Molecular Vision 2012; 18:1189-1196 <http://www.molvis.org/molvis/v18/a125>
`Received 1 March 2012 | Accepted 3 May 2012 | Published 6 May 2012
`
`© 2012 Molecular Vision
`
`The preservative polyquaternium-1 increases cytoxicity and NF-
`kappaB linked inflammation in human corneal epithelial cells
`
`Tuomas Paimela,1 Tuomas Ryhänen,1 Anu Kauppinen,1 Liisa Marttila,2 Antero Salminen,3,4
`Kai Kaarniranta1,2
`
`(The first two authors contributed equally to the work)
`
`1Department of Ophthalmology, Institute of Clinical Medicine, University of Eastern Finland, Kuopio, Finland; 2Department of
`Ophthalmology, Kuopio University Hospital, Kuopio, Finland; 3Department of Neurology, Institute of Clinical Medicine, University
`of Eastern Finland, Kuopio, Finland; 4Department of Neurology, Kuopio University Hospital, Kuopio, Finland
`
`Purpose: In numerous clinical and experimental studies, preservatives present in eye drops have had detrimental effects
`on ocular epithelial cells. The aim of this study was to compare the cytotoxic and inflammatory effects of the preservative
`polyquaternium-1 (PQ-1) containing Travatan (travoprost 0.004%) and Systane Ultra eye drops with benzalkonium
`chloride (BAK) alone or BAK-preserved Xalatan (0.005% latanoprost) eye drops in HCE-2 human corneal epithelial cell
`culture.
`Methods: HCE-2 cells were exposed to the commercial eye drops Travatan, Systane Ultra, Xalatan, and the preservative
`BAK. Cell viability was determined using colorimetric MTT (3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium
`bromide) assay and by release of lactate dehydrogenase (LDH). Induction of apoptosis was measured with a using a
`colorimetric caspase-3 assay kit. DNA binding of the nuclear factor kappa B (NF-κB) transcription factor, and productions
`of the proinflammatory cytokines, interleukins IL-6 and IL-8, were determined using an enzyme-linked immunosorbent
`assay (ELISA) method.
`Results: Cell viability, as measured by the MTT assay, declined by up to 50% after exposure to Travatan or Systane Ultra
`solutions which contain 0.001% PQ-1. BAK at 0.02% rather than at 0.001% concentration evoked total cell death signs
`on HCE-2 cells. In addition, cell membrane permeability, as measured by LDH release, was elevated by sixfold with
`Travatan and by a maximum threefold with Systane Ultra. Interestingly, Travatan and Systane Ultra activated NF-κB and
`elevated the secretion of inflammation markers IL-6 by 3 to eightfold and IL-8 by 1.5 to 3.5 fold, respectively, as analyzed
`with ELISA.
`Conclusions: Eye drops containing PQ-1 evoke cytotoxicity and enhance the NF-κB driven inflammation reaction in
`cultured HCE-2 cells. Our results indicate that these harmful effects of ocular solutions preserved with PQ-1 should be
`further evaluated in vitro and in vivo.
