86%, 12.02 ± 2.37%, 5.53 ± 1.46% and 0.35 ± 0.11%, respectively, for UVB-unexposed cells in the absence of CL. There were not differences in these percentages with respect to UVB-unexposed cells with both CLs. When cells without CL were exposed to UV-B, percentage of live cells, compared to unexposed cells, significantly decreased to 48.07 ± 5.42% (p50.001); whereas cells in early and late apoptosis, and dead cells increased to 21.87 ± 3.37% (p50.05), 28.70 ± 2.58% (p50.001) and 1.22 ± 0.08% (p50.001), respectively. We did not find any significant differences between exposed cells without CL and with non-UV-blocking CL. However, when HCE cells were covered with UV-blocking CL and exposed to UV radiation, percentages of live, early and late apoptotic, and dead cells were 72.35 ± 4.41% (p50.05), 16.90 ± 2.72%, 10.15 ± 1.82% (p50.001) and 0.42 ± 0.07% (p50.01), respectively, compared to exposed cells without CL. Same statistical differences were found when exposed HCE cells covered with UV-blocking CL were also compared to exposed HCE cells covered with nonUV-blocking CL.
Effect of Contact Lens with UV Filter on Oxidative Stress Figure 4 shows intracellular ROS production induced by UV-B radiation for each group. Fluorescence intensity data, expressed in arbitrary units, were normalized to total protein levels. The presence of CLs (non-UV-blocking or UV-blocking CLs) did not affect ROS production in UVB-unexposed cells. The UV-B radiation significantly increased the production
Curr Eye Res Downloaded from informahealthcare.com by Nyu Medical Center on 07/25/15 For personal use only.
796 A. Abengo´zar-Vela et al.
FIGURE 3 Effect of UV-blocking contact lens (CL) on UVB-exposed HCE cells apoptosis. (A) Representative flow cytometric dot plots of cell apoptosis analysis. Dot plot of annexin V-FITC versus propidium iodide shows levels of live cells (region H3), early (region H4) and late (region H2) apoptotic cells, and dead cells (region H1). (B) Bar graphs representing the percentage of gated cells in each region. Each bar represents the mean percent of cells ± SEM from three independent experiments. Differences were significant at *p50.05, **p50.01, ***p50.001, compared to unexposed cell groups; +p50.05, ++p50.01, +++p50.001, compared to exposed cells without CL; and #p50.05, ##p50.01, compared to exposed cells with non-UV-blocking CL.
Current Eye Research
Curr Eye Res Downloaded from informahealthcare.com by Nyu Medical Center on 07/25/15 For personal use only.
In Vitro Model to Test UV-Blocking CLs 797
FIGURE 4 Effect of UV-blocking contact lens (CL) on UVB-exposed HCE cells reactive oxygen species (ROS) production. Data are expressed as relative fluorescence value in arbitrary units (a.u.) normalized to total amount of protein in micrograms (mg). Each bar represents mean ± SEM from three independent experiments. Differences were significant at *p50.05 compared to corresponding UVB-unexposed cell group.
of intracellular ROS (p50.05) in both cells without CL and cells with non-UV-blocking CL after 1 h of incubation, compared to UV-unexposed cells. ROS production was not significantly increased in UV-B irradiated cells covered with UV-blocking CL compared to UV-unexposed cells.
DISCUSSION It is well known that both acute and chronic UV-B exposure can lead to several ocular surface diseases like photokeratitis6 or pterygium.12 Previous reports have shown that UV-B radiation causes both DNA and mitochondrial degradations decreasing ocular surface cell viability. Moreover, it is increasingly recognized that UV-B radiation is also involved in corneal cell death, increasing the number of apoptotic cells and producing nuclear fragmentation and loss of tissue cohesion.21,31 Indeed, the first clinical signs of photokeratitis are due to damage or loss of epithelial cells by shedding and apoptosis.8 This fact results in loss of the stratified epithelium to the basement membrane exposing nerve fibers leading to acute pain. Nowadays CLs include UV-blocking agents that minimize the ocular damage by exposure to UV light. Published studies on the protection of these CLs are in vivo studies that require a large investment in time and money and can lead to ethical problems associated with the use of animals. !
2015 Informa Healthcare USA, Inc.
Therefore, we proposed a cheaper, quicker and reliable alternative method to replace animal experiments. Our data demonstrate that UV-blocking CL protects corneal epithelial cells from apoptosis that leads to increase of cell viability after 72 h of recovery when cells are exposed to a relatively high dose of UV-B. The irradiance of the UV-B lamp used in this study was 680 mW/cm2, which is three times higher than the daily maximum irradiance in Valladolid (Spain) occurring in July, 202 mW/cm2.32 In addition, the UV-B dose used in this study is similar to average value of solar UV-B radiation reaching the human cornea, that is 0.187 J/cm2 for 1 h exposure.33 Thus, the irradiance of the lamp used in this experiment is environmentally and energetically relevant and the UV-B radiation dose used in this work is equivalent to 1 h exposure time to sunlight condensed into 5 min and 30 s. Our viability analysis by alamarBlueÕ assay demonstrated that UV-exposed cells covered with UV-blocking CL maintain their viability after 72 h of incubation. Our results showed that UV radiation decreased alamarBlueÕ fluorescence readings from exposed cells in the absence of CL or presence of nonUV-blocking CL after 24 h of culture. Moreover, there was no cellular recovery during the entire culture period of cells exposed to UV-B radiation with nonUV-blocking CL. This fact indicates that UV-B radiation affects metabolic activity due to the loss of appropriate cytoplasmic milieu, and non-UV-blocking CL did not protect cytoplasmic milieu from UV-B radiation. However, when HCE cells with UV-blocking CL were irradiated, alamarBlueÕ fluorescence readings increased after 72 h of incubation. Thus, UV-blocking CL protects cytoplasmic milieu from UV-B radiation. Similar to our own findings, Lin et al. found in an in vivo model of UV-B damage that corneal epithelial cells maintained normal cellular properties when corneas were covered with UV-blocking CL for 7 days of exposure.25 They demonstrated that UV radiation reduced corneal thickness in the presence or absence of nonUV-blocking CL along with a loss of p63+ basal cells, CK-5+ epithelial cells and Ki-67+ proliferative basal cells. However, UV-blocking CL was able to keep corneal thickness and all epithelial cell population maintained their cellular properties after UV irradiation. In addition, Giblin et al. found a swelling and haziness of the corneal epithelium along with severe loss of corneal epithelial cells in rabbit eyes irradiated with UV-B for 5 days. These adverse effects were reverted when rabbit corneas were covered with UV-blocking CL.20 Collectively, these results suggest that UV-blocking CL may maintain corneal epithelial cells healthy when UV-B radiation reaches cornea. We found that UV-B radiation induced apoptosis in corneal epithelial cells, as other in vitro and in vivo
Curr Eye Res Downloaded from informahealthcare.com by Nyu Medical Center on 07/25/15 For personal use only.
