Protection of a lens of an eye against the simulated diabetic cataract
Катаракта является очень распространенным осложнением при диабете. Мы подвергли бычие линзы действию высокой концентрации глюкозы (450 мг.%) в условиях культуры в течение двух недель и исследовали повреждения в линзе и возможную защиту специальными антиоксидантами N ацетил L цистеином (NAC) и цинков...
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Фізико-хімічний інститут ім. О.В. Богатського НАН України
2010
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irk-123456789-232442013-02-13T03:24:40Z Protection of a lens of an eye against the simulated diabetic cataract Bormusov, E. Dovrat, A. Chevion, M. Медицина транспорта Катаракта является очень распространенным осложнением при диабете. Мы подвергли бычие линзы действию высокой концентрации глюкозы (450 мг.%) в условиях культуры в течение двух недель и исследовали повреждения в линзе и возможную защиту специальными антиоксидантами N ацетил L цистеином (NAC) и цинковым комплексом desferrioxamine (DFO), селективным хелатором для железа. Исследовалось оптическое качество линз и окисление эпителия с дихлорфлюоресцеином (DCF), а также оценивались изменения в 2D гель электрофоретическом профиле белков хрусталика. Наблюдались изменения при высоком содержании глюкозы в фокусном расстоянии линзы, повышении окисления. NAC И Zn DFO почти полностью защищали линзы; DFO показывал только частичную защиту. Результаты демонстрировали, что антиоксиданты могут защищають хрусталик повреждающего действия высоких концентраций глюкозы. Комбинация NAC и ZnDFO действовала более эффективно. Катаракта є дуже поширеним ускладненням при діабеті. Ми піддали кришталик ока бика дії високої концентрації глюкози (450 мг %) в умовах культури протягом двох тижнів і досліджували пошкодження в лінзі і можливий захист спеціальними антиоксидантами N ацетил L цистеином (NAC) і цинковим комплексом desferrioxamine (DFO), селективним хелатором для заліза. Досліджувалася оптична якість лінз і окислення епітелію з дихлорфлюоресцеїном (DCF), а також оцінювалися зміни в 2D гель електрофоретичному профілі білків кришталика. Спостерігалися зміни при високому вмісті глюкози у фокусній відстані лінзи, підвищенні окислення. NAC І Zn DFO майже повністю захищали лінзи; DFO показував тільки частковий захист. Результати демонстрували, що антиоксиданти можуть защищають кришталик від ушкоджувальної дії високих концентрацій глюкози. Комбінація NAC і ZNDFO діяла ефективніше. 2010 Article Protection of a lens of an eye against the simulated diabetic cataract / E. Bormusov, A. Dovrat, M. Chevion // Актуальні проблеми транспортної медицини. — 2010. — № 4. — С. 68-79. — Бібліогр.: 44 назв. — англ. 1818-9385 http://dspace.nbuv.gov.ua/handle/123456789/23244 617.741-004.1 en Актуальні проблеми транспортної медицини Фізико-хімічний інститут ім. О.В. Богатського НАН України |
| institution |
Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| collection |
DSpace DC |
| language |
English |
| topic |
Медицина транспорта Медицина транспорта |
| spellingShingle |
Медицина транспорта Медицина транспорта Bormusov, E. Dovrat, A. Chevion, M. Protection of a lens of an eye against the simulated diabetic cataract Актуальні проблеми транспортної медицини |
| description |
Катаракта является очень распространенным осложнением при диабете. Мы подвергли бычие линзы действию высокой концентрации глюкозы (450 мг.%) в условиях культуры в течение двух недель и исследовали повреждения в линзе и возможную защиту специальными антиоксидантами N ацетил L цистеином (NAC) и цинковым комплексом desferrioxamine (DFO), селективным хелатором для железа. Исследовалось оптическое качество линз и окисление эпителия с дихлорфлюоресцеином (DCF), а также оценивались изменения в 2D гель
электрофоретическом профиле белков хрусталика. Наблюдались изменения при высоком содержании глюкозы в фокусном расстоянии линзы, повышении окисления. NAC И Zn DFO почти полностью защищали линзы; DFO показывал только частичную защиту. Результаты демонстрировали, что антиоксиданты могут защищають хрусталик повреждающего действия высоких концентраций глюкозы. Комбинация NAC и ZnDFO действовала более эффективно. |
