Hypoxia and recovery perturb free radical processes and antioxidant potential in common carp (Cyprinus carpio) tissues

The effects of hypoxia exposure and subsequent normoxic recovery on the levels of lipid peroxides (LOOH), thiobarbituric acid reactive substances (TBARS), carbonylproteins, total glutathione levels, and the activities of six antioxidant enzymes were measured in brain, liver, kidney and skeletal muscle of the common carp Cyprinus carpio. Hypoxia exposure (25% of normal oxygen level) for 5h generally decreased the levels of oxidative damage products, but in liver TBARS content were elevated. Hypoxia stimulated increases in the activities of catalase (by 1.7-fold) and glutathione peroxidase (GPx) (by 1.3-fold) in brain supporting the idea that anticipatory preparation takes place in order to deal with the oxidative stress that will occur during reoxygenation. In liver, only GPx activity was reduced under hypoxia and reoxygenation while other enzymes were unaffected. Kidney showed decreased activity of GPx under aerobic recovery but superoxide dismutase (SOD) and catalase responded with sharp increases in activities. Skeletal muscle showed minor changes with a reduction in GPx activity under hypoxia exposure and an increase in SOD activity under recovery. Responses by antioxidant defenses in carp organs appear to include preparatory increases during hypoxia by some antioxidant enzymes in brain but a more direct response to oxidative insult during recovery appears to trigger enzyme responses in kidney and skeletal muscle.     

Oxidative stress and antioxidant capacity of a terrestrially hibernating hatchling turtle

Hatchlings of the painted turtle, Chrysemys picta, hibernate terrestrially and can survive subfreezing temperatures by supercooling or by tolerating the freezing of their tissues. Whether supercooled or frozen, an ischemic hypoxia develops because tissue perfusion is limited by low temperature and/or freezing. Oxidative stress can occur if hatchlings lack sufficient antioxidant defenses to minimize or prevent damage by reactive oxygen species. We examined the antioxidant capacity and indices of oxidative damage in hatchling C. picta following survivable, 48 h bouts of supercooling (−6°C), freezing (−2.5°C), or hypoxia (4°C). Samples of plasma, brain, and liver were collected after a 24 h period of recovery (4°C) and assayed for Trolox-equivalent antioxidant capacity (TEAC), thiobarbituric acid reactive substances (TBARS), and carbonyl proteins. Antioxidant capacity did not vary among treatments in any of the tissues studied. We found a significant increase in TBARS in plasma, but not in the brain or liver, of frozen/thawed hatchlings as compared to untreated controls. No changes were found in the concentration of TBARS or carbonyl proteins in supercooled or hypoxia-exposed hatchlings. Our results suggest that hatchling C. picta have a well-developed antioxidant defense system that minimizes oxidative damage during hibernation.     

Supercooling and freeze-tolerance in the European wall lizard,Podarcis muralis,with a revisional history of the discovery of freeze-tolerance in vertebrates

Summary Wall lizards were collected in the fall of 1988 from a population introduced in 1951 into Cincinnati, OH. They were acclimated to 5 °C for several weeks prior to testing at sub-zero temperatures. Eleven super-cooled lizards were removed from the cooling chamber prior to crystallization after between 15 min and 26 h at body temperatures ranging from -2.2 to -5.9 °C. With the exception of one individual supercooled to-5.0 °C, all lizards recovered fully. The crystallization temperatures of 15 lizards which froze ranged from -0.6 to -6.4 °C. Frozen lizards were stiff with a distinct blue color, which faded upon thawing at 3 °C. The ice contents of frozen lizards were determined calorimetrically and/or estimated from a theoretical model, the two methods being generally in close agreement. Remarkably, five individuals recovered fully from exposures as long as 2 h and with as much as 28% of their body water frozen. Although these animals are not as tolerant as certain other vertebrates they are clearly able to withstand freezing under some circumstances. Failure to survive freezing was attributed either to excessive ice accumulation during a prolonged freeze or to excessive supercooling prior to freezing, which induced a large initial surge of ice formation upon crystallization. Our results accord with those of Weigmann (1929). We accordingly recognize him as the first to demonstrate freeze-tolerance in vertebrates, and we further recognize P. muralis as the first vertebrate known to survive freezing.     

