The ageing lens and cataract: a model of normal and pathological ageing

Cataract is a visible opacity in the lens substance, which, when located on the visual axis, leads to visual loss. Age-related cataract is a cause of blindness on a global scale involving genetic and environmental influences. With ageing, lens proteins undergo non-enzymatic, post-translational modification and the accumulation of fluorescent chromophores, increasing susceptibility to oxidation and cross-linking and increased light-scatter. Because the human lens grows throughout life, the lens core is exposed for a longer period to such influences and the risk of oxidative damage increases in the fourth decade when a barrier to the transport of glutathione forms around the lens nucleus. Consequently, as the lens ages, its transparency falls and the nucleus becomes more rigid, resisting the change in shape necessary for accommodation. This is the basis of presbyopia. In some individuals, the steady accumulation of chromophores and complex, insoluble crystallin aggregates in the lens nucleus leads to the formation of a brown nuclear cataract. The process is homogeneous and the affected lens fibres retain their gross morphology. Cortical opacities are due to changes in membrane permeability and enzyme function and shear-stress damage to lens fibres with continued accommodative effort. Unlike nuclear cataract, progression is intermittent, stepwise and non-uniform.     

Redox status of the eye lens

The aim of this work was to study the regional variation of some antioxidant systems in calf lens. Specific lens regions of nearly same age were obtained by a microsectioning technique, and the concentration of reduced and oxidized glutathione, protein sulfhydryl groups and iron were measured in each lens region. The concentration of reduced glutathione, the major redox buffer in lens, exponentially decreased from the cortical regions to the nucleus. In contrast, the concentration of protein sulfhydryl groups gradually increased from the cortex toward the nucleus. The protein-bound disulfides remained constant throughout the lens. Iron was concentrated in the outer cortical region. The results show that the most dynamic redox-active zone in the lens is the subcapsular cortical region where the oxidant flux meets a highly reducing environment containing a potent redox catalyst.     

Inhibition of ascorbic acid-induced modifications in lens proteins by peptides.

The effects of three dipeptides L-phenylalanyl-glybine, glycyl-L-phenylalanine,and aspartame (L-aspartyl-L-phenylalanine, methyl ester) as inhibitors of the ascorbic acid-induced modifications in lens proteins were studied. Their efficiency was compared to that of two known inhibitors--aminoguanidine and carnosine. The tested dipeptides diminished protein carbonyl content by 32-58% and most moderated the formation of chromophores, as measured by the absorbency at 325 nm of the glycated proteins. The appearance of non-tryptophan fluorescence (excitation 340 nm/emission 410 nm) was observed for proteins glycated with ascorbic acid. All of the dipeptides examined, as well as aminoguanidine, decreased this glycation-related fluorescence. The potential inhibitors prevented the intensive formation of very high molecular weight aggregates. A competitive mechanism of their inhibitory effect was proposed, based on the reactivity of individual substances toward ascorbic acid. These findings indicate that they have a potential for use as alternatives for aminoguanidine as an anti-glycation agent.     

Relative suppression of the sodium-dependent Vitamin C transport in mouse versus human lens epithelial cells

Vitamin C is a major antioxidant and UV absorbent in the human lens. In the rodent lens, the levels are very low for unknown reasons. Searching for clues to explain this suppression, we investigated the comparative uptake of Vitamin C in cultured human and mouse lens epithelial cells. When compared to human HLE-B3 lens epithelial cells, ¹⁴C-ASA uptake was 4- to 10-fold impaired in confluent mouse lens 17EM15 (p < 0.0001) and 21EM15 (p < 0.001) cells, respectively. High glucose concentrations reduced the uptake by 30–50% in all cells (p < 0.005). Incubation of cells with 6-deoxy-6-fluoro-ascorbic (F-ASA), i.e. a probe specific for the sodium-dependent Vitamin C uptake (SVCT2), revealed a 10-fold uptake suppression into mouse 17EM15 relative to human HLE-B3 and JAR choriocarcinoma cells (a control), that could be overcome by overexpressing hSVCT2 using two different promoter constructs. The relative Vitamin C uptake differences suggest either low expression of SVCT2, molecular differences between the transporters themselves or their biological regulation, since a recent study has shown that exogenous feeding of ascorbic acid to rats increased only modestly lenticular uptake (Mody et al., Acta Ophthalmol Scand 83: 228–223, 2005). Elucidation of the mechanism by which SCVT2 activity is suppressed in mouse lens may help unravel a major question of evolutionary significance for night vision in nocturnal animals.     

