Download Effects of benzalkonium chloride on growth and survival of

Effects of Benzalkonium Chloride on Growth and
Survival of Chang Conjunctival Cells
Magdalena De Saint Jean,13 Frangoise Brignole,2 Anne-France Bringuier,1
Alain Bauchet,4 Gerard Feldmann,1 and Christophe Baudouin^
PURPOSE. The
aim of this study was to investigate the action of benzalkonium chloride (BAC), used
as a preservative in most ophthalmic topical solutions, on epithelial conjunctival cells in vitro.
METHODS. A continuous human conjunctival cell line (Wong-Kilbourne derivative of Chang conjunctiva) was exposed to BAC solutions at various concentrations (0.1%-0.0001%) during a period
of 10 minutes. Cells were examined before treatment and 3, 24, 48, and 72 hours later, after
reexposure to normal cell culture conditions. Cell number and viability were assessed with crystal
violet and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide colorimetric assays. The
expression of the apoptotic marker Apo 2.7, nuclear antigen p53, membrane proteins Fas and Fas
ligand, and DNA content was studied by flow cytometry. Morphologic aspects of cell nuclei were
analyzed on slides with a nucleic acid-specific dye, 4',6'-diamidino-2-phenyIindole dihydrochloride. Cytoskeleton was labeled with a monoclonal anti-pancytokeratin antibody. In addition,
apoptosis was measured by DNA electrophoresis assays in agarose gel.
Cell exposure to 0.1% and 0.05% BAC induced cell lysis immediately after treatment. All
cells (100%) treated with 0.01% BAC died in a delayed manner within 24 hours, with most of the
characteristics of apoptosis (chromatin condensation and DNA fragmentation, reduction in cell
volume, expression of the apoptotic marker Apo 2.7, and apoptotic changes in DNA content).
Aliquots of 0.005%, 0.001%, 0.0005%, and 0.0001% BAC induced growth arrest and apoptotic cell
death in a dose-dependent manner between 24 and 72 hours after treatment. The expressions of
Fas and p53 did not vary after BAC treatment. Fas ligand was always negative.
RESULTS.
CONCLUSIONS. These results
suggest that BAC induces cell growth arrest and death at a concentration
as low as 0.0001%. The mode of BAC-induced cell death is dose-dependent. Cells die by necrosis
after BAC treatment at high concentrations and by apoptosis if low concentrations of BAC are
applied. This new aspect of in vitro toxicity of BAC could in part explain some ocular surface
disorders observed in patients undergoing long-term topical treatments with preservative-containing drugs. (Invest Ophthalmol Vis Sci. 1999;40:6l9-630)
B
enzalkonium chloride (BAC) is the most commonly used
preservative in many available ophthalmic solutions,
nebulizer compounds, and nasal sprays. It is a cationic
detergent whose surface-active structure is responsible for its
very rapid and prolonged incorporation into cell lipid membranes. ' The charged part of the molecule interacts with membrane proteins in a very specific high-affinity manner, for example, with guanine nucleotide triphosphate- binding proteins
(G heterotrimeric proteins), thereby influencing a variety of
cell processes.2 For instance, BAC has been shown to have
antiproliferative properties in many cellular systems, affecting
the DNA synthetic phase of the cell cycle.3"5 In skin, BAC
From the 'Laboratoire de Biologie Cellulaire, INSERM U327, Faculte de Medecine Xavier Bichat, Universite Paris VII; the 2Services
d'Immunohematologie et M'Ophthalmologie, Hopital Ambroise Pare,
AP-HP, Universite Paris V, Boulogne; and the ''Departement de Statistique et Informatique, Hopital Ambroise Pare, AP-HP, Universite Paris
V, Boulogne, France.
Submitted for publication October 6, 1998; accepted November
12, 1998.
Proprietary interest category: N.
