|Molecular Vision 2005;
Received 18 August 2004 | Accepted 18 January 2005 | Published 19 January 2005
Effect of the pyridoindole antioxidant stobadine on development of experimental diabetic cataract and on lens protein oxidation in rats: comparison with vitamin E and BHT
Zuzana Kyselova,1 Andrej
Gajdosik,1 Alena Gajdosikova,1 Olga Ulicna,2 Danica
Mihalova,1 Cimen Karasu,3 Milan
1Institute of Experimental Pharmacology, Slovak Academy of Sciences, Bratislava, Slovakia; 2Pharmacobiochemistry Laboratory, 3rd Department of Internal Medicine, Faculty of Medicine, Comenius University, Bratislava, Slovakia; 3Department of Pharmacology, Faculty of Medicine, Gazi University, Ankara, Turkey
Correspondence to: Milan Stefek, Institute of Experimental Pharmacology, Slovak Academy of Sciences, Dubravska Cesta 9, 841 04, Bratislava, Slovak Republic; Phone: +421-2-59 410 667; FAX: +421-2-54 775 928; email: firstname.lastname@example.org
Purpose: The aim of this study was to investigate the effect of dietary supplementation with the pyridoindole antioxidant stobadine on the development of diabetic cataract in rats. The findings were compared with the effect of the natural antioxidant vitamin E and the well known phenolic synthetic antioxidant butylated hydroxytoluene.
Methods: Streptozotocin induced diabetic male Wistars rats were fed for 18 weeks a standard diet or a diet supplemented with stobadine (0.05% w/w), vitamin E (0.1% w/w), butylated hydroxytoluene (BHT, 0.4% w/w), or a mixture of stobadine (0.05% w/w) and vitamin E (0.1% w/w). The progress of cataract was monitored biweekly by ophthalmoscopic inspection. Plasma glucose and body weight were recorded regularly. At the end of the experiment, the content of free sulfhydryl and carbonyl was determined in total lens proteins and in the stobadine group plasma levels of malondialdehyde were also measured.
Results: Long term treatment of diabetic animals with stobadine (STB), vitamin E, or BHT led to a marked delay in the development of advanced stages of cataract. At the end of the experiment, the visual cataract score was significantly decreased in the diabetic groups treated with stobadine or BHT, while vitamin E had no significant effect. Unexpectedly, combined treatment with STB+vitamin E advanced the progression of the higher stages of cataract, though without affecting the overall visual cataract score. Neither of the antioxidants exerted an effect on the glycemic state or body weight of the animals. Biochemical analyses of eye lens proteins showed significant diminution of sulfhydryl groups and elevation of carbonyl groups in diabetic animals in comparison to healthy controls. Dietary supplementation with any of the antioxidants studied did not influence the levels of these biomarkers significantly. Nevertheless, in diabetic animals, stobadine supplementation significantly attenuated plasma levels of malondialdehyde, an index of systemic oxidative damage.
Conclusions: The results are in accordance with the postulated pro-oxidant role of chronic hyperglycemia, however, the direct oxidative free radical damage of eye lens proteins does not seem to be the key mechanism effective in the development of diabetic cataract. Sugar cataractogenesis appears to be a complex process, in which multiple mechanisms may be involved, including consequences of the overt oxidative stress in diabetes (e.g., protein modifying potential of toxic aldehydes generated as byproducts of carbohydrate autoxidation and lipid peroxidation). The ability of stobadine to attenuate lipoxidation reactions in diabetes may account, at least partly, for its observed anticataract action. Mechanisms involving reduction of mitochondrial damage by stobadine are also discussed.
Cataractogenesis is one of the earliest secondary complications of diabetes mellitus. Since extracellular glucose diffuses into the lens uncontrolled by the hormone insulin, the lens is one of the most affected body parts in diabetes mellitus. We believe that diabetic cataract is a disorder that requires a biochemical and pharmacological, rather than a surgical solution. First of all, tight metabolic control remains the primary intervention in prevention of lens opacification. However, pharmacological blockade of biochemical events triggered by disposal of excess glucose could be required.