Activities of Antioxidant Systems During Germination of Chenopodium rubrum Seeds

The activities of superoxide-dismutase (SOD), catalase (CAT) and peroxidase (POD), and concentrations of glutathione and ascorbate have been studied during the first stages of germination in Chenopodium rubrum L. seeds. The highest CAT and SOD activity was found prior to radicle protrusion, while POD activity was maximal at the time of radicle protrusion and seedling development, new POD isozymes simultaneously appearing. The concentrations of total, reduced and oxidized glutathione showed similar changes during germination, the highest values being detected at the time of radicle protrusion. Ascorbic acid was present in the seeds in a detectable concentration only at the time preceding radicle protrusion, while its oxidized form dehydroascorbic acid was detected during the whole germination period studied. Gibberellic acid (GA₃, 160 M) had no effect on germination percentage, but in presence of GA₃, SOD and CAT activity notably increased prior to radicle protrusion, and oxidized glutathione concentration decreased in further germination.     

Glycation of human lens proteins from diabetic and (nondiabetic) senile cataract patients

Glycation (nonenzymatic glycosylation) in the human lens (cortex and nucleus) in senile (nondiabetic) and diabetic cataracts was studied by measuring the extent of early and late glycation products, the content of free -amino groups and the formation of disulfide bonds in the soluble lens proteins. There was a significant (p<0.001) increase in early and late glycation in the lens nucleus compared to the cortex in both the senile and diabetic groups. Overall these changes were much larger in the diabetic group. The concentration of free -amino groups was decreased in the senile nucleus as well as in the diabetic nucleus when compared with the senile and diabetic cortex (p<0.001). Disulfide bond content was in the order of diabetic nucleus > diabetic cortex > senile nucleus > senile cortex. Glycation of the lens proteins is a generalized feature which is enhanced in the diabetic lens compared to senile lens proteins and is associated with a decrease in free -amino groups and an increase in disulfide bonds formation in the lens proteins.     

牛角花齿蓟马为害对紫花苜蓿AsA、GSH含量及相关代谢酶活性的影响

为了明确非酶抗氧化物质抗坏血酸(AsA)、还原型谷胱甘肽(GSH)及相关代谢酶抗坏血酸过氧化物酶(APX)、谷胱甘肽还原酶(GR)在紫花苜蓿(Medicago sativa L.)对牛角花齿蓟马Odontothrips loti Haliday为害的抗性中的作用,测定了不同牛角花齿蓟马虫口密度下抗、感蓟马苜蓿无性系R-1、I-1的AsA、GSH含量及APX、GR活性的变化。结果表明:受牛角花齿蓟马为害后,R-1无性系在低虫口密度(1、3头/枝条)下,AsA、GSH含量和GR活性均上升,在高虫口密度(5、7头/枝条)下,AsA含量和GR活性先升高后下降,GSH含量上升后保持稳定;I-1无性系的AsA、GSH含量先升高后下降,GR活性在为害后期呈上升趋势;R-1、I-1无性系的APX活性均先上升后下降,但R-1无性系APX活性的上升速率及下降速率小于I-1无性系。说明AsA、GSH含量及APX、GR活性的升高可能是紫花苜蓿对牛角花齿蓟马诱导抗性的一种表现,但I-1无性系对蓟马为害的应激反应滞后于R-1无性系。在牛角花齿蓟马为害后期,R-1无性系体内的AsA、GSH含量及APX、GR活性仍处于较高水平,也说明了R-1无性系对牛角花齿蓟马为害的抗性较I-1无性系强。     

Effect of in ovo feeding of vitamin C on antioxidation and immune function of broiler chickens

