Quaternary Ammoniums and Other Preservatives

Contribution in Oxidative Stress and Apoptosis

on Chang Conjunctival Cells
Caroline Debbasch,1,2 Francoise Brignole,3 Pierre-Jean Pisella,1,2 Jean-Michel Warnet,1
Patrice Rat,1 and Christophe Baudouin2
PURPOSE. To investigate some of the toxicity mechanisms of 10
preservatives currently used in ophthalmic solutions in vitro.
METHODS. A continuous human conjunctival cell line was
treated with different concentrations of various preservatives
for 15 minutes and for 15 minutes followed by 24 hours of cell
recovery: three benzalkonium chlorides (BACs) with different
hydrocarbon chain length, benzododecinium bromide (BOB),
cetrimide (Cet), phenylmercuric nitrate (PM), thimerosal (thi),
methyl parahydroxybenzoate (MPHB), chlorobutanol (clb),
and EDTA. An inhibition study was then conducted using a
1-hour vitamin E pretreatment followed by a 15-minute BAC
treatment. Membrane integrity was assessed using a neutral red
test and chromatin condensation with a Hoechst 33342 test.
Reactive oxygen species were measured using dichlorofluorescein diacetate test for H2O2 production and hydroethidine test
.
for O
2 production. These tests were performed using microplate cold light cytofluorometry. Cell size and DNA content
were also analyzed using flow cytometry. Confocal microscopy
was used to explore morphologic changes.
RESULTS. A significant decrease of membrane integrity with
chromatin condensation was observed with all the quaternary
ammoniums tested at concentrations of 0.005% and higher.
The effect was amplified after 24 hours of cell recovery. The
other preservatives tested did not decrease membrane integrity. H2O2 production was observed with all the preservatives,
.
whereas O
2 production was significantly higher with the quaternary ammoniums at 0.005% and 0.01%, compared with the
other preservatives. Flow cytometry results confirmed the cytotoxicity observed with cold light cytofluorometry.
CONCLUSIONS. The quaternary ammoniums tested (BAC, BOB,
and Cet) were the most cytotoxic preservatives in the current
model. An apoptotic mechanism appeared to be present at low
concentrations of quaternary ammoniums, whereas a necrotic
process appeared at higher concentrations. Superoxide anions
may play an important role in tissue damage induced by preservatives in ocular surface disorders. (Invest Ophthalmol Vis
Sci. 2001;42:642 652)

reservatives are used in most ophthalmic preparations,

including eye drops and contact lens solutions. Although
topically administered medications are increasingly used with

From the 1Unit of Cellular Pharmacotoxicology, Centre Hospitalier National dOphtalmologie des Quinze-Vingts, the Toxicology
Laboratory, University of Paris-V; and the 2Ophthalmology and 3Immunohematology Services, Ho
pital Ambroise Pare, Assistance Publique
Ho
pitaux de Paris, University of Paris-V, Boulogne, France.
Submitted for publication August 17, 2000; revised October 27,
2000; accepted November 2, 2000.
Commercial relationships policy: N.
Corresponding author: Christophe Baudouin, Service dOphtalmologie, Ho
pital Ambroise Pare, AP-HP, Universite Paris-V, 9 avenue
Charles de Gaulle, 92104 Boulogne cedex, France.
debbasch@caramail.com

642

apparent safety and good tolerance, there is growing evidence

that long-term use of topical drugs can induce changes in the
ocular surface and may often produce damage to conjunctival
and corneal epithelial cells. There have been several reports of
the toxic effects of prolonged topical treatments, partly due to
the preservatives associated in the formulation of such treatments.13 In the eye, preservative turnover is very slow, and
quaternary ammonium molecules can be retained in ocular
tissues up to 7 days.4 The lipophilic nature of some preservatives causes them to bind to the ocular tissues immediately
after topical application. Previous studies by Burstein3 have
shown that topically applied benzalkonium chloride (BAC), the
most commonly used preservative in ophthalmic solutions, can
cause morphologic disruption of the corneal epithelium at
high concentrations.3 In addition, there is evidence that clinical concentrations of BAC may change the ionic resistance of
the cornea by intercalating into cellular membranes, which
results in increased permeability.5 Three types of mechanisms
have been described: detergent effects causing loss of tear film
stability, toxic effects to the corneal and conjunctival epithelia,
and immunoallergic reactions.2,6,7 Furthermore, repeated
doses of preserved eye drops can lead to a cumulative effect,
because the preservatives are in prolonged contact with the
epithelium. Several studies have confirmed the participation of
preservatives in induction of ocular surface inflammation,8,9
allergy,6 fibrosis,10 and dry eye syndrome.11,12 Preservatives
are also suspected of strongly increasing the risk of failure of
trabeculectomy in glaucoma.1316
In vitro models have been developed to predict the cytotoxic potential of preservatives. These models were essentially
based on corneal epithelial cells17,18 or on other epithelial
systems with characteristics similar to those of the superficial
layer of the corneal epithelium (MadinDarby canine kidney
cells).19 The human continuous conjunctival cell line has also
been useful for ocular toxicological studies.20 22 We recently
showed that BAC is a strong proapoptotic agent in Changs
conjunctival cells.23
The purpose of this study was to investigate, by flow cytometry and microplate cold light cytofluorometry, the cytotoxicity of 10 of the most common preservatives used in
ophthalmic solutions. Because stimulation of reactive oxygen
species (ROS) constitutes one of the mechanisms of cytotoxicity, we investigated ROS production induced by preservatives
in a well-adapted cellular model for in vitro cytotoxicity. Moreover, the protective effect of vitamin E against the cytotoxicity
of preservatives was explored. The new technique of microplate cold light cytofluorometry for cytotoxicity assays has
been validated in Chang conjunctival cells in previous studies2325 and allows the use of numerous fluorescent probes
directly on living cells. Their specificity, sensitivity and standardization ensure that they are well-adapted to cellular heterogeneity and comply with the requirements of cellular pharmacotoxicology screening procedures. Thus, this study could
be better performed physiologically on live rather than dead
cells, because labile markers can be significantly affected by
Investigative Ophthalmology & Visual Science, March 2001, Vol. 42, No. 3
Copyright Association for Research in Vision and Ophthalmology

