Download Ability of Dorzolamide Hydrochloride and Timolol Maleate to Target

LABORATORY SCIENCES
Ability of Dorzolamide Hydrochloride
and Timolol Maleate to Target Mitochondria
in Glaucoma Therapy
Sergio Claudio Saccà, MD; Sebastiano La Maestra, PhD; Rosanna T. Micale, PhD; Patrizia Larghero, PhD;
Giorgia Travaini, PhD; Barbara Baluce, PhD; Alberto Izzotti, MD, PhD
Objective: To test the ability of dorzolamide hydrochloride and timolol maleate to display antioxidant effects.
presence of mitochondria-containing subcellular fractions and in young human TM cells with functional
mitochondria.
Methods: Antioxidant activity was tested in whole tra-
becular meshwork (TM) tissue as collected from corneal donors’ biopsy specimens, young (third passage) and
old (10th passage) human TM cells, and acellular systems composed of pure DNA and subcellular fractions
containing or devoid of mitochondria. Oxidative stress
was induced by hydrogen peroxide. Monitored end points
included DNA fragmentation as evaluated by the halo test,
oxidative DNA damage in terms of 8-hydroxy-2⬘deoxyguanosine, and mitochondrial function as evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide test.
Results: The antioxidant effect of dorzolamide and timo-
lol were observed on TM biopsy specimens and human
TM cells exposed to hydrogen peroxide. As evaluated in
cell subfractions, timolol displays antioxidant activity regardless of mitochondria presence. Conversely, the antioxidant activity of dorzolamide was maximized in the
G
Author Affiliations:
Ophthalmology Unit,
Department of Head/Neck
Pathologies, San Martino
Hospital (Dr Saccà), and
Department of Health Sciences,
Faculty of Medicine, University
of Genoa (Drs La Maestra,
Micale, Larghero, Travaini, and
Izzotti), Genoa, and
Department of Hygiene, Public
Health, and Preventive
Medicine, University of
Messina, Messina (Dr Baluce),
Italy.
Conclusions: The antioxidant effect of timolol was di-
rect. The antioxidant effect of dorzolamide involves mitochondria and is likely to be exerted mainly during the
early glaucoma phases when the mitochondrial damage
in the TM tissue still occurs at low levels.
Clinical Relevance: Timolol has an antioxidant effect
on the entire cell, whereas dorzolamide exerts protective activity toward oxidative stress only in the presence
of intact mitochondria (ie, in endothelial cells that are
younger when the cellular damage is still limited). The
important role of mitochondrial damage in primary openangle glaucoma is supported by the finding that mutant
myocilin impairs mitochondrial functions in human TM
meshwork cells.
Arch Ophthalmol. 2011;129(1):48-55
LAUCOMA, THE MOST COM-
mon cause of irreversible
blindness worldwide,1 is a
syndrome characterized
by a progressive optic atrophy that results from retina ganglion cell
death. The degenerative form of glaucoma
(ie, primary open-angle glaucoma [POAG])
is a complex disease that affects various eye
structures, including trabecular meshwork (TM) cells2,3 and their extracellular
matrix,4 blood vessels,5 and neurons located in the retina and the geniculate nuclei.6 Many risk factors and pathogenic
mechanisms are able to induce damage in
these structures during POAG. These
mechanisms include intraocular pressure
(IOP) increase,7 vascular damage,5 autoimmunity,8 metalloprotease activation,9 endothelin release,10 and nitric oxide delivery.11 Recent experimental evidence suggests
that long-lasting oxidative stress is a major
pathogenic mechanism for POAG.12,13 Free
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48
radicals trigger a variety of injurious pathways, finally resulting in glaucomatous optic neuropathy.14
The entire wall of the anterior chamber
of the eye, which is formed by the cornea,
the iris, and the TM between them, is coated
with endothelial cells.15 These cells constitute the inner component of the TM and are
in direct contact with oxidizing agents contained in the aqueous humor, such as hydrogen peroxide.14,16,17 The TM endothelial cells regulate aqueous outflow by actively
releasing ligands that, binding to Schlemm
canal endothelial cells, increase transendothelial flow, thereby facilitating the egress
of aqueous humor.18 The TM pores contribute to only 10% of the total aqueous outflow resistance,19 with most of the aqueous humor outflow being regulated by an
active mechanism.20
Our previous studies demonstrated increased oxidative DNA damage in the TM
of patients with glaucoma compared with
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A
B
Sham
Hydrogen Peroxide
Timolol Maleate and Hydrogen Peroxide
Dorzolamide Hydrochloride and Hydrogen Peroxide
Figure 1. Fragmentation of DNA as evaluated by the halo test. A, Fragmentation of DNA according to the nuclear spreading factor (ie, the ratio between the area of
the external nuclear halo [red circle] and that of the inner nucleus [black circle]). B, Human trabecular meshwork endothelial cells unexposed (sham) and exposed
to 25µM hydrogen peroxide by itself or in the presence of timolol maleate or dorzolamide hydrochloride (fluorescent microscopy, original magnification ⫻400).
