Superoxide dismutase 3-mediated cell survival and proliferation

This dissertation studies the signaling events mediated by the extracellular superoxide dismutase (SOD3). SOD3 is an antioxidant enzyme which converts the harmful superoxide into hydrogen peroxide. Overproduction of these reactive oxygen species (ROS) in the cellular environment as a result of tissue injury or impaired antioxidant defense system has detrimental effects on tissue integrity and function. However, especially hydrogen peroxide is also an important signaling agent. Ischemic injury in muscle causes acute oxidative stress and inflammation. We investigated the ability of SOD3 to attenuate ischemia induced inflammation and to promote recovery of skeletal muscle tissue. We found that SOD3 can downregulate the expression of several inflammatory cytokines and cell adhesion molecules thus preventing the accumulation of oxidant-producing inflammatory cells. Secondly, SOD3 was able to promote long-term activation of the mitogenic Erk pathway, but increased only briefly the activity of pro-survival Akt pathway at an early stage of ischemic inflammation, thus reducing apoptosis. SOD3 is a prominent antioxidant in the thyroid gland where oxidative stress is constantly present. We investigated the role of SOD3 in normal thyroid follicular cells and the changes in its expression in various hyperproliferative disorders. We first showed that SOD3 is TSH-responsive which indicated its participation in thyroid function. Its principal function seems to be in follicular cell proliferation since knockdown cells were deficient in proliferation. Additionally, it was overexpressed in goiter tissue. However, SOD3 was consistently downregulated in thyroid cancer cell lines and tissues. In conclusion, SOD3 is involved in tissue maintenance, cell proliferation and inflammatory cell migration. Its mechanisms of action are the activation of known proliferation/survival pathways, inhibition of apoptosis and regulation of adhesion molecule expression.

Oxidative metabolism and muscle biochemical profile of polo horses supplemented with an ADE vitamin complex

Horses used for the game of polo experience abrupt and frequent changes in exercise intensity. To meet this variable energy demand, the horses use both aerobic and anaerobic pathways in varying proportions and intensities. In this context, there must be a balance between the formation of reactive oxygen species (ROS) and the action of antioxidants to prevent oxidative stress and its consequences. The effect of supplementation with an ADE vitamin complex on oxidative metabolism was evaluated in 18 crossbred horses randomly divided between a treated group (TG) and a control group (CG). The TG animals received the ADE vitamin complex (1mL/50 kg of body weight) by deep intramuscular injection at 30 and 15 days before the game. The CG horses received 10ml of saline by the same administration route and schedule. During the polo match, the animals played for a total of 7.5 min. Blood samples were collected on the same days as the treatments were administered, and immediately before and at 15, 90 and 180 minutes after the game. The concentrations of creatine phosphokinase (CK), lactate dehydrogenase (LDH), lactate, glucose, aspartate aminotransferase (AST), glutathione (GSH), superoxide dismutase (SOD) and malondialdehyde (MDA) were measured in the blood samples. After the game, the TG demonstrated higher levels of AST, lactate and glucose than the CG, suggesting more efficient energy use by the treated animals. The higher GSH and lower lactate levels in the TG before the game suggest the presence of a greater antioxidant supply in the treated animals. The maintenance of the MDA levels indicates that neither of the groups exhibited oxidative stress.

Polygala extraaxillaris: oxidative stress in Brachiaria decumbens mediated by volatile oils

The volatile oils extracted from the roots of Polygala extraaxillaris were analyzed to assess whether they increase oxidative stress in Brachiaria decumbens var. Piatã, as well as to assess their effect on cellular division and cytotoxicity in laboratory. Six concentrations were used (0%, 0.35%, 0.65%, 1.25%, 0.65%, and 5.0%) with four repetitions of 25 seeds. The substance 1-(2-hydroxyphenyl) - ethanone was identified as the major constituent of the volatile oils. The results showed that the highest concentrations of the oils resulted in an increase in the oxidative stress in B. decumbens, as well as alteration in germination and growth, with a consequent reduction in the process of cellular division, causing changes in the growth standard and antioxidant defense.