Characterization of sarcolemma and sarcoplasmic reticulum isolated from skeletal muscle of the freeze tolerant wood frog, Rana sylvatica: the beta(2)-adrenergic receptor and calcium transport systems in control, frozen and thawed states

In freeze tolerant wood frog Rana sylvatica, the freeze-induced liberation of glucose plays a critical role in survival in response to sub-zero temperature exposure. We have shown that the glycaemic response is linked to selective changes in the expression of hepatic adrenergic receptors through which catecholamines act to produce their hepatic glycogenolytic effects. The purpose of the present study was to determine if skeletal muscle, another catecholamine-sensitive tissue with glycogenolytic potential, displayed similar or different changes. In order to achieve these objectives, skeletal muscle derived from Rana sylvatica was studied in control, frozen and thawed states. In isolated sarcolemmal fractions, freezing effected an 88% decrease in beta(2)-adrenergic receptor expression but was without effect on the calcium pump; while thawing resulted in a recovery of the beta(2)-adrenergic receptor to 60% of control levels and a 2.4-fold increase in calcium transport. In isolated sarcoplasmic reticular fractions, freezing effected a 52% decrease in calcium binding and a 92% decrease in oxalate-stimulated calcium uptake; while thawing elicited partial normalization to control levels to 70% with respect to calcium binding and to 47% with respect to calcium uptake. Freezing and thawing were associated with increases and decreases, receptively, in blood glucose levels but were without effect on skeletal muscle glycogen content. Thus these muscle changes in Rana sylvatica in freezing and thawing are not linked to glycogen breakdown, are different from those previously seen in liver, and may provide a role in recovery of muscle function during thawing by protecting glycogen stores for contraction and maximizing extracellular calcium for excitation-contraction coupling in the frozen state. The involvement of thyroid hormone in triggering these muscle changes is discussed.     

Freeze-thaw effects on metabolic enzymes in wood frog organs.

To determine whether episodes of natural freezing and thawing altered the metabolic makeup of wood frog (Rana sylvatica) organs, the maximal activities of 28 enzymes of intermediary metabolism were assessed in six organs (brain, heart, kidney, liver, skeletal muscle, gut) of control (5 degrees C acclimated), frozen (24 h at -3 degrees C), and thawed (24 h back at 5 degrees C) frogs. The enzymes assessed represented pathways including glycolysis, gluconeo-genesis, amino acid metabolism, fatty acid metabolism, the TCA cycle, and adenylate metabolism. Organ-specific responses seen included (a) the number of enzymes affected by freeze-thaw (1 in gut ranging to 17 in heart), (b) the magnitude and direction of response (most often enzyme activities decreased during freezing and rebounded with thawing but, liver showed freeze-specific increases in several enzymes), and (c) the response to freezing versus thawing (enzyme activities in gut and kidney changed during freezing, whereas most enzymes in skeletal muscle responded to thawing). Overall, the data show that freeze-thaw implements selected changes to the maximal activities of various enzymes of intermediary metabolism and that these may aid organ-specific responses that alter fuel use during freeze-thaw, support cryoprotectant metabolism, and aid organ endurance of freeze-induced ischemia.     

L-malate reverses oxidative stress and antioxidative defenses in liver and heart of aged rats

The intracellular levels of antioxidant and free radical scavenging enzymes are gradually altered during the aging process. An age-dependent increase of oxidative stress occurring throughout the lifetime is hypothesized to be the major cause of aging. The current study examined the effects of L-malate on oxidative stress and antioxidative defenses in the liver and heart of aged rats. Sprague-Dawley male rats were randomly divided into four groups, each group consisting of 6 animals. Group Ia and Group IIa were young and aged control rats. Group Ib and Group IIb were young and aged rats treated with L-malate (210 mg/kg body weight per day). L-malate was orally administrated via intragastric canula for 30 days, then the rats were sacrificed and the liver and heart were removed to determine the oxidant production, lipid peroxidation and antioxidative defenses of young and aged rats. Dietary L-malate reduced the accumulation of reactive oxygen species (ROS) and significantly decreased the level of lipid peroxidation in the liver and heart of the aged rats. Accordingly, L-malate was found to enhance the antioxidative defense system with an increased activity of antioxidant enzymes, such as superoxide dismutase (SOD) and glutathione peroxidase (GPx) and increased glutathione (GSH) levels in the liver of aged rats, a phenomenon not observed in the heart of aged rats. Our data indicate that oxidative stress was reversed and the antioxidative defense system was strengthened by dietary supplementation with L-malate.     