Reprint requests: Christophe Baudouin, Service d'Ophthalmologie,
Hopital Ambroise Pare, 9 avenue Charles de Gaulle, 92,104 Boulogne,
Cedex France.
application induces activation of CD 1-positive epidermal Langerhans cells6 and mast cells.7' 8 Moreover, it promotes activation of lipooxygenases and synthesis and secretion of eicosanoids, of inflammatory mediators,9 and of many cytokines
such as interleukin (IL)-la, tumor necrosis factor-a, and IL-8,10
resulting in irritation, delayed hypersensibility, and allergic
reactions. In the eye, BAC turnover is very slow, and this
molecule is retained in ocular tissues up to 48 hours after a
single drop administration.' Its ocular cytotoxicity was demonstrated in many in vivo and in vitro models.""' 3 It affects
surface microvilli in rabbits and cat corneas,' 4 ' 5 induces a
release of lactate dehydrogenase and albumin by corneal
cells,"' 6 and alters electroretinogram amplitudes when injected into the subconjunctival space of pigmented rabbits.17
Several studies have confirmed the participation of preservatives such as BAC in the induction of ocular surface inflammation, l8 ' 9 ' allergy,20'21 fibrosis, and dry eye syndrome.22'23 In
one case, BAC was even incriminated as inducing endothelial
damage that required corneal transplantation when applied to
a patient with preexisting keratoconjunctivitis sicca.24 Finally,
some authors imputed to BAC a possible responsibility in the
failure of filtering surgery after several years of use of preservative-containing topical ophthalmic compounds.25"29
Investigative Ophthalmology & Visual Science, March 199.9, Vol. 40, No. 3
Copyright © Association for Research in Vision and Ophthalmology
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De Saint Jean et al.
In toxicological textbooks, BAC is variably classified as a
moderate to extreme irritant as determined by eye or skin
irritating tests.30"32 The scores are based on in vivo Draize test
and its variants33"35 with subjective grading of irritation of
ocular tissues (e.g., cornea, conjunctiva, iris). However, these
classic methods are imprecise and tend to overestimate the
immediate ocular reaction and to underestimate delayed consequences of the tested substances.
Many efforts were made to develop in vitro models to
predict a cytotoxic potential of preservatives. Because of the
very important, painful, and sight-threatening reactions of the
cornea to toxic substances, these models are essentially based
on corneal epithelial cells 1213 ' 36 or on some other epithelial
systems with characteristics similar to those of the superficial
layer of the corneal epithelium (Madin-Darby Canine Kidney
cells).37 Little is known about the mechanisms of preservativeinduced toxic effects on conjunctival cells. However, some
authors have shown their deleterious influence on cell viability
and proliferation, exclusively by quantitative in vitro assays.38"41
The aim of this study was to investigate the mechanisms of
conjunctival cell damage induced by BAC, with a qualitative
approach in addition to the quantification of cell survival
and death. We used a human continuous conjunctival cell
line previously used in toxicological ocular studies in
vitro.3'39'40'42"46 We investigated immediate and delayed actions of BAC on cell survival, proliferation, morphologic alterations, and immunologic expression of molecules associated
with programmed cell death such as Fas receptor,47'48 Fas
ligand,47"49 p53, 50 ' 5 ' and Apo 2.7.52"54 We thus showed that
BAC induces two different patterns of cell death (apoptosis and
necrosis) in a dose-dependent manner, with very rapid action
at high concentrations and delayed action at lower concentrations.
MATERIALS AND METHODS
Reagents
Eagle's minimum essential medium, fetal calf serum, and trypsin-EDTA were purchased from GIBCO-BRL (Paisley, Scotland). Benzalkonium chloride, 4',6'-diamidino-2-phenylindole
dihydrochloride (DAPQ, and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) were from Sigma (St. Louis,
MO). Monoclonal antibodies specific for the following human
antigens were used: anti-Fas (UB2, fluorescein isothiocyanate
[FITC]-conjugated; Immunotech, Marseilles, France), anti-Fas
ligand (NOK-1, purified; Pharmingen, San Diego, CA), anti-7A6/
Apo 2.7 (2.7A6A3, purified and phycoerythrin-conjugated; Immunotech), anti-pancytokeratin against cytokeratins 5, 6, 8,
and 17 (MNF116, purified; DAKO, Glostrup, Denmark), and
anti-p53 (DO7, FITC-conjugated; Pharmingen). Control antibodies (mouse FITC-conjugated IgGl and IgG2«, mouse phycoerythrin-conjugated IgGl) were purchased from Immunotech. A goat anti-mouse FITC-conjugated antibody was
obtained from DAKO. Staining solutions for cell cycle (DNAPrep Stain) were from Coulter (Miami, FL).
