Oxidative stress, superoxide production, and apoptosis of neutrophils in dogs with chronic kidney disease

Oxidative stress is a key component in the immunosuppression of chronic kidney disease (CKD), and neutrophil function may be impaired by oxidative stress. To test the hypothesis that in uremic dogs with CKD, oxidative stress is increased and neutrophils become less viable and functional, 18 adult dogs with CKD were compared with 15 healthy adult dogs. Blood count and urinalysis were done, and the serum biochemical profile and plasma lipid peroxidation (measurement of thiobarbituric acid reactive substances) were determined with the use of commercial reagents. Plasma total antioxidant capacity (TAC) was measured with a spectrophotometer and commercial reagents, superoxide production with a hydroethidine probe, and the viability and apoptosis of neutrophils with capillary flow cytometry and the annexin V-PE system. The plasma concentrations of cholesterol (P = 0.0415), creatinine (P < 0.0001), and urea (P < 0.0001) were significantly greater in the uremic dogs than in the control dogs. The hematocrit (P = 0.0004), urine specific gravity (P = 0.015), and plasma lipid peroxidation (P < 0.0001) were significantly lower in the dogs that were in late stages of CKD than in the control group. Compared with those isolated from the control group, neutrophils isolated from the CKD group showed a higher rate of spontaneous (0.10 ± 0.05 versus 0.49 ± 0.09; P = 0.0033; median ± standard error of mean) and camptothecin-induced (18.53 ± 4.06 versus 44.67 ± 4.85; P = 0.0066) apoptosis and lower levels of superoxide production in the presence (1278.8 ± 372.8 versus 75.65 ± 86.6; P = 0.0022) and absence (135.29 ± 51.74 versus 41.29 ± 8.38; P = 0.0138) of phorbol-12-myristate-13-acetate stimulation. Thus, oxidative stress and acceleration of apoptosis occurs in dogs with CKD, the apoptosis diminishing the number of viable neutrophils and neutrophil superoxide production.

Antioxidant effects of quercetin and naringenin are associated with impaired neutrophil microbicidal activity

Naringenin and quercetin are considered antioxidant compounds with promising activity against oxidative damage in human cells. However, no reports have described their effects on reactive oxygen species (ROS) production by phagocytes during microbicidal activity. Thus, the present study evaluated the effects of naringenin and quercetin on ROS production, specifically hypochlorous acid (HOCl), and their involvement in the microbicidal activity of neutrophils. Naringenin and quercetin inhibited HOCl production through different systems, but this inhibition was more pronounced for quercetin, even in the cell-free systems. With regard to the microbicidal activity of neutrophils, both naringenin and quercetin completely inhibited the killing of Staphylococcus aureus. Altogether, these data indicate that the decrease in the oxidant activity of neutrophils induced by these compounds directly impaired the microbicidal activity of neutrophils. Naringenin and quercetin exerted their effects by controlling the effector mechanisms of ROS production, with both positive and negative effects of these antioxidant agents in oxidative stress conditions and on ROS in the microbicidal activity of phagocytes. The present results challenge the traditional view of antioxidants as improvers of pathological conditions. © 2013 Francielli de Cássia Yukari Nishimura et al.

Mensuração do superóxido e apoptose neutrofílica em cães azotêmicos e urêmicos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Efeito das toxinas urêmicas guanidina e ácido guanidinoácetico sobre o metabolismo oxidativo e apoptose em neutrófilos de cães

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Circulating leukocyte heat shock protein 70 (HSP70) and oxidative stress markers in rats after a bout of exhaustive exercise

A novel method to measure oxidative stress resulting from exhaustive exercise in rats is presented. In this new procedure we evaluated the erythrocyte antioxidant enzymes, catalase ( CAT) and glutathione reductase (GR), the plasma oxidative attack markers, reactive carbonyl derivatives (RCD) and thiobarbituric reactive substances (TBARS). Muscular tissue damage was evaluated by monitoring plasma creatine kinase (CK) and plasma taurine ( Tau) concentrations. Also, we monitored total sulphydryl groups (TSG) and uric acid (UA), and the level of the 70 kDa heat shock protein (HSP70) in leukocytes as a marker of oxidative stress. In the study we found a correspondence between erythrocyte CAT and GR activities and leukocyte HSP70 levels, principally 3 h after the acute exercise, and this suggested an integrated mechanism of antioxidant defense. The increase in levels of plasma Tau was coincident with the increasing plasma levels of CK and TBARS, principally after two hours of exercise. Thus tissue damage occurred before the expression of any anti-oxidant system markers and the monitoring of Tau, CK or TBARS may be important for the estimation of oxidative stress during exhaustive exercise. Furthermore, the integrated analyses could be of value in a clinical setting to quantify the extent of oxidative stress risk and reduce the need to perform muscle biopsies as a tool of clinical evaluation.

