Ebselen protects Chinese hamster ovary cells from radiation-induced apoptosis

Summary

Radiation-induced apoptosis in chinese hamster ovary (CHO) cell lines is characterized by endonucleolytic cleavage of cellular DNA and changes of cell morphology within hours after radiation exposure. We investigated the capacity of ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one], a seleno-organic compound with selenium-dependent glutathione peroxidase (GPx) activity, to protect cells from radiation-induced apoptosis. This phenomenon was studied by the quantitation of apoptotic cells and DNA gel electrophoresis after 6 Gy X-ray exposure. We also measured the activity of GPx and membrane lipid peroxidation. It was observed that 20 µM ebselen efficiently blocked apoptotic cell formation and DNA fragmentation 48 h post irradiation. Furthermore the data demonstrated that lipid peroxides increased significantly in irradiated cells and ebselen inhibited this process by elevating the cellular GPx activity. The results presented here indicate the requirement of free radicals for radiation-induced apoptosis and ultimately may yield insight necessary for designing protocols to modulate the process of radiation-induced apoptosis with antioxidant agents that scavenge radiation-induced free radicals.     

Effect of zinc supplementation on glutathione peroxidase activity and selenium concentration in the serum, liver and kidney of rats chronically exposed to cadmium

It was investigated whether the ability of zinc (Zn) to prevent cadmium (Cd)-induced lipid peroxidation may be connected with its impact on glutathione peroxidase (GPx) activity and selenium (Se) concentration. GPx and Se were determined in the serum, liver and kidney of the rats that received Cd (5 or 50 mg/L) or/and Zn (30 mg/L) in drinking water for 6 months in whose the protective Zn impact was noted (Rogalska J, Brzóska MM, Roszczenko A, Moniuszko-Jakoniuk J. Enhanced zinc consumption prevents cadmium-induced alterations in lipid metabolism in male rats. Chem Biol Interact 2009;177:142-52). Moreover, dependences between these parameters, and indices of lipid peroxidation (F(2)-isoprostane, lipid peroxides, oxidized low density lipoprotein cholesterol) as well as concentrations of Cd and Zn were estimated. The supplementation with Zn during the exposure to 5 mg Cd/L entirely antagonized the Cd-induced increase in GPx activity and Se concentration in the liver and kidney, but not in the serum. Zn administration during the treatment with 50 mg Cd/L totally or partially prevented from the Cd-caused decrease in GPx activity and Se concentration in the serum, liver and kidney. At the higher level of Cd exposure, GPx activity in the serum and tissues positively correlated with Se concentration. Moreover, numerous correlations were noted between GPx and/or Se and the indices of lipid peroxidation. The results indicate that the protective impact of Zn against the Cd-induced lipid peroxidation during the relatively high exposure might be connected with its beneficial influence on Se concentration and GPx activity in the serum and tissues, whereas this bioelement influence at the moderate exposure seems to be independent of GPx and Se.     

Inhibitory Effects of Ebselen on Lipid Peroxidation in Rat Liver Microsomes

The effects of ebselen(2-pheny1-1,2-benzoisoselenazol-3(2H)-one), a synthetic seleno-organic compound with glutathione peroxidase-like activity were investigated on lipid peroxidation in rat liver microsomes. Ebselen inhibited malondialdehyde production coupled to the lipid peroxidation stimulated by either ADP-iron-ascorbate or CC1₄. The inhibitory activity of ebselen on each system was strongly increased by a 5-min preincubation with liver microsomes; the IC₅₀ values against ADP-Fe-ascorbate-stimulated and CC1₄-stimulated lipid peroxidation were 1.6/jM and 70 μM respectively. Ebselen also inhibited the endogenous lipid peroxidation with a NADPH-generating system, but it slightly stimulated the endogenous activity of ADP-Fe-ascorbate-stimulated lipid peroxidation (without a NADPH-generating system). Furthermore, ebselen inhibited oxygen uptake coupled to the lipid peroxidation by ADP-Fe-ascorbate and NADPH-ADP-iron; the IC₅₀ values were 2.5μM AND 20.3 μM respectively. Ebselen also prolonged the lag-time of onset of ADP-Fe-ascorbate-stimulated lipid peroxidation significantly, but not that observed with NADPH-ADP-Fe-stimulated lipid peroxidation.     

