Adriane Belló-Klein

Myocardial antioxidant and oxidative stress changes due to sex hormones.

The purpose of the present study was to examine myocardial antioxidant and oxidative stress changes in male and female rats in the presence of physiological sex hormone concentrations and after castration. Twenty-four 9-week-old Wistar rats were divided into four groups of 6 animals each: 1) sham-operated females, 2) castrated females, 3) sham-operated males, and 4) castrated males. When testosterone and estrogen levels were measured by radioimmunoassay, significant differences were observed between the castrated and control groups (both males and females), demonstrating the success of castration. Progesterone and catalase levels did not change in any group. Control male rats had higher levels of glutathione peroxidase (50%) and lower levels of superoxide dismutase (SOD, 14%) than females. Control females presented increased levels of SOD as compared to the other groups. After castration, SOD activity decreased by 29% in the female group and by 14% in the male group as compared to their respective controls. Lipid peroxidation (LPO) was assessed to evaluate oxidative damage to cardiac membranes by two different methods, i.e., TBARS and chemiluminescence. LPO was higher in male controls compared to female controls when evaluated by both methods, TBARS (360%) and chemiluminescence (46%). Castration induced a 200% increase in myocardial damage in females as determined by TBARS and a 20% increase as determined by chemiluminescence. In males, castration did not change LPO levels. These data suggest that estrogen may have an antioxidant role in heart muscle, while testosterone does not.

D-2-hydroxyglutaric acid induces oxidative stress in cerebral cortex of young rats.

Large amounts of d‐2‐hydroxyglutaric acid (DGA) accumulate in d‐2‐hydroxyglutaric aciduria (D‐2‐OHGA), an inherited neurometabolic disorder characterized by severe neurological dysfunction and cerebral atrophy. Despite the significant brain abnormalities, the neurotoxic mechanisms of brain injury in this disease are virtually unknown. In this work, the in vitro effect of DGA on various parameters of oxidative stress was investigated; namely chemiluminescence, thiobarbituric acid‐reactive substances (TBA‐RS), total radical‐trapping antioxidant potential (TRAP), total antioxidant reactivity (TAR) and the activities of the antioxidant enzymes catalase, glutathione peroxidase and superoxide dismutase in cerebral cortex from 30‐day‐old‐rats. DGA significantly increased chemiluminescence and TBA‐RS and decreased TAR values in the cortical supernatants. In contrast, TRAP and the antioxidant enzyme activities were not altered by the metabolite. Furthermore, the DGA‐induced increase of TBA‐RS was fully prevented by the free radical scavengers ascorbic acid plus Trolox (water‐soluble α‐tocopherol) and attenuated by the inhibitor of nitric oxide synthase Nω‐nitro‐L‐arginine methyl ester (L‐NAME), suggesting the role of superoxide, hydroxyl and nitric oxide radicals in this action. The data indicate a stimulation of lipid peroxidation through the production of free radicals and a reduction of the brain capacity to efficiently modulate the damage associated with the enhanced generation of free radicals by DGA. In the case that these findings also occur in human D‐2‐OHGA, it is feasible that oxidative stress may be involved in the pathophysiology of the brain injury observed in patients with this disease.

Total antioxidant capacity is impaired in different structures from aged rat brain

Our data support a disproportion between free radicals levels and scavenging systems activity in different cerebral regions of the aging rat. We investigated the total reactive antioxidant potential and reactivity levels, which represent the total antioxidant capacity, in different cerebral regions of the aging rat (cortex, striatum, hippocampus and the cerebellum). In addition, we have determined several oxidative stress parameters, specifically the free radicals levels, the macromolecules damage (lipid peroxidation and carbonyl content), as well as the antioxidant enzymes activities in different cerebral areas from young (2 months‐old), mature adult (6 months‐old) and old (24 months‐old) male Wistar rats. Free radicals levels, determined by 2′,7′‐dichlorofluorescein diacetate probe, were higher in striatum, cerebellum and hippocampus from aged rats. There was an age‐related increase in lipoperoxidation in hippocampus and cerebral cortex. In the cerebellum, a high activity of superoxide dismutase and a decrease of catalase activity were observed. The striatum exhibited a significant catalase activity decrease; and glutathione peroxidase activity was diminished in the hippocampus of mature and aged rats. There was a marked decrease of total antioxidant capacity in hippocampus in both reactivity and potential levels, whereas striatum and cerebral cortex displayed a reduction on reactivity assay. We suggest that age‐related variations of total antioxidant defenses in brain may predispose structures to oxidative stress‐related neurodegenerative disorders.

