Prolonged swimming promotes cellular oxidative stress and p66Shc phosphorylation, but does not induce oxidative stress in mitochondria in the rat heart.

Exercise-induced changes in p66Shc-dependent signaling pathway are still not fully understood. The p66Shc protein is one of the key players in cell signaling, particularly in response to oxidative stress. Therefore, the aim of this study was to investigate the effect of prolonged swimming on the phosphorylation of p66Shc as well as the induction of mitochondrial and cellular oxidative stress in rat hearts. Male Wistar rats were divided into a sedentary control group and an exercise group. The exercised rats swam for 3 hours and were burdened with an additional 3% of their body weight. After the cessation of exercise, their hearts were removed immediately for experiments. The exercise protocol caused increased levels of the following oxidative stress parameters in cardiac cells: DNA damage, protein carbonyls, and lipid dienes. There was also increased phosphorylation of p66Shc without any alterations in Akt and extracellular signal-regulated kinases. Changes in the ferritin L levels and the L to H subunit ratio were also observed in the exercised hearts compared with the control hearts. Despite increased phosphorylation of p66Shc, no significant increase was observed in either mitochondrial H2O2 release or mitochondrial oxidative stress markers. Regardless of the changes in phosphorylation of p66Shc, the antioxidant enzyme activities (superoxide dismutase and catalase) and anti-apoptotic (Bcl2), and pro-apoptotic (Bax) protein levels were not affected by prolonged swimming. Further studies are required to investigate whether p66Shc phosphorylation is beneficial or detrimental to cardiac cells after exercise cessation.

Protective effect of alpha-lipoic acid on cypermethrin-induced oxidative stress in Wistar rats.

Cypermethrin (CY), a class II pyrethroid pesticide, is globally used to control insects in the household and in agriculture. Despite beneficial roles, its uncontrolled and repetitive application leads to unintended effects in non-target organisms. In light of the relevant anti-oxidant properties of alpha-lipoic acid (ALA), in the work described herein we tested the effect of a commercially available ALA formulation on cypermethrin CY)-induced oxidative stress in Wistar rats. The rats were orally administered with 53.14 mg/kg of ALA and 35.71 mg/kg of CY for 60 days. The treatment with CY did not induce changes in either locomotor activities or in body weight. Differences were observed on superoxide dismutase (SOD), catalase (CAT) and lipid peroxidation that were re-established by ALA treatment at similar levels of the placebo group. Furthermore, ALA formulation increased glutathione (GSH) level and glutathione peroxidase (GPx) activity. Because of the widespread use of CY, higher amounts of pesticide residues are present in food, and a diet supplementation with ALA could be an active free radical scavenger protecting against diseases associated with oxidative stress.

The primary role of glutathione against nuclear DNA damage of striatum induced by permethrin in rats.

Pyrethroids are one of the most widely used class of insecticides and their toxicity is dominated by pharmacological actions upon the CNS. This study reports as the subchronic treatment (60 days) with permethrin (PERM) (1/10 of LD(50)) induced nuclear DNA damage in rat striatum cells. Comet assay outcomes showed that PERM produced single- and double-strand breaks in striatum cells, the DNA damage was not related to oxidation at pyrimidine and purine bases. Vitamin E (280 mg/kg body weight/day) and vitamin E+coenzyme Q(10) (10 mg/kg/3 ml) supplementation could protect PERM treated rats against nuclear DNA damage. With the aim to evaluate the cause of nuclear DNA damage observed in striatum of rat treated with PERM, in vitro studies on striatum submitochondrial particles (SMPs) and on striatum cells treated with 10 muM PERM alone or plus 16 or 32 nM GSH were performed. SMPs incubated with PERM showed a decrease in superoxide anion release from the electron transport chain by inhibition of mitochondrial complex I. The effect could be related to the decrease of membrane fluidity measured in the hydrophilic-hydrophobic region of the mitochondrial membrane. This result discarded the involvement of the mitochondrial reactive oxygen species in the nuclear DNA damage. On the contrary, GSH played a crucial role on striatum since it was able to protect the cells against nuclear DNA damage induced by PERM. In conclusion our outcomes suggested that nuclear DNA damage of striatum cells was directly related to GSH depletion due to PERM insecticide.

