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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-Weis 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.
Initial verification of the hypothesis has been done primarily by rat and mouse lens organ culture studies under ambient as well as 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 GSH and ATP as well as in terms of apoptotic cell death. In vivo studies on the possible prevention of oxidative stress and cataract formation has been done by administering pyruvate and caffeine orally in drinking water as well as by their topical application using diabetic and galactosemic animal models.
Photosensitized damage to lens has been found to be significantly prevented by ascorbate and pyruvate caused by exposure to visible as well as UVA. Caffeine has been found be effective against UVA as well as UVB. Oral or topical application of pyruvate has been found to inhibit the formation of cataracts induced by diabetes as well as galactosemia. Caffeine has also been found to inhibit cataract induced by sodium selenite as well as high levels of galactose. Studies with diabetes are in progress.
Various in vitro and in vivo studies summarized in the review strongly support the hypothesis that the 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 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 stress has now been well accepted to be involved in the pathogenesis of many aging diseases such as atherosclerosis, myocardial infarction, Alzheimer's disease, Parkinson's disease, schizophrenia, bipolar disorders, generalized tissue sclerosis and many vision impairing and blinding diseases such as cataracts, glaucoma and retinopathies etc. While all these diseases inflict significant functional disabilities, the vision impairment due to cataract has one of the most adverse impacts on life, functionally as well as economically. Its prevalence is also very high. Everyone encounters it soon after the 40's when deterioration in vision perception due to lens changes becomes functionally noticeable and continues to decline thereafter. As summarized in Figure 1 A, cataracts account for about 49% of vision impairment in the world (1). Seemingly, the occurrence of the disease is somewhat higher in women than in men. Hence, one of the major thrust of eye research, at least till recently, has been directed towards understanding the pathogenesis of the disease and its possible therapeutic prevention. The thrust of such research however appears to have been decreased somewhat due to the advancements in surgical techniques of removing the cataractous lenses and its replacement by a synthetic implant. While this leads to an immediate satisfactory recovery of vision, the overall prevalence of the disease continues to remain high and essentially unchanged due to the inevitable replacement of the old cases by new ones. Therefore, surgery alone is unable to cope up with the problem. In addition, like with any surgery, there is always the possibility of inherent post-surgical complications which in this case can consist of a posterior capsular tear and consequent penetration of vitreous in the anterior chamber, cystoid macular edema, and often noted detachment of vitreous from the retina with photopsia and appearance of floaters (2, 3). The eye also has been reported now to be more prone to retinal detachment with significant loss of vision (4, 5). Removal of cataract has also been understood to hasten the appearance of macular changes associated with aging and diabetes. Hence it would be desirable to develop therapeutic measures to prevent or delay the process of cataract formation. Several experimental (6–8) and epidemiological studies (9, 10) strongly suggest the possibility that this can be achieved by use of antioxidants and certain metabolic agonists.
It has been known for a long time that the incidence of cataract is high in countries with excessive sun light. This has now been well established by compilation of the data by WHO (1), reproduced in Table 1 and figure 1A. As would be further obvious by referring to Figures 1B and 1C, most of the countries with high incidence of cataracts are located in areas of the world with relatively higher UV index (erythemal index) shifting annually between 0 to 35 degree north and south. Cataracts extracted from such countries are also known to be more strongly pigmented with yellow to brown coloration, than those extracted from patients native to the countries in higher latitudes (11–13). It has been suggested that such coloration as well as its higher incidence in countries with higher solar light intensities are due to the penetration of UV in the eye and consequent photo oxidation of proteins (11–13). This has been particularly ascribed to photo oxidation of tryptophan moieties. However, the absorption maxima of tryptophan and other aromatic amino acids present free as well as part of the lens proteins is around 280 nm, wavelengths which do not penetrate the eye. Light only of wavelengths greater than 300 nm appear to do so. Therefore the correlation between the incidence data and the solar irradiation zones should be related to the pathophysiological effect of light primarily of wavelengths greater than 300 nm and beyond-although the contribution of visible light has received less attention. Many previous studies have suggested that the effect of UV on cataract formation involves photo-sensitization by tryptophan derived products generated metabolically, chiefly the kynurenine derivatives (11, 14). The absorption maxima of free kynurenine and hydroxyl kynurenine are near 368 nm (15), with a molar extinction coefficient of 365O M−1 Cm−1. This is subject to minor shifts when present in combination with proteins, and with changes in pH. It increases to 4300 M−1 Cm−1 when metabolically converted to 3-hydroxy glucoside.
