The Effect of Hydrogen Peroxide on the Growth of Microscopic Mycelial Fungi Isolated from Habitats with Different Levels of Radioactive Contamination

The effect of hydrogen peroxide (10^?9–10^?1 M) on the mycelial growth of the fungi Alternaria alternata, Cladosporium cladosporioides, Mucor hiemalis, and Paecilomyces lilacinus has been studied. The growth of fungi isolated from habitats with a background level of radioactive contamination was stopped by H₂O₂ concentrations equal to 10^?3 and 10^?2 M, whereas the growth of fungi that were isolated from habitats with high levels of radioactive contamination was only arrested by 10^?1 M H₂O₂. The response of the different fungi to hydrogen peroxide was of three types: (1) a constant growth rate of fungal hyphae at H₂O₂ concentrations between 10^?9 and 10^?4 M and a decrease in this rate at 10^?3 M H₂O₂, (2) a gradual decrease in the growth rate as the H₂O₂ concentration was increased, and (3) an increase in the growth rate as the H₂O₂ concentration was increased from 10^?6 to 10^?5 M. The melanin-containing species A. alternata and C. cladosporioides exhibited all three types of growth response to hydrogen peroxide, whereas the light-pigmented species M. hiemalis and P. lilacinus showed only the first type of growth response. A concentration of hydrogen peroxide equal to 10^?1 M was found to be lethal to all of the fungi studied. The most resistant to hydrogen peroxide was found to be the strain A. alternata 56, isolated from the exclusion zone of the Chernobyl Nuclear Power Plant.     

Regulation of T-cell activation and T-cell growth factor (TCGF) production by hydrogen peroxide

Activated macrophages are known to release a variety of immunoregulatory substances including the low-molecular-weight substances hydrogen peroxide and lactate. We report here that lactate but not hydrogen peroxide is capable of supporting a substantial production of T-cell growth factor (TCGF) in cultures of accessory cell-depleted splenic T-cell populations after stimulation with concanavalin A. Hydrogen peroxide and its biosynthetic precursor superoxide anion (O2-) mediate, however, a strong augmentation of the TCGF production by accessory cell-depleted T-cell populations in the presence of lactate. Lactate inhibits the incorporation of [3H]thymidine in short-term cultures (18-26 hr) of accessory cell-depleted T cells. This confirms the rule that (optimal) production of T-cell growth factor requires a growth inhibitory signal. Concentrations of hydrogen peroxide which augment TCGF production most effectively (i.e., 1 X 10(-5) M) do not inhibit the incorporation of [3H]thymidine; and higher concentrations (3 X 10(-5)-1 X 10(-4) M) of hydrogen peroxide inhibit both the production of TCGF and the incorporation of [3H]thymidine. In agreement with the augmenting effect of hydrogen peroxide on TCGF production, it was observed that the proliferative response in mixed lymphocyte cultures is suppressed by catalase and augmented by 1 X 10(-5) M H2O2. Proliferative and cytotoxic responses in mixed lymphocyte cultures with an external source of interleukin 2 (IL-2) in contrast, are not augmented by 1 X 10(-5) M H2O2. The relatively high concentration of 1 X 10(-4) M hydrogen peroxide was found to inhibit the proliferative responses in mixed lymphocyte cultures with or without external IL-2 but not the cytotoxic response in the presence of IL-2. This indicates that CTL precursor cells may be relatively resistant against H2O2.     