IOVS, March 2001, Vol. 42, No. 3

the use of extraction techniques. Therefore, we analyzed, in
the Changs human continuous conjunctival cell line, the immediate and delayed actions of different concentrations of
preservatives on membrane integrity, cell size, DNA condensation, and ROS production. Variations of labile markers (ROS)
were therefore instantaneously detected, thereby providing
reliable data. To our knowledge, this is the first report to
describe the relation between oxidative stress and apoptosis
after preservatives treatments and to compare in a similar
biologic way the cytotoxicity of 10 different preservatives.
Results thus obtained on the human conjunctival cell line may
contribute to a better understanding of preservative cytotoxicity.

MATERIALS

AND

METHODS

Conjunctival Cell Line

WongKilbourne-derived human conjunctival epithelial cells, an established cell line (WongKilbourne derivative of Chang conjunctiva,
clone 1-5c-4, American Type Culture Collection [ATCC] certified cell
line [CCL], 20.2), were cultured under standard conditions (humidified
atmosphere of 5% CO2 at 37C) in Dulbeccos minimum essential
medium (DMEM; Eurobio, Les Ulis, France) supplemented with 10%
fetal bovine serum (Dominique Dutscher, Brumath, France), 1% glutamine (Eurobio), 0.1% ampicillin (Panpharma, Fouge`res, France), and
2% kanamycin (Bristol Myers Squibb, Paris, France). Cells from passages 6 through 17 (after ATCC initial passage 65) were used in all
experiments. Normal culture development was assessed daily by
phase-contrast microscopy. Confluent cultures were removed by gentle trypsin incubation, and cells were counted. They were then seeded
into 96-well culture plates for microtitration analysis (5,000 cells per
well; [Nunc, Roskild, Denmark] and plated in 15-cm2 flasks (Nunc) for
flow cytometric analyses. Cultures were kept at 37C for 24 hours.
After subconfluence was attained (culture surface covering nearly
70%), cells were exposed to the different formulations. Because this
cell line spontaneously undergoes apoptosis at 100% confluence,25
70% of confluence was thus chosen to avoid any artifact in membrane
integrity assays.

Preservative Treatments
Five different preparations of quaternary ammonium molecules were
examined. Three formulations of BAC were tested: (1) C14 benzalkonium chloride, alkyl dimethylbenzylammonium chloride (100%
14-carbon alkyl; BAC100); (2) C14/C12 benzalkonium chloride (58%
14-carbon alkyl; 32% 12-carbon alkyl; BAC58); and (3) C14/C12
benzalkonium chloride (32% 14-carbon alkyl; 58% 12-carbon alkyl;
BAC32). Their antiseptic action is effective at low concentrations
and is due to the hydrocarbon chain length. The maximum activity
is obtained with the C14 molecules and the minimum with the C8
and C18 preservatives.26 In eye drops, the precise composition of
BAC is almost never known, because a mixture of C12 and C14
chains is mostly used. We therefore decided to compare the eventual difference in toxic effects of various preparations of BAC that
have different antiseptic activities.
Benzododecinium bromide (BOB) and cetrimide (Cet) were also
tested. The five quaternary ammoniums were each tested at concentrations of 0.00001%, 0.0001%, 0.001%, 0.005%, and 0.01%, the concentration used in most eye drops being 0.01%.
Five other preservatives were tested at five concentrations: (1)
phenylmercuric nitrate (PM) at concentrations ranging between
0.000001% and 0.001%, its usual concentration being 0.001%; (2)
thimerosal (thi) at concentrations ranging between 0.000004% and
0.004%, usually used at 0.004%; (3) methyl parahydroxybenzoate
(MPHB) at concentrations ranging between 0.00003% and 0.03%, its
usual concentration being 0.03%; (4) chlorobutanol (clb) at concentrations ranging between 0.00005% and 0.05%, its usual concentration
being 0.5%, because high concentrations of this drug are required to