The proteins of the lens are extremely long lived and there is virtually no protein turnover, which provides great opportunities for post-translational modification to occur. Multiple mechanisms have been implicated in the development of cataract in diabetes [1-3]. To date, the exact sequence of events which lead to opacification has not been clearly defined. Thus the relationship of the opacity to the initiating event may be obscure.
Free radical production is increased in the diabetic lens, while natural antioxidant defenses are compromised and thus increased oxidative stress results. Shifts in redox balances due to derangement in energy metabolism of carbohydrates and lipids also contribute to the overt oxidative stress in the diabetic individual. Human studies, in vitro, and in vivo animal experiments strongly suggest that there is an association between increased oxidative stress and the development of cataract [4-8]. Antioxidant supplementation was found to inhibit the development of cataract in experimentally induced diabetes in rats [9-13]. Several clinical studies pointed to a diminution of cataract incidence following adequate supply of antioxidants in food [14-16].
Numerous studies proved stobadine (STB), a novel synthetic pyridoindole, to be an efficient antioxidant . Recently, under conditions of an experimental glycation model in vitro, stobadine was found to protect bovine serum albumin against glyco-oxidative damage . Using a model of streptozotocin-diabetic rats in vivo, stobadine was found to attenuate pathological changes in the diabetic myocardium [19,20] and kidneys [20,21], to decrease matrix collagen cross-linking , and to reduce plasma cholesterol and triglyceride levels in diabetic animals [19,22]. Stobadine treatment normalized calcium homeostasis in the diabetic heart and liver  and produced a beneficial effect on leukocyte function in diabetic rats . These findings, along with its high oral bioavailability , efficient detoxification pathways [25,26], and toxic safety [27,28] render stobadine a promising agent in prevention of late diabetic complications.
Butylated hydroxytoluene (BHT), a well known synthetic antioxidant, was found in numerous studies to delay or prevent sugar cataractogenesis in rats [10,29-31]. In their clinical studies, Robertson et al. [32,33] and Rouhiainen  showed a significantly reduced risk of cataract after increased intake of the natural antioxidant vitamin E. Equivocal effects of vitamin E were described in its relation to sugar cataract development in rodents; a significant prevention of cataractogenesis [9,11,35] or an improvement of lens biochemical indices, yet without affecting the visual cataract score [36,37].
The present study investigated the effect of dietary supplementation with stobadine, vitamin E and buthylated hydroxytoluene (BHT) on the progression of eye lens opacification in streptozotocin-diabetic rats during a period of 18 weeks. The effect of combined stobadine and vitamin E treatment was also studied. At the end of the experiment, markers of lens protein glyco-oxidation were determined and effects of the antioxidants evaluated.
This investigation conforms with the Guide for the Care and Use of Laboratory Animals. The study was approved by the Ethics Committee of the Institute and performed in accordance with the Principles of Laboratory Animal Care (NIH publication 83-25, revised 1985) and the Slovak law regulating animal experiments (decree 289, part 139, July 9, 2003). Male Wistar rats, 8-9 weeks old, weighing 200-230 g, were used. The animals came from the Breeding Facility of the Institute of Experimental Pharmacology, Dobra Voda (Slovak Republic). Experimental diabetes was induced by a single intravenous dose of streptozotocin (STZ, 55 mg/kg). STZ was dissolved in 0.1 mol/l citrate buffer, pH 4.5. The animals were fasted overnight prior to STZ administration. Control animals received 0.1 mol/l citrate buffer. Water and food were available immediately after dosing. Ten days after STZ administration, all animals having a plasma glucose level >15 mmol/l were considered diabetic and were randomly divided into the following experimental groups (n=8): untreated diabetic rats (group D); diabetic rats treated with STB (0.05% w/w stobadine dipalmitate, group D+STB); diabetic rats treated with vitamin E (0.1% w/w D,L-alpha-tocopheryl acetate, group D+E); diabetic rats treated with BHT (0.4% w/w t-butyl hydroxytoluene, group D+BHT); and diabetic rats treated with the mixture of STB (0.05% w/w stobadine dipalmitate) and vitamin E (0.1% w/w D,L-alpha-tocopheryl acetate, group D+STB+E). Five control groups (C, C+STB, C+E, C+BHT, and C+STB+E) corresponding to the diabetic experimental groups were created (n=4). The animals were treated for a period of 18 weeks beginning 10 days after either vehicle or STZ injection. During the experiment the animals were housed in groups of two in cages of the type T4 Velaz (Prague, Czech Republic) with bedding composed of wood shavings (changed daily). Tap water and pelleted standard diet KKZ-P-M (Dobra Voda, Slovak Republic) were available ad libitum. The animal room was air conditioned and the environment was continuously maintained at a temperature of 23±1 °C and a relative humidity of 40-70%.