Conjunctival Cell Line Culture
A human conjunctival cell line (Wong-Kilbourne derivative of
Chang conjunctiva, clone l-5c-4, American Type Culture Collection [ATCC] CCL-20.2) was cultured under standard condi-
IOVS, March 1999, Vol. 40, No. 3
tions (5% CO2, 95% humidified air, 37°C) in Eagle's minimal
essential medium supplemented with 5% fetal calf serum, 2
mM L-glutamine, 50 mg/ml streptomycin, and 50 IU/ml penicillin. Cells from passages 6 to 17 (after ATCC initial passage
65) were used in all experiments. Cells were plated at a density
of 10,000 cells/well in 96-well plates (Falcon; Becton Dickinson Lab ware, Plymouth, England) for analysis of viability and
cell proliferation. Cells were plated in 75-cm2 flasks (Falcon)
for flow cytometric analysis and DNA isolation and on 20-mm2
permanox chamber slide systems (Lab-Tek; Nalge Nunc International, Naperville, IL), 25,000 cells per chamber, for morphologic and immunocytologic studies. Cells were treated
with BAC at least 24 hours after the passage (1:4 split ratio at
confluence) when approximately 60% of confluence was
reached.
BAC Treatment
Benzalkonium chloride was dissolved in serum-free medium at
the following concentrations: 0.1% (1 mg/ml), 0.05% (500
jLtg/ml), 0.01% (100 jag/ml), 0.005% (50 jug/ml), 0.001% (10
jag/ml), and 0.0001% (1 /Ltg/ml). Cells were treated for 10
minutes. After this time, BAC-containing medium was removed, cells were rinsed twice with culture medium, and
normal cell culture conditions were restored. The cells were
divided into two groups: the first treated only once and the
second reexposed to BAC 24 hours after the initial treatment.
The second BAC application was meant to mimic a repetitive
character of topical drug treatment. It was undertaken under
the same conditions as the first one, and cells were examined
after 3 hours, 24 hours, 48 hours, and 72 hours of recovery
period under normal cell culture conditions.
Cell Number and Cell Viability Assays
All assays were conducted using 96-well microtiter plates. At
the 24th, 48th, and 72nd hours after treatment, cells were
stained with crystal violet to determine the relative cell number
as described previously.55 Briefly, the cells were rinsed twice
with sterile phosphate-buffered saline (PBS; pH 7.4) and then
fixed in 70% cold ethanol for 10 minutes at room temperature.
A 0.5% crystal violet solution (100 jul/well) was added. The
relative cell number was determined by eluting the dye from
stained cells with 33% acetic acid, and absorbance was measured at 540 nm on an enzyme-linked immunosorbent assay
multiwell reader (iEMS Reader; Labsystems, Franklin, MA). Cell
viability was assessed with MTT assay as described previously.56 MTT is bioreduced in metabolically active cells into a
colored formazan product insoluble in tissue culture medium.
At times indicated above, 5 mg/ml MTT solution was added to
the culture medium (10 jul per 100 /ml of medium), and plates
were incubated at 37°C for 4 hours. After this period, the liquid
was carefully discarded. Acid-isopropanol (0.04N HC1 in isopropanol) was added (100 jul/well) and mixed thoroughly to
dissolve all formazan crystals. Then plates were rapidly read on
an enzyme-linked immunosorbent assay plate reader at 570
nm.
In both experiments, the background absorbance was
determined on wells without cells, containing the dye solution.
At each time point, values of relative cell number and viability
values were the mean of three to six determinations.
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Nuclear DNA Isolation and Electrophoresis
Twenty-four hours after a single BAC treatment, DNA was
isolated from adherent cells cultured in 75-cm2 flasks by proteinase K-phenol method as described previously. 57 DNA samples were treated with 50 jag/ml DNAase-free RNase, extracted
twice with phenol/chloroform, precipitated with ethanol, and
dissolved in 10 mM Tris-HCl, pH 7.6, and 1 mM EDTA. DNA
samples (10 jag) were fractionated by electrophoresis on 1%
agarose gels and visualized by staining with ethidium bromide
(0.5 jig/ml).
Morphologic Procedures
Cells were analyzed 24 hours after a single BAC treatment.
Cells cultured on chamber slides were rinsed twice with PBS
and fixed and permeabilized with 4% paraformaldehyde/0.3%
Triton X-100 in PBS (10 minutes at 4°C). Nonspecific sites were
blocked with 0.3% bovine serum albumin in PBS. Then slides
were incubated for 45 minutes with an anti-pancytokeratin
antibody and Apo 2.7-purified antibody used at a 1:50 dilution.