798 A. Abengo´zar-Vela et al. assays have demonstrated previously.21,30 In this study, apoptosis was assessed by flow cytometry using annexin V-FITC and PI. This double staining allowed us to distinguish not only between live, apoptotic, and dead cells, but also between early and late apoptotic stages.34 We found that the percentage of apoptotic cells increased when HCE cells were exposed to UV-B in the absence of CL or presence of non-UV-blocking CL. Moreover, there was a higher increase of late than early apoptotic cells after UV-B radiation. It indicates that early apoptotic epithelial cells preserve their plasma membrane integrity to retain the potentially harmful cellular contents inside while late apoptotic epithelial cells release cellular content into the extracellular space. Thus, UV-B radiation promotes a leakage of intracellular molecules that may exacerbate the inflammatory response induced by UV radiation. On the other hand, UV-blocking CL was able to reduce percentage of apoptotic and dead cells. Percentage of HCE cells in late apoptosis was lower than the percentage of cells in early apoptosis when HCE cells where covered with UV-blocking CL and exposed to UV-B radiation. In addition, live cells found in irradiated HCE cells in the absence of CL or presence of non-UV-blocking CL were not viable as alamarBlueÕ assay pointed out previously. However, when irradiated HCE cells were covered with UV-blocking CL, a high cell percentage was alive and they were totally viable after 72 h incubation. It indicates that UVblocking CL can prevent morphological changes of the cell membrane and DNA fragmentation by UV-B radiation keeping cells healthy. UV-blocking CL also prevents other apoptotic events. Chandler et al. reported that UV-B radiation increased caspase-3 activity and TUNEL positive cells in rabbit corneas, even when they were covered with non-UV-blocking CL.4 The use of UV-blocking CL decreased apoptosis in the cornea as evidenced by reduction in caspase-3 activity and in TUNEL-positive cells.4 All these findings demonstrate that UV-blocking CL can prevent apoptosis at different stages in UV-irradiated cornea, however more studies are necessary to know if CL with UV filter can protect human cornea from apoptosis. The eye and particularly cornea and conjunctiva are constantly under oxidative stress due to their continuous exposure to light and oxygen. Oxidative stress is caused by the generation of uncontrolled ROS like superoxide (O 2 ), hydroxyl radical (HO ) or hydrogen peroxide (H2O2). Indeed, UV-B radiation is one of the major ROS inducer on the ocular surface. Although the ocular surface have antioxidative enzymes that protect against ROS,35 UV-B can also inactivate the antioxidant enzyme system in the cornea,36 and may exacerbate ROS-induced ocular surface diseases. We have found that UV-blocking CL tends to decrease UV-induced ROS production in HCE cell line. Our in vitro data agree with Ibrahim et al. who investigated UV-induced oxidative damage
markers in a soft contact lens mouse model.23 They found an increase of hexanoyl-lysine, 4-hydroxynonenal and 8-hydroxy-20 -deoxyguanosine, advanced lipid and DNA oxidation products, in the mouse cornea. These levels of oxidative stress markers were reduced by UV-blocking CL, but not by CL without UV-blocking filter. That indicates radiation-induced ROS react with lipids and DNA in cornea and UV-blocking CL can protect corneal epithelial cells from oxidative damage. This in vitro model aims to be a valid alternative to current in vivo models for the study of UV-blocking CLs. Data found in this in vitro model are in agreement with previous studies conducted in different in vivo models. Our in vitro model can be easily performed and requires a minimum investment of time and money. In addition, this model eliminates the use of animals in experimentation fulfilling the first premise of the three Rs of humane animal experimentation, which are: replace, reduce and refine. Therefore, it can be concluded that this in vitro model could be a valid alternative to in vivo models. This study has some limitations that must be considered. Firstly, we only tested UV-blocking SiHCL. However, some new UV-blocking hydrogel CLs have been released nowadays to the market. Secondly, this test was performed using a monolayer rather than a stratified structure. And finally the protective effect of UV-blocking CL has only been tested in a corneal epithelial cell line but fitted CLs cover and protect not only corneal epithelial cells but also other cell types, such as conjunctival cells or keratocytes. Thus, more studies are necessary to know whether UV-blocking CL may protect other cell types from UV-B irradiation. In conclusion, this study reveals that a CL with a UV filter can minimize UV-induced damage in HCE cells compared to non-UV filter CLs. Our in vitro data agree with previous in vivo studies about the effect of UV-blocking CL on corneal cells regarding viability, apoptosis and ROS production. Hence, this in vitro approach may be useful in order to test new ophthalmic materials, mainly new UV-blocking CLs, as alternative method quicker, cheaper and ethically less controversial than in vivo studies.
DECLARATION OF INTEREST The authors declare that they have no conflict of interest. All CLs were kindly provided by both manufacturers.
REFERENCES 1. Bergmanson JP, Sheldon TM. Ultraviolet radiation revisited. CLAO J 1997;23:196–204. Current Eye Research
Curr Eye Res Downloaded from informahealthcare.com by Nyu Medical Center on 07/25/15 For personal use only.
