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.     

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 antioxi-dant function is meager at present. Hence, we have examined the effectivekiess 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.     

Oxidative stress to rat lens in vitro: Protection by taurine

The concentration of taurine is high in the lens. However, its function therein remains unknown. Studies from other tissues suggest that in addition to several other modes of action, it acts as an antioxidant. We therefore hypothesize that taurine may be a part of the antioxidant defense mechanisms involved in protecting the lens against oxidative stress and consequent cataract formation. In these studies, the protective effect of taurine was examined using lens culture system with menadione as an oxidant. Inclusion of this compound in the incubation medium was found to have several adverse effects on the lens, such as a decrease in its ability to accumulate rubidium against a concentration gradient and fall in the levels of glutathione, ATP and an increase in water insoluble proteins. All these deleterious effects were attenuated significantly by addition of physiological amounts of taurine to the menadione-containing medium.     

Intraocular penetration of pyruvate following its topical administration in mice

Previous studies from our laboratory have demonstrated that pyruvate, an endogenous α-keto acid metabolite, has a protective effect against oxidative stress induced damage to the ocular tissues including the lens, in which in addition to exerting its protective effect against tissue damage caused by oxyradicals generated under organ culture, it is also found effective in preventing actual cataract formation in vivo in animal models undergoing direct oxidative stress as well as in diabetes. In the latter studies, pyruvate was administered mixed with diet and drinking water. However, with the view of the desirability of treating eye diseases by topical administration of the pharmacological agents, the present studies were conducted to determine the penetrability of pyruvate through the cornea to the aqueous humor and the lens following its topical administration as its ester, ethyl pyruvate (EP). These experiments were done in CD-1 mice. After instillation of the drops in the conjunctival cul-de-sac, aqueous humor samples were aspirated at the desired times and analyzed for pyruvate. In a separate group of animals, analyses were done also in the lens. Analyses were done spectrophotometrically by monitoring the decrease in absorption of NADH due to the reduction of pyruvate to lactate by lactate dehydrogenase. The levels of pyruvate were found to be significantly elevated in both the aqueous humor as well as the lens, the peak concentrations being 4.7 and 3.6 mM, respectively. Such levels have been previously shown to be effective in exerting its antioxidant effects. The results are therefore considered pharmacological significant from the point of view of its potential use for topical treatment of cataracts induced by oxidative stress and diabetes.     

Attenuation of galactose-induced cataract by pyruvate

Data in the present paper demonstrate a significant inhibition in the progress of sugar cataract formation by systemic administration of pyruvate. The formation of the cataract was induced by feeding young rats a diet containing 30% galactose. All animals fed this diet developed nuclear lens opacity by the end of 30 days. This was delayed if the diet and water contained, in addition, 2% sodium pyruvate. The incidence of cataract in the latter group was 0% at day 30 and only 25% at day 55. Physiologically, the inhibition was associated with the prevention of lens membrane damage as reflected by its ability to maintain transport of rubidium ions against a concentration gradient; decreased tissue hydration as indexed by the lens wet weight; inhibition of protein glycation, and higher levels of ATP. Since pyruvate, being a normal tissue metabolite, is likely to be non-toxic, the findings are considered useful for further pharmacological studies with this and other similar metabolites, relevant to protection against various secondary complications of diabetes and galactosemia.     

Attenuation of galactose-induced cataract by pyruvate

Data in the present paper demonstrate a significant inhibition in the progress of sugar cataract formation by systemic administration of pyruvate. The formation of the cataract was induced by feeding young rats a diet containing 30% galactose. All animals fed this diet developed nuclear lens opacity by the end of 30 days. This was delayed if the diet and water contained, in addition, 2% sodium pyruvate. The incidence of cataract in the latter group was 0% at day 30 and only 25% at day 55. Physiologically, the inhibition was associated with the prevention of lens membrane damage as reflected by its ability to maintain transport of rubidium ions against a concentration gradient; decreased tissue hydration as indexed by the lens wet weight; inhibition of protein glycation, and higher levels of ATP. Since pyruvate, being a normal tissue metabolite, is likely to be non-toxic, the findings are considered useful for further pharmacological studies with this and other similar metabolites, relevant to protection against various secondary complications of diabetes and galactosemia.     