Photochemistry and photobiology of actinic erythema: defensive and reparative cutaneous mechanisms

Sunlight is part of our everyday life and most people accept it as beneficial to our health. With the advance of our knowledge in cutaneous photochemistry, photobiology and photomedicine over the past four decades, the terrestrial solar radiation has become a concern of dermatologists and is considered to be a major damaging environmental factor for our skin. Most photobiological effects (e.g., sunburn, suntanning, local and systemic immunosuppression, photoaging or dermatoheliosis, skin cancer and precancer, etc.) are attributed to ultraviolet radiation (UVR) and more particularly to UVB radiation (290-320 nm). UVA radiation (320-400 nm) also plays an important role in the induction of erythema by the photosensitized generation of reactive oxygen species (singlet oxygen (1O2), superoxide (O2.-) and hydroxyl radicals (.OH)) that damage DNA and cellular membranes, and promote carcinogenesis and the changes associated with photoaging. Therefore, research efforts have been directed at a better photochemical and photobiological understanding of the so-called sunburn reaction, actinic or solar erythema. To survive the insults of actinic damage, the skin appears to have different intrinsic defensive mechanisms, among which antioxidants (enzymatic and non-enzymatic systems) play a pivotal role. In this paper, we will review the basic aspects of the action of UVR on the skin: a) photochemical reactions resulting from photon absorption by endogenous chromophores; b) the lipid peroxidation phenomenon, and c) intrinsic defensive cutaneous mechanisms (antioxidant systems). The last section will cover the inflammatory response including mediator release after cutaneous UVR exposure and adhesion molecule expression

Alternative oxidase in the branched mitochondrial respiratory network: an overview on structure, function, regulation, and role

Plants and some other organisms including protists possess a complex branched respiratory network in their mitochondria. Some pathways of this network are not energy-conserving and allow sites of energy conservation to be bypassed, leading to a decrease of the energy yield in the cells. It is a challenge to understand the regulation of the partitioning of electrons between the various energy-dissipating and -conserving pathways. This review is focused on the oxidase side of the respiratory chain that presents a cyanide-resistant energy-dissipating alternative oxidase (AOX) besides the cytochrome pathway. The known structural properties of AOX are described including transmembrane topology, dimerization, and active sites. Regulation of the alternative oxidase activity is presented in detail because of its complexity. The alternative oxidase activity is dependent on substrate availability: total ubiquinone concentration and its redox state in the membrane and O2 concentration in the cell. The alternative oxidase activity can be long-term regulated (gene expression) or short-term (post-translational modification, allosteric activation) regulated. Electron distribution (partitioning) between the alternative and cytochrome pathways during steady-state respiration is a crucial measurement to quantitatively analyze the effects of the various levels of regulation of the alternative oxidase. Three approaches are described with their specific domain of application and limitations: kinetic approach, oxygen isotope differential discrimination, and ADP/O method (thermokinetic approach). Lastly, the role of the alternative oxidase in non-thermogenic tissues is discussed in relation to the energy metabolism balance of the cell (supply in reducing equivalents/demand in energy and carbon) and with harmful reactive oxygen species formation.

Nonspecific blockade of vascular free radical signals by methylated arginine analogues

Methylated arginine analogues are often used as probes of the effect of nitric oxide; however, their specificity is unclear and seems to be frequently overestimated. This study analyzed the effects of NG-methyl-L-arginine (L-NMMA) on the endothelium-dependent release of vascular superoxide radicals triggered by increased flow. Plasma ascorbyl radical signals measured by direct electron paramagnetic resonance spectroscopy in 25 rabbits increased by 3.8 ± 0.7 nmol/l vs baseline (28.7 ± 1.4 nmol/l, P<0.001) in response to papaverine-induced flow increases of 121 ± 12%. In contrast, after similar papaverine-induced flow increases simultaneously with L-NMMA infusions, ascorbyl levels were not significantly changed compared to baseline. Similar results were obtained in isolated rabbit aortas perfused ex vivo with the spin trap a-phenyl-N-tert-butylnitrone (N = 22). However, in both preparations, this complete blockade was not reversed by co-infusion of excess L-arginine and was also obtained by N-methyl-D-arginine, thus indicating that it is not related to nitric oxide synthase. L-arginine alone was ineffective, as previously demonstrated for NG-methyl-L-arginine ester (L-NAME). In vitro, neither L-arginine nor its analogues scavenged superoxide radicals. This nonspecific activity of methylated arginine analogues underscores the need for careful controls in order to assess nitric oxide effects, particularly those related to interactions with active oxygen species.