After washing in PBS, a secondary antibody, FITC-conjugated
goat anti-mouse, was applied (1:100) for 30 minutes. For DAPI
nuclear staining, cells were fixed and permeabilized for 10
minutes in ice-cold 70% ethanol then washed in PBS and
stained with DAPI at a concentration of 0.5 mg/ml for 5
minutes at room temperature. After staining, the slides were
washed extensively and mounted in Quantafluor Mounting
Medium (Kallestad, Chaska, MN) before examination. A Leica
DML microscope (Leica, Heiklelberg, Germany) was used for
visualization. Morphologic analysis was performed in a masked
manner by the same investigator during the whole experimental procedure. Fractions of apoptotic cells and those presenting
abnormal cytoskeleton were estimated after counting cells in
three different visual fields (magnification, X40).
Flow Cytometry
Expression of Apoptosis-Related Molecules. All antibodies were used as recommended by suppliers. For flow
cytometric analysis of Fas expression, cells were harvested
with trypsin-EDTA, pelleted, washed twice in PBS, and incubated for 30 minutes with FITC-conjugated anti-Fas antibody
(20 ix\/5 X 105 cells) and FITC-conjugated mouse IgGl (20
jitl/5 X 105 cells) as a negative control. For Apo 2.7 labeling,
the cells were fixed and permeabilized with 4% paraformaldehyde/0.3% Triton X-100 in PBS (10 minutes at 4°C) and then
incubated for 30 minutes with phycoerythrin-conjugated Apo
2.7 antibody (20 ptl/106 cells) 54 and phycoerythrin-conjugated
mouse IgGl (20 JU-1/5 X 105 cells) as an isotypic control. For
p53 labeling, the cells were fixed and permeabilized for 5
minutes with 1% paraformaldehyde in PBS, followed by 100%
cold methanol (10 minutes at -20°C). 5 8 Labeling of p53 was
done with FITC-conjugated anti-p53 antibody (10 ptl/106 cells)
and with FITC-conjugated mouse IgG2« (20 jul/106 cells) as a
negative control.
Labeling of Fas ligand was done with anti-Fas ligandpurified antibody (10 /Ltg/ml) and with purified mouse IgGl (10
/xg/ml) as a negative control. All flow cytometric measurements were performed on a FACScan flow cytometer (Becton
Dickinson, Mountain View, CA) equipped with an argon laser
emitting at 488 nm, using Lysis II software for data analysis.
Forward and side scatters, FITC-fluorescence (FL1, 525 nm
band-pass), phycoerythrin-fluorescence (FL-2, 575-nm band-
Benzalkonium Chloride and Conjunctiva! Cells
621
pass), and propidium iodide fluorescence (FL3, 630-nm bandpass) were measured. At least 10,000 events were collected
per sample. The FACS data are reported as mean fluorescence
intensities.
DNA Content Flow Cytometric Analysis. At die 24th
hour of the recovery period after a single BAC treatment, cells
were trypsinized, washed with cold PBS, and fixed with 70%
ethanol in PBS at — 20°C. After 12 hours, samples were washed
with cold PBS, stained with DNA-Prep Stain (Coulter) containing
propidium iodide and RNA-ase III-A for 30 minutes at room temperature according to manufacturer's instructions, and then
stored in the dark before analysis (within 24 hours) with a FACScan. The sub-Gl region was determined by a gate defined in the
controls and excluding the debris as described previously.59
Statistical Analysis
Flow cytometric results were calculated as arithmetic mean ±
SEM, and significance was determined using the Student's
unpaired t-test with P < 0.05 regarded as significant. Results of
colorimetric assays were calculated as arithmetic mean ± SEM,
and significance values were calculated by means of the twoway ANOVA with P < 0.05 regarded as significant. All experiments in this study were at least duplicated.
RESULTS
Cell Viability and Cell Number Assays
Figure 1 shows changes in cell viability measured with MIT
mitochondrial reduction assay. Cell viability decreased significantly in a dose-dependent manner after a single (Fig. 1A) or
double (Fig. IB) 10-minute treatment with BAC at 0.0001%,
0.0005%, 0.005%, 0.001%, 0.005%, 0.01%, and 0.05% (P <
0.001 at all time points after the last treatment).
Relative cell number was assessed with a crystal violet
colorimetric assay. Cell number was significantly decreased at
the 48th and 72nd hours after a single treatment with 0.01%
BAC (P < 0.001) and 0.001% BAC (P < 0.05 at the 48th hour,
P < 0.01 at the 72nd, and at the 96th hour after treatment),
whereas the proliferation ratio was not modified or increased
(P < 0.05 at the 72nd hour after treatment) in the sample
treated with 0.0001% BAC (Fig. 2A). In samples treated twice,
0.001% and 0.0001% BAC induced a significant decrease (P <
0.001) in cell number, respectively, at the 24th and 48th hours
after the second treatment (Fig. 2B).