Photodamage to the lens in vitro: Implications of the Haber-Weiss reaction

Studies have been conducted to examine the implications of photochemical of O₂⁻ and its derivatization to H₂O₂ and OH· in the physiology of the lens in vitro. Physiological status was determined by measuring the uptake of rubidium by the intact tissue when cultured in riboflavin-containing medium, in dark and light, and in the presence and absence of various scanvengers. In the presence of light, the uptake of rubidium in the lens was greatly diminished; this suggests photodamage to the tissue. MnSOD and ferricyanide protected against this photochemical damage. The damaging process was thus initiated by the generation of O₂⁻. The tissue damage was also attenuated by catalase, ferrocyanide, and mannitol. These results, therefore, suggest the participation of hydrogen peroxide and the subsequent Haber-Weiss reaction in the photodamaging process.     

Quercetin inhibits hydrogen peroxide-induced oxidation of the rat lens

Cataract results from oxidative damage to the lens. The mechanism involves disruption of the redox system, membrane damage, proteolysis, protein aggregation and a loss of lens transparency. Diet has a significant impact on cataract development, but the individual dietary components responsible for this effect are not known. We show that low micromolar concentrations of the naturally-occurring flavonoid, quercetin, inhibit cataractogenesis in a rat lens organ cultured model exposed to the endogenous oxidant hydrogen peroxide. Other phenolic antioxidants, (+)epicatechin and chlorogenic acid, are much less effective. Quercetin was active both when incubated in the culture medium together with hydrogen peroxide, and was also active when the lenses were pre-treated with quercetin prior to oxidative insult. Quercetin protected the lens from calcium and sodium influx, which are early events leading to lens opacity, and this implies that the non-selective cation channel is protected by this phenolic. It did not, however, protect against formation of oxidized glutathione resulting from H2O2 treatment. The results demonstrate that quercetin helps to maintain lens transparency after an oxidative insult. The lens organ culture/hydrogen peroxide (LOCH) model is also suitable for examining the effect of other dietary antioxidants.     

Pyruvate secreted by human lymphoid cell lines protects cells from hydrogen peroxide mediated cell death

Reactive oxygen species (ROS) released from polymorphonuclear leukocytes and macrophages could cause DNA damage, but also induce cell death. Therefore inhibition of cell death must be an important issue for accumulation of genetic changes in lymphoid cells in inflammatory foci. Scavengers in the post culture medium of four lymphoid cell lines, lymphoblastoid cell lines (LCL), Raji, BJAB and Jurkat cells, were examined. Over 80% of cultured cells showed cell death 24 h after xanthine (X)/xanthine oxidase (XOD) treatment, which was suppressed by addition of post culture medium from four cell lines in a dose-dependent manner. H2O2 but not O2*- produced by the X/XOD reaction was responsible for the cytotoxity, thus we used H2O2 as ROS stress thereafter. The H2O2-scavenging activity of post culture media from four cell lines increased rapidly at the first day and continued to increase in the following 2-3 days for LCL, Raji and BJAB cells. The scavenging substance was shown to be pyruvate, with various concentrations in the cultured medium among cell lines. Over 99% of total pyruvate was present in the extracellular media and less than 1% in cells. alpha-Cyano-4-hydroxycinnamate, a specific inhibitor of the H+-monocarbohydrate transporter, increased the H2O2-scavenging activity in the media from all four cell lines via inhibition of pyruvate re-uptake by cultured cells from the media. These findings suggest that lymphoid cells in inflammatory foci could survive even under ROS by producing pyruvate, so that accumulation of lymphoid cells with DNA damage is possible.     

In vitro Damage to Rat Lens by Lumazine and Xanthine Oxidase: Prevention by Superoxide Dismutase

Intact rat lenses incubated with lumazine and xanthine oxidase are physiologically damaged as evidenced by a decrease in the net accumulation of rubidium ions against a concentration gradient. Superoxide dismutase protected the tissue against this damage. These experiments, therefore, demonstrate the susceptibility of the lens tissue to O₂^?_? injury under ambient and nonphotochemical conditions, suggesting a possible implication of this radical in the tissue in vivo and eventual cataract formation. The lumazine/xanthine oxidase system which is known to cause oxygen reduction predominantly by the monovalent route, producing superoxide, appears quite suitable to evaluate the toxicity of O₂^?_? to the tissues in vitro.     

