Effect of ambient oxygen concentration on lipofuscin accumulation in cultured rat heart myocytes--a novel in vitro model of lipofuscinogenesis

The objective of this study was to elucidate the factors involved in the accumulation of lipofuscin in post-mitotic cells. The hypothesis that oxidative stress accelerates the rate of lipofuscin accumulation was tested by examining the effects of 5%, 20%, and 40% ambient oxygen concentration on lipofuscin content in cultured rat cardiac myocytes. Lipofuscin was quantified by microspectrofluorometry at 7 and 12 days of in vitro age. Lipofuscin-emitted yellow autofluorescence increased in direct relationship to ambient oxygen concentration with age. Transmission electron microscopic examination of the cells after 3, 8, and 12 days in culture indicated a progressive time and oxygen dependent increase in the frequency and size of lipofuscin organelles. The results are interpreted to suggest that oxidative stress is one of the causal factors in the accumulation of lipofuscin.     

Unilateral eyestalk ablation reduces neurolipofuscin accumulation rate in the contralateral eyestalk of a crustacean, Pacifastacus leniusculus

Use of the lipofuscin ageing method as a crustacean fisheries research tool requires a calibration of tissue lipofuscin concentration to chronological age that is applicable to the natural population under investigation. Current approaches, involving known-age individuals or analysis of cohorts in neurolipofuscin concentration frequency distributions of the wild population, have advantages and disadvantages. A possible alternative that could be applied to individuals of unknown age involves initial biopsy of lipofuscin-loaded tissue from an eyestalk followed, after a known time period, by sampling of the second eyestalk, providing two successive lipofuscin measurements from the same individual and, thus, the neurolipofuscin accumulation rate in the intervening period. We tested the feasibility of this approach by examining the effect of eyestalk removal itself on subsequent lipofuscin accumulation in the remaining eyestalk using known-age individuals of a convenient decapod model, the signal crayfish, Pacifastacus leniusculus. By comparison with untreated controls, a 61% reduction in average neurolipofuscin accumulation rate in the remaining eyestalk occurred. It is hypothesized that this represents either slowed lipofuscinogenesis due to reduced oxidative metabolism or glycosylation, or increased lipofuscin loss due to enhanced proteolytic or phagocytic activity. It is recommended that the proposed ablation technique not be used for calibration of lipofuscin-based age determinations due to its unpredictable effect on lipofuscin accumulation.     

Lipofuscin-formation in retinal pigment epithelial cells is reduced by antioxidants.

The accumulation of lipofuscin by retinal pigment epithelium may be an important feature in the pathogenesis of age-related macular degeneration, suggesting the possibility that this common cause of blindness might be prevented or delayed by antioxidants. In support of this idea, we now report significantly reduced formation of lipofuscin when the antioxidant substances lutein, zeaxanthin, lycopene (carotenoids), or alpha-tocopherol were added to rabbit and bovine (calf) retinal pigment epithelial (RPE) cells exposed to normobaric hyperoxia (40%) and photoreceptor outer segments. Rabbit and calf RPE cells were grown for 2 weeks with addition of one of the test substances every 48 h. The cellular uptake of carotenoids and alpha-tocopherol was assayed by HPLC after 2 weeks. The lipofuscin-content was measured by static fluorometry (rabbit cells) or by image analysis (calf cells). Both rabbit and calf RPE showed similar results with significantly lower amounts of lipofuscin in antioxidant-treated cells. The effect of carotenoids is especially interesting, since the result is not dependent on their protective effect against photo-oxidative reactions. The chain-breaking abilities of these antioxidants in peroxidative reactions of lipid membranes and quenching of free radicals seem to be of importance for inhibition of lipofuscin formation.     