In Vitro Model to Test UV-Blocking CLs 799 2. Chaney EK, Sliney DH. Re-evaluation of the ultraviolet hazard action spectrum – the impact of spectral bandwidth. Health Phys 2005;89:322–332. 3. Kolozsvari L, Nogradi A, Hopp B, Bor Z. UV absorbance of the human cornea in the 240- to 400-nm range. Invest Ophthalmol Vis Sci 2002;43:2165–2168. 4. Chandler HL, Reuter KS, Sinnott LT, Nichols JJ. Prevention of UV-induced damage to the anterior segment using class I UV-absorbing hydrogel contact lenses. Invest Ophthalmol Vis Sci 2010;51:172–178. 5. Podskochy A. Protective role of corneal epithelium against ultraviolet radiation damage. Acta Ophthalmol Scand 2004;82:714–717. 6. Blumthaler M, Ambach W, Daxecker F. On the threshold radiant exposure for keratitis solaris. Invest Ophthalmol Vis Sci 1987;28:1713–1716. 7. Ringvold A. Cornea and ultraviolet radiation. Acta Ophthalmol (Copenh) 1980;58:63–68. 8. Cullen AP. Photokeratitis and other phototoxic effects on the cornea and conjunctiva. Int J Toxicol 2002;21:455–464. 9. Young AR. Acute effects of UVR on human eyes and skin. Prog Biophys Mol Biol 2006;92:80–85. 10. Taylor H. The biological effects of UV-B on the eye. Photochem Photobiol 1989;50:489–492. 11. Coroneo MT. Pterygium as an early indicator of ultraviolet insolation: a hypothesis. Br J Ophthalmol 1993;77:734–739. 12. Di Girolamo N, Kumar RK, Coroneo MT, Wakefield D. UVB-mediated induction of interleukin-6 and -8 in pterygia and cultured human pterygium epithelial cells. Invest Ophthalmol Vis Sci 2002;43:3430–3437. 13. Taylor HR, West SK, Rosenthal FS, Munoz B, Newland HS, Emmett EA. Corneal changes associated with chronic UV irradiation. Arch Ophthalmol 1989;107:1481–1484. 14. Bergmanson JP, Soderberg PG. The significance of ultraviolet radiation for eye diseases. A review with comments on the efficacy of UV-blocking contact lenses. Ophthalmic Physiol Opt 1995;15:83–91. 15. Delcourt C, Carriere I, Ponton-Sanchez A, Lacroux A, Covacho MJ, Papoz L. Light exposure and the risk of cortical, nuclear, and posterior subcapsular cataracts: the pathologies oculaires liees a l’age (POLA) study. Arch Ophthalmol 2000;118:385–392. 16. West SK, Rosenthal FS, Bressler NM, Bressler SB, Munoz B, Fine SL, Taylor HR. Exposure to sunlight and other risk factors for age-related macular degeneration. Arch Ophthalmol 1989;107:875–879. 17. Efron N, Morgan PB, Woods CA, International Contact Lens Prescribing Survey Consortium. An international survey of daily disposable contact lens prescribing. Clin Exp Optom 2013;96:58–64. 18. Moore L, Ferreira JT. Ultraviolet (UV) transmittance characteristics of daily disposable and silicone hydrogel contact lenses. Cont Lens Anterior Eye 2006;29:115–122. 19. Andley UP, Malone JP, Townsend RR. Inhibition of lens photodamage by UV-absorbing contact lenses. Invest Ophthalmol Vis Sci. 2011;52:8330–8341. 20. Giblin FJ, Lin LR, Leverenz VR, Dang L. A class I (senofilcon A) soft contact lens prevents UVB-induced ocular effects, including cataract, in the rabbit in vivo. Invest Ophthalmol Vis Sci 2011;52:3667–3675.
!
2015 Informa Healthcare USA, Inc.
21. Youn HY, McCanna DJ, Sivak JG, Jones LW. In vitro ultraviolet-induced damage in human corneal, lens, and retinal pigment epithelial cells. Mol Vis 2011;17:237–246. 22. Giblin FJ, Lin L, Simpanya MF, Leverenz VR, Fick CE. A class I UV-blocking (senofilcon A) soft contact lens prevents UVA-induced yellow fluorescence and NADH loss in the rabbit lens nucleus in vivo. Exp Eye Res 2012; 102:17–27. 23. Ibrahim OM, Kojima T, Wakamatsu TH, Dogru M, Matsumoto Y, Ogawa Y, et al. Corneal and retinal effects of ultraviolet-B exposure in a soft contact lens mouse model. Invest Ophthalmol Vis Sci 2012;53:2403–2413. 24. Bergmanson JP, Pitts DG, Chu LW. The efficacy of a UV-blocking soft contact lens in protecting cornea against UV radiation. Acta Ophthalmol (Copenh) 1987;65: 279–286. 25. Lin DP, Chang HH, Yang LC, Huang TP, Liu HJ, Chang LS, et al. Assessment of ultraviolet B-blocking effects of weekly disposable contact lenses on corneal surface in a mouse model. Mol Vis 2013;19:1158–1168. 26. Contreras-Ruiz L, de la Fuente M, Parraga JE, Lopez-Garcia A, Fernandez I, Seijo B, et al. Intracellular trafficking of hyaluronic acid-chitosan oligomer-based nanoparticles in cultured human ocular surface cells. Mol Vis 2011;17:279–290. 27. Gorbet MB, Tanti NC, Jones L, Sheardown H. Corneal epithelial cell biocompatibility to silicone hydrogel and conventional hydrogel contact lens packaging solutions. Mol Vis 2010;16:272–282. 28. Reinoso R, Martin-Sanz R, Martino M, Mateo ME, Blanco-Salado R, Calonge M, Corell A. Topographical distribution and characterization of epithelial cells and intraepithelial lymphocytes in the human ocular mucosa. Mucosal Immunol 2012;5:455–467. 29. Freitas M, Lima JL, Fernandes E. Optical probes for detection and quantification of neutrophils’ oxidative burst: a review. Anal Chim Acta 2009;649:8–23. 30. Podskochy A, Gan L, Fagerholm P. Apoptosis in UV-exposed rabbit corneas. Cornea 2000;19:99–103. 31. Bilbao J, Salvador Gonzalez P, De Miguel Castrillo A. UV-B climatology in central spain. Int J Climatol 2008;28: 1933–1941. 32. Zigman S. Environmental near-UV radiation and cataracts. Optometry Vision Sci 1995;72:899–901. 33. O’Brien J, Wilson I, Orton T, Pognan F. Investigation of the alamar blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J Biochem 2000;267: 5421–5426. 34. van Engeland M, Nieland LJ, Ramaekers FC, Schutte B, Reutelingsperger CP. Annexin V-affinity assay: a review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry 1998;31:1–9. 35. Chen Y, Mehta G, Vasiliou V. Antioxidant defenses in the ocular surface. Ocul Surf 2009;7:176–185. 36. Cejkova J, Stipek S, Crkovska J, Ardan T. Changes of superoxide dismutase, catalase and glutathione peroxidase in the corneal epithelium after UVB rays: histochemical and biochemical study. Histol Histopathol 2000;15: 1043–1050.
ORIGINAL ARTICLE
In Vitro Model for Predicting the Protective Effect of Ultraviolet-Blocking Contact Lens in Human Corneal Epithelial Cells
Curr Eye Res Downloaded from informahealthcare.com by Nyu Medical Center on 07/25/15 For personal use only.
