Effects of intermittent hypoxia different regimes on mitochondrial lipid peroxidation and glutathione-redox balance in stressed rats

The purpose of this study was to compare the influence of two regimes of intermittent hypoxia (IH) [repetitive 5 cycles of 5 min hypoxia (7% O₂ or 12% O₂ in N₂) followed by 15 min normoxia, daily for three weeks] on oxidative stress protective systems in liver mitochondria. To estimate the effectiveness of hypoxia adaptation at the early and late preconditioning period, we exposed rats to acute 6-h immobilization at the 1^st and 45^th days after cessation of IH. We showed that severity of hypoxic episodes during IH might initiate different adaptive programs. Moderate hypoxia during IH prevents mitochondrial glutathione pool depletion induced by immobilization stress, maintains GSH-redox cycle via activation of glutathione peroxidase, glutathione-S-transferase, glutathione reductase, NADP⁺-dependent isocitrate dehydrogenase, and increases Mn-SOD activity. Such regimen of hypoxic preconditioning caused the decrease of mitochondrial superoxide anion generation as well as of basal and stimulated in vitro lipid peroxidation and this protective effect remained for 45 days under renormoxic conditions. Hypoxic adaptation in a more severe regimen exerted beneficial effects on the mitochondrial antioxidant defense system only at its later phase.     

Antioxidant effect of caeruloplasmin on microsomal lipid peroxidation

Microsomal NADPH-dependent lipid peroxidation catalyzed by ADP-Fe³⁺ was inhibited by the addition of caeruloplasmin. The antioxidant effect of caeruloplasmin was independent of the superoxide anion (O^?₂ scavenging activity. Since caeruloplasmin enhanced the function of ADP-Fe³⁺ acting as electron acceptor for microsomal electron transport system, the antioxidant effect of caeruloplasmin is considered to depend on the ferroxidase activity.     

Effect of cryopreservation on sperm parameters, lipid peroxidation and antioxidant enzymes activity in fowl semen

The aim of the present study was to determine the influence of chicken semen cryopreservation on sperm parameters, lipid peroxidation and antioxidant enzymes activities. Pooled semen from 10 Black Minorca roosters was used in the study. Semen samples were subjected to cryopreservation using the “pellet” method and dimethylacetamide (DMA) as a cryoprotectant. In the fresh and the frozen-thawed semen sperm membrane integrity (SYBR-14/propidium iodide (PI)), acrosomal damage (PNA-Alexa Fluor^®488) and mitochondrial activity (Rhodamine 123) were assessed using flow cytometry. Malondialdehyde (MDA) concentration, catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were determined in sperm cells and seminal plasma by spectrophotometry. All sperm characteristics evaluated using flow cytometry were affected by cryopreservation. After freezing-thawing, there was significant (P < 0.01) reduction in sperm membrane integrity, sperm acrosome integrity and mitochondrial activity. Following cryopreservation, MDA concentration significantly increased in chicken seminal plasma and spermatozoa (P < 0.01, P < 0.05). The CAT activity in seminal plasma significantly decreased (P < 0.05), while intracellular activity of this enzyme did not significantly change in frozen-thawed semen. In seminal plasma of frozen-thawed semen the significant increase (P < 0.01) in GPx activity was detected. Whereas GPx activity in spermatozoa remained statistically unchanged after thawing. The SOD activity significantly increased (P < 0.01) in cryopreserved seminal plasma with simultaneous decrease (P < 0.01) of its activity in cells. In conclusion, this is probably the first report describing the level of antioxidant enzymes in frozen-thawed avian semen. The present study showed that the activity of CAT, GPx and SOD in chicken semen was affected by cryopreservation, what increased the intensity of lipid peroxidation (LPO). Catalase appeared to play an important role in the sperm antioxidant defense strategy at cryopreservation since, opposite to SOD and GPx, its content was clearly reduced by the cryopreservation process. Change in the antioxidant defense status of the chicken spermatozoa and surrounding seminal plasma might affect the semen quality and sperm fertilizing ability.     