Age-dependent effect of oxidative stress on cardiac sarcoplasmic reticulum vesicles

The oxidative stress hypothesis of aging suggests that accumulation of oxidative damage is a key factor of the alterations in physiological function during aging. We studied age-related sensitivity to oxidative modifications of proteins and lipids of cardiac sarcoplasmic reticulum (SR) isolated from 6-, 15- and 26-month-old rats. Oxidative stress was generated in vitro by exposing SR vesicles to 0.1 mmol/l FeSO4/EDTA + 1 mmol/l H2O2 at 37 degrees C for 60 min. In all groups, oxidative stress was associated with decreased membrane surface hydrophobicity, as detected by 1-anilino-8-naphthalenesulfonate as a probe. Structural changes in SR membranes were accompanied by degradation of tryptophan and significant accumulation of protein dityrosines, protein conjugates with lipid peroxidation products, conjugated dienes and thiobarbituric acid reactive substances. The sensitivity to oxidative damage was most pronounced in SR of 26-month-old rat. Our results indicate that aging and oxidative stress are associated with accumulation of oxidatively damaged proteins and lipids and these changes could contribute to cardiovascular injury.     

Lipofuscin: mechanisms of age-related accumulation and influence on cell function

The accumulation of lipofuscin within postmitotic cells is a recognized hallmark of aging occurring with a rate inversely related to longevity. Lipofuscin is an intralysosomal, polymeric substance, primarily composed of cross-linked protein residues, formed due to iron-catalyzed oxidative processes. Because it is undegradable and cannot be removed via exocytosis, lipofuscin accumulation in postmitotic cells is inevitable, whereas proliferative cells efficiently dilute it during division. The rate of lipofuscin formation can be experimentally manipulated. In cell culture models, oxidative stress (e.g., exposure to 40% ambient oxygen or low molecular weight iron) promotes lipofuscin accumulation, whereas growth at 8% oxygen and treatment with antioxidants or iron-chelators diminish it. Lipofuscin is a fluorochrome and may sensitize lysosomes to visible light, a process potentially important for the pathogenesis of age-related macular degeneration. Lipofuscin-associated iron sensitizes lysosomes to oxidative stress, jeopardizing lysosomal stability and causing apoptosis due to release of lysosomal contents. Lipofuscin accumulation may also diminish autophagocytotic capacity by acting as a sink for newly produced lysosomal enzymes and, therefore, interfere with recycling of cellular components. Lipofuscin, thus, may be much more directly related to cellular degeneration at old age than was hitherto believed.     

A novel hypothesis of lipofuscinogenesis and cellular aging based on interactions between oxidative stress and autophagocytosis.

Based on a series of experiments, using cultured postmitotic neonatal rat cardiac myocytes as a model system, we present a novel hypothesis of lipofuscin formation. This hypothesis proposes that lipofuscin is formed within secondary lysosomes due to an interplay of two processes, the production of partially reduced oxygen species by mitochondria and the autophagocytotic degradation within secondary lysosomes. Specifically, it is proposed that H2O2 generated by mitochondria and other organelles permeates into the lumen of secondary lysosomes, which contain iron derived from cellular structures undergoing intralysosomal degradation. The interaction between reactive ferrous iron and H2O2 results, via Fenton-type mechanisms, in the generation of hydroxyl free radicals (OH), inducing lipid peroxidation and eventually leading to intermolecular cross-linking and lipofuscin formation. Additionally, mitochondria undergoing intralysosomal decomposition might continue for a certain period to produce superoxide anion radicals (O2-) and thus also H2O2. This model of lipofuscinogenesis could satisfactorily explain the variations observed in the rates of lipofuscinogenesis among different postmitotic cell types in various species. Such variations might arise from a variety of factors including differences in the efficiency of the 'anti-oxidative shield', rate of H2O2 generation, amount of chain-breaking antioxidants, mode of intralysosomal iron chelation, rate of autophagocytosis as well as degree of efficiency of the intralysosomal hydrolytic enzymes.     

Age-related differences in oxidative protein-damage in young and senescent fibroblasts

Aging is accompanied by an accumulation of oxidized proteins and cross-linked modified protein material. The intracellular formation and accumulation of highly oxidized and cross-linked proteins, the so-called lipofuscin, is a typical sign of senescence. However, little is known whether the lipofuscin accumulation during aging is related to environmental conditions, as oxidative stress, and whether the accumulation of oxidized proteins and lipofuscin is preferentially taking place in the cytosol or the nucleus and finally, what is the role of lysosomes in this process.Therefore, we investigated human skin fibroblasts in an early stage of proliferation (“young cells”) and in a late stage (“senescent cells”). Such cells were compared for the amount of protein carbonyls and lipofuscin and their distribution within the cytosol and the nucleus. Furthermore, cells were exposed to single and repeated doses of hydrogen peroxide and paraquat, measuring the same set of parameters. In addition to that the role of the proteasome to degrade oxidized proteins in young and senescent cells was tested. Furthermore, detailed microscopic analysis was performed testing the intracellular distribution of lipofuscin. The results clearly demonstrated that repeated/chronic oxidative stress induces a senescence-like phenotype of the distribution of oxidized proteins as well as of lipofuscin. It could be demonstrated that most of the lipofuscin is located in lysosomes and that senescent cells contain less lysosomes not lipofuscin-laden in comparison to young cells.     