Antonio Abengo´zar-Vela1,2, Cristina Arroyo1, Roberto Reinoso1,2, Amalia Enrı´quez-de-Salamanca1,2, Alfredo Corell1,2 and Marı´a Jesu´s Gonza´lez-Garcı´a1,2 1
Instituto de Oftalmobiologı´a Aplicada (IOBA), Universidad de Valladolid, Valladolid, Spain and 2 CIBER-BBN, Valladolid, Spain
ABSTRACT Purpose: To develop an in vitro method to determine the protective effect of UV-blocking contact lenses (CLs) in human corneal epithelial (HCE) cells exposed to UV-B radiation. Materials and Methods: SV-40-transformed HCE cells were covered with non-UV-blocking CL, UV-blocking CL or not covered, and exposed to UV-B radiation. As control, HCE cells were covered with both types of CLs or not covered, but not exposed to UV-B radiation. Cell viability at 24, 48 and 72 h, after UV-B exposure and removing CLs, was determined by alamarBlueÕ assay. Percentage of live, dead and apoptotic cells was also assessed by flow cytometry after 24 h of UV-B exposure. Intracellular reactive oxygen species (ROS) production after 1 h of exposure was assessed using the dye H2DCF-DA. Results: Cell viability significantly decreased, apoptotic cells and intracellular ROS production significantly increased when UVB-exposed cells were covered with non-UV-blocking CL or not covered compared to non-irradiated cells. When cells were covered with UV-blocking CL, cell viability significantly increased and apoptotic cells and intracellular ROS production did not increase compared to exposed cells. Conclusions: UV-B radiation induces cell death by apoptosis, increases ROS production and decreases viable cells. UV-blocking CL is able to avoid these effects increasing cell viability and protecting HCE cells from apoptosis and ROS production induced by UV-B radiation. This in vitro model is an alternative to in vivo methods to determine the protective effect of UV-blocking ophthalmic biomaterials because it is a quicker, cheaper and reliable model that avoids the use of animals. Keywords: Apoptosis, contact lens, corneal epithelial cells, oxidative stress, ultraviolet light
INTRODUCTION
radiation that reaches the earth is mainly UV-A and -B. Even though the UV-B radiation represents less than 1% of the total radiation reaching the earth’s surface, it is a radiation of high energy level and it has the greatest potential ocular damage.2 The eye is one of the organs more susceptible to UV light damage. The human cornea has a high absorption coefficient for UV-B radiation,3 acting as UV-B filter that absorbs approximately up to 92% of
The ultraviolet radiation (UV) is a part of the electromagnetic spectrum that covers the wavelength range from 100 to 400 nm, and is further divided into the following bands: UV-vacuum from 100 to 200 nm; UV-C from 200 to 280 nm; UV-B from 280 to 315; and UV-A from 315 to 400 nm.1 UV-C radiation is fully absorbed in the upper atmosphere. Thus, the UV
Received 27 June 2014; revised 12 August 2014; accepted 18 August 2014; published online 6 October 2014 Correspondence: Marı´a Jesu´s Gonza´lez-Garcı´a, Instituto de Oftalmobiologı´a Aplicada (IOBA), Universidad de Valladolid, Paseo de Bele´n 17, Valladolid 47011, Spain. Tel: +34 983184756. E-mail: [email protected]
792
Curr Eye Res Downloaded from informahealthcare.com by Nyu Medical Center on 07/25/15 For personal use only.
In Vitro Model to Test UV-Blocking CLs 793 UV-B at 300 nm.4 In addition, corneal epithelium absorbs a substantial amount of the UV-B radiation that reaches the cornea, protecting the deeper corneal layers and eye structures from UV-B radiation.3,5 The result of this light absorption is the damage to the corneal epithelium by UV-B radiation. One of the most common acute effect of UV-B irradiation on the cornea is photokeratitis,6,7 characterized by both apoptosis (programmed cell death) and inflammation in corneal epithelium.8,9 Not only does acute UV exposure produce adverse ocular effects; but also chronic UV irradiation is one of the most important risk factor for ocular surface disorders. It is involved in solar keratopathy,10 pterygium,11,12 and ocular melanoma in conjunctiva,10,13 even in cataract formation14,15 and retina degeneration.16 Currently, silicone hydrogel (SiH) contact lenses (CLs) have quickly become the most popular type of soft CLs prescribed for daily wear representing 14.1% of daily disposable lens fits and 48.8% of reusable lens fits.17 In addition, today’s advanced techniques have allowed some manufacturers to incorporate different UV absorbing monomers into their SiHCLs. The different classes of UV-blocking CLs are labeled as Class I and Class II, according to the America National Standards Institute (ANSI). Class I UV filter blocks 90% of UV-A and 99% of UV-B, and Class II UV filter blocks 70% UV-A and 95% of UV-B, with 380 nm as upper limit of UV-B.18 Thus, there is a need to evaluate and compare the performance of these UV-blocking CLs. Some in vitro and in vivo studies have evidenced that UV-absorbing ophthalmic biomaterials have protective effects on cornea, lens and retina.19–23 However, only in vivo experiments have tested the protective effect of UV-blocking CLs on corneal cells.4,20,23–25 The aim of this study was to develop a suitable in vitro method as alternative to in vivo models to examine the protective effect of UV-blocking ophthalmic biomaterials, mainly CLs, when human corneal epithelial cells were exposed to UV-B radiation. Cellular viability, apoptosis and oxidative stress were analyzed.
MATERIALS AND METHODS Cell Line and Culture Medium SV-40-transformed human corneal epithelial (HCE) cells, a gift from Dr. Arto Urtti (University of Helsinki, Finland), were cultured in Dulbecco’s modified Eagle’s medium/nutrient mixed F12 (DMEM/F-12) L-glutamine supplemented with 15% fetal bovine serum (FBS), 10 ng/mL human epidermal growth factor (EGF), 0.5% dimethyl sulfoxide (DMSO), !
2015 Informa Healthcare USA, Inc.
0.1 mg/mL cholera toxin, 5 mg/mL insulin, 100 U/mL penicillin and 0.1 mg/mL streptomycin (all reagents from Sigma, St. Louis, MO), as previously described.26 The cells were maintained at 37 C with a 5% CO2 humidified atmosphere. When culture reached confluence, cells were harvested by trypsin and seeded into white 24-well plates with clear bottom (Perkin Elmer, Waltham, MA, USA) at a density of 60,000 cells per well; except for cell viability assay where cell density was 35,000 cells per well. Cells from passage 50 to 61 were used in this study.
Contact Lenses Two types of CLs were tested in this study: non-UVblocking SiHCL (Lotrafilcon B; AirOptixÕ ; CIBA Vision, Duluth, GA); and class I UV-blocking SiHCL (Senofilcon A; AcuvueÕ OasysÕ ; Johnson & Johnson Vision Care, Limerick, Ireland). Technical details of CLs studied are summarized in Table 1. UV spectra for both CLs have been previously described,18 showing that UV-B transmittance for Lotrafilcon-B and Senofilcon-A CLs was 62.89% and 0.03% of UV-B radiation, respectively, being UV-A transmittance for Lotrafilcon-B and Senofilcon-A CLs 80.71% and 18.35%, respectively.18
Exposure of Cells to UV-B Radiation HCE cells were seeded and growth until preconfluence. Then, medium was removed, and phenol redfree Roswell Park Memorial Institute (RPMI) 1640 Medium (Sigma, St. Louis, MO) was added to each well. Prior to sink CLs in the well, CLs were gently soaked for 10 s with RPMI in order to avoid decreasing viability of HCE cells due to CLs borate-buffered packaging solution.27 Then, CLs were totally immersed in culture medium and placed on top of cell monolayer with concave surface facing upwards, covering more than 94.5% of the well surface area (well diameter 14.8 mm). Cells in the presence/ absence of CLs were then exposed to UV-B light generated from 6 W UV lamps (Vilber Lourmat, Marne-la-Valle´e, France) with peak intensity at 312 nm (range 280–380 nm; UV-C cut-off filter TABLE 1 Nominal parameters of CLs used in this study. Material Brand Company Base curve (mm) Diameter (mm) Central thickness (mm) Power (D) Water content (%) Dk: oxygen permeability UV-Blocking
Lotrafilcon B Õ
AirOptix CIBA Vision 8.6 14.2 0.08 –1.00 33 110 No
Senofilcon A AcuvueÕ OasysÕ Johnson & Johnson 8.8 14.0 0.07 –1.00 38 103 Class I
794 A. Abengo´zar-Vela et al. by cells to resorufin, a fluorescence compound. This conversion occurs intracellularly by mitochondrial, microsomal and cytosolic oxidoreductases.28 AlamarBlueÕ can distinguish metabolically active cells because non-viable cells have lower innate metabolic activity and thus generate a proportionally lower signal than healthy cells.
