Carbaryl-induced effects on glutathione content, glutathione reductase and superoxide dismutase activity of the cyanobacterium Nostoc muscorum

The carbamate insecticide carbaryl, at concentrations of 10 mg/l and above, significantly stimulated glutathione reductase (GR) and superoxide dismutase (SOD) activity in the cyanobacterium Nostoc muscorum. A low content of total glutathione (GSH + GSSG), decreased photosynthetic activity, and an increased level of H₂O₂ was observed in pesticide treated cyanobacteria. As no glutathione peroxidase was observed in this species, stimulation of GR and SOD activity, higher production of H₂O₂, and low glutathione level was attributed to the utilization of GSH to remove H₂O₂ spontaneously and nonenzymatically under conditions of pesticide toxicity.     

Fusicoccin stimulates the production of H2O2 in sycamore cell cultures and induces alternative respiration and cytochrome c leakage from mitochondria

Fusicoccin (FC) is a well known toxin acting as a 14-3-3 protein-mediated activator of the plasma membrane H⁺-ATPase and it has been widely used to study the regulatory mechanism and the physiological role of this enzyme's activity. Recently, FC has been shown to induce other responses similar to those occurring under a stress condition, perhaps not strictly dependent on the activation of proton extrusion. In this paper we report that in cultured sycamore ( Acer pseudoplatanus L.) cells FC induces H₂O₂ overproduction as well as other novel, presumably related responses, such as the activation of the alternative oxidase and the leakage of cytochrome c from the mitochondria, accompanied by a decrease of the cytochrome pathway capacity. The relationship between H₂O₂ production and other phenomena has also been studied by means of exogenously added H₂O₂.     

The oxidative stress response in Enterococcus faecalis: relationship between H2O2 tolerance and H2O2 stress proteins

The hydrogen peroxide (H₂O₂) stress response in Enterococcus faecalis ATCC19433 was investigated. A 2·4 mmol l⁻¹ H₂O₂ pretreatment conferred protection against a lethal concentration (45 mmol l⁻¹) of this agent. The relatively high concentrations of H₂O₂ used for adaptation and challenge treatments in Ent. faecalis emphasised the strong resistance towards oxidative stress in this species. Various stresses (NaCl, heat, ethanol, acidity and alkalinity) induced weak or strong H₂O₂ cross-protection. This paper describes the involvement of protein synthesis in the active response to lethal dose of H₂O₂, in addition to the impressive enhancement of synthesis of five H₂O₂ stress proteins. Combined results suggest that these proteins might play an important role in the H₂O₂ tolerance response.     

Drought-induced spikelet sterility is associated with an inefficient antioxidant defence in rice panicles

Water stress-induced spikelet sterility limits rice production under upland conditions. The causes of spikelet sterility under drought stress are poorly understood. In this study the role of antioxidant defence management in drought-induced spikelet sterility was investigated in two rice ( Oryza sativa ) genotypes differing in drought resistance. Drought-resistant N22 genotype showed less water stress-induced spikelet sterility when compared to the susceptible N118 genotype under upland conditions. The N22 panicles maintained higher RWC and turgor potential and lower H₂O₂ levels across the developmental stages under water stress than that of N118 panicles. Drought-induced enhancement in superoxide dismutase (SOD, EC 1.15.1.1) activity coupled with higher ascorbate (AsA), glutathione (GSH) content and enhanced ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2) activities resulted in lower H₂O₂ levels in N22 panicles. In contrast, insufficient enhancement in SOD, APX and GR activities resulted in relatively higher H₂O₂ levels under water stress in N118 panicles. The N22 panicles exhibited a higher number of SOD and APX isozymes in comparison with N118 panicles that might provide better reactive oxygen species scavenging. Hence it is concluded that well-equipped antioxidant defence plays an important role in minimizing water stress-induced spikelet sterility in upland rice.     