| format |
Article |
| author |
Bormusov, E. Dovrat, A. Chevion, M. |
| author_facet |
Bormusov, E. Dovrat, A. Chevion, M. |
| author_sort |
Bormusov, E. |
| title |
Protection of a lens of an eye against the simulated diabetic cataract |
| title_short |
Protection of a lens of an eye against the simulated diabetic cataract |
| title_full |
Protection of a lens of an eye against the simulated diabetic cataract |
| title_fullStr |
Protection of a lens of an eye against the simulated diabetic cataract |
| title_full_unstemmed |
Protection of a lens of an eye against the simulated diabetic cataract |
| title_sort |
protection of a lens of an eye against the simulated diabetic cataract |
| publisher |
Фізико-хімічний інститут ім. О.В. Богатського НАН України |
| publishDate |
2010 |
| topic_facet |
Медицина транспорта |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/23244 |
| citation_txt |
Protection of a lens of an eye against the simulated diabetic cataract / E. Bormusov, A. Dovrat, M. Chevion // Актуальні проблеми транспортної медицини. — 2010. — № 4. — С. 68-79. — Бібліогр.: 44 назв. — англ. |
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Актуальні проблеми транспортної медицини |
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2025-07-03T02:40:15Z |
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2025-07-03T02:40:15Z |
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| fulltext |
АКТУАЛЬНЫЕ ПРОБЛЕМЫ ТРАНСПОРТНОЙ МЕДИЦИНЫ № 4 (22), 2010 г.
68
ACTUAL PROBLEMS OF TRANSPORT MEDICINE #4 (22), 2010
Резюме
ОСОБЕННОСТИ ВТОРИЧНОЙ
ПРОФИЛАКТИКИ ИШЕМИЧЕСКОЙ
БОЛЕЗНИ СЕРДЦА У РАБОЧИХ
ТРАНСПОРТА
Телятников О.В.
Обследовано 52 больных со стено?
кардией напряжения ИИ?ИИИ ФК на
фоне артериальной гипертензии,
атерогеноq дислипопротеинемии и сер?
дечной недостаточности, которые прини?
мали аторвастатин в суточных дозах 10 и
20 мг, что привело к повышению уровня
NO?синтазы, уровня нитратов и снижения
нитритов на фоне базисной терапии, в
сравнении с базисной терапией без
аторвастатина.
Ключевые слова: статины, NО#синтаза,
нитраты, нитриты, ишемическая бо#
лезнь сердца.
Summary
THE FEATURES OF SECOND
PROPHYLAXIS OF ISCHEMIC HEART
TROUBLE FOR TRANSPORT WORKERS
Telyatnikov O.V.
Examinated 52 patients with
Stenocardia of tention II?III Functional
Classes (FC) with arterial hipertention
(AH),heart insufficiency and atherogenic
dislipoproteinemia, who took atorvastatin
(daily dose?20 mg), what led to increase of
NO?sуntase activity, content of nitrites had
decreased, content of nitrates had
increased. In another group of patients, that
didn’t receive statins, wasn’t observed
increase of activity NO?sуntase in blood
plasma.
Key words: statins, NO#sуntase, nitrites,
nitrates, ischemic heart disease
Впервые поступила в редакцию 29.08.2010 г.
Рекомендована к печати на заседании
редакционной коллегии после рецензирования
Abstract
Oxidative stress represents a
mechanism which could lead to diabetic
cataract. We exposed bovine lenses in culture
conditions for two weeks to high glucose
concentration (450 mg%) and investigated
the damage to the lens and possible
protection by special antioxidants ? N?acetyl?
L?cysteine (NAC) and the zinc complex of
desferrioxamine (DFO), a selective chelator
for iron. We monitored the optical quality of
the lenses and the oxidation of the epithelium
with dichlorofluorescein (DCF) assay, as well
as the changes in lens proteins profile by 2D
gel electrophoresis. Under high glucose
changes in lens focal length, increased
oxidation, and changes in lens crystalline
were observed. NAC and Zn? DFO nearly
completely protected the lenses; DFO
showed only partial protection. The results
demonstrated that antioxidants should be
considered as treatment modality protecting
the lens from high glucose damage. It is
proposed that a combination of NAC and Zn/
DFO could prove highly efficient.