`
`Benzalkonium chloride (BAK) is the most commonly
`used preservative in ophthalmic drops. BAK has cytotoxic
`effects and it has been shown to induce inflammation on the
`ocular surface cells in numerous in vitro and in vivo models
`[1-11]. Since the clinical treatment of glaucoma or dry eye
`syndrome usually requires a long-term topical drug therapy,
`ocular side effects may be potentiated by the use of preserved
`ocular
`drops
`[4,6,7,12]. Polyquaternium-1
`0.001%
`(Polyquad®, PQ-1) is a detergent-type preservative derived
`from BAK. PQ-1 was formulated in the mid of 1980s by Alcon
`as a preservative for contact lens storage solutions. Nowadays,
`it is being increasingly used as a preservative in ophthalmic
`drops for glaucoma and artificial tear solutions. Recent
`findings reveal that PQ-1 has detrimental effects on cell
`
`Correspondence to: Kai Kaarniranta, Department of Ophthalmology,
`University of Eastern Finland, P.O. Box 1627, 70211 Kuopio,
`Finland; Phone: +358-17-172485; FAX: +358-17-172486; email:
`kai.kaarniranta@uef.fi
`
`membrane integrity and it induces cytotoxicity in ocular
`surface cells [13,14]. Cell membrane damage may activate
`inflammation and cytotoxicity via Toll-like receptors (TLRs)
`that are a class of proteins playing a key role in the innate
`immune system [15]. TLRs are classical inducers of nuclear
`factor kappa B (NF-κB) transcription factor that is a
`ubiquitous inducible transcription factor, and the master
`regulator of acute and chronic immune responses, cellular
`proliferation and cell death. The NF-κB protein complex is
`maintained in the cytoplasm in an inactive state by the
`presence of inhibitory kappa proteins (IκBs). In response to
`various stress stimuli, IκB kinase (IKK) can phosphorylate
`IκB proteins which, in turn, leads to NF-κB activation through
`the formation of a heterodimer of p50 and p65 NF-κB subunits
`which is then translocated to the nucleus where it triggers the
`activation of many inflammatory genes, such as interleukins
`IL-6 and IL-8 [15].
`This study explored the hypothesis that PQ-1 might have
`some detrimental effects on ocular surface cells. Therefore,
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`Molecular Vision 2012; 18:1189-1196 <http://www.molvis.org/molvis/v18/a125>
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`© 2012 Molecular Vision
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`cytotoxicity, cellular permeability and the inflammatory
`effects of commercial eye drops containing PQ-1 0.001%
`(Travatan and Systane Ultra) or an eye drop with BAK 0.02%
`(Xalatan) and pure BAK 0.001% or 0.02% were analyzed
`using HCE-2 human corneal epithelial cell cultures.
`
`METHODS
`Cell culturing and treatments: Cells used in this study were
`human corneal epithelial cells (HCE-2) obtained from
`American Type Culture Collection (ATCC, Manassas, VA).
`The cells were grown to confluency on 12-well plates
`(Cellstar®; Greiner Bio-One GmbH, Frickenhausen Germany)
`in a standard cell culture incubator (humidified CO2 10%
`athmosphere and 37 °C). Keratinocyte- Serum Free Medium
`(SFM, with bovine pituitary extract and epidermal growth
`factor, cat. no. 17005–042; Life Technologies, Invitrogen,
`GIBCO®, Paisley, UK) containing insulin 0.005 mg/ml (cat.
`no. I-6634; Sigma-Aldrich, Steinheim, Germany), fetal
`bovine serum 10% (FBS, cat. no. CH30160.03; Thermo
`Scientific, Hyclone, Logan, UT) and penicillin 100U/ml +
`streptomycin 100 µg/ml (cat. no. DE17–602E; Lonza, Basel,
`Switzerland) was used as the culture medium. Fresh medium
`was supplied to the cells every other day and the cells were
`subcultured twice a week using 0.25% trypsin-EDTA (cat. no.
`25200056; Life Technologies, Invitrogen) to detach the cells
`from plates.
`The cells were exposed to the treatments (see below) for
`5, 15, and 30 min, and then kept in the cell culture medium
`for 24 h, except for NF-κB activity test for 6 h, on 12 well
`plates before being analyzed. For every well 100,000 cells
`were seeded and cultured for 48 h before of exposures. Culture
`medium volume was 1 ml per dish, while for exposures used
`volume was 0.5 ml per dish. The cells were washed once with
`keratinocyte-SFM medium (without any supplements) before
`and after treatment to prevent any extra protein precipitation
`caused by BAK. The treatments were: 0-control (normal cell
`culture medium), Travatan
`(40 µg/ml
`travoprost,
`polyquaternium-1/polidronium
`chloride
`0.001%
`as
`preservative; Alcon, Hünenberg, Switzerland), Systane Ultra
`(artificial tear drops, Polyquaternium-1/polidronium chloride
`0.001% as preservative; Alcon), BAK 0.001% and 0.02% v/
`v aqueous solution (FeF Chemicals A/S, Køge, Denmark), and
`Xalatan (0.005% latanoprost, BAK 0.02% as preservative;
`Pfizer, New York City, NY).