Level of hydrogen peroxide in electrochemically activated solutions and study of its effect on growth of Escherichia coli

The formation of hydrogen peroxide in catholytes and anolytes of electrochemically activated solutions: bidistilled water and solutions of sodium chloride and nutrition medium M9 was studied. The concentration of hydrogen peroxide was determined by the method of enhanced chemiluminescence in a system peroxidase-luminol-p-iodophenol. It was shown that the concentration of hydrogen peroxide depends on the ionic content of the solution and varies from a few fractions of a micromole in catholytes of bidistilled water and sodium chloride solutions (10(-5) divided by 10(-2) M) to 20-25 microM in catholytes of medium M9. The concentration of H2O2 in anolytes of various solutions was 15-20 times lower than in the corresponding catholytes and was equal to a few nanomoles in bidistilled water and a few micromoles in medium M9. The biological activity of the catholyte of medium M9 was determined from changes in the growth of E. coli cells. It was found that this catholyte stimulates the cell growth. The stimulating effect was 20-25% and did not change after the decomposition of hydrogen peroxide in the catholyte by catalase. The addition of H2O2 at the corresponding concentration to the inactivated nutrient medium produced no stimulating effect. These data suggest that hydrogen peroxide formed in the catholyte of nutrient medium M9 does not affect its biological activity.     

Synergistic killing of Escherichia coli by near-UV radiation and hydrogen peroxide: distinction between recA-repairable and recA-nonrepairable damage.

Wild-type cells and six DNA repair-deficient mutants (lexA, recA, recB, recA, recB, polA1, and uvrA) of Escherichia coli K-12 were treated with near-ultraviolet radiation plus hydrogen peroxide (H2O2). At low H2O2 concentrations (6 X 10(-6) to 6 X 10(-4) M), synergistic killing occurred in all strains except those containing a mutation in recA. This RecA-repairable damage was absent from stationary-phase cells but increased in logarithmic cells as a function of growth rate. At higher H2O2 concentrations (above 6 X 10(-4) M) plus near-ultraviolet radiation, all strains, including those with a mutation in recA, were synergistically killed; thus, at high H2O2 concentrations, the damage was not RecA repairable.     

Effect of hydrogen peroxide on morphological characteristics and resistance of wheat calluses to the stinking smut fungus

We studied the effect of hydrogen peroxide on morphological characteristics and resistance of common wheat calluses (Triticum aestivum L.) to Tilletia caries Till. The induction of the defense response and morphogenesis in calluses depended on H2O2 concentration. A correlation was revealed between the elevated concentration of hydrogen peroxide in wheat calluses and high activity of oxalate oxidase in the cell wall. Administration of H2O2 into the callus culture medium was followed by rhizogenesis, induced the formation of dense regions, and inhibited fungal growth on calluses. Hydrogen peroxide at high concentrations was less potent in inhibiting the growth of fungi. A relationship was found between oxalate oxidase activity, H2O2 concentration, and morphogenetic and defense responses of calluses induced by exogenous hydrogen peroxide. These data suggest that the induction of H2O2 generation is one of the approaches to increase callus resistance.     

Superoxide and hydrogen peroxide formation during enzymatic oxidation of DOPA by phenoloxidase

Generation of superoxide anion and hydrogen peroxide during enzymatic oxidation of 3-(3,4-dihydroxyphenyl)-DL-alanine (DOPA) has been studied. The ability of DOPA to react with O2*- has been revealed. EPR spectrum of DOPA-semiquinone formed upon oxidation of DOPA by O2*- was observed using spin stabilization technique of ortho-semiquinones by Zn2+ ions. Simultaneously, the oxidation of DOPA by O2*- was found to produce hydrogen peroxide (H2O2). The analysis of H2O2 formation upon oxidation of DOPA by O2*- using 1-hydroxy-3-carboxy-pyrrolidine (CP-H), and SOD as competitive reagents for superoxide provides consistent values of the rate constant for the reaction between DOPA and O2*- being equal to (3.4+/-0.6)x10(5) M(-1) s(-1).The formation of H2O2 during enzymatic oxidation of DOPA by phenoloxidase (PO) has been shown. The H2O2 production was found to be SOD-sensitive. The inhibition of H2O2 production by SOD was about 25% indicating that H2O2 is produced both from superoxide anion and via two-electron reduction of oxygen at the enzyme. The attempts to detect superoxide production during enzymatic oxidation of DOPA using a number of spin traps failed apparently due to high value of the rate constant for DOPA interaction with O2*-.     