Introduction
Cataract is a highly prevalent
complication in diabetes. Several
mechanisms explaining the formation of
diabetic cataract have been proposed,
including those that involve oxidative stress,
as a causative factor. The “Free Radical
УДК 617.741#004.1
PROTECTION OF A LENS OF AN EYE AGAINST THE SIMULATED
DIABETIC CATARACT
Elvira Bormusov, Ahuva Dovrat and Mordechai Chevion
Rappaport Faculty of Medicine, Technion, Haifa, Israel Faculties of Medicine and
Dental Medicine, the Hebrew University of Jerusalem
Key words: diabetic cataract, protection of a lens
ACTUAL PROBLEMS OF TRANSPORT MEDICINE #4 (22), 2010
АКТУАЛЬНЫЕ ПРОБЛЕМЫ ТРАНСПОРТНОЙ МЕДИЦИНЫ № 4 (22), 2010 г.
69
Theory of Aging”, first proposed in 1956 by
Denham Harman [1] (reviewed by Harman
2003[2]), suggests that aging results from
accumulation of changes caused by highly
reactive oxygen?derived species (ROS),
including free radicals, known to induce cell
damage [3]. Continuous exposure of the lens
to oxidative stress has been shown to lead
to reduced lens clarity and cataract formation
[4]. Since the epithelial layer contains the
bulk of the metabolic enzymes, and since
labile iron has been incriminated as a
necessary factor for injurious oxidative
stress, the damage to the epithelial layer can
precede and account for the development of
lens opacity [5]. Indeed, the levels of
redoxactive iron were found elevated in the
advanced forms of cataract [6].
Desferrioxiamine (Desferal®, DFO) is
a selective high affinity iron chelator,
rendering iron as a stable ferric complex [7].
DFO has been often used as a means of
reducing tissue oxidative stress and injury.
Reddan et al. demonstrated that DFO
protects cultured rabbit lens epithelial cell
from oxidative insult [8]. Avunduk et al.
showed the effectiveness of DFO in
preventing cataractous changes in rat lenses
following in vivo exposure of rats to oxidative
stress [7]. We prepared the complex of DFO
with zinc and gallium [9?13]. These
complexes are similar to the ferrioxamine
(Fe(III)?DFO complex), but show three
additional advantages: (i) they better infiltrate
into cells, (ii) are less toxic than DFO alone,
and (iii) act via the combination of both ‘push’
and ‘pull’ mechanisms. Thus, we examined
the protective effect of these DFO complexes
on cataract formation in bovine lens, under
conditions simulating the diabetic state. We
have used an additional alternative strategy
for curbing ROS?induced injury and
protection of the lens. This involved the
employment of N?acetyl cysteine (NAC), to
scavenge free radicals [14?16] and to
replenish reduced sulfhydryl residues [14?
19]. NAC is a precursor of glutathione – the
major source of cellular sulfhydryl groups and
acts as a potent an anti?inflammatory agent.
Experimentally, we incubated intact bovine
lenses in culture, in the presence of high
glucose concentrations (450 mg %),
mimicking the diabetic state, and examined
the protection bestowed by the DFO
complexes and NAC, on the injurious
processes on the lenses. We used a unique
system of intact bovine lenses, maintained
for a long?term, under culture conditions.
This system allows for direct exposure to a
pre?set glucose concentration, and
monitoring the effects on lens transparency
with a highly sensitive optical method [20].
The system can detect early optical damage
to lenses, which cannot be detected by other
methods often used. At the completion of the
culture period lens epithelium and lens
proteins were analyzed. It was anticipated
that the extent of protection exerted on the
lens, in this system, will be indicative of the
involvement of iron as a catalyst and of free
radicals as causative agents, in the damage
to the diabetic lens.
Materials and Methods
Lens organ culture system:
Lenses were excised in a delicate
operation from eyes obtained from 1?year?
old male calves under sterile conditions, 2?
4 hours after enucleating. From each animal
one eye is used for experimental treatment
and the other eye serves as control. Each
lens is placed in a specially designed culture
container, which we have developed [21].