Hypoimmunity and numerous stresses are two major challenges in broiler industry. Nutrient intervention at the specific time of embryonic stage is a feasible way to improve animal performance. This study was conducted to investigate the possible effects of in ovo feeding (IOF) of vitamin C at embryonic age 15^th day (E15) on growth performance, antioxidation and immune function of broilers. A total of 240 broiler fertile eggs were randomly divided into two groups (0 and 3 mg injected dose of vitamin C at E15), and new-hatched chicks from each treatment were randomly allocated into six replicates with 10 chicks per replicate after incubation. The results indicated that in ovo vitamin C injection improved the hatchability (P < 0.05) and increased immunoglobulin M (IgM) (at the broiler’s age 1^st day, D1), IgG and IgM concentrations (D21), as well as lysozyme activity (D21, P < 0.05) and total antioxidant capacity (D42, P < 0.01) in plasma of broilers. On D21, the splenic expression level of DNA methyltransferase 1 (DNMT1) was up-regulated in vitamin C (VC) group, whereas interleukin (IL)-6, interferon-γ, ten-eleven translocation protein 1 and thymine-DNA glycosylase were down-regulated (P < 0.05). On D42, in ovo vitamin C injection up-regulated splenic expression levels of DNMT1, DNA methyltransferase 3B (DNMT3B) and growth arrest and DNA-damage-inducible protein beta (P < 0.05), whereas down-regulated splenic expression levels of IL-6, tumour necrosis factor-α and methyl-CpG-binding domain protein 4 (P < 0.05). Our findings suggested that IOF of 3 mg vitamin C at E15 could improve, to some extent, the antioxidant activity and immune function in plasma, corresponding with the lower expression of pro-inflammatory cytokines in spleen. However, IOF of vitamin C leading to the changes in the expression of DNA methyltransferases and demethylases may suggest an increased trend of DNA methylation level in spleen and whether DNA methylation variation is associated with the lower expression of pro-inflammatory cytokines in spleen warrants future study.     

Iron (II) Ions Induced Oxidation of Ascorbic Acid and Glucose

Lipid peroxidation (LPO) of polyunsaturated fatty acids (PUFAs) is suspected to be involved in the generation of chronic diseases. A model reaction for LPO is the air oxidation of PUFAs initiated by Fe²⁺ and ascorbic acid. In the course of such model reactions glycolaldehyde (GLA) was detected as main aldehydic product. Since it is difficult to explain the generation of GLA by oxidation of PUFAs, it was suspected that GLA might be derived by oxidation of ascorbic acid. This assumption was verified by treatment of ascorbic acid with Fe²⁺.

Produced aldehydic compounds were trapped by addition of pentafluorobenzylhydroxylamine hydrochloride (PFBHA-HCl), trimethylsilylated and finally identified by gas chromatography/mass spectrometry (GC/MS). Oxidation of ascorbic acid with O₂ in presence of iron ions produced not only glycolaldehyde (GLA), but also glyceraldehyde (GA), dihydroxyacetone (DA) and formaldehyde. Glyoxal (GO) and malondialdehyde (MDA) were detected as trace compounds.

The yield of the aldehydic compounds was increased by addition of lipid hydroperoxides (LOOH) or H₂O₂. The buffer influenced the reaction considerably: Iron ions react with Tris buffer by producing dihydroxyace-tone (DA). Since ascorbic acid is present in biological systems and Fe²⁺ ions are obviously generated by cell damaging processes, the production of GLA and other aldehydic components might add to the damaging effects of LPO.

Glucose suffers also oxidation to short-chain aldehydic compounds in aqueous solution, but this reaction requires addition of equimolar amounts of Fe²⁺ together with equimolar amounts of H₂O₂ or 13-hydroperoxy-9-cis-11-trans-octadecadienoic acid (13-HPODE). Therefore this reaction, also influenced by the buffer system, seems to be not of biological relevance.     