that of unaffected control individuals.21,22 Therefore, we decided to investigate whether glaucoma medications such
as dorzolamide hydrochloride and timolol maleate have a
direct, intrinsic antioxidant effect on the TM.
Dorzolamide is a topical carbonic anhydrase (CA) inhibitor that displays significant IOP-lowering activity and
vasoactive effect.23 This issue was tested under various experimental conditions including the use of whole TM, human TM (HTM) cells, and mixtures of subcellular components. Experiments were performed directly on HTM
fragments collected from corneal donors. To explore the
mechanism of the detected antioxidant effect, experiments were also performed in HTM cells, representing the
first line of interaction with the aqueous humor in the anterior chamber of the eye. We also performed experiments in mitochondria-containing subcellular fractions because dorzolamide recognizes the CA enzyme as a main
target. In humans, 16 different CA isoforms were isolated,
which included CAI and CAII (cytosolic) and CA VA/VB
(mitochondrial isoforms).24 In particular, the cytosolic enzyme CAII plays a pivotal role in the regulation of IOP, with
its inhibition being an efficient method for regulating aqueous humor dynamics.25 The antioxidant activity of dorzolamide was compared with that of a ␤-blocking agent (ie,
timolol), whose antioxidant effects on endothelial cells have
been previously reported.26
Dorzolamide and timolol were tested for their ability
to counteract oxidative stress as induced by hydrogen peroxide at different doses. The end points monitored included oxidative DNA damage, DNA fragmentation, and
mitochondrial function. The level of oxidative DNA damage was tested by analyzing 8-hydroxy-2⬘-deoxyguanosine (8-oxo-dG), the most abundant DNA oxidative lesions demonstrated in the TM of patients with glaucoma.21
The 8-oxo-dG results from the interaction between the
hydroxyl radical OH and the C2 of guanine, resulting in
a hydroxylated guanine that, if unrepaired by specific gly-
cosylases, may cause G→A transversions.27 Fragmentation of DNA was evaluated by the halo test. Mitochondrial function was evaluated by analyzing the reduction
of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) salts. The MTT test is an indicator of cell viability and mitochondrial activity, 2 functions that are altered in glaucoma.
METHODS
TM SAMPLE COLLECTION
AND TREATMENT
Fresh ocular specimens collected from 5 corneal donors with
no history of ocular diseases were obtained from the Genoa Lions Eye Bank–Melvin Jones Foundation of Genoa, Italy. The
human samples were obtained and processed according to the
tenets of the Declaration of Helsinki.
The pigmented black stripe containing the complete TM,
from the Schwalbe line to the scleral spur and including the
Schlemm canal and the TM pigmented band, was cut away
(Figure 1). The TM tissue collected from each study participant was divided in 8 fragments and suspended in phosphatebuffered saline (PBS; pH 7.4) containing 10mM D-glucose to
evaluate oxidative stress as induced by hydrogen peroxide in
the absence or presence of dorzolamide and timolol before treatment for 30 minutes at 37°C.
Dorzolamide was provided in its pure form from Merck &
Co, Inc (Rahway, New Jersey). Timolol, devoid of denaturating preservatives such as benzalkonium chloride or similar
preservatives, was used as a commercially available drug
(Farmila-Thea Pharmaceuticals, Thissen, Belgium). Preliminary dose-response experiments were performed by means of
the halo test to determine that dorzolamide and timolol at the
dosage used did not induce toxic effects.