`Cell viability:
`MTT assay—The cytotoxicity of exposure was
`measured with MTT-assay [16]. Color of MTT tetrazole salt
`was measured with a spectrophotometer at the wavelength of
`570 nm. Briefly, fresh MTT solution (10 mg/ml in 1× PBS)
`was added (1:20 volume of medium) and the cells were
`incubated for 1.5 h. The cells were lysed and purple formazan
`dissolved into the solution by overnight incubation with MTT-
`lysis buffer (20% SDS, 50% N,N-dimethylformamide, 2%
`
`acetic acid, 25mM HCl; the volume of medium + volume of
`MTT-salt solution).
`LDH assay—The permeability of cellular membranes
`following the exposures was determined by measuring the
`amount of released lactate dehydrogenase (LDH) enzyme
`from HCE-2 cells. The commercial CytoTox 96® -kit (cat.
`no. G1780; Promega, Fitchburg, WI) was used according to
`the manufacturer’s instructions. Maximum LDH release of
`HCE-2 cells was determined by lysing HCE-2 cells for 45 min
`(lysis buffer provided within the assay), and subsequently
`measuring the LDH from the culture medium. Absorbance
`values after the colorimetric reaction were measured at the
`wavelength of 490 nm with a reference wavelength of 655 a
`BIO-RAD Model 550 microplate reader (BIO-RAD,
`Hercules, CA).
`Caspase-3—The levels of an apoptosis marker caspase-3
`(active form) were measured from cell lysates using a
`colorimetric assay kit (cat. no. CASP-3-C; Sigma-Aldrich).
`Caspase-3 hydrolyses the peptide substrate Ac-DEVD-pNA
`(acetyl-Asp-Glu-Val-Asp p-nitroanilide) releasing pNA (p-
`nitroaniline) which can be measured at the wavelength of 405
`nm. The assay was performed according to the instructions of
`the manufacturer. Caspase-3 (Product Code C5974; Sigma)
`was used as a positive control, and the Assay Buffer provided
`with the kit served as a negative control.The absorbance
`values were measured using a BIO-RAD Model 550
`microplate reader (BIO-RAD).
`Inflammation:
`IL-6 and IL-8 assays—The concentrations (pg/ml) of
`IL-6 were measured from cell culture medium samples in
`duplicates by BD OptEIA™ Human IL-6 ELISA Set (cat. no.
`555220; BD Biosciences, San Diego, CA). The assay was
`performed according to the instructions of manufacturer. For
`determining the IL-8 concentrations (pg/ml), the BD
`OptEIA™ Human IL-8 ELISA Set (cat. no. 555244; BD
`Biosciences) was used. The absorbance values after the
`colorimetric reaction were measured at the wavelength of 450
`nm with a reference wavelength of 655 nm using a BIO-RAD
`Model 550 microplate reader (BIO-RAD).
`NF-κB assay—NF-κB p65 ELISA kit (cat. no. EKS-446)
`was obtained from Enzo Life Sciences (Lausen, Switzerland)
`and used to measure p65 subunit after the binding to DNA.
`The cells were lysed in 25% glycerol, 0.42 M NaCl, 1.5 mM
`MgCl2, 0.2 mM EDTA, 20 mM HEPES before analyses, and
`the assay was performed according to the manufacturer’s
`instructions using 10 μg of protein per well. The luminescence
`signal was measured using VICTOR™ 1420 multilabel
`counter (PerkinElmer/Wallac, Turku, Finland).
`Statistical analysis: Statistical analyses were conducted with
`GraphPad Prism (Graphpad Software, San Diego, CA).
`Differences between groups were analyzed with the one-way
`ANOVA test followed by Dunnett’s post hoc tests. P-values
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`© 2012 Molecular Vision
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`Figure 1. Level of cytotoxicity in HCE-2 cells analyzed by MTT assay. Columns represent the viability of cells (mean±SD. The viability of
`control cells is set as 100%. One-Way ANOVA, followed by Dunnett’s post hoc test, evaluated the statistical differences (n=6, *0.01<p≤0.05,
`**0.001<p≤0.01, ***p≤0.001, ns=not significant). Experiments were repeated three times.