Oxidative modification of human low-density lipoprotein by horseradish peroxidase in the absence of hydrogen peroxide

Heme-peroxidases, such as horseradish peroxidase (HRP), are among the most popular catalysts of low density lipoprotein (LDL) peroxidation. In this model system, a suitable oxidant such as H₂O₂ is required to generate the hypervalent iron species able to initiate the peroxidative chain. However, we observed that traces of hydroperoxides present in a fresh solution of linoleic acid can promote lipid peroxidation and apo B oxidation, substituting H₂O₂.

Spectral analysis of HRP showed that an hypervalent iron is generated in the presence of H₂O₂ and peroxidizing linoleic acid. Accordingly, careful reduction of the traces of linoleic acid lipid hydroperoxide prevented formation of the ferryl species in HRP and lipid peroxidation. However, when LDL was oxidized in the presence of HRP, the ferryl form of HRP was not detectable, suggesting a Fenton-like reaction as an alternative mechanism. This was supported by the observation that carbon monoxide, a ligand for the ferrous HRP, completely inhibited peroxidation of LDL.

These results are in agreement with previous studies showing that myoglobin ferryl species is not produced in the presence of phospholipid hydroperoxides, and emphasize the relevance of a Fenton-like chemistry in peroxidation of LDL and indirectly, the role of pre-existing lipid hydroperoxides.     

Role of glutathione S-transferases in protection against lipid peroxidation. Overexpression of hGSTA2-2 in K562 cells protects against hydrogen peroxide-induced apoptosis and inhibits JNK and caspase 3 activation

The physiological significance of the selenium-independent glutathione peroxidase (GPx) activity of glutathione S-transferases (GSTs), associated with the major Alpha class isoenzymes hGSTA1-1 and hGSTA2-2, is not known. In the present studies we demonstrate that these isoenzymes show high GPx activity toward phospholipid hydroperoxides (PL-OOH) and they can catalyze GSH-dependent reduction of PL-OOH in situ in biological membranes. A major portion of GPx activity of human liver and testis toward phosphatidylcholine hydroperoxide (PC-OOH) is contributed by the Alpha class GSTs. Overexpression of hGSTA2-2 in K562 cells attenuates lipid peroxidation under normal conditions as well as during the oxidative stress and confers about 1.5-fold resistance to these cells from H(2)O(2) cytotoxicity. Treatment with 30 microm H(2)O(2) for 48 h or 40 microm PC-OOH for 8 h causes apoptosis in control cells, whereas hGSTA2-2-overexpressing cells are protected from apoptosis under these conditions. In control cells, H(2)O(2) treatment causes an early (within 2 h), robust, and persistent (at least 24 h) activation of JNK, whereas in hGSTA2-2-overexpressing cells, only a slight activation of JNK activity is observed at 6 h which declines to basal levels within 24 h. Caspase 3-mediated poly(ADP-ribose) polymerase cleavage is also inhibited in cells overexpressing hGSTA2-2. hGSTA2 transfection does not affect the function of antioxidant enzymes including GPx activity toward H(2)O(2) suggesting that the Alpha class GSTs play an important role in regulation of the intracellular concentrations of the lipid peroxidation products that may be involved in the signaling mechanisms of apoptosis.     

Association of classical semen parameters, sperm DNA fragmentation index, lipid peroxidation and antioxidant enzymatic activity of semen in ram-lambs