Based on the observation that exposure of animals (16) as well as humans to high oxygen atmosphere (17) and the relatively wide prevalence of cataract in areas of the world with excessive sunlight, we hypothesized that the high incidence of cataracts in countries with excessive light could be due to photochemical generation of reactive oxygen species (ROS) including superoxide and its derivatization to other potent entities such as hydrogen peroxide, hydroxyl radicals and singlet oxygen, in the aqueous and the lens and consequent oxidative damage (18–20). This can happen even by photosensitization in blue light (20)- light and oxygen acting synergistically in the presence of an appropriate photosensitizer. One of the earliest compounds known to be biologically strongly active photodynamically is acridine (21), pharmacologically used as acriflavine for external would healing. Hence in initial examination of the hypothesis proposing that a continued photochemical generation of reactive oxygen might indeed be involved in causing oxidative stress to the lens we used riboflavin, known to be present as such as well as FAD and FMN in cornea (22), aqueous humor (23) and lens (24). Because of its high absorption maxima at 360, 450, 460 and 475 nm with extinction coefficients varying between 12,500 and 11,000 M−1 Cm−1 (25), values which are about 3 times higher than that of kynurenine, we thought it would photosensitize the lens more strongly and would do so in near UV as well as under visible light. In addition, we thought that it should be capable of acting as a pseudocatalyst, converting molecular oxygen to its more reactive forms cyclically and thus be able to perpetuate the generation of ROS, in visible as well as in near UV, by integration of the following reactions. As shown below, the system will continue to do so as long as light enters the eye, and oxygen and reducing equivalents remain available to consistently reduce activated riboflavin molecules. Only a trace amount of this or other photosensitizer would be required.
We first tested the effectiveness of the above photochemical system in causing lens damage in the rat lens organ culture system, measuring the transport of rubidium ions against its electro chemical gradient in the presence and absence of various scavengers of ROS and noticing the status of its transparency (19, 20). As shown in figure 2, when lenses were cultured in medium 199 containing 50 μM riboflavin phosphate for an overnight period of 18 hours in dark, the concentration of the rubidium ions in the lens rose to about 22 fold higher than in the medium-showing its accumulation in the lens against a concentration gradient. This value was similar to the value obtained with lenses incubated in the basal medium with riboflavin but in dark. Incubation in light using a day light tube adjusted to an intensity of 50 ft. candle was found highly injurious to the tissue, the distribution ratio of rubidium ions in this case being reduced to 5. That this was related to the generation of ROS such as superoxide and hydrogen peroxide was evident by the preventive effect of fericytochrome c as well as catalase. Significance of superoxide and hydrogen peroxide generation in the photo damage to the lens was further obvious by using the above system of incubation with potassium ferricyanide and potassium ferrocyanide (26). Addition of the ferricyanide to the medium which scavenges superoxide was nearly fully protective. Addition of ferrocyanide was also protective (Table 2). This is explained by the re-oxidation of the ferrocyanide to ferricyanide by the hydrogen peroxide produced simultaneously by the dismutation of superoxide going on in the same medium. Thus, the two agents work in concert, one scavenging superoxide and the other scavenging hydrogen peroxide. The lenses also remained very clear.
The importance of the superoxide generation in the oxidative cascade leading to lens opacification and cataract formation has now been further substantiated by studies with SOD knock out mice showing that the knockouts develop spontaneous cataracts (27, 28). However despite the presence of these enzymes human cataract development with aging and certain diseases such as diabetes still is a fact suggesting that the effectiveness of SOD and other antioxidant enzymes could be limited due to several reasons such as their deactivation with aging and their selective distribution and availability in various cellular compartments. Also, being macromolecules, they cannot penetrate to certain sensitive sites of oxidation buried in proteins and in nucleic acids. In addition, the lens is surrounded and bathed by aqueous and the vitreous humors, fluids that lack the enzymatic defenses. Therefore the lens cell membranes which remain continuously exposed to a photochemical oxidative environment due to the continued light penetration during the long periods of photopic vision remain susceptible to photo damage. Investigations were hence conducted to find out additional modes involved in protecting lens against oxidative stress.
