Shambhu D. Varma

Oxidative stress on lens and cataract formation: role of light and oxygen

The mechanism of oxidative damage to the lens through intraocular photochemical generation of superoxide and its derivatization to other oxidants such as singlet oxygen, hydroxyl radical and hydrogen peroxide has been studied. Rat lenses when organ cultured aerobically in TC 199 containing additional amounts of riboflavin were damaged as demonstrated by an inhibition of the uptake of Rb 86 against a concentration gradient. The pump was not affected by light if the culture was conducted in the basal TC 199. However, light was observed to induce significant peroxidative degradation of the tissue lipids even in the basal medium, the degradation being indicated by the formation of malonaldehyde. Both the inhibition of the pump as well as the peroxidative degradation of the tissue lipids, were attenuated considerably by scavengers of superoxide and hydrogen peroxide. In addition, the lipid degradation was prevented by vitamins C and E. The results suggest that the photodynamic injury to the lens cation pump as well as to membrane lipids is incumbent upon an initial generation of superoxide and its derivatization to other oxidants. Thus, the ocular lens is susceptible to oxidative insult and physiological damage through photocatalytic generation of various oxygen radicals. Large concentrations of ascorbic acid in the aqueous humor seems to be able to provide significant protection against such an insult. Thus, this may be one of the functions of high concentration of ascorbic acid in the aqueous humor. The implication of oxidative stress has also been examined in the genesis of cataracts in vivo. Treatment with vitamin E of the Emory mouse led to a decrease in the rate of cataract progression suggesting that at least in some instances an oxidative stress could participate in the formation of cataracts. Oxygen radicals may inflict damage at multifarious biochemical sites. Human lens lipids were also shown to have an absorption maxima at 239 nm indicating their susceptibility to oxidative degradation. In addition the lipid extract has fluorescence similar to that of lipofuscins. The levels of MDA were higher in the brunescent cataracts as compared to that in the nonbrunescent cataracts. The implications of oxidative stress towards the genesis of cataracts in humans is being explored further.

Photoperoxidation in lens and cataract formation: preventive role of superoxide dismutase, catalase and vitamin C.

Exposure of rat lens to fluorescent daylight (150 ft candles) under tissue culture conditions led to a substantial lipid peroxidation as evidenced by the formation of malonaldehyde (MDA). MDA content of lenses incubated overnight in presence of such light was approximately sixfold of that in the control lenses cultured in the dark. These cultures were maintained in physiological medium resembling aqueous humor which does not contain any additional photoactive component. Thus, the lens in its physiological surroundings is susceptible to photoperoxidation by light of wavelengths which freely penetrate the eye. Photoperoxidation could be thwarted by superoxide dismutase, catalase, and ascorbate, suggesting that the observed peroxidative degradation is initiated by photocatalytic generation of superoxide and its subsequent derivation to other potent oxidants. These studies provide for the first time suggestive evidence that senile cataract development may in part be linked to the in vivo photochemical generation of superoxide and other potent oxidants in the aqueous humor and lens derived from the ambient oxygen and light; and ascorbate which is maintained at high levels in this fluid by virtue of its active transport from plasma, is physiologically important in preventing the deleterious action of these potent oxidants. The studies thus indicate for the first time the possibilities of a hitherto unrecognized role of ascorbate against cataracts and other age-, light- and oxygen-dependent ocular abnormalities, In addition, the study re-emphasizes the role of tissue catalase and superoxide dismutase in the prevention of photoperoxidative damages to the tissue.

PREVENTION OF SELENITE CATARACT BY VITAMIN C

Studies have been conducted to determine the efficacy of vitamin C in the prevention of cataracts induced by selenite. Administration of the latter to rat pups results in the development of advanced cataracts within 5 days. Treatment with ascorbate had a significant preventive effect. The observations indicate that selenite cataract is due to an oxidative stress to the lens. In addition, the findings are in conformity with our view that ascorbate functions as an anticataractogenic substance.

Protection against Superoxide Radicals in Rat Lens

Studies have been conducted to determine the mechanisms by which rat ocular tissues may protect themselves against the toxic reactions initiated by superoxide radicals. Such radicals may be formed in the transparent ocular tissues by photochemical as well as metabolic oxidations. A proof of the existence of SOD, the enzyme which dismutates O2 – to relatively less toxic H2O2, has been obtained for the first time in the case of lens and cornea. The activity of the enzyme in lens was lower as compared to that in cornea and retina. The amount of protein giving an activity of one unit varied between 456–600 μg in cornea and 45–92 μg in retina. It is possible that the low activity of SOD in the lens is compensated for by high ascorbate and glutathione. Ascorbate effectively scavenged the superoxide radicals at 10––6 m. The effectiveness of glutathione was also observed to lie in the same range.

PREVENTION OF INTRACELLULAR OXIDATIVE STRESS TO LENS BY PYRUVATE AND ITS ESTER

Pyruvate is a well-known scavenger of hydrogen peroxide (H2O2). In addition, it scavenges superoxide radical (O2.-). However, evidence on its intracellular antioxidant function is meager at present. Hence, we have examined the effectiveness of this metabolite and its ethyl ester against intracellular oxidative damage to the lens under organ culture. Menadione, a redoxcycling quinone, was used to generate the reactive oxygen species (ROS). It was found to inhibit lens metabolism as evidenced by a decrease of ATP. Additionally, tissue oxidation was apparent by loss of glutathione (GSH), and increase in the level of oxidized glutathione (GSSG), coupled with increase of the urea soluble proteins (water insoluble). The overall physiological damage was apparent by the inhibition of the Na+-K+-ATPase dependent cation pump, as evidenced by a decreased rubidium transport. These deleterious effects were attenuated by pyruvate and ethyl-pyruvate. The later was found to be more effective.