The objective was to determine relationships among classical semen characteristics, sperm chromatin structure assay (SCSA), lipid peroxidation and antioxidant enzymatic activity in ram-lamb semen. Fifty-seven ram-lambs were electroejaculated, and routine semen evaluation was conducted (as part of a breeding soundness evaluation). The percentage of sperm DNA fragmentation index (%DFI) and the percentage of sperm with abnormally high DNA stainability (HDS; immature spermatozoa) were determined by SCSA using the metachromatic properties of acridine orange. Semen was centrifuged at 800 x g for 15 min to separate spermatozoa and seminal plasma and the aliquots were stored at -70 degrees C until analyzed. Lipid peroxidation, superoxide dismutase (SOD), and glutathione peroxidase (GPx) levels in seminal plasma and spermatozoa were measured by spectrophotometric assays. The classical semen parameters were negatively related to lipid peroxidation and GPx activity in spermatozoa; motility and morphology were negatively related to %DFI (P < 0.05). Based on Kruskal-Wallis pair-wise comparison of median values among breeding soundness outcome groups, %DFI was lower in the satisfactory group compared to other groups (P < 0.05) and the lipid peroxidation and GPx activity in seminal plasma and spermatozoa were lower in satisfactory and questionable groups (P < 0.05). However, the SOD was lower in the unsatisfactory group (P < 0.05). In summary, classical semen parameters were negatively related to % DFI, lipid peroxidation and GPx activity in ram-lamb spermatozoa and seminal plasma. There were indications that SOD and GPx have crucial protective roles against the toxic effect of reactive oxygen species (ROS) in ram-lamb semen.     

Efficacy of the antioxidant ebselen in experimental uveitis.

Inflammation results in the production of free radicals. In a model of experimental uveitis upon subcutaneous injection of endotoxin to Lewis rats, i.e., endotoxin-induced experimental uveitis (EIU), we have evaluated the status of the antioxidant capacity of ocular tissues. EIU results in a decrease of glutathione (GSH) content and glutathione peroxidase (GPx) activity in whole eye homogenates 24-h after endotoxin administration. Furthermore, an increase in malondialdehyde (MDA) content was observed in these same samples, thus confirming the involvement of oxidative stress in the pathophysiology of the process. In view of the ability of the antioxidant ebselen as GPx enzyme mimic, we tested the effect of the oral treatment with two doses of 100 mg/kg body weight of ebselen (first dose administered at the same time of endotoxin, and the second after 12 h). Ebselen administration normalized the GSH and MDA contents and protected the GPx activity of the EIU rat eyes. The GPx activity in the eye homogenate of the treated rats could be completely acounted for by the ebselen-dependent GPx-like activity, i.e., GPx activity measured in the acidic supernatant of the homogenate after neutralization. Unmodified ebselen was detected in whole eye homogenates, thus it shows for the first time the penetration of ebselen through the blood-aqueous and blood-retina barrier. The results herein may allow the proposal of ebselen as a suitable antiinflammatory agent in ocular tissues.     

Peroxynitrite reductase activity of selenoprotein glutathione peroxidase: a computational study

The peroxynitrite reductase activity of selenoprotein glutathione peroxidase (GPx) has been investigated using density functional theory calculations for peroxynitrite/peroxynitrous acid (ONOO-/ONOOH) substrates through two different "oxidation" and "nitration" pathways. In the oxidation pathway for ONOO-, the oxidation of GPx and the subsequent formation of the selenenic acid (E-Se-OH) occur through a concerted mechanism with an energy barrier of 4.7 (3.7) kcal/mol, which is in good agreement with the computed value of 7.1 kcal/mol for the drug ebselen and the experimentally measured barrier of 8.8 kcal/mol for both ebselen and GPx. For ONOOH, the formation of the E-Se-OH prefers a stepwise mechanism with an overall barrier of 6.9 (11.3) kcal/mol, which is 10.2 (11.2) kcal/mol lower than that for hydrogen peroxide (H2O2), indicating that ONOOH is a more efficient substrate for GPx oxidation. It has been demonstrated that the active site Gln83 residue plays a critical role during the oxidation process, which is consistent with the experimental suggestions. The nitration of GPx by ONOOH produces a nitro (E-Se-NO2) product via either of two different mechanisms, isomerization and direct, having almost the same barrier heights. A comparison between the rate-determining barriers of the oxidation and nitration pathways suggests that the oxidation of GPx by ONOOH is more preferable than its nitration. It was also shown that the rate-determining barriers remain the same, 21.5 (25.5) kcal/mol, in the peroxynitrite reductase and peroxidase activities of GPx.     