Vitamin C recycling and function in human monocytic U-937 cells

The uptake, recycling, and function of ascorbic acid was evaluated in cultured U-937 monocytic cells. Dehydroascorbic acid, the two-electron oxidized form of the vitamin, was taken up on the glucose transporter and reduced to ascorbate to a much greater extent than ascorbate itself was accumulated by the cells. In contrast to dehydroascorbic acid, ascorbate entered the cells on a sodium- and energy-dependent transporter. Intracellular ascorbate enhanced the transfer of electrons across the cell membrane to extracellular ferricyanide. Rates of ascorbate-dependent ferricyanide reduction were saturable, fivefold greater than basal rates, and facilitated by intracellular recycling of ascorbate. Whereas reduction of dehydroascorbic acid concentrations above 400 microM consumed reduced glutathione (GSH), even severe GSH depletion by 1-chloro-2,4-dinitrobenzene was without effect on the ability of the cells to reduce concentrations of dehydroascorbic acid likely to be in the physiologic range (< 200 microM). Dialyzed cytosolic fractions from U-937 cells reduced dehydroascorbic acid to ascorbate in an NADPH-dependent manner that appeared due to thioredoxin reductase. However, thioredoxin reductase did not account for the bulk of dehydroascorbic acid reduction, since its activity was also decreased by treatment of intact cells with 1-chloro-2,4-dinitrobenzene. Thus, U-937 cells loaded with dehydroascorbic acid accumulate ascorbate against a concentration gradient via a mechanism that is not dependent on GSH or NADPH, and this ascorbate can serve as the major source of electrons for transfer across the plasma membrane to extracellular ferricyanide.     

Hemin-mediated Hemolysis in Erythrocytes： Effects of Ascorbic Acid and Glutathione

In the present work,we investigated the effect of ascorbic acid and glutathione on hemolysisinduced by hemin in erythrocytes.Ascorbic acid not only enhanced hemolysis,but also induced formationof thiobarbituric acid-reactive substances in the presence of hemin.It has been shown that glutathioneinhibits hemin-induced hemolysis by mediating hemin degradation.Erythrocytes depleted of glutathionebecame very sensitive to oxidative stress induced by hemin and ascorbic acid.H_2O_2 was involved in hemin-mediated hemolysis in the presence of ascorbic acid.However,a combination of glutathione and ascorbicacid was more effective in inhibiting hemolysis induced by hemin than glutathione alone.Extracellular andintracellular ascorbic acid exhibited a similar effect on hemin-induced hemolysis or inhibition of hemin-induced hemolysis by glutathione.The current study indicates that ascorbic acid might function as anantioxidant or prooxidant in hemin-mediated hemolysis,depending on whether glutathione is available.     

Combination of glycemic and oxidative stress in lens: implications in augmentation of cataract formation in diabetes

It is well known that the incidence of cataract is higher in diabetics as compared to non-diabetics. Its rate of maturation is also faster in the diabetics. The precise mechanism of this acceleration is not clearly understood. It is hypothesized that this could be a result of the combination of the metabolic and oxidative stress induced by glycemia itself with the age-associated increase in ambient generation of oxyradical species. In the current studies, we have investigated this possibility using the galactose cataract model. Galactosemia was induced by feeding rats a 50% galactose diet. The increased susceptibility of the glycemic lenses to physiological damage by reactive oxygen species (ROS) was studied by incubating them in Tyrode in the absence and presence of menadione. The resulting physiological damage to the lens was assessed initially in terms of its ability to maintain Na⁺-K⁺ ATPase dependent active transport of potassium ions, as represented by the uptake of rubidium ions. Subsequently, the level of ATP, indexing the general metabolic status, and the level of glutathione (GSH), indexing the status of antioxidant reserve, were also determined. The uptake of rubidium in the normal lenses incubated in the presence of the quinone was depressed to more than 50% of the controls run in the basal medium. A similar depression existed in the galactosemic lenses in comparison to the normal lenses. However, in the presence of menadione, the inhibition of the uptake was accentuated further in the case of galactosemic lenses, the uptake here being only 20% of the normal controls. Similarly, the galactosemic lenses were also more susceptible to menadione dependent decrease in ATP and GSH.     

Vitamin C content in plants is modified by insects and influences susceptibility to herbivory

Analysis of a diverse cross‐sample of plant‐insect interactions suggests that the abundance of vitamin C (L ‐ascorbic acid, ascorbate or AsA) in plants influences their susceptibility to insect feeding. These effects may be mediated by AsAs roles as an essential dietary nutrient, as an antioxidant in the insect midgut, or as a substrate for plant‐derived ascorbate oxidase, which can lead to generation of toxic reactive oxygen species. Ascorbate can also influence the efficacy of plant defenses such as myrosinases and tannins, and alter insects' susceptibility to natural enemies. Conversely, herbivores appear to influence both de novo synthesis and redox cycling of AsA in their host plants, thereby potentially altering the nutritional value of crops and their susceptibility to pests. The recent development of genetically modified crops with enhanced AsA content provides both an impetus and a tool set for further studies on the role of AsA in plant‐insect interactions.     

