Enrique Martínez-Ballarín

Acute Exercise Increases Plasma Total Antioxidant Status and Antioxidant Enzyme Activities in Untrained Men

Antioxidant defences are essential for cellular redox regulation. Since free-radical production may be enhanced by physical activity, herein, we evaluated the effect of acute exercise on total antioxidant status (TAS) and the plasma activities of catalase, glutathione reductase, glutathione peroxidase, and superoxide dismutase and its possible relation to oxidative stress resulting from exercise. Healthy untrained male subjects (n = 34) performed three cycloergometric tests, including maximal and submaximal episodes. Venous blood samples were collected before and immediately after each different exercise. TAS and enzyme activities were assessed by spectrophotometry. An increase of the antioxidant enzyme activities in plasma was detected after both maximal and submaximal exercise periods. Moreover, under our experimental conditions, exercise also led to an augmentation of TAS levels. These findings are consistent with the idea that acute exercise may play a beneficial role because of its ability to increase antioxidant defense mechanisms through a redox sensitive pathway.

Melatonin reduces membrane rigidity and oxidative damage in the brain of SAMP8 mice.

We evaluated the autophagy-lysosomal pathway and membrane fluidity in brain cells and mitochondrial membranes obtained from senescence-accelerated (SAMP(8)) and senescence-resistant (SAMR(1)) mice at 5 and 10 months of age. Moreover, we studied whether chronic treatment from age 1 to 10 months with melatonin stabilizes membrane fluidity. Fluidity was measured by polarization changes of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene-p-toluene sulfonate. Results showed that in untreated animals at 5 months of age, synaptosomal and mitochondrial fluidity was decreased in SAMP(8) compared to SAMR(1), as was the cathepsin D/B ratio, indicating dysfunction of the autophagy-lysosomal pathway. Moreover, we detected synaptosomal rigidity and programmed cell death capability in both groups at 10 months of age. Mitochondrial fluidity, however, did not show a significant age-dependent change but was lower in SAMP(8) than in SAMR(1) at the 5- and 10-month time points. Melatonin administration prevented rigidity in the mitochondrial membrane and seemed to decrease age-related autophagy-lysosomal alterations. These data suggest that melatonin may act to slow down the aging process because of its ability to enhance membrane fluidity and maintain structural pathways.

N-acetylserotonin suppresses hepatic microsomal membrane rigidity associated with lipid peroxidation.

N-acetylserotonin, the immediate precursor of melatonin in the tryptophan metabolic pathway in the pineal gland, has been reported to be an antioxidant. The aim of this work was to test the effect of N-acetylserotonin in stabilizing biological membranes against oxidative stress. Hepatic microsomal membranes from male adult rats were incubated with N-acetylserotonin (0.001-3 mM) before inducing lipid peroxidation using FeCl(3), ADP and NADPH. Control experiments were done by incubating microsomal membranes with N-acetylserotonin in the absence of lipid peroxidation-inducing drugs. Membrane fluidity was assessed by fluorescence spectroscopy and malonaldehyde plus 4-hydroxyalkenals concentrations were measured to estimate the degree of lipid peroxidation. Free radicals induced by the combination of FeCl(3)+ADP+NADPH produced a significant decrease in the microsomal membrane fluidity, which was associated with an increase in the malonaldehyde plus 4-hydroxyalkenals levels. These changes were suppressed in a concentration-dependent manner when N-acetylserotonin was added in the incubation buffer. In the absence of lipid peroxidation, N-acetylserotonin (0.001-3 mM) did not change membrane fluidity nor malonaldehyde plus 4-hydroxyalkenals levels. These results suggest that the protective role of N-acetylserotonin in preserving optimal levels of fluidity of the biological membranes may be related to its ability to reduce lipid peroxidation.

