Vitex negundo attenuates calpain activation and cataractogenesis in selenite models

Experimental Eye Research 88 (2009) 575–582

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Vitex negundo attenuates calpain activation and cataractogenesis in selenite models B.N. Rooban a, Y. Lija a, P.G. Biju a, V. Sasikala a, V. Sahasranamam b, Annie Abraham a, * a b

Department of Biochemistry, University of Kerala, Kariavattom, Thiruvananthapuram-695581, Kerala, India Regional Institute of Ophthalmology, Medical College, Thiruvananthapuram-695037, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 8 January 2008 Accepted in revised form 13 November 2008 Available online 6 December 2008

Recent investigations have shown that phytochemical antioxidants can scavenge free radicals and prevent various diseases. Cataract is the leading cause of blindness and is associated with oxidative damage of the lens. Selenite-induced cataract in rat pups is an excellent mimic of oxidative stressinduced cataract. Selenite cataract is associated with oxidative stress, loss of calcium homeostasis, calpain activation and protein insolubilization in the lens. Our present study focuses on the isolation of flavonoids from Vitex negundo and to assess its efficacy in preventing these changes in the lens of selenite-induced cataract models. Eight-day-old Sprague-Dawley rat pups were used for the study and divided into four groups: Control (G I), Sodium selenite-induced (G II), Sodium selenite þ quercetin treated (G III), Sodium selenite þ flavonoids from Vitex negundo (FVN) (G IV). Cataract was induced by a single subcutaneous injection of Sodium selenite (4 mg/Kg body weight) on the 10th day. Treatment groups received quercetin (1.0 mg/Kg body weight) and FVN (1.0 mg/Kg body weight) intraperitoneally from 8th to 15th day. Cataract was visualized from the 16th day. Morphological examination of the rat lenses revealed no opacification in G I and mild opacification in G III and G IV (stage 1) whereas dense opacification in G II (stage 4–6). The activities of superoxide dismutase (SOD), catalase, Ca2þATPase, concentration of reduced glutathione (GSH) and protein sulfhydryl content were significantly increased in G III and G IV compared to G II, while decreased activities of calpains, lower concentration of calcium and thiobarbituric acid reactive substances (TBARS) were observed in G III and IV as compared to G II. Lens protein profile of water soluble proteins showed normal levels of expression in treated groups compared to that of selenite-induced rats. These results indicate good antioxidant and therapeutic potential of FVN in modulating biochemical parameters against selenite-induced cataract, which have been reported in this paper for the first time. Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: antioxidants flavonoids calpains oxidative stress quercetin selenite cataract Vitex negundo

1. Introduction Age-related cataract remains a major cause of blindness and its prevalence in developing countries is much more than that in the developed ones (Nirmalan et al., 2003). At present the most effective treatment of cataract is the surgical extirpation of the lens, but it possesses postoperative complications (Toda et al., 2007; Bockelbrink et al., 2008). Hence, it is important to look into alternative pharmacological measures for the treatment of this disorder. Selenite-induced cataract (oxidative stress model) is an extremely rapid and convenient model of nuclear cataract in rats (Ostadalova et al., 1978). Major events of selenite cataract are loss of calcium homeostasis, reactive oxygen species generation, lipid peroxidation,

* Corresponding author. Tel.: þ91 471 2418078/2532220, 9447246692 (mobile); fax: þ91 471 2307158. E-mail address: [email protected] (A. Abraham). 0014-4835/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2008.11.020

calpain activation, insolubilization of proteins, decreased water soluble proteins and GSH (Shearer et al., 1997). Ionic homeostasis plays an essential role in lens transparency and the loss of Ca2þ homeostasis has been implicated in cataract (Gupta et al., 2004). Pure nuclear cataracts which account for approximately 30% of cataracts, exhibit a normal internal ionic content while lenses with cortical cataract (pure or mixed) have increased lenticular calcium (Duncan and Bushell, 1975). Levels of Calcium in the lens are maintained in the sub-micromolar range by membrane Ca2þATPase (Liu et al., 2002). Calpains (EC 3.4.22.17) are a family of calcium dependent neutral cysteine proteases and at least four proteolytically active calpains are expressed in rodent lenses (Ma et al., 1999). These include ubiquitous calpains 1(m-calpain) and 2 (m-calpain) and lens-specific Lp82 and Lp85 (Shih et al., 2006). Oxidative stress has been identified as one of the major causes of age-related diseases including cataract (Salganik, 2001). Lens antioxidant status and lipid peroxidation products have been implicated in human cataract (Donma et al., 2002; Ganea and Harding, 2006).

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Vitex negundo, a deciduous shrub belonging to the family of Verbenaceae, is reported to have anti-inflammatory, ophthalmic, analgesic and anti-histaminic properties (Dharmasiri et al., 2003). However, there are no previous reports on the flavonoids of Vitex negundo preventing oxidative stress-induced cataract. In the present study, an attempt has been made to determine efficacy of flavonoids of Vitex negundo (FVN) in modulating or preventing selenite-induced cataractogenesis. Quercetin at a concentration of 1.0 mg/Kg body weight was used as a reference flavonoid because of its established antioxidant property (Hibatallah et al., 1999). Protective effects of quercetin on galactose induced cataract (Mohan et al., 1998) and hydrogen peroxide induced cataract (Sanderson et al., 1999; Cornish et al., 2002) have been reported. This is the first report on the effect of flavonoids from Vitex negundo to protect against calpain-induced proteolysis and oxidative stress in selenite-induced cataractogenesis. 2. Materials and methods

ophthalmoscope and later on with the naked eye. The development of cataract was assessed weekly for three weeks by slit-lamp illumination. At the final examination, the pupils were dilated with Tropicamide 0.5% and Phenylephrine hydrochloride 2.5%. Each stage was graded and identified with the help of an expert ophthalmologist. Classification of the cataract stages was based on a scale of 1 through 6 (Hiraoka and Clark, 1995). Stage 0 ¼ normal clear lens; Stage 1 ¼ initial sign of posterior subcapsular or nuclear opacity involving tiny scatters; Stage 2 ¼ slight nuclear opacity with swollen fibers or posterior subcapsular scattering foci; Stage 3 ¼ diffuse nuclear opacity with cortical scattering; Stage 4 ¼ partial nuclear opacity; Stage 5 ¼ nuclear opacity not involving lens cortex; Stage 6 ¼ mature dense opacity involving the entire lens. On the 30th day, rats were euthanized by sodium pentothal injection, lenses were excised and the experiments conducted. All ethical guidelines were followed for the conduct of animal experiments in strict compliance with the Institutional Animal Ethical Committee and Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India.