Heparinized blood samples were taken from rat tails after overnight fasting of the animals. Plasma glucose levels were measured using the commercial Glucose (Trinder) kit (Sigma, St. Louis, MO).
For detection of malondialdehyde (MDA) in plasma samples, the modified 2,4-dinitrophenylhydrazine (DNPH) derivatization method was used . Plasma samples (250 μl) taken from heparinized blood were hydrolyzed for 30 min with 0.5 μl 6 M NaOH at 60 °C. After subsequent precipitation with 200 μl of 30% TCA, the samples were centrifuged (7,000 rpm/10 min) and 300 μl of the supernatant derivatized with 30 μl of 5 M DNPH in 12 M HCl at laboratory temperature for 10 min. Then the samples were extracted twice with 2 ml of n-hexane, evaporated to dryness and finally analyzed by HPLC on a reversed phase C(18) column with UV detection (310 nm).
For stobadine determination, plasma samples (1 ml) were diluted with isotonic saline up to 3 ml and precipitated with 1 ml of ice cold 30% TCA. After subsequent centrifugation (3,000 rpm/15 min), the pH was adjusted to 10-11 with 5 M NaOH. These alkaline solutions, after addition of a known amount of the internal standard N-propyl analog of stobadine, were extracted with chloroform (3x2.5 ml). These extracts were treated with anhydrous sodium sulfate and then evaporated to dryness under N2 at 37 °C. The residues were dissolved in 25 μl of methanol and analyzed by GC (GC HP 5890) using an HP-5 (12 m x 0.2 mm x 0.33 μm) column and the HP 5970 Mass Selective Detector.
Evaluation of cataract development
The progress of cataract was monitored biweekly by a hand held ophthalmoscope equipped with a slit lamp by an individual without prior knowledge of affiliation of the animal to an experimental group. Eye inspection was preceded by topical administration of 1% mydriacyl drops. Cataract formation was scored essentially according to the classification of Ao et al.  or Suryanarayana et al.  as follows: clear normal lens (O), peripheral vesicles (I), peripheral vesicles and cortical opacities (II), diffuse central opacities (III), and mature cataract (IV). Cataract formation was considered complete (grade IV) when the red fundus reflex was no longer visible through any part of the lens and the lens appeared dull white to the naked eye.
At the end of week 18, the rats were killed and the eye globes were excised. The lenses were then dissected, rinsed with ice cold saline, and frozen in saline for preservation. Each pair of lenses was homogenized in a glass homogenizer with a teflon pestle in 1.2 ml of ice cold phosphate buffer (20 mM, pH 7.4) saturated with nitrogen. The total homogenate was used for further analyses.
Total protein concentration in the lens homogenate was determined by the Lowry method  using bovine serum albumin as a standard.
The content of free carbonyl in the total lens proteins was determined by the procedure of Levine et al.  using the 2,4-dinitrophenylhydrazine (DNPH) reagent. Two 0.2-ml aliquots of lens homogenate containing 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 mM DNPH dissolved in 2 M HCl as a sample or with 0.5 ml of 2 M 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 the sample was 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 M guanidine. Any insoluble material was removed by centrifugation at 3,000 rpm for 15 min. The difference spectrum of the DNPH derivatized samples compared to HCl controls was scanned at 322-370 nm on a Hewlett Packard 8452A Diode Array Spectrophotometer. Carbonyl content was calculated from the absorbance readings, using 22,000 M-1.cm-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 blanks.