Effect of different organotins on DNA of mollusk (Scapharca inaequivalvis) erythrocytes assessed by the comet assay.

The alkaline comet assay, employing a single-cell gel-electrophoresis, is a rapid, simple and sensitive technique for visualizing and measuring DNA damage leading to strand breakage in individual cells. In this study, we report data about the effect of different organotin compounds (MBTC, DBTC and TBTC) on DNA from erythrocytes of the Scapharca inaequivalvis bivalve mollusc. Our results show significant DNA damage after 30 min in vitro incubation with 10microM of organotins. Since TBTC turned out to be the most genotoxic compound, followed by MBTC and DBTC, we exposed the molluscs to 50ppb of TBTC for 11 days. A significant increase of comet parameters was measured in our experimental conditions. The use of the comet test as a high-throughput screening assay to monitor the effect of environmental pollutants on marine organisms has been proposed.

Lead-induced changes in human erythrocytes and lymphocytes.

In the present work we studied, by chemiluminescence measurements, the influence of lead on the production of reactive oxygen species (ROS) in haemolysates obtained from human erythrocytes incubated in the presence of different concentrations of lead acetate. Moreover, we evaluated the modification of proteins and lipids in human erythrocyte and lymphocyte membranes by using the fluorescence probes N-(1-pyrene)maleimide (PM), laurdan and pyrene. No significant changes in chemiluminescence were detected for erythrocytes incubated with 1-10 microM lead acetate for 3 h at 37 degrees C. By increasing the lead acetate concentration in cell suspensions up to 50 microM for the same incubation time, the percentage of chemiluminescence inhibition was ca. 20%. It was shown that, after incorporating fluorescence probes in the membrane lipid bilayer of erythrocytes and lymphocytes treated with 10 and/or 50 microM lead acetate, the total fluorescence intensity and the excimer to monomer intensity ratio of PM decreased and the generalized fluorescence polarization of laurdan decreased by 10-15%. The pyrene excimerization coefficient (kappa(ex)) increased by 20% (in comparison with a magnitude of kappa(ex) for white membranes isolated from intact erythrocytes) with 6-10 microM lead acetate for 3 h at 37 degrees C. The data obtained suggest that the effect of low concentrations of lead acetate does not cause production of ROS in erythrocytes in vitro, but can change the physicochemical state of proteins and lipids in erythrocyte and lymphocyte membranes. This effect is important because it influences the enzymatic activity and the functionality of receptors and channels present at the plasma membrane level, thus modulating the molecular composition of the intracellular space and cell functions.

Vibrio cholerae periplasmic superoxide dismutase: isolation of the gene and overexpression of the protein.

Superoxide dismutases are ubiquitous enzymes which play an important role in protecting cells against oxidative damage and which have also been shown to contribute to the pathogenicity of many bacterial species. Here we demonstrate that Vibrio cholerae, the causative agent of cholerae, expresses an active periplasmic Cu,Zn superoxide dismutase. Moreover, we have set up an expression system yielding large amounts of V. cholerae recombinant Cu,Zn superoxide dismutase in the periplasm of Escherichia coli and a procedure to obtain the enzyme in a highly purified form. Unlike the bovine enzyme, V. cholerae Cu,Zn superoxide dismutase has been proved to be highly resistant to inactivation by hydrogen peroxide. This property, which appears to be common to other bacterial enzymes of this class, might improve the ability of Cu,Zn superoxide dismutase to protect bacteria against the reactive oxygen species produced by phagocytes.

Oxidative inactivation of calcineurin by Cu,Zn superoxide dismutase G93A, a mutant typical of familial amyotrophic lateral sclerosis.