Exercise training protects against contraction-induced lipid peroxidation in the diaphragm

Endurance exercise training promotes a small but significant increase in antioxidant enzyme activity in the costal diaphragm (DIA) of rodents. It is unclear if these training-induced improvements in muscle antioxidant capacity are large enough to reduce oxidative stress during prolonged contractile activity. To test the hypothesis that training-related increases in DIA antioxidant capacity reduces contraction-induced lipid peroxidation, we exercise trained adult female Sprague-Dawley (n = 7) rats on a motor-driven treadmill for 12 weeks at approximately 75% maximal O2 consumption (90 min/day). Control animals (n = 8) remained sedentary during the same 12-week period. After training, DIA strips from animals in both experimental groups were excised and subjected to an in vitro fatigue contractile protocol in which the muscle was stimulated for 60 min at a frequency of 30 Hz, every 2 s, with a train duration of 330 m. Compared to the controls, endurance training resulted in an increase (P < 0.05) in diaphragmatic non-protein thiols and in the activity of the antioxidant enzyme superoxide dismutase. Following the contractile protocol, lipid peroxidation was significantly lower (P < 0.05) in the trained DIA compared to the controls. These data support the hypothesis that endurance exercise training-induced increases in DIA antioxidant capacity protect the muscle against contractile-related oxidative stress.     

Enhancement of lipid peroxidation in rat liver on acute exposure to styrene and acrylamide a consequence of glutathione depletion

Lipid peroxidation, glutathione level and activity of glutathione-S-transferase were studied in liver and brain of rats 4 and 3 h after a single i.p. administration of 0, 25, 75, 100 mg/kg acrylamide or 0, 50, 100, 200, 600 mg/kg styrene, respectively. In liver both acrylamide and styrene caused an increase in lipid peroxidation and decrease in glutathione contents and activity of glutathione-S-transferase in a dose dependent manner, while in brain only acrylamide produced a decrease in glutathione content. The decrease in glutathione content was not always associated with increase of lipid peroxidation. The enhancement of lipid peroxidation occurred only when glutathione contents were depleted to certain critical levels. No effect of acrylamide or styrene was seen on lipid peroxidation under in vitro conditions. The addition of glutathione in the incubation mixture significantly inhibited the rate of lipid peroxidation of liver homogenates of acrylamide and styrene treated animals.The results suggest that enhancement of lipid peroxidation in liver on exposure to acrylamide or styrene is a consequence of depletion of glutathione to certain critical levels. The inhibition of glutathione-S-transferase activity by acrylamide and styrene suggests that detoxication of these neurotoxic compounds could be suppressed following acute exposure.     

Cold-stress-induced modulation of antioxidant defence: role of stressed conditions in tissue injury followed by protein oxidation and lipid peroxidation

The aim of this study was to determine the effects of cold stress on antioxidant enzyme activities and examine protein oxidation and lipid peroxidation in various tissues (brain, liver, kidney, heart and stomach). Twenty male Wistar rats (3 months old) weighing 220 ± 20 g were used. The rats were randomly divided into two groups of ten: the control group and the cold stress group. Cold stress was applied to the animals by maintaining them in a cold room (5 °C) for 15 min/day for 15 days. Blood samples were taken for measuring plasma corticosterone levels. Tissues were obtained from each rat for measuring the antioxidant enzyme activities, protein oxidation and lipid peroxidation. Corticosterone levels were increased in the cold stress group. Copper, zinc superoxide dismutase activities were increased in the brains, livers and kidneys, whereas they decreased in the hearts and stomachs of rats in the cold stress group. Catalase activities were increased in the brains, livers, kidneys and hearts, whereas they decreased in the stomachs of rats in the cold stress group. Selenium-dependent glutathione peroxidase activities were increased in the brain, liver, heart and stomach. Reduced glutathione levels were decreased, while levels of protein carbonyl, conjugated diene and thiobarbituric-acid-reactive substances were increased in all tissues of the cold stress group. These results lead us to conclude that cold stress can disrupt the balance in an oxidant/antioxidant system and cause oxidative damage to several tissues by altering the enzymatic and non-enzymatic antioxidant status, protein oxidation and lipid peroxidation.     