Alterations in antioxidant enzymes and oxidative damage in experimental diabetic rat tissues: Effect of vanadate and fenugreek (Trigonella foenum graecum)

With the premise that oxygen free radicals may be responsible for the severity and complications of diabetes, the level of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) as well as the oxidative damage were examined in the tissues of control, diabetic and treated rats. After three weeks of diabetes, the activity of CAT was significantly increased in heart in diabetes (about 6-fold) but decreased in liver. The SOD activity decreased significantly in liver but increased in brain. The activity of GPx decreased significantly in liver and increased in kidney. A significant increase was observed in oxidative damage in heart and kidney and a small increase in brain with decrease in liver and muscle. Vanadate and fenugreek (Trigonella foenum graecum) administration to diabetic animals showed a reversal of the disturbed antioxidant levels and peroxidative damage. Results suggest that oxidative stress play a key role in the complications of diabetes. Vanadate and fenugreek seeds showed an encouraging antioxidant property and can be valuable candidates in the treatment of the reversal of the complications of diabetes.     

Effects of low selenium diets on antioxidant status and MPTP toxicity in mice

To investigate the role of chronic oxidative stress in MPTP neurotoxicity, C57BL mice were maintained 6–8 weeks on diets deficient in nutrients essential to cellular antioxidant defenses, selenium (Se) and alpha-tocopherol (vit E), and the effects on tissue antioxidant status and MPTP toxicity were evaluated relative to controls on supplemented diets. Activities of the major antioxidant enzymes, glutathione peroxidase (GPx), catalase, and superoxide dismutase, and levels of malondialdehyde as a marker for oxidative stress, were measured in brain, lung, liver and blood. Caudate depletion of dopamine and its metabolites served as a measure of MPTP neurotoxicity. For mice on the Se deficient diet, levels of the selenoenzyme GPx decreased from 50% in brain to 90% in blood. No compensatory changes in the activities of the other antioxidant enzymes were observed and addition of vit E to the diet did not alter antioxidant enzyme activities or malondialdehyde levels. In animals not treated with MPTP, the Se deficient diet significantly increased malondialdehyde only in liver. No protective effect of the antioxidant supplements against caudate depletion of dopamine and its metabolites was observed. However, malondialdehyde levels were increased in the brains of MPTP treated mice on the low Se diets, suggesting the possibility of secondary oxidative damage to tissues accompanying the destruction of substantia nigra neurons by MPTP.     

Glutathione status and antioxidant enzymes in a crocodilian species from the swamps of the Brazilian Pantanal

In a previous study oxidative damage markers - lipid peroxidation and protein oxidation - were determined in organs of wild Caiman yacare captured in winter-2001 and summer-2002 at various developmental stages. An increase in oxidative damage occurred in the hatchling-juvenile transition (but not in the juvenile-adult transition) and winter-summer transition (in juveniles), suggesting that oxidative stress is associated with development and season. Herein the effect of development and season on glutathione (GSH) metabolism and the effect of development on the activity of antioxidant enzymes (catalase, glutathione peroxidase, glutathione reductase and glutathione S-transferase) and glucose 6-phosphate dehydrogenase were analyzed. The ratio GSSG:GSH-eq increased in lung, liver, kidney and brain by 1.8- to 4-fold in the embryo/hatchling to juvenile transition. No changes occurred in juvenile-adult transition. GSSG:GSH-eq across seasons was significantly elevated in summer. Total-glutathione content was mostly stable in various organs; in liver it increased in the embryo-juvenile transition. Enzyme activities were only determined in summer-animals (embryos, hatchlings and juveniles). For most antioxidant enzymes, activities increased from embryo/hatchling to juvenile in liver and Kidney. In lung, there was an inverse trend for enzyme activities and total glutathione content. Thus, increased metabolic rates during early caiman growth - in embryo-juvenile transition - appears to be related to redox imbalance as suggested by increased GSSG:GSH-eq and activation of antioxidant defenses. Differences in oxidative stress across seasons were related with summer-winter nocturnal temperatures. These results, as a whole, were interpreted in the context of ecological biochemistry.     

Deep Undercooling in Woody Taxa Growing North of the -40 degrees C Isotherm

The freezing of deep undercooled water in cold-hardened 3-year-old stems of 16 woody taxa was studied in mid-January by differential thermal analysis. The initiation temperature and the size of the low temperature exotherm (LTE) were compared for nonthawed, thawed, and freeze-killed stems. In general, the initiation temperature of the LTE for nonthawed stems occurred at a lower temperature than for thawed stems and freeze-killed stems. In some cases, no LTE was detected in nonthawed stems although a LTE was detected after thawing. The size of the LTE increased after thawing the stem and also after the stem was freeze killed. The LTE observed in one species disappeared upon exposure to continuous low sub-zero temperatures. Results suggest that undercooling which subsequently results in the LTE in woody stems is due to the cell wall and the plasma membrane. During periods of prolonged freezing, cellular water migrates from the cells which undercool to extracellular ice. This results in a concentration of cell solutes which lowers the homogeneous nucleation temperature of the cell sap. The cold hardiness of nonthawed and thawed stems was compared by a controlled freeze test. In general, thawing had little effect on the survival temperature whereas it had a marked effect on the initiation of the LTE.     