Influence of low dozes of ionizing radiation on accumulation of melanin pigments and catalase and superoxidedismutase activities in Cladosporium cladosporioides

Influence of low dozes of ionizing radiation on melanin pigments synthesis and activity of antioxidant enzymes catalase and superoxidedismutase of two strains of Cladosporium cladosporioides 4, (isolated from radioactive soil) and 396 (control) were investigated. It was shown, that in C. cladosporioides 4 under the exposure of ionizing radiation an increase of melanin synthesis in a stationary growth phase and increase of superoxidedismutase activity in a logarithmic phase were observed; in the control strain C. cladosporioides 396 activation of melanin synthesis and superoxide dismutase activity in both growth phases was revealed. It was established that in C. cladosporioides 4 the endocellular catalase activity in a logarithmic phase is 3.2 times higher, than in control strain. Under the action of ionizing radiation a 2-fold increase of this enzyme activity unlike the control strain in which the activity inhibition was revealed. The obtained results testify to the complex response of antioxidant systems and melanin to the action of low dozes of radiation which depends on the growth phase and presence of radioadaptation properties in the investigated fungi.     

Peroxidase-mediated toxicity to schistosomula of Schistosoma mansoni

Guinea pig eosinophil peroxidase (EPO) was capable of killing schistosomula of Schistosoma mansoni in vitro when combined with hydrogen peroxide and a halide. Killing was measured by 51Cr release, by microscopic evaluation of viability, and by reinfection experiments in mice. Parasite killing was dependent on each component of the EPO-H2O2-halide system, was completely inhibited by catalase and azide, and was partially inhibited by cyanide. The EPO-mediated system required 10(-4) M H2O2 and 10(-4) M iodide at pH 7.0, and the schistosomula were killed with exposure to this system of less than 30 min at 37 degrees C. At pH 6.0, the EPO-mediated system showed significant cidal activity with 10(-6) M iodide. Canine neutrophil peroxidase (myeloperoxidase [MPO]) was also able to kill schistosomula in vitro in the presence of 10(-4) M H2O2 and 10(-4) iodide at pH 7.0 and pH 6.0. Physiologic concentrations of chloride (0.1 M) could substitute for iodide at pH 7.0 and pH 6.0 as the halide cofactor; however, at pH 7.0, a higher concentration of enzyme was required. These findings with isolated enzyme systems are compatible with a role for peroxidase in the host defense against schistosomula.     

Decomposition of H2O2 by human cataractous lenses

It was shown that human lens opacity was accompanied by the decrease in the lens ability to cleave H2O2 (10(-4) M), added to the lens-surrounding medium. The rate of peroxide decomposition at the stage of mature cataract in isolated human lenses was 3.5 times lower than that of the control human lenses (transparent lens, initial cataract). Specific catalase inhibitor--3-amino,IH-1,2,4-triazole showed no significant influence on the rate of H2O2 cleavage. Reduced glutathione (10 microM) added to the lens incubation medium induced a sharp increase in the rate of H2O2 detoxication. The results indicate that reduced glutathione metabolism is of primary importance in the maintenance of anti-peroxide activity in the lens.     

A comparison of the effects of ocular preservatives on mammalian and microbial ATP and glutathione levels