Oxidative modification of human low-density lipoprotein by horseradish peroxidase in the absence of hydrogen peroxide

Heme-peroxidases, such as horseradish peroxidase (HRP), are among the most popular catalysts of low density lipoprotein (LDL) peroxidation. In this model system, a suitable oxidant such as H₂O₂ is required to generate the hypervalent iron species able to initiate the peroxidative chain. However, we observed that traces of hydroperoxides present in a fresh solution of linoleic acid can promote lipid peroxidation and apo B oxidation, substituting H₂O₂.

Spectral analysis of HRP showed that an hypervalent iron is generated in the presence of H₂O₂ and peroxidizing linoleic acid. Accordingly, careful reduction of the traces of linoleic acid lipid hydroperoxide prevented formation of the ferryl species in HRP and lipid peroxidation. However, when LDL was oxidized in the presence of HRP, the ferryl form of HRP was not detectable, suggesting a Fenton-like reaction as an alternative mechanism. This was supported by the observation that carbon monoxide, a ligand for the ferrous HRP, completely inhibited peroxidation of LDL.

These results are in agreement with previous studies showing that myoglobin ferryl species is not produced in the presence of phospholipid hydroperoxides, and emphasize the relevance of a Fenton-like chemistry in peroxidation of LDL and indirectly, the role of pre-existing lipid hydroperoxides.     

Taurine protects against carbon tetrachloride toxicity in the cultured neurons and in vivo

Carbon tetrachloride (CCl4) has been found to induce cellular damage by generating oxygen free radicals. A study was carried out to investigate the effects of taurine (extracted from Pegasus laternarius Cuvier) on CCl4 intoxicated cultured neurons. CCl4 application (0.4 mmol x l(-1), 0.8 mmol x l( -1), 1.2 mmol x l (-1) and 1.6 mmol x l(-1 )) increased the lipid peroxidation product and decreased glutathione peroxidase (GPx) activity significantly in a concentration dependent manner. Pretreatment of cultures with taurine (10 micromol x l(-1), 30 micromol x l(-1) and 60 micromol x l(-1)) prevented the loss of GPx activity and lipid peroxidation. The effects of three different dosages of taurine (10 mg/kg body wt., 20 mg/kg body wt. and 40 mg/ kg body wt.) for 45 days on the activities of superoxide dismutase and glutathione peroxidase were examined in the cerebrum, cerebellum and medulla of normal and CCl4 treated mice. Treatment of mice with taurine provided protection against CCl4 toxicity as was evident by lipid peroxide status. Taurine was not so successful at inducing the activity of SOD in normal animals except in the medulla where it could increase the activity of SOD (p < 0.05). Taurine induces the GPx activity in a dose dependent manner in all regions of the brain studied. Also in the CCl4 poisoned mice taurine could augment the status of GPx activity in a dose dependent manner. Hence it is concluded that taurine can protects neurons from the oxidative stress induced by CCl4.     

Aluminium-induced enhancement of ageing-related biochemical and electrophysiological parameters in rat brain regions

We investigated alterations in the ageing-related parameters: multiple-unit action potentials, Na(+), K(+)-ATPase activity, glutathione-s-transferase (GST) activity, glutathione peroxidase (GPx) activity, lipid peroxidation and lipofuscin contents in the brain regions cerebral cortex, striatum, hippocampus and thalamus, resulting from the chronic administration of aluminium chloride (AlCl3) in drinking water to rats of 6 and 12 months of age. Aluminium treatment significantly depressed Na(+), K(+)-ATPase, GST and GPx activities, elevated lipid peroxidation and lipofuscin contents, and produced intense epileptiform activity in the electroencephalograms of the studied brain regions together with a concomitant increase in the multiple-unit action potentials (MUA) indicating a vigorous neuronal epileptic hyperactivity. Taken together the aluminium-induced alterations in these parameters are indicative of an accelerated ageing process.     