Liposome-mediated augmentation of catalase in alveolar type II cells protects against H2O2 injury

Oxidant injury to the alveolar epithelium can be mediated by exposure to oxidant gases such as O2 at high concentrations and O3, inflammatory cell-derived reactive O2 species, and the intracellular metabolism of xenobiotics such as paraquat. An in vitro model of alveolar epithelial oxidant injury was developed based on exposure of cultured rat type II pneumocytes to superoxide and hydrogen peroxide (H2O2) enzymatically generated in the culture medium. Cytotoxicity was assessed by the release of lactate dehydrogenase (LDH) into the culture medium, which was a more reliable indicator of damage than release of 51Cr by prelabeled cells. Incubation of cells for 6-8 h with xanthine plus xanthine oxidase and glucose plus glucose oxidase induced the release of greater than 50% of total intracellular LDH. Oxidant exposure also resulted in significant detachment of cells from culture dishes. Modulation of oxidant damage was accomplished using liposomes as vectors for the delivery of catalase. Treatment of cells with catalase liposomes for 2 h resulted in augmentation of cellular catalase specific activities up to 631% of controls. Catalase was partitioned into intracellular and surface-associated compartments in catalase liposome-treated cells. Partial and complete protection against oxidant injury, induced by xanthine plus xanthine oxidase and glucose plus glucose oxidase, respectively, was achieved by pretreatment of cells with catalase liposomes. LDH release during oxidant exposure was inversely related to augmentation of cellular catalase activities. Catalase liposome-treated cells also exhibited an enhanced ability to scavenge enzymatically generated H2O2 from the culture medium. These observations suggest a useful approach to modulation of alveolar injury induced by reactive O2 species.     

Energy substrates and the completion of spontaneous meiotic maturation

This study was carried out to examine how different combinations of pyruvate and glucose affect spontaneous meiotic maturation of cumulus-cell-enclosed mouse oocytes (CEO) to metaphase II (MII). Most experiments used an open system in which oocytes were cultured in 1 ml medium in plastic tubes. Initial experiments examined the dose response effects of pyruvate or glucose alone in the presence or absence of 2 mM glutamine. When medium lacked both pyruvate and glucose, more than 91% of the oocytes died in glutamine-free medium during 15 h of culture; viability was restored with the addition of glutamine, but only 11% of the CEO reached MII. In the absence of glutamine, 62-68% of oocytes completed maturation in 0.23-2.3 mM pyruvate, while 44-60% MII was observed in 0.55-27.8 mM glucose. The addition of glutamine to these cultures had a general suppressive effect on the completion of maturation. When glucose was added to pyruvate-containing cultures, the combination of 1 mM pyruvate/5.5 mM glucose was most effective in supporting maturation (about 90% MII), with little effect of glutamine. No further increase in maturation was observed when glucose was increased five-fold (to 27.8 mM). The positive effect of glucose was in part attributed to stimulation of glycolysis and increased production of pyruvate, since a reduced culture volume (8 microl), which allows the accumulation of secreted pyruvate, improved maturation in glucose-containing, but not pyruvate-containing, medium, and FSH, which stimulates glycolysis, increased progression to MII in glucose-containing, but not pyruvate-containing, medium. Yet these results also suggest that glucose has a beneficial effect on maturation apart from simple provision of pyruvate. The pyruvate effect was directly on the oocyte, because denuded oocytes responded more effectively than CEO to this energy substrate. The highest percentage of MII oocytes (96-97%) occurred in microdrop cultures containing glucose but lacking glutamine. These results indicate that glutamine supports oocyte viability but is not an adequate energy source for the completion of spontaneous meiotic maturation and may be detrimental. In addition, while pyruvate and glucose alone can each support meiotic progression of CEO to MII, optimal maturation requires the provision of both substrates to the culture medium when a large volume (1 ml) is used. It is concluded that careful attention to specific energy substrate supplementation and culture volume is important to optimise spontaneous meiotic maturation in vitro.     

The effects of "oxygen radicals" generated in the medium on lenses in organ culture: inhibition of damage by chelated iron

Rat lenses in organ culture were exposed to activated species of oxygen generated in the culture medium either by xanthine oxidase and hypoxanthine or by riboflavin and visible light, two systems which have been shown to produce superoxide and H2O2. In each case there was marked damage to carrier-mediated transport systems of the lens. Under standard culture conditions this damage was strongly inhibited by catalase, but not by superoxide dismutase (SOD). By the addition to the medium of chelated iron, hydroxyl radicals were produced in a Fenton reaction with a concomitant decrease in H2O2 levels. With both oxygen radical-generating systems, the addition of chelated iron strongly inhibited lens damage. This inhibitory effect could be reversed by the addition of SOD with the chelated iron. Under such conditions SOD converts superoxide anion to H2O2, thereby preventing reduction of the chelated iron and thus stopping the generation of hydroxyl radicals. Increased lens damage following addition of SOD to the iron-containing systems correlated with higher H2O2 concentrations, and was inhibited by catalase. These findings suggest that, when generated in the fluids surrounding the lens, H2O2 poses a much greater oxidative stress for the lens than do the superoxide or hydroxyl free radicals.     