2.1. Chemicals & solvents 2.4. Analytical methods All the biochemicals and quercetin were purchased from Sigma Chemical Company, St Louis, MO, USA and other chemicals and solvents of analytical grade were from SRL Chemicals, India. Protein marker for SDS-PAGE was purchased from GENEI- Bangalore, India. 2.2. Extraction, isolation and estimation of flavonoids Fresh leaves of Vitex negundo were collected from Trivandrum, India during early summer. A specimen of the collected material was verified with the herbarium of Tropical Botanical Garden and Research Institute, Thiruvananthapuram, India (Accession No.17038 P.S Jyothish) and authenticated by an expert. The shade dried plant material –leaves (1 Kg) was crushed for extraction. This was taken in a round-bottomed flask and 80% methanol added to cover the material and refluxed in a water bath at 65  C for 24 h. The supernatant was removed and the extraction was repeated twice. The extract was decanted, filtered and concentrated to remove the solvent in a rotor evaporator. Extract was cleared off low polarity contaminants such as fats, terpenes, chlorophyll and xanthophyll by repeated extraction with Petroleum ether (60– 80  C), Benzene and Ethyl acetate respectively. Ethyl acetate extract contained a bulk of polyphenols (59 g/Kg), which was evaporated in vacuum and the flavonoid content (405 mg/Kg) was determined by the method of Eskin et al. (1978) using quercetin as reference standard. Flavonoids obtained was dissolved in phosphate-buffered saline (prepared in sterile water) and used for the in vivo studies. 2.3. In vivo experiments Neonatal rat pups of Sprague-Dawley strain initially weighing 10–12 g on the 8th day were used for the study. The pups were housed along with their mother in polypropylene cages in rooms maintained at 25  1  C. The animals were maintained on a standard laboratory animal diet (Hindustan Lever Ltd., India) and provided water ad libitum throughout the experimental period. Animals were grouped as G I Control (normal laboratory diet), G II (normal laboratory diet þ sodium selenite), G III (normal laboratory diet þ sodium selenite þ quercetin) and G IV (normal laboratory diet þ sodium selenite þ FVN) with eight rats in each group. The neonatal rat pups in experimental groups (II–IV) received a single subcutaneous injection of sodium selenite (4 mg/Kg body weight) on the 10th day. The treatment groups (III & IV) received intraperitoneal injection of quercetin and FVN at the concentration 1.0 mg/Kg body weight from the 8th day upto the 15th day. Cataract could be visualized from the 16th day with the help of an

2.4.1. DPPH radical scavenging assay The free radical scavenging activity of FVN was measured in terms of hydrogen donating or radical-scavenging ability using the stable radical DPPH by the method of Blois (1958). 0.1 mM solution of DPPH in ethanol was prepared and 0.1 ml of this solution was added to 3.0 ml of test solution (FVN) in water at different concentrations (10–100 mg/ml) and quercetin (reference control) was tested at similar concentrations in water. 30 min later, the absorbance was measured at 517 nm. Lower the absorbance of the reaction mixture, higher the free radical scavenging activity. A system devoid of the compound served as control. The antioxidant activity of the compound was expressed as IC50. The IC50 value was defined as the concentration (in mg/ml) of extract that inhibited the formation of DPPH radicals by 50%. 2.4.2. Assay of SOD activity SOD activity in the lens samples was measured by the method of Kakkar et al. (1984). The assay mixture contained 1.2 ml sodium pyrophosphate buffer (0.052 M, pH 8.3), 0.1 ml of 186 mM PMS, 0.3 ml of 300 mM NBT, 0.2 ml of 780 mM NADH, 1.0 ml homogenate (lens homogenized in 0.25 M sucrose buffer) and water to a final volume of 3.0 ml. Reaction was started by the addition of NADH and incubated at 30  C for 1 min. The reaction was stopped by the addition of 1.0 ml glacial acetic acid and the mixture stirred vigorously. 4.0 ml n-butanol was added to the mixture and shaken well. The mixture was allowed to stand for 10 min, centrifuged, and the butanol layer was taken out and the absorbance was measured at 560 nm against a butanol blank. A system devoid of enzyme served as the control. One unit activity is defined as the enzyme concentration required for inhibition of chromogen production by 50% in 1 min. 2.4.3. Assay of catalase activity Catalase activity in the lens samples was measured by the method of Aebi (1984). Reaction mixture containing 2.0 ml of homogenate (lens homogenized in 50 mM phosphate buffer, pH 7.0) and 1.0 ml of 30 mM hydrogen peroxide (in 50 mM phosphate buffer, pH 7.0) were prepared. A system devoid of the substrate (hydrogen peroxide) served as the control. Reaction was started by the addition of the substrate and decrease in absorbance monitored at 240 nm for 30 s at 25  C. The difference in absorbance per unit time was expressed as the activity. One unit is defined as the amount of enzyme required to decompose 1.0 mmole of hydrogen peroxide per minute at pH 7.0 and 25  C.

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2.4.4. Estimation calcium and selenium level Calcium and selenium concentrations in the lens samples were analyzed flame photometrically after digestion in Nitric acid: Perchloric acid mixture (5:1).

centrifuged at 10,000  g for 20 min at 4  C. The supernatant was collected; the pellets were washed thrice with the same buffer and supernatants pooled together. The obtained supernatant comprised of the water-soluble lens proteins and was designated as WSF.