Relationship between active oxygen species and cardenolide production in cell cultures of Digitalis thapsi: effect of calcium restriction

Elimination of calcium ions from the medium of undifferentiated cell cultures of Digitalis thapsi increased cardenolide production and induced extracellular H₂O₂ accumulation, as measured by the quenching of pyranine fluorescence. The addition of catalase reduced the response and the inclusion of superoxide dismutase enhanced the loss of fluorescence. This suggested that, besides H₂O₂, the superoxide anion was also formed before dismutating to H₂O₂. Additionally, exogenous H₂O₂ or superoxide dismutase stimulated cardenolide production whereas the addition of catalase markedly reduced it. These results point to a connection between H₂O₂ and cardenolide formation. The absence of calcium did not alter the levels of lipid peroxidation products; however, changes in the antioxidant system of D. thapsi cells were observed. Catalase activity was extremely low in control cultures and remained unaltered upon calcium elimination. Ascorbate peroxidase activity was not modified in calcium-free cultures. By contrast, calcium deprivation stimulated superoxide dismutase activity and strongly inhibited glutathione reductase activity. Also, a significant decrease in reduced glutathione was observed. These responses were emulated by treatment of the cultures with the glutathione biosynthesis inhibitor buthionine sulfoximine and by ethyleneglycol-bis-β-aminoethyl ether and LaCl₃. All these results indicate that the depletion of extracellular calcium induces changes in the redox state of cells and suggest that this alteration stimulates cardenolide formation in D. thapsi cultures.     

Senescence-related changes in the antioxidant status of ginkgo and birch leaves during autumn yellowing

The antioxidant status of birch and ginkgo leaves during autumnal senescence was characterized by the activities of catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX) and superoxide dismutase (SOD). The contents of leaf H₂O₂ and ascorbate were used as indicators of oxidative stress. Degradation of chlorophyll (chl) during natural senescence was not accompanied either by an increase of H₂O₂ or by a decrease of reduced ascorbate. A transient decrease of reduced ascorbate in ginkgo and birch leaves in early senescence was accompanied by CAT inactivation. The activity of ionically-bound PODs was stimulated in late senescence in both species, when more than 30% of chl was degraded. Induction of MnSOD in both species and new isoforms of CuZnSOD in birch in late senescence was accompanied by the disappearance of other CuZnSOD isoforms in birch and FeSOD in ginkgo. The role of antioxidative enzymes in keeping ascorbate reduced and endogenous H₂O₂ at low levels in senescent leaves of deciduous trees was discussed.     

Inhibition of Respiration in Sea Urchin Spermatozoa Following Interaction with Fixed Unfertilized Eggs V. Inhibition of Electron Transport in a Span of Mitochondrial Respiratory Chain between Cytochrome b and Cytochrome c in Sea Urchin Spermatozoa, Induced by the Interaction with Glutaraldehyde Fixed Eggs

In the sea urchin, Anthocidaris crassispina , α and γ peaks of reduced cytochrome b were distinctly observed but no peaks of cytochrome a and cytochrome c were found in the difference spectra between H₂O₂ oxidized and the aerobic suspensions of the immotile spermatozoa, which were obtained by an incubation of the suspension of spermatozoa and the glutaraldehyde-fixed eggs for 15 min at 20°C. A similar profile of difference spectrum was also obtained between the aerobic sperm suspension containing antimycin A and the H₂O₂ oxidized one. In Hemicentrorus pulcherrimus , faint peaks of reduced cytochrome a and cytochrome c , as well as evident peaks of cytochrome b , were also found in the difference spectra between aerobic suspension of the fixed-egg-reacted spermatozoa and the H₂O₂ oxidized one. In intact swimming spermatozoa of A. crassispina as well as H. pulcherrimus , no peaks of reduced cytochromes were found under aerobic condition. These results suggest that the inhibition of sperm respiration by the fixed eggs is due, at least in part, to the blockage of electron transport in a span between cytochrome b and cytochrome c.     