Dietary antioxidants prevent age-related retinal pigment epithelium actin damage and blindness in mice lacking αvβ5 integrin

In the aging human eye, oxidative damage and accumulation of pro-oxidant lysosomal lipofuscin cause functional decline of the retinal pigment epithelium (RPE), which contributes to age-related macular degeneration. In mice with an RPE-specific phagocytosis defect due to lack of αvβ5 integrin receptors, RPE accumulation of lipofuscin suggests that the age-related blindness we previously described in this model may also result from oxidative stress. Cellular and molecular targets of oxidative stress in the eye remain poorly understood. Here we identify actin among 4-hydroxynonenal (HNE) adducts formed specifically in β5(-/-) RPE but not in neural retina with age. HNE modification directly correlated with loss of resistance of actin to detergent extraction, suggesting cytoskeletal damage in aging RPE. Dietary enrichment with natural antioxidants, grapes or marigold extract containing macular pigments lutein/zeaxanthin, was sufficient to prevent HNE-adduct formation, actin solubility, lipofuscin accumulation, and age-related cone and rod photoreceptor dysfunction in β5(-/-) mice. Acute generation of HNE adducts directly destabilized actin but not tubulin cytoskeletal elements of RPE cells. These findings identify destabilization of the actin cytoskeleton as a consequence of a physiological, sublethal oxidative burden of RPE cells in vivo that is associated with age-related blindness and that can be prevented by consuming an antioxidant-rich diet.     

Topographical localization of lipofuscin pigment in the brain of the aged fat‐tailed dwarf lemur (Cheirogaleus Medius) and grey lesser mouse lemur (Microcebus Murinus): Comparison to iron localization

The present study was undertaken to explore the distribution of lipofuscin in the brain of cheirogaleids by autofluorescence and compare it to other studies of iron distribution. Aged dwarf (Cheirogaleus medius) and mouse (Microcebus murinus) lemurs provide a reliable model for the study of normal and pathological cerebral aging. Accumulation of lipofuscin, an age pigment derived by lipid peroxidation, constitutes the most reliable cytological change correlated with neuronal aging. Brain sections of four aged (8–15 year old) and 3 young (2–3 year old) animals were examined. Lipofuscin accumulation was observed in the aged animals but not in the young ones. Affected regions include the hippocampus (granular and pyramidal cells), where no iron accumulation was observed, the olfactory nucleus and the olfactory bulb (mitral cells), the basal forebrain, the hypothalamus, the cerebellum (Purkinje cells), the neocortex (essentially in the pyramidal cells), and the brainstem. Even though iron is known to catalyse lipid oxidation, our data indicate that iron deposits and lipofuscin accumulation are not coincident. Different biochemical and morphological cellular compartments might be involved in iron and lipofuscin deposition. The nonuniform distribution of lipofuscin indicates that brain structures are not equally sensitive to the factors causing lipofuscin accumulation. The small size, the rapid maturity, and the relatively short life expectancy of the cheirogaleids make them a good model system in which to investigate the mechanisms of lipofuscinogenesis in primates. Am. J. Primatol. 49:183–193, 1999. © 1999 Wiley‐Liss, Inc.     

The influence of natural inhibitors of radical reactions on autooxidation of adrenaline]

In the presence of EDTA soluble antioxidants (1 muM reduced glutathion, 3 muM cystein, 1 muM ascorbic acid) inhibited the autoxidation of epinephrine at pH 10.2; as to alpha-tocopherol (40 muM) and the oxidative forms of these antioxidants - they were ineffective. The inhibitory effect of superoxide dismutase was much greater than that of the antioxidants testsd. The appearance of adrenochrome during the free radical autoxidation of epinephrine proceeded without any participation of the hydroxyl radical.     