Prooxidant and cytotoxic action of N-acetylcysteine and glutathione combined with vitamin Bl2b

We studied the prooxidant and cytotoxic action of thiols N-acetylcystein (NAC) and glutathione (GSH) combined with vitamin Bl2b. The synergism of action of the thiols and Bl2b resulted in human carcinoma cell damage was found. It was shown that GSH and NAC in physiological doses combined with Bl2b caused the initiation of apoptosis. It was established that prooxidant action of the thiols combined with vitamin Bl2b, i. e. generation and accumulation of hydrogen peroxide in culture medium, led to intracellular oxidative stress and injury of cell redox system. These effects were completely abolished by nonthiol antioxidants catalase and pyruvate. The chelators of iron phenanthroline and deferoxamine did not suppress the H2O2 accumulation in culture medium but significantly inhibited the cell death induced by the thiols combined with Bl2b. Therefore, the thiols GSH and NAC widely used as antioxidants, in combination with vitamin Bl2b show prooxidant characteristics and induce, with the participation of intracellular iron, apoptotic HEp-2 cell death.     

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 Dulbecco's Modified Eagle's 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.     

Pyruvate secreted by human lymphoid cell lines protects cells from hydrogen peroxide mediated cell death

Reactive oxygen species (ROS) released from polymorphonuclear leukocytes and macrophages could cause DNA damage, but also induce cell death. Therefore inhibition of cell death must be an important issue for accumulation of genetic changes in lymphoid cells in inflammatory foci. Scavengers in the post culture medium of four lymphoid cell lines, lymphoblastoid cell lines (LCL), Raji, BJAB and Jurkat cells, were examined. Over 80% of cultured cells showed cell death 24 h after xanthine (X)/xanthine oxidase (XOD) treatment, which was suppressed by addition of post culture medium from four cell lines in a dose-dependent manner. H₂O₂ but not O^·-₂ produced by the X/XOD reaction was responsible for the cytotoxity, thus we used H₂O₂ as ROS stress thereafter. The H₂O₂-scavenging activity of post culture media from four cell lines increased rapidly at the first day and continued to increase in the following 2–3 days for LCL, Raji and BJAB cells. The scavenging substance was shown to be pyruvate, with various concentrations in the cultured medium among cell lines. Over 99% of total pyruvate was present in the extracellular media and less than 1% in cells. α-Cyano-4-hydroxycinnamate, a specific inhibitor of the H⁺-monocarbohydrate transporter, increased the H₂O₂-scavenging activity in the media from all four cell lines via inhibition of pyruvate re-uptake by cultured cells from the media. These findings suggest that lymphoid cells in inflammatory foci could survive even under ROS by producing pyruvate, so that accumulation of lymphoid cells with DNA damage is possible.     

Substitution of glutamine by pyruvate to reduce ammonia formation and growth inhibition of mammalian cells

In mammalian cell culture technology glutamine is required for biomass synthesis and as a major energy source together with glucose. Different pathways for glutamine metabolism are possible, resulting in different energy output and ammonia release. The accumulation of ammonia in the medium can limit cell growth and product formation. Therefore, numerous ideas to reduce ammonia concentration in cultivation broths have been developed. Here we present new aspects on the energy metabolism of mammalian cells. The replacement of glutamine (2 mM) by pyruvate (10 mM) supported cell growth without adaptation for at least 19 passages without reduction in growth rate of different adherent commercial cell lines (MDCK, BHK21, CHO-K1) in serum-containing and serum-free media. The changes in metabolism of MDCK cells due to pyruvate uptake instead of glutamine were investigated in detail (on the amino acid level) for an influenza vaccine production process in large-scale microcarrier culture. In addition, metabolite profiles from variations of this new medium formulation (1-10 mM pyruvate) were compared for MDCK cell growth in roller bottles. Even at very low levels of pyruvate (1 mM) MDCK cells grew to confluency without glutamine and accumulation of ammonia. Also glucose uptake was reduced, which resulted in lower lactate production. However, pyruvate and glutamine were both metabolized when present together. Amino acid profiles from the cell growth phase for pyruvate medium showed a reduced uptake of serine, cysteine, and methionine, an increased uptake of leucine and isoleucine and a higher release of glycine compared to glutamine medium. After virus infection completely different profiles were found for essential and nonessential amino acids.     