DNA Fragmentation Assays
Figure 3 shows results of a DNA electrophoretic assay after BAC
treatment. No fragmentation was observed after electrophoresis
of DNA of untreated cells (lane 2). After a 24-hour recovery
period, DNA electrophoresis of cells treated with 0.0001% BAC
(10 minutes) showed some weak fragmentation (lane 3). Ladder
pattern was moderate in 0.001% BAC-treated cells (lane 4),
whereas cells treated with 0.01% BAC showed a characteristic
apoptotic ladder pattern, documenting DNA fragmentation into
nucleosomal and oligonucleosomal fragments (lane 5). Treatment
with 0.1% BAC induced a continuous smear trace of DNA, confirming abundant cell lysis (lane 6).
Morphologic Analysis by Fluorescence
Microscopy
Cells were examined 24 hours, after a single treatment with
BAC. As demonstrated by anti-cytokeratin labeling, approxi-
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120
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T
Time after treatment (h)
96
Time after treatment (h)
B
FIGURE 1. Viability of conjunctival cells exposed to BAC, as determined by MTT reduction assay. The
values in dashed rectangles are not significantly different from each other. (A) Cell viability at different
time points after a single treatment with BAC. A double mark on the x axis indicates a change in the time
scale. Three hours after treatment the viability values of cells treated with concentrations of 0.05%, 0.01%,
or 0.005% BAC decreased significantly compared with control (P < 0.001). From the 24th hour of the
recovery period, all viability values were significantly decreased when compared with control (P < 0.001).
(B) Cell viability after a double treatment with BAC. At all time points, viability values are significantly
decreased when compared with control (P < 0.001).
mately 75% of cells treated with 0.01% BAC presented
shrunken cytoskeleton compared with nontreated cells (Fig.
4A). AS shown in Figure 4B, with DAPI staining these cells
presented chromatin condensation and fragmentation and re-
duced nuclear size when compared with control cells. Chromatin clumps were peripheral or had a central disposition.
Cells treated with 0.001% BAC showed mildly diminished
cell and nuclear sizes. Chromatin condensation was less fre-
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Benzalkonium Chloride and Conjunctival Cells
IOVS, March 1999, Vol. 40, No. 3
623
1,8 -r
••—Control
*
0.0001%
*••'"••
0.001%
0.01%
Time after treatment (h)
0-7 T
Control
0.0001%
48
Time after treatment (h)
A
0.001%
•
0.01%
72
FIGURE 2. Relative cell number as determined with crystal violet colorimetric assay after a single BAC
treatment (A) and after a double BAC treatment (B). There is no significant difference between the values
in dashed rectangles.
quent, and many cells died without usual figures of apoptosis.
There was neither cell size reduction nor chromatin condensation in 0.0001% BAC-treated cells (data not shown).
Moreover all BAC samples presented an increased expression of Apo 2.7 as seen in the 0.001% BAC-treated cells
(Fig. 4C).
Flow Cytometry
Cell Size Analysis. Alteration of cell volume after BAC
treatment was confirmed with FACScan analysis of forward
scatter performed 24 hours after a single BAC treatment (Fig.
5). Cells treated with 0.001% BAC had a 15% reduction of cell
volume in comparison with untreated cells, and there was the
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De Saint Jean et al.
1
2 3 4 56
FIGURE 3. Nuclear DNA samples are isolated from cells 24 hours after
treatment with BAC. lane 1: DNA molecular markers (OX174/#aeffl)Lane 2: Control cells. Lane 3- Very slight DNA fragmentation in
0,0001% BAC-treated cells. Lane 4: 0.001% BAC-treated cells presented moderate DNA fragmentation. Lane 5: Apoptotic laddering
pattern in 0.01% BAC-treated cells. Lane 6: Continuous smear characteristic of necrosis in cells treated with 0.1% BAC.
appearance of a distinct population of small cells, whose size
was comparable to that of 0.01% BAC-treated cells. There was
a global reduction of cell volume (30% in comparison with
untreated cells) in cells treated with 0.01% BAC. Cells treated
with 0.1% BAC were observed in the debris gate, which is
suggestive of cell lysis. There was no modification in cell
volume in 0.0001% BAC-treated samples (data not shown). All
results are plotted on the graph shown in Figure 5B.
