Middle-age abolishes cardioprotection conferred by thioredoxin-1 in mice.

Thioredoxin-1 (Trx1) has cardioprotective effects on ischemia/reperfusion (I/R) injury, although its role in ischemic postconditioning (PostC) in middle-aged mice is not understood. This study aimed to evaluate if combining two cardioprotective strategies, such as Trx1 overexpression and PostC, could exert a synergistic effect in reducing infarct size in middle-aged mice. Young or middle-aged wild-type mice (Wt), transgenic mice overexpressing Trx1, and dominant negative (DN-Trx1) mutant of Trx1 mice were used. Mice hearts were subjected to I/R or PostC protocol. Infarct size, hydrogen peroxide (H2O2) production, protein nitration, Trx1 activity, mitochondrial function, and Trx1, pAkt and pGSK3ß expression were measured. PostC could not reduce infarct size even in the presence of Trx1 overexpression in middle-aged mice. This finding was accompanied by a lack of Akt and GSK3ß phosphorylation, and Trx1 expression (in Wt group). Trx1 activity was diminished and H2O2 production and protein nitration were increased in middle-age. The respiratory control rate dropped after I/R in Wt-Young and PostC restored this value, but not in middle-aged groups. Our results showed that Trx1 plays a key role in the PostC protection mechanism in young but not middle-aged mice, even in the presence of Trx1 overexpression.

Time course of systemic oxidative stress and inflammatory response induced by an acute exposure to Residual Oil Fly Ash.

It is suggested that systemic oxidative stress and inflammation play a central role in the onset and progression of cardiovascular diseases associated with the exposure to particulate matter (PM). The aim of this work was to evaluate the time changes of systemic markers of oxidative stress and inflammation, after an acute exposure to Residual Oil Fly Ash (ROFA). Female Swiss mice were intranasally instilled with a ROFA suspension (1.0mg/kg body weight) or saline solution, and plasma levels of oxidative damage markers [thiobarbituric acid reactive substances (TBARSs) and protein carbonyls], antioxidant status [reduced (GSH) and oxidized (GSSG) glutathione, ascorbic acid levels, and superoxide dismutase (SOD) activity], cytokines levels, and intravascular leukocyte activation were evaluated after 1, 3 or 5h of exposure. Oxidative damage to lipids and decreased GSH/GSSG ratio were observed in ROFA-exposed mice as early as 1h. Afterwards, increased protein oxidation, decreased ascorbic acid content and SOD activity were found in this group at 3h. The onset of an adaptive response was observed at 5h after the ROFA exposure, as indicated by decreased TBARS plasma content and increased SOD activity. The observed increase in oxidative damage to plasma macromolecules, together with systemic antioxidants depletion, may be a consequence of a systemic inflammatory response triggered by the ROFA exposure, since increased TNF-α and IL-6 plasma levels and polymorphonuclear leukocytes activation was found at every evaluated time point. These findings contribute to the understanding of the increase in cardiovascular morbidity and mortality, in association with environmental PM inhalation.

Impaired cardiac mitochondrial function and contractile reserve following an acute exposure to environmental particulate matter.

BACKGROUND: It has been suggested that mitochondrial function plays a central role in cardiovascular diseases associated with particulate matter inhalation. The aim of this study was to evaluate this hypothesis, with focus on cardiac O2 and energetic metabolism, and its impact over cardiac contractility. METHODS: Swiss mice were intranasally instilled with either residual oil fly ash (ROFA) (1.0 mg/kg body weight) or saline solution. After 1, 3 or 5 h of exposure, O2 consumption was evaluated in heart tissue samples. Mitochondrial respiration, respiratory chain complexes activity, membrane potential and ATP content and production rate were assessed in isolated mitochondria. Cardiac contractile reserve was evaluated according to the Langendorff technique. RESULTS: Three hours after ROFA exposure, tissue O2 consumption was significantly decreased by 35% (from 1180 +/- 70 to 760 +/- 60 ng-at O/min g tissue), as well as mitochondrial rest (state 4) and active (state 3) respiration, by 30 and 24%, respectively (control state 4: 88 +/- 5 ng-at O/min mg protein; state 3: 240 +/- 20 ng-at O/min mg protein). These findings were associated with decreased complex II activity, mitochondrial depolarization and deficient ATP production. Even though basal contractility was not modified (control: 75 +/- 5 mm Hg), isolated perfused hearts failed to properly respond to isoproterenol in ROFA-exposed mice. Tissue O2 consumption rates positively correlated with cardiac contractile state in controls (r2 = 0.8271), but not in treated mice (r2 = 0.1396). GENERAL SIGNIFICANCE: The present results show an impaired mitochondrial function associated with deficient cardiac contractility, which could represent an early cardiovascular alteration after the exposure to environmental particulate matter.