2.4.5. Assay of Ca2þATPase activity Ca2þATPase activity in the lens samples was measured by the method of Rorive and Kleinzeller (1974). To the assay mixture in both set of tubes added 0.1 ml of the above supernatant. To the control tubes added 0.2 ml of the substrate, ATP. All the tubes were incubated for 30 min in a water bath at 37  C. The enzyme activity is stopped by adding 2 ml of 10% TCA. Then 0.2 ml of ATP is added to control tubes and the same kept in ice for 20 min. All the tubes were then centrifuged at 2500 rpm for 10 min and the supernatant collected. The protein free supernatant was analyzed for inorganic phosphate. For that 3 ml of the supernatant was treated with 1 ml of ammonium molybdate and 0.4 ml ANSA. The color developed was read at 680 nm after 20 min.

2.4.10. Casein zymography Calpain activity was studied zymographically by the method of Raser et al. (1995). 8% gels (1 mm thickness) co-polymerized with 0.1% alkali denatured casein were prerun with zymography running buffer containing 25 mM Tris (pH 8.3), 192 mM Glycine, 1 mM EGTA and 1 mM DTT for 15 min at 4  C. Samples were loaded (40 mg/well) and following run completion, gel was incubated in zymography development buffer containing 20 mM Tris (pH 7.4), 10 mM DTE and 2 mM calcium, at room temperature for 24 h. Gels were stained with Coomassie Brilliant Blue. Upon destaining, calpain activity developed as clear bands against a dark background indicating proteolysis of casein.

2.4.6. Estimation of GSH content Lens glutathione in reduced form (GSH) was assayed by the method of Sedlak and Lindsay (1968). Lenses were homogenized in cold 20 mM EDTA solution on ice. After deproteinization with 5% TCA, an aliquot of the supernatant was allowed to react with 150 mM DTNB [5, 50 -Dithiobis-(-2-nitrobenzoic acid)]. The product was detected and quantified spectrophotometrically at 416 nm. Pure GSH was used as standard for establishing the calibration curve. 2.4.7. Estimation of protein sulfhydryl content Protein sulfhydryl content of lens proteins was determined using the Ellman0 s procedure modified by Grattagliano et al. (1996). Aliquots of total lens homogenate of approximately 3 mg of protein was precipitated with equal volume of 4% sulfosalicylic acid (SSA) and 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 6.0) and read spectrophotometrically at 412 nm and 530 nm before and after 30 min incubation in the dark with 50 ml of 10 mM DTNB. Content of protein sulfhydryls was calculated using a calibration curve prepared with reduced glutathione. 2.4.8. Estimation of the level of thiobarbituric acid reactive substance (TBARS) TBARS concentration in the lens samples was estimated by the method of Niehaus and Samuelsson (1968). Briefly, lenses were homogenized in 0.1 M Tris–HCl buffer (pH. 7.5). 1 ml of the homogenate was combined with 2 ml of TCA–TBA–HCl reagent 15% trichloroacetic acid (TCA) and 0.375% thiobarbituric acid (TBA) in 0.25 N HCl] and boiled for 15 min. Precipitate was removed after cooling by centrifugation at 1000  g for 10 min and absorbance of the sample was read at 535 nm against a blank without tissue homogenate. 2.4.9. SDS-PAGE Analysis of lens proteins SDS-PAGE was carried out in Hoeffer Slab gel apparatus using the method of Laemmli (1970) with some modification. A 12% gel of 7  8 cm and 1.0 mm thickness was used. Electrophoretic mobility depends on both molecular charge and size, so that the resulting protein pattern is characteristic of the specimen. All reagents and gels were made using Laemmli system. Protein concentration of the samples was estimated by the method of Lowry et al. (1951) and 40 ml (80 mg protein) was added each well. For the preparation of water soluble fraction, decapsulated lenses were homogenized in ice-cold PBS (pH 7.4) in a glass homogenizer. The homogenate was

2.4.11. Immunoblot of Lp82 Soluble proteins of rat lens (40 mg/well) were separated on a 12% SDS-gel. The proteins were electroblotted onto a nitrocellulose membrane (0.45 mm, Bio-Rad) under cold condition for 1 h at 100 V. The membrane was blocked overnight at 4  C with 2% BSA (Bovine Serum Albumin, Fraction V prepared in Tris buffered saline-Tween-20). After rinsing the membrane with Tris buffered saline Tween (TBST), it was incubated for 2 h with 1:1000 diluted rabbit anti-Lp82 primary antibody. The membrane was washed three times (15 min each) with TBST and incubated with 1:5000 diluted goat anti-rabbit IgG secondary antibody coupled to alkaline phosphatase for 1 h. The membrane was washed three times again (15 min each) with TBST buffer and developed with the BCIP/NBT substrate which developed into purple blue insoluble precipitates indicating the presence of Lp82. b-Actin was used as a loading control with rabbit anti-b-actin primary antibody at 1:1000 dilution. 2.4.12. Estimation of protein value Protein values of the samples were determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard. 2.5. Statistical analysis All statistical calculations were carried out with Statistical Package for Social Sciences (SPSS) software program (version 10.0 for Windows). The values are expressed as the mean  SD. The data were statistically analyzed using analysis of variance (ANOVA) and significant difference of means was determined using Duncan’s multiple range tests at the level of p < 0.05 (Steel et al., 1996). 3. Results Preliminary studies were done to assess the toxicity of the plant material and to fix the dose before the main experiment. Evaluation of toxicity parameters – activities of Glutamate Oxaloacetate Transaminase (GOT) and Glutamate Pyruvate Transaminase (GPT)which are common indicators of toxicity in serum and liver confirmed FVN was non toxic (data not given). A dose-response study was also carried out using FVN at different concentrations (0.5, 1.0, 1.5 mg/Kg body weight) in selenite-induced rats to fix the dose of FVN and 1.0 mg/Kg body weight was found to be the most effective. Effective dose was fixed by measuring the concentrations of GSH and TBARS in the lens. Concentration of GSH remained high at doses 1.0 and 1.5 mg/Kg with no significant difference, while 0.5 mg/Kg treatment showed a decrease. Concentration of TBARS was found to be decreased at doses of 1.0 and 1.5 mg/Kg with no significant difference while the concentration was comparatively