Synthesis and characterization of a triphenylphosphonium-conjugated peroxidase mimetic. Insights into the interaction of ebselen with mitochondria

Mitochondrial production of peroxides is a critical event in both pathology and redox signaling. Consequently their selective degradation within mitochondria is of considerable interest. Here we have explored the interaction of the peroxidase mimetic ebselen with mitochondria. We were particularly interested in whether ebselen was activated by mitochondrial glutathione (GSH) and thioredoxin, in determining whether an ebselen moiety could be targeted to mitochondria by conjugating it to a lipophilic cation, and in exploring the nature of ebselen binding to mitochondrial proteins. To achieve these goals we synthesized 2-[4-(4-triphenylphosphoniobutoxy) phenyl]-1,2-benzisoselenazol)-3(2H)-one iodide (MitoPeroxidase), which contains an ebselen moiety covalently linked to a triphenylphosphonium (TPP) cation. The fixed positive charge of TPP facilitated mass spectrometric analysis, which showed that the ebselen moiety was reduced by GSH to the selenol form and that subsequent reaction with a peroxide reformed the ebselen moiety. MitoPeroxidase and ebselen were effective antioxidants that degraded phospholipid hydroperoxides, prevented lipid peroxidation, and protected mitochondria from oxidative damage. Both peroxidase mimetics required activation by mitochondrial GSH or thioredoxin to be effective antioxidants. Surprisingly, conjugation to the TPP cation led to only a slight increase in the uptake of ebselen by mitochondria due to covalent binding of the ebselen moiety to proteins. Using antiserum against the TPP moiety we visualized those proteins covalently attached to the ebselen moiety. This analysis indicated that much of the ebselen present within mitochondria is bound to protein thiols through reversible selenenylsulfide bonds. Both MitoPeroxidase and ebselen decreased apoptosis induced by oxidative stress, suggesting that they can decrease mitochondrial oxidative stress. This exploration has led to new insights into the behavior of peroxidase mimetics within mitochondria and to their use in investigating mitochondrial oxidative damage.     

Lipid peroxidation-mediated oxidative stress and dopamine neuronal apoptosis in the substantia nigra during development.

The cellular pathways underlying naturally occurring neuronal apoptosis in the rat substantia nigra (SN) during the perinatal period remain largely unknown. Determining the mediators of this process in development may shed light on causes of premature neuronal death in adult neurodegenerative disorders, including the loss of dopamine neurons in Parkinson's disease. In the present study, we investigated whether lipid peroxidation-mediated oxidative stress mediates developmental death of nigral neurons by (1) establishing the profile of lipid peroxidation and other oxidative stress markers throughout the postnatal period both in the SN and striatum, and (2) examining whether the inhibitor of lipid peroxidation, alpha-tocopherol, protects these neurons from death. In addition to monitoring, the level of lipid peroxidation throughout development, we also measured the activities of three antioxidant enzymes, namely superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx). We have shown that lipid peroxidation and SOD activity progressively increased from postnatal day (PND) 3 to PND 42 in both SN and striatum. During this period, GPx activity remained stable, while catalase activity transiently increased at PND 8 only in the SN. Furthermore, alpha-tocopherol treatment from embryonic day 18 to PND 2 did not reduce the number of apoptotic neurons at PND 3. These results do not support the hypothesis that lipid peroxidation-mediated oxidative stress is the major mediator of nigral dopamine neuronal apoptosis during the perinatal period.     

The Correlation between Selenium-Dependent Glutathione Peroxidase Activity and Oxidant/Antioxidant Balance in Sera of Diabetic Patients with Nephropathy

Background:Oxidative stress is an imbalance between free radical''s production and the body''s ability to counteract or detoxify their harmful effects through neutralization by antioxidants, oxidative stress is thought to be involved in the pathogenesis of diabetic nephropathy. One of the key enzymatic antioxidants is glutathione peroxidase (GPx), which plays an important protective function in diabetes complications, by reducing the rising state of oxidative stress and removing toxicity from peroxides and converting them into a non-toxic substance. The objective of this research was to evaluate the rule of glutathione peroxidase in regulate oxidants/antioxidants levels diabetic patients with nephropathy.Methods:In a case-control study, we assessed serum GPx activity (Se-Dependent, non-selenium dependent and total GPx), total oxidant, lipid peroxidation, total antioxidant, and catalase in healthy control subjects (group 1), in diabetic patients without diabetic nephropathy (group 2) and diabetic patients with nephropathy (group 3).Results:GPx activity was significantly lower in T2D patients with and without nephropathy compared to healthy subject’s control. Total oxidants and lipids peroxidation have a negative correlation with the GPx and other antioxidants.Conclusion:Decreased GPx activity indicate a relationship between GPx activity and diabetic nephropathy.Key Words: Diabetic Nephropathy, Glutathione Peroxidase, Oxidative Stress, Reactive Oxygen Species, Selenium     

