Nicorandil protects cardiac mitochondria against permeability transition induced by ischemia-reperfusion

Ischemia followed by reperfusion is known to negatively affect mitochondrial function by inducing a deleterious condition termed mitochondrial permeability transition. Mitochondrial permeability transition is triggered by oxidative stress, which occurs in mitochondria during ischemia-reperfusion as a result of lower antioxidant defenses and increased oxidant production. Permeability transition causes mitochondrial dysfunction and can ultimately lead to cell death. A drug able to minimize mitochondrial damage induced by ischemia-reperfusion may prove to be clinically effective. We aimed to analyze the effects of nicorandil, an ATP-sensitive potassium channel agonist and vasodilator, on mitochondrial function of rat hearts and cardiac HL-1 cells submitted to ischemia-reperfusion. Nicorandil decreased mitochondrial swelling and calcium uptake. It also decreased reactive oxygen species formation and thiobarbituric acid reactive substances levels, a lipid peroxidation biomarker. We thus confirm previous reports that nicorandil inhibits mitochondrial permeability transition and demonstrate that nicorandil inhibits this process by preventing oxidative damage and mitochondrial calcium overload induced by ischemia-reperfusion, resulting in improved cardiomyocyte viability. These results may explain the good clinical results obtained when using nicorandil in the treatment of ischemic heart disease.

Horseradish peroxidase compound I as a tool to investigate reactive protein-cysteine residues: from quantification to kinetics

Proteins containing reactive cysteine residues (protein-Cys) are receiving increased attention as mediators of hydrogen peroxide signaling. These proteins are mainly identified by mining the thiol proteomes of oxidized protein-Cys in cells and tissues. However, it is difficult to determine if oxidation occurs through a direct reaction with hydrogen peroxide or by thiol-disulfide exchange reactions. Kinetic studies with purified proteins provide invaluable information about the reactivity of protein-Cys residues with hydrogen peroxide. Previously, we showed that the characteristic UV-Vis spectrum of horseradish peroxidase compound I, produced from the oxidation of horseradish peroxidase by hydrogen peroxide, is a simple, reliable, and useful tool to determine the second-order rate constant of the reaction of reactive protein-Cys with hydrogen peroxide and peroxynitrite. Here, the method is fully described and extended to quantify reactive protein-Cys residues and micromolar concentrations of hydrogen peroxide. Members of the peroxiredoxin family were selected for the demonstration and validation of this methodology. In particular, we determined the pK(a) of the peroxidatic thiol of rPrx6 (5.2) and the second-order rate constant of its reactions with hydrogen peroxide ((3.4 +/- 0.2) x 10(7) M(-1) s(-1)) and peroxynitrite ((3.7 +/- 0.4) x 10(5) M(-1) s(-1)) at pH 7.4 and 25 degrees C. (C) 2011 Elsevier Inc. All rights reserved.

Long-term intermittent feeding, but not caloric restriction, leads to redox imbalance, insulin receptor nitration, and glucose intolerance

Calorie restriction is a dietary intervention known to improve redox state, glucose tolerance, and animal life span. Other interventions have been adopted as study models for caloric restriction, including nonsupplemented food restriction and intermittent, every-other-day feedings. We compared the short- and long-term effects of these interventions to ad libitum protocols and found that, although all restricted diets decrease body weight, intermittent feeding did not decrease intra-abdominal adiposity. Short-term calorie restriction and intermittent feeding presented similar results relative to glucose tolerance. Surprisingly, long-term intermittent feeding promoted glucose intolerance, without a loss in insulin receptor phosphorylation. Intermittent feeding substantially increased insulin receptor nitration in both intra-abdominal adipose tissue and muscle, a modification associated with receptor inactivation. All restricted diets enhanced nitric oxide synthase levels in the insulin-responsive adipose tissue and skeletal muscle. However, whereas calorie restriction improved tissue redox state, food restriction and intermittent feedings did not. In fact, long-term intermittent feeding resulted in largely enhanced tissue release of oxidants. Overall, our results show that restricted diets are significantly different in their effects on glucose tolerance and redox state when adopted long-term. Furthermore, we show that intermittent feeding can lead to oxidative insulin receptor inactivation and glucose intolerance. (C) 2011 Elsevier Inc. All rights reserved.