`
`Figure 2. Released amount of lactate dehydrogenase (LDH) from HCE-2 cells. Columns represent the mortality (mean±SD) of cells, total
`cellular death being 100%. One-Way ANOVA, followed by Dunnett’s post hoc test, evaluated the statistical differences (n=6, *0.01<p≤0.05,
`**0.001<p≤0.01, ***p≤0.001, ns=not significant). Experiments were repeated three times.
`
`below 0.05 were considered significant. There were six
`parallel samples in every analysis.
`
`RESULTS
`Cellular viability: MTT analysis revealed the extensive
`toxicity associated with BAK 0.02% containing Xalatan and
`BAK 0.02% (Figure 1). Both solutions evoked total cell death
`even after 5 min of exposure (followed by 24 h recovery in
`normal medium). At the same time point, the viability of cells
`exposed to Travatan, Systane Ultra and BAK 0.001% was near
`to the control. At later time points (15 and 30 min exposure +
`24 h recovery) Travatan, Systane Ultra, and BAK 0.001%
`exposures reduced cellular viability in a time dependent
`manner. Based on the MTT analysis results and the extensive
`protein precipitation caused by the higher concentration of
`
`BAK (0.02%) and Xalatan, they were excluded from further
`experiments.
`Cellular permeability analysis, measured via the release
`of LDH, indicated that exposure to Travatan (preserved with
`PQ-1) was the most harmful (Figure 2). After 5 min exposure
`(followed by 24 h recovery in normal medium) the mortality
`was approx. 25%, which increased at later time points (15 and
`30 min) up to 50%. In addition Systane Ultra and BAK
`(0.001%) were slightly toxic to HCE-2 cells as reflected in the
`decline in cellular viability from 10% to 20%. However,
`Systane Ultra seemed to be slightly more toxic at the 30 min
`time point.
`Caspase-3 levels were elevated after 5 min of exposure
`to Travatan (followed by 24 h recovery in normal medium)
`when compared to control cells (Figure 3). Moreover, the
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`Figure 3. Active caspase-3 analysis in HCE-2 cells. Columns represent the proportion of caspase-3 from positive control which was set to be
`100% (mean±SD). One-Way ANOVA, followed by Dunnett’s post hoc test, evaluated the statistical differences (n=6, *0.01<p≤0.05,
`**0.001<p≤0.01, ***p≤0.001, ns=not significant). Experiments were repeated three times.
`
`Figure 4. Interleukin-6 secretion from HCE-2 cells analyzed by ELISA. Columns represent the amount of IL-6 pg/ml (mean±SD). One-Way
`ANOVA, followed by Dunnett’s post hoc test, evaluated the statistical differences (n=6, *0.01<p≤0.05, **0.001<p≤0.01, ***p≤0.001, ns=not
`significant). Experiments were repeated three times.
`
`exposure for 15 and 30 min to Travatan and Systane Ultra
`solutions increased the level of caspase-3, although the
`increase was not statistically significant.
`Inflammation: Travatan, Systane Ultra and BAK 0.001%
`increased IL-6 levels already after a 5 min exposure when
`compared to the controls (Figure 4). There was approximately
`three times more IL-6 released into the culture medium of the
`Travatan and Systane Ultra-treated cells. After 15 min of
`exposure to Travatan and Systane Ultra, the levels were 8
`times higher than the controls. For comparison, BAK 0.001%
`increased the amount of IL-6 by only about twofold in
`response to 15 min´ treatment. The 30 min exposure amplified
`the levels even more in Systane Ultra (~10×) and in BAK
`0.001%-treated (~4×) samples, whereas in Travatan exposed
`
`samples the IL-6 levels were the same as in the 15 min
`samples. The response of interleukin-8 was similar to that seen
`with IL-6 (Figure 5), although the elevations were not as
`dramatic (with Travatan ~50%–300%, Systane Ultra ~0%–
`350% and no elevation with BAK 0.001%). NF-κB levels
`were increased statistically significantly in the Systane Ultra
`(15 and 30 min exposures) and Travatan (30 min exposure)
`treated cells (Figure 6). BAK 0.001% did not have any effect
`on the NF-κB expression.