Alteration in the ubiquitin structure and function in the human lens: a possible mechanism of senile cataractogenesis

High-performance liquid chromatography purification followed by mass spectrometry analyses highlighted that human senile cataractous lens includes a 8182 Da species which is absent in the normal lens, whereas a 8566/8583 Da species is present in both lenses. Western blot analysis identified both species as ubiquitin. The species at lower molecular weight is a shorter form due to the cleavage of the C-terminal residues 73-76. As it is the last amino acid of ubiquitin which is involved in the protein degradation mechanism, we suggest that this structure modification compromises the function of ubiquitin and consequently the physiologically occurring degradation of the lens proteins.     

Fluorescence resonance energy transfer study of subunit exchange in human lens crystallins and congenital cataract crystallin mutants

Lens α-crystallin is an oligomeric protein with a molecular mass of 500–1000 kDa and a polydispersed assembly. It consists of two types of subunits, αA and αB, each with a molecular mass of 20 kDa. The subunits also form homo-oligomers in some other tissues and in vitro. Their quaternary structures, which are dynamic and characterized by subunit exchange, have been studied by many techniques, including fluorescence resonance energy transfer (FRET) and mass spectrometry analysis. The proposed mechanism of subunit exchange has been either by dissociation/association of monomeric subunits or by rapid equilibrium between oligomers and suboligomers. To explore the nature of subunit exchange further, we performed additional FRET measurements and analyses using a fluorescent dye-labeled W9F αA-crystallin as the acceptor probe and Trp in other crystallins (wild-type and R116C αA, wild-type and R120G αB, wild-type and Q155* βB2) as the donor probe and calculated the transfer efficiency, Förster distance, and average distance between two probes. The results indicate only slight decreased efficiency and increased distance between two probes for the R116C αA and R120G αB mutations despite conformational changes.     

The antioxidant system in diapausing and active red mason bee Osmia bicornis

The red mason bee Osmia bicornis L. (Hymenoptera: Megachilidae) belongs to a group of insects that undergo an obligatory diapause in the imago stage. Red mason bees (O. bicornis) consume oxygen during overwintering diapause, and reactive oxygen species can be produced despite substantial inhibition of metabolism. These are first studies to investigate the antioxidant system in diapausing red mason bees and to compare the antioxidant systems of overwintering and active imagines. The present study analyzes total antioxidant status, glutathione and ascorbic acid levels, and the activity of superoxide dismutase, catalase and peroxidase in overwintering (October to March) and active (April) female and male O. bicornis. Diapause phases (prediapause, diapause and post‐diapause) cannot be distinguished based on the parameters of the antioxidant system of the bees. During overwintering, a significant decrease is noted only in ascorbic acid content. The remaining antioxidants remain fairly stable, which indicates the absence of oxidative stress in diapausing specimens. The analyzed parameters distinguish diapausing bees from active insects. Excluding total antioxidant status, the evaluated parameters are significantly higher in active individuals than in overwintering specimens. Sex‐related differences are found only for catalase, the level of which is consistently higher in males than in females.     

Free Radical Scavengers and Photosynthetic Pigments in Pinus Cembra L. Needles as Affected by Ozone Exposure

The goal of this study was the characterization of the antioxidative protection system of current and 1-year-old needles of a cembran pine (Pinus cembra L.) and its possible responses to elevated concentrations of atmospheric O₃. Twigs of a mature cembran pine at the alpine timberline (1950 m a.s.l.) were exposed in climate-controlled twig chambers for 91 d to charcoal-filtered air (CF), ambient air O₃ concentration (A), and two-fold ambient air O₃ concentration (2A). Additionally, a chamberless control group (AA) was used to examine chamber effects. At the end of the fumigation period the contents of free radical scavengers and photosynthetic pigments were measured in the needles. Independent from O₃ exposure, total ascorbate and -tocopherol contents were higher in 1-year-old needles compared to the current flush while the opposite was found for glutathione. The amounts of pigments and antioxidants in P. cembra needles were comparable to those in other conifers growing at high-elevation sites. The only hint toward O₃ induced changes in the composition of antioxidants was an increase in the glutathione redox state toward more oxidation in 1-year-old needles upon exposure to A or AA conditions, but not upon 2A exposure. Chlorophyll and carotenoid contents were not affected by O₃ neither in current- nor in previous-year needles. The de-epoxidation state of the xanthophyll cycle pigments, however, was significantly increased in 1-year-old needles under A and AA compared to the CF control, but not under 2A. Hence, Pinus cembra, which is well adapted to the extreme environment of the timberline ecotone, exhibited only marginal biochemical changes in response to elevated O₃.     

