Anaysa Paola Bolin

ROS production in neutrophils from alloxan-induced diabetic rats treated in vivo with astaxanthin

BACKGROUND Astaxanthin (ASTA) is a carotenoid which has powerful antioxidant, anti-tumor, anti-diabetic, anti-inflammatory and cardioprotective properties. The present study investigated the effect of daily ASTA intake on oxidative stress and the functional properties of neutrophils from alloxan-induced diabetic rats. METHODS Neutrophils isolated from ASTA-fed rats (30days, 20mg ASTA/kg of body weight - BW) induced to diabetes by alloxan treatment (i.p. 75mg/BW) were assessed by: production of superoxide and hydrogen peroxide, nitric oxide, basal calcium release, oxidative damage (TBARS and carbonyls content), and activities of major antioxidant enzymes. RESULTS Our results show that diabetes promotes a significant oxidative stress in neutrophils. The production of superoxide was significantly increased in neutrophils from diabetic rats and treatment with ASTA was not effective in reducing superoxide levels. At the same time, a reduction in the activity of total superoxide dismutase enzyme was observed, which was not restored after treatment with ASTA. At resting conditions, neutrophils have a higher basal production of hydrogen peroxide, which is enhanced following PMA-stimulation. Treatment with ASTA does not restore values to the basal levels. The indicators of oxidative damage to biomolecules showed that diabetic rats significantly increased the lipid and protein damage, but this change was reversed after treatment with ASTA. CONCLUSION Our results show that diabetes condition promotes a marked oxidative stress in neutrophils and treatment with ASTA for 30days at a dose of 20mg/kg of BW partially reverses those deleterious effects.

Cytokines and Oxidative Stress Status Following a Handball Game in Elite Male Players

Background. Handball is considered an intermittent sport that places an important stress on a players aerobic and anaerobic metabolism. However, the oxidative stress responses following a handball game remain unknown. We investigated the responses of plasma and erythrocyte antioxidant system and oxidative stress biomarkers following a single handball game. Methods. Fourteen male elite Brazilian handball athletes were recruited in the present study. Blood samples were taken before, immediately, and 24 hours after the game. Results. After the game and during 24 hours of recovery, the concentration of all oxidative stress indices changed significantly in a way indicating increased oxidative stress in the blood (thiol groups and reduced glutathione decreased, whereas TBARS and plasma antioxidant capacity was increased) as well as in erythrocyte (increased levels of TBARS and protein carbonyls). Erythrocyte antioxidant enzyme activities were also significantly changed by handball. Muscle damage indices (creatine kinase and lactate dehydrogenase) increased significantly after exercise. In addition, IL-6 increased after the game, whereas TNF-α decreased during recovery. Conclusion. This study demonstrates that a single handball game in elite athletes induces a marked state of oxidative stress evidenced by the oxidative modification in plasma and erythrocyte macromolecules, as well as by changes in the enzymatic and nonenzymatic antioxidant system.

Testosterone suppresses oxidative stress in human neutrophils

The in vitro effect of testosterone on human neutrophil function was investigated. Blood neutrophils from healthy male subjects were isolated and treated with 10 nM, 0.1 and 10 µM testosterone for 24 h. As compared with untreated cells, the testosterone treatment produced a significant decrease of superoxide production as indicated by the measurement of extra‐ and intracellular superoxide content. An increment in the production of nitric oxide was observed at 0.1 and 10 µM testosterone concentrations, whereas no effect was found for 10 nM. Intracellular calcium mobilization was significantly increased at 10 nM, whereas it was reduced at 10 µM testosterone. There was an increase in phagocytic capacity at 10 nM and a decrease of microbicidal activity in neutrophils treated with testosterone at 10 µM. Glutathione reductase activity was increased by testosterone treatment, whereas no effect was observed in other antioxidant enzyme activities. An increase in the content of thiol groups was observed at all testosterone concentrations. Lipid peroxidation in neutrophils evaluated by levels of TBARS was decreased at 10 nM and 0.1 µM testosterone. These results indicate the antioxidant properties of testosterone in neutrophils as suggested by reduction of superoxide anion production, and lipid peroxidation, and by the increase in nitric oxide production, glutathione reductase activity and the content of thiol groups. Therefore, the plasma levels of testosterone are important regulators of neutrophil function and so of the inflammatory response. Copyright

Oxidative stress and antioxidant status response of handball athletes: Implications for sport training monitoring☆

The chronic exposure to regular exercise training seems to improve antioxidant defense systems. However, the intense physical training imposed on elite athletes may lead to overtraining associated with oxidative stress. The purpose of the present study was to investigate the effect of different training loads and competition on oxidative stress, biochemical parameters and antioxidant enzymatic defense in handball athletes during 6-months of monitoring. Ten male elite handball athletes were recruited to the study. Blood samples were collected four times every six weeks throughout the season. During most intense periods of training and competitions there were significant changes in plasma indices of oxidative stress (increased TBARS and decreased thiols). Conversely, chronic adaptations to exercise training demonstrated a significant protective effect against oxidative stress in erythrocyte (decrease in TBARs and carbonyl group levels). Erythrocyte antioxidant enzyme activities were significantly increased, suggesting a training-induced antioxidant adaptation. Biomarkers of skeletal muscle damage were significantly increased during high-intensity training period (creatine kinase, lactate dehydrogenase and aspartate aminotransferase). No significant changes were observed in plasma IL-6, TNF-α and uric acid, whereas a significant reduction was found in the IL-1β concentration and gamma-glutamyl transferase activity. Oxidative stress and antioxidant biomarkers can change throughout the season in competitive athletes, reflecting the physical stress and muscle damage that occurs as the result of competitive handball training. In addition, these biochemical measurements can be applied in the physiological follow-up of athletes.

