Temporal Relationship between Lens Protein Oxidation and Cataract Development in

Streptozotocin-Induced Diabetic Rats

Z. KYSELOVA, S. J. GARCIA1 , A. GAJDOSIKOVA, A. GAJDOSIK AND M. STEFEK

Current evidence supports the view that cataractogenesis is a multifarious process, in which
combination of more closely linked events induces subtle post-translational modifications in
the lens structural proteins, enhancing their aggregation, fragmentation, and precipitation,
resulting eventually in lens opacification

Chronic hyperglycemia is a major determinant in the development of secondary complications of

diabetes, including diabetic cataract.

Although the etiology of cataract is not fully understood, oxidative damage to the constituents of
the eye lens is considered to be a major mechanism in the initiation and progression of various
types of cataracts, including diabetic cataract.8

STZ menjadi katarak

The data suggest that, although onset of cataract due to STZ-induced hyperglycemia was not
affected,

mengapa katarak
Chronic elevation of blood glucose in diabetes presents a severe risk factor for development of
cataract as one of the earliest secondary complications of diabetes mellitus.

Cataract is considered a major cause of visual impairment in diabetic patients as the incidence and
progression of cataract is elevated in patients with diabetes mellitus [5, 6]. The association between
diabetes and cataract formation has been shown in clinical epidemiological and basic research
studies. Due to increasing numbers of type 1 and type 2 diabetics worldwide, the incidence of
diabetic cataracts steadily rises. Even though cataract surgery, the most common surgical
ophthalmic procedure worldwide, is an effective cure, the elucidation of pathomechanisms to delay
or prevent the development of cataract in diabetic patients remains a challenge. Furthermore,
patients with diabetes mellitus have higher complication rates from cataract surgery [7]. Both
diabetes and cataract pose an enormous health and economic burden, particularly in developing
countries, where diabetes treatment is insufficient and cataract surgery often inaccessible [8].

Mengapa protein karbonil?

Accumulation of protein free carbonyls has been closely related to the development of both
senile and diabetic cataracts (Altomare et al. 1996, Vendemiale et al. 1999, Boscia et al.
2000, Chevion et al. 2000).

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In their study on human cataractous lenses, Boscia et al. (2000) identified a threshold of
protein oxidation above which clinically significant cataracts developed. They found protein
sulfhydryls below and protein carbonyls above their specific thresholds to be predictive for
the presence of cataract.

Increase of free carbonyls and decrease of free

sulfhydryls beyond the above mentioned values resulted in further steep increase in the rate of
advanced cataract development.

the presence of the increased

levels of free carbonyls in proteins of the cataractous diabetic eye lens can be interpreted as
a result of oxidative insult initiated by hyperglycemia.
Dosis STZ

Experimental diabetes was induced by a single i.v. dose of streptozotocin (STZ, 55 mg/kg).

Curcumin and Turmeric Delay Streptozotocin-Induced Diabetic Cataract in Rats

Palla Suryanarayana, Megha Saraswat, Tiruvalluru Mrudula, T. Prasanna Krishna,
Kamala Krishnaswamy, and G. Bhanuprakash Reddy (2005) Investigative Ophthalmology &
Visual Science

Wistar-NIN rats were selected and diabetes was induced by streptozotocin (35 mg/kg body weight,
intraperitoneally)

Mean : 40-45 mg/kgbb i.p

Observasi katarak
The progress of cataract was monitored weekly by an individual without prior knowledge of
affiliation of an animal to an experimental group. Eyes were inspected by hand-held slit-lamp,
preceded by topical administration of 1% mydriacyl drops. Cataract formation was scored on
the basis of 0 for absence of advanced cataract (clear lenses and cataractous lenses still
transparent) and 1 for presence of advanced cataract (opaque lens). No attempt was made to
grade the cataract, only a binomial paradigm was used: present or absent. Cataract formation was
considered complete when the red fundus reflex was no longer visible through any part of
the lens (Follansbee et al. 1997, Agardh et al. 2000).

Klasifikasi katarak
Eyes were examined every week using a slit lamp biomicroscope (Kowa Portable, Japan) on
dilated pupils.

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Initiation and progression of lenticular opacity was graded into five categories as follows:
Stage 0 - clear, clear lenses and no vacuoles present;
Stage 1 - vacuoles cover approximately one half of the surface of the anterior pole, forming a
subcapsular cataract;
Stage 2 - some vacuoles have disappeared and the cortex exhibits a hazy opacity;
Stage 3 - a hazy cortex remained and dense nuclear opacity is present
Stage 4 - a mature cataract is observed as a dense opacity in both cortex and nucleus.