Expression of Apoptosis-Related Molecules. FACSscan analysis of expression of the apoptotic marker Apo 2.7 is
illustrated in Figure 6. There was a dose-dependent relation
between the intensity of cell expression of Apo 2.7 and concentrations of BAC (r = 0.99, P = 0.0083). Untreated cells
were negative to Apo 2.7, except for a minority of slightly
positive cells (2%). There were 44% Apo 2.7-positive cells after
treatment with 0.0001% BAC (mean fluorescence FL1 11), 69%
Apo 2.7-positive cells after treatment with 0.001% BAC (mean
FL1 12), and 89% in the 0.01% BAC-treated samples (mean FL1
19). The expressions of Fas (Fig. 7) and p53 (data not shown)
were slight and constant, without any variations after BAC
treatment. Fas ligand was always negative (data not shown).
DNA Content Histograms
We measured apoptotic cell population at the 24th hour after
10 minutes of BAC treatment. Figure 8 shows that the appearance of sub-Gl apoptotic population and the number of cells in
this region were dependent on BAC concentrations. Normal
untreated cells presented a 4% sub-Gl population. The population of sub-Gl cells was 18% in 0.0001% BAC-treated cells,
32% in 0.001% BAC-treated cells, and 54% in 0.01% BACtreated cells.
IOVS, March 1999, Vol. 40, No. 3
DISCUSSION
Our results reveal new aspects of BAC toxicity and suggest a
specific dose-dependent action on cell viability and proliferation. In this study we used a human continuous conjunctival
cell line that provides constant cell culture conditions, as
opposed to primary or secondary cell cultures of conjunctival
epithelium, very often contaminated with fibroblasts. Potential
disadvantages of our model that make impossible a direct
extrapolation to in vivo data are the existence of variables such
as drug diffusion, stratified character of conjunctival barrier,
and proper characteristics of the Chang cell line (some enzymatic activities, possible contamination by HeLa cells), which
differ from normal epithelium. 60 However, because Chang
cells are derived from human conjunctiva, this model has been
used as one approach to understand some disorders of ocular
surface. Chang conjunctival cells have been used for in vitro
ocular toxicological studies 3 ' 39 ' 4 "' 4243 and for other investigations concerning production of cytokines, growth factors, and
their receptors. 4 ''~ /i6 Moreover, some recent findings put into
evidence the common characteristics of this cell line and the
conjunctival epithelium. The two cellular systems normally
express Fas but not HLA DR 61 " 63 and overexpress these proteins in inflammatory conditions (the cell line treated with
interferon-y52 or the inflammatory epithelium of patients with
Sjogren's syndrome 63 ). Hence, we have used the Chang cell
line to evaluate the toxicity of preservatives, taking into consideration all limits of this model that dictate caution in'the
extrapolation of the results to ocular surface disorders present
in patients treated long term.
Previous investigations showed the noxious effects of low
concentrations of BAC on cellular homeostasis. A 1-hour application of BAC solutions ranging from 0.0013% to 0.0007% on
epithelial corneal cells in vitro has been shown to produce a
50% decrease in cell viability, a 70% increase in intracellular
calcium concentration, and a significant decrease in intracellular pH (from 7.39 to 7.17 to 7.24). 36 It is noteworthy that pH
and calcium changes are common apoptosis inducers in other
cellular systems 64 ' 65 and occurred, in the experiments cited
above, in a delayed manner after treatment (between 0.5 and 4
hours), which is an argument for cell death by apoptosis.
In our model, after a short application (10 minutes) of
0.1% and 0.05% BAC, viability of treated cells rapidly decreased
between to and the third hour after treatment. Cells showed
characteristics of immediate abundant lysis, with membrane
debris observed in culture supernatants and nonhomogeneous
and very low cell volumes on flow cytometric analysis graphs.
The continuous smear traces seen in DNA electrophoresis
assay confirmed the necrotic character of cell alterations.