Curr Eye Res Downloaded from informahealthcare.com by Nyu Medical Center on 07/25/15 For personal use only.
Cell Apoptosis
FIGURE 1 The emission spectrum of the UV-B lamp used for this in vitro model.
included; Figure 1) for 5 min and 30 s. The lamp was placed on a stand where it emits uniform irradiance of 680 mW/cm2 at 15 cm, according to the manufacturer instruction. UV-B radiant exposure used was 200 mJ/ cm2; calculated with the following formula: H = Et. Here H is the radiant exposure (J/cm2), E is the irradiance (W/cm2) and t is the exposure time (s). Control cells were not irradiated. After irradiation, all CLs were removed, phenol red-free RPMI was replaced with the supplemented DMEM/F12 culture medium used for cell growth, and cells were incubated at 37 C for specific times according to the experiment carried out. Experiments were repeated three times and were always performed with new CLs.
Cell Viability Cell viability was assessed by alamarBlueÕ test (Life Technologies, Carlsbad, CA) following the manufacturer‘s instructions with some modifications. Briefly, after UV-B exposure and removing all CLs and phenol red-free RPMI, cells were maintained for 24 h of recovery in supplemented DMEM/F12 cell culture medium. Then, medium was discarded and attached cells were washed with phosphate buffered saline (PBS; Life Technologies), and 1 mL of alamarBlueÕ solution (10 % v/v in supplemented culture medium) was added at 24, 48 and 72 h of culture. Cells were incubated with alamarBlueÕ for 4 h at 37 C. Finally, alamarBlueÕ solution from each sample was collected and fluorescence at 560 nmex/590 nmem was measured by UV/Vis spectrophotometry (SpectraMaxÕ M5, Molecular Devices, Sunnyvale, CA). Samples were assayed in duplicates. AlamarBlueÕ , or resazurin, is a non-toxic and nonfluorescent compound that is reduced continuously
Percentages of live, early and late apoptotic, and dead cells were assessed by flow cytometry. After UV-B exposure and 24 h of recovery in supplemented DMEM/F12 culture medium, cell supernatants were collected and centrifuged, and well-attached cells were trypsinized and collected. Subsequently, detached cells from wells and cells from supernatants were pooled. After that, cell suspensions were washed with 2 mL of Cell Wash Solution (BD Biosciences, San Jose, CA), centrifuged at 500 g for 5 min, and stained with an annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) commercial kit (BeckmanCoulter, Fullerton, CA) as described previously.29 The flow cytometry analysis was performed with a Cytomics FC 500 Cytometer (Beckman-Coulter) using 488 nm excitation with an argon-ion laser for FITC and PI. The data collected were analyzed using the Cytomics CXP software program (BeckmanCoulter). At least 15,000 HCE cells were analyzed for each condition. Controls included cross-reactivity of the fluorescence signals of each channel. Flow cytometry results were expressed as means ± SEM.
Reactive Oxygen Species Production UV-induced intracellular reactive oxygen species (ROS) production was determined using 20 ,70 -dihydrodichlorofluorescein diacetate (H2DCFDA; Sigma) probe. Non-fluorescent H2DCF-DA passively diffuses into cells, where intracellular esterases cleave it to the polar non-fluorescent compound H2DCF. Then, H2DCF is oxidized to highly fluorescent DCF by intracellular ROS. Cells were seeded in 24 well-plates in supplemented DMEM/F12 medium. When cells reached preconfluence, medium was discarded, serum-free DMEM/F12 medium was added and cells were maintained in it for 24 h. After that, medium was removed and cells were loaded with 10 mM H2DCF-DA solution for 30 min. Then, medium was replaced with phenol red-free RPMI, CLs were immersed in wells with the concave side facing up, and cells were exposed to 200 mJ/cm2 UV-B radiation. Control cells were placed under the UV lamp, but were not irradiated. CLs were then removed and cells were cultured for 1 h at 37 C. Finally, fluorescence was measured by UV/Vis spectrophotometry at 498 nmex/522 nmem.30 After fluorescence was read, medium was removed and well plates Current Eye Research
In Vitro Model to Test UV-Blocking CLs 795 with attached cells were stored at 80 C before determining the protein content of the cell lysates by using the bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Rockford, IL), according to manufacturer’s instructions. Samples were performed in duplicates and measurements were from three independent experiments. Data were expressed as mean fluorescence in arbitrary units (a.u.) normalized to total amount of protein (mg) per well.
Curr Eye Res Downloaded from informahealthcare.com by Nyu Medical Center on 07/25/15 For personal use only.
Statistical Analysis Statistical differences for cell viability assay between each CL group at each time point tested were assessed by two-way repeated-measures analyses of variance (ANOVA) followed by the Bonferroni post-hoc test, and differences between time points were assessed by one-way ANOVA followed by the Bonferroni post-hoc test. Cell apoptosis and ROS production data were analyzed using Student’s unpaired t-test for comparing UVB-unexposed cells with exposed cells and statistical differences between groups were assessed using one-way ANOVA followed by the Bonferroni post-hoc test. p Values below 0.05 were considered significant.
RESULTS Effect of Contact Lens with UV Filter on Cell Viability Viability of HCE cells over time after exposure to UV-B radiation in presence of either UV-blocking CL or non-UV-blocking CL is shown in Figure 2. Any of
FIGURE 2 Effect of UV-blocking contact lens (CL) on UVB-exposed HCE cells viability at 24, 48 and 72 h after UV irradiation. Data are expressed as fluorescence intensity at 590 nm in arbitrary units (a.u.). Each bar represents the mean ± SEM from three independent experiments. *Denotes significantly different compared to corresponding UVB-unexposed cell group: *p50.05, ***p50.001. +Denotes significantly different comparing non-UV-blocking CL group versus UV-blocking CL group for UV-B exposed cells: +++ p50.001. #Denotes significantly different compared to 24 h data: #p50.05, ##p50.01, ###p50.001. !