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B.N. Rooban et al. / Experimental Eye Research 88 (2009) 575–582 Table 1 Concentration of selenite in lens. Parameters

Group I

Group II

Group III

Group IV

Selenite content (ng/mg lens)

0.110  0.006

0.440  0.004a

0.430  0.002a

0.410  0.006a

Group I – Control, Group II – Selenite treated group, Group III – Selenite þ quercetin, Group IV – Selenite þ FVN treated group. a Statistically significant difference at p < 0.05 when compared with Group I.

dependent inhibition of DPPH activity with an IC50 value of 34 mg/ ml, whereas that for quercetin was 20 mg/ml (Fig. 1). 3.2. Selenite concentration in lens

Fig. 1. DPPH radical scavenging activity of Vitex negundo and quercetin in vitro. Ability of the compounds to scavenge DPPH free radical generation is expressed as % inhibition taking concentrations from 10–100 mg/ml. The value obtained for FVN significantly differs from that for quercetin, at p < 0.05.

higher for 0.5 mg/Kg treatment. From this study 1.0 mg/Kg body weight was fixed as the minimal effective dose of FVN for the production of maximum beneficial effects (data not given). Quercetin was used as a reference flavonoid to compare with FVN in the in vivo study. 3.1. Free radical scavenging activity In vitro free radical scavenging activity (DPPH) was conducted to ascertain the antioxidant potency of the FVN. DPPH is a stable free radical at room temperature and accepts an electron or hydrogen radical to become more stable diamagnetic molecule. The reduction capability of DPPH radicals was determined by the decrease in its absorbance at 517 nm, which is induced by antioxidants. The flavonoid fraction of Vitex negundo exhibited a significant dose

Lens Selenite concentration after 24 h post induction was assessed for all groups as a separate study to rule out the selenite sequestering effect of FVN and quercetin. Selenite-induced group showed significantly elevated levels of selenite compared to control. FVN and quercetin treated groups showed no significant reduction in selenite level compared to selenite-induced group (Table 1). 3.3. Transparency of the lens Selenite administration resulted in large nuclear opacities at 3–4 days post injection in 100% cases. In our study, slit lamp examinations indicated the development of cataract at stages 4–6 in selenite-induced group compared to the control. Quercetin and FVN prevented the maturation of cataract in the treated groups (Fig. 2). 50% of the lenses in G III were clear and remaining lens showed cataract stages 1–3. 62.5% of the lenses in G IV demonstrated normal state and the remaining 37.5% showed stage 1 cataract which is an indication of the anticataractous potential of FVN (Table 2). 3.4. Calcium homeostasis and Ca2þATPase activity For the evaluation of the effect of flavonoid fraction of Vitex negundo on the activity of the vital calcium regulator in the lens and the calcium balance, activity of Ca2þATPase and the concentration of

Fig. 2. Lens opacification of experimental animals (A) Lenses of G I exhibited no opacification. (B) Lenses of G II exhibited dense opacification. (C–G III & D–G IV) exhibited mild or no opacification.

B.N. Rooban et al. / Experimental Eye Research 88 (2009) 575–582 Table 2 Slit lamp examinations. Groups

Stage 0 Stage 1 Stage 2 Stage 3 Stage 4 Stage 5 Stage 6 Mean Stage

I (n ¼ 16) II (n ¼ 16) III (n ¼ 16) IV (n ¼ 16)

8 – 4 5

– – 1 3

– – 2 –

– – 1 –

– 2 – –

– 2 – –

– 4 – –

– 2.63  0.18a 0.50  0.07 0.19  0.01

Group I – Control, Group II – Selenite treated group, Group III – Selenite þ quercetin, Group IV – Selenite þ FVN treated group. n ¼ pair of eyes. a Statistically significant different when compared with III & IV (p < 0.05).

calcium were evaluated. Analysis of these parameters in G II revealed that selenite induction alters the activity of Ca2þATPase and calcium level in lens. Activity of Ca2þATPase was decreased significantly in the G II rats compared to that of control group whereas quercetin and Vitex negundo treated groups showed a significant increase as compared to that of selenite-induced group (Table 3). Concentration of calcium was elevated significantly followed by selenite induction in the G II rats than control group. Quercetin and Vitex negundo administration inhibited the activation of calcium followed by selenite induction in the treated group (Table 3).

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calpain activation led to protein degradation and aggregation resulting in lens opacification. G I showed the normal protein profile of the WSF. Two bands in G II were found to have lower expression intensity in WSF and correspond to 22.0 kDa and 30.5 kDa respectively. These bands in G III and G IV showed expression near to normal levels in G I (Fig. 3). 3.8. Calpain system in lens Zymographic analysis shows the activation of calpain 2 and Lp82. The zymogram for calpain exhibited activities of calpain 2 and Lp82 as caseinolytic clear bands in G II. Treatment groups III and IV showed a marked decline in the intensity of clearings but were marginally greater than G I (Fig. 4a). Activation of Lp82 by cellular calcium causing break down of lens crystallins would lead to autolysis and contribute to the sudden fall in Lp82 protein. Lp82 gave an activity band close to the start of the gel while calpain 2 was further down. Lp82 proteins in the lens samples were visualized by an immunoblot (Fig. 4b). G II was found to show lower levels of Lp82 proteins as compared to G III and G IV. Levels of Lp82 protein was comparable among G I, G III and G IV.