Protective effects of polysaccharide from Euphorbia kansui (Euphorbiaceae) on the swimming exercise-induced oxidative stress in mice

The present study examined the effects of derivatives of galactosides and glucosides in a polysaccharide extract from Euphorbia kansui (Euphorbiaceae) on exercise-induced oxidative stress in mice. Exhaustive swimming exercise significantly increases the degree of lipid peroxidation in terms of malondialdehyde content and reduces the antioxidant activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx). Our findings revealed that chronic oral treatment with the extract elevates enzymatic activities of SOD and GPx accompanied by a corresponding decrease in malondialdehyde. The antioxidative activities of these compounds against exercise-induced oxidative stress are correlated with various activities such as reducing the production of superoxide and hydroxyl radicals, inhibiting lipid peroxidation, enhancing antioxidative defenses, and increasing the production of SOD and GPx activity and expression in different tissues. These compounds may be involved in glycogen metabolism to meet the requirement of working skeletal muscles and act as antioxidants by terminating the chain reaction of lipid peroxidation to maintain the morphological stability of mitochondria in spinal motor neurons. These observations suggest that E. kansui has antioxidative and antifatigue properties and can be given as prophylactic and (or) therapeutic supplements for increasing antioxidant enzyme activities and preventing lipid peroxidation during strenuous exercise.     

The antioxidant status during maturation of reticulocytes to erythrocytes in type 2 diabetics

Free radical-induced lipid peroxidation has been associated with numerous disease processes including diabetes mellitus. The extent of lipid peroxidation (LPO) and antioxidant defense system [i.e., levels of glutathione (GSH), glucose-6-phosphate dehydrogenase (G6PDH), glutathione reductase (GR), glutathione peroxidase (GPx), glutathione-S-transferase (GST), and catalase (CAT)] were evaluated in reticulocytes and erythrocytes of type 2 diabetic males and age-matched controls. Type 2 diabetics have shown increased lipid peroxidation and decreased levels of GSH, GR, GPx, G6PDH, and GST both in reticulocytes and erythrocytes compared to controls, indicating the presence of oxidative stress and defective antioxidant systems in these patients. CAT activity is found to be enhanced in both the reticulocytes and erythrocytes of diabetics, with a greater percentage enhancement in reticulocytes. The extent of increase in lipid peroxidation is greater in erythrocytes compared to reticulocytes in these patients. Furthermore, the maturation of reticulocytes to erythrocytes resulted in decreased GSH and decreased activities of all antioxidant enzymes (except CAT) both in normals and type 2 diabetes individuals, indicating decreased scavenging capacity as reticulocytes mature to erythrocytes. These maturational alterations are further intensified in type 2 diabetics. The present study reveals that the alterations in lipid peroxidation and antioxidant system lean toward early senescence of erythrocytes in type 2 diabetic patients.     

Effects of cadmium exposure on lipid peroxidation and the antioxidant system in fourth-instar larvae of Propsilocerus akamusi (Diptera: Chironomidae) under laboratory conditions

Enzymatic antioxidants such as selenium-dependent glutathione peroxidase (GPx), glutathione transferase (GST), glutathione reductase (GR), and superoxide dismutases (SOD), as well as the concentration of hydrogen peroxide (H2O2) and malondialdehyde (MDA, an indicator of lipid peroxidation) were determined to identify which antioxidant enzymes participate in the efficient scavenging of ROS generated upon exposure to high doses of Cd2+ in fourth-instar Propsilocerus akamusi (Tokuna) (Diptera: Chironomidae) larvae after 72-h exposure. A significant increase in MDA levels and a change in GR and GPx activities in the Cd(2+)-treated P. akamusi were observed. The MDA in 25.0 and 50.0 mmol/liter treatments was significantly higher than that of the control dose after 72 h exposure. GPx activity was significantly induced by Cd2+ exposure only in the 50.0-mmol/liter treatment with a 0.59-fold increase in the control. All doses of Cd2+ significantly suppressed GR activity compared with the findings for the control dose, with an inhibited rate up to 0.55-fold in the 25.0 mmol/liter Cd2+ treatment. SOD and GST activities were not altered. The results indicate that Cd2+ can induce oxidative stress as indicated by the changes in lipid peroxidation and antioxidant status. For P. akamusi, an increase in the dose that the threshold needed for defense (namely, MDA level and GPx activity) activation was achieved. From this, organisms can be hypothesized to enable cells to avoid oxidant stress up to a certain extent where damage is again measurable (higher Cd2+ concentration).     

