Deepa Trivedi

Lycopene attenuates oxidative stress induced experimental cataract development: an in vitro and in vivo study.

OBJECTIVES Lycopene, a nutritional antioxidant, was evaluated for its anticataract potential to further establish its role in cataract prevention. METHODS The ability of lycopene to modulate the biochemical parameters was investigated by in vitro studies. Enucleated rat lenses were maintained in organ culture containing Dulbeccos Modified Eagles Medium alone or in addition with 100 microM selenite and served as the normal and control groups, respectively. For the test group, the control medium was supplemented with 10 microM lycopene. The lenses were incubated for 24 h at 37 degrees C. At the end of the incubation period, the lenses were examined for morphologic variation, and biochemical parameters such as reduced glutathione, the lipid peroxidation product malondialdehyde, and the antioxidant enzymes glutathione peroxidase, glutathione S-transferase, superoxide dismutase, and catalase were estimated. In vivo selenite cataract was induced in 9-d-old rats by subcutaneous injection of sodium selenite (25 micromoles/kg of body weight). The rats in the test group were injected with lycopene (200 microg/kg body weight, intraperitoneally) 4 h before the selenite challenge. The incidence of cataract was observed when the rats first opened their eyes. Galactose cataract was induced in rats by feeding 30% galactose in the diet. Rats in the test group were fed orally with 200 microg/kg of lycopene daily, and rats in the control group received only vehicle. Cataract stages were graded at regular intervals. RESULTS A fall (25%) in the glutathione level and a rise (32%) in the malondialdehyde content were observed in control as opposed to normal lenses. Lycopene supplementation in the medium significantly (P < 0.001) restored glutathione and malondialdehyde levels. A significant decrease in the activity of antioxidant enzymes also was observed in the control lenses. A significant restoration in the activities of superoxide dismutase (P < 0.05) and catalase and glutathione S-transferase (P < 0.01), with no effect on glutathione peroxidase, was observed in the lycopene-supplemented group. Lycopene also reduced the incidence of selenite cataract. Only 9% of the eyes in the test group developed dense nuclear opacity as opposed to 83% in the control group. A significant delay in the onset and progression of galactose cataract was observed with oral feeding of lycopene. Only 35% of the eyes developed mature cataract as opposed to 100% in the control group. CONCLUSIONS Lycopene protects against experimental cataract development by virtue of its antioxidant properties, and it may be useful for prophylaxis or therapy against cataracts.

Green Tea (Camellia sinensis) Protects against Selenite-Induced Oxidative Stress in Experimental Cataractogenesis

Cataract is the leading cause of blindness worldwide. It is a multifactorial disease primarily associated with oxidative stress produced by free radicals. The protection offered by various antioxidants in cataract development is well established. Polyphenolic compounds present in green tea (Camellia sinensis) are reported to possess antioxidant property in various pathological conditions. The present study was undertaken to evaluate the anticataract potential of green tea leaf (GTL) extract in the development of lens opacification. Enucleated rat lenses were randomly divided into normal, control and treated groups and incubated for 24 h at 37°C. Oxidative stress was induced by sodium selenite in the culture medium of the two groups (except the normal group). The medium of the treated group was additionally supplemented with GTL extract. After incubation, lenses were subjected to glutathione and malondialdehyde estimation. Enzyme activity of superoxide dismutase, catalase and glutathione peroxidase was also measured in different sets of the experiment. In vivo cataract was induced in 9-day-old rat pups of both control and treated groups by a single subcutaneous injection of sodium selenite. The treated pups were injected GTL extract intraperitoneally prior to selenite challenge and continued for 2 consecutive days thereafter. Cataract incidence was evaluated on 16th postnatal day by slit lamp examination. There was positive modulation of biochemical parameters in the organ culture study. Green tea was also found to reduce the incidence of selenite cataract in vivo. The results suggest that green tea possesses significant anticataract potential and acts primarily by preserving the antioxidant defense system.

Ocimum sanctum Modulates Selenite-Induced Cataractogenic Changes and Prevents Rat Lens Opacification