Evidence that OGG1 glycosylase protects neurons against oxidative DNA damage and cell death under ischemic conditions

7,8-Dihydro-8-oxoguanine DNA glycosylase (OGG1) is a major DNA glycosylase involved in base-excision repair (BER) of oxidative DNA damage to nuclear and mitochondrial DNA (mtDNA). We used OGG1-deficient (OGG1(-/-)) mice to examine the possible roles of OGG1 in the vulnerability of neurons to ischemic and oxidative stress. After exposure of cultured neurons to oxidative and metabolic stress levels of OGG1 in the nucleus were elevated and mitochondria exhibited fragmentation and increased levels of the mitochondrial fission protein dynamin-related protein 1 (Drp1) and reduced membrane potential. Cortical neurons isolated from OGG1(-/-) mice were more vulnerable to oxidative insults than were OGG1(+/+) neurons, and OGG1(-/-) mice developed larger cortical infarcts and behavioral deficits after permanent middle cerebral artery occlusion compared with OGG1(+/+) mice. Accumulations of oxidative DNA base lesions (8-oxoG, FapyAde, and FapyGua) were elevated in response to ischemia in both the ipsilateral and contralateral hemispheres, and to a greater extent in the contralateral cortex of OGG1(-/-) mice compared with OGG1(+/+) mice. Ischemia-induced elevation of 8-oxoG incision activity involved increased levels of a nuclear isoform OGG1, suggesting an adaptive response to oxidative nuclear DNA damage. Thus, OGG1 has a pivotal role in repairing oxidative damage to nuclear DNA under ischemic conditions, thereby reducing brain damage and improving functional outcome. Journal of Cerebral Blood Flow & Metabolism (2011) 31, 680-692; doi:10.1038/jcbfm.2010.147; published online 25 August 2010

Generation and suppression of singlet oxygen in hair by photosensitization of melanin

We have studied the spectroscopic properties of hair (white, blond, red, brown, and black) under illumination with visible light, giving special emphasis to the photoinduced generation of singlet oxygen ((1)O(2)). Irradiation of hair shafts (lambda(ex)>400 nm) changed their properties by degrading the melanin. Formation of C3 hydroperoxides in the melanin indol groups was proven by (1)H NMR. After 532-nm excitation, all hair shafts presented the characteristic (1)O(2) emission (lambda(em) = 1270 nm), whose intensity varied inversely with the melanin content. (1)O(2) lifetime was also shown to vary with hair type, being five times shorter in black hair than in blond hair, indicating the role of melanin as a (1)O(2) suppressor. Lifetime ranged from tenths of a nanosecond to a few microseconds, which is much shorter than the lifetime expected for (1)O(2) in the solvents in which the hair shafts were suspended, indicating that (1)O(2) is generated and suppressed inside the hair structure. Both eumelanin and pheomelanin were shown to produce and to suppress (1)O(2), with similar efficiencies. The higher amount of (1)O(2) generated in blond hair and its longer lifetime is compatible with the stronger damage that light exposure causes in blond hair. We propose a model to explain the formation and suppression of (1)O(2) in hair by photosensitization of melanin with visible light and the deleterious effects that an excess of visible light may cause in hair and skin. 2011 Published by Elsevier Inc.

Co-stressors chilling and high light increase photooxidative stress in diuron-treated red alga Kappaphycus alvarezii but with lower involvement of H(2)O(2)

Diuron is one of the most commonly found N-phenylurea herbicides in marine/estuarine waters that promotes toxic effects by inhibiting photosynthesis and affecting the production of reactive oxygen species (ROS) in autotrophs. Since photo- and thermoacclimation are also ROS-mediated processes, this work evaluates a hypothetical additive effect of high light (HL) and chilling (12 degrees C) on 50 nM diuron toxicity to the highly-photosynthetically active apices of the red alga Kappaphycus alvarezii. Additive inhibition of photosynthesis was mainly evidenced by significant decreases of quantum yield of photosystem II and electron transfer rates upon co-stressors exposure to diuron-treated algae. Under extreme 12 degrees C/HL/diuron conditions, unexpected lower correlations between H(2)O(2) concentrations in seawater and radical-sensitive protein thiols were concomitantly measured with the highest indexes of photoinhibition (parameter beta). Altogether, these data support the hypothesis that co-stressors chilling/HL additively inhibit photosynthesis in diuron-exposed K. alvarezii but with less involvement of H(2)O(2) in injury effects than with only chilling or HL. (C) 2010 Elsevier Inc. All rights reserved.

