Kavita R. Hegde

Role of ultraviolet irradiation and oxidative stress in cataract formation-medical prevention by nutritional antioxidants and metabolic agonists.

Purpose:Cataract is a significant cause of visual disability with relatively high incidence. It has been proposed that such high incidence is related to oxidative stress induced by continued intraocular penetration of light and consequent photochemical generation of reactive oxygen species, such as superoxide and singlet oxygen and their derivatization to other oxidants, such as hydrogen peroxide and hydroxyl radical. The latter two can also interact to generate singlet oxygen by Haber-Weiss reaction. It has been proposed that in addition to the endogenous enzymatic antioxidant enzymes, the process can be inhibited by many nutritional and metabolic oxyradical scavengers, such as ascorbate, vitamin E, pyruvate, and xanthine alkaloids, such as caffeine. Methods:Initial verification of the hypothesis has been done primarily by rat and mouse lens organ culture studies under ambient as well as ultraviolet (UV) light irradiation and determining the effect of such irradiation on its physiology in terms of its efficiency of active membrane transport activity and the levels of certain metabolites such as glutathione and adenosine triphosphate as well as in terms of apoptotic cell death. In vivo studies on the possible prevention of oxidative stress and cataract formation have been conducted by administering pyruvate and caffeine orally in drinking water and by their topical application using diabetic and galactosemic animal models. Results:Photosensitized damage to lens caused by exposure to visible light and UVA has been found to be significantly prevented by ascorbate and pyruvate. Caffeine has been found be effective against UVA and UVB. Oral or topical application of pyruvate has been found to inhibit the formation of cataracts induced by diabetes and galactosemia. Caffeine has also been found to inhibit cataract induced by sodium selenite and high levels of galactose. Studies with diabetes are in progress. Conclusions:Various in vitro and in vivo studies summarized in this review strongly support the hypothesis that light penetration into the eye is a significant contributory factor in the genesis of cataracts. The major effect is through photochemical generation of reactive oxygen species and consequent oxidative stress to the tissue. The results demonstrate that this can be averted by the use of various antioxidants administered preferably by topical route. That they will be so effective is strongly suggested by the effectiveness of pyruvate and caffeine administered topically to diabetic and galactosemic animals.

Oxidative Damage to Lens in Culture: Reversibility by Pyruvate and Ethyl Pyruvate

It is generally believed that prophylactic intake of antioxidants is beneficial in delaying the onset of some aging manifestations such as cataract. However, whether such a supplementation will also be effective if the pathophysiogical process has already set in remains a largely open question. We examined this possibility with lens changes leading to cataract formation, since cataract genesis is intimately related to a continued generation of reactive oxygen species (ROS) in the aqueous humor. Since the formation of cataract is a well-defined progressive disease, starting with an early refractive change and leading to gradual enhancement of opacification, we hypothesized that even a belated start with an appropriate anti-oxidant could halt the pathological process and delay cataract maturation and vision impairment. Using lens cultures, we tested this hypothesis with pyruvate, known to be an effective and highly potent ROS scavenger. Adding pyruvate to the culture medium after lenses had sustained a 50% damage was significantly effective in preventing progress. This was apparent by better maintenance of the active rubidium transport activity in these lenses compared to controls without pyruvate treatment. Glutathione levels were also higher in the pyruvate group.

Oxidative damage to mouse lens in culture. Protective effect of pyruvate

Studies have been conducted to examine the feasibility of preventing oxyradical-dependent oxidative stress to mouse lens in culture, using pyruvate as an antioxidant. The extent of oxidative damage to the tissue was assessed by measurement of the status of Na(+)-K(+) ATPase dependent active transport of rubidium 86Rb(+). The tissue levels of adenosine triphosphate (ATP), glutathione (GSH), malonaldehyde (MDA) and catalase were also determined. While the measurement of 86Rb(+) uptake provides an assessment of the integrity of the primary active transport system, measurement of the other components reflects the status of intracellular oxidative stress. ATP measurement also reflected on the overall status of metabolic integrity. Incubation of the lens with xanthine (XA)/xanthine oxidase (XO) system had an adverse effect on all these parameters. Incorporation of pyruvate was strikingly protective. The protective effect of pyruvate is apparently due to its ability to scavenge ROS generated in the medium with the possibility of its action on tissue metabolism as well. The findings are hence considered useful for further studies on the prevention of oxidative stress to tissues by exogenous supplementation with pyruvate, specially the human lens where the biochemistry of its antioxidant mechanisms is similar to the mouse lens, contrary to the rat lens.