Hepatoprotective treatment attenuates oxidative damages induced by carbon tetrachloride in rats

The present study evaluated the hepatoprotective effect of an N-acetyl or-methionine + choline chloride + caffeine + thiamine hydrochloride + nicotinamide + pyridoxine hydrochloride compound at doses of 0.2, 0.6 and 1.0 mL/kg of b.w., and the assessment was done by the investigation of serum-enzymatic activity, metabolic functions of the liver and histophatological changes in female Wistar rats, which were subjected to experimental intoxication with CCl4. One hundred and nineteen rats were randomly distributed into 17 groups, performing five different treatments, being evaluated seven animals per treatment in four periods: 2, 4, 6 and 8 days after CCl4-induced intoxication. Treated rats with the hepatoprotective medicine (HM) presented a significant reduction in infiltration of inflammatory cells, steatosis, necrosis and liver congestion when compared to non-treated rats (control). Beside these results, the treatment showed a positive effect on circulatory alterations in the intoxicated animals, with reduction of spleen and renal congestion, as well as, promotion of a significant improvement in ALT, AST, LDH, ALP, GGT enzymatic serum activity reduction and in recovering liver function regarding the metabolism of urea, triglycerides and glucose. These findings indicate therapeutic usefulness of the compound when administered at dose 0.6 and 1.0 mL/kg of b.w. in female Wistar rats. (C) 2010 Elsevier GmbH. All rights reserved.

Dose-dependent effect of Resveratrol on bladder cancer cells: Chemoprevention and oxidative stress

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cigarette smoke exposure severely reduces peripheral insulin sensitivity without changing GLUT4 expression in oxidative muscle of Wistar rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Energy Expenditure, Lipid Profile, Oxidative Stress, and Cardiac Energy Metabolism After Growth Hormone Treatment in Obese Young Rats

Obesity is rampant in modern society and growth hormone (GH) could be useful as adjunct therapy to reduce the obesity-induced cardiovascular damage. To investigate GH effects on obesity, initially 32 male Wistar rats were divided into two groups (n = 16): control (C) was fed standard-chow and water and hyper-caloric (H) was fed hypercaloric chow and 30% sucrose in its drinking water. After 45 days, both C and H groups were divided into two subgroups (n = 8): C + PL was fed standard-chow, water and received saline subcutaneously; C + GH was fed standard-chow, water, and received 2 mg/kg/day GH subcutaneously; H + PL was fed hypercaloric diet, 30% sucrose in its drinking water, and received saline subcutaneously; and H + GH was fed hypercaloric diet, 30% sucrose in its drinking water, and received GH subcutaneously. After 75 days of total experimental period, H + PL rats were considered obese, having higher body weight, body mass index, Lee-index, and atherogenic index (AI) compared to C + PL. Obesity was accompanied by enhanced myocardial lipid hydroperoxide (LH) and lactate dehydrogenase (LDH), as well of depressed energy expenditure (RMR) and oxygen consumption(VO(2))/body weight. H + GH rats had higher fasting RMR, as well as lower AI and myocardial LH than H + PL. Comparing C + GH with C + PL, despite no effects on morphometric parameters, lipid profile, myocardial LH, and LDH activity, GH enhanced fed RMR and myocardial pyruvate dehydrogenase. In conclusion, the present study brought new insights into the GH effects on obesity related cardiovascular damage demonstrating, for the first time, that GH regulated cardiac metabolic pathways, enhanced energy expenditure and improved the lipid profile in obesity condition. Growth hormone in standard fed condition also offered promising therapeutic value enhancing pyruvate-dehydrogenase activity and glucose oxidation in cardiac tissue, thus optimizing myocardial energy metabolism.