3.5. Antioxidant status in the lens 4. Discussion To evaluate the effect of FVN on the regulation of antioxidant status in the lens, activities of antioxidant enzymes- superoxide dismutase and catalase were screened. These factors were chosen because of their role as defense enzymes against oxidative stress in lens. In the presence of sodium selenite, the activities of superoxide dismutase and catalase were reduced significantly in G II lenses in comparison to that of normal lenses. However, treatment with quercetin and FVN showed significant reversal in the loss of activity and maintained at levels close to the control (Table 4). For the further evaluation of the antioxidant status, concentration of GSH was analyzed. Selenite induction (G II) produced a significant fall in GSH in comparison with normal lenses. Treatment with quercetin and FVN significantly restored the GSH concentration (Table 4). 3.6. Levels of the indicators of lipid (TBARS) and protein (sulfhydryl content) oxidation in lens It is well known that the level of TBARS reflects the overall tissue lipid peroxidation (Zoric, 2003; Varma and Hegde, 2004). The level of TBARS in the lens of animals treated with selenite alone (G II) was higher than that of control, quercetin and FVN treated groups. These results confirm that the selenite-induced elevation of lipid peroxidation was significantly inhibited by quercetin and FVN (Table 5). The level of protein sulfhydryl content is the important indicator of tissue protein oxidation. In the selenite-induced rats, protein sulfhydryl content was relatively decreased compared to the normal. However, in rats administered quercetin and FVN, lens protein sulfhydryl content was greater than the concentration observed in the G II (Table 5). 3.7. SDS-PAGE profile of lens protein The relative changes in the water soluble fraction (WSF) of lens proteins were detected by SDS-PAGE analysis. In selenite cataract,

Preventive protection as a therapeutic approach against cataract has generated considerable interest in recent years. We have used selenite-induced cataract as a model to evaluate the therapeutic efficacy of FVN. Selenite levels in lens were measured in all groups to determine if quercetin and FVN treatment induced modulation was a result of selenite being sequestered and not available in lens to induce cataract. Levels in selenite-induced and quercetin and FVN treated groups showed no significant changes, indicating no adverse effect of treatment on selenite availability to lens. The antioxidant potential of FVN was assessed by measuring the DPPH free radical scavenging capacity based on its ability to donate a hydrogen ion (Kumazawa et al., 2002). FVN exhibited an IC50 of 34 mg/ml, indicating appreciable antioxidant activity for the fraction. The level of selenite reaching the lens is critical for the development of cataract. Selenite levels in lens showed statistically significant increase in selenite injected group over control. FVN and quercetin treated groups showed similar levels to selenite-induced (G II) group, clearly indicating that treatment had no effect on selenite accumulation in lens. Selenite manifests its effect on lens by inducing oxidative stress. Oxidative damage of the critical sulfhydryl groups of proteins could lead to the inactivation of membrane proteins like Ca2þATPase. Previous studies show that Ca2þATPase is particularly sensitive to oxidative stress and results in a loss of their activity (Ahuja et al., 1999). Decrease in the activity of Ca2þATPase and accumulation of Ca2þ are considered as essential features in selenite cataract formation (Wang et al., 1993). In rodent lenses, the calcium influx was observed to cause activation of calcium dependent proteases, which partially degrade the crystallins and thereby resulting in the protein insolubilization (Shearer et al., 1997). In FVN treated group, lower levels of calcium and higher levels of Ca2þATPase activity were observed attributing to its protective effect. Previous studies

Table 3 Concentrations of calcium and Ca2þATPase activity. Parameters

Group I

Group II

Group III

Group IV

Ca2þATPase (mmoles phosphate liberated/mg protein/h) Calcium (mmole/g dry tissue)

3.30  0.02 0.410  0.002

2.10  0.01* 0.970  0.004*

2.90  0.01** 0.740  0.003**

3.10  0.02** 0.610  0.002**

Group I – Control, Group II – Selenite treated group, Group III – Selenite þ quercetin, Group IV – Selenite þ FVN treated group. Comparison of Group II against Group I (*significantly different at p < 0.05), comparison of Group III & IV against Group II (**significantly different at p < 0.05).

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Table 4 Antioxidant status in the lens. Parameters

Group I

Group II

Group III

Group IV

SOD (units/mg protein) 11.22  0.41 5.25  0.12* 10.14  0.31 ** 11.00  0.35** Catalase(units/mg protein) 6.30  0.25 3.10  0.18* 5.70  0.20** 6.01  0.21** 15.37  0.54 8.40  0.18* 13.61  0.17** 14.90  0.34** GSH(mg/g wet tissue) Group I – Control, Group II – Selenite treated group, Group III – Selenite þ quercetin, Group IV – Selenite þ FVN treated group. Comparison of Group II against Group I (*significantly different at p < 0.05), comparison of Group III & IV against Group II (**significantly different at p < 0.05).

on cataract using Disulfiram and Verapamil hydrochloride also reported similar effects (Nabekura et al., 2003; Ettl et al., 2004). The antioxidant enzymes, SOD and catalase play a critical role in protecting cells from oxidative stress. Selenite administration was found to decrease the activities of these enzymes in the lens (Shearer et al., 1997), accompanied by the generation of free radical species in the aqueous humor and significant reduction in nicotinamide adenine dinucleotide phosphate (reduced form) (Harding, 1970). Restoration of levels of activity of the antioxidant enzymes in quercetin and FVN treated group could be attributed to their antioxidant effect (Table 3). Similar results were reported by others as well as our research group (Padmaja and Raju, 2004; Gupta et al., 2005; Lija et al., 2006). The antioxidant, reduced glutathione is found in unusually high levels in lens which maintains protein thiol groups in the reduced state and prevents the cross linking of soluble crystallins (Reddy, 1990). The mechanism of GSH loss in selenite model is by a non-enzymatic reaction of GSH with selenite which results in the formation of the selenium derivative, GS–Se– SG. Oxidation of GS–Se–SG by a single electron transfer to oxygen results in formation of superoxide anion as an intermediate (Seko et al., 1989). Supplementation of GSH or maintenance of its level in lens may help to maintain its protective ability against oxidative stress and lead to slower age-related loss of antioxidant activity of lens and eventually to delay the onset of cataract (Harding, 2001). We found the FVN treatment to prevent the above process and maintained the GSH in its active form. Similar results were reported with the use of antioxidant therapy in streptozotocin-induced diabetic cataract (Suryanarayana et al., 2005) and selenite cataract (Elanchezhian et al., 2007).