Pp2a: At The Crossroads Between Growth and Defence In Plants

Living organisms manage their resources in well evolutionary-preserved manner to grow and reproduce. Plants are no exceptions, beginning from their seed stage they have to perceive environmental conditions to avoid germination at wrong time or rough soil. Under favourable conditions, plants invest photosynthetic end products in cell and organ growth to provide best possible conditions for generation of offspring. Under natural conditions, however, plants are exposed to a multitude of environmental stress factors, including high light and insufficient light, drought and flooding, various bacteria and viruses, herbivores, and other plants that compete for nutrients and light. To survive under environmental challenges, plants have evolved signaling mechanisms that recognise environmental changes and perform fine-tuned actions that maintain cellular homeostasis. Controlled phosphorylation and dephosphorylation of proteins plays an important role in maintaining balanced flow of information within cells. In this study, I examined the role of protein phosphatase 2A (PP2A) on plant growth and acclimation under optimal and stressful conditions. To this aim, I studied gene expression profiles, proteomes and protein interactions, and their impacts on plant health and survival, taking advantage of the model plant Arabidopsis thaliana and the mutant approach. Special emphasis was made on two highly similar PP2A-B regulatory subunits, B’γ and B’ζ. Promoters of B’γ and B’ζ were found to be similarly active in the developing tissues of the plant. In mature leaves, however, the promoter of B’γ was active in patches in leaf periphery, while the activity of B’ζ promoter was evident in leaf edges. The partially overlapping expression patterns, together with computational models of B’γ and B’ζ within trimeric PP2A holoenzymes suggested that B’γ and B’ζ may competitively bind into similar PP2A trimmers and thus influence each other’s actions. Arabidopsis thaliana pp2a-b’γ and pp2a-b’γζ double mutants showed dwarfish phenotypes, indicating that B’γ and B’ζ are needed for appropriate growth regulation under favorable conditions. However, while pp2a-b’γ displayed constitutive immune responses and appearance of premature yellowings on leaves, the pp2a-b’γζ double mutant supressed these yellowings. More detailed analysis of defense responses revealed that B’γ and B’ζ mediate counteracting effects on salicylic acid dependent defense signalling. Associated with this, B’γ and B’ζ were both found to interact in vivo with CALCIUM DEPENDENT PROTEIN KINASE 1 (CPK1), a crucial element of salicylic acid signalling pathway against pathogens in plants. In addition, B’γ was shown to modulate cellular reactive oxygen species (ROS) metabolism by controlling the abundance of ALTERNATIVE OXIDASE 1A and 1D in mitochondria. PP2A B’γ and B’ζ subunits turned out to play crucial roles in the optimization of plant choices during their development. Taken together, PP2A allows fluent responses to environmental changes, maintenance of plant homeostasis, and grant survivability with minimised cost of redirection of resources from growth to defence.

The effect of porphyrins on normal and transformed mouse cell lines in the presence of visible light

Photodynamic therapy consists of the uptake of a photosensitizing dye, often a porphyrin, by tumor tissue and subsequent irradiation of the tumor with visible light of an appropriate wavelength matched to the absorption spectrum of the photosensitizing dye. This class of molecules produces reactive oxygen species when activated by light, resulting in a direct or indirect cytotoxic effect on the target cells. Photodynamic therapy has been used in the treatment of cancer but the technology has a potential for the treatment of several disease conditions mainly because of its selectivity. However, it is not clear why the porphyrins are retained preferentially by abnormal tissue. This paper describes a study of the effect of the association of porphyrin and visible light on two mouse fibroblast cell lines: A31, normal cells and B61, an EJ-ras transformed variant of A31. Two water-soluble porphyrins were used, a positively charged one, tetra(N-methyl-4-pyridyl)porphyrin chloride, and a negatively charged one, tetra(4-sulfonatophenyl)porphyrin-Na salt (TPPS4) in order to assess the effect on cell survival. The results suggest that the B61 cell line is more sensitive to incubation with the anionic porphyrin (TPPS4) followed by light irradiation and that the anionic porphyrin is more efficient in killing the cells than the cationic porphyrin.

Effect of L-arginine, dimercaptosuccinic acid (DMSA) and the association of L-arginine and DMSA on tissue lead mobilization and blood pressure level in plumbism

Lead (Pb)-induced hypertension is characterized by an increase in reactive oxygen species (ROS) and a decrease in nitric oxide (NO). In the present study we evaluated the effect of L-arginine (NO precursor), dimercaptosuccinic acid (DMSA, a chelating agent and ROS scavenger), and the association of L-arginine/DMSA on tissue Pb mobilization and blood pressure levels in plumbism. Tissue Pb levels and blood pressure evolution were evaluated in rats exposed to: 1) Pb (750 ppm, in drinking water, for 70 days), 2) Pb plus water for 30 more days, 3) Pb plus DMSA (50 mg kg-1 day-1, po), L-arginine (0.6%, in drinking water), and the combination of L-arginine/DMSA for 30 more days, and 4) their respective matching controls. Pb exposure increased Pb levels in the blood, liver, femur, kidney and aorta. Pb levels in tissues decreased after cessation of Pb administration, except in the aorta. These levels did not reach those observed in nonintoxicated rats. All treatments mobilized Pb from the kidney, femur and liver. Pb mobilization from the aorta was only effective with the L-arginine/DMSA treatment. Blood Pb concentrations in Pb-treated groups were not different from those of the Pb/water group. Pb increased blood pressure starting from the 5th week. L-arginine and DMSA treatments (4th week) and the combination of L-arginine/DMSA (3rd and 4th weeks) decreased blood pressure levels of intoxicated rats. These levels did not reach those of nonintoxicated rats. Treatment with L-arginine/DMSA was more effective than the isolated treatments in mobilizing Pb from tissues and in reducing the blood pressure of intoxicated rats.