Exercise Training Improves Baroreflex Sensitivity Associated With Oxidative Stress Reduction in Ovariectomized Rats

The protection from coronary events that young women have is sharply reduced at menopause. Oxidative stress and baroreflex sensitivity impairment of the circulation have been demonstrated to increase cardiovascular risk. On the other hand, exercise training has been indicated as a nonpharmacological treatment for many diseases. The aim of the present study was to test the hypothesis that exercise training can improve baroreflex sensitivity associated with reduction in oxidative stress in ovariectomized rats, an experimental model of menopause. Exercise training was performed on a treadmill for 8 weeks. Arterial pressure and baroreflex sensitivity, which were evaluated by tachycardic and bradycardic responses to changes in arterial pressure, were monitored. Oxidative stress was evaluated by chemiluminescence and superoxide dismutase and catalase antioxidant enzyme activities. Exercise training reduced resting mean arterial pressure (112±2 vs 122±3 mm Hg in the sedentary group) and heart rate (325±4 vs 356±12 bpm in the sedentary group) and also improved baroreflex sensitivity (tachycardic response, 63% and bradycardic response, 58%). Myocardium (25%) and gastrocnemius muscle (48%) chemiluminescence were reduced, and myocardial superoxide dismutase (44%) and gastrocnemius catalase (97%) activities were enhanced in trained rats in comparison with sedentary rats. Myocardium chemiluminescence was positively correlated with systolic arterial pressure (r=0.6) and inversely correlated with baroreflex sensitivity (tachycardic response, r=−0.8 and bradycardic response, r=−0.7). These results indicate that exercise training in ovariectomized rats improves resting hemodynamic status and reflex control of the circulation, probably associated with oxidative stress reduction, suggesting a homeostatic role for exercise training in reducing cardiovascular risk in postmenopausal women.

3-Hydroxyglutaric acid induces oxidative stress and decreases the antioxidant defenses in cerebral cortex of young rats.

Glutaryl-CoA dehydrogenase deficiency (GDD) is an inherited neurometabolic disorder biochemically characterized by tissue accumulation of glutaric, 3-hydroxyglutaric (3-OHGA) and glutaconic acids and clinically by severe neurological symptoms and cerebral atrophy whose pathophysiology is poorly known. In the present study we investigated the effect of 3-OHGA, considered the main neurotoxin in GDD, on the lipoperoxidation parameters chemiluminescence and thiobarbituric acid-reactive species (TBA-RS), and on the amount of nitric oxide metabolites in cerebral cortex of young rats. Total radical-trapping antioxidant potential (TRAP), which reflects the tissue antioxidant defenses, was also examined. We observed that 3-OHGA significantly increased chemiluminescence, TBA-RS and nitric oxide metabolites, in contrast to TRAP, which was decreased by the metabolite. The data indicate a stimulation of lipid peroxidation and free radical production, and a reduction of the tissue antioxidant defenses caused by the metabolite. In case these findings also occur in the human condition, it may be presumed that oxidative stress is involved in the brain damage observed in GDD.