The culture medium consists of M199 with
Earl’s balanced salt solution, supplemented
with 5.96g/L HEPES, 3% dialyzed fetal calf
serum and antibiotics (penicillin 100 U/ml
and streptomycin 0.1 mg/ml). Our intact lens
culture system mimics the lens conditions
inside the eye and makes it possible to keep
lenses for long?term studies for several
weeks in order to test the effects of
potentially damaging agents. The lenses
were incubated at 35°C.
Experimental treatments were initiated
after pre?incubation for 24 hours. Damaged
lenses and their matched controls from the
contra lateral eyes were excluded prior to
experimental treatment. The culture medium
is replaced every 24 hours.
Lens optical quality monitoring system
An automated scanning laser system
[21] was used for daily testing of both treated
and control lenses. A 670 nm diode laser with
the beam parallel to the axis of the lens is
directed towards the cultured lens along one
АКТУАЛЬНЫЕ ПРОБЛЕМЫ ТРАНСПОРТНОЙ МЕДИЦИНЫ № 4 (22), 2010 г.
70
ACTUAL PROBLEMS OF TRANSPORT MEDICINE #4 (22), 2010
meridian. After passing through the lens, the
laser beam is refracted and the system
determines the back vertex focal length for
every beam position. Each scan consists of
measurements of the same beam from 22
different points across the lens. A lens of
good optical quality is able to focus the laser
beam from various locations. When the lens
is damaged, its ability to focus the laser beam
at various locations is altered.
High glucose concentration (simulating
diabetes): Lenses were exposed to 450 mg%
glucose in the culture medium, which
simulates diabetes conditions.
Preparation of lens epithelial samples
for dichlorofluorescein (DCF) assay
There were preparations of a forward
monolayer epithelium bovine lenses from all
experiments. For this purpose the capsule
opened and crystalline lens fibers were
cleaned. On the object�plate here was only
a capsule and a cellular monolayer
epithelium.
Reactive Oxygen Species (ROS) was
discovered by flow of epithelial cells lens
labeled with 5�(and 6�)chloromethyl�2',7'�
dichlorodihydrofluorescein diacetate, acetyl
ester (CM�H
2
DCFDA,
C6827) to measure
the component level of
cellular oxidation in the
cells of lens
epithelium. When is
added oxidation to
cells this reagent
undergoes and
converts in the
fluorescent isomer.
The fluorescent signal
was detected with a
f l u o r e s c e n c e
microscope, using
sources of excitement
and filters,
corresponding to for
fluorescein.
Protein analysis
using two�dimensional
polyacrylamide gel
electrophoresis was
done according to
[22].
Protein concentration was measured
by the micro�method of Lowry [23]
Results and Discussion
The experiments included 78 intact
bovine lenses, which were divided into eight
different treatment groups. These groups
include lenses incubated, for 12 days, with
high glucose levels (450 mg %) with or
without each one of the antioxidants tested
(NAC, Zn/DFO and DFO), as well as control
lenses. Lens optical quality was analyzed
every 24 hours. Figure1 demonstrates the
changes in the optical quality of the lenses
along the incubation period. In the control
group no significant change in the Back
Vertex Distance (BVD) with time of
incubation, was observed. Lenses incubated
with high glucose showed fluctuations in the
BVD along the incubation period, which
indicate changes in lens volume. High
fluctuations appear also in lenses treated
with high glucose and NAC. The group
exposed to ZnDFO and to DFO alone,
demonstrated reduced optical changes
representing smaller lens injury. The lenses
show almost no volume changes.
Other studies also demonstrated
Fig. 1. Lens optical quality during the twelve days of incubation of intact lenses in cul-
ture conditions, demonstrated by "Back Vertex Distance". Control lenses show almost
no change in Back Vertex Distance with time in culture (red), while glucose treated
lenses show variability in Back Vertex Distance which reflect changes in lens volume.
ACTUAL PROBLEMS OF TRANSPORT MEDICINE #4 (22), 2010
АКТУАЛЬНЫЕ ПРОБЛЕМЫ ТРАНСПОРТНОЙ МЕДИЦИНЫ № 4 (22), 2010 г.
71
changes in lens optical quality in diabetes
[24] measured lens opacity in diabetic
patients using a back�light scattering
quantification system. Lens opacity was
significantly higher in diabetic patients than
in the control group, and showed correlation
with glycated hemoglobin levels. Freel [25]
compared cytoplasmic textures from a
variety of human and animal lenses in
electron microscope images in order to relate
the extent of roughness with the extent of
opacification. Lens cytoplasms exhibiting the
greatest roughness correlated with the
greatest light scattering.