Impact of lactate in the perfusate on function and metabolic parameters of isolated working rat heart

The goal of this study was to investigate the effect of 1 mM exogenous lactate on cardiac function, and some metabolic parameters, such as glycolysis, glucose oxidation, lactate oxidation, and fatty acid oxidation, in isolated working rat hearts. Hearts from male Sprague-Dawley rats were isolated and perfused with 5 mM glucose, 1.2 mM palmitate, and 100 μU/ml insulin with or without 1 mM lactate. The rates of glycolysis, glucose, lactate, and fatty acid oxidation were determined by supplementing the buffer with radiolabeled substrates. Cardiac function was similar between lactate+ and lactate− hearts. Glycolysis was not affected by 1 mM lactate. The addition of lactate did not alter glucose oxidation rates. Interestingly, palmitate oxidation rates almost doubled when 1 mM lactate was present in the perfusate. This study suggests that subst rate supply to the heart is crucially important when evaluating the data from metabolic studies.     

Fructose induced deactivation of glucose-6-phosphate dehydrogenase activity and its prevention by pyruvate: Implications in cataract prevention

Glucose-6-phosphate dehydrogenase (G6PDH) is an important lens enzyme diverting about 14% of the tissue glucose to the hexose monophosphate shunt pathway. The main function of such a pronounced activity of the enzyme is to support reductive biosyntheses, as well as to maintain a reducing environment in the tissue so as to prevent oxy-radical induced damage and consequent cataract formation. Sugars are one of the well-known cataractogenic agents. Several reports suggest that the cataractogenic effect of the sugars in diabetes as well as in normal aging is initiated by the glycation of the proteins including the enzymes and subsequent formation of more complex and biologically inactive or harmful structures. In a diabetic lens the concentration of fructose exceeds significantly the concentration of glucose, suggesting that the contribution of fructosylation may be greater than that of glucosylation. These studies were undertaken to examine further the possibility that in addition to glycation, generation of oxygen free radicals by fructose and consequent oxidative modifications in certain enzymes may be an important participant in the cataractogenic process. This hypothesis was tested by using G6PDH. The enzyme was incubated with various levels of fructose (0–20 mM) and its activity determined as a function of time. This led to a significant loss of its activity, which was prevented by superoxide dismutase, catalase, mannitol and myoinositol. Most interestingly, pyruvate at levels between 0.2 and 1.0 mM also offered substantial protection. Hence, the results, while elucidating further the mechanism of enzyme deactivation by sugars such as fructose, also demonstrate the possibility of therapeutic prevention of cataracts by pyruvate and other such keto acids, in diabetes and other disabilities involving oxygen free radicals in the pathogenetic process.     

Protective effect of seminal plasma proteins on the degradation of ascorbic acid

The objectives of this study were to determine ascorbic acid stability and its effect on antiproteinase activity of seminal plasma in the presence of an oxidant. Effect of seminal plasma, and additives: glutathione, albumin, hydrogen peroxide and Tris buffer, on ascorbic acid degradation was investigated by UV absorbance. Antiproteinase against trypsin amidase activity was measured spectrophotometrically using N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) as substrate. Ascorbic acid was destroyed much more rapidly with the addition of hydrogen peroxide than in Tris buffer at pH 8.2 alone. Seminal plasma protected ascorbic acid more efficiently than glutathione and albumin alone. The protective effect of seminal plasma on ascorbic acid degradation may closely relate to the function of ascorbic acid in reproductive system of scurvy-prone animals including teleost fish. Within the range of 1–8 mM concentrations, ascorbic acid had a pro-oxidant action on seminal plasma antiproteinase activityin vitro when they were incubated with hydrogen peroxide.Abbreviations AA Ascorbic acid - BAPNA N-benzoyl-DL-arginine-p-nitroanilide - DMSO dimethyl sulfoxide - GSH glutathione - H₂O₂ hydrogen peroxide