Increased oxidized low density lipoprotein associated with high ceruloplasmin activity in patients with active acromegaly.

OBJECTIVE: Active acromegaly is associated with increased mortality from cardiovascular causes. Several studies have shown increased atherogenic risk factors and biomarkers of inflammation and atherosclerosis in association with growth hormone excess. The aim of this study was to evaluate oxidized low density lipoprotein (oxLDL) levels and some modulators of LDL oxidative modification in patients with acromegaly. DESIGN: Open transversal study. PATIENTS: Fifteen patients with active acromegaly and 15 controls were studied. MEASUREMENTS: We evaluated the levels of oxLDL, thiobarbituric acid reactive substances (TBARS), ceruloplasmin, bilirubin, uric acid and total reactive antioxidant potential, and the activities of ceruloplasmin, myeloperoxidase, superoxide distmutase, paraoxonase 1, and platelet activating factor acethylhydrolase. Statistical analysis was performed including body mass index as a covariate or as a fixed variable. RESULTS: Patients with acromegaly showed significantly higher levels of oxLDL (120 +/- 19 vs. 86 +/- 20 U/l, P < 0.001) and endothelin (P < 0.05), increased ceruloplasmin activity (P < 0.01) and a trend towards higher values in TBARS concentration (P = 0.07) in comparison to healthy controls. OxLDL was positively associated with GH, IGF-I and its binding protein 3 (r = 0.63, P < 0.001; r = 0.53, P < 0.01; and r = 0.56, P < 0.01; respectively). OxLDL showed direct associations with endothelin-1 (r = 0.53, P < 0.01) and ceruloplasmin activity (r = 0.43, P < 0.05). The other parameters evaluated were similar in both groups. CONCLUSIONS: The increase in plasma oxLDL levels, a direct marker of the plaque formation, could constitute a link between atherosclerosis and active acromegaly. LDL oxidation would not be the consequence of diminished antioxidant defences, but of an enhancement in prooxidant factors like ceruloplasmin.

Antioxidant status in the aqueous humour of patients with glaucoma associated with exfoliation syndrome.

PURPOSE: To establish the antioxidant status of the aqueous humour in glaucoma associated with exfoliation syndrome (XFG) and to compare it to primary open-angle glaucoma (POAG) and cataract patients. METHODS: Patients were diagnosed with POAG, XFG, or cataract (n=25 for each group). Total reactive antioxidant potential (TRAP) was measured by chemiluminescence. Ascorbic acid levels and the activities of catalase, glutathione peroxidase (GPx), and superoxide dismutase (SOD) were measured spectrophotometrically.ResultsTRAP value was lower in XFG (28+/-2 microM Trolox) than in POAG (55+/-8 microM Trolox; P<0.001). TRAP values in both glaucomas were lower than the cataract value (124+/-5 microM Trolox; P<0.001). A decrease in ascorbic acid was measured in XFG (230+/-20 microM) compared with POAG (415+/-17 microM; P<0.001). Ascorbic acid in both glaucomas was lower than in cataract (720+/-30 microM; P<0.001). A significant increase in GPx was found in XFG (30+/-2 U/ml) compared with POAG (16+/-3 U/ml). GPx activity in both glaucomas was increased when compared with cataracts (6+/-2 U/ml; P<0.001). A significant increase of 67% in SOD activity was observed in the glaucoma group vscataract group (27+/-3 U/ml; P<0.001), but no changes were found between both glaucomas. CONCLUSIONS: The antioxidant status of the aqueous humour may play a role in the pathophysiology of both glaucomas.

Decreased oxidative stress in prehepatic portal hypertensive rat livers following the induction of diabetes.