Antioxidant status and lipid peroxidation in type II diabetes mellitus

Diabetes Mellitus (DM), a state of chronic hyperglycaemia, is a common disease affecting over 124 million individuals worldwide. In this study, erythrocyte glutathione levels, lipid peroxidation, superoxide dismutase, catalase, and glutathione peroxidase and some extracellular antioxidant protein levels of patients with type II diabetes mellitus and healthy controls were investigated. Thirty-eight patients (21 males; with age of mean +/- SD, 53.1+/-9.7 years) and 18 clinically healthy subjects (10 males; with age of mean +/- SD, 49.3+/-15.2 years) were included in the study. Levels of erythrocyte lipid peroxidation, serum ceruloplasmin and glucose levels, HbA1C levels, and erythrocyte catalase activity were significantly increased, whereas serum albumin and transferrin levels, erythrocyte glutathione levels, and glutathione peroxidase activity were significantly decreased compared to those of controls. There was no significant difference in superoxide dismutase activity compared to controls. The results suggest that the antioxidant deficiency and excessive peroxide-mediated damage may appear in non-insulin dependent diabetes mellitus.     

Cellular response to bioactive lipid peroxidation products

Reactive aldehydes, such as 4-hydroxy-2-nonenal, have been implicated as inducers in generating intracellular reactive oxygen species and activation of stress signaling pathways, that integrate with other signaling pathways to control cellular responses to the extracellular stimuli. Here, I briefly summarize a novel signaling pathway in cellular response, in which aldehyde-stimulated detoxification response is mediated by cyclooxygenase metabolites. These findings argue that lipid mediators could induce a cellular process that represents a cellular defense program against toxic compounds.     

Normobaric hyperoxic stress in budgerigars: enzymic antioxidants and lipid peroxidation

The effects of acute (3 h), repeated acute (3 exposures each of 3 h) and chronic (72 h) normobaric hyperoxic exposure in budgerigars (Melopsittacus undulatus) were evaluated by monitoring the effects on pulmonary enzymic antioxidants, and indicators of lipid peroxidation. All durations of oxygen exposure resulted in significant respiratory alkalosis and elevated pulmonary and blood glutathione peroxidase concentrations. The concentrations of other pulmonary enzymic antioxidants including glutathione reductase and superoxide dismutase were not significantly altered by oxygen exposure. Pulmonary concentrations of the lipid peroxidation markers malonaldehyde and 4-hydroxyalkenal were not significantly elevated following oxygen exposure. Plasma concentrations of 8-epi isoprostane F(2alpha) were significantly elevated following both acute and repeated acute exposure. The results indicate that in budgerigars, both acute and chronic oxygen exposure can result in significant alteration in respiratory function and increased production of reactive oxygen species.     