Purpose: To study the effect of Ocimum sanctum (OS) on selenite-induced morphological and biochemical changes in isolated rat lenses as well as on cataract incidence in rat pups. Methods: Transparent rat lenses were divided into normal, selenite-only, and four treated groups. Selenite-only and treated group lenses were subjected to oxidative stress in vitro by incorporating sodium selenite (100 μ M) in the culture medium. The effect of OS (70, 140, 280, and 560 μ g/ml) was studied on the levels of reduced glutathione (GSH) and thiobarbituric acid reacting substances (TBARS) in selenite-challenged lenses. The lowest concentration of OS offering significant modulation on these two parameters was determined. Subsequently, the effect of prior and cotreatment with the lowest effective concentration of OS was studied on TBARS, GSH, and on lens antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GSHPx), catalase (CAT), and glutathione-S-transferase (GST). Changes in lens protein profiles under different incubation conditions were analyzed by SDS gel-electrophoresis. In vivo, cataract was induced by a single subcutaneous injection of sodium selenite (25 μ mole/kg b.w.) to 9-day-old rat pups. The anticataract effect of OS (5 and 10 mg/kg b.w.) injected intraperitoneally 4 hr prior to selenite challenge was evaluated by the presence of lens nuclear opacity in rat pups on the 16th postnatal day. Insolubilization of lens proteins post–selenite injection was monitored for 4 days. Results: The lenses in the selenite-only group developed cortical opacities in 24 hr. OS showed different degrees of positive modulation in selenite-induced morphological as well as biochemical changes. The lowest effective dose of OS that significantly modulated glutathione and thiobarbituric acid reacting substances was found to be 140 μ g/ml. At this dose, a significant increase in antioxidant enzyme levels and preservation of normal lens protein profile was observed. OS at the dose of 70 μ g/ml did not show any significant protection with respect to either morphology or biochemistry of lenses. In vivo, 5 and 10 mg/kg of OS reduced the incidence of selenite cataract by 20% and 60%, respectively, and prevented protein insolubilization as well. Conclusions: Aqueous extract of OS possesses potential anticataract activity against selenite-induced experimental cataractogenesis. The protective effect was supported by restoration of the antioxidant defense system and inhibition of protein insolubilization of rat lenses as well.

Lycopene prevents sugar-induced morphological changes and modulates antioxidant status of human lens epithelial cells.

Cataract is a multifactorial disease. Osmotic stress, together with weakened antioxidant defence mechanisms, is attributed to the changes observed in human diabetic cataract. Epidemiological studies provide evidence that nutritional antioxidants slow down the progression of cataract. The usefulness of lycopene, a dietary carotenoid, in the pathogenesis of human cataracts has not been studied so far. Since the epithelium is the metabolic unit of the lens, the effect of lycopene on galactose-induced morphological changes and antioxidant status of human lens epithelial cells (HLEC) in culture was evaluated in the present study. HLEC of fresh cadaver eyes obtained from an eye bank were cultured in medium supplemented with fetal calf serum (200 ml/l). On confluency, the cells were subcultured in medium containing either 30 mm-d-galactose or 30 mm-d-galactose+lycopene (5, 10 or 20 microm) for 72 h. The cells were observed under the phase-contrast microscope and transmssion electron microscope for any morphological changes and then harvested for the estimation of various biochemical variables. Malondialdeyde, glutathione and antioxidant enzymes were significantly altered in the control as compared with the normal cultures. Vacuolization was also observed in the presence of galactose. Addition of lycopene confers significant protection against these changes in HLEC.

Pyruvate Inhibits Galactosemic Changes in Cultured Cat Lens Epithelial Cells

An attempt was made to maintain cat lens epithelial cells (CLEC) in culture and study the morphology, growth and survival of these cells in vitro. The influence of incorporation of galactose (30 mM) into the culture medium on the morphology and biochemistry of CLEC in the primary culture was then investigated. To establish the effect of galactose on CLEC, various biochemical parameters associated with galactosemic cataract such as aldose reductase (AR), Na+K+ATPase, glutathione, polyol and soluble/insoluble proteins were estimated after 24 h of incubation. The effect of pyruvate (5 mM), a ‘physiological antioxidant’, on the changes induced by galactose in CLEC was studied. CLEC in culture showed regular hexagonal cells with prominent nuclei. The CLEC culture attained confluency in 11 days during primary culture and semiconfluency in 14 days in two subsequent passages. Vacuolization and significantly raised AR activity, polyol levels and insoluble protein contents were observed; they had no effect on Na+K+ATPase and soluble protein after 24 h of incubation in the culture medium with galactose. Supplementation of pyruvate (5 mM) resulted in a lesser number of vacuoles together with a positive modulation of these parameters.

Pyruvate modulates antioxidant status of cultured human lens epithelial cells under hypergalactosemic conditions

Lens epithelial cells are the metabolic unit of the lens and antioxidant enzymes are mainly concentrated here. The purpose of this study was to maintain human lens epithelial cells (HLEC) in culture and examine the status of antioxidant enzymes (glutathione peroxidase (GSHPx), catalase (CAT), glutathione-S-transferase (GST)), lipid peroxidation product malondialdehyde (MDA) and glutathione (GSH) levels in these cells under normal as well as hypergalactosemic (30 mM galactose) conditions. Further, effect of pyruvate, a physiological antioxidant has also been evaluated on these parameters. For conducting experiments, anterior capsule specimens obtained from fresh cadaver eyes from eye bank were cultured in Dulbeccos Modified Eagles Medium (DMEM) supplemented with 20% fetal calf serum. Upon confluency, the cells were subcultured in three separate flasks containing DMEM alone (normal group), DMEM + 30 mM D-galactose (control group), DMEM + 30 mM D-galactose + 5 mM pyruvate (test group) and incubated for 24 or 72 h. These cells were observed under the phase contrast microscope for any morphological changes and harvested for the estimation of various antioxidant parameters. Our results show significant weakened antioxidant defense in HLEC when incubated in the presence of galactose as compared to normal. Addition of pyruvate significantly modulated levels of GSH, MDA, GSHPx, CAT and GST.