The 5HT1a receptor agonist 8-Oh DPAT induces protection from lipofuscin accumulation and oxidative stress in the retinal pigment epithelium

Age-related macular degeneration (AMD), a major cause of blindness in the elderly, is associated with oxidative stress, lipofuscin accumulation and retinal degeneration. The aim of this study was to determine if a 5-HT(1A) receptor agonist can reduce lipofuscin accumulation, reduce oxidative damage and prevent retinal cell loss both in vitro and in vivo. Autophagy-derived and photoreceptor outer segment (POS)-derived lipofuscin formation was assessed using FACS analysis and confocal microscopy in cultured retinal pigment epithelial (RPE) cells in the presence or absence of the 5-HT(1A) receptor agonist, 8-OH DPAT. 8-OH DPAT treatment resulted in a dose-dependent reduction in both autophagy- and POS-derived lipofuscin compared to control. Reduction in autophagy-induced lipofuscin was sustained for 4 weeks following removal of the drug. The ability of 8-OH DPAT to reduce oxidative damage following exposure to 200 μM H(2)O(2) was assessed. 8-OH DPAT reduced superoxide generation and increased mitochondrial superoxide dismutase (MnSOD) levels and the ratio of reduced glutathione to the oxidized form of glutathione in H(2)O(2)-treated cells compared to controls and protected against H(2)O(2)-initiated lipid peroxidation, nitrotyrosine levels and mitochondrial damage. SOD2 knockdown mice, which have an AMD-like phenotype, received daily subcutaneous injections of either saline, 0.5 or 5.0 mg/kg 8-OH DPAT and were evaluated at monthly intervals. Systemic administration of 8-OH DPAT improved the electroretinogram response in SOD2 knockdown eyes of mice compared to knockdown eyes receiving vehicle control. There was a significant increase in the ONL thickness in mice treated with 8-OH DPAT at 4 months past the time of MnSOD knockdown compared to untreated controls together with a 60% reduction in RPE lipofuscin. The data indicate that 5-HT(1A) agonists can reduce lipofuscin accumulation and protect the retina from oxidative damage and mitochondrial dysfunction. 5-HT(1A) receptor agonists may have potential as therapeutic agents in the treatment of retinal degenerative disease.     

Antioxidant modulation of oxidant-stimulated uptake and release of arachidonic acid in eicosapentaenoic acid-supplemented human lymphoma U937 cells

Omega-3 polyunsaturated fatty acids (PUFA) are increasingly finding use as treatments for a variety of medical conditions. PUFA supplementation can, however, result in increased oxidative stress causing elevated turnover rate of membrane phospholipids, impairment of membrane integrity and increased formation of inflammatory mediators. The aim of this study was to determine which antioxidant compounds were most effective in ameliorating the stimulation of phospholipid turnover by oxidative stress. U937 cells were supplemented with eicosapentaenoic acid and either ascorbic acid, alpha-tocopherol, beta-carotene or astaxanthin prior to being challenged with oxidant. Although all antioxidants were found to be effective in decreasing oxidant-stimulated peroxide formation, only alpha-tocopherol significantly decreased oxidant-stimulated release of 3H-labeled arachidonic acid (AA), while ascorbic acid markedly increased release. All antioxidants except alpha-tocopherol decreased oxidant-stimulated 3H-AA uptake. Our data suggest that antioxidants are not equally effective in combating the effects of oxidative stress upon membrane phospholipid turnover, and that optimal protection will require mixtures of antioxidants.     

Dependence of protective effect of alpha-tocopherol on its concentration and time of action on PC12 cell under conditions of oxidative stress

At the short-term incubation (0.5 and 1.5 h) of cells of the PC12 neuronal line with alpha-tocopherol, its protective effect against the cytotoxic hydrogen peroxide action was increased with rise of its concentration in samples; the protection was practically absent at action of nanomolar antioxidant concentrations, but was well expressed at its micromolar concentrations. These data agree with the concept that alpha-tocopherol increases the cell living activity by reacting directly with free radicals, which leads to formation of the less reactive compounds deprived of non-paired electron. The evidence is obtained that at the long-term action on PC12 cells, alpha-tocopherol not only in micro-, but also in nanomolar concentrations increases statistically significantly the cell living activity under conditions of oxidative stress. As follows from the obtained data, an important role in realization of the alpha-tocopherol protective effect at the long-term incubation with it seems to be played by modulation by this antioxidant of activity of protein kinase activated by extracellular signaling, phosphatidylinosite 3-kinase, and protein kinase C.     