Major determinants of photoinduced cell death: Subcellular localization versus photosensitization efficiency

We present a study on whether and to what extent subcellular localization may compete favorably with photosensitization efficiency with respect to the overall efficiency of photoinduced cell death. We have compared the efficiency with which two cationic photosensitizers, namely methylene blue (MB) and crystal violet (CV), induce the photoinduced death of human cervical adenocarcinoma (HeLa) cells. Whereas MB is well known to generate singlet oxygen and related triplet excited species with high quantum yields in a variety of biological and chemical environments (i.e., acting as a typical type II photosensitizer), the highly mitochondria-specific CV produces triplet species and singlet oxygen with low yields, acting mostly via the classical type I mechanism (e.g., via free radicals). The findings described here indicate that the presumably more phototoxic type II photosensitizer (MB) does not lead to higher degrees of cell death compared to the type I (CV) photosensitizer. In fact, CV kills cells with the same efficiency as MB, generating at least 10 times fewer photoinduced reactive species. Therefore, subcellular localization is indeed more important than photochemical reactivity in terms of overall cell killing, with mitochondrial localization representing a highly desirable property for the development of more specific/efficient photosensitizers for photodynamic therapy applications. (C) 2011 Elsevier Inc. All rights reserved.

A ditryptophan cross-link is responsible for the covalent dimerization of human superoxide dismutase 1 during its bicarbonate-dependent peroxidase activity

Unlike intermolecular disulfide bonds, other protein cross-links arising from oxidative modifications cannot be reversed and are presumably more toxic to cells because they may accumulate and induce protein aggregation. However, most of these irreversible protein cross-links remain poorly characterized. For instance, the antioxidant enzyme human superoxide dismutase 1 (hSod1) has been reported to undergo non-disulfide covalent dimerization and further oligomerization during its bicarbonate-dependent peroxidase activity. The dimerization was shown to be dependent on the oxidation of the single, solvent-exposed TrP(32) residue of hSod1, but the covalent dimer was not isolated nor was its structure determined. In this work, the hSod1 covalent dimer was isolated, digested with trypsin in H(2)O and H(2)(18)O, and analyzed by UV-Vis spectroscopy and mass spectrometry (MS). The results demonstrate that the covalent dimer consists of two hSod1 subunits cross-linked by a ditryptophan, which contains a bond between C3 and N1 of the respective Trp(32) residues. We further demonstrate that the cross-link cleaves under usual MS/MS conditions leading to apparently unmodified Trp(32), partially hinders proteolysis, and provides a mechanism to explain the formation of hSod1 covalent trimers and tetramers. This characterization of the covalent hSod1 dimer identifies a novel oxidative modification of protein Trp residues and provides clues for studying its occurrence in vivo. (C) 2010 Elsevier Inc. All rights reserved.

Effect of flavonoids on 2 `-deoxyguanosine and DNA oxidation caused by singlet molecular oxygen

Antioxidant potential is generally investigated by assaying the ability of a compound to protect biological systems from free radicals. However, non-radical reactive oxygen species can also be harmful. Singlet molecular oxygen ((1)O(2)) is generated by energy transfer to molecular oxygen. The resulting (1)O(2) is able to oxidize the nucleoside 2`-deoxyguanosine (dGuo), which leads to the formation of 8-oxo-7,8-dihydro-2`-deoxyguanosine (8-oxodGuo) and spiroiminodihydantoin 2`-deoxyribonucleoside diastereomers (dSp) in an aqueous solution. The main objective of the present study was to verify whether the presence of flavonoids (flavone, apigenin, quercetin, morin and catechin) at different concentrations could protect dGuo from (1)O(2) damage. Of the tested flavonoids, flavone possessed antioxidant activity, as determined by a decrease in the formation of both products. Apigenin, morin, quercetin and catechin all increased the formation of 8-oxodGuo at a concentration of 100 mu M. The quantification of plasmid strand breaks after treatment with formamidopyrimidine-DNA glycosylase showed that flavone protected and quercetin and catechin enhanced DNA oxidation. Our results show that compounds, such as flavonoids, may affect the product distribution of (1)O(2)-mediated oxidation of dGuo, and, in particular, high concentrations of flavonoids with hydroxyl groups in their structure lead to an increase in the formation of the mutagenic lesion 8-oxodGuo. (C) 2010 Elsevier Ltd. All rights reserved.