`
`DISCUSSION
`The ocular toxicity associated with BAK has been known for
`decades [17]. Preservative-free ophthalmic compounds are
`safer and better tolerated than BAK-preserved solutions [18],
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`Figure 5. Interleukin-8 secretion from HCE-2 cells analyzed by ELISA. Columns represent the amount of IL-8 pg/ml (mean±SD).. One-Way
`ANOVA, followed by Dunnett’s post hoc test, evaluated the statistical differences (n=6, *0.01<p≤0.05, **0.001<p≤0.01, ***p≤0.001, ns=not
`significant). Experiments were repeated three times.
`
`Figure 6. Binding of NF-κB (p65) to DNA 6 h after stimulation in HCE-2 cells. Columns represent the amount of NF-κB (mean±SD), control
`is set as 100%. One-Way ANOVA, followed by Dunnett’s post hoc test, evaluated the statistical differences (n=6, *0.01<p≤0.05,
`**0.001<p≤0.01, ***p≤0.001, ns=not significant). Experiments were repeated three times.
`
`but antimicrobial preservatives are required for the use of
`multi-dosage medicine containers. New preservatives, such as
`polyquaternium-1 (PQ-1), have been developed as alternative
`to BAK [19]. PQ-1, a detergent-type preservative derived
`from BAK, has been used in artificial tears since the 1980s,
`and recent studies have assessed its suitability as a
`preservative for prostaglandin analogs used in antiglaucoma
`therapy [20]. The main disadvantage associated with PQ-1 is
`its tendency to reduce the density of conjunctival goblet cells,
`thereby decreasing the aqueous tear film production [21].
`Despite being less toxic to the corneal surface than BAK, PQ-1
`is known to induce damage to corneal epithelial cells as well
`[22]. This is the first study to demonstrate that while PQ-1
`
`induces cytotoxicity, it also induces inflammation in an NF-
`kB-dependent manner in corneal epithelial cells.
`Not only preservatives, drugs and excipients also may
`evoke potential cytotoxicity [23]. Several in vitro studies have
`shown that active antiglaucoma compounds do not exert
`cytotoxic effects [24,25]. On the contrary, recent studies have
`reported controversial results of protective effects of
`prostaglandin analogs against BAK-induced toxicity in
`human conjuctival cells [26-28]. The discrepancies in these
`studies might be due to the different BAK concentrations used
`as well as different cell line characteristics [22]. The drug
`solutions containing PQ-1 as preservative as well as BAK
`0,001% showed cytotoxicity in a time-dependent manner in
`corneal epithelial cells, measured via the MTT assay, whereas
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`the LDH assay revealed that exposure to Travatan (which
`contains PQ-1) evoked the most prominent LDH release of
`these three exposures as compared non-treated cells. The
`observed LDH increase may be a result of the drug i.e.,
`travoprost, changing cell membrane integrity and function
`[23]. However, it is important to notice that LDH release does
`not always mean increased cytotoxicity, when analyzed by
`sensitive ELISAs.
`Our findings with human corneal epithelial cells are in
`agreement with Brasnu et al. [23] who reported similar
`toxicity in conjunctiva-derived epithelial cells after 15 min of
`exposure to BAK 0.001%. However, in the present study it
`was found that PQ-1 0.001% containing eye drops showed
`similar or higher cytotoxic effects as BAK 0.001% after 15
`min in cytotoxicity assays. The hypothesis of reduced cellular
`toxicity of PQ-1 is based on its larger size (approximately 27
`times larger than BAK molecule) which results in its
`diminished capability of passing into the cornea [22,29]. In
`addition, PQ-1 lacks the hydrophobic domain present in the
`BAK molecule [22]. In earlier studies, the reduced cytotoxic
`effects of PQ-1 in comparison to BAK were explained by the
`latter’s corneal permeability [30]. Contrary to earlier results,
`the results from our study suggest that PQ-1 exerts almost as
`extensive cytotoxicity as BAK, in a time dependent manner
`i.e., depending on the cytotoxicity assay being used.