ECL cells of the rat stomach: development of lipofuscin in response to sustained gastrin stimulation

Ageing cells, especially post-mitotic cells, are known to accumulate pigments, i.e. highly electron-dense material, referred to as ceroid or lipofuscin. This material is formed as a consequence of autophagocytosis and peroxidation of the products undergoing degradation. The present study describes the development of lipofuscin in the ECL cells of the rat stomach. These cells produce and secrete histamine in response to gastrin. They are rich in secretory vesicles, which fuse to form vacuoles in hypergastrinaemic rats. Hypergastrinaemia was induced by continuous infusion of human Leu¹⁵-gastrin-17 for 6 days or by daily treatment with omeprazole for 10 weeks. Either treatment caused both vacuoles and lipofuscin bodies to appear in large numbers; the vacuoles disappeared promptly after interruption of the hypergastrinaemia, whereas the lipofuscin bodies remained. Antrectomy-evoked hypogastrinaemia was associated with a reduced number and volume density of lipofuscin bodies. Treatment with α-fluoromethylhistidine, an irreversible inhibitor of the histamine-forming enzyme, resulted in depletion of ECL-cell histamine and was found to prevent the omeprazole-evoked formation of vacuoles and lipofuscin. The numbers of both vacuoles and lipofuscin bodies were well-correlated with the serum gastrin concentration, suggesting that gastrin stimulates the development not only of vacuoles but also of lipofuscin, perhaps through enhanced autophagocytosis and/or oxidative stress. Thus, lipofuscin bodies may develop from vacuoles, and both vacuoles and lipofuscin bodies may reflect the efforts of overstimulated ECL cells to cope with the excessive formation of secretory products.     

Early Onset of Lipofuscin Accumulation in Dystrophin-Deficient Skeletal Muscles of DMD Patients and mdx Mice

Lipofuscin, the so-called ageing pigment, is formed by the oxidative degradation of cellular macromolecules by oxygen-derived free radicals and redox-active metal ions. Usually it accumulates in post-mitotic, long-lived cells such as neurons and cardiac muscle cells. In contrast, it is rarely seen in either normal or diseased skeletal muscle fibres. In this paper, we report that lipofuscin accumulates at an early age in both human and murine dystrophic muscles. Autofluorescent lipofuscin granules were localized, using confocal laser scanning microscopy and electron microscopy, in dystrophin-deficient skeletal muscles of X chromosome-linked young Duchenne muscular dystrophy (DMD) patients and of mdx mice at various ages after birth. Age-matched normal controls were studied similarly. Autofluorescent lipofuscin granules were observed in dystrophic biceps brachii muscles of 2-7-year-old DMD patients where degeneration and regeneration of myofibres are active, but they were rarely seen in age-matched normal controls. In normal mice, lipofuscin first appears in diaphragm muscles nearly 20 weeks after birth but in mdx muscles it occurs much earlier, 4 weeks after birth, when the primary degeneration of dystrophin-deficient myofibres is at a peak. Lipofuscin accumulation increases with age in both mdx and normal controls and is always higher in dystrophic muscles than in age-matched normal controls. At the electron microscopical level, it was confirmed that the localisation of autofluorescent granules observed by light microscopy in dystrophin-deficient skeletal muscles coincided with lipofuscin granules in myofibres and myosatellite cells, and in macrophages accumulating around myofibres and in interstitial connective tissue. Our results agree with previous biochemical and histochemical data implying increased oxidative damages in DMD and mdx muscles. They indicate that dystrophin-deficient myofibres are either more susceptible to oxidative stress, or are subjected to higher intra- or extracellular oxidative stress than normal controls, or both.     