Effects of cucumber mosaic virus infection on electron transport and antioxidant system in chloroplasts and mitochondria of cucumber and tomato leaves

We examined the responses of the photosynthetic and respiratory electron transport and antioxidant systems in cell organelles of cucumber ( Cucumis sativus L.) and tomato ( Lycopersicon esculentum Mill.) leaves to infection of cucumber mosaic virus (CMV) by comparing the gas exchange, Chl fluorescence, respiratory electron transport, superoxide dismutase (SOD, EC 1.15.1.1) and ascorbate–glutathione (AsA–GSH) cycle enzymes and the production of H₂O₂ in chloroplasts, mitochondria and soluble fraction in virus-infected and non-infected leaves. Long-term CMV infection resulted in decreased photosynthesis and respiration rates. Photosynthetic electron flux to carbon reduction, respiratory electron transport via both complex I and complex II and also the Cyt respiration rate all significantly decreased, while photosynthetic alternative electron flux and alternative respiration significantly increased. These changes in electron transport were accompanied by a general increase in the activities of SOD/AsA–GSH cycle enzymes followed by an increased H₂O₂ accumulation in chloroplasts and mitochondria. These results demonstrated that disturbance of photosynthetic and respiratory electron transport by CMV also affected the antioxidative systems, thereby leading to oxidative stress in various organelles.     

Cytoprotective effect of polypeptide from Chlamys farreri on neuroblastoma (SH-SY5Y) cells following H2O2 exposure involves scavenging ROS and inhibition JNK phosphorylation

Oxidative stress has long been linked to cell death in many neurodegenerative conditions. Treatment with antioxidants is a promising approach for slowing disease progression. In this study, we used the neuroblastoma SH-SY5Y cells as an in vitro model to first assess the effect of polypeptide from Chlamys farreri (PCF), a natural marine antioxidant, on H₂O₂-induced neuronal cell death. Pre-treatment of SH-SY5Y cells with PCF inhibited H₂O₂-induced cell death in a concentration-dependent manner. In parallel, intracellular reactive oxygen species generation and lipid peroxidation were inhibited by PCF. Under severe H₂O₂ insult, PCF promoted endogenous antioxidant defense components including glutathione peroxidase, catalase, superoxide dismutase, and glutathione. PCF also protected DNA from oxidative damage and enhanced the removal of 8-oxo-7,8-dihydro-2'-deoxyguanosine from DNA. Further, we found that PCF potentially prevented H₂O₂–induced cell apoptosis. When investigated mitogen-activated protein kinase signaling pathway, we found that pre-treatment of cells with PCF significantly blocked H₂O₂–induced phosphorylation of c- Jun N-terminal kinase of the mitogen-activated protein kinase family. However, PCF had little inhibitory effect on the H₂O₂–induced activation of extracellular signal-regulated kinase. Taken together, these data demonstrate that PCF prevents oxidative stress-induced reactive oxygen species production and c- Jun N-terminal kinase activation and may be useful in the treatment of neurodegenerative diseases.     

Sinorhizobium meliloti rpoE2 is necessary for H2O2 stress resistance during the stationary growth phase

RpoE2 is an extracytoplasmic σ factor produced by Sinorhizobium meliloti during stationary growth phase. Its inactivation affected the synthesis of the superoxide dismutase, SodC, and catalase, KatC. The absence of SodC within the cell did not result in an increased sensitivity to extracellular superoxides. In contrast, the absence of KatC affected the resistance of S. meliloti to H₂O₂ during the stationary growth phase. A katC strain behaved as an rpoE2 strain during an H₂O₂ challenge, suggesting that the H₂O₂ sensitivity of the rpoE2 strain resulted only from the lack of KatC in this strain.     

Hydrogen Peroxide Induces a Long-Lasting Inhibition of the Ca2+-Dependent Glutamate Release in Cerebrocortical Synaptosomes Without Interfering with Cytosolic Ca2+

Abstract: We studied the action of H₂O₂ on the exocytosis of glutamate by cerebrocortical synaptosomes. The treatment of synaptosomes with H₂O₂ (50–150 µ M ) for a few minutes results in a long-lasting depression of the Ca²⁺-dependent exocytosis of glutamate, induced by KCl or by the K⁺-channel inhibitor 4-aminopyridine. The energy state of synaptosomes, as judged by the level of phosphocreatine and the ATP/ADP ratio, was not affected by H₂O₂, although a transient decrease was observed after the treatment. H₂O₂ did not promote peroxidation, as judged by the formation of malondialdehyde. In indo-1-loaded synaptosomes, the treatment with H₂O₂ did not modify significantly the KCl-induced increase of [Ca²⁺]_i. H₂O₂ inhibited exocytosis also when the latter was induced by increasing [Ca²⁺]_i with the Ca²⁺ ionophore ionomycin. The effects of H₂O₂ were unchanged in the presence of superoxide dismutase and the presence of the Fe³⁺ chelator deferoxamine. These results appear to indicate that H₂O₂, apparently without damaging the synaptosomes, induces a long-lasting inhibition of the exocytosis of glutamate by acting directly on the exocytotic process.     