Tkachov [26] described
histomorphological changes in the
cataractous lens of diabetic patients using
Scheimpflug densitometry and light
microscopy. Their study revealed smaller cell
density of the lens, larger cell area of lens
epithelium, and a lower nucleusplasma ratio
in cataractous lenses of diabetics compared
to clear non�diabetic lenses. In our study at
the end of the culture period, lenses were
photographed by inverted microscope.
Figure 2a shows a photograph of a control
lens with clear sutures and homogeneous
epithelial cell layer. Figure 2b demonstrates
the severe damage to the lens, when
incubated in the presence of high glucose,
which is indicated by the blisters under and
between the epithelial cells.
Photographs of the lens epithelial layer
with higher magnification (Figure 3) show that
for the controls an intact lens epithelial layer
is observed (Figure 3a). Glucose treated
lenses show swollen epithelial cells with
blisters at the cells borders (Figure 3b). Each
of the three antioxidants used in the study
reduces the glucose�induced damage to the
lens epithelium (Figures 3c�3e).
The antioxidant protective effect of
each of the three agents were compared
using the 5,6� chloromethyl�2',7'�
dichlorodihydrofluorescein diacetate (DCF)
assay. Formation of ROS in the epithelium
was monitored and detected, by
fluorescence, in intact bovine epithelial cells
layers, from the different treatment groups.
Figure 4a shows a molecule of non
fluorescent (reduced and acetylated) DCF.
The di�acetyl ester of DCF is cell
permeable and undergoes hydrolysis within
cells, thus remain trapped within the cells.
While the DCF is a poorly fluorescent
molecule, upon its oxidation by ROS DCF is
converted to a highly fluorescent compound
that can be easily monitored. Figure 4b
demonstrates control lens epithelium after
incubation of the intact lens for 12 days in
culture. There is almost no fluorescence in
the cells; minor fluorescence can be
detected in the cells nuclei. On the other
hand, glucose treated lenses epithelium
show very high fluorescence, in the swollen
epithelial cells (Figure 4c).
DFO prevents the cytosolic oxidation,
but high oxidation was observed within the
nuclei (Figure 4d). ZnDFO provided better
protection against oxidation than DFO or
NAC. The epithelium from glucose�plus�
Fig 2a - Inverted Microscope photograph of control lens
after 12 days incubation in organ culture conditions.
(Magnification x25) Note the clear lens with lens sutures.
Fig 2b - Inverted Microscope photograph of glucose
treated lens (450 mg%), after 12 days incubation in organ
culture conditions. The photo show high glucose damage
as bubbles at the lens surface. (Magnification x25).
АКТУАЛЬНЫЕ ПРОБЛЕМЫ ТРАНСПОРТНОЙ МЕДИЦИНЫ № 4 (22), 2010 г.
72
ACTUAL PROBLEMS OF TRANSPORT MEDICINE #4 (22), 2010
ZnDFO treated lenses look like the controls
(Figure 4e). Glucose and NAC treated lens
epithelium showed swollen cells with less
oxidation than in glucose�alone treated lens
epithelium (Figure 4f).
Fig 4d – Glucose and DFO treated lens
epithelium after 12 days in organ culture
conditions. DFO reduced the oxidation levels
in the cells. Note that the main oxidation
remained in the nuclei of the cells
In order to follow the effects of
exposure to high glucose together with the
antioxidants on the cells nuclei, the nuclei
were stained with propidium iodide, an
intercalating DNA staining fluorescent agent
(Figure 5). Control lens epithelium show
Fig 3a. Inverted microscope photograph of control
lens epithelium after 12 days incubation in organ
culture conditions. (Magnification x 100) Note the
similar size of the cells and the borders between
the cells
Fig 3b. Inverted microscope photograph of glucose
treated lens epithelium after 12 days incubation in
organ culture conditions. (Magnification x 100) note
the swelling cells with different size and the
bubbles between the cells
Fig 3c Inverted microscope photograph of glucose
and DFO treated lens epithelium after 12 days in-
cubation in organ culture conditions. (Magnification
x 100) glucose damage was reduced by DFO
treatment
.