Fig. 3. SDS PAGE profile of lens protein. M: lane showing the molecular weight marker. L1: showing the protein profile of normal rats. L2: showing the protein profile of selenite induced rats. Arrows indicating lower expression intensity of protein at 22.0 kDa and 30.5 kDa. L3 & L4 are the protein profile of quercetin and FVN treated rats respectively showing normal protein profile.

Table 5 Levels of the indicators of lipid (TBARS) and protein (sulfhydryl content) oxidation in lens. Parameters

Group I

Group II

Group III

Group IV

TBARS 9.67  0.24 15.59  0.57* 10.19  0.38** 9.10  0.31** (n mol/g wet tissue) 18.62  0.35 11.14  0.18* 16.34  0.20** 18.12  0.25** Protein sulfhydryl (mg/g wet tissue) Group I – Control, Group II – Selenite treated group, Group III- Selenite þ quercetin, Group IV – Selenite þ FVN treated group. Comparison of Group II against Group I (*significantly different at p < 0.05), comparison of Group III & IV against Group II (**significantly different at p < 0.05).

Free radical induced lipid peroxidation is one of the basic mechanisms of lens opacity (Awasthi et al., 1996) in selenite models. Lower levels of TBARS in the FVN treated group is an indication to the prevention of lipid peroxidation. Similar results were reported by (Gupta et al., 2003; Ertekin et al., 2004; Geraldine et al., 2006). One of the pathological events leading to protein precipitation is mediated through disulfide cross-linking of protein via sulfhydryl oxidation, leading to higher molecular weight aggregate formation, protein precipitation and lens opacification (Dong et al., 2000). Seleniteinduced group had significantly lower level of protein sulfhydryl content over control. In FVN administered group, protein sulfhydryl content was found to be increased, again confirming its protective effect against oxidative damage. Selenite cataract is characterized by marked decline in water soluble protein through protein insolubilization (Shearer et al., 1997). Similar changes were also observed in human cataract (Srivastava and Srivastava, 2003). The diminished expression of two bands of WSF in selenite-induced group could therefore be interpreted as due to the insolubilization caused either by excessive proteolysis by calpains or structural alterations brought about by sulfhydryl oxidation. FVN treated group had the expression profile

Fig. 4. (a) Zymography of calpains G I: showing zymographic analysis of normal rats. G II: zymography of selenite induced rats showing higher intensity of calpain 2 and Lp82 activity. G III & G IV zymography or quercetin and FVN treated groups showing lesser activation index. (b) Immunoblot of Lp82 G I: showing immunoblot analysis of normal rats. G II: immunoblot of selenite induced rats showing lower level of Lp82 protein. G III & G IV showing normal level of Lp82 protein in quercetin and FVN treated rats compared to control. Beta actin at 43 kDa was used a loading control.

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of these bands similar to control, suggesting a protective effect against proteolysis and insolubilization of proteins. Calpains are Ca2þ activated neutral proteases and crystallins are their excellent substrates (Shearer et al., 1996). Though not found in humans, Lp82 is of significant importance in rodent cataractous models (Nakamura et al., 2000) and is over activated in selenite model. In the present study, zymographic analysis of Lp82 and the ubiquitous calpain 2 showed bands of higher intensity (activity) in selenite-induced group. Activation of calpains was found to be in tandem with rise in levels of Ca2þ as described above. Treatment with quercetin and FVN was found to exhibit a lesser activation index, which was comparable with the level of Ca2þ observed. Hence, it could be inferred that the near normal levels of Ca2þ and Ca2þATPase activity following treatment has a direct bearing on the observed decrease in activation of Lp82 and calpain 2. Immunoblot of Lp82 was carried out to know the level of its protein in relation to activation. Major loss of Lp82 protein along with increased activity has been reported during selenite-induced cataract, which might be explained by the process of auto-proteolytic degradation (Shearer et al., 1998). In our study, the level of Lp82 protein was found to be reduced in selenite-induced group which exhibited a high level of enzyme activity. Quercetin and FVN treated groups exhibited near normal protein levels and enzyme activity, confirming a positive protective effect of treatment. The protective effect could be attributed to the excellent antioxidant activity of FVN at the lens level, which is supported by our previous reported observations (Biju et al., 2007). In conclusion, intraperitoneal administration of FVN showed anticataractogenic activity against selenite-induced cataract in experimental animals and the effect observed was appreciable compared to the reference flavonoid, quercetin. This study using the flavonoids of Vitex negundo has shown remarkable results in protection against selenite cataract. Further studies are required to identify the active flavonoids in the fraction and their individual efficacy. Acknowledgements We gratefully acknowledge the financial assistance from Kerala State Council for Science Technology and Environment (KSCSTE), Government of Kerala, India, as a research grant (Order No: (T) 17/ R&D augmentation/04/KSCSTE, dated 20-2-2004) to Dr. Annie Abraham. Authentication of the plant material by Mrs. Gayathri Devi. V, Scientist, Regional Research Institute, Poojapura, Trivandrum, Kerala, India is duly acknowledged. References Aebi, H., 1984. Catalase in vitro. In: Packer, L. (Ed.), Meth. Enzymol, vol. 105. Newyork Academic Press, pp. 121–126. Ahuja, R.P., Borchman, D., Dean, W.L., Paterson, C.A., Zeng, J., Zhang, Z., FergusonYankey, S., Yappert, M.C., 1999. Effect of oxidation on Ca2þ-ATPase activity and membrane lipids in lens epithelial microsomes. Free Radic. Biol. Med. 27,177–185. Awasthi, S., Srivastava, S.K., Piper, J.T., Singhal, S.S., Chaubey, M., Awasthi, Y.C., 1996. Curcumin protects against 4-hydroxy-2-noneal induced cataract. Am. J. Clin. Nutr. 64, 761–766. Bockelbrink, A., Roll, S., Ruether, K., Rasch, A., Greiner, W., Willich, S.N., 2008. Cataract surgery and the development or progression of age-related macular degeneration: a systematic review. Surv. Ophthalmol. 53, 359–367. Biju, P.G., Rooban, B.N., Lija, Y., Gayathri Devi, V., Sahasranamam, V., Abraham., Annie, 2007. Drevogenin D prevents selenite-induced oxidative stress and calpain activation in cultured rat lens. Mol. Vis. 13, 1121–1129. Blois, M.S., 1958. Antioxidant determination by the use of a stable free radical. Nature 181, 1199–1200. Cornish, K.M., Williamson, G., Sanderson, J., 2002. Quercetin metabolism in the lens: role in inhibition of hydrogen peroxide induced cataract. Free Radic. Biol. Med. 33, 63–70. Dharmasiri, M.G., Jayakody, J.R.A.C., Galhena, G., Liyanage, S.S.P., Ratnasooriya, W.D., 2003. Anti-inflammatory and analgesic activities of mature fresh leaves of Vitex negundo. J. Ethnopharmacol. 87, 199–206.