Oxidative burst and expression of germin/oxo genes during wounding of ryegrass leaf blades: comparison with senescence of leaf sheaths

Two bursts of H₂O₂ production have been detected by in situ 3,3'-diaminobenzidine (DAB) staining after cutting of Lolium perenne L. leaf blades. The first burst, which occurred immediately after wounding was inhibited by Na-diethydithiocarbamate (DIECA), a Cu/Zn–superoxide dismutase (SOD) inhibitor. The second burst, which was initiated several hours later, coincided with the induction of oxalate oxidase (G-OXO) activity detected in vitro or visualized in situ by the α-chloronaphtol assay. Four genes encoding G-OXO have been identified from cDNA obtained from wounded L. perenne L . leaf blades. Comparison of protein sequences revealed more than 91% homology in the coding region between G-OXOs of the true cereals and G-OXOs of ryegrass, which is a Gramineae belonging to the tribe of Festucaceae. The wound-dependent increase of G-OXO activity in floated cut leaf blades was the result of differential induction of the four g-oxo genes. The involvement of G-OXOs in wound-induced H₂O₂ production coincided with the presence in leaf tissues of oxalate throughout the period of increase of G-OXO synthesis. Moreover, expression of g-oxo genes was enhanced by an exogenous supply of H₂O₂ or methyljasmonate (MeJa). Expression of the four g-oxo genes was also induced after in planta stinging of leaf blades. The pattern of their expression in planta was identical to that occuring in senescing leaf sheaths. These results emphasize the importance of G-OXOs in H₂O₂ production in oxalate-producing plant species such as ryegrass. G-OXOs might be crucial during critical events in the life of plants such as cutting and senescence by initiating H₂O₂-mediated defences against pathogens and foraging animals.     

Drought acclimation confers oxidative stress tolerance by inducing co-ordinated antioxidant defense at cellular and subcellular level in leaves of wheat seedlings

Wheat ( Triticum aestivum L.) seedlings of a drought-resistant cv. C306 were subjected to severe water deficit directly or through stress cycles of increasing intensity with intermittent recovery periods (drought acclimation). The antioxidant defense in terms of redox metabolites and enzymes in leaf cells, chloroplasts, and mitochondria was examined in relation to ROS-induced membrane damage. Drought-acclimated seedlings modulated growth by maintaining favorable turgor potential and RWC and were able to limit H₂O₂ accumulation and membrane damage as compared with non-acclimated plants during severe water stress conditions. This was due to systematic upregulation of H₂O₂-metabolizing enzymes especially ascorbate peroxidase (APX, EC 1.11.1.11) and by maintaining ascorbate–glutathione redox pool in acclimated plants. By contrast, failure in the induction of APX and ascorbate–glutathione cycle enzymes makes the chloroplast susceptible to oxidative stress in non-acclimated plants. Non-acclimated plants protected the leaf mitochondria from oxidative stress by upregulating superoxide dismutase (SOD, EC 1.15.1.1), APX, and glutathione reductase (GR, EC 1.6.4.2) activities. Rewatering led to rapid enhancement in all the antioxidant defense components in non-acclimated plants, which suggested that the excess levels of H₂O₂ during severe water stress conditions might have inhibited or downregulated the antioxidant enzymes. Hence, drought acclimation conferred enhanced oxidative stress tolerance by well-co-ordinated induction of antioxidant defense both at the chloroplast and at the mitochondrial level.     