Preservatives and Conjunctival Cells

643

produce antimicrobial effects, and we could not obtain this concentration because other excipients are needed to make clb soluble; and
(5) EDTA at concentrations ranging between 0.00001% and 0.01%, the
most common concentration used being 0.01%.
An inhibition study was performed using a 1-hour vitamin E pretreatment followed by a 15-minute BAC 0.001% treatment.
All preservatives and vitamin E were provided by Transphyto,
Clermont-Ferrand, France. All dilutions were realized in culture medium. The complete culture medium was used as a negative control.
Durations of cell treatments with preservatives were chosen as a
compromise between in vitro and in vivo data currently available on
preservatives and in line with our previous work using the same
conjunctival cell line. In vitro, it has been demonstrated that a 100second application of 0.007% BAC produces lysis of 50% of conjunctival cells.27 A 1-hour application of 0.0013% to 0.007% BAC solution
on epithelial corneal cells also produces a 50% decrease in membrane
integrity.28 In vivo, in corneal and conjunctival tissues, BAC has a
half-life of 20 hours for the epithelium and 11 hours for the total
conjunctiva.4
In the present study two incubation times were therefore applied
to control and treated cells: 15 minutes of treatment and 15 minutes
followed by 24 hours of cell recovery in normal culture medium, as
performed in our previous studies.2325 The 24-hour cell recovery
period was also tested as a way of approaching the clinical conditions
in which the conjunctival tissue may recover after eye drop instillation.

Experimental Procedures
Experiments were performed using microplate cold light fluorometry,
which allows fluorometric detection (280 870 nm) with high sensitivity (picograms to femtograms per milliliter) and specificity. Fluorometry was performed with a microplate cytofluorometer29 (Fluorolite
1000; Dynex; Cergy Pontoise, France). According to the recommendations of the European Centre for the Validation of Alternative Methods
(ECVAM), three cellular markers were evaluated: cellular viability,
cellular proliferation, and cellular metabolism with ROS production.30
To complete these results, cell size and DNA content were also
analyzed by flow cytometry. All flow cytometric measurements were
performed on a commercially available flow cytometer (EPICS XL;
Beckman Coulter, Miami, FL) equipped with an argon laser emitting at
488 nm, using software provided by the manufacturer (EPICS XL
system II; Beckman Coulter) for data analysis.
Microplate Cold Light Fluorometry. This new and original
technique allows direct use of numerous fluorescent probes directly
on living cells and allows analysis of 96 wells in less than 1 minute.
Furthermore, each cell sample can be considered sufficiently similar to
the other samples.29 All fluorescent probes were added to live cells, in
mostly physiological conditions, because this method allows detection
of the fluorescent signal directly in the microplate cytofluorometer.
Four different tests were used according to previously validated
methods in a Changs cell line20,22 and other cell systems.31,32 Briefly,
membrane integrity, closely correlated with cellular viability, was evaluated with neutral red (Fluka, Ronkonkoma, NY) using fluorometric
detection (excitation, 535 nm; emission, 600 nm). Neutral red was
used at 50 g/ml. In accordance with the validated protocol of Borenfreund and Puerner,33 200 l per well of medium containing neutral
red was added to living cells, and the microplates were incubated for
3 hours at 37C in atmosphere with 5% CO2. The neutral red fluorescence was measured as previously described.29
H2O2 was detected with the 2,7-dichlorofluorescein diacetate
(DCFH-DA; Molecular Probes, Eugene, OR) dye added to live cells
before any treatment, as previously described.32 This probe is a nonfluorescent cell-permanent compound currently used in flow cytometry that we adapted to microplate cytometry. Once inside the cell, it
is cleaved by endogenous esterases and can no longer pass out of the
cell. The de-esterified product becomes the fluorescent compound
2,7-dichlorofluorescein on oxidation by ROS. The fluorescent signal
detected (excitation, 490 nm; emission, 535 nm) has been demonstrated to be proportional to ROS production.31,32

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.
O
2 was detected using hydroethidine (Molecular Probes). It was
.
oxidized to the fluorescent ethidium cation by O
2 , allowing the cation
to bind to nuclear DNA with an extensive fluorescent enhancement.34
The probe was used on cells at 5 M after 10 minutes (excitation, 485
nm; emission, 600 nm).
Hoechst 33342 (Molecular Probes) is a specific UV fluorescent
probe (excitation, 360 nm; emission, 450 nm). It specifically reacts
with the DNA, at adenine and thymine levels, by intercalation after 30
minutes.35,36 This probe was used on cells at a final concentration of 10
g/ml. One microliter of propidium iodide (Sigma, St Louis, MO) at 0.5
mg/ml was added to the Hoechst 33342 solution to control necrosis of
cells. In all experiments, the background fluorescence was determined
on wells without cells but containing the dye solution and was deduced from all control and treated wells.
Microplate cold light cytofluorometry results were obtained in
fluorescence units and were expressed as a percentage of the control.
Wells containing cells with complete culture medium but without any
treatment were used as the control. Each drug concentration was
tested in six wells, and each experiment was performed in triplicate.
Statistical comparisons were performed using an analysis of variance
(ANOVA) test to compare the five quaternary ammoniums. The Mann
Whitney test and the z correlation test at a 0.05 level of significance
were also performed (Statview IV for Windows; Abacus, Berkeley, CA).
Flow Cytometric Analysis. Alteration of cell size after preservative treatments was confirmed with flow cytometric analysis of
forward scatter on a linear mode performed 15 minutes after the
treatment. Cells were trypsinized, washed with cold phosphate-buffered saline (PBS), and analyzed for size. At least 3000 cells were
analyzed per sample.
DNA Content. After 15 minutes of treatment, cells were
trypsinized, washed with cold PBS, and fixed 10 minutes with 95%
ethanol in PBS at 20C. Samples were washed with cold PBS, stained
with propidium iodide at room temperature for 20 minutes, and
analyzed on the flow cytometer. The sub-G1 region was determined by
a gate defined in the controls in the whole-cell population, as described
previously.20,37
Immunocytology. In parallel, standard immunofluorescence
was performed to assess morphologic patterns of cells. Cells were
cultured on slides and treated with preservatives for 15 minutes. They
were washed with PBS and fixed as previously described. Phalloidin
(Alexa 488; 200 units/ml, Molecular Probes) was then added to explore
for F-actin. After 30 minutes of incubation, cells were washed in PBS.
Propidium iodide was added to mark cell nuclei before examination
with a confocal epifluorescence microscope (E800 PCM 2000; Nikon,
Tokyo, Japan).