Effects of BAC concentrations of 0.01% to 0.0001% were
progressive and delayed, and cell viability and proliferation
were altered in a dose-dependent time course: relatively rapidly for 0.01% BAC (between t 0 and the 24th hour) and more
gradually (between t 0 and the 72nd hour) for BAC concentrations less than or equal to 0.005%. The morphologic analysis
showed intact cellular structures with a global decrease in cell
and nuclear volumes (cells treated with 0.001% and 0.01%
BAC), chromatin condensation (0.001% BAC- and 0.01% BACtreated cells), and a high expression of the apoptotic marker
Apo 2.7. This expression was specific, not related to cell
membrane damage (even permeabilized, normal cells remained negative to Apo 2.7), and closely correlated with BAC
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Benzalkonium Chloride and Conjunctiva! Cells
625
FIGURE 4. Morphologic analysis of cells 24 hours after a single BAC treatment. (A) Cytoskeleton labeling with anti-pancytokeratin antibody. Left:
cytoskeleton of normal cells (magnification, X400). Right: shrunken cytoskeleton of cells treated witli 0.01% BAC (magnification, X400 for both). (B)
DAP1 nuclear staining of the cells cultured on slides. Left: normal ceU nuclei (magnification, XI000). Middle: nuclei of cells treated with 0.01% BAC
showing a characteristic apoptotic peripheral condensation and fragmentation of chromatin (magnification, X1000). Right: nuclei of cells treated witli
0.01% BAC showing a central chromatin condensation (magnification, X1000). (C) Ininiunocytologic expression of Apo 2.7. Left: untreated cells are
negative or weakly positive. Right: some 0.001% BAC-treated cells show a strong expression of Apo 2.7. Nuclei are counterstained with DAP1.
concentrations. Cell viability decreased progressively after
treatment;, excluding a necrotic process. The apoptotic ladder
pattern seen in DNA electrophoresis assays and a sub-Gl peak
on flow cytometric analysis of DNA content histograms, the
very hallmarks of apoptosis66 confirmed the presence of programmed cell death also with a dose-dependent intensity. The
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10VS, March 1999, Vol. 40, No. 3
Control
BAC 0.001%
mean FS 310
mean FS 355
1023
1023
FSC-Height
FSC-Height
BAC 0.01%
mean FS 260
M1
M1
BAC 0.1%
M1
mean FS 61
1023
1023
FSC-Height
Control
M1
FSC-Height
0.001%
0.01%
0.1%
BAC concentration (%)
B
FIGURE 5. (A) Flow cytometric analysis of cell size. "Forward scatter" parameter (FS) is represented on
the x axis. Upper left: untreated cells (mean FS = 355). Upper right: 0.001% BAC-treated cells (mean FS =
310). Note the appearance of small cells to the left of the arrow. These small cell fractions expressed Apo
2.7 at very high intensities (72% with mean FL1 52, data not shown). The empty black graph represents
size profile of untreated cells. Lower left: 0.01% BAC-treated cells. Note the shift to the left, toward lower
FS (mean FS = 260). The empty black graph profile of untreated cells. Lower right: 0.1% BAC-treated cells.
(B) Graph of flow cytometric analysis of cell size (parameter FS) 24 hours after a single BAC treatment.
relatively low intensity of DNA laddering in the case of 0.001%
BAC- and 0.0001% BAC-treated cells, with a corresponding
low percentage of sub-Gl cells, is compatible with the small
number of late apoptotic cells. In fact, some authors have
reported that the intensity of nuclear changes (chromatin condensation and fragmentation) is related to the dose of proapoptotic substances.67 Toxicity of BAC was delayed, slow, and
prolonged, probably because of incorporation and persistence
of BAC molecules in cell membranes; consequently, at one
time point, only a very small fraction of the whole cell population accomplishes the apoptotic process, with complete
DNA fragmentation, thereby rendering difficult its detection by
gel electrophoresis or by flow cytometry. Furthermore, BAC
cellular effects were cumulative in time, which was confirmed
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Benzalkonium Chloride and Conjunctival Cells
IOVS, March 1999, Vol. 40, No. 3
CM
O
a
627
100 -,
80 -
>
w
o
a
tn
"55
o
0.0001%
0.001%
0.01%
BAC concentration (%)
FIGURE 6. Percentage of cells expressing the apoptotic marker Apo 2.7 as studied by flow cytometry.
Normal untreated cells are negative to the apoptotic marker Apo 2.7 except for a minority of cells (2%).
Twenty-four hours after a single treatment (10-minute) with 0.0001%, 0.001%, or 0.01% BAC 44%, 69%, and
89%, respectively, of cells expressed the apoptotic marker Apo 2.7.
with a progressive decrease in cell viability (MTT assay) after a
single treatment.