Myoglobin-H(2)O(2) catalyzes the oxidation of beta-ketoacids to alpha-dicarbonyls: Mechanism and implications in ketosis

Acetoacetate (AA) and 2-methylacetoacetate (MAA) are accumulated in metabolic disorders such as diabetes and isoleucinemia. Here we examine the mechanism of AA and MAA aerobic oxidation initiated by myoglobin (Mb)/H(2)O(2). We propose a chemiluminescent route involving a dioxetanone intermediate whose thermolysis yields triplet alpha-dicarbonyl species (methylglyoxal and diacetyl). The observed ultraweak chemiluminescence increased linearly on raising the concentration of either Mb (10-500 mu M) or AA (10-100 mM). Oxygen uptake studies revealed that MAA is almost a 100-fold more reactive than AA. EPR spin-trapping studies with MNP/MAA revealed the intermediacy of an alpha-carbon-centered radical and acetyl radical. The latter radical, probably derived from triplet diacetyl, is totally suppressed by sorbate, a well-known quencher of triplet carbonyls. Furthermore, an EPR signal assignable to MNP-AA(center dot) adduct was observed and confirmed by isotope effects. Oxygen consumption and a-dicarbonyl yield were shown to be dependent on AA or MAA concentrations (1-50 mM) and on H(2)O(2) or tert-butOOH added to the Mb-containing reaction mixtures. That ferrylMb is involved in a peroxidase cycle acting on the substrates is suggested by the reaction pH profiles and immunospin-trapping experiments. The generation of radicals and triplet dicarbonyl products by Mb/H(2)O(2)/beta-ketoacids may contribute to the adverse health effects of ketogenic unbalance. (C) 2011 Elsevier Inc. All rights reserved.

Tempol ameliorates murine viral encephalomyelitis by preserving the blood-brain barrier, reducing viral load, and lessening inflammation

Multiple sclerosis (MS) is a progressive inflammatory and/or demyelinating disease of the human central nervous system (CNS). Most of the knowledge about the pathogenesis of MS has been derived from murine models, such as experimental autoimmune encephalomyelitis and vital encephalomyelitis. Here, we infected female C57BL/6 mice with a neurotropic strain of the mouse hepatitis virus (MHV-59A) to evaluate whether treatment with the multifunctional antioxidant tempol (4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy) affects the ensuing encephalomyelitis. In untreated animals, neurological symptoms developed quickly: 90% of infected mice died 10 days after virus inoculation and the few survivors presented neurological deficits. Treatment with tempol (24 mg/kg, ip, two doses on the first day and daily doses for 7 days plus 2 mM tempol in the drinking water ad libitum) profoundly altered the disease outcome: neurological symptoms were attenuated, mouse survival increased up to 70%, and half of the survivors behaved as normal mice. Not Surprisingly, tempol substantially preserved the integrity of the CNS, including the blood-brain barrier. Furthermore, treatment with tempol decreased CNS vital titers, macrophage and T lymphocyte infiltration, and levels of markers of inflammation, such as expression of inducible nitric oxide synthase, transcription of tumor necrosis factor-alpha and interferon-gamma, and protein nitration. The results indicate that tempol ameliorates murine viral encephalomyelitis by altering the redox status of the infectious environment that contributes to an attenuated CNS inflammatory response. overall, our study supports the development of therapeutic strategies based on nitroxides to manage neuroinflammatory diseases, including MS. (C) 2009 Elsevier Inc. All rights reserved.

Mitochondrial ATP-sensitive K(+) channels as redox signals to liver mitochondria in response to hypertriglyceridemia

We have recently demonstrated that hypertriglyceridemic (HTG) mice present both elevated body metabolic rates and mild mitochondrial uncoupling in the liver owing to stimulated activity of the ATP-sensitive potassium channel (mitoK(ATP)). Because lipid excess normally leads to cell redox imbalance, we examined the hepatic oxidative status in this model. Cell redox imbalance was evidenced by increased total levels of carbonylated proteins, malondialdehydes, and GSSG/GSH ratios in HTG livers compared to wild type. In addition, the activities of the extramitochondrial enzymes NADPH oxidase and xanthine oxidase were elevated in HTG livers. In contrast, Mn-superoxide dismutase activity and content, a mitochondrial matrix marker, were significantly decreased in HTG livers. isolated HTG liver mitochondria presented lower rates of H(2)O(2) production, which were reversed by mitoK(ATP) antagonists. In vivo antioxidant treatment with N-acetylcysteine decreased both mitoKATP activity and metabolic rates in HTG mice. These data indicate that high levels of triglycerides increase reactive oxygen generation by extramitochondrial enzymes that promote MitoK(ATP) activation. The mild uncoupling mediated by mitoK(ATP) increases metabolic rates and protects mitochondria against oxidative damage. Therefore, a biological role for mitoK(ATP) is a redox sensor is shown here for the first time in an in vivo model of systemic and cellular lipid excess, (C) 2009 Elsevier Inc. All rights reserved.