581

Dong, D., Lu, A., Liu, Y., Jia, W., Hou, W., 2000. The early biochemical changes of cataractous lenses of rats cultured in vitro. Zhonghua Yan. Ke. Za. Zhi. 36, 344–721. Donma, O., Yorulmaz, E.O., Pekel, H., Sugugul, N., 2002. Blood and lens lipid peroxidation and antioxidant status in normal individuals, senile and diabetic cataractous patients. Curr. Eye Res. 25, 9–16. Duncan, G., Bushell, A.R., 1975. Ion analysis of cataractous lenses. Exp. Eye Res. 20, 223–230. Elanchezhian, R., Ramesh, E., Sakthivel, M., Isai, M., Geraldine, P., 2007. Acetyl-Lcarnitine prevents selenite-induced cataractogenesis in an experimental animal model. Curr. Eye Res. 32, 961–971. Eskin, N.A.M., Hoehn, E., Frenkel, C., 1978. A simple and rapid quantitative method for total phenolo. J. Agric. Food Chem. 26, 973–974. Ertekin, M.V., Kocer, I., Karslioglu, I., Taysi, S., Gepdiremen, A., Sezen, O., Balci, E., Bakan, N., 2004. Effects of oral Ginkgo biloba supplementation on cataract formation and oxidative stress occurring in lenses of rats exposed to total cranium radiotherapy. Jpn. J. Ophthalmol. 48, 499–502. Ettl, A., Daxer, A., Gottinger, W., Schmid, E., 2004. Inhibition of experimental diabetic cataract by topical administration of RSverapamil hydrochloride. Br. J. Ophthalmol. 88, 44–77. Ganea, E., Harding, J.J., 2006. Glutathione-related enzymes and the eye. Curr. Eye Res. 31, 1–11. Geraldine, P., Brijit Sneha, B., Elanchezhian, R., Ramesh, E., Kalavathy, C.M., Kaliamurthy, J., Thomas, P.A., 2006. Prevention of selenite induced cataractogenesis by acetyl-L-carnitine: an experimental study. Exp. Eye Res. 83, 1340–1349. Grattagliano, I., Vendemiale, G., Sabba, C., Buonamico, P., Altomare, E., 1996. Oxidation of circulating proteins in alcoholics: role of acetaldehyde and xanthine oxidase. J. Hepatol. 25, 28–36. Gupta, S.K., Trivedi, D., Srivastava, S., Joshi, S., Halder, N., Verma, S.D., 2003. Lycopene attenuates oxidative stress induced experimental cataract development: an in vitro and in vivo study. Nutrition 19, 794–799. Gupta, P.D., Johar, K., Vasavada, A., 2004. Causative and preventive action of calcium in cataractogenesis. Acta Pharmacol. Sin. 25, 1250–1256. Gupta, S.K., Srivastava, S., Trivedi, D., Joshi, S., Halder, N., 2005. Ocimum sanctum modulates selenite-induced cataractogenic changes and prevents rat lens opacification. Curr. Eye Res. 30, 583–591. Harding, J.J., 1970. Free and protein bound glutathione in normal and cataractous human lenses. Biochem. J. 117, 957–960. Harding, J.J., 2001. Can drugs or micronutrients prevent cataract? Drugs Aging 18, 473–486. Hibatallah, J., Carduner, C., Poelman, M.C., 1999. In-vivo and in-vitro assessment of the free-radical-scavenger activity of Ginkgo flavone glycosides at high concentration. J. Pharm. Pharmacol 51, 1435–1440. Hiraoka, T., Clark, J.I., 1995. Inhibition of lens opacification during the early stages of cataract formation. Invest. Ophthalmol. Vis. Sci. 36, 2550–2555. Kakkar, P., Das, B., Viswanathan, P.N., 1984. A modified spectrophotometric assay of superoxide dismutase. Indian J. Biochem. Biophys. 21, 130–132. Kumazawa, S., Taniguchi, M., Suzuki, Y., Shimura, M., Kwon, M., Nakayama, T., 2002. Antioxidant activity of polyphenols in carob pods. J. Agric. Food Chem. 50, 373–377. Laemmli, U.K., 1970. Cleavage of structural proteins during assemble of head of bacteriophage T4. Nature 227, 680–685. Lija, Y., Biju, P.G., Reeni, A., Cibin, T.R., Sahasranamam, V., Abraham., Annie, 2006. Modulation of selenite cataract by the flavonoid fraction of Emilia sonchifolia in experimental animal models. Phytother. Res. 20, 1091–1095. Liu, L., Paterson, C.A., Borchman, D., 2002. Regulation of sarco/endoplasmic Ca2þATPase expression by calcium in human lens cells. Exp. Eye Res. 5, 583–590. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265–275. Ma, H., Hata, I., Shih, M., Fukiage, C., Nakamura, Y., Azuma, M., Shearer, T.R., 1999. Lp82 is the dominant form of calpain in young mouse lens. Exp. Eye Res. 68, 447–456. Mohan, N., Gupta, S.K., Agnihotri, S., Joshi, S., Uppal, R.K., 1998. Anticataract action of topical quercetin and myricetin in galactosemic rats. Med. Sci. Res. 16, 685–686. Nabekura, T., Koizumi, Y., Nakao, M., Tomohiro, M., Inomata, M., Ito, Y., 2003. Delay of cataract development in hereditary cataract UPL rats by disulfiram and aminoguanidine. Exp. Eye Res. 76, 169–174. Nakamura, Y., Fukiage, C., Shih, M., Ma, H., David, L.L., Azuma, M., Shearer, T.R., 2000. Contribution of calpain Lp82-induced proteolysis to experimental cataractogenesis in mice. Invest. Ophthalmol. Vis. Sci. 41, 1460–1466. Niehaus Jr., W.G., Samuelsson, B., 1968. Formation of malonaldehyde from phospholipid arachidonate during microsomal lipid peroxidation. Eur. J. Biochem. 6, 126–130. Nirmalan, P.K., Krishnadas, R., Ramakrishnan, R.D., Thulasiraj, R., Katz, J., Tielsch, J.M., Robin, A.L., 2003. Lens opacities in a rural population of southern India: the Aravind comprehensive eye study. Invest. Ophthalmol. Vis. Sci. 44, 4639–4643. Ostadalova, I., Babicky, A., Obenberger, J., 1978. Cataract induced by administration of a single dose of sodium selenite to suckling rats. Experientia 34, 222–223. Padmaja, S., Raju, T.N., 2004. Antioxidant effect of curcumin in selenium induced cataract of Wistar rats. Indian J. Exp. Biol. 42, 601–603. Raser, K.J., Posner, A., Wang, K.K., 1995. Casein zymography: a method to study mucalpain, m-calpain, and their inhibitory agents. Arch. Biochem. Biophys. 319, 211–216.