2015 Informa Healthcare USA, Inc.
the two types of SiHCLs altered the viability of UV-unexposed control cells at any time point measured. When HCE cells in the absence of CL or in the presence of the non-UVB-blocking CL were irradiated with 200 mJ/cm2, their viability dramatically decreased after 24, 48 and 72 h of recovery, compared to UV-unexposed cells (p50.001). In contrast, when cells were covered with UV-blocking CL and exposed to UV-B radiation, viability increased over time with a significant difference at 48 h (p50.05), compared to its control. Cell viability values for exposed cells covered with UV-blocking CL were significantly higher than those found for exposed cells covered with non-UV-blocking CL at all time points (p50.001).
Effect of Contact Lens with UV Filter on Apoptosis To further confirm the protective effect of a UV-blocking filter in the CL against UV-B exposure, apoptosis assay by flow cytometry was carried out in HCE cells after 24 h of UV exposure (Figure 3). Percentages of live, early and late apoptotic, and dead cells were 81.95 ± 3.86%, 12.02 ± 2.37%, 5.53 ± 1.46% and 0.35 ± 0.11%, respectively, for UVB-unexposed cells in the absence of CL. There were not differences in these percentages with respect to UVB-unexposed cells with both CLs. When cells without CL were exposed to UV-B, percentage of live cells, compared to unexposed cells, significantly decreased to 48.07 ± 5.42% (p50.001); whereas cells in early and late apoptosis, and dead cells increased to 21.87 ± 3.37% (p50.05), 28.70 ± 2.58% (p50.001) and 1.22 ± 0.08% (p50.001), respectively. We did not find any significant differences between exposed cells without CL and with non-UV-blocking CL. However, when HCE cells were covered with UV-blocking CL and exposed to UV radiation, percentages of live, early and late apoptotic, and dead cells were 72.35 ± 4.41% (p50.05), 16.90 ± 2.72%, 10.15 ± 1.82% (p50.001) and 0.42 ± 0.07% (p50.01), respectively, compared to exposed cells without CL. Same statistical differences were found when exposed HCE cells covered with UV-blocking CL were also compared to exposed HCE cells covered with nonUV-blocking CL.
Effect of Contact Lens with UV Filter on Oxidative Stress Figure 4 shows intracellular ROS production induced by UV-B radiation for each group. Fluorescence intensity data, expressed in arbitrary units, were normalized to total protein levels. The presence of CLs (non-UV-blocking or UV-blocking CLs) did not affect ROS production in UVB-unexposed cells. The UV-B radiation significantly increased the production
Curr Eye Res Downloaded from informahealthcare.com by Nyu Medical Center on 07/25/15 For personal use only.
796 A. Abengo´zar-Vela et al.
FIGURE 3 Effect of UV-blocking contact lens (CL) on UVB-exposed HCE cells apoptosis. (A) Representative flow cytometric dot plots of cell apoptosis analysis. Dot plot of annexin V-FITC versus propidium iodide shows levels of live cells (region H3), early (region H4) and late (region H2) apoptotic cells, and dead cells (region H1). (B) Bar graphs representing the percentage of gated cells in each region. Each bar represents the mean percent of cells ± SEM from three independent experiments. Differences were significant at *p50.05, **p50.01, ***p50.001, compared to unexposed cell groups; +p50.05, ++p50.01, +++p50.001, compared to exposed cells without CL; and #p50.05, ##p50.01, compared to exposed cells with non-UV-blocking CL.
Current Eye Research
Curr Eye Res Downloaded from informahealthcare.com by Nyu Medical Center on 07/25/15 For personal use only.
In Vitro Model to Test UV-Blocking CLs 797
FIGURE 4 Effect of UV-blocking contact lens (CL) on UVB-exposed HCE cells reactive oxygen species (ROS) production. Data are expressed as relative fluorescence value in arbitrary units (a.u.) normalized to total amount of protein in micrograms (mg). Each bar represents mean ± SEM from three independent experiments. Differences were significant at *p50.05 compared to corresponding UVB-unexposed cell group.
of intracellular ROS (p50.05) in both cells without CL and cells with non-UV-blocking CL after 1 h of incubation, compared to UV-unexposed cells. ROS production was not significantly increased in UV-B irradiated cells covered with UV-blocking CL compared to UV-unexposed cells.
DISCUSSION It is well known that both acute and chronic UV-B exposure can lead to several ocular surface diseases like photokeratitis6 or pterygium.12 Previous reports have shown that UV-B radiation causes both DNA and mitochondrial degradations decreasing ocular surface cell viability. Moreover, it is increasingly recognized that UV-B radiation is also involved in corneal cell death, increasing the number of apoptotic cells and producing nuclear fragmentation and loss of tissue cohesion.21,31 Indeed, the first clinical signs of photokeratitis are due to damage or loss of epithelial cells by shedding and apoptosis.8 This fact results in loss of the stratified epithelium to the basement membrane exposing nerve fibers leading to acute pain. Nowadays CLs include UV-blocking agents that minimize the ocular damage by exposure to UV light. Published studies on the protection of these CLs are in vivo studies that require a large investment in time and money and can lead to ethical problems associated with the use of animals. !
2015 Informa Healthcare USA, Inc.
Therefore, we proposed a cheaper, quicker and reliable alternative method to replace animal experiments. Our data demonstrate that UV-blocking CL protects corneal epithelial cells from apoptosis that leads to increase of cell viability after 72 h of recovery when cells are exposed to a relatively high dose of UV-B. The irradiance of the UV-B lamp used in this study was 680 mW/cm2, which is three times higher than the daily maximum irradiance in Valladolid (Spain) occurring in July, 202 mW/cm2.32 In addition, the UV-B dose used in this study is similar to average value of solar UV-B radiation reaching the human cornea, that is 0.187 J/cm2 for 1 h exposure.33 Thus, the irradiance of the lamp used in this experiment is environmentally and energetically relevant and the UV-B radiation dose used in this work is equivalent to 1 h exposure time to sunlight condensed into 5 min and 30 s. Our viability analysis by alamarBlueÕ assay demonstrated that UV-exposed cells covered with UV-blocking CL maintain their viability after 72 h of incubation. Our results showed that UV radiation decreased alamarBlueÕ fluorescence readings from exposed cells in the absence of CL or presence of nonUV-blocking CL after 24 h of culture. Moreover, there was no cellular recovery during the entire culture period of cells exposed to UV-B radiation with nonUV-blocking CL. This fact indicates that UV-B radiation affects metabolic activity due to the loss of appropriate cytoplasmic milieu, and non-UV-blocking CL did not protect cytoplasmic milieu from UV-B radiation. However, when HCE cells with UV-blocking CL were irradiated, alamarBlueÕ fluorescence readings increased after 72 h of incubation. Thus, UV-blocking CL protects cytoplasmic milieu from UV-B radiation. Similar to our own findings, Lin et al. found in an in vivo model of UV-B damage that corneal epithelial cells maintained normal cellular properties when corneas were covered with UV-blocking CL for 7 days of exposure.25 They demonstrated that UV radiation reduced corneal thickness in the presence or absence of nonUV-blocking CL along with a loss of p63+ basal cells, CK-5+ epithelial cells and Ki-67+ proliferative basal cells. However, UV-blocking CL was able to keep corneal thickness and all epithelial cell population maintained their cellular properties after UV irradiation. In addition, Giblin et al. found a swelling and haziness of the corneal epithelium along with severe loss of corneal epithelial cells in rabbit eyes irradiated with UV-B for 5 days. These adverse effects were reverted when rabbit corneas were covered with UV-blocking CL.20 Collectively, these results suggest that UV-blocking CL may maintain corneal epithelial cells healthy when UV-B radiation reaches cornea. We found that UV-B radiation induced apoptosis in corneal epithelial cells, as other in vitro and in vivo
Curr Eye Res Downloaded from informahealthcare.com by Nyu Medical Center on 07/25/15 For personal use only.