Fig 3d. Inverted microscope photograph of glucose
and ZnDFO treated lens epithelium after 12 days
incubation in organ culture conditions. (Magnifica-
tion x 100) glucose damage was reduced by
ZnDFO treatment
Fig 3e Inverted microscope photograph of glucose and
NAC treated lens epithelium after 12 days incubation in
organ culture conditions. (Magnification x 100) glucose
damage was reduced by NAC treatment
ACTUAL PROBLEMS OF TRANSPORT MEDICINE #4 (22), 2010
АКТУАЛЬНЫЕ ПРОБЛЕМЫ ТРАНСПОРТНОЙ МЕДИЦИНЫ № 4 (22), 2010 г.
73
intact cells and intact nuclei (Figure 5a);
glucose treated lens epithelium show swollen
cells with broken nuclei (Figure 5b). DFO and
NAC slightly reduced the damage to the cells
nuclei (Figures 5c &5e), while Zn/DFO totally
prevent the damage. The epithelium from
lenses treated with glucose in the presence
of ZnDFO looks the same as the controls
(Figure 5d).
The injurious effects of high glucose
and the protective effects of the three
antioxidants under study, on the profile of
lens soluble proteins and their intactness
were examined by 2D gel electrophoresis
analysis (Figure 6). Protein profile of control
lens soluble proteins is demonstrated in
Figure 6a. The molecular weight of lens
soluble proteins is below 32kDa. Incubation
of the lenses in the presence of 450mg%
glucose for 12 days reduce the amount of
Fig 4a. Non-fluorescent DCF
Fig 4b. Control lens epithelium after 12 days in or-
gan culture conditions. Note the low fluorescence
of the epithelial cells
Fig 4c.Glucose treated lens epithelium after 12
days in organ culture conditions. Note the high
fluoresce in the epithelial cells which indicate high
oxidation
Fig 4d. Glucose and DFO treated lens epithelium
after 12 days in organ culture conditions. DFO re-
duced the oxidation levels in the cells. Note that
the main oxidation remained in the nuclei of the
cells
Fig 4e. Glucose and ZnDFO treated lens epithe-
lium after 12 days in organ culture conditions.
ZnDFO prevents the oxidation in the cells and the
cells looks like the controls
Fig 4f . Glucose and NAC treated lens epithelium
after 12 days in organ culture conditions.
АКТУАЛЬНЫЕ ПРОБЛЕМЫ ТРАНСПОРТНОЙ МЕДИЦИНЫ № 4 (22), 2010 г.
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ACTUAL PROBLEMS OF TRANSPORT MEDICINE #4 (22), 2010
soluble proteins mainly at the basic area of
the gel, the area of gamma crystalline (Figure
6b) Incubation of the glucose treated lenses
in the presence of DFO partially protect lens
proteins as demonstrated in Figure 6c.
Almost no protection to lens soluble proteins
was provided in the presence of ZnDFO
(Figure 6d). On the other handfull protection
from glucose damage was provided in the
presence of NAC. Lenses incubated in high
glucose in the presence of NAC show the
same soluble protein profile as the controls.
(Figure 6e).
We have demonstrated a role for
oxidative damage in diabetic cataract
formation. This is in accord with previous
proposals [27�29]. Thus, the possible use of
antioxidant agents in cataract prevention and
treatment is highly appealing. Future
investigations should consider this possibility
in human subjects. It is likely that NAC and
Zn/DFO (rather than DFO alone) could
provide a beneficial outcome.
In summary, we used a unique
Fig 5a. Control lens epithelium after 12 days in or-
gan culture conditions
Fig 5b. Glucose treated lens epithelium after 12
days in organ culture conditions
Fig 5c. Glucose and DFO treated lens epithelium
after 12 days in organ culture conditions
Fig 5d . Glucose and ZnDFO treated lens epithe-
lium after 12 days in organ culture conditions
Fig 5e. Glucose and NAC treated lens epithelium
after 12 days in organ culture conditions
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АКТУАЛЬНЫЕ ПРОБЛЕМЫ ТРАНСПОРТНОЙ МЕДИЦИНЫ № 4 (22), 2010 г.