Preparat lensa
At the indicated time intervals, the rats were killed, and the eye globes were excised. The
lenses were then dissected, rinsed with ice-cold saline and preserved deep-frozen under saline.
Each pair of lenses was homogenized in a glass homogenizer with a teflon pestle in 1.2 ml of
ice-cold phosphate buffer (20 mmol/l, pH 7.4) saturated with nitrogen. The total homogenate
was used for further analyses.

Pengukuran karbonil
Content of free carbonyls in the total lens proteins was determined by the procedure of Levine
et al. (1990) using the 2,4-dinitrophenylhydrazine (DNPH) reagent. Two aliquots of lens
homogenate of approximately 3 mg of proteins were precipitated with equal volume of 10%
trichloroacetic acid (TCA), and after centrifugation, the pellets were treated with 0.5 ml of 10
mmol/l DNPH dissolved in 2 mol/l HCl as a sample or with 0.5 ml of 2 mol/l HCl as a control
blank. The reaction mixtures were allowed to stand for 1 h at room temperature with stirring
at 10-min intervals. Next, 0.5 ml of ice-cold 20% TCA was added and left on ice for 15 min.
The precipitated proteins were subsequently washed three times with 1 ml of ethanol-ethyl
acetate (1:1). The washed pellets were dissolved overnight in 1.8 ml of 6 mol/l guanidine.
Any insoluble material was removed by centrifugation at 3000 rpm for 15 min. The difference
spectrum of the DNPH derivatives vs. HCl controls was scanned at 322-370 nm on Hewlett
Packard 8452A Diode Array Spectrophotometer. Carbonyl content was calculated from the
absorbance readings, using 22 000 l.mol-1cm-1 as the molar absorption coefficient. The final
values were normalized to actual protein amount determined on the basis of absorbance
readings at 280 nm of parallel HCl-treated control blank samples.

Kemunculan katarak pada rattus

the appearance of advanced cataract in diabetic rats became apparent after 10 weeks.

The onset of cataract was observed after 4 weeks by slit lamp

examination.

The onset of cataract was observed after 4 weeks by slit lamp examination.

Protein karbonil
The diabetic state led to an increase in free carbonyl groups of lens proteins as measured
by absorbance of DNPH bound to total lens proteins (Fig. 1b).

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kenapa DNPH
Lysine, arginine, proline, histidine and tryptophan are the amino acid residues most likely
to form carbonyl derivatives as a result of direct metal-catalyzed oxidation (Stadtman and
Berlett 1997). The conventional assay of protein carbonyls measures spectrophotometrically
the binding of 2,4-dinitrophenylhydrazine (DNPH) (Levine et al. 1990, Reznick and Packer
1994).

The
DNPH assay was found selective enough to discriminate between protein-bound carbonyls
produced by metal-catalyzed oxidations and those formed in the early glycation steps.

Ekstrak kejibeling
Hasil slit lamp microscope observations
indicated that these supplements delayed the progression
and maturation of cataract.

Diabetes causes increased oxidative stress in various tissues, as evidenced by increased levels of
oxidized DNA, proteins, and lipids, which are thought to play an important role in the pathogenesis
of various diabetic complications.9 Several studies have suggested that intake of antioxidant-rich
foods may slow the progression of cataract.10–13

Dietary intervention, particularly the use of traditional foods and medicines derived from natural
sources, is the mainstay in the management of diabetes. In this context, there has been a growing
interest in recent times in identifying as many dietary/ spice sources as possible for their ability to
control diabetes. 12,14,15 Nevertheless, most studies of natural sources have focused only on their
ability to maintain blood glucose levels, and have not been investigated for their beneficial effects
on secondary complications of diabetes such as cataract, retinopathy, nephropathy, and
neuropathy. Therefore, we have been interested in investigating various dietary sources for their
potential to prevent the secondary complications of diabetes, such as cataract.16–18

cara mengambil lensa mata

lenses were dissected by the posterior approach and stored at _70°C until further analysis.

Mekanisme katarak menjadi DM

Various biochemical parameters related to cataractogenesis such asoxidative stress/antioxidant
system, the polyol pathway, protein oxidation, protein content, and crystallin distribution were
studied.

Pathogenesis katarak diabetic

The enzyme aldose reductase (AR) catalyzes the reduction of glucose to sorbitol through the polyol
pathway, a process linked to the development of diabetic cataract. Extensive research has focused
on the central role of the AR pathway as the initiating factor in diabetic cataract formation. It has
been shown that the intracellular accumulation of sorbitol leads to osmotic changes resulting in
hydropic lens fibers that degenerate and form sugar cataracts [9, 10].