Effect of therapeutic plasma concentrations of non-steroidal anti-inflammatory drugs on the production of reactive oxygen species by activated rat neutrophils

The release of reactive oxygen specie (ROS) by activated neutrophil is involved in both the antimicrobial and deleterious effects in chronic inflammation. The objective of the present investigation was to determine the effect of therapeutic plasma concentrations of non-steroidal anti-inflammatory drugs (NSAIDs) on the production of ROS by stimulated rat neutrophils. Diclofenac (3.6 µM), indomethacin (12 µM), naproxen (160 µM), piroxicam (13 µM), and tenoxicam (30 µM) were incubated at 37ºC in PBS (10 mM), pH 7.4, for 30 min with rat neutrophils (1 x 10(6) cells/ml) stimulated by phorbol-12-myristate-13-acetate (100 nM). The ROS production was measured by luminol and lucigenin-dependent chemiluminescence. Except for naproxen, NSAIDs reduced ROS production: 58 ± 2% diclofenac, 90 ± 2% indomethacin, 33 ± 3% piroxicam, and 45 ± 6% tenoxicam (N = 6). For the lucigenin assay, naproxen, piroxicam and tenoxicam were ineffective. For indomethacin the inhibition was 52 ± 5% and diclofenac showed amplification in the light emission of 181 ± 60% (N = 6). Using the myeloperoxidase (MPO)/H2O2/luminol system, the effects of NSAIDs on MPO activity were also screened. We found that NSAIDs inhibited both the peroxidation and chlorinating activity of MPO as follows: diclofenac (36 ± 10, 45 ± 3%), indomethacin (97 ± 2, 100 ± 1%), naproxen (56 ± 8, 76 ± 3%), piroxicam (77 ± 5, 99 ± 1%), and tenoxicam (90 ± 2, 100 ± 1%), respectively (N = 3). These results show that therapeutic levels of NSAIDs are able to suppress the oxygen-dependent antimicrobial or oxidative functions of neutrophils by inhibiting the generation of hypochlorous acid.

The oxidation of apocynin catalyzed by myeloperoxidase: Proposal for NADPH oxidase inhibition

Apocynin has been used as an efficient inhibitor of the NADPH oxidase complex and its mechanism of inhibition is linked to prior activation through the action of peroxidascs. Here we studied the oxidation of apocynin catalyzed by myeloperoxidase (MPO) and activated neutrophils. We found that apocynin is easily oxidized by MPO/H2O2 or activated neutrophils and has as products dimer and trimer derivatives. Since apocynin impedes the migration of the cytosolic component p47phox to the membrane and this effect could be related to its conjugation with essential thiol groups, we studied the reactivity of apocynin and its MPO-catalyzed oxidation products with glutathione (GSH). We found that apocynin and its oxidation products do not react with GSH. However, this thiol compound was efficiently oxidized by the apocynin radical during the MPO-catalyzed oxidation. We suggest that the reactivity of apocynin radical with thiol compounds could be involved in the inhibitory effect of this methoxy-catechol on NADPH oxidase complex. (c) 2006 Elsevier B.V. All rights reserved.

Direct observation of oxidative stress on the cell wall of Saccharomyces cerevisiae strains with atomic force microscopy

We imaged pores on the surface of the cell wall of three different industrial strains of Saccharomyces cerevisiae using atomic force microscopy. The pores could be enlarged using 10 mM diamide, an SH residue oxidant that attacks surface proteins. We found that two strains showed signs of oxidative damage via changes in density and diameter of the surface pores. We found that the German strain was resistant to diamide induced oxidative damage, even when the concentration of the oxidant was increased to 50 mM. The normal pore size found on the cell walls of American strains had diameters of about 200nm. Under conditions of oxidative stress the diameters changed to 400nm.This method may prove to be a useful rapid screening process (45-60 min) to determine which strains are oxidative resistant, as well as being able to screen for groups of yeast that are sensitive to oxidative stress. This rapid screening tool may have direct applications in molecular biology (transference of the genes to inside of living cells) and biotechnology (biotransformations reactions to produce chiral synthons in organic chemistry.

Trypanocidal action of eupomatenoid-5 is related to mitochondrion dysfunction and oxidative damage in Trypanosoma cruzi

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)