The effect of one year's swimming exercise on oxidant stress and antioxidant capacity in aged rats

The effect of exercise on oxidant stress and on alterations in antioxidant defense in elderly has been investigated extensively. However, the impact of regularly performed long-term physical activity starting from adulthood and prolonged up to the old age is not yet clear. We have investigated the changes in the activities of antioxidant enzymes - superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) - and lipid peroxidation in various tissues of rats which had performed (old-trained) or had not performed (old-control) regular swimming exercise for one year. These animals were compared with young-sedentary rats. Increased lipid peroxidation was observed with ageing in all tissues (heart, liver, kidney, striated muscle) and swimming had no additional effect on this elevation of lipid peroxidation. Heart and striated muscle SOD activites, and striated muscle CAT activity increased as a consequence of ageing, whereas kidney and liver CAT activities, as well as GPx activities in kidney, liver, lung and heart were significantly decreased compared to young controls. Lung and heart SOD, liver CAT activities as well as GPx activities in liver, lung and heart were increased significantly in rats which performed exercise during ageing, compared to the old-control group. These findings suggest that lifelong exercise can improve the antioxidant defense in many tissues without constituting any additional oxidant stress.     

Role of Glutathione Peroxidase 4 in Glutamate-Induced Oxytosis in the Retina

Purpose

The purpose of the present study was to investigate the role of glutathione peroxidase 4 (GPx4) in glutamate-induced oxytosis in the retina.

Methods

For in vitro studies, an immortalized rat retinal precursor cell line R28 was used. Cells were transfected with siRNA specifically silencing GPx4 or with scrambled control siRNA. Lipid peroxidation was evaluated by 4-hydroxy-2-nonenal (4-HNE) immunostaining. Cytotoxicity and cell death were evaluated using an LDH activity assay and annexin V staining, respectively. Cells transfected with GPx4 siRNA or control siRNA were treated with glutamate (1 or 2 mM), and the cytotoxicity was evaluated using the LDH activity assay. For in vivo studies, retinal ganglion cell damage was induced by intravitreal injection of 25-mM N-methyl-D-aspartate (NMDA, 2 μL/eye) in GPx4^+/+ and GPx4^+/− mice. The evaluation of lipid peroxidation (4-HNE immunostaining), apoptosis (TUNEL staining), and cell density in the ganglion cell layer (GCL) were performed at 12 h, 1 day, and 7 days after the NMDA injection.

Results

GPx4 knockdown significantly increased LDH activity by 13.9-fold (P < 0.01) and increased peroxidized lipid levels by 3.2-fold in R28 cells (P < 0.01). In cells transfected with scrambled control siRNA, treatment with glutamate at 1 or 2 mM did not increase LDH activity; whereas, in cells transfected with GPx4 siRNA, glutamate treatment significantly increased LDH activity (1.52-fold, P < 0.01). GPx4^+/− mice exhibited higher levels of lipid peroxidation in retinas treated with NMDA than GPx4^+/+ mice (1.26-fold, P < 0.05). GPx4^+/− mice had more TUNEL-positive cells induced by NMDA in GCL (1.45-fold, P < 0.05). In addition, the cell density in GCL of GPx4^+/− mice was 19% lower than that in GPx4^+/+ mice after treatment with NMDA (P < 0.05).

Conclusion

These results suggest that defective GPx4 expression is associated with enhanced cytotoxicity by glutamate-induced oxytosis in the retina.