The TM tissue fragments as collected from each study participant were treated as follows: group 1, control individuals
(untreated); group 2, hydrogen peroxide (25µM and 100µM)
for 5 minutes; group 3, timolol (500µM) for 30 minutes fol-
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A
8-oxo-dG/105 Nucleotides
10
50µM
100µM
∗∗
8
∗∗
∗∗
6
∗
∗
††
4
†
∗
††
∗
∗
††
††
2
0
Sham
Hydrogen
Hydrogen
Hydrogen
Peroxide
Peroxide
Peroxide
Dorzolamide Timolol Dorzolamide
Maleate Plus Timolol
Maleate
EVALUATION OF DNA FRAGMENTATION
BY HALO TEST IN TM FRAGMENTS
8-oxo-dG/105 Nucleotides
B
Hydrogen
Peroxide
noDrop Technologies, Inc, Wilmington, Delaware). The
8-oxo-dG was detected by trifluoracetic acid enrichment, 32Ppostlabeling, monodirectional thin layer chromatography, and
electronic autoradiography, as previously reported21,28 (Figure 1).
Electronic autoradiography was used to identify 32P-labeled
8-oxo-dG; it was quantified by calculating the emitted ␤radiation using a 32P imager (Instant Inmager; Packard Bioscience Co, Meriden, Connecticut). Positive reference standards
were obtained by incubating calf thymus DNA with 1mM copper sulfate and 50mM hydrogen peroxide or using an authentic 8-oxo-dG reference standard (National Cancer Institute
Chemical Carcinogen Reference Standard Repository, Midwest Research Institute, Kansas City, Missouri). Samples without DNA were used as the negative control.
C
Figure 2. Inhibition of oxidative damage as induced by hydrogen peroxide in
trabecular meshwork (TM) specimens by timolol maleate and dorzolomide
hydrochloride. A, Histograms indicate mean (SD) 8-hydroxy-2⬘-deoxyguanosine
(8-oxo-dG) amounts in human TM fragments in the presence of 50µM
(light blue columns) and 100µM hydrogen peroxide (dark blue columns).
A significant antioxidant effect of dorzolamide and timolol is detected.
B, Corresponding oxidative DNA damage (8-oxo-dG) as detected by
phosphorus 32–postlabeling in human TM fragments collected from corneal
donors under basal conditions or after exposure to hydrogen peroxide by itself
or in the presence of dorzolamide and/or timolol. C, Fragments of TM as
collected from ocular ring specimens removed from corneal donors. The
pigmented black stripe (circled) containing the complete meshwork, from the
Schwalbe line to the scleral spur and including the Schlemm canal and the
pigmented band of the trabecular meshwork, was cut away and incubated with
oxidizing agents in the presence or absence of dorzolamide and timolol.
*P⬍.05 and ** P⬍.01 vs sham; †P⬍.05 and ††P⬍.01 vs hydrogen
peroxide.
Evaluation by the halo test of DNA fragmentation in TM fragments
took place, as described by Sestili and Cantoni29 (Figure 1). After
treatments, as previously described, TM fragments were washed
twice with PBS. Cells from treated TM fragments were obtained
after type 1A collagenase (Sigma Chemical Company) digestion
for 20 minutes at 37°C and centrifugation at 1200g for 10 minutes.
Cells were resuspended at a concentration of 2.0⫻104 cells/
100µL in 1.5% low-melting agarose-PBS and 5mM editic acid
(pH 7.2) at 37°C and sandwiched between an agarose-coated
slide and a coverslip. After gelling, the coverslips were removed and the slides immersed in a lysis buffer (2.5M sodium
chloride, 100mM editic acid, 10mM Tris, 1% sodium lauroyl
sarcosinate, 5% dimethylsulfoxide, 1% Triton X100, and 0.02M
sodium hydroxide), pH 12.5, for 20 minutes on ice. The slides
were then incubated for 15 minutes in an alkaline hypotonic
buffer (0.1M sodium hydroxide and 1mM editic acid), pH 12.5,
washed with 0.4M Tris hydrochloride, pH 7.5, and stained with
10 µg/mL of ethidium bromide or SYBR Green dye (Invitrogen Corporation, Carlsbad, California) for 5 minutes. Fluorescent-labeled DNA was visualized using an Olympus BX51TF
fluorescence microscope equipped with a digital camera (Camedia C-4040; Olympus America Inc, Center Valley, Pennsylvania). Images of at least 100 randomly selected nuclei were
acquired and analyzed by ImageJ software (National Institutes
of Health, Bethesda, Maryland; http://rsb.info.nih.gov
/nih-image/). Damage to DNA was expressed as a nuclear spreading factor, which is the ratio between the area of the outer halo
(total fluorescent area minus nucleus area) and that of the
nucleus (nucleus area) (Figure 2). A nuclear spreading factor greater than 1 indicates the occurrence of nuclear DNA
fragmentation.