Effect of oxygen and peroxide on Bacteroides fragilis cell and phage survival after treatment with DNA damaging agents

Abstract Bacteroides fragilis Bf-2 cells were more sensitive to far-UV radiation, N -methyl- N '-nitrosoguanidine, ethylmethane sulphonate, acriflavine and mitomycin C under aerobic conditions than under anaerobic conditions. The opposite effect was observed with H₂O₂-treated cells and exposure to O₂ enhanced the survival of H₂O₂-treated cells. Pretreatment of cells with sublethal concentrations of H₂O₂ also increased the survival of H₂O₂-treated cells. Reactivation of UV- and X-irradiated and methylmethane sulphonate and H₂O₂-treated phage b-1 was induced by O₂ and H₂O₂ in B. fragilis .     

Amyloid β-Mediated Oxidative and Metabolic Stress in Rat Cortical Neurons: No Direct Evidence for a Role for H2O2 Generation

Abstract: H₂O₂ and free radical-mediated oxidative stresses have been implicated in mediating amyloid β(1–40) [Aβ(1–40)] neurotoxicity to cultured neurons. In this study, we confirm that addition of the H₂O₂-scavenging enzyme catalase protects neurons in culture against Aβ-mediated toxicity; however, it does so by a mechanism that does not involve its ability to scavenge H₂O₂. Aβ-mediated elevation in intracellular H₂O₂ production is suppressed by addition of a potent H₂O₂ scavenger without any significant neuroprotection. Three intracellular biochemical markers of H₂O₂-mediated oxidative stress were unchanged by Aβ treatment: (a) glyceraldehyde-3-phosphate dehydrogenase activity, (b) hexose monophosphate shunt activity, and (c) glucose oxidation via the tricarboxylic acid cycle. Ionspray mass spectra of Aβ in the incubation medium indicated that Aβ itself is an unlikely source of reactive oxygen species. In this study we demonstrate that intracellular ATP concentration is compromised during the first 24-h exposure of neurons to Aβ. Our results challenge a pivotal role for H₂O₂ generation in mediating Aβ toxicity, and we suggest that impairment of energy homeostasis may be a more significant early factor in the neurodegenerative process.     

Response of antioxidant systems to NaCl stress in the halophyte Cakile maritima

The effects of salt stress on antioxidative activities were investigated in a coastal halophyte, Cakile maritima . Two Tunisian accessions, Jerba and Tabarka, were compared. Plants were subjected to 100, 200, or 400 m M NaCl for 20 days. Parameters of oxidative stress [malondialdehyde (MDA), electrolyte leakage (EL), and hydrogen peroxide (H₂O₂) concentration], activities of several enzymes [superoxide dismutase (SOD), catalase (CAT), peroxydase (POD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR)], and antioxidant molecules (ascorbate, ASC, and glutathione, GSH) were determined. Growth of Jerba plants was improved at 100 m M NaCl as compared to that of control. Tabarka growth was inhibited by salt at all NaCl concentrations. The relative salt tolerance of Jerba was associated with high antioxidant enzyme activities and glutathione content, together with low MDA content, EL, and H₂O₂ concentration. Lower antioxidant activities and higher MDA content, EL, and H₂O₂ concentration were found in Tabarka. As a whole, these data suggest that the capacity to limit oxidative damage is important for salt tolerance of C. maritima .     

Is hydrogen peroxide a second messenger of salicylic acid in systemic acquired resistance?

Elevated levels of salicylic acid (SA) are required for the induction of systemic acquired resistance (SAR) in plants. Recently, a salicylic acid-binding protein (SABP) isolated from tobacco was shown to have catalase activity. Based on this finding elevated levels of hydrogen peroxide (H₂O₂) were postulated to act as a second messenger of SA in the SAR signal transduction pathway. A series of experiments have been carried out to clarify the role of H₂O₂ in SAR-signaling. No increase of H₂O₂ was found during the onset of SAR. Induction of the SAR gene, PR-1, by H₂O₂ and H₂O₂-inducing chemicals is strongly suppressed in transgenic tobacco plants that express the bacterial salicylate hydroxylase gene, indicating that H₂O₂ induction of SAR genes is dependent on SA accumulation. Following treatment of plants with increasing concentrations of H₂O₂, a dose-dependent accumulation of total SA species was found, suggesting that H₂O₂ may induce PR-1 gene expression through SA accumulation. While the results do not support a role for H₂O₂ in SAR signaling, it is suggested that SA inhibition of catalase activity may be important in tissues undergoing a hypersensitive response.