Dapsone induces oxidative stress and impairs antioxidant defenses in rat liver

Dapsone (DDS) is currently used in the treatment of leprosy, malaria and in infections with Pneumocystis jirovecii and Toxoplasma gondii in AIDS patients. Adverse effects of DDS involve methemoglobinemia and hemolysis and, to a lower extent, liver damage, though the mechanism is poorly characterized. We evaluated the effect of DDS administration to male and female rats (30 mg/kg body wt, twice a day, for 4 days) on liver oxidative stress through assessment of biliary output and liver content of reduced (GSH) and oxidized (GSSG) glutathione, lipid peroxidation, and expression/activities of the main antioxidant enzymes glutathione peroxidase, superoxide dismutase, catalase and glutathione S-transferase. The influence of DDS treatment on expression/activity of the main DDS phase-II-metabolizing system, UDP-glucuronosyltransferase (UGT), was additionally evaluated. The involvement of dapsone hydroxylamine (DDS-NHOH) generation in these processes was estimated by comparing the data in male and female rats since N-hydroxylation of DDS mainly occurs in males. Our studies revealed an increase in the GSSG/GSH biliary output ratio, a sensitive indicator of oxidative stress, and in lipid peroxidation, in male but not in female rats treated with DDS. The activity of all antioxidant enzymes was significantly impaired by DDS treatment also in male rats, whereas UGT activity was not affected in any sex. Taken together, the evidence indicates that DDS induces oxidative stress in rat liver and that N-hydroxylation of DDS was the likely mediator. Impairment in the activity of enzymatic antioxidant systems, also associated with DDS-NHOH formation, constituted a key aggravating factor.     

Effect of Garlic Supplementation on Erythrocytes Antioxidant Parameters, Lipid Peroxidation, and Atherosclerotic Plaque Formation Process in Oxidized Oil-Fed Rabbits

Effect of garlic supplementation on blood antioxidant status, lipid peroxidation, and coronary plaque formation process was investigated in oxidized oil-fed rabbits. Eighteen adult male mixed European rabbits were given a balanced diet (21 g% protein, 34 g% fat, 45 g% carbohydrate), which contained isocaloristic addition of nonoxidized or oxidized rapeseed oil in the presence and absence of garlic. The experiment lasted 24 weeks. At the beginning and every 6 weeks, rabbits were weighed, and blood was taken. To evaluate the antioxidant status of the rabbits, erythrocytes malondialdehyde (MDA) concentration, total superoxide dismutase (t-SOD), and glutathione peroxidase (GPX) activations were determined. After the experiment was completed, aortas were dissected for histological examinations. Changes in the contents of the above parameters and histological examinations showed that oxidized rapeseed, oil administered to rabbits, caused the development of atherosclerotic changes and disturbed antioxidant status. The addition of garlic in such diets inhibited atherosclerotic changes in the aorta wall, and it is related to the homeostatic activity of antioxidative enzymes and lipid peroxidation.     

Antioxidant properties of resveratrol and piceid on lipid peroxidation in micelles and monolamellar liposomes

The antioxidant activities of trans-resveratrol (trans-3,5,4′-trihydroxystilbene) and trans-piceid (trans-5,4′-dihydroxystilbene-3-O-β-d-glucopyranoside), its more widespread glycosilate derivative, have been compared measuring their inhibitory action on peroxidation of linoleic acid (LA) and the radical scavenging ability towards different free radicals (such as DPPH) and radical initiators. It has been found that the two stilbenes have similar antioxidant capacity, while the comparison with BHT (2,6-di-tert-butyl-4-methylphenol) and -tocopherol (vitamin E, vit. E), taken as reference, points out a slower but prolonged protective action against lipid peroxidation. Furthermore, piceid appears more efficacious than resveratrol as a consequence of the reaction of the latter with its radical form.

The DSC profiles of phosphatidylcholine liposomes of various chain lengths, and EPR measurements of spin labelled liposomes demonstrated that the susceptible hydroxyl group of these compounds are located in the lipid region of the bilayer close to the double bonds of polyunsatured fatty acids, making these stilbenes particularly suitable for the prevention and control of the lipid peroxidation of the membranes.     

Toxicity of 1,2-dibromoethane in isolated hepatocytes: Role of lipid peroxidation