UV-B-Induced Damage to the Lens In Vitro: Prevention by Caffeine

Ultraviolet (UV) irradiation is one of the significant risk factors in the genesis of cataracts. Pathogenetically, the process can be triggered by the intraocular generation of various reactive species of oxygen that are well known to be initiated by the penetration of light, especially of the UV frequencies. The contribution of UV exposure in the etiology of this disease is likely to increase further due to ozone depletion in the upper atmosphere. The present studies were undertaken to examine if the UV effects can be attenuated with the xanthine-based alkaloids primarily present in tea and coffee. We have examined this possibility by in vitro lens culture studies with caffeine. As expected, mice lenses incubated in Tyrode solution exposed to UV at 302 nm are physiologically damaged, as evidenced by the inhibition of the active transport of (86)Rb(+), an ion acting as a surrogate of the K(+). There was a simultaneous decrease in the levels of adenosine triphosphate and glutathione. The addition of caffeine to the medium prevented such deleterious effects. That caffeine and perhaps other xanthinoids have a protective effect against cataract formation induced by UV has hence been demonstrated for the first time.

Prevention of cataract by pyruvate in experimentally diabetic mice.

Previous studies have demonstrated that administration of pyruvate prevents cataract formation in diabetic rats. It is known that the induction of cataractous process in this case is initiated by aldose reductase (AR) catalyzed synthesis and accumulation of excessive sorbitol in the lens fibres and epithelium and their consequent osmotic hydration. Synthesis of this and other polyols is competitively inhibited by pyruvate. The objective of the present investigations was hence to determine whether pyruvate would have a similar protective effect in species where cataract formation is relatively independent of sorbitol synthesis such as in humans where the lens AR activity is extremely low, especially with glucose as a substrate. The Km of AR for glucose is known to be very high. The possible protective effect of pyruvate in the low AR models was conceived on the basis of our previous findings suggesting that it can also exert substantial antiglycating as well as antioxidant effects. The present studies have hence been conducted with mice, a species known to be low in lens AR, similar to that in humans. As stipulated, pyruvate administration has indeed been found to offer a significant protection against development of diabetic cataract in this model also. The effect correlated with the inhibition of protein glycation as well as of oxidative stress. The latter was apparent by the prevention of the loss of glutathione known to be associated with diabetes. Although there was a small but noticeable increment in the sorbitol content of the diabetic lenses, this was osmotically insignificant. Even this increase was prevented by pyruvate. The magnitude of the elevation in the contents of glycated proteins and the depression in the level of glutathione were, on the contrary, highly pronounced, suggesting a more prominent role of the latter factors. In addition, the possibility of a direct metabolic support it could offer to the tissue is also imminent by its effect on the maintenance of ATP, as shown earlier. The present studies are therefore considered more relevant to the pathogenesis of cataract in human diabetics and its possible prevention by endogenous compounds with antiglycating and antioxidant properties. Inhibition of cataract formation by pyruvate in an animal model with low lens AR, similar to that in humans, has been shown for the first time. (Mol Cell Biochem 269: 115–120, 2005)

Inhibition of Selenite Induced Cataract by Caffeine

Purpose:  The objective of the investigation was to study possible inhibition of oxidative stress and cataract formation by caffeine in vivo.