582

B.N. Rooban et al. / Experimental Eye Research 88 (2009) 575–582

Reddy, V.N., 1990. Glutathione and its function in the lens-an overview. Exp. Eye Res. 50, 771–778. Rorive, G., Kleinzeller, A., 1974. Ca2þ-activated ATPase from renal tubular cells. In: Packer, L. (Ed.), Meth. Enzymol., vol. 32. Newyork Academic Press, pp. 303–306. Salganik, R.I., 2001. The benefits and hazards of antioxidants: controlling apoptosis and other protective mechanisms in cancer patients and the human population. J. Am. Coll. Nutr. 20, 464–472. Sanderson, J., McLauchlan, W.R., Williamson, G., 1999. Quercetin inhibits hydrogen peroxide-induced oxidation of the rat lens. Free Radic. Biol. Med. 26, 639–645. Sedlak, J., Lindsay, R.H., 1968. Estimation of total, protein bound, and nonprotein sulfhydryl groups in tissue with Ellman’s reagent. Anal. Biochem. 25, 192–205. Seko, Y., Saito, Y., Kitahara, J., Imura, N., 1989. Active oxygen generation by the reaction of selenite with reduced glutathione in vitro. In: Wendel, A. (Ed.), Selenium Biology and Medicine. Newyork. Springer-Verlag, pp. 70–73. Shearer, T.R., Shih, M., Mizuno, T., David, L., 1996. Crystallins from rat lens are especially susceptible to calpain-induced light scattering compared to other species. Curr. Eye Res. 15, 860–868. Shearer, T.R., Ma, H., Fukiage, K., Azuma, M., 1997. Selenite nuclear cataract: review of the model. Mol. Vis. 38, 1–14.

Shearer, T.R., Ma, H., Shih, M., Hata, I., Fukiage, C., Nakamura, Y., Azuma, M., 1998. Lp82 calpain during rat lens maturation and cataract formation. Curr. Eye Res. 17, 1037–1043. Shih, M., Ma, H., Nakajima, E., David, L.L., Azuma, M., Shearer, T.R., 2006. Biochemical properties of lens-specific calpain Lp85. Exp. Eye Res. 82, 146–152. Srivastava, O.P., Srivastava, K., 2003. BetaB2-crystallin undergoes extensive truncation during aging in human lenses. Biochem. Biophys. Res. Commun. 301, 44–49. Steel, R.G.D., Torrie, J.H., Dickey, D.A., 1996. Principles and Procedures of Statistics. McGraw Hill, New York. Suryanarayana, P., Saraswat, M., Mrudula, T., Krishna, T.P., Krishnaswamy, K., Reddy, G.B., 2005. Curcumin and turmeric delay streptozotocin-induced diabetic cataract in rats. Invest. Ophthalmol. Vis. Sci. 4, 2092–2099. Toda, J., Kato, S., Oshika, T., Sugita, G., 2007. Posterior capsule opacification after combined cataract surgery and vitrectomy. J. Cataract Refract. Surg. 33, 104–107. Varma, S.D., Hegde, K.R., 2004. Effect of alpha-ketoglutarate against selenite cataract formation. Exp. Eye Res. 79, 913–918. Wang, Z., Bunce, G.E., Hess, J.L., 1993. Selenite and Ca2þ homeostasis in the rat lens: effect on Ca2þATPase and passive Ca2þ transport. Curr. Eye Res. 12, 213–218. Zoric, L., 2003. Parameters of oxidative stress in the lens, aqueous humor and blood in patients with diabetes and senile cataracts. Srp. Arh. Celok. Lek. 131, 137–142.