Svitlana Kovtun

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.

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.

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.

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.

Attenuation and delay of diabetic cataracts by antioxidants: effectiveness of pyruvate after onset of cataract.

Cataract is one of the most significant vision-impairing complications of diabetes. The present study examined the feasibility of inhibiting cataract formation by treatment with pyruvate, a metabolite known to effectively scavenge reactive species of oxygen and inhibit protein glycation, both known to be involved in the genesis of diabetic cataracts. In addition, pyruvate stimulates tissue metabolism, which is depressed with the onset of cataract formation. The objective of our experiments was to determine if this compound could be effective in offsetting the progress of cataract, specifically if administered after the diabetes-induced lens changes have begun, as opposed to the previous reports wherein it has been reported to delay cataract formation if administered prophylactically with the immediate onset of diabetes. Diabetes was induced by intraperitoneal administration of streptozotocin to mice. Lens transparency was assessed by slit lamp examination and its photography. ATP was determined enzymatically by reacting it with luciferin-luciferase mixture and measuring the fluorescence intensity. The findings described herein are in accordance with this possibility. The incidence of cataract in the group of diabetic animals, where treatment with pyruvate was initiated after the initial lens changes set in, was significantly lower at all times of observation in comparison to the untreated diabetic group. In addition, the severity of opacities in the pyruvate-treated group, when present, was much minor, the transparency of these cases being close to that in the control animals. The ophthalmic findings are supported biochemically by ATP levels, which were significantly higher in the pyruvate group in comparison to the untreated group. The present findings emphasize the clinical usefulness of initiating treatment with anti-oxidants and metabolic agonists even when the lens changes are detected at the time of the diabetes diagnosis. The latter usually comes much later than the onset of visual aberrations. Prophylaxis is not an absolute requirement.

Sulforaphane-induced transcription of thioredoxin reductase in lens: possible significance against cataract formation

Purpose Sulforaphane is a phytochemically derived organic isothiocyanate 1-isothiocyanato-4-methylsulfinyl-butane present naturally in crucifers, including broccoli and cauliflower. Biochemically, it has been reported to induce the transcription of several antioxidant enzymes. Since such enzymes have been implicated in preventing cataract formation triggered by the intraocular generation of oxy-radical species, the purpose of this investigation was to examine whether it could induce the formation of antioxidant enzymes in the eye lens. Thioredoxin reductase (TrxR) was used as the target of such induction. Methods Mice lenses were cultured for an overnight period of 17 hours in medium 199 fortified with 10% fetal calf serum. Incubation was conducted in the absence and presence of sulforaphane (5 μM). Subsequently, the lenses were homogenized in phosphate-buffered saline (PBS), followed by centrifugation. TrxR activity was determined in the supernatant by measuring the nicotinamide adenine dinucleotide phosphate (reduced) (NADPH)-dependent reduction of 5,5′-dithiobis-2-nitrobenzoic acid (DTNB). Non-specific reduction of DTNB was corrected for by conducting parallel determinations in the presence of aurothiomalate. The reduction of DTNB was followed spectrophotometrically at 410 nm. Results The activity of TrxR in the lenses incubated with sulforaphane was found to be elevated to 18 times of that observed in lenses incubated without sulforaphane. It was also noticeably higher in the lenses incubated without sulforaphane than in the un-incubated fresh lenses. However, this increase was much lower than that observed for lenses incubated with sulforaphane. Conclusion Sulforaphane has been found to enhance TrxR activity in the mouse lens in culture. In view of the protective effect of the antioxidant enzymes and certain nutrients against cataract formation, the findings suggest that it would, by virtue of its ability to enhance the activity of such enzymes, prevent the tissue against oxidative stress that leads to cataract formation. Additional studies with the activities of other antioxidant enzymes such as quinone oxidoreductase and the levels of Nrf2 are in progress.

Prevention of cataract in diabetic mice by topical pyruvate

Background: It has been previously reported that oral administration of sodium pyruvate inhibits oxidative stress and cataract formation in diabetic animals. With a view to exploring the clinical usefulness of these findings, this study examined its preventive effect when administered topically as an eye drop. Methods: Diabetes was induced by intraperitoneal injections of streptozotocin. At the onset of diabetes, an eye drop preparation containing 2.5% sodium pyruvate was administered six times a day at 90-minute intervals. Treatment was continued for 6 weeks. Cataract formation was monitored ophthalmoscopically after mydriasis with 1% tropicamide eye drops. Subsequently, the treated and untreated diabetic animals and the age-matched normal controls were euthanized, their eyes enucleated, and the lenses isolated for biochemical assessment of protein glycation and glutathione levels. Results: Treatment with pyruvate eye drops was found to be significantly effective in inhibiting protein glycation. Glutathione levels were also better maintained. In addition, ophthalmoscopic examination revealed that the incidence of cataract in the pyruvate-treated group was only 12% as compared with the untreated diabetics in whom the incidence was 73%. Cataracts at this stage were largely equatorial. Conclusion: The results demonstrate that topical application of pyruvate can potentially be useful in attenuating or preventing cataract formation induced by diabetes and other conditions of oxidative stress.