RESULTS
Cellular Viability and Membrane
Integrity Evaluation
Membrane integrity significantly decreased after 15 minutes of
treatment with all the quaternary ammoniums tested (Fig. 1A).
This toxicity appeared at concentrations of 0.005% and 0.01%,
and membrane integrity decreased between 20% and 36% of
the control value (P 0.001 compared with control for all
preservatives). After 24 hours of cell recovery (Fig. 1B), significant cellular damage was found at 0.001% and above. The
same decrease of membrane integrity was observed with
BAC100, BAC58, and BAC32, ranging between 70% with 0.001%
BAC to 30% with 0.01% BAC. With BOB, this decrease varied
from 56% at 0.001% to 32% at 0.01%, and with Cet, it varied
from 57% at 0.001% to 31% at 0.01%. No significant difference
was found when the five quaternary ammoniums were compared using ANOVA.
Clb, EDTA, organomercurials (thi and PM), and MPHB did
not decrease membrane integrity after 15 minutes or after 24
hours of cell recovery for all the concentrations tested (data

FIGURE 1. Membrane integrity evaluation with neutral red test after

treatment with different concentrations of various quaternary ammoniums. (A) Fifteen minutes of treatment: There was a significant decrease of membrane integrity after 0.005% and 0.01% treatments. (B)
Fifteen minutes of treatment followed by 24 hours of cell recovery:
Significant cellular damage was found at concentrations of 0.001% and
higher. (A, B) *P 0.001 compared with control.

not shown). In Table 1 we present the mean values obtained

with all the concentrations tested for Clb , EDTA, organomercurials, and MPHB (no significant differences between the five
concentrations tested) and the mean values obtained with
0.005% and 0.01% BAC100, BAC58, BAC32, BOB, and Cet.

H2O2 Production with DCFH-DA Test

Significant ROS production was observed with all the quaternary ammoniums tested, even at lowest concentrations such as
0.00001% (Fig. 2). Maximum production was observed at
0.001% for BAC100 (mean fluorescence, 169% of the control),
BAC58 (mean fluorescence, 220% of the control), BAC32 (mean
fluorescence, 176% of the control), and Cet (mean fluorescence, 235% of the control). At higher concentrations, H2O2

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645

TABLE 1. Membrane Integrity Evaluations

Control (n 60)
Quaternary ammoniums,
0.005 and 0.01% (n 60)
Chlorobutanol (n 60)
EDTA (n 60)
Organomercurials (n 120)
MPHB (n 60)

15-Minute
Treatment

15-Minute
Treatment
Followed by
24 Hours of
Cell Recovery

100

100

24 4.3*
112 4.3
107 9.5
110 16.3
113 7.5

28 3.8*
97 10.7
92 16.3
91 18.8
101 11.3

Data are expressed as mean percentages of control (SD). Quaternary ammoniums: means of BAC, BOB, and Cet; organomercurials:
means of thi and PM; clb, EDTA, organomercurials, MPHB: means of all
concentrations tested.
* P 0.0001 compared with control and all other preservatives.
No difference between all other preservative groups.

production decreased, possibly because of the cytotoxicity

demonstrated by cell viability analysis. The maximum observed
with BOB appeared at 0.01% (mean fluorescence, 255% of the
control). Clb, EDTA, organomercurials, and MPHB also showed
significant H2O2 production, but the maximal production was
observed with thi (Fig. 3).
.
O
2 Production with Hydroethidine Test
.
Quaternary ammoniums induced an O
2 synthesis at 0.005%
and 0.01% with no difference among the three BAC formulations, BOB, and Cet, according to ANOVA. The maximum was
observed for all the quaternary ammoniums at 0.01%: 172%
with BAC100, 201% with BAC58, 172% with BAC32, 172% with
.
BOB, and 159% with Cet (Fig. 4). Significant O
2 production
was also observed with thi at 0.004% (mean fluorescence,
124%, P 0.001 compared with control) and clb at 0.05%
(mean fluorescence, 126%, P 0.001 compared with control),
but it was significantly less than the production observed with
quaternary ammoniums (P 0.0004 with thi compared with
quaternary ammoniums; P 0.005 with clb compared with
quaternary ammoniums; Fig. 5).