CELL LINE CULTURE AND TREATMENT
lowed by hydrogen peroxide (50µM and 100µM) for 5 minutes; group 4, dorzolamide (500µM) for 30 minutes followed
by hydrogen peroxide (25µM and 100µM) for 5 minutes; and
group 5, dorzolamide (500µM) and timolol (500µM) for 30 minutes followed by hydrogen peroxide (25µM and 100µM) for 5
minutes. Monitored end points included oxidative DNA damage as evaluated by phosphorus 32 (32P)–postlabeling and DNA
fragmentation as evaluated by the halo test.
ANALYSIS OF 8-OXO-dG
BY 32P-POSTLABELING
After treatment, the TM fragments were homogenized and DNA
extracted (GenePure; Sigma Chemical Company, St Louis, Missouri) was quantified by fiber optic spectrophotometry (Na-
The HTM cells isolated from the juxtacanalicular and corneoscleral region were supplied by ScienCell Research Laboratories (Carlsbad, California). The HTM cells were grown in polyL-lysine–coated flask culture (2 µg/cm2) in fibroblast medium
with 2% (vol/vol) fetal bovine serum, 1% (vol/vol) fibroblast
growth supplement, and 1% (vol/vol) penicillin-streptomycin
solution. Cells were maintained at 37°C in a humidified atmosphere with 5% carbon dioxide to reach semiconfluence (80%90%). The medium was supplied with fresh culture medium
every 24 hours. When an 80% to 90% of confluence was attained, the monolayers were subcultured or used for experiments by means of trypsin (Sigma Chemical Company).
The cells were pretreated with drugs before oxidative stress
as induced by hydrogen peroxide as previously described for
HTM biopsies (groups 1-8). However, dorzolamide and timo-
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lol were used at 250µM and hydrogen peroxide at 10 and 25µM,
respectively, for 10 minutes. These doses and experimental conditions have been selected to avoid any toxic effect, as evaluated by the trypan blue survival test.
After incubation, the medium was removed and cells were
washed twice in phosphate buffer, pH 7.4, gently scraped, and
suspended (20⫻103 cells per well) in complete medium. Monitored end points included DNA fragmentation (evaluated by
the halo test), as previously described, and mitochondrial function (per the MTT test). The effects of drugs and oxidizing agents
on HTM cells at various aging stages were comparatively evaluated in cells at the third vs 10th passage.
ANALYSIS OF MITOCHONDRIAL FUNCTION
BY MTT TEST IN HTM CELLS
Mitochondrial function was evaluated by analyzing the reduction of MTT salts to purple formazan crystals occurring in cells
with functional mitochondria owing to a specific dehydrognase. The HTM cells were seeded in 24-well microtiter tissue
culture plates (3 ⫻ 104 cells per well) and incubated in complete medium. After 48 hours the monolayer cells were pretreated with dorzolamide and timolol, followed by oxidative
stress induced by hydrogen peroxide. Experimental group and
drug pretreatments were performed as described. After treatment, the medium was removed and the monolayer was washed
with PBS solution (pH 7.4) containing 10mM D-glucose. The
MTT solution (0.5 mg/mL) was added, and the plates were incubated for 3 hours at 37°C in 5% carbon dioxide. After this
period, the supernatant was discarded and the solubilization
solution (HEPES buffer [Sigma Chemical Company] 50mM/
ethanol 1:9, pH 8) was added. The MTT reduction was detected by quantifying its reduced derivative by evaluating absorbance at 590 nm using a fiber optic spectrophotometer
(NanoDrop Technologies, Inc).30 Results are expressed as the
percentage of decrease in the ability of reducing MTT in treated
cells compared with 100% referred to untreated controls.
Statistical analyses were performed using the t test for unpaired data (StatView software, version 3.0; Abacus Concepts,
Berkley, California). Reported results are mean (SE) of at least
3 independent experiments as performed for each experimental condition tested.
hydrogen peroxide (50µM); group 5, DNA, S12, and hydrogen
peroxide; group 6, DNA, S105, and hydrogen peroxide; group 7,
DNA, hydrogen peroxide, and dorzolamide (500µM); group 8,
DNA, S12, hydrogen peroxide, and dorzolamide; group 9, DNA,
S105, hydrogen peroxide, and dorzolamide; group 10, DNA, hydrogen peroxide, and timolol (500µM); group 11, DNA, S12, hydrogen peroxide, and timolol; group 12, DNA, S105, hydrogen
peroxide, and timolol; group 13, DNA, hydrogen peroxide, dorzolamide, and timolol; group 14, DNA, S12, hydrogen peroxide,
dorzolamide, and timolol; and group 15, DNA, S105, hydrogen
peroxide, dorzolamide, and timolol.