Treatment of isolated hepatocytes with 1,2-dibromoethane (DBE) caused a concentration dependent depletion of cellular glutathione (GSH) content and a parallel increase in the covalent binding of reactive intermediates to cell proteins, as a consequence of the haloalkane activation. The reduction of the hepatocyte GSH content, induced by DBE, stimulated the onset of lipid peroxidation, as measured by malondialdehyde (MDA) accumulation. N-Acetylcysteine (1 mM) was found to partially prevent GSH loss and to inhibit MDA formation, whereas equal concentrations of cysteine and methionine were ineffective on these respects. The stimulation of the peroxidative reactions appeared to be also associated with an increase in the leakage of lactate dehydrogenase (LDH) from the cells, indicative of a severe hepatocyte injury. Antioxidants such as -tocopherol, N,N′-phenyl-phenylenediamine (DPPD) and promethazine, as well as N-acetylcysteine reduced MDA formation to various extents and also protect against LDH release, yet without interfering with the covalent binding of DBE reactive intermediates to hepatocyte proteins. These results suggest the involvement of lipid peroxidation, consequent to GSH depletion, in the pathogenesis of liver cell necrosis due to DBE.     

Glutathione-dependent protection by rat liver microsomal protein against lipid peroxidation

GSH is an important cellular defense against oxidant injury. Its effect in the rat liver microsomal lipid peroxidation system has been examined. Incubation of fresh rat liver microsomes with ascorbic acid and ADP-chelated iron leads to the peroxidation of microsomal lipids (production of thiobarbituric acid-reactive substances and destruction of polyunsaturated fatty acids) following a 2 to 5 min lag. Addition of 0.1 mM GSH to the system lengthened the lag period by 5 to 15 min without affecting the rate or the extent of lipid peroxidation. GSH could not be replaced in prolonging the lag by cysteine, mercaptoethanol, dithiothreitol, propylthiouracil, or GSSG. The GSH effect on the lag was abolished by heating or trypsin digestion of the microsomes, indicating that microsomal protein is required for its expression. Progressively longer lags were observed as the GSH concentration was increased from 0.1 to 5 mM, but there was no evidence of GSH oxidation as a consequence of the protection against lipid peroxidation. GSH protected against heat inactivation of the microsomal protein responsible for the GSH effect. Experiments with an oxygen electrode revealed that the GSH protection did not alter the ratio of O₂ consumed to thiobarbituric acid-reactive substances produced. This implicated free radical scavenging as the mechanism of protection. These results indicate the existence of a GSH-dependent rat liver microsomal protein which scavenges free radical. This protein may be an important defense against free radical injury to the microsomal membrane.     

Inhibition of copper-induced lipid peroxidation by sinapic acid and its derivatives in correlation to their effect on the membrane structural properties

The efficiencies of sinapic acid and its derivatives syringic acid, syringaldehyde, three sinapoyl esters (ethyl, propyl, butyl sinapates), 4-vinylsyringol and sinapine were investigated for prevention of lipid peroxidation in correlation with their interactions with model lipid membrane systems. Significant antioxidant activities of propyl and butyl sinapates were seen by fluorimetric assay in phosphatidylcholine liposomes as model membrane using C11-BODIPY^581/591 lipophilic fluorescent probe. The sinapic acid esters also had the highest impact on membrane structural properties, as observed by differential scanning calorimetry and fluorescence polarisation measurements. The greatest protection of phospholipids from peroxidation by these esters correlated well with their polarity and insertion into the lipid bilayer.     

Weak antioxidant defenses make the heart a target for damage in copper-deficient rats

Copper deficiency causes more salient pathologic changes in the heart than in the liver of rats. Although oxidative stress has been implicated in copper deficiency-induced pathogenesis, little is known about the selective toxicity to the heart. Therefore, we examined the relationship between the severity of copper deficiency-induced oxidative damage and the capacity of antioxidant defense in heart and liver to investigate a possible mechanism for the selective cardiotoxicity. Weanling rats were fed a purified diet deficient in copper (0.4 μg/g diet) or one containing adequate copper (6.0 μg/g diet) for 4 weeks. Copper deficiency induced a 2-fold increase in lipid peroxidation in the heart (thiobarbituric assay) but did not alter peroxidation in the liver. The antioxidant enzymatic activities of superoxide dismutase, catalase, and glutathione peroxidase were, respectively, 3-, 50- and 1.5-fold lower in the heart than in the liver, although these enzymatic activities were depressed in both organs by copper deficiency. In addition, the activity of glutathione reductase was 4 times lower in the heart than in the liver. The data suggest that a weak antioxidant defense system in the heart is responsible for the relatively high degree of oxidative damage in copper-deficient hearts.     