Combined astaxanthin and fish oil supplementation improves glutathione-based redox balance in rat plasma and neutrophils

The present study aimed to investigate the effects of daily (45 days) intake of fish oil (FO; 10mg EPA/kg body weight (BW) and 7 mg DHA/kg BW) and/or natural ASTA (1mg ASTA/kg BW) on oxidative stress and functional indexes of neutrophils isolated from Wistar rats by monitoring superoxide (O(2)(-)), hydrogen peroxide (H(2)O(2)), and nitric oxide (NO()) production compared to the progression of auto-induced lipid peroxidation and Ca(2+) release in activated neutrophils. Furthermore, phagocytic capacity, antioxidant enzyme activities, glutathione-recycling system, and biomarkers of lipid and protein oxidation in neutrophils were compared to the redox status. Our results show evidence of the beneficial effects of FO+ASTA supplementation for immune competence based on the redox balance in plasma (significant increase in GSH-dependent reducing power), non-activated neutrophils (increased activity of the glutathione-recycling enzymes GPx and GR) and PMA-activated neutrophils (lower O(2)(-), H(2)O(2), and NO() generation, reduced membrane oxidation, but higher phagocytic activity). Combined application of ASTA and FO promoted hypolipidemic/hypocholesterolemic effects in plasma and resulted in increased phagocytic activity of activated neutrophils when compared with ASTA or FO applied alone. In PMA-activated neutrophils, ASTA was superior to FO in exerting antioxidant effects. The bulk of data reinforces the hypothesis that habitual consumption of marine fish (e.g. salmon, which is a natural source of both astaxanthin and fish oil) is beneficial to human health, in particular by improving immune response and lowering the risk of vascular and infectious diseases.

Comparative effect of fucoxanthin and vitamin C on oxidative and functional parameters of human lymphocytes.

The aim of this study was to evaluate the effects of FUCO alone or combined with vitamin C on different features of lymphocyte function related to ROS/RNS (reactive oxygen/nitrogen species) production. For this purpose we have evaluated the cytotoxicity of increasing concentrations of FUCO and vitamin C, the proliferative capacity of stimulated T- and B-lymphocytes, superoxide anion radicals (O(2)), hydrogen peroxide (H(2)O(2)) and nitric oxide (NO) production, antioxidant enzyme activities and the indexes of oxidative damage in proteins (carbonyl and thiol content). We have also evaluated the release of inflammatory cytokines and glucose-6-phosphate dehydrogenase (G6PDH) activity. Healthy human lymphocytes were acutely treated in vitro with FUCO (2 μM) with or without vitamin C (100 μM). Results revealed that human lymphocytes treated with FUCO at 2μM did not present any significant alteration in the proliferation of T- and B-lymphocytes at both resting and stimulated conditions. Moreover, FUCO used at low concentrations showed more pro-oxidant than antioxidant effects, which were recognized by the increased H(2)O(2) and increased NO production. Anti-inflammatory activity of FUCO was confirmed by significantly increased IL-10 and decreased TNF-α production. Vitamin C increased T-lymphocyte proliferation, whereas vitamin C plus FUCO promoted a reduction in the proliferation rate of these cells. All groups decreased pro-inflammatory cytokine TNF-α and increased anti-inflammatory IL-10 production although only vitamin C decreased IFN-γ either alone or when combined with FUCO. Overall, the combination of the antioxidants had more antioxidant and anti-inflammatory effects than when they were applied alone.

Green tea polyphenols change the profile of inflammatory cytokine release from lymphocytes of obese and lean rats and protect against oxidative damage