We have hypothesized that is offered by ascorbate as one of the protective compounds (20). The concentration of this substance is about 20 fold higher in the aqueous than in the blood. Consequently, it is high in the lens also. Such a high concentration is maintained by its active transport from the plasma across the blood aqueous barrier. As shown in Table 3 its concentration in the aqueous and the lens is significantly higher in comparison to many other tissues and fluids (29). Additionally, as summarized in Table 4 its level in the diurnal animals is significantly higher than that in the nocturnal animals, pointing out to a natural biological adaptation in response to the photochemical environment in the aqueous and lens of the diurnal animals (29). The initial verification of the ascorbate hypothesis was done by lens culture experiments directly in light in the riboflavin containing medium (20). As shown in figure 3 addition of ascorbate to the medium was significantly effective in preventing photochemical damage to lens transport pump. The results on experiments with peroxide generated by the xanthine-xanthine oxidase system in dark are summarized in Table 5 (30). As summarized in Table 6, the rate constant of its reaction with the hydroxyl radical is significantly high and hence is controlled essentially only by its diffusion (31). However, while ascorbate could be directly effective in protecting the tissue by scavenging ROS in the aqueous milieu, its effectiveness in the lipid environ of the tissue could become partial by limiting its availability primarily to the aqueous region. Vitamin E, which is lipid soluble, could therefore be the compound of choice to protect the tissue lipids. This is indicated by preventive effect of this nutrient when fed to Emory mice, diabetic rats and UV-exposed rabbits (32, 33, 6)
However, the availability of such nutritional antioxidants can be varied, depending upon the agricultural seasons, dietary variations and many physiological factors such as the effectiveness of their gastrointestinal absorption. The efficiency of their transport through the blood aqueous barrier can also decline with age. Interestingly, its concentration in the aqueous humor of cataractous patients have been reported to be lower than normal (34). In addition, there seems to be some correlation with the type of cataract and metabolic status of the patients such as status of tissue glycolysis.
We therefore hypothesized that pyruvate, produced normally by tissue metabolism can also participate in the delay of cataracts. It is well known to react with various ROS generating acetate and carbon dioxide (35–39). The rate constants of these reactions as summarized below are also sufficiently high.
As shown in figure 4, its incorporation in the medium of incubation was found highly effective against oxidative modification of the proteins inflicted by incubation of the tissue in presence of hydrogen peroxide (40). It was also found effective in preventing oxidative damage to the rat lens in terms of the decreased ability to carry out active transport of rubidium ions when incubated with xanthine and xanthine oxidase (41) or in riboflavin containing medium exposed to day light (42). Similar effects have been observed also in the case of mouse lens (43) where the incubation medium contained xanthine oxidase as well as uricase (Figure 5). More recently, we have demonstrated that its protective effect, in addition to being exerted by its ROS scavenging activity can also be attributed to its ability to stimulate the payoff portion of tissue glycolysis (44), and thereby help in the maintenance of tissue levels of ATP, required for supporting the biosynthetic activities in addition to active transport. In all its reactions with various ROS, the products generated are acetate, carbon dioxide and water. Hence no toxic products are generated, unlike the case with ascorbate and vitamin E where products of reaction containing a keto group (dehydroascorbate and tocopherones) can potentially act as protein cross-linking agents. They can also become pro-oxidants in presence of certain metal ions. Pyruvate on the other hand, in addition to its radical scavenging properties, also inhibits competitively the process of protein glycation by sugars, an important reaction involved with aging and cataract formation, particularly in diabetes (45). Encouraged with these beneficial properties, experiments have also been done to examine its effectiveness in preventing cataract formation in vivo using galactosemic and diabetic animal models. Maintaining rats as well mice on water containing 2% sodium pyruvate has been found to be significantly effective in preventing cataract formation (7, 46–48). As shown in Figures 6 & 7, lenses in both the species maintained on pyruvate-containing water and food were much more transparent than in the group receiving regular diet and water.
In addition to its effectiveness against diabetic cataract formation, we also demonstrated that it is effective in preventing oxyradical induced lens damage triggered by UVA irradiation (49). The inhibition of rubidium uptake induced by ROS was significantly prevented by pyruvate. The biochemical damage reflected by the decrease in the levels of GSH was also substantially minimized (Figure 8). The frank appearance of cataracts (Figure 9) as well as the associated histological changes (Figures 10, ,11)11) was also prevented.
Therefore, these and several other studies with antioxidants strongly suggest that cataract is a pharmacologically preventable disease. However, it would be necessary to carry on such treatment only topically and not systemically. This is because of the greater effectiveness of compounds when administered topically. In addition, it is desirable to use this route to avoid toxicity of the compounds which will then be required to be given in larger amounts if one uses the oral route. However achieving this topically is problematic because of the instability of most of the antioxidants when compounded pharmaceutically. In addition, in the eye they get photolyzed due to continued light penetration, especially during the long period of photopic vision. It was therefore decided to investigate cataract prevention by antioxidants and free radical scavenging compounds that are relatively more stable and well tolerated physiologically by the human body.