75
experimental lens culture system equipped
with highly sensitive and reproducible
detection monitoring instrumentation. When
lenses were exposed to diabetic�like
conditions, under incubation for 12 days in
culture, an oxidative damage developed. This
was in contrast to incubation of lenses under
normo�glycemic medium, where the lenses
remained clear and un�affected. When NAC
was included in the incubation medium, no
protein alterations were developed. Likewise
Zn/DFO provided complete protection
against oxidative injury. DFO alone provided
good, but limited, protection. We propose
that these antioxidants, in particularly NAC
and Zn/DFO should be considered as
preventative treatment in diabetic patients, in
order to delay or avoid, and possible treat
ocular opacity. Indeed, In vitro and in vivo
studies have demonstrated that NAC, a thiol
compound that is used clinically possesses
not only antioxidant properties, but also anti�
inflammatory and vasodilatory properties.
NAC acts as a cysteine pro�drug and a GSH
Fig 6a. Lens soluble protein profile (2D gel electrophore-
sis) of control lens after 12 day incubation in culture con-
ditions.
Fig 6b. Lens soluble protein profile (2D gel electro-
phoresis) of glucose (450 mg%) treated lens after
12 day incubation in culture conditions. Note the
missing proteins mainly at the basic side of the gel,
the area of gamma crystalline
Fig 6c. Lens soluble protein profile (2D gel electrophore-
sis) of glucose (450 mg%) + DFO treated lens after 12
day incubation in culture conditions. Note the missing
proteins mainly at the basic side of the gel, the area of
gamma crystalline
Fig 6d. Lens soluble protein profile (2D gel electro-
phoresis) of glucose (450 mg%) + ZnDFO treated
lens after 12 day incubation in culture conditions.
Note the missing proteins mainly at the basic side of
the gel, the area of gamma crystalline
Fig 6e. Lens soluble protein profile (2D gel electrophoresis)
of glucose (450 mg%) + NAC treated lens after 12 day in-
cubation in culture conditions. Note the protective effect of
NAC on lens proteins.
АКТУАЛЬНЫЕ ПРОБЛЕМЫ ТРАНСПОРТНОЙ МЕДИЦИНЫ № 4 (22), 2010 г.
76
ACTUAL PROBLEMS OF TRANSPORT MEDICINE #4 (22), 2010
precursor [17]. It can reduce disulfide bonds
in proteins[14] [18], scavenge free radicals
[15] and bind metals to form complexes [19].
This can explain our results, where the
unusual protective effect of NAC on lenticular
epithelium, was observed.
Further research is needed to examine
whether within the intact animal NAC will
prove as efficient. This is since
pharmacokinetics studies have shown that
NAC undergoes extensive first pass
metabolism in the liver and kidneys resulting
in low concentrations of ‘free’ NAC in the
plasma [30] and [31].
In earlier studies using the same
model with non�diabetic lenses, we
demonstrated that lens periphery is the
most sensitive area first damaged by
exposure to hyperbaric oxygen tension.
While DFO proved only partially beneficial in
salvaging the lens periphery from oxidative
damage, Zn/DFO led to complete protective
benefit to the lens anterior pole, the part
most relevant for sight preservation [32, 33].
These observations were in accord with
other publications demonstrating the
beneficial effects of iron chelating agents
against oxidative insult�induced tissue
damage [7, 8, 34�37]. Typically, high dose
of DFO mesylate (Desferal®) was
administred, subcutaneously (10 mg/kg of
body weight), for lomg term(up to 90
consecutive days), prevented cataractous
changes in rats. The disadvantage of DFO
in these studies stems from its limited tissue
penetration [38, 39]. Indeed, Zn/DFO easily
infiltrates into cells and tissues [9, 32, 40]
and is, thus, a better protective drug, as is
evident also under conditions that simulate
diabetes.
The unusual activity of Zn/DFO is
based on the combination of ‘push and pull’
mechanisms [10, 41]. Desferrioxamine
neutralized the redox activity of iron by
binding labile ferric iron and ‘pulling out’ iron
from its (low affinity) binding sites. Zinc ion
is liberated from the complex during the
exchange of the zinc with iron, and the ‘free’
zinc acts as a secondary antioxidant,
‘pushing out’ additional iron from binding
sites, thus, inhibiting the catalysis of the
formation of highly reactive ROS. The Zn/
DFO complex carries additional attributes
which make it more effective – it infiltrates
into cells, and eliminates the toxicity of free
DFO.