This study aimed to investigate whether green tea polyphenols (GT) modulate some functional parameters of lymphocytes from obese rats. Male Wistar rats were treated with GT by gavage (12 weeks/5 days/week; 500 mg/kg of body weight) and obesity was induced by cafeteria diet (8 weeks). Lymphocytes were obtained from mesenteric lymph nodes for analyses. In response to the cafeteria diet we observed an increase in activity of the metabolic enzyme hexokinase, ROS production, MnSOD, CuZnSOD and GR enzyme activities and proliferation capacity of the cells (baseline), whereas IL-10 production was decreased. Obese rats treated with GT decreased cell proliferation (under ConA stimulation). Hexokinase and G6PDH activity, ROS production and MnSOD, CuZnSOD, GPx and GR enzymes remained increased, accompanied by an increase in Nrf2 mRNA level. There was a decrease in pro-inflammatory IL-2, IL-6, IL-1β, TNF-α cytokines that were accompanied by a decrease in the mRNA level of TRL4 while IL-10 production was increased in obese rats treated with GT. GT treatment of lean rats showed similar results to that of obese rats treated with GT, indicating that the effects of GT are independent of diet. Foxp3 and IRF4 mRNA levels were increased by GT. In conclusion, cafeteria diet modulated the function of lymphocytes from lymph nodes, increasing ROS production and decreasing anti-inflammatory IL-10, which could contribute to the inflammatory state in obesity. GT reduced ROS production, improving the redox status and reducing pro-inflammatory cytokine production by lymphocytes, suggesting that GT treatment may be driving lymphocytes to a more anti-inflammatory than pro-inflammatory microenvironment.

Glycolaldehyde impairs neutrophil biochemical parameters by an oxidative and calcium-dependent mechanism-Protective role of antioxidants astaxanthin and vitamin C

AIM The present study examined the effects of glycolaldehyde (GC) on biochemical parameters of human neutrophils and whether the antioxidant astaxanthin associated with vitamin C can modulate these parameters. METHODS Neutrophils from healthy subjects were treated with GC (1mM) followed or not by the antioxidants astaxanthin (2 μM) and vitamin C (100 μM). We examined the phagocytic capacity, hypochlorous acid, myeloperoxidase (MPO) and glucose-6-phosphate dehydrogenase (G6PDH) activities, cytokines and [Ca(2+)](i). Also, superoxide anion, hydrogen peroxide, nitric oxide production, antioxidant enzyme activities and glutathione-recycling system were evaluated. RESULTS GC promoted a marked reduction on the phagocytic capacity, maximal G6PDH and MPO activities, hypochlorous acid production and release of IL-1β, IL-6 and TNF-α cytokines. Some impairment in the neutrophils biochemical parameters appears to be mediated by oxidative stress through ROS/RNS production and calcium reduction. Oxidative stress was evidenced by reduction in the activities of the main antioxidant enzymes, GSH/GSSG ratio and in the increment of O(2)(-) and H(2)O(2) and NO. CONCLUSIONS Treatment of cells with the combination of the antioxidants astaxanthin and vitamin C was able to restore some neutrophils function mainly by decreasing ROS/RNS production and improving the redox state. Overall, our findings demonstrate that GC modulates several neutrophils biochemical parameters in vitro.

Changes in lymphocyte oxidant/antioxidant parameters after carbonyl and antioxidant exposure

During normal B- and T-cell life, processes including activation, proliferation, signaling pathways and apoptosis are markedly dependent on ROS generation. However, these cells can also suffer the effect of oxidant overproduction. Thus, the purpose of the present study was to examine the possible pro-oxidant effects of MGO/high glucose and antioxidant effects of astaxanthin associated with vitamin C on some oxidative and antioxidant parameters of human lymphocytes in vitro. Lymphocytes from healthy subjects were treated with 20mM of glucose and 30 μM MGO followed or not by the addition of the antioxidants astaxanthin (2 μM) and vitamin C (100 μM) for up to 24h. We examined superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase (G6PDH) activities, GSH/GSSG ratio and total thiol and carbonyl content. Oxidative parameters included superoxide anion, hydrogen peroxide and nitric oxide production. The association of astaxanthin and vitamin C proved to be a powerful antioxidant in human lymphocytes as showed by the marked reduction in superoxide anion, and hydrogen peroxide production as well as increased GSH content, GSH/GSSG ratio, GPx and GR activities. The antioxidant association showed to be more potent than their individual application. High glucose and methylglyoxal did not promote oxidative stress in human lymphocytes, since neither the oxidative parameters nor the antioxidant defense system was altered. According to these results, new therapies with the association of astaxanthin and vitamin C may be helpful to improve the immune function of patients with exacerbated production of ROS.

Polyphenol-rich green tea extract improves adipose tissue metabolism by down-regulating miR-335 expression and mitigating insulin resistance and inflammation

Obesity leads to changes in miRNA expression in adipose tissue, and this modulation is linked to the pathophysiology of the disease. Green tea (GT) is a natural source of polyphenols that have been shown to confer health benefits, particularly preventing metabolic diseases. Here, we investigated if the beneficial effects of GT in obesity results from changes in the miRNA profile in white adipose tissue. GT treatment [500 mg/body weight (BW)/12 weeks] increased energy expenditure of high-fat diet-fed mice (16 weeks), leading to reduced weight gain, decreased adiposity, reduced inflammation and improved insulin sensitivity. These phenotypes were associated with a decrease in the expression of miR-335 in the adipose tissue. miR-335 was up-regulated by TNF-α in adipocytes and, in turn, down-regulated genes involved in insulin signaling and lipid metabolism. On the other hand, GT inhibited TNF-α effect. In conclusion, miR-335 serves as a link between inflammation and impaired metabolism in adipose tissue, providing an important mechanistic insight into the molecular basis underlying GT action during obesity.