`In vitro study does not mimic precisely the in vivo
`conditions; in vivo tears may provide additional protection as
`well as diminishing the direct contact time of cytotoxic agents
`to corneal cells. However, it has been proposed that detergent-
`type agents, such as BAK, can be absorbed into ocular tissues
`i.e., they can exert effects long after contact with the
`preservative has ceased
`[27,28]. Low concentrations
`(0.001%) of BAK can induce growth arrest and apoptosis in
`conjunctival cells in vitro for many hours after exposure to the
`compound [31]. In addition, BAK has induced necrosis when
`present at moderate concentrations, although apoptosis seems
`to be triggered low detergent concentrations (0.0001% to
`0.001%) in a dose-dependent manner [31]. In a rabbit model
`it was reported that the cell-permeable preservative BAK can
`accumulate in the conjunctiva and cornea of the eye, and may
`therefore continue to act long after the actual drug is no longer
`present in tears [28,32]. It is not known whether PQ-1 exerts,
`at least in part, the same prolonged effects. However, the
`increase in caspase-3 activity induced by the PQ-1-containing
`eye drops after 15 min incubation was also observed at the
`other two time points although these increases did not reach
`statistical significance. Interestingly, BAK 0.001% did not
`affect the caspase-3 levels. The 5, 15, and 30 min incubations
`used in our study may mimic the innate contact of corneal
`exposure as well as in multi-treatment conditions, and these
`exposure times are in concordance with the in vitro schedules
`used in cytotoxic preservative corneal research.
`The apoptosis and inflammation pathways are closely
`linked through their common mediators and transduction
`
`signals. It is a generally established dogma that apoptosis does
`not
`induce
`inflammation [33]. However, some pro-
`inflammatory cytokines, such as tumor necrosis factor alpha
`(TNF-α), interferon gamma (IFN- γ), and IL-1, can induce
`apoptosis [34,35] while IL-6 and IL-8 have been shown to
`inhibit apoptosis through several mechanisms [36,37]. The
`rapid induction of IL-6 mRNA expression detected in corneal
`tissues is evidence that IL-6 is closely involved in the host
`corneal response during inflammatory conditions [38]. Our
`findings revealed that Travatan and Systane Ultra (which both
`have PQ-1) were able to induce the expression of IL-6 and
`IL-8 but not IL-1beta (data not shown). BAK 0.001% slightly
`increased IL-6, but had no effect on the IL-8 level. We
`observed a trend toward a caspase-3 (apoptosis marker)
`increase in a time-dependent manner with Travatan and
`Systane Ultra. In addition, we found a statistically significant
`NF-kB induction by eye drops containing PQ-1, indicating
`that the increase in IL-6 and IL-8 expression was being
`mediated via NF-kB activation. These data suggest that PQ-1
`induces inflammatory rather than caspase-3 -mediated
`apoptosis in corneal epithelial cells and this mechanism is
`associated with activation of NF-kB.
`In conclusion, the cationic polymer polyquad (PQ-1
`0.001%) preservative was less cytotoxic in vitro than the
`highest commercial BAK (0.02%) concentrations. However,
`PQ-1 containing eye drops did induce even stronger
`inflammatory response than did BAK 0.001% exposure in
`human corneal epithelial cells. Further studies will be needed
`to elucidate the exact mechanism of this action, and whether
`these effects occur also in vivo, especially in conjunction with
`long-lasting therapy.
`ACKNOWLEDGMENTS
`This work was supported by the Academy of Finland, the
`EVO grant 5503726, the Finnish Cultural Foundation and its
`North Savo Fund, the Finnish Eye Foundation, Finnish
`Funding Agency for Technology and Innovation, and the
`Päivikki and Sakari Sohlberg Foundation. We thank Dr. Pertti
`Pellinen for critical comments and Dr Ewen MacDonald for
`checking the language.
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`Articles are provided courtesy of Emory University and the Zhongshan Ophthalmic Center, Sun Yat-sen University, P.R. China.
`The print version of this article was created on 3 May 2012. This reflects all typographical corrections and errata to the article
`through that date. Details of any changes may be found in the online version of the article.
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