It is a well accepted notion that oxygen
and ROS play key roles in senile cataract
formation. This view is supported by former
observations demonstrating the rapid
development of cataract under conditions of
high oxygen load in humans treated by
hyperbaric oxygen [42]. Oxygen is also
believed to be one of the potential causative
agents for the development of nuclear
cataract following vitrectomy [43, 44]. Under
normal clinical circumstances the effects of
oxygen�load accumulate over many years of
exposure to relatively low oxygen loads. We
have shown that increasing the partial
pressure of oxygen, for a relatively short
time causes a sharp increase in the damage
to the lens.
Conclusions
Based on the independent effects of
NAC and Zn/DFO, we propose to use their
combination as a means of prevention and/
or treatment of cataract, in diabetic patients.
This combination can be applied by topically
drops or systemically by ingestion. It is
anticipated that in both ways the
antioxidants will get access to the lens
through the posterior chamber of the
aqueous humor. While the risk of using such
a combination is very limited, further
investigation is needed in order to
crystallize, in detail, the efficiency and
protocol/s of treatment of human subjects.
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Реферат
ЗАЩИТА ХРУСТАЛИКА ГЛАЗА ОТ
ИМИТИРОВАННОЙ ДИАБЕТИЧЕСКОЙ
КАТАРАКТЫ
Эльвира Бормусова, Аува Доврат и
Мордехай Чевион
Катаракта является очень распрос�
траненным осложнением при диабете.
Мы подвергли бычие линзы действию вы�
сокой концентрации глюкозы (450 мг.%)
в условиях культуры в течение двух не�
дель и исследовали повреждения в лин�
зе и возможную защиту специальными
антиоксидантами � N�ацетил�L�цистеи�
ном (NAC) и цинковым комплексом
desferrioxamine (DFO), селективным хела�
тором для железа. Исследовалось опти�
ческое качество линз и окисление эпите�
лия с дихлорфлюоресцеином (DCF), а
также оценивались изменения в 2D гель�
электрофоретическом профиле белков
хрусталика. Наблюдались изменения при
высоком содержании глюкозы в фокус�
ном расстоянии линзы, повышении окис�
ления. NAC И Zn DFO почти полностью
защищали линзы; DFO показывал только
частичную защиту. Результаты демонст�
рировали, что антиоксиданты могут за�
щищають хрусталик повреждающего
действия высоких концентраций глюко�
зы. Комбинация NAC и ZnDFO действо�
вала более эффективно.
Ключевые слова: хрусталик, диабети�
ческая катаракта
Резюме
ЗАХИСТ КРИШТАЛИКА ОКА ВІД
ІМІТОВАНОЇ ДІАБЕТИЧНОЇ КАТАРАКТИ.
Ельвіра Бормусова, Аува Доврат і
Мордехай Чевіон
Катаракта є дуже поширеним уск�
ладненням при діабеті. Ми піддали криш�
талик ока бика дії високої концентрації
глюкози (450 мг %) в умовах культури
протягом двох тижнів і досліджували по�
шкодження в лінзі і можливий захист
спеціальними антиоксидантами � N�аце�
тил�L�цистеином (NAC) і цинковим комп�
лексом desferrioxamine (DFO), селектив�
ним хелатором для заліза. Досліджувала�
ся оптична якість лінз і окислення епіте�
лію з дихлорфлюоресцеїном (DCF), а та�
кож оцінювалися зміни в 2D гель�елект�
рофоретичному профілі білків криштали�
ка. Спостерігалися зміни при високому
вмісті глюкози у фокусній відстані лінзи,
підвищенні окислення. NAC І Zn DFO май�
же повністю захищали лінзи; DFO пока�
зував тільки частковий захист. Результа�
ти демонстрували, що антиоксиданти мо�
жуть защищають кришталик від ушкоджу�
вальної дії високих концентрацій глюко�
зи. Комбінація NAC і ZNDFO діяла ефек�
тивніше.
Ключові слова: кришталик ока, діабе�
тична катаракта
Впервые поступила в редакцию 22.07.2010 г.
Рекомендована к печати на заседании
редакционной коллегии после рецензирования
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