Occupational airborne contamination in south Brazil: 1. Oxidative stress detected in the blood of coal miners

Reactive oxygen species and nitrogen species have been implicated in the pathogenesis of coal dust-induced toxicity. The present study investigated several oxidative stress biomarkers (Contents of lipoperoxidation = TBARS, reduced = GSH, oxidized = GSSG and total glutathione = TG, alpha-tocopherol, and the activities of glutathione S-transferase = GST, glutathione reductase = GR, glutathione peroxidase = GPx, catalase = CAT and superoxide dismutase = SOD), in the blood of three different groups (n = 20 each) exposed to airborne contamination associated with coal mining activities: underground workers directly exposed, surface workers indirectly exposed, residents indirectly exposed (subjects living near the mines), and controls (non-exposed subjects). Plasma TBARS were increased and whole blood TG and GSH levels were decreased in all groups compared to controls. Plasma alpha-tocopherol contents showed approximately half the values in underground workers compared to controls. GST activity was induced in workers and also in residents at the vicinity of the mining plant, whilst CAT activity was induced only in mine workers. SOD activity was decreased in all groups examined, while GPx activity showed decreased values only in underground miners, and GR did not show any differences among the groups. The results showed that subjects directly and indirectly exposed to coal dusts face an oxidative stress condition. They also indicate that people living in the vicinity of the mine plant are in health risk regarding coal mining-related diseases.

Double-strand DNA cleavage induced by oxindole-Schiff base copper(II) complexes with potential antitumor activity

Some oxindole-Schiff base copper(II) complexes have already shown potential antitumor activity towards different cells, inducing apoptosis in a process modulated by the ligand, and having nuclei and mitochondria as main targets. Here, three novel copper(II) complexes with analogous ligands were isolated and characterized by spectroscopic techniques, having their reactivity compared to the so far most active complex in this class. Cytotoxicity experiments carried out toward human neuroblastoma SH-SY5Y cells confirmed its proapoptosis property. DNA cleavage studies were then performed in the presence of these complexes, in order to verify the influence of ligand structural features in its nuclease activity. All of them were able to cause double-strand DNA scissions, giving rise to nicked circular Form II and linear Form III species, in the presence of hydrogen peroxide. Additionally, DNA Form II was also detected in the absence of peroxide when the most active complex, [Cu(isaepy)(2)](2+) 1, was used. In an effort to better elucidate their interactions with DNA, solutions of the different complexes titrated with DNA had their absorption spectra monitored. An absorbance hyperchromism observed at 260 nm pointed to the intercalation of these complexes into the DNA structure. Further, investigations of 2-deoxy-D-ribose (DR) oxidation catalyzed by each of those complexes, using 2-thiobarbituric acid reactive species (TBARS) method, and detection of reactive oxygen species (ROS) formation by spin-trapping EPR, suggested that their mechanism of action in performing efficiently DNA cleavage occurs preferentially, but not only by oxidative pathways. (C) 2007 Elsevier Inc. All rights reserved.

Mild mitochondrial uncoupling in mice affects energy metabolism, redox balance and longevity

Caloric restriction is the most effective non-genetic intervention to enhance lifespan known to date. A major research interest has been the development of therapeutic strategies capable of promoting the beneficial results of this dietary regimen. In this sense, we propose that compounds that decrease the efficiency of energy conversion, such as mitochondrial uncouplers, can be caloric restriction mimetics. Treatment of mice with low doses of the protonophore 2,4-dinitrophenol promotes enhanced tissue respiratory rates, improved serological glucose, triglyceride and insulin levels, decrease of reactive oxygen species levels and tissue DNA and protein oxidation, as well as reduced body weight. Importantly, 2,4-dinitrophenol-treated animals also presented enhanced longevity. Our results demonstrate that mild mitochondrial uncoupling is a highly effective in vivo antioxidant strategy, and describe the first therapeutic intervention capable of effectively reproducing the physiological, metabolic and lifespan effects of caloric restriction in healthy mammals.