Peroxide Damage to the Eye Lens In Vitro Prevention by Pyruvate

The ability of pyruvate to protect the eye lens against physiological damage by hydrogen peroxide has been studied. The physiological damage was estimated in terms of a decrease in the ability of the lens to transport rubidium against an electrochemical gradient under organ culture conditions. Peroxide was either added directly to the culture medium or generated therein by incorporation of xanthine and xanthine oxidase. In both these cases, addition of pyruvate to the medium led to a greater accumulation of rubidium by the lens. The net accumulation of this cation in the presence of 1 to 5 mM pyruvate from the medium containing peroxide (0.2 to 0.45 mM) was very close to that observed in the absence of peroxide. The protective effect was thus substantial. The mechanism of the pyruvate effect has been discussed, and seems to be related to the scavenging of peroxide by pyruvate.

Inhibition of human lens aldose reductase by flavonoids, sulindac and indomethacin

The inhibition of human lens aldose reductase by flavonoids has been studied. Quercetin, the major pentahydroxyflavone, was observed to inhibit human lens aldose reductase by 50% at a concentration of 5 X 10(-6) M. The inhibitory activity of its 3-O-glucoside was similar to that of the parent aglycon. Glycosidation with L-sugar (quercitrin and guaijaverin), however, improved the inhibitory activity (the IC50 values being 1 X 10(-6) M and 2.5 X 10(-6) M respectively). The improvement in inhibitory activity with glycosidation with L-sugar was also apparent from the high inhibitory activity of myricitrin as compared to myricetin, although the improvement in this case of hexahydroxy flavone glycosidation was significantly less than in the case of penthahydroxy flavone glycosidation. The structure-activity relationship observed for human lens enzyme was similar to that reported previously for rat lens enzyme. Inhibitory activity on the whole however, was lower with human lens enzyme. Some known inhibitors of cyclo-oxygenase such as indomethacin, aspirin and sulindac also inhibited human lens aldose reductase. Thus, an inhibitor of one of the enzymes may actually inhibit both and, when administered, may exert mixed physiological effects.

Ascorbic acid and the eye lens.

Exposure of mice to hyperbaric oxygen leads to an inhibition of the mitotic activity in the germinative epithelium of the lens. This is followed by an eventual development of cataracts. Cataracts have also been observed in human beings treated with hyperbaric oxygen for different afflictions. The lens damage and cataract formation appears to be due to in situ generation of active radicals and other active species of oxygen. These oxygen derivatives may also contribute to the multifactorial process of senile cataract formation in human beings. This hypothesis is based on in vitro experiments with rat lenses cultured in medium generating oxygen radicals, the generation of the radicals being accomplished either photochemically or enzymatically. The ability of the lens to transport rubidium and amino acids from such a medium is adversely affected. This is a recognized index of the damage to the tissue physiology. Scavengers of active oxygen species have been found to protect against this damage. Ascorbate, present in concentrations similar to that in the primate aqueous and lens, is also protective. The studies, therefore, point to an antioxidant and perhaps an anti-cataract effect of ascorbate. Pyruvate is another agent useful in this regard.

Radio-isotopic determination of hydrogen peroxide in aqueous humor and urine

The concentrations of hydrogen peroxide in the aqueous humor and urine of several animal species and humans have been determined. The determinations are based on peroxide-dependent decarboxylation of I-[14C]-alpha-ketoglutaric acid and measurement of the resulting 14CO2 by quantitating the radioactive disintegration. The levels of H2O2 in most animals varied between 5.0 and 41 microM for aqueous, and 115 and 187 microM for urine. The levels of peroxide in the urine of steer, cat and baboon were lower and fell out of the above range. In the aqueous of humans with cataracts, the levels ranged from 33 to 324 microM, the overall average being 189 +/- 88 microM. The source of such high levels in the aqueous of cataract patients is currently being studied.

PHOTOPEROXIDATION OF LENS LIPIDS: PREVENTION BY VITAMIN E

Abstract— Light of visible frequency was observed to initiate peroxidative degradation of lipids of rat lenses when the latter were maintained in organ culture. The extent of degradation was monitored by measurement of malanaldehyde. This photodegradative process, which we believe is triggered by light catalyzed generation of superoxide and its subsequent transformation to other potent oxidants. was observed to be thwarted substantially if the medium of organ culture was fortified with 10−3 and 107M vitamin E (α‐tochopherol). These studies suggest that vitamin E may be metabolically beneficial by protecting light exposed tissues, such as those in the eye against photoperoxidativc damage concomitant to light‐catalyzed generation of oxygen‐free radicals. The findings appear relevant to age‐associated pathogenesisof cataracts and their possible attenuation. In addition, they provide a basis of pathogenesis in other ocular tissues such as the macula known to undergo age‐dependent degeneration.