Protection against reactive oxygen species during mouse preimplantation embryo development: role of EDTA, oxygen tension, catalase, superoxide dismutase and pyruvate

Oxidative damage due to the production of reactive oxygen species (ROS) is one of a number of culture-induced stresses which may compromise preimplantation embryo development in vitro. Ethylenediaminetetraacetic acid (EDTA), reduced oxygen tension, superoxide dismutase (SOD) and catalase (CAT) offer protection against oxidative stress, but few attempts have been made to determine which of these agents, or which combination, is the most effective. In particular, no systematic investigation of their actions and interactions has been made using a multifactorial experimental design. Murine zygotes were cultured in the presence or absence of 10 miccroM EDTA, SOD (100-7,000 U/ml) and CAT (50-100 U/ml) at atmospheric (20%) and reduced (5%) oxygen tensions. Blastocyst formation and hatching rates (at various time points), and cell numbers were recorded, whilst parallel groups of embryos had their consumption of pyruvate, a hydrogen peroxide scavenger, measured. All parameters interacted significantly and affected blastocyst formation, hatching rate and cell numbers but the effect of EDTA was the most pronounced. There were beneficial effects of 5% O2, CAT and SOD, while 20% O2 had a deleterious effect on development. EDTA improved blastocyst formation and hatching rates but paradoxically led to a reduction in cell number. 5% O2 was the next most significant parameter to enhance embryo development and also increased cell numbers. No differences in pyruvate uptake were apparent between the various treatment groups. The results suggest that embryo culture in EDTA-free medium under 5% O2 provides the most practical and physiological conditions for in vitro murine embryo culture.     

Differential effect of l-histidine in human lymphocytes damaged by different oxygen radical producing systems

The effect of histidine on damage induced by oxygen radicals was studied in peripheral blood lymphocytes treated with free oxygen radical-inducing agents: hydrogen peroxide, xanthine oxidase plus hypoxanthine, bleumycin and γ-rays. l-Histidine, at a concentration of 1 mM, was found to potentiate both cell killing and inhibition of PHA-stimulated cell division brought about by hydrogen peroxide or xanthine oxidase plus hypoxanthine. In contrast, l-histidine did not affect γ-ray- or bleomycin-induced cell killing and inhibition of PHA-stimulated cell division. We suggest that l-histidine potentiation of cell damage is mainly mediated by interaction of the amino acid with hydrogen peroxide and/or iron rather than with other reactive oxygen species. In addition, these results also indicate that hydrogen peroxide produced by γ-radiation- or bleomycin-treated cells plays no role in the toxic effects elicited by these agents.     

Vitamin B12b enhances the cytotoxicity of dithiothreitol

It has been found previously that vitamin B12b amplifies significantly the cytotoxic effects of ascorbic acid by catalyzing the formation of reactive oxygen species, and the antioxidant dithiothreitol (DTT), in contrast to catalase, does not prevent the cytotoxicity. Therefore, in this study we examined whether B12b is able to enhance the cytotoxicity of DTT. It was revealed that B12b strongly increases the cytotoxic effect of DTT. Vitamin B12b added to DTT catalyzed the generation and drastic accumulation of hydrogen peroxide in culture medium to a concentration of 260 microM within 7 min. The extracellular oxidative burst induced by the combination of B12b and DTT (DTT + B12b) was accompanied by intracellular oxidative stress, the destabilization of lysosomes, and damage to DNA. The accumulation of DNA lesions led to the initiation of apoptotic cell death, including the activation of caspase-3 and the release of cytochrome c. The antioxidants pyruvate and catalase completely prevented the DTT + B12b-induced oxidative stress and cell death. The iron chelators desferrioxamine and phenanthroline prevented the geno- and cytotoxic action of the combination although they did not reduce the exogenous oxidative burst, indicating a key role for intracellular iron in the cytotoxicity of the combination. Thus, vitamin B12b dramatically enhances the cytotoxicity of DTT, catalyzing the generation of hydrogen peroxide and inducing extra- and intracellular oxidative stress, early destabilization of lysosomes, and iron-dependent DNA damage.