Calcineurin is a serine/threonine phosphatase involved in a wide range of cellular responses to calcium mobilizing signals. Previous evidence supports the notion of the existence of a redox regulation of this enzyme, which might be relevant for neurodegenerative processes, where an imbalance between generation and removal of reactive oxygen species could occur. In a recent work, we have observed that calcineurin activity is depressed in two models for familial amyotrophic lateral sclerosis (FALS) associated with mutations of the antioxidant enzyme Cu,Zn superoxide dismutase (SOD1), namely in neuroblastoma cells expressing either SOD1 mutant G93A or mutant H46R and in brain areas from G93A transgenic mice. In this work we report that while wild-type SOD1 has a protective effect, calcineurin is oxidatively inactivated by mutant SOD1s in vitro; this inactivation is mediated by reactive oxygen species and can be reverted by addition of reducing agents. Furthermore, we show that calcineurin is sensitive to oxidation only when it is in an 'open', calcium-activated conformation, and that G93A-SOD1 must have its redox-active copper site available to substrates in order to exert its pro-oxidant properties on calcineurin. These findings demonstrate that both wild-type and mutant SOD1s can interfere directly with calcineurin activity and further support the possibility of a relevant role for calcineurin-regulated biochemical pathways in the pathogenesis of FALS.

Hemoglobin components from trout (Salmo irideus): determination of their peroxidative activity.

The peroxidative activity of trout hemoglobins, HbI and HbIV, which differ in their conformation, was compared with that of HbA. Artificial substrates (guaiacol and dopamine) and more physiological substrates such as model lipid membranes containing unsaturated fatty acids were used. The results indicate that all the hemoglobin molecules assayed show different levels of peroxidative activity. The capability to act as peroxidases is greater in HbIV than in HbI and HbA. In contrast, native globins did not show peroxidase activity. The different peroxidative activity of the Hbs is discussed in relation to stability both vs. protein oxidation and protein dissociation. The results confirm the view that hemoglobin may be of importance in establishing the life span of the erythrocyte itself.

Calcineurin activity is regulated both by redox compounds and by mutant familial amyotrophic lateral sclerosis-superoxide dismutase.

Calcineurin (CN) is a protein phosphatase involved in a wide range of cellular responses to calcium-mobilizing signals, and a role for this enzyme in neuropathology has been postulated. We have investigated the possibility that redox modulation of CN activity is relevant to neuropathological conditions where an imbalance in reactive oxygen species has been described. We have monitored CN activity in cultured human neuroblastoma SH-SY5Y cells and obtained evidence that CN activity is promoted by treatment with ascorbate or dithiothreitol and impaired by oxidative stress. Evidence for the existence of a redox regulation of this enzyme has been also obtained by overexpression of wild-type antioxidant Cu,Zn superoxide dismutase (SOD1) that promotes CN activity and protects it from oxidative inactivation. On the contrary, overexpression of mutant SOD1s associated with familial amyotrophic lateral sclerosis (FALS) impairs CN activity both in transfected human neuroblastoma cell lines and in the motor cortex of brain from FALS-transgenic mice. These data suggest that CN might be a target in the pathogenesis of SOD1-linked FALS.

A free cysteine residue at the dimer interface decreases conformational stability of Xenopus laevis copper,zinc superoxide dismutase.

The two Cu,Zn superoxide dismutases from the amphibian Xenopus laevis (denoted XSODA and XSODB) display different heat sensitivities, XSODA being more thermolabile than XSODB. In this study, we have investigated the contribution of a free cysteine residue located close to the subunit interface of XSODA to its lower thermal stability. We have found that mutation of residue Cys 150 to Ala in XSODA makes the thermal stability of this enzyme comparable to that of the wild-type XSODB isoenzyme, while the introduction of a cysteine residue in the same position of XSODB renders this enzyme variant much more heat-sensitive. Differential scanning calorimetry experiments showed that XSODA has a melting temperature about 8.5 degrees C lower than that of XSODB. On the contrary, the melting temperature of XSODACys150Ala is very close to that of XSODB, while the melting temperature of XSODBSer150Cys is even lower than that of wild-type XSODA. These data indicate that the free cysteine residue present in XSODA affects not only the reversibility of unfolding of the enzyme but also its conformational stability. We suggest that the large effect of the Cys 150 residue on XSODA stability might be due to incorrect disulfide bond formation or disulfide bond interchange during heat-induced unfolding rather than to alteration of the interaction between the enzyme subunits.