1. Oxidative stress (OS) is a biological entity indicated as being responsible for several pathologies, including diabetes. Diabetes can also be associated with human cirrhosis. Portal hypertension (PH), a major syndrome in cirrhosis, produces hyperdynamic splanchnic circulation and hyperaemia. The present study was designed to investigate the occurrence of OS in prehepatic PH rat livers following the induction of diabetes. 2. Five groups of rats were used: control, sham operated, chronic diabetes (induced with a single dose of streptozotocin at 60 mg/kg, i.p.), prehepatic PH and chronic diabetic plus prehepatic PH. The occurrence of OS was determined in liver homogenates by measuring hydroperoxide-initiated chemiluminescence and the activity of anti-oxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase). 3. Prehepatic PH produced a significant increase in hydroperoxide-initiated chemiluminescence in the liver compared with control and sham-operated rats, whereas the liver in chronic diabetic rats showed no difference. However, chemiluminescence values decreased almost by 50% in the chronic diabetic plus prehepatic PH group. Concomitantly, the activities of the anti-oxidant enzymes in chronic diabetes, prehepatic PH and chronic diabetic plus prehepatic PH groups were decreased (P < 0.05 vs control and sham-operated groups). 4. Livers from the chronic diabetic group did not show any evidence of the occurrence of OS, whereas the prehepatic PH group showed the occurrence of OS. The association of PH and chronic diabetes resulted in a significant decrease in the occurrence of OS, which could be explained by an anti-oxidant response to an OS.

Higher antioxidant defences in plasma and low density lipoproteins from rugby players.

BACKGROUND: Even if physical activity constitutes a well-known antiatherogenic factor, the precise mechanisms underlying this protective effect are not completely clear. MATERIALS AND METHODS: Lipid and antioxidant profiles were evaluated in 15 well-trained rugby players and 15 sedentary controls. Lipoprotein fractions were separated by sequential ultracentrifugation and alpha-tocopherol content was determined in each fraction by high-performance liquid chromatography. Susceptibility to in vitro oxidation was also measured in intermediate and low density lipoproteins isolated from both groups of subjects as the production of conjugated dienes. RESULTS: Although the sportsmen were not receiving any special diet or vitamin supplementation they showed a slightly improved lipoprotein profile, mainly represented by increased high density lipoprotein-cholesterol levels (P < 0.05), and an enhanced antioxidant status. The latter was evidenced by an increment in total radical antioxidant potential (P < 0.001), higher ascorbic acid (P < 0.005) and alpha-tocopherol (P < 0.05) plasma concentrations, and elevated activities of superoxide dismutase (P < 0.001) and arylesterase (P < 0.01). Moreover, only the fraction of intermediate and low density lipoproteins from rugby players presented higher alpha-tocopherol content in comparison with sedentary controls (484 +/- 67 vs. 377 +/- 123 microg dL(-1), respectively; P < 0.01). Nevertheless, the susceptibility to in vitro oxidation of this lipoprotein fraction was not different between both groups. CONCLUSIONS: Given that intermediate density and low density lipoproteins represent the most atherogenic fraction, this finding, in combination with the improved lipid and antioxidant status, would add to the link between regular physical activity and protection against cardiovascular disease.

Evaluation of total reactive antioxidant potential (TRAP) of tissue homogenates and their cytosols.

This study evaluates the possibility of obtaining total reactive antioxidant potential (TRAP) indexes in homogenates and their cytosolic fractions by a procedure based on the quenching of luminol luminescence induced by the thermolysis of 2,2'-azo-bis(2-amidinopropane). Measurements were performed in rat brain, liver, kidney, and heart homogenates. TRAP indexes can be easily determined both in homogenates and their cytosolic fractions. The results obtained indicate that heart homogenates are the least and liver homogenates the most protected of the systems considered. Glutathione is the measured antioxidant that contributes the most to TRAP values, while uric acid makes a significant contribution only in liver. A calculation of theoretical TRAP values from the measured concentrations of the main antioxidants (glutathione, uric acid, ascorbic acid, and alpha-tocopherol) for the different homogenates shows that, in most tissues (liver, brain, and kidney), nearly 50% of the experimentally determined TRAP values are not accounted for. This difference is mainly due to the contribution of proteins to the measured TRAP.

Time course and quantitative analysis of the adaptive responses to 85% oxygen in the rat lung and heart.