DNA Condensation Evaluation

All quaternary ammoniums and clb molecules induced a concentration-dependent increase of the fluorescence ratio after
15 minutes of treatment, although all the quaternary ammoniums tested did not produce the same intensity of fluorescence
compared with control (Figs. 6A, 6B). Mean fluorescence increased from 122% to 547% with BAC100, from 129% to 452%

FIGURE 2. H2O2 production evaluation after 15 minutes of treatment

with quaternary ammoniums. *P 0.001 compared with control.

FIGURE 3. H2O2 production evaluation after 15 minutes of treatment

with preservatives. *P 0.001 compared with control. Bar shading: 1,
1/1000 dilution; 2, 1/100 dilution; 3, 1/10 dilution; 4, 1/2 dilution; and
5, the higher concentration tested for all preservatives: 0.004% for thi,
0.001% for PM, 0.03% for MPHB, 0.05% for Clb, and 0.01% for EDTA.

with BAC58, from 100% to 361% with BAC32, from 109% to

367% with BOB, from 119% to 360% with Cet, and from 150%
to 260% with clb. Chromatin condensation observed with all
the quaternary ammoniums tested at 0.005% and 0.01% was
significantly higher than that with clb (mean fluorescence,
370% with quaternary ammoniums, 162% with clb; P 0.0001
for all concentrations tested). As shown in Figure 7 with
Hoechst staining, cells treated with quaternary ammoniums
showed, in a concentration-dependent manner, chromatin
condensation and fragmentation typical of apoptosis, when
compared with control cells. We present in Table 2 the mean
values obtained with all the concentrations tested for organomercurials, MPHB, and EDTA (no significant difference among
the five concentrations tested and among the four preservatives, according to ANOVA). The chromatin condensation observed was significantly less than that observed with the quaternary ammoniums molecules (mean fluorescence, 119% with
EDTA and MPHB and 128% with organomercurials versus 370%
with quaternary ammoniums, with P 0.0022 when EDTA or
MPHB were compared with quaternary ammoniums and P
0.0002 when organomercurials were compared with quaternary ammoniums).
After 24 hours of cell recovery (Fig. 6C), the quaternary
ammoniums still induced a marked increase of Hoechst fluorescence at 0.001% and higher, ranging from 165% with
BAC100 to 415% with BAC32. No significant difference was
observed between the five quaternary ammoniums. Quaternary ammoniums at 0.005% and 0.01% induced a significant
chromatin condensation compared with EDTA, organomercurials, and MPHB (mean fluorescence, 299% with quaternary
ammoniums at all concentrations of 0.005% and 0.01%, 121%
with EDTA, 118% with organomercurials, 117% with MPHB,

.
FIGURE 4. O
2 production evaluation after 15 minutes of treatment
with quaternary ammoniums. *P 0.001 compared with control.

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.
FIGURE 5. O
2 production evaluation after 15 minutes of treatment
with preservatives. *P 0.001 compared with control. Key to shading
is in Figure 3.

P 0.0001 for all values). The chromatin condensation observed with clb was less significant (mean fluorescence, 241%,
P 0.05) compared with quaternary ammonium molecules
(Table 2).

Cell Size Analysis

Concentration-dependent toxicity was confirmed by flow cytometry by the alteration of cell size after quaternary ammonium treatment (Fig. 8). Cells treated with 0.01% BAC58 had a
49% reduction of cell size in comparison with untreated cells.
No difference was observed among the five quaternary ammoniums. The other preservatives tested did not show any alteration of cell volume (data shown only for MPHB and three
concentrations of BAC58).

DNA Content
We measured sub-G1 cell population after 15 minutes of treatment with the different preservatives. Normal untreated cells
showed a 17% 5% sub-G1 population. The population of
sub-G1 cells was 42% 3%, 54% 7%, and 74% 9% for
0.0001%, 0.001%, and 0.01% quaternary ammonium treatments, respectively (Fig. 9). The other preservatives tested did
not show any significant increase in the apoptotic cell population (sub-G1 population varied from 17% to 23%; data shown
only for MPHB and three concentrations of BAC58).

Morphologic Changes
A concentration-dependent cell retraction was observed after
treatment with quaternary ammoniums molecules, whereas no
morphologic change was observed with the other preservatives tested (Fig. 10).