Effect of freezing-thawing rates on the functional state and ionic permeability of rat liver mitochondria

The effects of various rats of freezing-thawing reactions on the functional state and ionic permeability of rat liver mitochondria were studied. The degree of mitochondrial damage during the freezing -- thawing process depended on the rate of thawing rather than on that of freezing. The mitochondria which were slowly or rapidly frozen down to --196 degrees and subsequently slowly thawed revealed a higher membrane permeability for K+ Na+ and H+ and a more than 2-fold increase of the ATPase activity and the maximal rate of NADH oxidation via the antimycin-insensitive pathway in the presence of cytochrome c. This was concomitant with a complete inhibition of the ATP-synthetase activity and a marked inhibition of the respiratory chain function due to the efflux of cytochrome c from the inner mitochondrial membrane. After freezing and rapid thawing the functional activity of mitochondria changed insignificantly. A comparison of different cryoeffects demonstrated that the minimal damaging effects were exerted by rapid freezing -- rapid thawing, when the mitochondria partly restored their ability for oxidative phosphorylation.     

The effects of selenium on oxidative stress biomarkers in the freshwater characid fish matrinxã, Brycon cephalus (Günther, 1869) exposed to organophosphate insecticide Folisuper 600 BR (methyl parathion)

Methyl parathion (MP), an organophosphate widely applied in agriculture and aquaculture, induces oxidative stress due to free radical generation and changes in the antioxidant defense system. The antioxidant roles of selenium (Se) were evaluated in Brycon cephalus exposed to 2 mg L(-1) of Folisuper 600 BR (MP commercial formulation - MPc, 600 g L(-1)) for 96 h. Catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), glutathione S-transferase (GST), reduced glutathione (GSH) and lipid peroxidation (LPO) levels in the gills, white muscle and liver were evaluated in fish fed on diets containing 0 or 1.5 mg Se kg(-1) for 8 weeks. In fish treated with a Se-free diet, the MPc exposure increased SOD and CAT activities in all tissues. However, the GPx activity decreased in white muscle and gills whereas no alterations were observed in the liver. MPc also increased GST activity in all tissues with a concurrent decrease in GSH levels. LPO values increased in white muscle and gills and did not change in liver after MPc exposure. A Se-supplemented diet reversed these findings, preventing increases in LPO levels and concurrent decreases in GPx activity in gills and white muscle. Similarly, GSH levels were maintained in all tissue after MPc exposure. These results suggest that dietary Se supplementation protects cells against MPc-induced oxidative stress.     

Sodium selenite enhances glutathione peroxidase activity and DNA strand breaks in hepatoma induced by N-nitrosodiethylamine and promoted by phenobarbital

An element/compound that acts as an antioxidant as well as, can increase the oxidative stress offers a new approach in differentiation therapy. Experiments were carried out to determine the effect of selenite on DNA damage and glutathione peroxidase (GPx) activity in N-nitrosodiethylamine (DEN) induced, phenobarbital promoted rat hepatoma. Supra-nutritional level of selenite (4 ppm) was supplemented at either, before-initiation/after-initiation and/or during entire period of the study. At the end of experiment period (20 weeks), extent of DNA damage (alkaline comet assay), selenium concentration, and GPx activity were assessed on nodular tissue (NL) cells, surrounding liver (SL) cells, and whole liver tissue (control) cells. Hepatic selenium level and GPx activity were decreased in DEN and PB-administered animals, whereas the DNA damage was found to be increased in both NL and SL cells compared with control group. However, the DNA damage is more in SL cells than in NL cells. Pre-supplementation of selenite did not show any difference in DNA (strand breaks) damage, selenium, and GPx activity. Increased hepatic selenium concentration and GPx activity were observed in both NL and SL cells in post-supplementation and entire period of selenite supplemented animals compared to DEN + PB treated animals. However, DNA damage was increased in NL but decreased in SL cells. Supplementation of selenite alone for 16 or 20 weeks had shown increased DNA damage, selenium concentration, and GPx activity compared to normal control animals. In summary, cancer bearing animals increased DNA damage and decreased Se level and GPx activity in NL and SL cells and other organs in cancer bearing animals, supplementation of Se further provoked DNA damage (no change in pretreatment) in NL cells, however it decreased DNA damage SL cells and other organs (kidney, lungs, and spleen). On the other hand Se levels and GPx activity were increased in NL and SL cells and other organs of Se-supplemented rats (no difference in group 3 animals). These results demonstrate that, in addition to chemopreventive and chemotherapeutic role of selenite, it also prevents cellular DNA damage induced in cancerous condition.     