The phenomenon of oxygen tolerance (resistance to 100% O(2) in rats previously exposed to 85% O(2)) constitutes one of the few models of adaptive responses to oxidative stress in mammals. In vitro studies suggest that reactive oxygen species mediate this response. To test this hypothesis in vivo, we followed the time course of oxidative stress, enzyme induction, and edema in the lung, heart and liver of rats exposed to 85% O(2) for 1 to 5 days. Interestingly, not only the lung, but also the heart of rats exposed to 85% O(2) showed increases in the production of O(*-)(2) (aconitase inactivation) early during the exposure. Increases in O(*-)(2) were associated to oxidative stress (increased in situ chemiluminescence) and transient edema in both tissues. Both the lung and heart displayed induction of superoxide dismutase and reversion of the oxidative stress and damage. The adaptive response in the heart was faster and more efficient, suggesting that this tissue is at a more critical risk when exposed to elevated O(2) concentrations.

Peripheral markers of oxidative stress in probable Alzheimer patients.

BACKGROUND: The current research on Alzheimer's disease is mainly focused in the post-mortem characterization of pathological and biochemical alterations in the brain. The finding of peripheral markers that could be associated with the changes observed in the Alzheimer's brain would be of interest in this field. The aim of the present study was to evaluate the state of different peripheral markers of oxidative stress in probable Alzheimer patients and compare them with a group of healthy individuals. DESIGN: The determinations made include the plasma total antioxidant capacity (TRAP) and tert-butyl hydroperoxide-initiated chemiluminescence and catalase activity in erythrocytes from 18 patients with probable Alzheimer's disease and 18 matched control subjects with normal cognitive function. RESULTS: TRAP was decreased in Alzheimer patients by 24% (control group 308 micromol L-1 Trolox, SEM 34, n = 18). tert-Butyl hydroperoxide-initiated chemiluminescence and catalase activity showed an increase in erythrocytes from Alzheimer patients by 52% (control group 116 700 cps mg-1 haemoglobin, SEM 6690) and 75% (control group 2.55 pmol mg-1 protein, SEM 0.39, n = 18) respectively. CONCLUSION: Oxidative stress in the blood of probable Alzheimer patients could be a reflection of the brain condition and suggests that oxygen free radicals could be partially responsible of the damage observed in this disease.

Soccer players under regular training show oxidative stress but an improved plasma antioxidant status.

Physical activity is known to induce oxidative stress in individuals subjected to intense exercise. In this study, we investigated the lipoprotein profile and the plasma antioxidant status in a group of soccer players engaged in a regular training programme. As was expected for aerobic exercise, high-density lipoprotein-cholesterol (HDL-C) and HDL3-C levels were significantly increased in the sportsmen (P<0.05). Total plasma antioxidant capacity was 25% higher in sportsmen than in controls (P<0.005). Accordingly, plasma hydrosoluble antioxidant levels (ascorbic acid and uric acid) were found to be significantly elevated in the soccer players (P<0.005). In addition, these subjects showed high concentrations of alpha-tocopherol in plasma compared with controls (P<0.005). Furthermore, an increase in plasma superoxide dismutase activity was also observed in relation to exercise (P<0.01). The elevation in plasma activities of antioxidant enzymes and the higher levels of free radical scavengers of low molecular mass may compensate the oxidative stress caused by physical activity. High levels of high-density lipoprotein in plasma may offer additional protection by inhibiting low-density lipoprotein oxidation and thus liposoluble antioxidant consumption. Therefore, soccer players under regular training show an improved plasma antioxidant status in comparison to sedentary controls.

Oxidative stress and in vivo chemiluminescence in mouse skin exposed to UVA radiation.