Correlation between the Different Tests

Performed on Preservatives
Membrane integrity and chromatin condensation showed significant negative correlation (r 0.863, P 0.0001; Fig. 11).
Poor but significant correlation was found between H2O2 production and membrane integrity or chromatin condensation
(H2O2 production versus membrane integrity: r 0.417, P
0.0003; H2O2 production versus chromatin condensation: r
0.269, P 0.0104). However, this correlation was only due
to the two highest concentrations of quaternary ammoniums
tested. A significant negative correlation was shown with mem.
brane integrity and O
2 production (r 0.551, P 0.0001).
The results obtained with the neutral red probe after 15 minutes of treatment were confirmed after 24 hours of cell recovery (r 0.770, P 0.0001). At the same time, chromatin
.
condensation was well correlated with the O
2 production (r

FIGURE 6. Chromatin condensation evaluation with Hoechst 33342

test after treatment with different concentrations of various preservatives. Fifteen minutes of treatment with (A) quaternary ammoniums: a
significant concentration-dependent increase in fluorescence was observed, indicating an increase in chromatin condensation; (B) clb: a
significant increase in fluorescence was found for all concentrations
tested; and (C) quaternary ammoniums followed by 24 hours of cell
recovery: chromatin condensation was still observed at 0.001% and
higher concentrations. *P 0.001 compared with control.

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647

FIGURE 7. Hoechst 33342 nuclear

staining of the cultured cells. (A)
Normal cell nuclei. Nuclei of cells
treated with (B) 0.03% MPHB: no
modifications compared with control; (C) 0.00001%, (D) 0.005%, and
(E) 0.01% BAC showing a characteristic apoptotic peripheral condensation and fragmentation of chromatin,
in a concentration-dependent manner. Magnification, 40.

0.738, P 0.0001) and was associated with the decrease of

membrane integrity after 24 hours of cell recovery (r
.
0.697, P 0.0001). H2O2 and O
2 productions, however,
were not correlated.

Inhibition Study
No cytotoxicity was observed after a 1-hour vitamin E treatment (Fig. 12). Membrane integrity and chromatin condensation were not altered, compared with complete culture medium alone. No ROS production was detected. After a 1-hour
vitamin E pretreatment followed by a 15-minute BAC 0.001%
treatment, there was no alteration of membrane integrity
(mean fluorescence, 88% with BAC 0.001% versus 120% with a
vitamin E pretreatment followed by a BAC treatment; P
0.001 compared with BAC). Significant decreases in chromatin
.
condensation, H2O2, and O
2 production were observed compared with BAC. However, after a vitamin E pretreatment
followed by a BAC treatment, H2O2 production was increased
(mean fluorescence, 146% with vitamin E with BAC versus
105% with BAC alone; P 0.001 compared with BAC),
.
whereas no significant difference was observed when O
2 productions were compared.

DISCUSSION
Cationic agents are used in pharmaceutical preparations for
antimicrobial preservation because of their ability to lyse miTABLE 2. Chromatin Condensation Evaluations

Control (n 60)
Quaternary ammoniums,
0.005 and 0.01% (n 60)
Clb (n 60)
EDTA (n 60)
Organomercurials (n 120)
MPHB (n 60)

15-Minute
Treatment

15-Minute
Treatment
Followed by
24 Hours of
Cell Recovery

100

100

370 18.2*
162 16.3*
119 10.2
128 18.1
119 18.0

299 19.3*
241 20.7*
121 10.4
118 17.6
117 17.7

See Table 1 for explanation of data.

crobial cellular membranes. The quaternary ammonium cationic surfactants (BAC, BOB, Cet) we tested have detergent-like
properties that may cause cell damage by emulsification of the
cell wall lipids. Numerous clinical and biologic side effects of
surfactant preservatives have been described, such as ocular
irritation (with lacrimation, hyperemia, photophobia, and
edema), punctate keratitis, gray corneal epithelial haze,
pseudomembrane formation, decreased corneal epithelial microvilli, and cytotoxicity to the corneal epithelial cells.1,2,5 In
addition, cell permeability caused by quaternary ammoniums is
potentiated when EDTA is used. BAC disrupts the bacterial
external membrane, and EDTA disorganizes the cell envelope.38
Organomercurials include thi, phenylmercuric nitrate or
acetate, and mercuric oxide. They bind to the cell membrane
protein sulfhydryl groups, causing an increase in cellular permeability. Adverse ocular side effects due to these preservatives are rare. The most striking side effect is mercurial deposits in various ocular tissues. These compounds have also
induced allergic reactions in sensitized persons; erythema,
edema, and hyperemia of the eyelids; or conjunctivitis. Thi,
however, has been shown to produce cytotoxicity for corneal
epithelial cells.39 44
Clb is mainly known to induce cytotoxicity in corneal epithelial cells. In previous studies, it was reported that occasional
use (twice daily up to 12 days) of a clb-preserved artificial tear
resulted in only modest exfoliation of corneal epithelial cells
(i.e., up to a maximum of 14%).45 These changes were reversible, and it was therefore suggested that the eye could adapt to
repeated use of these preserved artificial tears.46,47
Our results revealed new aspects of preservative toxicity.
After a 15-minute application of all the quaternary ammoniums
tested, membrane integrity of treated cells was altered,
whereas there was no variation of membrane integrity with the
other preservatives. The difference was significant among all
the quaternary ammoniums tested at 0.005% and 0.01% and
among the other preservatives, even at the highest concentrations (P 0.002 compared with quaternary ammoniums).
After a 0.01% quaternary ammonium treatment, cells showed
characteristics of immediate abundant lysis, with membrane
debris and low cell size on flow cytometric analysis graphs.
The effects of all the quaternary ammoniums tested were
progressive. There was a 75% decrease of membrane integrity

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IOVS, March 2001, Vol. 42, No. 3

FIGURE 8. Flow cytometric analysis

of cell size after 15 minutes of treatment: (A) Control, (B) MPHB 0.03%,
(C) BAC 104%, (D) BAC 103%, and
(E) BAC 102%. FS, forward scatter.

after a treatment with quaternary ammoniums at 0.005% and

0.01% that persisted after 24 hours. No significant difference
was observed among the five quaternary ammoniums analyzed,
and the three BAC tested were similar despite supposed differ-

ences in their antimicrobial activities. In contrast, the other

preservatives tested showed no alteration of membrane integrity, even at high concentrations and even after 24 hours of cell
recovery.