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In the lens, sorbitol is produced faster than it is converted to fructose by the enzyme sorbitol
dehydrogenase. In addition, the polar character of sorbitol prevents its intracellular removal
through diffusion. The increased accumulation of sorbitol creates a hyperosmotic effect that results
in an infusion of fluid to countervail the osmotic gradient. Animal studies have shown that the
intracellular accumulation of polyols leads to a collapse and liquefaction of lens fibers, which
ultimately results in the formation of lens opacities [9, 11]. These findings have led to the “Osmotic
Hypothesis” of sugar cataract formation, emphasizing that the intracellular increase of fluid in
response to AR-mediated accumulation of polyols results in lens swelling associated with complex
biochemical changes ultimately leading to cataract formation [9, 10, 12].

Furthermore, studies have shown that osmotic stress in the lens caused by sorbitol accumulation
[13] induces apoptosis in lens epithelial cells (LEC) [14] leading to the development of cataract
[15]. Transgenic hyperglycemic mice overexpressing AR and phospholipase D (PLD) genes
became susceptible to develop diabetic cataract in contrast to diabetic mice overexpressing PLD
alone, an enzyme with key functions in the osmoregulation of the lens [16]. These findings show
that impairments in the osmoregulation may render the lens susceptible to even small increases of
ARmediated osmotic stress, potentially leading to progressive cataract formation. The role of
osmotic stress is particularly important for the rapid cataract formation in young patients with type
1 diabetes mellitus [17, 18] due to the extensive swelling of cortical lens fibers [18]. A study
performed by Oishi et al. investigated whether AR is linked to the development of adult diabetic
cataracts [19]. Levels of AR in red blood cells of patients under 60 years of age with a short
duration of diabetes were positively correlated with the prevalence of posterior subcapsular
cataracts. A negative correlation has been shown in diabetic patients between the amount of AR
in erythrocytes and the density of lens epithelial cells, which are known to be decreased in diabetics
compared to nondiabetics suggesting a potential role of AR in this pathomechanism
[20].

The polyol pathway has been described as the primary mediator of diabetes-induced oxidative
stress in the lens [21]. Osmotic stress caused by the accumulation of sorbitol induces stress in the
endoplasmic reticulum (ER), the principal site of protein synthesis, ultimately leading to the
generation of free radicals. ER stress may also result from fluctuations of glucose levels initiating
an unfolded protein response (UPR) that generates reactive oxygen species (ROS) and causes
oxidative stress damage to lens fibers [22]. There are numerous recent publications that describe
oxidative stress damage to lens fibers by free radical scavengers in diabetics. However, there is no
evidence that these free radicals initiate the process of cataract formation but rather accelerate
and aggravate its development.

Hydrogen peroxide (H2O2) is elevated in the aqueous humor of diabetics and induces the
generation of hydroxyl radicals (OH–) after entering the lens through processes described as
Fenton reactions [23]. The free radical nitric oxide (NO•), another factor elevated in the diabetic
lens [24] and in the aqueous humor [25], may lead to an increased peroxynitrite formation, which
in turn induces cell damage due to its oxidizing properties. Furthermore, increased glucose levels
in the aqueous humor may induce glycation of lens proteins, a process resulting in the generation
of superoxide radicals (O2 −) and in the formation of advanced glycation endproducts (AGE) [26].

5
By interaction of AGE with cell surface receptors such as receptor for advanced glycation
endproducts in the epithelium of the lens further O2 − and H2O2 are generated [27].

In addition to increased levels of free radicals, diabetic lenses show an impaired antioxidant
capacity, increasing their susceptibility to oxidative stress. The loss of antioxidants is exacerbated
by glycation and inactivation of lens antioxidant enzymes like superoxide dismutases [28].
Copper-zink superoxide dismutase 1 (SOD1) is the most dominant superoxide dismutase
isoenzyme in the lens [29], which is important for the degradation of superoxide radicals (O2 −)
into hydrogen peroxide (H2O2) and oxygen [30]. The importance of SOD1 in the protection
against cataract development in the presence of diabetes mellitus has been shown in various in
vitro and in vivo animal studies [31–33]. In conclusion, a variety of publications support the
hypothesis that the initiating mechanism in diabetic cataract formation is the generation of polyols
from glucose by AR, which results in increased osmotic stress in the lens fibers leading to their
swelling and rupture.