Aberrant copper chemistry as a major mediator of oxidative stress in a human cellular model of amyotrophic lateral sclerosis.

We have investigated the response to oxidative stress in a model system obtained by stable transfection of the human neuroblastoma cell line SH-SY5Y with plasmids directing constitutive expression of either wild-type human Cu,Zn superoxide dismutase or a mutant of this enzyme (H46R) associated with familial amyotrophic lateral sclerosis. We report that expression of mutant H46R Cu,Zn superoxide dismutase induces a selective increase in paraquat sensitivity that is reverted by addition of D-penicillamine. Furthermore, expression of this mutant enzyme affects the activity of the endogenous wild-type enzyme both in basal conditions and in copper overloading experiments. Our data indicate that aberrant metal chemistry of this mutant enzyme is the actual mediator of oxidative stress and that concurrent impairment of the activity of wild-type endogenous enzyme compromises the cell's ability to respond to oxidative stress.

Steady-state fluorescence and circular dichroism of trout hemoglobins I and IV interacting with tributyltin.

The effect of tributyltin chloride (TBTC) on rainbow trout (Salmo irideus) hemoglobin I (HbI) and hemoglobin IV (HbIV) was characterized by the steady-state fluorescence of intrinsic and extrinsic fluorescent probes. The fluorescence emission spectrum (lambdaex 280 nm) is greatly increased in intensity by the presence of the organotin in both proteins. Circular dichroism spectra in the same samples show a small decrease in theta222, a measure correlated with the percentage of the alpha-helical content. Morever, important changes in near-UV, Soret, and visible regions of CD were induced by TBTC. The correlation of data obtained with trout hemoglobins (HbI and HbIV) with similar measurements on globins suggests that the presence of heme is necessary for the interaction of the organotin compound with the proteins.

Voltage-activated sodium currents in a cell line expressing a Cu,Zn superoxide dismutase typical of familial ALS.

The whole-cell configuration of the patch-clamp recording was used to study the voltage-dependent Na+ currents in a model system for the familial form of amyotrophic lateral sclerosis (ALS) associated with mutations in Cu,Zn superoxide dismutase. Here we report that the amplitude of voltage-gated Na+ currents is significantly reduced in cell lines expressing mutant Cu,Zn superoxide dismutase G93A when compared with the parental, untransfected cell line and to a cell line expressing the wild-type enzyme. This effect is associated with a shift toward positive values of the steady-state inactivation curve of the Na+ currents. These results indicate that expression of a Cu,Zn superoxide dismutase typical of patients affect with familial ALS influence the functionality of the voltage-dependent Na+ channels; this effect may contribute to the pathogenesis of the disease.

The effect of indolinic and quinolinic nitroxide radicals on trout erythrocytes exposed to oxidative stress.