Protective effect of β-carbolines and other antioxidants on lipid peroxidation due to hydrogen peroxide in rat brain homogenates

Tryptoline and pinoline are two beta-carbolines isolated from the nervous system of mammals. We investigated the ability of these compounds to prevent lipid peroxidation induced by hydrogen peroxide in rat brain homogenates. We also compared their effects with other known antioxidants including melatonin, trolox and ascorbic acid. Lipid peroxidation was assessed by measuring malonaldehyde (MDA) and 4-hydroxy-alkenals (4-HDA) concentrations in the brain homogenates. Incubation with hydrogen peroxide (5 mM) increased MDA+4-HDA levels, which were totally prevented by tryptoline, pinoline, melatonin and trolox in a concentration-dependent manner. By contrast, higher MDA-4-HDA concentrations compared with control experiments were found after incubation with ascorbic acid, thus reflecting an increase of lipid peroxidation induced by this compound. Although in vivo studies are needed, the data suggest that these beta-carbolines may be potential neuroprotective agents because of their antioxidant activities.

Melatonin improves deferoxamine antioxidant activity in protecting against lipid peroxidation caused by hydrogen peroxide in rat brain homogenates.

Deferoxamine (DF) is an antioxidant molecule because of its ability to chelate iron. This study compared the ability of DF alone or in combination with melatonin, 5-methoxytryptophol or pinoline in preventing lipid peroxidation due to hydrogen peroxide (H(2)O(2)) in rat brain homogenates. Malondialdehyde (MDA) and 4-hydroxyalkenals (4-HDA) in the homogenates were measured as indices of lipid peroxidation. Incubation of homogenates with DF reduced, in a dose-dependent manner, MDA+4-HDA formation due to H(2)O(2). When melatonin, 5-methoxytryptophol or pinoline were added to the incubation medium, the efficacy of DF in preventing lipid peroxidation was enhanced. These cooperative effects between DF, melatonin, and related pineal products may be important in protecting tissues from the oxidative stress due to iron overload.

Melatonin and Structurally-Related Compounds Protect Synaptosomal Membranes from Free Radical Damage

Since biological membranes are composed of lipids and proteins we tested the in vitro antioxidant properties of several indoleamines from the tryptophan metabolic pathway in the pineal gland against oxidative damage to lipids and proteins of synaptosomes isolated from the rat brain. Free radicals were generated by incubation with 0.1 mM FeCl3, and 0.1 mM ascorbic acid. Levels of malondialdehyde (MDA) plus 4-hydroxyalkenal (4-HDA), and carbonyl content in the proteins were measured as indices of oxidative damage to lipids and proteins, respectively. Pinoline was the most powerful antioxidant evaluated, with melatonin, N-acetylserotonin, 5-hydroxytryptophan, 5-methoxytryptamine, 5-methoxytryptophol, and tryptoline also acting as antioxidants.

Melatonin reduces protein and lipid oxidative damage induced by homocysteine in rat brain homogenates

Numerous data indicate that hyperhomocysteinemia is a risk factor for cardio‐ and cerebrovascular diseases. At least in part, homocysteine (HCY) impairs cerebrovascular function because it generates large numbers of free radicals. Since melatonin is a well‐known antioxidant, which reduces oxidative stress and decreases HCY concentrations in plasma, the aim of this study was to investigate the effect of melatonin in preventing HCY‐induced protein and lipid oxidation in rat brain homogenates. Brain homogenates were obtained from Sprague–Dawley rats and were incubated with or without HCY (0.01–5 mM) or melatonin (0.01–3 mM). Carbonyl content of proteins, and malondialdehyde (MDA) and 4‐hydroxyalkenals (4‐HDA) concentrations in the brain homogenates were used as an index of protein and lipid oxidation, respectively. Under the experimental conditions used, the addition of HCY (0.01–5 mM) to the homogenates enhanced carbonyl protein and MDA+4‐HDA formation. Melatonin reduced, in a concentration‐dependent manner, protein and lipid oxidation due to HCY in the brain homogenates. These data suggest that preserving proteins from oxidative insults is an additional mechanism by which melatonin may act as an agent in potentially decreasing cardiovascular and cerebrovascular diseases related to hyperhomocysteinemia. J. Cell. Biochem. 102: 729–735, 2007.