Vitamin E supplementation in the mitigation of carbon tetrachloride induced oxidative stress in rats

Oxidative stress is implicated in the pathophysiology of a number of chronic diseases including atherosclerosis, diabetes, cataracts and accelerated aging. The aim of this study was to elucidate the protective role of vitamin E supplementation when oxidative stress is induced by CCl4 administration, using the rat as a model. Rats were fed diets for four weeks either with or without dl-alpha-tocopherol acetate supplementation. Half of the rats (n = 9) from each of the diet groups were then challenged with CCl4 at the completion of the four week diet period. Plasma levels of 8-iso-PGF(2alpha), antioxidant micronutrients and antioxidant enzyme activities were measured to examine changes in oxidative stress subsequent to the supplementation of dl-alpha-tocopherol in the diet. Plasma alpha-tocopherol (vitamin E) concentrations were higher for the groups supplemented with dl-alpha-tocopherol acetate, however the supplemented diet group that was subsequently challenged with CCl4 had significantly lower (p <0.001) plasma alpha-tocopherol concentration than the dl-alpha-tocopherol acetate diet group that was not challenged with CCl4. Total plasma 8-iso-PGF(2alpha) concentration was elevated in diet groups challenged with CCl4, however, the concentration was significantly lower (p <0.001) when the diet was supplemented with dl-alpha-tocopherol acetate. The antioxidant enzymes were not influenced by either dietary alpha-tocopherol manipulation or by the inducement of oxidative stress with CCl4. Plasma concentrations of trans-retinol (vitamin A) were reduced by CCl4 administration in both the dl-alpha-tocopherol acetate supplemented and unsupplemented diet groups. The results of this study indicate that dl-alpha-tocopherol acetate supplementation was protective of lipid peroxidation when oxidative stress is induced by a pro-oxidant challenge such as CCl4.     

Proteasome inhibition by lipofuscin/ceroid during postmitotic aging of fibroblasts.

We have studied the effects of hyperoxia and of cell loading with artificial lipofuscin or ceroid pigment on the postmitotic aging of human lung fibroblast cell cultures. Normobaric hyperoxia (40% oxygen) caused an irreversible senescence-like growth arrest after about 4 wk and shortened postmitotic life span from 1-1/2 years down to 3 months. During the first 8 wk of hyperoxia-induced 'aging', overall protein degradation (breakdown of [(35)S]methionine metabolically radiolabeled cell proteins) increased somewhat, but by 12 wk and thereafter overall proteolysis was significantly depressed. In contrast, protein synthesis rates were unaffected by 12 wk of hyperoxia. Lysosomal cathepsin-specific activity (using the fluorogenic substrate z-FR-MCA) and cytoplasmic proteasome-specific activity (measured with suc-LLVY-MCA) both declined by 80% or more over 12 wk. Hyperoxia also caused a remarkable increase in lipofuscin/ceroid formation and accumulation over 12 wk, as judged by both fluorescence measurements and FACscan methods. To test whether the association between lipofuscin/ceroid accumulation and decreased proteolysis might be causal, we next exposed cells to lipofuscin/ceroid loading under normoxic conditions. Lipofuscin/ceroid-loaded cells indeed exhibited a gradual decrease in overall protein degradation over 4 wk of treatment, whereas protein synthesis was unaffected. Proteasome specific activity decreased by 25% over this period, which is important since proteasome is normally responsible for degrading oxidized cell proteins. In contrast, an apparent increase in lysosomal cathepsin activity was actually caused by a large increase in the number of lysosomes per cell. To test whether lipofuscin/ceroid could in fact directly inhibit proteasome activity, thus causing oxidized proteins to accumulate, we incubated purified proteasome with lipofuscin/ceroid preparations in vitro. We found that proteasome is directly inhibited by lipofuscin/ceroid. Our results indicate that an accumulation of oxidized proteins (and lipids) such as lipofuscin/ceroid may actually cause further increases in damage accumulation during aging by inhibiting the proteasome.     