The purpose of this study was to evaluate the ability of indolinic and quinolinic nitroxide radicals to protect trout (Salmo irideus) erythrocytes against oxidative stress. By using laurdan as a fluorescence probe, it was observed that the nitroxides inhibited the shift towards a gel phase of liposomes prepared with phospholipids extracted from trout erythrocyte membranes prior to the hemolytic event. In addition, the presence of 100 microM nitroxides in these liposomes protected the latter against lipid peroxidation determined by monitoring conjugated diene formation. However, the short chain analogue of the indolinic nitroxide and the quinolinic nitroxide had a negative effect on trout hemolysis, contrary to what has already been observed in previous studies on human RBCs (red blood cells). The half-time (t1/2) of the hemolytic process was 174 +/- 4.02 min for the former and 184 +/- 4.30 min for the latter compared to the control, 283 +/- 5.05 min. Furthermore, the nitroxides remarkably increased the autoxidation rate of both trout and human hemoglobin to met-Hb. Even though protection at the membrane level is conferred by the nitroxides during the early stages of lipid peroxidation, their antioxidative ability might be overwhelmed at a later stage by other mechanisms such as the increased autoxidation of hemoglobin in the presence of the nitroxides, thus giving a possible explanation for the early induction of hemolysis induced by the nitroxides. The superoxide scavenging ability of all the nitroxides used was also evaluated through chemiluminescence.

Antioxidant activities of different hemoglobin derivatives.

The antioxidant activity of hemoglobin was examined by studying both its peroxidase activity and its interaction with the superoxide anion. The peroxidase activity of both the subunits (alpha and beta) was reduced with respect to the alpha 2 beta 2 tetramer and heme-oxidation was found to be associated with a decrease in this activity. Lucigenin-amplified chemiluminescence experiments have shown that at low pH, the presence of hemoglobin reduces the level of superoxide anion generated by the xanthine/xanthine oxidase system (met-Hb is more efficient in reducing the level of O2- than oxy-hemoglobin). These results confirm that hemoglobin may be of importance in providing protection against oxidative damage to erythrocytes.

Expression of a Cu,Zn superoxide dismutase typical of familial amyotrophic lateral sclerosis induces mitochondrial alteration and increase of cytosolic Ca2+ concentration in transfected neuroblastoma SH-SY5Y cells.

We have set up a model system for familial amyotrophic lateral sclerosis (FALS) by transfecting human neuroblastoma cell line SH-SY5Y with plasmids directing constitutive expression of either wild-type human Cu,Zn superoxide dismutase (Cu,ZnSOD) or a mutant of this enzyme (G93A) associated with FALS. We have tested mitochondrial function and determined cytosolic Ca2+ concentration in control cells (untransfected) and in cells expressing either wild-type Cu,ZnSOD or G93A. We report that G93A induces a significant loss of mitochondrial membrane potential, an increased sensitivity toward valinomycin and a parallel increase in cytosolic Ca2+ concentration. The above phenomena are not related to total Cu,ZnSOD content and activity in the cell.

Effect of Val 73 --> Trp mutation on the reaction of "cambialistic" superoxide dismutase from Propionibacterium shermanii with hydrogen peroxide.

The H2O2 inactivation of the "cambialistic" superoxide dismutases from Propionibacterium shermanii, which is active with either iron or manganese at the active site, has been studied in the native and Val 73 --> Trp mutant enzymes. The wild-type iron-containing form of this enzyme is much more resistant to treatment with H2O2 with respect to the other metal-specific Fe superoxide dismutase isoenzymes. After incubation with high amounts of H2O2 the enzyme maintains more than 40% of the initial activity. The activity of the Val 73 --> Trp mutant drastically decreases to less than 5% of the initial activity after incubation with hydrogen peroxide. Amino acid analysis of the H2O2-treated mutant enzyme evidenced the loss of the Trp 73 residue which is shown to play a critical role in the stabilization of the monomer fold of the enzyme. On the other hand, the manganese-containing wild-type and mutant enzymes were completely resistant toward H2O2 demonstrating the specific role of iron in the inactivation process.

Effect of organotin compounds on trout hemoglobins.

The stability of trout hemoglobin was examined in the presence of some organotin compounds. Tributyltin chloride (TBTC) and triphenyltin chloride (TPTC) protect HbI most efficently from the oxidation. On the other hand, the same compounds accelerate the precipitation process in HbIV to a great extent. Parahydroxymercuribenzoate (PMB), an agent blocking free SH-groups of the protein, abolished the ability of TPTC to decrease the oxidation rate of HbI.