Myocardial antioxidant enzyme activities and concentration and glutathione metabolism in experimental hyperthyroidism

Hyperthyroidism was induced in rats by l-thyroxine administration (12 mg/L in drinking water, 4 weeks). Animals were assessed hemodynamically, and heart, lung, and liver morphometry were performed. Lipid peroxidation (LPO) and protein oxidation (carbonyls) were measured in heart homogenates. It was quantified glutathione (GSH) metabolism, and antioxidant enzyme activities its and protein expression (by Western blot). At the end of treatment, it was observed cardiac hypertrophy, elevation of left ventricular systolic and end diastolic pressures, lung and liver congestion. LPO and carbonyls were increased in the hyperthyroid group, and GSH was decreased by 46% in the fourth week. Myocardial oxidative stress time course analysis revealed that it was increased in the second week of treatment. Antioxidant enzyme activities elevation was accompanied by protein expression induction in the hyperthyroid group in the fourth week. These results imply that hyperthyroidism generates myocardial dysfunction associated with oxidative stress inducing antioxidant enzyme activities and protein expression.

Induction of oxidative stress by L-2-hydroxyglutaric acid in rat brain.

L‐2‐hydroxyglutaric acid (LGA) is the biochemical hallmark of L‐2‐hydroxyglutaric aciduria (L‐OHGA), an inherited neurometabolic disorder characterized by progressive neurodegeneration with cerebellar and pyramidal signs, mental deterioration, epilepsy, and subcortical leukoencephalopathy. Because the underlying mechanisms of the neuropathology of this disorder are virtually unknown, in this study we tested the in vitro effect of LGA on various parameters of oxidative stress, namely, chemiluminescence, thiobarbituric acid‐reactive substances (TBA‐RS), protein carbonyl formation (PCF), total radical‐trapping antioxidant potential (TRAP), total antioxidant reactivity (TAR), and the activities of the antioxidant enzymes catalase, glutathione peroxidase, and superoxide dismutase in cerebellum and cerebral cortex of 30‐day‐old rats. LGA significantly increased chemiluminescence, TBA‐RS, and PCF measurements and markedly decreased TAR values in cerebellum, in contrast to TRAP and the activity of the antioxidant enzymes, which were not altered by the acid. Similar but less pronounced effects were provoked by LGA in cerebral cortex. Moreover, the LGA‐induced increase of TBA‐RS was significantly attenuated by melatonin (N‐acetyl‐5‐methoxytryptamine) and by the combinations of ascorbic acid plus Trolox (soluble α‐tocopherol) and of superoxide dismutase plus catalase but not by the inhibitor of nitric oxide synthase Nω‐nitro‐L‐arginine methyl ester (L‐NAME), creatine, or superoxide dismutase or catalase alone in either cerebral structure. The data indicate that LGA provokes oxidation of lipids and proteins and reduces the brain capacity to modulate efficiently the damage associated with an enhanced production of free radicals, possibly by inducing generation of superoxide and hydroxyl radicals, which are trapped by the scavengers used. Thus, in case these findings can be extrapolated to human L‐OHGA, it may be presumed that oxidative stress is involved in the pathophysiology of the brain damage observed in this disorder.

Exercise Training Reduces Sympathetic Modulation on Cardiovascular System and Cardiac Oxidative Stress in Spontaneously Hypertensive Rats