Lens thiol depletion by peroxynitrite. Protective effect of pyruvate

Pyruvate (PY) is known to be a potent scavenger of H2O2 by undergoing its peroxidative decarboxylation. While doing so, it also inhibits ·OH generation, in addition to its direct ·OH scavenging effect. We now hypothesize that PY would also be decarboxylated by cleaving the -O-O- bond in peroxynitrite (ONOO−) (PN), with the effect of protecting tissues against NOx induced damage. We have verified this by measuring 14CO2 formation on incubation of 1-14C-PY with 3-morpholinosydnonimine (SIN-1). Its protective effect against PN induced thiol depletion was initially assessed by determining its ability to inhibit oxidation of pure GSH. This was further evaluated by incubating lens homogenate with SIN-1 with or without PY. As conceived, PY did inhibit PN induced loss of protein as well as non-protein -SH. The findings therefore appear potentially useful to protect against nitrite induced damage to the lens and other tissues known to occur with aging and certain diseases such as diabetes.

Kynurenine-induced photo oxidative damage to lens in vitro: protective effect of caffeine

Photochemical generation of reactive species of oxygen in the lens and aqueous and consequent physiological damage to the tissue has been implicated in the genesis of human cataracts. The present studies were undertaken to examine the feasibility of possible prevention of such damage to the lens initiated by UV activation of kynurenine, a well-known photosensitizer in the human lens. The studies were done by organ culturing intact mouse lenses in medium containing kynurenine and exposed to UVA. Tissue damage was assessed by the inhibition of its ability to carry active transport of rubidium ions and the associated decrements in the levels of GSH and ATP. These deleterious effects were significantly prevented by caffeine, an alkaloid present in many common beverages and known to chemically deactivate the said oxygen derivatives. Further studies on the pharmacological significance of the findings are hence in progress.

Inhibition of glycolysis in the retina by oxidative stress: prevention by pyruvate

Intraocular generation of reactive oxygen species (ROS) with consequent oxidative stress has been shown to be a significant factor in the pathogenesis of many vision-impairing diseases such as cataracts and retinal degenerations. Previous studies have shown that pyruvate can inhibit such oxidative stress. This is attributable to its property of scavenging various ROS and consequently inhibiting many of the apparent toxic reactions such as lipid peroxidation and loss of tissue thiols. It is hence expected that ROS will have an adverse effect on tissue metabolism also. The present investigations were hence undertaken to study the possibility that while scavenging ROS, the compound could be effective also in preventing the inhibition of tissue metabolism as well. Since glycolysis constitutes the major bioenergetic source of the retina, the objective of the present studies was to ascertain if the effects of pyruvate are indeed reflected in the maintenance of this pathway even when the tissue is exposed to ROS. This hypothesis was examined by incubating retinal explants in ROS-generating medium in the absence and presence of pyruvate and measuring 3H2O generated from 5-3H glucose. In addition, the lactate generated was also measured. As hypothesized, ROS-induced inhibition of glycolysis indexed by the decrease in 3H2O as well as lactate formation was significantly prevented by pyruvate. This effect was also reflected by the elevation of NAD/NADH ratio, a major pacemaker of glycolysis.

Prevention of Oxidative Damage to Lens by Caffeine

PURPOSE The primary objective of this study was to examine the possibility of inhibiting oxidative damage to the lens in vitro by caffeine. METHODS Oxidative damage was inflicted by incubating mouse lenses in Tyrode medium containing 0.1 mM Fe(8)Br(8), an iron complex soluble in aqueous medium. Parallel incubations were conducted in the presence of caffeine (5 mM). RESULTS Lenses incubated in the medium containing Fe(8)Br(8) undergo oxidative stress, as evidenced by the inhibition of Na(+)-K(+) ATPase-driven rubidium transport and the loss of tissue glutathione and ATP. These effects were prevented in presence of caffeine. That the effects are due to the oxyradicals produced was ascertained further by parallel studies with Tempol (5 mM), a well-known scavenger of reactive oxygen species (ROS) with its activity being more pronounced with hydroxyl radicals as compared to other ROS. CONCLUSIONS Caffeine was found to be effective in preventing oxidative stress to the lens induced by iron under ambient conditions. The protective effect is attributable to its ability to scavenge ROS, particularly the hydroxyl radical.