The fact that in our model the expression of apoptotic
molecules such as Fas and p53 remained unchanged suggests
mechanisms of apoptosis different from those implying Fas, Fas
ligand, and p53 pathways. An increase in cellular calcium
concentration demonstrated previously with BAC36'68 could
explain the programmed cell death by Fas-independent activation of caspases.6? Some authors have reported BAC-induced
reversible DNA damage, an important argument for BAC-induced programmed cell death.70 Nevertheless, in our system
p53 remained unchanged, so this apoptotic way is most unlikely.
We ascertained that in different conditions of concentration, BAC can induce two distinct patterns of cell death, apoptosis, and necrosis. Many other chemical substances could
induce necrosis at high concentrations and apoptosis at low
ones.71'72 Some histologic scores of drug toxicity distinguish
cell degeneration with nuclear shrinkage and chromatin condensation (characteristics of apoptosis) as opposed to cell lysis.
Both patterns can be induced by the same substance under
different conditions. Our observations of BAC action are consistent with these well-known aspects of drug toxicity.
In vivo, the pathways of apoptosis and inflammation are
closely related by common mediators and transduction signals.
In fact, it is known that human dendritic cells and, thus, their
epithelial form Langerhans' cells, which are abundantly found
in human conjunctiva, are capable of phagocytosing apoptotic
bodies and presenting apoptotic antigens that stimulate major
histocompatibility complex class I-restricted cytotoxic T lymphocytes.73 By direct interaction with Langerhans' cells,
apoptotic cells can elicit tolerogenic but also stimulator}'
• % of cells positive for
Fas
I I mean fluorescence
Control
0.001%
0.01%
BAC concentration (%)
FIGURE 7. Graph represents a flow cytometric analysis of expression of Fas antigen/CD95. Cells were
analyzed 24 hours after a single BAC treatment. The intensity of fluorescence reflects the intensity of Fas
expression after comparison with a control isotype-matched antibody. There was no significant variation
of Fas expression after BAC treatment.
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De Saint Jean et al.
IOVS, March 1999, Vol. 40, No. 3
BAC 0.0001%
Control
10'
10 2
FL2-Height
10 1
BAC 0.001%
BAC 0.01%
10
FIGURE 8. FACScan analysis of DNA content 24 hours after cell treatment with 0.0001% BAC (upper
right), 0.001% BAC (lower left), and 0.01% BAC (lower right). A control (untreated cells) DNA histogram
is presented on the upper left panel. The number of cells is represented as a function of fluorescence (FL).
After exclusion of debris, 4% (control), 18% (0.0001% BAC), 32% (0.001% BAC), and 54% (0.01% BAC) of
apoptotic cells (sub-Gl population) were detected.
responses.73 In normal cell turnover of the conjunctiva, apoptotic cells could induce a tolerance to tissue-restricted selfantigens because of their immunosuppressive properties.74
It is generally established that apoptosis does not induce
the inflammatory reaction.66 However, during inflammation,
some cytokines such as tumor necrosis factor-a, interferon-y,
and IL-1 are capable of inducing apoptosis,75'76 and the two
processes are known to be closely related in many pathologies
of ocular surface.77 For example, in Sjogren's syndrome, cytokine-producing lymphocytes induce lacrimal gland inflammation and, at the same time, prime the lacrimal acinar cells for
apoptotic cell death.77'78 Long-term use of topical preserved
drugs has been associated with conjunctival metaplasia, stromal infiltrates and the epithelial expression of inflammationdependent molecules (HLA DR), and apoptotic markers (Apo
2.7).61'79'80 The role of active compounds in this pathologic
process is not well established, even though some in vivo and
in vitro studies40'81 have demonstrated the lack of noxious,
tissular or cellular, side effects. Thus, our hypothesis is that
preservative-induced tissular aggression could promote, by
common mediators, apoptosis and inflammation, with a subsequent bidirectional interaction and mutual potentialization between the two processes, perpetuating the tissue injury. However, this assumption should be confirmed with other studies,
because an in vitro model can never exactly reflect in vivo
reality. With regard to the antiseptic action of drug adjuvants,
some data reported a bacterial contamination of multiuse ophthalmic solutions despite the presence of preservatives.82'83 In
conclusion, with all available data considered, preservatives
should be avoided, if possible, in chronic ocular diseases such
as glaucoma, dry eye, or allergy, because the risk to worsen
patient symptoms or to compromise the issue of the affection
(failure of the surgery in glaucoma) could not be excluded.
Some ophthalmic solutions are now available in single-use
doses and could in some cases successfully substitute for the
classic multi-dose preparations. Finally, all aspects of potential
preservative-induced toxicity should be taken into consideration in elaboration of new topical drugs.
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