The content of sulfhydryl groups in lens proteins was determined using Ellman's procedure as modified by Altomare et al. . Aliquots (0.2 ml) of total lens homogenate of approximately 3 mg of proteins were precipitated with an equal volume of 4% sulfosalicylic acid (SSA). The pellets obtained after centrifugation were washed with 1 ml of 2% SSA to remove free thiols. The washed pellets were dissolved in 0.2 ml of 6 M guanidine (pH 7.4) and read spectrophotometrically at 412 nm and 530 nm, before and after 30 min incubation in the dark with 50 μl of 10 mM DTNB (5,5'-dithiobis[2-nitrobenzoic acid]). The content of protein sulfhydryls was calculated using a calibration curve prepared with reduced glutathione.
Streptozotocin (STZ), 2,4-dinitrophenylhydrazine (DNPH), and DTNB (5,5'-dithiobis[2-nitrobenzoic acid]) were obtained from Sigma. Other chemicals were purchased from local commercial sources and were of analytical grade quality.
Statistical analysis of the data was done using the unpaired Student's t-test and the Mann-Whitney U-test.
Blood glucose and body weight
The average blood glucose levels of the control and diabetic experimental groups at the beginning and at the end of the 18 week experiment are given in Table 1. Persistent hyperglycemia, on average over 15 mmol/l, was recorded in diabetic animals throughout the whole experiment. At the end of the experiment, the weights of diabetic rats were significantly lower as compared with those in the control group (Table 2). Administration of the antioxidants did not significantly affect blood glucose and body weights either in control or diabetic animals.
As shown in Figure 1A, in untreated diabetic rats, appearance of advanced stages of cataract (stage III and IV) became apparent after 10 weeks. In stobadine treated diabetic animals (Figure 1B), the advanced stages of cataract formation were postponed by 6 weeks, while a 4 week delay was observed in the diabetic groups administered vitamin E (Figure 1C) or BHT (Figure 1D). The values of the average cataract score were significantly (Mann-Whitney U-test) lower in treated than untreated diabetic groups (1) for STB through weeks 10-18, with the exception of week 16, (2) for BHT over weeks 12-18, yet (3) for vitamin E only in week 12 (Figure 1, Table 3). At the end of the experiment (week 18), the untreated diabetic group showed a mean cataract score value of 3.5±0.2, whereas mean score values of 2.8±0.3, 3.1±0.3, and 1.9±0.5 were seen in STB, vitamin E and BHT treated diabetic groups, respectively (p<0.05 for D+STB compared to D; not significant for D+E compared to D (p=0.1120); p<0.01 for D+BHT compared to D, Mann-Whitney U-test.)
Unexpectedly, in diabetic animals treated with the combination of STB and vitamin E, advanced stages of cataract III and IV appeared as early as in week 12 (Figure 2). In spite of the fact that in this experimental group and in untreated diabetic animals progression of the average cataract score values was very close through weeks 12 to 18 (see Table 3), development of the final stage IV was apparently faster in the diabetic rats treated with the combination of STB and vitamin E.
None of the control rats untreated or administered the antioxidants studied developed lens opacity.
Changes in free carbonyls and sulfhydryls of lens proteins
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 (Figure 3A). At week 18 of the experiment, significantly higher values of protein free carbonyl were observed in the lenses of diabetic animals compared to healthy controls (p<0.05). The increase in DNPH reactive carbonyl was accompanied by a decrease in protein free sulfhydryl groups titratable by DTNB (Figure 3B, p<0.001). The individually administered antioxidants studied did not significantly affect lens protein levels of free carbonyl and sulfydryl, either in diabetic (Figure 3) or control (data not shown) animals. However, administration of the mixture STB+E to diabetic rats significantly stimulated depletion of free sulfhydryl groups in the total eye lens proteins (p<0.05, Student's t-test).
Plasma levels of MDA and stobadine
At the end of the experiment (week 18), significantly (p<0.05) elevated plasma levels of MDA were observed in diabetic rats in comparison with healthy controls (Figure 4). Stobadine supplementation to diabetic animals resulted in decrease of plasma MDA level from 3.1±0.3 to 2.2±0.2 μmol/l, (p<0.05). Effects of other antioxidants were not evaluated.