Mouse skin was exposed to UVA radiation (320-400 nm). The in vivo chemiluminescence of the skin was measured after irradiation. Chemiluminescence showed a maximum 13-fold increase (control emission, 10 +/- 1 cps cm-2) after 45-60 min of exposure to UVA, with no further increase with 60 min additional exposure. Spectral analysis of the emitted chemiluminescence showed that the principal species emitted in the 400-500 nm range. Topical application with alpha-tocopherol (10% v/w) and beta-carotene (1 mM) greatly reduced the UVA-induced skin chemiluminescence. Thiobarbituric acid reactive substance (TBARS) levels were increased by 130% in skin homogenates after 2 h of exposure to UVA (control value, 77 +/- 14 nmol malonaldehyde equivalents (g tissue)-1). The activities of antioxidant enzymes in skin homogenates were decreased after 2 h of irradiation: the superoxide dismutase (SOD) activity (control value, 181 +/- 10 U SOD (g tissue)-1) was decreased by 40% and the catalase activity (control value, 1.34 +/- 0.14 pmol (g tissue)-1) was decreased by 45%. In vivo chemiluminescence appears to be a suitable method for following the kinetics of the oxidative stress processes and for testing the effect of topical application with antioxidant and photoprotective agents.

Hydrogen peroxide increases Na+/K(+)-ATPase function in alveolar type II cells.

We have studied the regulation of Na+/K(+)-ATPase function in alveolar type II cells submitted to oxidative stress. Alveolar type II cells were isolated from Sprague Dawley rats and suspended in Dulbecco's modified Eagle's medium. 500 muM xanthine plus 0.5 or 5 mU/ml xanthine oxidase (group 1 and 2, respectively) were added to the cell suspensions. Following various exposure times the reaction was stopped by adding allopurinol and cells were processed to assay H2O2 steady state concentrations, enzymatic activity of catalase and Na+/K(+)-ATPase function. Hydrogen peroxide production by the xanthine-xanthine oxidase system reached maximal values at 30 min of incubation in both groups. H2O2 steady state concentration increased 2- and 10-fold, respectively. Catalase activity was not changed after slight oxidative stress (group 1) but decreased in severe oxidative stress (group 2). Decreases in the Na+/K(+)-ATPase activity (10 and 60% for groups 1 and 2) were found during the first hour of exposure coinciding with the peak in H2O2 steady state concentration. This early inactivation was followed by progressive increases in the activity up to 70% over the control value in group 1, and to the control value in group 2. [3H]Ouabain binding studies showed that the increase in Na+/K(+)-ATPase activity after oxidative stress was due to an increase in the number of phosphorylated pump molecules in the plasma membrane of alveolar type II cells.

Oxidative stress in muscle and liver of rats with septic syndrome.

Sepsis, as infection associated to systemic manifestations, was produced in rats by cecal ligation and double perforation. Sham-operated rats were used as controls. The spontaneous chemiluminescence of rat adductor muscle and liver were measured at 6, 12, 24, and 30 h after the surgical procedure. Muscle chemiluminescence showed a maximal increase of about twofold (control emission 10 +/- 1 cps/cm2) after 6-12 h of sepsis, while liver chemiluminescence increased by about 80% (control emission: 11 +/- 1 cps/cm2) after 24 h of sepsis. The activities of muscle antioxidant enzymes were found maximally diminished after 12 h of sepsis: 46% decrease for Mn-superoxide dismutase, 83% decrease for catalase, and 55% decrease for glutathione peroxidase. In liver, only catalase activity showed a 52% decrease after 24 h of sepsis. State 3 oxygen uptake of muscle mitochondria with either malate-glutamate or succinate as substrates was 40% decreased after 12 h of sepsis in both cases. State 4 oxygen uptake of muscle mitochondria was not affected. The rate of H2O2 production of muscle mitochondria after 12 h of sepsis with either malate-glutamate or succinate as substrates was increased about 2.5 times but was not affected when assayed in the presence of as rotenone and antimycin. The oxygen uptake of liver mitochondria isolated from septic rats did not show differences as compared with those of control rats after 6 to 24 h of sepsis. Oxidative stress appears to occur in skeletal muscle early at the onset of the septic syndrome, with inhibition of active mitochondrial respiration and inactivation of antioxidant enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydrogen peroxide metabolism and oxidative stress in cortical, medullary and papillary zones of rat kidney.