At the end of the treatments, hydrogen peroxide was evaporated by heating at 50°C for 5 minutes and DNA was purified
by proteinase K (Boehringer Ingelheim GmbH, Mannheim, Germany) digestion and immunoaffinity column chromatography using a commercially available kit (GenePure) in the presence of antioxidant (dithiothreitol). Extracted DNA was
quantified by fiber optic spectrophotometry (NanoDrop Technologies Inc) evaluating absorbance at 260 and 280 nm. Purified DNA samples showed a 260/280 ratio greater than 1.75 and
less than 1.90, which was assumed as an indicator of DNA purity. Samples were stored at –80°C until 8-oxo-dG quantification by 32P-postlabeling, as previously described. All reported
experiments have been performed in 3 independent analyses,
and results are expressed as mean (SD).
RESULTS
EVALUATION OF DRUG ANTIOXIDANT
ACTIVITY IN TM BIOPSIES
Figure 2 shows an example of the results obtained by analyzing with 32P-postlabeling 8-oxo-dG formation in TM tissue undergoing oxidative stress and/or drug pretreatments.
Quantitative results are also reported in this figure. Dorzolamide was effective in protecting DNA of TM cells from
hydrogen peroxide used at high and very high concentrations. Timolol was protective only at high hydrogen peroxide concentrations. Similar results were obtained in TM
samples undergoing oxidative stress by analyzing DNA fragmentation by means of the halo test (Figure 3).
EVALUATION OF ANTIOXIDANT EFFECTS
IN CELLULAR SUBFRACTIONS
To obtain cellular subfractions, Sprague Dawley rats (Charles
River Laboratories International Inc, Wilmington, Massachusetts) were killed, their livers were removed and homogenized
in saccharose Tris 4/1 (vol/vol), and cellular subfraction pellets were collected after 12 000g centrifugation for 30 minutes
at 4°C (S12 fraction). Cellular subfraction pellets collected after 105 000g centrifugation for 30 minutes at 4°C (S105) were
also collected. The ribosomal protein S12 includes mitochondria, whereas S105 is the postmitochondrial cytosolic fraction
(ie, microsomes) devoid of mitochondria.
Cellular subfractions and DNA (calf thymus DNA; Sigma
Chemical Company) were exposed to oxidative stress in the
absence or presence of dorzolamide and timolol. In this manner, we analyzed the antioxidant ability of dorzolamide and timolol in the absence or presence of mitochondria.
In the presence of cellular subfractions, DNA was treated for
5 minutes and calculated after hydrogen peroxide addition at room
temperature in a final volume of 1 mL under the following experimental conditions: group 1, untreated DNA (100µg); group
2, untreated DNA (100µg) plus S12 (2-mg proteins); group 3,
untreated DNA plus S105 (200-mg proteins); group 4, DNA and
EVALUATION OF ANTIOXIDANT
ACTIVITY IN HTM CELLS
The HTM cells were severely affected by oxidative damage, as observed by analyzing all monitored end points.
Hydrogen peroxide induces oxidative DNA damage in
nuclear DNA, as demonstrated by the increased DNA fragmentation (Figure 4). Hydrogen peroxide also induced failure in mitochondrial function, as demonstrated by the decreased ability of these organelles to
reduce MTT salts (Table 1).
Dorzolamide and timolol exerted protective effects
(Figure 4 and Table 1). The antioxidant effect of dorzolamide at the low dose of hydrogen peroxide (10µM) was
higher (P⬍.01) than that displayed by timolol. At higher
doses of hydrogen peroxide (25µM), the protective effect
of the 2 drugs was comparable but lower than that displayed by dorzolamide at the low dose of oxidative stress.
No addictive effect was observed when the 2 drugs were
used in combination. Protective properties displayed by
dorzolamide were affected by cell aging because this drug
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played antioxidant effects independently of the cellular
subfraction used. When used in combination, the protective effect of the 2 drugs was slightly increased
(Table 2).
was effective in protecting young (third passage) but not
old (10th passage) cells.
EVALUATION OF ANTIOXIDANT ACTIVITY
ON DNA IN THE PRESENCE
OF CELLULAR SUBFRACTIONS
DISCUSSION
A remarkable oxidative effect on DNA was induced by
hydrogen peroxide. This effect was attenuated in the presence of S105 and, to a greater extent, of S12 (Table 2).