At the end of week 18, average levels of stobadine in deproteinized plasma in diabetic animals treated with food fortified with stobadine dipalmitate (0.05% w/w), were determined to be 5.6±0.8 ng/ml.
In diabetic animals, the antioxidants tested (when administered individually) apparently delayed the onset of advanced stages of cataract (stage III and IV). Considering values of the average cataract scores, stobadine or BHT treatment was found to be more effective than that of vitamin E for which a significant effect was seen only in week 12. The anticataract action of BHT was described by other authors in galactosemic and STZ-diabetic rats [10,29-31,43]. Several clinical studies pointed to a diminution of incidence of cataract after an adequate supply of antioxidant vitamins in food [14-16,32-34]. Equivocal effects of the natural antioxidant vitamin E have been described in its relation to sugar cataract development in rodents: a significant prevention of cataractogenesis [9,11,35], or an improvement of lens biochemical indices, without affecting the visual cataract score [36,37].
At the end of our experiment (week 18), biochemical analyses of eye lens proteins showed significant diminution of sulfhydryl groups and elevation of carbonyl groups in diabetic animals in comparison to healthy controls. These results point to the pro-oxidant role of hyperglycemia and are in agreement with findings of other authors (discussed in the following paragraphs). In their study on human cataractous lenses, Boscia et al.  identified a quantitative threshold of protein oxidation above which clinically significant cataracts develop.
A progressive decrease of protein sulfhydryls was observed during the development of diabetic and senile cataracts [8,44-46]. Sulfhydryl oxidation is thought to be one of the main pathological events leading, through disulfide cross-linking and molecular aggregation, to protein precipitation and lens opacification [47-52].
The presence of increased levels of free carbonyls in proteins of the cataractous diabetic eye lens, as described also by others [6,8,53,54], can be interpreted as a result of oxidative insult initiated by hyperglycemia, since the DNPH assay [42,55] was found to be selective enough to discriminate between protein bound carbonyls produced by metal catalyzed oxidations and those formed in the early glycation steps [56,57].
Dietary supplementation with any of the antioxidants studied in our experiments did not influence the levels of the biomarkers (>CO, sulfhydryl) significantly, in spite of the fact that a markedly decreased incidence of cataract was observed in diabetic groups treated with STB or BHT at week 18 when compared with untreated diabetic animals. The results indicate that oxidative modifications of eye lens proteins demonstrated by a decrease of sulfhydryl and increase of >CO groups may not be directly associated with the development of diabetic cataract. Cataractogenesis in diabetes seems to be a rather complex process, presumably including additional pathways, not connected with direct free radical oxidative modification of lens crystallins (e.g., mitochondrial damage). Damage of mitochondria of superficial cortex fiber cells at the lens equator where the first globular degeneration is seen in glucose cataract , followed by calcium release and activation of calcium sensitive calpain proteases [59-61], was suggested as a likely cause of lens opacification in diabetes. Mitochondrial oxidative damage of lens epithelial cells leading to severe depletion of intracellular ATP was reported to be one of the initiating events contributing to the formation of cataract. Beneficial effects of estrogen therapy against cataract in postmenopausal women was explained by stabilization of the mitochondrial membrane due to the antioxidant action of the hormone, independently of estrogen receptors [62,63]. Anticataract actions of the antioxidants studied in our experiment may be hypothetically explained by a plausible protection of lens mitochondria. At least for stobadine, other authors reported its ability to preserve fine mitochondrial structure in cerebral capillaries and neurons under conditions of global cerebral ischemia-reperfusion injury in dogs . Stobadine was found to ameliorate the mitochondrial energy production reduced in the diabetic kidney . Stobadine was also reported to prevent calcium accumulation in diabetic tissues .