In vivo hepatic oxidative stress because of carbon tetrachloride toxicity: protection by melatonin and pinoline

Abstract:  The protective in vivo effects of melatonin or pinoline on carbon tetrachloride (CCl4)‐induced oxidative damage were investigated in liver of rats and compared to rats injected only with CCl4 (5 mL/kg body weight). Hepatic cell membrane fluidity, monitored using fluorescence spectroscopy, exhibited a significant decrease in animals exposed to CCl4 compared to control rats. Increases in lipid and protein oxidation, as assessed by concentrations of malondialdehyde (MDA) and 4‐hydroxyalkenals (4‐HDA), and protein carbonylation, respectively, were also seen in hepatic homogenates of animals exposed to CCl4. The administration of melatonin (10 mg/kg body weight) or pinoline injected 30 min before and 1 hr after CCl4, fully prevented membrane rigidity and protein oxidation. However, treatment with melatonin was more effective in terms of reducing lipid peroxidation than pinoline, as the increases in MDA+4‐HDA levels because of CCl4 were reduced by 93.4% and 34.4% for melatonin or pinoline, respectively. Livers from CCl4‐injected rats showed several histopathological alterations; above all, there were signs of necrosis and ballooning degeneration. The concurrent administration of melatonin or pinoline reduced the severity of these morphological changes. On the basis of the biochemical and histopathological findings, we conclude that both melatonin and pinoline were highly effective in protecting the liver against oxidative damage and membrane rigidity because of CCl4. Therefore, these indoles may be useful as cotreatments for patients with hepatic intoxication induced by CCl4.

Melatonin and pinoline prevent aluminium-induced lipid peroxidation in rat synaptosomes

The serum concentrations of aluminum, a metal potentially involved in the pathogenesis of Alzheimers disease, increase with age. Also, intense and prolonged exposure to aluminum may result in dementia. Melatonin and pinoline are two well known antioxidants that efficiently reduce lipid peroxidation due to oxidative stress. Herein, we investigated the effects of melatonin and pinoline in preventing aluminum promotion of lipid peroxidation when the metal was combined with FeCl3 and ascorbic acid in rat synaptosomal membranes. Lipid peroxidation was estimated by quantifying malondialdehyde (MDA) and 4-hydroxyalkenal (4-HDA) concentrations in the membrane suspension. Under the experimental conditions used herein, the addition of aluminum (0.0001 to 1 mmol/L) enhanced MDA + 4-HDA formation in the synaptosomes. Melatonin and pinoline reduced, in a concentration-dependent manner, lipid peroxidation due to aluminum, FeCl3 and ascorbic acid in the synaptosomal membranes. These results suggest that the indoleamine melatonin and the beta-carboline pinoline may potentially act as neuroprotectant agents in the therapy of those diseases with elevated aluminum concentrations in the tissues.

Levels of membrane fluidity in the spinal cord and the brain in an animal model of amyotrophic lateral sclerosis

A mutant form of the copper/zinc superoxide dismutase (SOD1) protein is found in some patients with amyotrophic lateral sclerosis (ALS). Alteration of the activity of this antioxidant enzyme leads to an oxidative stress imbalance, which damages the structure of lipids and proteins in the CNS. Using fluorescence spectroscopy, we monitored membrane fluidity in the spinal cord and the brain in a widely used animal model of ALS, the SODG93A mouse, which develops symptoms similar to ALS with an accelerated course. Our results show that the membrane fluidity of the spinal cord in this animal model significantly decreased in symptomatic animals compared with age-matched littermate controls. To the best of our knowledge, this is the first report showing that membrane fluidity is affected in the spinal cord of a SODG93A animal model of ALS. Changes in membrane fluidity likely contribute substantially to alterations in cell membrane functions in the nervous tissue from SODG93A mice.