Dorzolamide and timolol were able to attenuate hydrogen peroxide effects to a similar extent. The antioxidant
effect of dorzolamide was remarkably increased in the
presence of the mitochondria containing S12 (Table 2).
This situation was not observed for timolol, which dis-
The results of this study provide evidence that dorzolamide and timolol display antioxidant effects, protecting the whole TM as collected from human donors. ReTable 1. Antioxidant Effects of Dorzolamide Hydrochloride
and Timolol Maleate vs Hydrogen Peroxide
Experimental Group
3
∗∗
Sham
Hydrogen peroxide, 10µM
Hydrogen peroxide, 25µM
Dorzolamide
Timolol
Dorzolamide and timolol
Dorzolamide and hydrogen
peroxide, 10µM
Timolol and hydrogen
peroxide, 10µM
Dorzolamide, timolol, and
hydrogen peroxide, 10µM
Dorzolamide and hydrogen
peroxide, 25µM
Timolol and hydrogen
peroxide, 25µM
Dorzolamide, timolol, and
hydrogen peroxide, 25µM
∗∗
∗
†
†
∗
†
NSF
2
1
Do Sh
a
Tim rzol m
Tim ol am
i
ol ol M de
ol
an alea
Do d
te
rz Ma
ol le
am at
id e
e
Hy
dr
og
en
Pe
Do rox
i
Tim rzol de
Tim ol am
i
ol ol M de
ol
an alea
Do d
te
rz Ma
ol le
am at
id e
e
0
100µM
Hydrogen Peroxide
Figure 3. Fragmentation of DNA as evaluated by the halo test in trabecular
meshwork fragments exposed to oxidative stress in the absence or presence
of dorzolamide hydrochloride and/or timolol maleate. The histogram reports
quantitative results expressed as mean (SD) nuclear spreading factor (NSF).
Both drugs show an antioxidant activity on trabecular meshwork. These data
confirm those previously obtained with analyzing by 8-hydroxy-2⬘-deoxyguanosine formation. The vertical line conveys DNA as evaluated by the halo
test calculating the NSF. *P ⬍.05 and **P ⬍ .01 vs sham; †P⬍ .05 vs hydrogen peroxide.
HTM Third
Passage,
Mean (SD), %
HTM 10th
Passage,
Mean (SD), %
100 (1.91)
70.03 (0.78) b
65.21 (1.21) b
93.56 (0.33)
89.27 (0.74) d
107.14 (2.56)
88.61 (1.10) d,e
100 (0.95) a
60.05 (0.68) b
55.11 (0.37) c
101.65 (0.35)
91.30 (0.60)
86.02 (0.67) d
58.17 (0.96) b
75.45 (1.13) d
54.52 (0.21) b
87.90 (1.39) d,e
54.05 (0.32) b
78.67 (1.43) d,f
51.82 (0.41) c
75.75 (0.90) d,f
50.88 (0.28) b
82.24 (1.76) d,f
52.29 (0.41) c
Abbreviations: HTM, human trabecular meshwork;
MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide.
a MTT 50% compared with sham third passage; P ⬍ .05 vs sham.
b P ⬍ .01 vs sham.
c P ⬍ .001 vs sham.
d P ⬍ .05 vs hydrogen peroxide.
e P ⬍ .01 vs hydrogen peroxide, 10µM.
f P ⬍ .001 vs hydrogen peroxide, 25µM.
∗
10
∗
∗
8
†
∗
∗
††
NSF
6
∗
§§
∗
§§
§§
∗
††
4
2
∗
10µM
Hydrogen Peroxide
en
Pe
Do rox
id
r
z
Ti
ol e
Tim mol am
i
o
de
l
ol
ol Ma
an lea
Do d
te
rz Ma
ol le
am at
id e
e
og
dr
Hy
en
Pe
Do rox
id
r
z
Ti
ol e
Tim mol am
id
o
l
e
ol
ol Ma
lea
a
Do nd
t
e
rz Ma
ol le
am at
id e
e
og
dr
Hy
Do Sh
a
Tim rzol m
Tim ol am
i
ol ol M de
ol
an alea
Do d
te
r z Ma
ol le
am at
id e
e
0
25µM
Hydrogen Peroxide
Figure 4. Fragmentation of DNA as evaluated by the halo test in human trabecular meshwork cells exposed to oxidative stress (10µM and 25µM hydrogen
peroxide concentration) in the absence or presence of dorzolamide hydrochloride and/or timolol maleate. Histogram reports quantitative mean (SD) results
expressed as nuclear spreading factor (NSF). The vertical line conveys DNA fragmentation as evaluated by the halo test calculating the NSF. *P ⬍ .001 vs sham;
†P ⬍ .05 and ††P ⬍.001 vs 10µM hydrogen peroxide; §P⬍.05, and §§P⬍ .001 vs 25µM hydrogen peroxide.