Screening and evaluation of antioxidant activity of some pyridazine derivatives

Antioxidants are compounds that can delay, inhibit, or prevent the oxidation of materials that can be oxidized by scavenging free radicals and help in diminishing oxidative stress. They belong to different chemical classes. Recently there are studies related to pyridazinone derivatives for their antioxidant activities. Since there are evidences implicates reactive oxygen species and nitric oxide as mediators of inflammation and/or tissue damage in inflammatory and arthritic disorders it was though that compounds that have both antioxidant and anti-inflammatory activities would have been essential for the inflammatory diseases. Based on these findings a series of 2H-pyridazine-3-one and 6-chloropyridazine analogues that have anti-inflammatory activity was tested in vitro on superoxide formation and effects on lipid peroxidation were determined against alpha-tocopherol. Most of the compounds have strong inhibitory effect on superoxide anion (between 84% - 99%) at 10(- 3) M concentration. In addition, these compounds showed similar activity to alpha-tocopherol at 10(- 3) M concentrations.     

Deltamethrin-induced oxidative stress and biochemical changes in tissues and blood of catfish (Clarias gariepinus): antioxidant defense and role of alpha-tocopherol

ABSTRACT: BACKGROUND: The pyrethroid class of insecticides, including deltamethrin, is being used as substitutes for organochlorines and organophosphates in pest-control programs because of their low environmental persistence and toxicity. This study was aimed to investigate the impact of commonly used pesticides (deltamethrin) on the blood and tissue oxidative stress level in catfish (Clarias gariepinus); in addition to the protective effect of alpha-tocopherol on deltamethrin induced oxidative stress. METHODS: Catfish were divided into three groups, 1st control group include 20 fish divided into two tanks each one contain 10 fish, 2nd deltamethrin group, where Fish exposed to deltamethrin in a concentration (0.75ug/l) and 3rd Vitamin E group, Fish exposed to deltamethrin and vitamin E at a dose of 12ug/l for successive 4 days. Serum, liver, kidney and Gills were collected for biochemical assays. Tissue oxidative stress biomarkers malondialdhyde (MDA) and catalase activity in liver, kidney and gills tissues, serum liver enzymes (ALT and AST), serum albumin, total protein, urea and creatinine were analysed. RESULTS: Our results showed that 48 h. exposure to 0.75 ug/l deltamethrin significantly (p<0.05) increased lipid peroxidation (MDA) in the liver, kidney and gills while catalase activity was significantly decreased in the same tissues. This accompanied by significant increase in serum ALT, AST activity, urea and creatinine and a marked decrease in serum albumin and total proteins. CONCLUSIONS: It could be concluded that deltamethrin is highly toxic to catfish even in very low concentration (0.75 ug/l). Moreover the effect of deltamethrin was pronounced in the liver of catfish in comparison with kidneys and gills. Moreover fish antioxidants and oxidative stress could be used as biomarkers for aquatic pollution, thus helping in the diagnosis of pollution. Adminstration of 12 ug/l alpha-tocopherol restored the quantified tissue and serum parameters, so supplementation of alpha-tocopherol consider an effective way to counter the toxicity of deltamethrin in the catfish.     

Fructose-mediated damage to lens alpha-crystallin: prevention by pyruvate

Post-translational modifications in lens crystallins due to glycation and oxidation have been suggested to play a significant role in the development of cataracts associated with aging and diabetes. We have previously shown that alpha-keto acids, like pyruvate, can protect the lens against oxidation. We hypothesize that they can also prevent the glycation of proteins competitively by forming a Schiff base between their free keto groups and the free -NH(2) groups of protein as well as subsequently inhibit the oxidative conversion of the initial glycation product to advanced glycation end products (AGE). The purpose of this study was to investigate these possibilities using purified crystallins. The crystallins isolated from bovine lenses were incubated with fructose in the absence and presence of pyruvate. The post-incubation mixtures were analyzed for fructose binding to the crystallins, AGE formation, and the generation of high molecular weight (HMW) proteins. In parallel experiments, the keto acid was replaced by catalase, superoxide dismutase (SOD), or diethylene triaminepentaacetic acid (DTPA). This was done to ascertain oxidative mode of pyruvate effects. Interestingly, the glycation and consequent formation of AGE from alpha-crystallin was more pronounced than from beta-, and gamma-crystallins. The changes in the crystallins brought about by incubation with fructose were prevented by pyruvate. Catalase, SOD, and DTPA were also effective. The results suggest that pyruvate prevents against fructose-mediated changes by inhibiting the initial glycation reaction as well as the conversion of the initial glycated product to AGE. Hence it is effective in early as well as late phases of the reactions associated with the formation of HMW crystallin aggregates.