Antioxidant properties of natural compounds used in popular medicine for gastric ulcers

There is evidence concerning the participation of reactive oxygen species in the etiology and physiopathology of human diseases, such as neurodegenerative disorders, inflammation, viral infections, autoimmune pathologies, and digestive system disorders such as gastrointestinal inflammation and gastric ulcer. The role of these reactive oxygen species in several diseases and the potential antioxidant protective effect of natural compounds on affected tissues are topics of high current interest. To consider a natural compound or a drug as an antioxidant substance it is necessary to investigate its antioxidant properties in vitro and then to evaluate its antioxidant functions in biological systems. In this review article, we shall consider the role of natural antioxidants derived from popular plants to reduce or prevent the oxidative stress in gastric ulcer induced by ethanol.

Recent advances in angiotensin II signaling

Angiotensin II (Ang II)* is a multifunctional hormone that influences the function of cardiovascular cells through a complex series of intracellular signaling events initiated by the interaction of Ang II with AT1 and AT2 receptors. AT1 receptor activation leads to cell growth, vascular contraction, inflammatory responses and salt and water retention, whereas AT2 receptors induce apoptosis, vasodilation and natriuresis. These effects are mediated via complex, interacting signaling pathways involving stimulation of PLC and Ca2+ mobilization; activation of PLD, PLA2, PKC, MAP kinases and NAD(P)H oxidase, and stimulation of gene transcription. In addition, Ang II activates many intracellular tyrosine kinases that play a role in growth signaling and inflammation, such as Src, Pyk2, p130Cas, FAK and JAK/STAT. These events may be direct or indirect via transactivation of tyrosine kinase receptors, including PDGFR, EGFR and IGFR. Ang II induces a multitude of actions in various tissues, and the signaling events following occupancy and activation of Ang receptors are tightly controlled and extremely complex. Alterations of these highly regulated signaling pathways may be pivotal in structural and functional abnormalities that underlie pathological processes in cardiovascular diseases such as cardiac hypertrophy, hypertension and atherosclerosis.

Glutathione peroxidase induction protects Saccharomyces cerevisiae sod1deltasod2delta double mutants against oxidative damage

Saccharomyces cerevisiae mutants deficient in superoxide dismutase genes (sod1delta, sod2delta and the double mutant) were subjected to H2O2 stress in the stationary phase. The highest sensitivity was observed in the sod2delta mutant, while the sod1deltasod2delta double mutant was not sensitive. Sod mutants had lower catalase activity (44%) than wild-type cells, independent of H2O2 stress. Untreated cells of sod1deltasod2delta double mutants showed increased glutathione peroxidase activity (126%), while sod1delta had lower activity (77%) than the wild type. Glutathione levels in sod1delta were increased (200-260%) after exposure to various H2O2 concentrations. In addition, the highest malondialdehyde levels could be observed without H2O2 treatment in sod1delta (167%) and sod2delta (225%) mutants. In contrast, the level of malondialdehyde in the sod1deltasod2delta double mutant was indistinguishable from that of the wild type. These results suggest that resistance to H2O2 by sod1deltasod2delta cells depends on the induction of glutathione peroxidase and is independent of catalase, and that glutathione is a primary antioxidant in the defense against H2O2 in stationary phase sod1delta mutants.