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cently, the antioxidant properties of antiglaucomatous
drugs have gained attention. Timolol has been reported
to display antioxidant protective effects in endothelial
cells.26 These results were recently confirmed by Miyamoto et al,31 who demonstrated that TM cells showed reduced sensitivity to hydrogen peroxide when cells were
treated with timolol. Our study provides evidence that dorzolamide displays antioxidant effects in TM and HTM cells.
Dorzolamide exerts more relevant antioxidant properties
than timolol when active mitochondria are present in the
experimental system. Mitochondria have been suggested
to play a major role in glaucoma development.32 The TM
POAG cells display defective mitochondrial function33 and
dysfunction in intracellular calcium regulation.34 A recent in vivo study35 performed in 235 individuals demonstrates that mitochondria undergo severe alterations and
progressive loss in the TM of patients with glaucoma compared with healthy controls.
Furthermore, the important role of mitochondrial damage in POAG is supported by the finding that mutant myocilin impairs mitochondrial functions in HTM cells36 and
may confer different sensitivity to oxidative stress depending on the mutation.37 Our data suggest that topical therapy with dorzolamide counteracts the adverse consequences of oxidative damage as occurring in whole TM
and in its endothelial component.
The antioxidant effects of dorzolamide are maximized in the presence of intact mitochondria, which areexpected to be pivotal intracellular targets for the antioxidant properties of this drug.38 Dorzolamide displays
more remarkable antioxidant effects in young HTM cells,
which have good mitochondrial function, than in older
cells, which have poor mitochondrial function. Accordingly, it is conceivable that the antioxidant effect of dorzolamide is maximized when the drug targets stillfunctional TM cells, whereas it is negligible when targeting
TM tissue devoid of significant mitochondrial function,
which characterizes advanced stages of glaucoma. It is
conceivable that dorzolamide therapy at early glaucoma
stages fully displays the multiple mechanisms of this drug,
including antioxidant effects. Conversely, the same drug
could be less effective when administered at late stages
of glaucoma.
Dorzolamide exerts its therapeutic effects through multiple mechanisms, including improvement of ocular perfusion,39-41 thus decreasing optic nerve sensitivity to IOPinduced damages. 42-46 Our results indicate that an
additional mechanism displayed by this drug is direct protection from oxidative stress exerted, targeting CA and
mitochondria. At the level of the central nervous system, CA inhibition exerts a relaxing effect directly on the
optic nerve vessels, as demonstrated by the finding that
dorzolamide induces dilatation of retinal arterioles.47 This
situation results in the reduction of retinal neural cell damage, thus supporting the view that dorzolamide can be
considered a neuroprotectant.33 This mechanism probably occurs through the protection against the induction of oxidative stress secondary to intracellular pH alterations.48 Oxidative stress is closely linked to acidification
in mitochondria and in the cytoplasm49 and is able to induce retinal ganglion cell death by apoptosis.50 Dorzolamide, as an antioxidant, could induce protection against
retinal ganglion cell loss. Similar mechanisms could contribute to the explanation of why dorzolamide but not
timolol increases blood flow in the optic nerve head and
choroid after 6 months of treatment.41 Timolol antioxidant effects are exerted through their own metabolism27
by inducing in endothelial cells the expression of peroxiredoxin-2 through the activation of the FOXO3a transcription factor.31
Oxidative stress plays a fundamental role in glaucoma pathogenesis, and its effect on TM has been dem-
Table 2. Effect of Oxidative Stress and Dorzolamide Hydrochloride and/or Timolol Maleate on 8-oxo-dG Formation
as Evaluated in the Presence of Cell Subfractions by Phosphorus 32 (32P)–Postlabeling
Experimental Group
Control individuals
Hydrogen peroxide
Dorzolamide and hydrogen peroxide
Timolol and hydrogen peroxide
Dorzolamide, timolol, and hydrogen peroxide
Treatment
8-oxo-dG 32P-Postlabeling