Another conceivable explanation may be consistent with an increased lipid peroxidation rate in diabetes mellitus [5,6,66-68]. Dyslipidemia, including hypertriglyceridemia, is considered a significant and independent risk factor for diabetic complications [68,69]. Decomposition of lipid peroxides initiates chain reactions that produce reactive carbonyl compounds. One of the byproducts of lipid peroxidation is the toxic compound malondialdehyde (MDA), whose involvement in cataractogenesis has been suggested, mainly due to its cross-linking ability [70-74]. Lens MDA may be the result of lipid peroxidation of the lens cell membranes or may represent the consequence of its migration from the readily peroxidizable retina or from the central body compartment [5,6,75-77]. According to the results presented here and the previous findings of Perkiner et al. , stobadine supplementation of diabetic animals significantly attenuated plasma levels of MDA, an index of systemic oxidative damage. In our previous papers we reported on the ability of stobadine to reduce oxidative damage of the diabetic heart and kidney tissue as measured by conjugated dienes [19,21] or MDA . Stobadine also caused a significant correction of hypercholesterolemia and hypertriglyceridemia in diabetic rats [19,22], consistent with beneficial effects of stobadine on dyslipidemia in a short term clinical trial in humans . The ability of stobadine to attenuate lipoxidation reactions in diabetes, and thus the production of toxic aldehydes, may account, at least partly, for its observed anticataract action.
In vivo, it is very likely that an antioxidant would not exert its effect by itself, but would act in combination with other antioxidants present (e.g., antioxidant vitamins). Such interactions may affect the final antioxidant capacity. In their pulse radiolytic study on stobadine, Steenken et al.  demonstrated the ability of a water soluble analog of alpha-tocopherol, Trolox, to recycle stobadine from its one electron oxidation product on giving a corresponding Trolox radical. Kagan et al.  reported that the antioxidant potency of stobadine in vivo might thus be increased by its interaction with other antioxidants having more negative redox potentials (e.g., vitamin E). Indeed, Rackova et al.  showed that when Trolox and stobadine were present simultaneously in liposomal incubations, Trolox spared stobadine markedly in a dose dependent manner. This sparing effect of Trolox was inferred by its direct interaction with stobadinyl radicals leading to stobadine regeneration.
By analogy, we anticipated an enhanced antioxidant and anticataract action of stobadine when administered to diabetic rats in a mixture with vitamin E. However, contrary to our expectations, the combined treatment STB+E increased the progression of the advanced stages of cataract. Moreover, biochemical analyses showed further decrease of lens protein free sylfhydryls in diabetic rats treated with the mixture STB+E in comparison with the untreated diabetic group, indicating a prooxidant effect of the STB+E cocktail. The reason for the observed detrimental effect of the antioxidant mixture STB+E is not yet known. The experimental data presented here were obtained by using synthetic D,L-alpha-tocopherol which may behave differently from its natural D-isomer. Nevertheless, the result is reminiscent of the studies by Clarke et al.  on RCS strain rats. They found that individually taken vitamin E, C, or A were effective in reducing cataract risk, while the combination of the three supplements potentiated cataract formation. Other authors described beneficial effects of the combination STB+E. Similar or even enhanced protection in comparison with treatments by individual antioxidants was observed in rodent diabetic kidney and brain , diabetic heart and liver , or peripheral nerves (unpublished). We can only hypothesize that in diabetic animals treated with the mixture STB+E, the resulting total antioxidant concentration in the lens may have exceeded the level above which the reduction potential of the antioxidant mixture to recycle transition metals in their lower valence may prevail (e.g., Fe3+ to Fe2+), thus driving the production of detrimental hydroxyl radicals via the Fenton reaction with endogenous hydrogen peroxide. A similar mechanism has been suggested for pro-oxidant action at elevated concentrations of vitamin E [83,84].
In conclusion, the results are in accordance with the postulated pro-oxidant role of chronic hyperglycemia. However, the direct oxidative free radical damage of eye lens proteins does not seem to be the key mechanism effective in the development of diabetic cataract. Sugar cataractogenesis appears to be a multifactorial process in which apart from the reported non-oxidative pathways and free radical directed protein oxidative modifications other mechanisms may be involved, including consequences of the overt oxidative stress in diabetes (e.g., protein modifying potential of toxic aldehydes generated as byproducts of carbohydrate auto-oxidation and lipid peroxidation). The ability of stobadine to attenuate lipoxidation reactions in diabetes may account, at least partly, for its observed anticataract action.
This investigation was supported by VEGA grants number 2/2050/22, APVT grant number 20-020802 and SBAG-SLOVAK-2 (103S180) grant.
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