The cortical, medullary and papillary regions of rat kidney were evaluated for a series of parameters related to hydrogen peroxide metabolism and oxidative stress. The rates of oxygen uptake, prostaglandin synthesis and malondialdehyde production by kidney slices were: 47, 0.003 and 0.051 mumol/h g wet wt., respectively, in cortex, 32, 0.023 and 0.035 in medulla and 22, 0.034 and 0.007 in papilla. The activities of superoxide dismutase, catalase and glutathione peroxidase were: 144 +/- 16 U/g wet wt., 880 +/- 100 pmol/g wet wt. and 177 +/- 16 U/g wet wt. in cortex; 97 +/- 9 U/g wet wt., 550 +/- 50 pmol/g wet wt. and 142 +/- 18 U/g wet wt. in medulla; and 23 +/- 2 U/g wet wt., 90 +/- 9 pmol/g wet wt. and 147 +/- 5 U/g wet wt. in papilla. Hydrogen peroxide steady-state concentrations were 0.09 +/- 0.01, 0.07 +/- 0.01 and 0.08 +/- 0.01 microM whereas alpha-tocopherol content was 21 +/- 2, 23 +/- 1 and 34 +/- 3 mumol/g wet wt. and hydroperoxide-initiated chemiluminescence was 22 +/- 2, 33 +/- 2 and 14 +/- 1 cpm. 10(-3)/mg prot for cortex, medulla and papilla, respectively. After 60 min ischemia-30 min reperfusion hydroperoxide-initiated chemiluminescence and hydrogen peroxide steady-state concentration increased by 30% and 60% in cortex and 80% and 60% in medulla, whereas alpha-tocopherol content decreased by 30%, 50% and 2% in cortex, medulla and papilla, respectively. The reperfusion/control ratio of hydroperoxide-initiated chemiluminescence and hydrogen peroxide steady-state concentrations in cortex and medulla indicate the occurrence of oxidative stress after ischemia-reperfusion. The lower sensitivity to oxidative stress found in papilla could be explained by the relatively high relationship of alpha-tocopherol content to hydrogen peroxide production rate in this sub-organ.

Oxidative stress in skeletal muscle during sepsis in rats.

Skeletal muscle is a target organ during sepsis; nevertheless, there is no evidence of a possible free radical overproduction with tissue damage in this situation. We studied Sprague Dawley female rats in two groups: a septic group with cecal ligation and double cecal perforation and a control group that was sham operated. Hind limb adductor muscles spontaneous chemiluminescence was measured at 2, 4, 6, 12, 24, and 30 hr after the surgical procedure as the expression of oxygen excited species generation. Muscle samples were also taken and activity of the principal antioxidant enzymes--superoxide dismutase (SOD), catalase, and glutathione peroxidase--as well as myeloperoxidase, an index of neutrophil infiltration was determined. CPK seric assays at 12 and 24 hr were used to reflect muscle injury and revealed high levels. Previously administered bovine superoxide dismutase was employed to prevent or attenuate oxidative stress. The results showed that light emission by rat skeletal muscle doubled from 4 to 12 hr of sepsis and could be attenuated with SOD pretreatment. Observed changes may be attributed to the production of oxygen free radicals that do not depend on local neutrophil infiltration. The detoxifying antioxidant enzyme activities in skeletal muscle were diminished (Mn SOD 46% at 6 hr, catalase 83% at 12 hr glutathione peroxidase 55% at 12 hr), which would also facilitate muscle septic damage.

Inhibition of microsomal lipid peroxidation by alpha-tocopherol and alpha-tocopherol acetate.

1. The antioxidant effects of alpha-tocopherol and alpha-tocopherol acetate were assayed for the (a) oxygen uptake, (b) chemiluminescence and (c) malondialdehyde formation, of tert-butyl hydroperoxide-supplemented rat liver microsomes. 2. Oxygen uptake was inhibited 60% by both alpha-tocopherol and alpha-tocopherol acetate with the half-maximal effect at 5 nmol tocopherol/mg protein. Chemiluminescence and malondialdehyde formation were equally inhibited 35% by both tocopherols with half-maximal effects at 2 nmol tocopherol/mg protein. 3. The rate of O2 uptake by tocopherol-supplemented microsomes was dependent on O2 concentration. A 60% inhibition by 5 nmol tocopherol/mg protein at 0.2 mM O2 is decreased to 5% inhibition at 0.6 mM O2. 4. The inhibition of O2 uptake, chemiluminescence and malondialdehyde formation indicate that both alpha-tocopherol and alpha-tocopherol acetate have similar effects as free radical traps in the hydrophobic domain of biomembranes. The different inhibition observed at different O2 concentrations indicate competition between vitamin E and O2 by unoxygenated lipid radicals.