The aim of this study was to investigate the mechanism of action of the preservative sodium chlorite (NaClO2), and the relationship with intracellular glutathione depletion. A detailed comparison of the dose responses of two cultured ocular epithelial cell types and four species of microorganism was carried out, and comparisons were also made with the quaternary ammonium compound benzalkonium chloride (BAK), and the oxidant hydrogen peroxide (H2O2). The viability of mammalian and microbial cells was assessed in the same way, by the measurement of intracellular ATP using a bioluminescence method. Intracellular total glutathione was measured by reaction with 5,5'-dithiobis-2-nitrobenzoic acid in a glutathione reductase-dependent recycling assay. BAK and H2O2 caused complete toxicity to conjunctival and corneal epithelial cells at approximately 25 ppm, in contrast to NaClO2, where > 100 ppm was required. The fungi Candida albicans and Alternaria alternata had a higher resistance to NaClO2 than the bacteria Staphyloccus aureus and Pseudomonas aeruginosa, but the bacteria were extremely resistant to H2O2. NaClO2 caused substantial depletion of intracellular glutathione in all cell types, at concentrations ranging from < 10 ppm in Pseudomonas, 25-100 ppm in epithelial cells, to > 500 ppm in fungal cells. The mechanisms of cytotoxicity of NaClO2, H2O2 and BAK all appeared to differ. NaClO2 was found to have the best balance of high antibacterial toxicity with low ocular toxicity. The lower toxicity of NaClO2 to the ocular cells, compared with BAK and H2O2, is in agreement with fewer reported adverse effects of application in the eye.     

Screening for Indian isolates of egg-parasitic fungi for use in biological control of fascioliasis and amphistomiasis in ruminant livestock

Wild isolates of the egg-parasitic fungi Paecilomyces lilacinus and Verticillium chlamydosporium, obtained from the organic environment of Durg, Chhattisgarh, India, were subjected to screening for in vitro growth using different media types, range of incubation temperature and pH, and their predatory activity to the eggs of Fasciola gigantica and Gigantocotyle explanatum. Maximum growth of P. lilacinus was obtained in corn-meal agar compared to any other media types. The preferred medium for growth of V. chlamydosporium was corn-meal agar, followed by potato-dextrose agar. After initial growth for 16 h of incubation, no growth was observed in water agar for both the fungi. Six different temperatures--4 degrees C, 10 degrees C, 18 degrees C, 26 degrees C, 34 degrees C and 40 degrees C--were used to observe growth profiles of the fungi in corn-meal agar medium. While no and very little growth of P. lilacinus and V. chlamydosporium was observed at 4 degrees C and 10 degrees C, respectively, growth profiles of both the fungi were optimal at 26-40 degrees C. A range of pH (pH 4-8) supported growth of both P. lilacinus and V. chlamydosporium. Full-grown plates of the fungi baited with viable eggs of F. gigantica and G. explanatum revealed that V. chlamydosporium was more vigorous in its egg-parasitic ability compared to P. lilacinus. Distortion of the eggs started on day 2-3 of egg baiting in culture plates of V. chlamydosporium, with complete distortion by day 7. On the contrary, P. lilacinus exhibited very limited egg-parasitic ability and some of the baited eggs even showed development of miracidia.     

Regulation of morphologicaland functional properties of astroglial cells by hydrogen peroxide

Effects of hydrogen peroxide on morphological characteristics, proliferation index, menadione-dependent lucigenin-enhanced chemiluminescence of C6 glioma cells were studied. It was established that H2O2 at 1 x 10(-8) - 5 x 10(-7) M concentrations acts as a regulator of morphological and functional properties of astrocytes by inducing their reactivation that is manifested as a cell body hypertrophy and an increase of proliferative activity and of menadione-dependent production of superoxide (O2- ). Cytodestructive action of hydrogen peroxide at a concentration higher than 1 microM on C6 glioma cells shows itself as a decrease of their proliferation index and the ability to generate O2- under menadione action. Using lipopolysaccharide B as a functional stimulator it has been shown that H2O2 modifies signaling pathways leading to the increase of mitotic activity of C6 glioma cells and decreases the yield of lucigenin-enhanced chemiluminescence of astrocytes under menadione action to the level of control values.     