Protection of plasmid DNA by a Ginkgo biloba extract from the effects of stannous chloride and the action on the labeling of blood elements with technetium-99m

Ginkgo biloba extract (EGb) is a phytotherapeutic agent used for the treatment of ischemic and neurological disorders. Because the action of this important extract is not fully known, assays using different biological systems need to be performed. Red blood cells (RBC) are labeled with technetium-99m (Tc-99m) and used in nuclear medicine. The labeling depends on a reducing agent, usually stannous chloride (SnCl2). We assessed the effect of different concentrations of EGb on the labeling of blood constituents with Tc-99m, as sodium pertechnetate (3.7 MBq), and on the mobility of a plasmid DNA treated with SnCl2 (1.2 µg/ml) at room temperature. Blood was incubated with EGb before the addition of SnCl2 and Tc-99m. Plasma (P) and RBC were separated and precipitated with trichloroacetic acid, and soluble (SF-P and SF-RBC) and insoluble (IF-P and IF-RBC) fractions were isolated. The plasmid was incubated with Egb, SnCl2 or EGb plus SnCl2 and agarose gel electrophoresis was performed. The gel was stained with ethidium bromide and the DNA bands were visualized by fluorescence in an ultraviolet transilluminator system. EGb decreased the labeling of RBC, IF-P and IF-RBC. The supercoiled form of the plasmid was modified by treatment with SnCl2 and protected by 40 mg/ml EGb. The effect of EGb on the tested systems may be due to its chelating action with the stannous ions and/or pertechnetate or to the capability to generate reactive oxygen species that could oxidize the stannous ion.

Elevated levels of erythrocyte-conjugated dienes indicate increased lipid peroxidation in schistosomiasis mansoni patients

Schistosoma mansoni causes liver disease by inducing granulomatous inflammation. This favors formation of reactive oxygen species, including superoxide ions, hydrogen peroxide and hydroxyl radicals all of which may induce lipid peroxidation. We have evaluated lipid peroxidation in 18 patients with hepatosplenic schistosomiasis mansoni previously treated with oxamniquine followed by splenectomy, ligature of the left gastric vein and auto-implantation of spleen tissue, by measuring levels of erythrocyte-conjugated dienes and plasma malondialdehyde (MDA). Age-matched, healthy individuals (N = 18) formed the control group. Erythrocyte-conjugated dienes were extracted with dichloromethane/methanol and quantified by UV spectrophotometry, while plasma MDA was measured by reaction with thiobarbituric acid. Patient erythrocytes contained two times more conjugated dienes than control cells (584.5 ± 67.8 vs 271.7 ± 20.1 µmol/l, P < 0.001), whereas the increase in plasma MDA concentration (about 10%) was not statistically significant. These elevated conjugated dienes in patients infected by S. mansoni suggest increased lipid peroxidation in cell membranes, although this was not evident when a common marker of oxidative stress, plasma MDA, was measured. Nevertheless, these two markers of lipid peroxidation, circulating MDA and erythrocyte-conjugated dienes, correlated significantly in both patient (r = 0.62; P < 0.01) and control (r = 0.57; P < 0.05) groups. Our data show that patients with schistosomiasis have abnormal lipid peroxidation, with elevated erythrocyte-conjugated dienes implying dysfunctional cell membranes, and also imply that this may be attenuated by the redox capacity of antioxidant agents, which prevent accumulation of plasma MDA.

Superoxide release and cellular gluthatione peroxidase activity in leukocytes from children with persistent asthma

Asthma is an inflammatory condition characterized by the involvement of several mediators, including reactive oxygen species. The aim of the present study was to investigate the superoxide release and cellular glutathione peroxidase (cGPx) activity in peripheral blood granulocytes and monocytes from children and adolescents with atopic asthma. Forty-four patients were selected and classified as having intermittent or persistent asthma (mild, moderate or severe). The spontaneous or phorbol myristate acetate (PMA, 30 nM)-induced superoxide release by granulocytes and monocytes was determined at 0, 5, 15, and 25 min. cGPx activity was assayed spectrophotometrically. The spontaneous superoxide release by granulocytes from patients with mild (N = 15), moderate (N = 12) or severe (N = 6) asthma was higher at 25 min compared to healthy individuals (N = 28, P < 0.05, Duncan test). The PMA-induced superoxide release by granulocytes from patients with moderate (N = 12) or severe (N = 6) asthma was higher at 15 and 25 min compared to healthy individuals (N = 28, P < 0.05 in both times of incubation, Duncan test). The spontaneous or PMA-induced superoxide release by monocytes from asthmatic patients was similar to healthy individuals (P > 0.05 in all times of incubation, Duncan test). cGPx activity of granulocytes and monocytes from patients with persistent asthma (N = 20) was also similar to healthy individuals (N = 10, P > 0.05, Kruskal-Wallis test). We conclude that, under specific circumstances, granulocytes from children with persistent asthma present a higher respiratory burst activity compared to healthy individuals. These findings indicate a risk of oxidative stress, phagocyte auto-oxidation, and the subsequent release of intracellular toxic oxidants and enzymes, leading to additional inflammation and lung damage in asthmatic children.