Chemically induced antioxidant-sensitive respiration. Relation to glutathione content and lipid peroxidation in the perfused rat liver

The interrelations between the hepatic chemically induced antioxidant-sensitive respiration and the contents of malondialdehyde (MDA) and of reduced glutathione (GSH), were studied in the isolated hemoglobin-free perfused rat liver. Antioxidant-sensitive respiration was induced by the infusion of agents such as ethanol, iron, xanthine or t-butyl hydroperoxide, or by phenylhydrazine pretreatment in vivo. The development of this respiratory component occurred concomitantly with high levels of MDA in the perfused livers, while those of GSH were diminished.     

Lipid Peroxidation and Antioxidant Enzyme Activities in Vascular and Alzheimer Dementias

It has been reported that oxidative stress may play a role in the pathogenesis of dementia of the Alzheimer type (AD) and the cerebral ischemia which causes vascular dementia (VD). We measured malondialdehyde (MDA) levels and superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) activities in blood samples from patients with AD and VD and in healthy non-demented controls (CTR) which similar ages to the patients, in order to evaluate the degree of oxidative stress in patients with AD and VD. A sample of 150 subjects consisting of 50 patients with AD; 50 patients with VD and 50 CTR, aged from 65 to 85 years on, was analyzed. Most of the changes observed were in SOD activity and MDA levels. Catalase activity were least affected. Significant differences were observed in SOD and GR activity between males and females in CRT and in patients with AD, but not in VD. We have found a decrease in antioxidant enzymes activities (SOD, CAT, GPx and GR) in patients with AD and VD and significant differences were observed between CRT and AD patients for ages from 65 to 74, 75 to 84 and from 85 years to 94 years in SOD activity and MDA levels (P < 0.001). MDA levels increase with age in VD, AD and CTR. No significant variation with respect to sex were detected, but significant variations in MDA levels were detected between CRT and patients with VD and AD (P < 0.001). We conclude that oxidative stress plays an important role in the brain damage for both AD and VD, being observed higher levels of oxidative stress for AD that for VD.     

Antioxidant responses and lipid peroxidation following intranasal 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration in rats: increased susceptibility of olfactory bulb

We evaluated an alternative method to investigate a possible involvement of environmental toxins in the pathology of Parkinson's disease (PD). There is considerable evidence supporting the role of oxidative stress in the toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin largely used to modeling PD in primates and rodents. We have recently demonstrated that rats treated with intranasal (i.n.) infusion of MPTP suffer from progressive signs of PD that are correlated with time-dependent degeneration in dopaminergic neurons. In the present study, we investigated the time-dependent (2 h to 7 days) effect of a single i.n. administration of MPTP (0.1 mg/nostril) on the glutathione-related antioxidant status and lipid peroxidation (TBARS) in the adult Wistar rat brain. The effects were more pronounced in the olfactory bulb at 6 h after i.n. MPTP administration, as indicated by an increase in TBARS and total glutathione (GSH-t) levels, and also in the gamma-glutamyl transpeptidase (GGT) activity. Increased levels of TBARS, GSH-t and GGT activity were also observed at 6 h post-MPTP infusion in some structures (e.g. striatum, hippocampus and prefrontal cortex). No difference regarding glutathione reductase activity was observed in any of the brain structures analyzed, while a marked decrease in glutathione peroxidase activity was specifically observed in the substantia nigra 7 days after MPTP treatment. These results demonstrate that a single i.n. infusion of MPTP in rats induces significant alterations in the brain antioxidant status and lipid peroxidation, reinforcing the notion that the olfactory system represents a particularly sensitive route for the transport of neurotoxins into the central nervous system that may be related to the etiology of PD.