BACKGROUND Spontaneously hypertensive rats (SHRs) show increased cardiac sympathetic activity, which could stimulate cardiomyocyte hypertrophy, cardiac damage, and apoptosis. Norepinephrine (NE)-induced cardiac oxidative stress seems to be involved in SHR cardiac hypertrophy development. Because exercise training (ET) decreases sympathetic activation and oxidative stress, it may alter cardiac hypertrophy in SHR. The aim of this study was to determine, in vivo, whether ET alters cardiac sympathetic modulation on cardiovascular system and whether a correlation exists between cardiac oxidative stress and hypertrophy. METHODS Male SHRs (15-weeks old) were divided into sedentary hypertensive (SHR, n = 7) and exercise-trained hypertensive rats (SHR-T, n = 7). Moderate ET was performed on a treadmill (5 days/week, 60 min, 10 weeks). After ET, cardiopulmonary reflex responses were assessed by bolus injections of 5-HT. Autoregressive spectral estimation was performed for systolic arterial pressure (SAP) with oscillatory components quantified as low (LF: 0.2-0.75 Hz) and high (HF: 0.75-4.0 Hz) frequency ranges. Cardiac NE concentration, lipid peroxidation, antioxidant enzymes activities, and total nitrates/nitrites were determined. RESULTS ET reduced mean arterial pressure, SAP variability (SAP var), LF of SAP, and cardiac hypertrophy and increased cardiopulmonary reflex responses. Cardiac lipid peroxidation was decreased in trained SHRs and positively correlated with NE concentrations (r = 0.89, P < 0.01) and heart weight/body weight ratio (r = 0.72, P < 0.01), and inversely correlated with total nitrates/nitrites (r = -0.79, P < 0.01). Moreover, in trained SHR, cardiac total nitrates/nitrites were inversely correlated with NE concentrations (r = -0.82, P < 0.01). CONCLUSIONS ET attenuates cardiac sympathetic modulation and cardiac hypertrophy, which were associated with reduced oxidative stress and increased nitric oxide (NO) bioavailability.

Exercise intensity influences cell injury in rat hippocampal slices exposed to oxygen and glucose deprivation

We evaluated the effects of two levels of daily forced exercise intensity (moderate and high) in the treadmill over cell susceptibility to oxygen and glucose deprivation (OGD) in hippocampal slices from Wistar rats. Moderate exercise decreased lactate dehydrogenase (LDH) release after OGD, while a significant increase in LDH release was observed in the high intensity group submitted to OGD. Our data corroborate the hypothesis that higher training intensity exacerbates brain damage, while a moderate intensity reduces the injury caused by in vitro ischemia.

Baroreflex sensitivity improvement is associated with decreased oxidative stress in trained spontaneously hypertensive rat

Background Baroreflex sensitivity (BRS) impairment has been associated with endothelial dysfunction and oxidative stress. Methods Because exercise training could improve endothelial function in spontaneously hypertensive rats (SHR), the effect of moderate exercise training on oxidative stress and BRS was investigated. Groups were divided into sedentary and trained Wistar–Kyoto rats (S-WK, n = 7 and T-WK, n = 6) and SHR (S-SHR and T-SHR, n = 9 each). Exercise training was performed on a treadmill (5 days/week, 60 min, 10 weeks), and the lactate threshold (20 m/min) was used to determine moderate intensity. Results Exercise training reduced mean arterial pressure in WK and SHR (S-WK 127 ± 4, T-WK 105 ± 5, S-SHR 169 ± 4 versus T-SHR 140 ± 4 mmHg; P < 0.01). Baroreflex bradycardic (S-WK −1.89 ± 0.15, T-WK −2.11 ± 0.37, S-SHR −0.80 ± 0.09 versus T-SHR −1.29 ± 0.10 bpm/mmHg; P < 0.0001) and tachycardic (S-WK 2.57 ± 0.19, T-WK 2.73 ± 0.21, S-SHR 1.18 ± 0.07 versus T-SHR 2.02 ± 0.10 bpm/mmHg; P < 0.0001) responses were significantly different between groups. Lipoperoxidation in erythrocytes (S-WK 11 320 ± 739, T-WK 10 397 ± 765, S-SHR 20 511 ± 1627 versus T-SHR 10 211 ± 589 counts per second (cps)/mg haemoglobin; P < 0.0001) and aortas (S-WK 12 424 ± 2219, T-WK 7917 ± 726, S-SHR 26 957 ± 1772 versus T-SHR 17 777 ± 1923 cps/mg protein; P < 0.0001) was reduced in T-SHR compared with S-SHR. Inverse correlations were observed between both bradycardic and tachycardic responses and lipoperoxidation in erythrocytes (r = 0.56 and r = −0.77, respectively; P < 0.01) and aortas (r = 0.77 and r = −0.80, respectively; P < 0.0001). Conclusion Our results indicate that exercise training decreases oxidative stress, which is related to an improvement in BRS in SHR.