798 A. Abengo´zar-Vela et al. assays have demonstrated previously.21,30 In this study, apoptosis was assessed by flow cytometry using annexin V-FITC and PI. This double staining allowed us to distinguish not only between live, apoptotic, and dead cells, but also between early and late apoptotic stages.34 We found that the percentage of apoptotic cells increased when HCE cells were exposed to UV-B in the absence of CL or presence of non-UV-blocking CL. Moreover, there was a higher increase of late than early apoptotic cells after UV-B radiation. It indicates that early apoptotic epithelial cells preserve their plasma membrane integrity to retain the potentially harmful cellular contents inside while late apoptotic epithelial cells release cellular content into the extracellular space. Thus, UV-B radiation promotes a leakage of intracellular molecules that may exacerbate the inflammatory response induced by UV radiation. On the other hand, UV-blocking CL was able to reduce percentage of apoptotic and dead cells. Percentage of HCE cells in late apoptosis was lower than the percentage of cells in early apoptosis when HCE cells where covered with UV-blocking CL and exposed to UV-B radiation. In addition, live cells found in irradiated HCE cells in the absence of CL or presence of non-UV-blocking CL were not viable as alamarBlueÕ assay pointed out previously. However, when irradiated HCE cells were covered with UV-blocking CL, a high cell percentage was alive and they were totally viable after 72 h incubation. It indicates that UVblocking CL can prevent morphological changes of the cell membrane and DNA fragmentation by UV-B radiation keeping cells healthy. UV-blocking CL also prevents other apoptotic events. Chandler et al. reported that UV-B radiation increased caspase-3 activity and TUNEL positive cells in rabbit corneas, even when they were covered with non-UV-blocking CL.4 The use of UV-blocking CL decreased apoptosis in the cornea as evidenced by reduction in caspase-3 activity and in TUNEL-positive cells.4 All these findings demonstrate that UV-blocking CL can prevent apoptosis at different stages in UV-irradiated cornea, however more studies are necessary to know if CL with UV filter can protect human cornea from apoptosis. The eye and particularly cornea and conjunctiva are constantly under oxidative stress due to their continuous exposure to light and oxygen. Oxidative stress is caused by the generation of uncontrolled ROS like superoxide (O 2 ), hydroxyl radical (HO ) or hydrogen peroxide (H2O2). Indeed, UV-B radiation is one of the major ROS inducer on the ocular surface. Although the ocular surface have antioxidative enzymes that protect against ROS,35 UV-B can also inactivate the antioxidant enzyme system in the cornea,36 and may exacerbate ROS-induced ocular surface diseases. We have found that UV-blocking CL tends to decrease UV-induced ROS production in HCE cell line. Our in vitro data agree with Ibrahim et al. who investigated UV-induced oxidative damage
markers in a soft contact lens mouse model.23 They found an increase of hexanoyl-lysine, 4-hydroxynonenal and 8-hydroxy-20 -deoxyguanosine, advanced lipid and DNA oxidation products, in the mouse cornea. These levels of oxidative stress markers were reduced by UV-blocking CL, but not by CL without UV-blocking filter. That indicates radiation-induced ROS react with lipids and DNA in cornea and UV-blocking CL can protect corneal epithelial cells from oxidative damage. This in vitro model aims to be a valid alternative to current in vivo models for the study of UV-blocking CLs. Data found in this in vitro model are in agreement with previous studies conducted in different in vivo models. Our in vitro model can be easily performed and requires a minimum investment of time and money. In addition, this model eliminates the use of animals in experimentation fulfilling the first premise of the three Rs of humane animal experimentation, which are: replace, reduce and refine. Therefore, it can be concluded that this in vitro model could be a valid alternative to in vivo models. This study has some limitations that must be considered. Firstly, we only tested UV-blocking SiHCL. However, some new UV-blocking hydrogel CLs have been released nowadays to the market. Secondly, this test was performed using a monolayer rather than a stratified structure. And finally the protective effect of UV-blocking CL has only been tested in a corneal epithelial cell line but fitted CLs cover and protect not only corneal epithelial cells but also other cell types, such as conjunctival cells or keratocytes. Thus, more studies are necessary to know whether UV-blocking CL may protect other cell types from UV-B irradiation. In conclusion, this study reveals that a CL with a UV filter can minimize UV-induced damage in HCE cells compared to non-UV filter CLs. Our in vitro data agree with previous in vivo studies about the effect of UV-blocking CL on corneal cells regarding viability, apoptosis and ROS production. Hence, this in vitro approach may be useful in order to test new ophthalmic materials, mainly new UV-blocking CLs, as alternative method quicker, cheaper and ethically less controversial than in vivo studies.
DECLARATION OF INTEREST The authors declare that they have no conflict of interest. All CLs were kindly provided by both manufacturers.
REFERENCES 1. Bergmanson JP, Sheldon TM. Ultraviolet radiation revisited. CLAO J 1997;23:196–204. Current Eye Research
Curr Eye Res Downloaded from informahealthcare.com by Nyu Medical Center on 07/25/15 For personal use only.