In this connection, we have recently observed that caffeine which is much more stable than ascorbate, vitamin E and pyruvate is highly effective in protecting the lens against UV-induced damage. This was initially ascertained by incubation lens studies under UVB (300nm) conduced in basal Tyrode medium pulsed with 86Rb+ without or with caffeine (50). Subsequent to incubation, they were examined for the state of their transparency, extent of 86Rb+ accumulation and the contents of ATP and GSH. As expected, incubation under UV was associated with the loss of transparency as monitored photographically (Figure 12). 86Rb+ accumulation was also inhibited, with associated loss of ATP and GSH. In the presence of caffeine, the lenses remained much more transparent. The extent of 86Rb+ accumulation as well as the contents of GSH and ATP was also significantly higher (Table 7). Further confirmation of the effectiveness of caffeine against oxyradical species was also done in dark where ROS was generated by Fe8Br8 a water soluble iron complex (51). It exerted similar inhibitory effects on the rubidium accumulation and loss of transparency. The contents of GSH and ATP were also decreased (Table 8). In order to confirm further that the effect of caffeine observed above is due to its ability to scavenge ROS, parallel studies were conducted with Tempol, a more conventional oxyradical scavenger which also was found effective (Table 9). However, since UV wavelength used in the above experiments were lower than the UV that normally enters the eye, further confirmation of the protective effect of caffeine against UV damage was obtained by lens culture experiments at 365 nm, maintaining similar irradiation intensity of 0.6 mW/sq. cm (52). As expected, the effect of such irradiation does not significantly affected -the rubidium pump damage. However addition of kynurenine to the medium significantly enhanced the damaging effect of UVA, the uptake of rubidium in this case being only 20% of the basal controls. Addition of caffeine was again highly protective, the uptake of rubidium in this case being 85% of the basal dark control. It also maintained the levels of ATP and GSH closer to the control (Table 10). Figure 13 represents the ESR spectroscopy of the medium exposed to UVA. As apparent, hydroxyl quartet signals were well noticeable. The signals were minimized significantly by addition of caffeine. Thus one of the primary modes of the caffeine effect appears to be related to its ability to scavenge the ROS produced in the medium by photo-irradiation. Hence studies are in progress to test its effectiveness in vivo. At present we are limited by the lack of an appropriate animal model of UV cataracts. However, since oxidative stress is an important factor in the genesis of cataracts in general including UV irradiation, we have examined the effectiveness of caffeine against cataract formation in selenite and galactosemic animal models (53–55). As will be obvious by reference to figure 14, administration of caffeine to sodium selenite treated rats was highly preventive, indicated by the inhibition of cataract formation as well as by the maintenance of the levels of ATP and GSH. Similar results were obtained with galactosemic rats where they were given caffeine mixed with the diet (54). However in view of the desirability of treating eye diseases with topical eye drops, further experiments with galactosemic animals have been done with caffeine eye drops (55), such mode of treatment being expected to be more efficacious while at the same time avoiding administration of the compound orally in larger amounts. Hence, in these experiments, the galactosemic animals were treated with eye drops containing about 280 μg of caffeine, 5 times/day and cataract formation was followed for 25 days. As indicated in Figure 15, the progress of cataract was significantly thwarted by caffeine treatment. The extent of opacity till the end of the experiment was only minor if any in the caffeine eye drops treated group. In the untreated (placebo) group cataract was highly advanced occupying the entire lens. The prevention of cataract was also evident by higher levels of GSH (Figure 16). The protective effect was further apparent by H&E histology (Figure 17). In the caffeine untreated group, the sub-epithelial and deeper cortical layers of the tissue had abundance of nucleated fiber cells representing aberrantly differentiating cells. The cells are also hydropic with the overall structure becoming non-homogeneous. On the contrary, lenses of the animals treated with caffeine eye drops maintained a normal structure consisting of a single layer of epithelial cells followed by well-aligned non-nucleated fiber cells. The central region of the tissue also remained homogenous. As we have previously established, the cataractogenic process eventually leads to apoptotic cell death. That was also found to be inhibited by caffeine. This was done by staining the lens sections with DAPI and F-dUTP (Figure 18). As expected, most of the nucleated cells are in the bow zone and the anterior epithelium in the normal tissue. In the case of galactose-fed animals, the distribution of DAPI-positive cells was highly abnormal, showing abundance of nucleated cells in the deeper cortex. That these cells are indeed apoptotic was proved by TUNEL staining using F-dUTP. In the galactosemic animals treated with caffeine, the apoptotic cells were essentially absent and the distribution of the cells was also normal as expected.
The anti-apoptotic effect of caffeine is also now being studied in lenses exposed directly to UV, preliminary results being similar to that in galactosemic animals. In conclusion, therefore, it has been shown that oxyradical generation is an important pathogenic factor in the genesis of cataracts induced concomitant to the penetration of UV as well as visible light. We have demonstrated that such cataract formation can potentially be prevented by treatment with compounds which can act as oxyradical scavengers when given in small amounts through topical eye drops. In view of the instability of ascorbate and vitamin E when incorporated into pharmaceutical preparations as well as in the eye due to photolytic decomposition, it is apparent that some more stable compounds such as the pyruvate esters and xanthine alkaloids such as caffeine might be found useful.
The work was supported by NEI, NIH through an RO1 grant EY01292 to SDV.
Supported by NIH NEI grant RO1 EY01292 to S. D. Varma
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