Oxidative damage during chagasic cardiomyopathy development: role of mitochondrial oxidant release and inefficient antioxidant defense

In this study, we evaluated the oxidant status and antioxidant defense capabilities of the heart during the course of Trypanosoma cruzi infection and disease development in a murine model system. Our data show that the extent of protein carbonylation and lipid peroxidation is increased in the heart, but not the skeletal muscle, of infected mice. The level of oxidative injury biomarkers in the myocardium consistently increased with chronic disease severity. The antioxidant defense constituted by catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GSR), and reduced glutathione was increased in murine heart and skeletal tissue in response to the stress of T. cruzi infection. After the initial burst, CAT, GPx, and GSR remained unresponsive to the severity of chronic tissue damage in chagasic hearts. The cardiac level of Mn(2+) superoxide dismutase (MnSOD) was diminished in chagasic mice. Our data suggest that the host responds to acute injuries by activating antioxidant defenses that are of sufficient magnitude to scavenge the reactive oxidants in skeletal tissue. The myocardia of infected mice, however, sustain increased oxidative injuries with disease progression. We surmise that MnSOD deficiencies, resulting in the increased release of mitochondrial free radicals, lead to sustained oxidative stress that exceeds the cardiac antioxidant defense capacity and contribute to persistent oxidative damage in chagasic myocardium.     

Stress-induced activation of the AMP-activated protein kinase in the freeze-tolerant frog Rana sylvatica

Survival in the frozen state depends on biochemical adaptations that deal with multiple stresses on cells including long-term ischaemia and tissue dehydration. We investigated whether the AMP-activated protein kinase (AMPK) could play a regulatory role in the metabolic re-sculpting that occurs during freezing. AMPK activity and the phosphorylation state of translation factors were measured in liver and skeletal muscle of wood frogs (Rana sylvatica) subjected to anoxia, dehydration, freezing, and thawing after freezing. AMPK activity was increased 2-fold in livers of frozen frogs compared with the controls whereas in skeletal muscle, AMPK activity increased 2.5-, 4.5- and 3-fold in dehydrated, frozen and frozen/thawed animals, respectively. Immunoblotting with phospho-specific antibodies revealed an increase in the phosphorylation state of eukaryotic elongation factor-2 at the inactivating Thr56 site in livers from frozen frogs and in skeletal muscles of anoxic frogs. No change in phosphorylation state of eukaryotic initiation factor-2alpha at the inactivating Ser51 site was seen in the tissues under any of the stress conditions. Surprisingly, ribosomal protein S6 phosphorylation was increased 2-fold in livers from frozen frogs and 10-fold in skeletal muscle from frozen/thawed animals. However, no change in translation capacity was detected in cell-free translation assays with skeletal muscle extracts under any of the experimental conditions. The changes in phosphorylation state of translation factors are discussed in relation to the control of protein synthesis and stress-induced AMPK activation.     

Burn-induced oxidative stress is altered by a low zinc status: kinetic study in burned rats fed a low zinc diet

As an initial subdeficient status of zinc, considered as an essential antioxidant trace element, is frequent in burned patients, we aim to assess the effects of low zinc dietary intakes on burn-induced oxidative stress, in an animal model. After 8 weeks of conditioning diets containing 80 ppm (control group) or 10 ppm of zinc (depleted group), Wistar rats were 20% TBSA burned and sampled 1-10 days after injury. Kinetic evolutions of zinc status, plasma oxidative stress parameters, and antioxidant enzymes were also studied in blood and organs. The zinc-depleted diet induced, before injury, a significant decrease in zinc bone level and the increase of oxidative stress markers without stimulation of antioxidant enzyme activity. After burn, more markedly in zinc depleted animals than in controls, zinc levels decreased in plasma and bone, while increasing in liver. The decrease of thiol groups and GSH/GSSG ratio and the depression of GPx activity in liver are also moderately emphasized. Nevertheless, depleted zinc status could not be considered as determining for oxidative damages after burn injury. Further investigations must also be done to enlighten the mechanism of beneficial effects of zinc supplementation reported in burned patients.     

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.