FIGURE 9. Flow cytometric analysis

of DNA content 15 minutes after cell
treatments: (A) Control (untreated
cells) DNA. The number of cells is
represented as a function of fluorescence (FL). The percentage of apoptotic cells (sub-G1 population) detected was (A) 20%, control; (B) 23%,
MPHB 0.03%; (C) 46%, BAC 104%;
(D) 53%, BAC 103%; and (E) 79%,
BAC 102%.

IOVS, March 2001, Vol. 42, No. 3

Preservatives and Conjunctival Cells

649

FIGURE 10. F-actin exploration using phalloidin. (A) Control cells and
cells treated with (B) BAC 104%,
(C) BAC 102%, and (D) Cet 102%.
A concentration-dependent decrease
of cell size associated with chromatin
condensation and cell disorganization was observed with quaternary
ammoniums tested at 102%. Magnification, 1000.

However, ROS evaluation showed that H2O2 production

increased with the noncytotoxic preservatives and with quaternary ammonium concentrations less than or equal to
0.001%, even though there was no alteration of cell viability,
whereas H2O2 production was decreased with quaternary am-

FIGURE 11. Correlations between the different tests performed. The

results obtained with all the preservatives tested are presented. The
different test compared are RN (neutral red): membrane integrity
evaluation; H2O2: hydrogen peroxide production evaluation; Hoechst:
.
chromatin condensation evaluation; and O
2 : peroxide anion production evaluation. The decrease of RN is correlated with an increase of
.
O
2 production and with an increase of Hoechst fluorescence. H2O2
variations cannot be interpreted.

moniums at high concentrations (0.005% and 0.01%), possibly

because of the decrease of cell viability observed for these two
.
concentrations. Concerning O
2 , there was a marked increase
in production with all quaternary ammoniums tested at 0.005%
and 0.01%, whereas a nonsignificant increase was observed at
.
the lowest concentrations. O
2 , but not H2O2, could therefore
play a role in the decrease of membrane integrity, because
.
these two parameters were well correlated. Furthermore, O
2
was also associated with chromatin condensationthese two
parameters being very significantly correlatedsuggesting that
.
O
2 may induce apoptosis or at least participate in epithelial
cell degeneration. In a cardiomyocyte model, ROS clearly induced apoptosis.48
Superoxide-generating systems have been demonstrated to
be cytotoxic for cultured cells, to degrade polysaccharides and
DNA, to promote peroxidation of membrane lipids, to alter

FIGURE 12. Evaluation of membrane integrity, chromatin condensation, and ROS production after a 1-hour vitamin E treatment and a
1-hour vitamin E treatment followed by a 15-minute BAC 0.001%
treatment. ***P 0.0001; **P 0.001; *P 0.05 compared with BAC
0.001%.

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Debbasch et al.