In Vitro Model to Test UV-Blocking CLs 799 2. Chaney EK, Sliney DH. Re-evaluation of the ultraviolet hazard action spectrum – the impact of spectral bandwidth. Health Phys 2005;89:322–332. 3. Kolozsvari L, Nogradi A, Hopp B, Bor Z. UV absorbance of the human cornea in the 240- to 400-nm range. Invest Ophthalmol Vis Sci 2002;43:2165–2168. 4. Chandler HL, Reuter KS, Sinnott LT, Nichols JJ. Prevention of UV-induced damage to the anterior segment using class I UV-absorbing hydrogel contact lenses. Invest Ophthalmol Vis Sci 2010;51:172–178. 5. Podskochy A. Protective role of corneal epithelium against ultraviolet radiation damage. Acta Ophthalmol Scand 2004;82:714–717. 6. Blumthaler M, Ambach W, Daxecker F. On the threshold radiant exposure for keratitis solaris. Invest Ophthalmol Vis Sci 1987;28:1713–1716. 7. Ringvold A. Cornea and ultraviolet radiation. Acta Ophthalmol (Copenh) 1980;58:63–68. 8. Cullen AP. Photokeratitis and other phototoxic effects on the cornea and conjunctiva. Int J Toxicol 2002;21:455–464. 9. Young AR. Acute effects of UVR on human eyes and skin. Prog Biophys Mol Biol 2006;92:80–85. 10. Taylor H. The biological effects of UV-B on the eye. Photochem Photobiol 1989;50:489–492. 11. Coroneo MT. Pterygium as an early indicator of ultraviolet insolation: a hypothesis. Br J Ophthalmol 1993;77:734–739. 12. Di Girolamo N, Kumar RK, Coroneo MT, Wakefield D. UVB-mediated induction of interleukin-6 and -8 in pterygia and cultured human pterygium epithelial cells. Invest Ophthalmol Vis Sci 2002;43:3430–3437. 13. Taylor HR, West SK, Rosenthal FS, Munoz B, Newland HS, Emmett EA. Corneal changes associated with chronic UV irradiation. Arch Ophthalmol 1989;107:1481–1484. 14. Bergmanson JP, Soderberg PG. The significance of ultraviolet radiation for eye diseases. A review with comments on the efficacy of UV-blocking contact lenses. Ophthalmic Physiol Opt 1995;15:83–91. 15. Delcourt C, Carriere I, Ponton-Sanchez A, Lacroux A, Covacho MJ, Papoz L. Light exposure and the risk of cortical, nuclear, and posterior subcapsular cataracts: the pathologies oculaires liees a l’age (POLA) study. Arch Ophthalmol 2000;118:385–392. 16. West SK, Rosenthal FS, Bressler NM, Bressler SB, Munoz B, Fine SL, Taylor HR. Exposure to sunlight and other risk factors for age-related macular degeneration. Arch Ophthalmol 1989;107:875–879. 17. Efron N, Morgan PB, Woods CA, International Contact Lens Prescribing Survey Consortium. An international survey of daily disposable contact lens prescribing. Clin Exp Optom 2013;96:58–64. 18. Moore L, Ferreira JT. Ultraviolet (UV) transmittance characteristics of daily disposable and silicone hydrogel contact lenses. Cont Lens Anterior Eye 2006;29:115–122. 19. Andley UP, Malone JP, Townsend RR. Inhibition of lens photodamage by UV-absorbing contact lenses. Invest Ophthalmol Vis Sci. 2011;52:8330–8341. 20. Giblin FJ, Lin LR, Leverenz VR, Dang L. A class I (senofilcon A) soft contact lens prevents UVB-induced ocular effects, including cataract, in the rabbit in vivo. Invest Ophthalmol Vis Sci 2011;52:3667–3675.
!
2015 Informa Healthcare USA, Inc.
21. Youn HY, McCanna DJ, Sivak JG, Jones LW. In vitro ultraviolet-induced damage in human corneal, lens, and retinal pigment epithelial cells. Mol Vis 2011;17:237–246. 22. Giblin FJ, Lin L, Simpanya MF, Leverenz VR, Fick CE. A class I UV-blocking (senofilcon A) soft contact lens prevents UVA-induced yellow fluorescence and NADH loss in the rabbit lens nucleus in vivo. Exp Eye Res 2012; 102:17–27. 23. Ibrahim OM, Kojima T, Wakamatsu TH, Dogru M, Matsumoto Y, Ogawa Y, et al. Corneal and retinal effects of ultraviolet-B exposure in a soft contact lens mouse model. Invest Ophthalmol Vis Sci 2012;53:2403–2413. 24. Bergmanson JP, Pitts DG, Chu LW. The efficacy of a UV-blocking soft contact lens in protecting cornea against UV radiation. Acta Ophthalmol (Copenh) 1987;65: 279–286. 25. Lin DP, Chang HH, Yang LC, Huang TP, Liu HJ, Chang LS, et al. Assessment of ultraviolet B-blocking effects of weekly disposable contact lenses on corneal surface in a mouse model. Mol Vis 2013;19:1158–1168. 26. Contreras-Ruiz L, de la Fuente M, Parraga JE, Lopez-Garcia A, Fernandez I, Seijo B, et al. Intracellular trafficking of hyaluronic acid-chitosan oligomer-based nanoparticles in cultured human ocular surface cells. Mol Vis 2011;17:279–290. 27. Gorbet MB, Tanti NC, Jones L, Sheardown H. Corneal epithelial cell biocompatibility to silicone hydrogel and conventional hydrogel contact lens packaging solutions. Mol Vis 2010;16:272–282. 28. Reinoso R, Martin-Sanz R, Martino M, Mateo ME, Blanco-Salado R, Calonge M, Corell A. Topographical distribution and characterization of epithelial cells and intraepithelial lymphocytes in the human ocular mucosa. Mucosal Immunol 2012;5:455–467. 29. Freitas M, Lima JL, Fernandes E. Optical probes for detection and quantification of neutrophils’ oxidative burst: a review. Anal Chim Acta 2009;649:8–23. 30. Podskochy A, Gan L, Fagerholm P. Apoptosis in UV-exposed rabbit corneas. Cornea 2000;19:99–103. 31. Bilbao J, Salvador Gonzalez P, De Miguel Castrillo A. UV-B climatology in central spain. Int J Climatol 2008;28: 1933–1941. 32. Zigman S. Environmental near-UV radiation and cataracts. Optometry Vision Sci 1995;72:899–901. 33. O’Brien J, Wilson I, Orton T, Pognan F. Investigation of the alamar blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J Biochem 2000;267: 5421–5426. 34. van Engeland M, Nieland LJ, Ramaekers FC, Schutte B, Reutelingsperger CP. Annexin V-affinity assay: a review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry 1998;31:1–9. 35. Chen Y, Mehta G, Vasiliou V. Antioxidant defenses in the ocular surface. Ocul Surf 2009;7:176–185. 36. Cejkova J, Stipek S, Crkovska J, Ardan T. Changes of superoxide dismutase, catalase and glutathione peroxidase in the corneal epithelium after UVB rays: histochemical and biochemical study. Histol Histopathol 2000;15: 1043–1050.