(8-oxo-dG Molecules/105 Nucleotides) a
DNA
DNA and S12
DNA and S105
DNA and hydrogen peroxide
DNA, S12, and hydrogen peroxide
DNA, S105, and hydrogen peroxide
DNA, hydrogen peroxide, and dorzolamide
DNA, S12, hydrogen peroxide, and dorzolamide
DNA, S105, hydrogen peroxide, and dorzolamide
DNA, hydrogen peroxide, and timolol
DNA, S12, hydrogen peroxide, and timolol
DNA, S105, hydrogen peroxide, and timolol
DNA, hydrogen peroxide, dorzolamide, and timolol
DNA, S12, hydrogen peroxide, dorzolamide, and timolol
DNA, S105, hydrogen peroxide, dorzolamide, and timolol
1.32 (0.21)
1.09 (0.02)
1.24 (0.19)
17.14 (2.05) b
8.02 (0.43) c
10.41 (0.26) b
7.02 (0.63) d
2.07 (0.15) e
6.34 (0.53) d
5.92 (0.46) d
5.28 (0.54) d
6.04 (0.33) d
4.80 (0.18) e
3.56 (0.14) d
4.85 (0.12) d
Abbreviations: 8-oxo-dG, 8-hydroxy-2⬘-deoxyguanosine; S12, cellular subfraction pellets collected after 12 000g centrifugation for 30 minutes at 4°C;
S105, cellular subfraction pellets collected after 105 000g centrifugation for 30 minutes at 4°C.
a Values are mean (SE) of 3 independent experiments.
b P ⬍ .01 vs controls.
c P ⬍ .05 vs controls.
d P ⬍ .05 vs hydrogen peroxide.
e P ⬍ .01 vs hydrogen peroxide exposed.
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onstrated.51 In fact, oxidative DNA damage is remarkably increased in this region in patients with glaucoma21,22
and was proposed to be responsible for diminishing TM
cellularity,52 altering TM intracellular cytoskeletal structures,17 and contributing to neuronal cell death in the optic nerve head.53 Neural degeneration extends beyond the
retinal ganglion cells, involving neurons of the lateral geniculate nucleus of the brain which, during glaucoma,
are affected by peroxynitrite-mediated oxidative cell injury6 and glutamate toxicity.54
Drugs that display antioxidant effects are, in principle, able to counteract many of these oxidative-related
mechanisms. Accordingly, evaluating the antioxidant potential of antiglaucomatous drugs is relevant when addressing their clinical use, taking into account that TM
is the most sensitive tissue of the anterior chamber to oxidative damage, as evaluated by ex vivo testing with hydrogen peroxide.55
In conclusion, reported results indicate that dorzolamide and timolol exert antioxidant protective effects
on HTM. The antioxidant properties of these drugs, developed to inhibit aqueous humor production by the ciliary epithelia exerted in the TM, are likely to be a major
determinant of their therapeutic properties. However, reported antioxidant effects are drug specific, not class specific. Further experiments will be performed to verify
whether these effects are shared by other ␤-blockers or
CA inhibitors. The antioxidant effects of dorzolamide were
exerted toward high and low hydrogen peroxide concentrations, whereas timolol was protective only toward low hydrogen peroxide concentrations. This difference is related to the different properties and
pharmacokinetic characteristics of these drugs.56 Timolol has direct antioxidant effects related to its own metabolism.21 Conversely, dorzolamide exerts protective activity mainly in the presence of intact mitochondria. These
findings suggest that dorzolamide should be used in the
treatment of glaucoma when TM damage is not advanced and the trabecular cells have a certain viability
and intact mitochondrial function.
Submitted for Publication: July 6, 2010; final revision
received July 9, 2010; accepted July 9, 2010.
Corresponding Author: Sergio Claudio Saccà, MD, Ophthalmology Unit, Department of Head/Neck Pathologies, San Martino Hospital, Viale Benedetto XV, 16132
Genoa, Italy ([email protected]).
Financial Disclosure: None reported.
Funding/Support: This study was supported by grants
from The Glaucoma Foundation.
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Ophthalmic Images
Traumatic Anterior Subluxation of Natural Lens
With Aniridia and Blood Lining Descemet Folds
Mathew Giegengack, MD
Shree K. Kurup, MD
A man was struck with a garden hose in the right eye (Figure). The trauma
resulted in limbal laceration, 360° iridodialysis, and expulsion of iris through
the wound with anterior lens dislocation. Seven days later, corneal edema
with Descemet folds containing blood trapped within were seen.
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