vascular permeability, and to potentiate inflammation.49 In

addition, these free radicals may play a significant role in the
generation of chemotactic factors and in augmentation of the
inflammatory response by inactivation of normally available
serum antiproteases that are known to neutralize the effects of
leukocytic proteases.50 It was also recently shown that topical
application of a free radical scavenger (ascorbic acid) decreased oxygen radical tissue damage after excimer keratectomy and reduced the acute inflammatory reaction efficiently.51 An inhibition study using a 1-hour vitamin E pretreatment followed by a 15-minute BAC 0.001% treatment was
.
conducted in our model. Chromatin condensation and O
2
production were significantly decreased compared with BAC
alone. The same levels of fluorescence were found after vitamin E treatment or vitamin E associated with BAC treatment.
H2O2 production was observed, whereas there was no alteration of membrane integrity. Reactive oxygen metabolites thus
appear to play an important role in cytotoxic effects and in the
amplification of the inflammatory process.
.
A slight O
2 production was also shown with clb and thi at
the highest concentrations tested, without any consequence
for membrane integrity, even though an increase of chromatin
condensation was observed with clb.
Only a few studies were performed to evaluate the extent of
oxygen radical damage to the ocular surface. Presence of oxygen free radicals has been demonstrated in the tear fluid of
patients with dry eye syndrome52 or in vivo after excimer laser
corneal surgery.5356 In a rabbit model, presence of lipid peroxidation was demonstrated in the superficial corneal stroma
after excimer surgery. The lipid peroxidation was hypothesized to be from oxygen free radicals generated by the infiltrating polymorphonuclear cells at the site of tissue damage.56
Furthermore, Shimmura et al.53 clearly identified the specific
species of radicals formed (OH.) by the excimer laser and the
cytotoxic effects on keratocytes in a contamination-free culture. They showed that hydroxyl radicals may be partly responsible for stromal fibroblast cell apoptosis after excimer laser
.
treatment. In our study, O
2 production was thus observed
when membrane integrity decreased (after 15 minutes and also
after 24 hours of cell recovery).
Whatever the mechanisms involved, these alterations observed with quaternary ammoniums occurred together with
morphologic modifications (cell size reduction, chromatin condensation, cytoskeleton retraction, increased sub-G1 population), all highly suggestive of the apoptotic process. According
to ECVAM recommendations, cellular DNA has been evaluated
using the Hoechst test. Hoechst 33342, a DNA fluorochrome,
unlike propidium iodide, is not excluded by live or apoptotic
cells. It has been observed that short exposure of cells to low
concentrations of Hoechst 33342 leads to strong labeling of
apoptotic cells.57 Live cells, however, require much longer
incubation with Hoechst to obtain a comparable fluorescence
intensity. Supravital uptake of Hoechst combined with exclusion of propidium iodide (to identify necrotic and late apoptotic cells) has been proposed as an assay of apoptosis.35 In the
present study, apoptosis was distinguished from necrosis using
the Hoechst 33342 and the neutral red tests. Cells in apoptosis
are characterized by decreased membrane integrity (detectable
using the neutral red test) and by chromatin condensation,
which increases Hoechst 33342 fluorescence.57,58 The combination of these two parameters does not occur in normal cells
or in cells undergoing necrosis. The quaternary ammoniums
tested showed a concentration-dependent increase of chromatin condensation (P 0.001 compared with the control at
0.005% and 0.01%) associated with a decrease of membrane
integrity. This result is in accordance with previous studies
conducted with quaternary ammoniums, in which different
techniques were used on the same cell line.25 This increase of

IOVS, March 2001, Vol. 42, No. 3

Hoechst 33342 fluorescence was also observed with clb at all
concentrations, although there was no alteration of the cell
.
cycle and of membrane integrity, only an increase in O
2 production at the higher concentration tested, whereas there was
a nonsignificant increase of fluorescence with EDTA, organomercurials, and MPHB. A concentration-dependent apoptotic
process was confirmed using flow cytometric analysis of DNA
content with quaternary ammoniums but was not obtained
with the other preservatives tested, even with clb.57
We have recently shown that BAC may induce two distinct
patterns of cell death: apoptosis and necrosis. Necrosis was
found to be induced by high concentrations, whereas apoptosis appeared at lower concentrations with an expression of the
apoptotic marker Apo 2.7 and typical apoptotic changes in
DNA content.21,25 A similar phenomenon may therefore occur
with the three different BAC solutions, BOB, and Cet.
Experimental models showed that BAC is, at least to a
large extent, responsible for toxic and/or immunoinflammatory effects on ocular structures.9,24 However, any extrapolation of information obtained from the WongKilbourne
cell line to the ocular surface must be made with caution.
The effects observed in vitro may be concentration dependent. Unlike the in vitro situation, in vivo there is almost
instantaneous dilution and continuous action of the lids.
Therefore, the concentrations used in vitro cannot be obtained in vivo, and consequently the toxic effects observed
in vitro may be less important in vivo. Furthermore, the
preocular mucin and glycocalyx, which is normally present
and protects the apical cell membrane in vivo may be absent
in the Chang cells in culture, explaining a higher susceptibility to these preservatives at the concentrations tested.
However, it was demonstrated that residual amounts of BAC
could also be detected in the conjunctival epithelium 9 days
after a single topical application.4 Furthermore, the presence of delayed tear clearance may elevate the tear concentration of these preservatives to toxic levels. This may explain why medicamentosus is common in patients with
delayed tear clearance.59 In addition, renewal of ocular
surface epithelia can explain, at least in part, the less important toxicity observed in vivo, although it has been
shown that preservatives induce corneal epithelial damage
and limbal and conjunctival infiltration by immunocompetent cells.60
Furthermore, interactions between inflammatory reactions
and apoptosis are present in the ocular surface.61 The present
study also showed a clear correlation between apoptosis and
superoxide anion production, which suggests that these reactive radical species may play an important role in the tissue
damage that occurs in ocular surface disorders. It remains to be
determined whether this free radical production constitutes
the origin or the consequence of the cytotoxicity of quaternary
ammoniums.
Inflammation, free radical production, and apoptosis therefore seem to be closely related processes in ocular cells. In the
near future, development of cytoprotective drugs, which could
protect the ocular surface from drug-induced toxicity, is of
great importance. Nevertheless, this study confirms that quaternary ammoniums, including the less extensively studied
BOB and Cet, should be avoided as far as possible, especially in
chronic ocular surface diseases such as glaucoma, dry eye, and
allergy. The other preservatives tested were found to be less
toxic and may thus be developed for prolonged use in eye
drops. Some preservative-free ophthalmic solutions are also
available in single-use doses or in multidoses.62,63 In the future,
they should be a successful substitute for the classic multidose
preparations.

IOVS, March 2001, Vol. 42, No. 3

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