Regulation of oxygen radical release from murine peritoneal macrophages by pharmacologic doses of PGE2

The ability of pharmacologic doses of PGE2 to alter the release of superoxide (O2-) and hydrogen peroxide (H2O2) from elicited peritoneal macrophages (M theta) was studied. Twice-daily administration of 200 or 100 micrograms of PGE2 to mice during accumulation of peritoneal M theta resulted in a significant reduction in M theta recovery and in the triggered release of H2O2, but not O2-. Cultivation of elicited M theta from normal mice with concentrations of PGE2 in excess of 10(-7) M for 24-48 h resulted in a significant reduction in the triggered release of H2O2, but not O2-. Cultivation for shorter periods of time or with lower concentrations of PGE2 failed to alter H2O2 release. This effect of PGE2 was reproduced by the phosphodiesterase inhibitor theophylline. The ability of PGE2 to inhibit H2O2 release in the presence of normal production of O2- was not prevented by the addition of superoxide dismutase. Cultivation of peritoneal M theta with 10(-5) M PGE2 for 48 h failed to increase intracellular catalase, although increased H2O2 scavenger activity was demonstrated. The inhibition of extracellular release of H2O2, but not O2-, by pharmacologic doses of PGE2 may be one mechanism for the anti-inflammatory action of this compound.     

A real-time electrochemical technique for measurement of cellular hydrogen peroxide generation and consumption: evaluation in human polymorphonuclear leukocytes

There has been a long-standing need for sensitive and specific techniques for hydrogen peroxide (H(2)O(2)) measurement. We describe the development and application of a highly sensitive electrochemical sensor, utilizing a membrane-coated platinum microelectrode, suitable for real-time measurement of hydrogen peroxide generation and consumption in biochemical or cellular systems. This sensor provides high sensitivity enabling measurement of hydrogen peroxide down to 5-10 nM concentrations. We demonstrate that it can be used to measure the magnitude and time course of H(2)O(2) generation from the NADPH oxidase in leukocytes as well as the rate of H(2)O(2) degradation. After human polymorphonuclear leukocytes (PMNs) were activated by phorbol 12-myristate acetate, H(2)O(2) concentration increased with time and reached a peak concentration, from 5 to 15 microM in PMNs prepared from different individuals, within 3 to 8 min, then decreased slowly. The H(2)O(2) concentration in the solution is less than the total H(2)O(2) generation from the activated PMNs because a part of H(2)O(2) generated is decomposed. H(2)O(2) in solution, generated from the PMNs, was rapidly consumed after the activated PMNs were treated with 10 microM diphenylene iodonium (DPI). The rate of H(2)O(2) consumption was measured following the addition of exogenous H(2)O(2). The total production of H(2)O(2) from the activated PMNs was calculated from the measured H(2)O(2) concentration and the rate of H(2)O(2) consumption. This technique enables sensitive and continuous real-time measurement of H(2)O(2) concentration and total H(2)O(2) generation in cellular or enzyme systems without addition of any detection reagents.     

Effect of hydrogen peroxide on neuron sensitivity to acetylcholine

The dose-dependent effect of hydrogen peroxide on snail neuromembrane chemosensitivity was studied by means of standard voltage-clamp method. Short-term exposure (7 min) of neurons to H(2)O(2) (10(-11)-10(-4) M) caused dose-dependent depression of Acetylcholine (Ach)-induced ionic currents in the membrane. The H(2)O(2)-induced depression of Ach-sensitivity of membrane was more pronounced in K(+)-free solution than in normal physiological solution and it disappeared in cold medium (5 degrees C). The H(2)O(2) (10(-11)-10(-4) M) decreased membrane electrical conductivity and cell volume. The dose-dependent decrease in Ach-sensitivity of the snail neuromembrane by H(2)O(2) may be due to a decrease in the number of functionally active membrane receptors caused by a decrease in membrane active surface. H(2)O(2)-induced decrease in Ach-sensitivity has a metabolic but Na(+)-K(+) pump independent character, the nature of which is the subject for current investigation.     

Fungi of Orobanche aegyptiaca in Nepal with Potential as Biological Control Agents

A survey of pathogens of the root-parasitic weed Orobanche aegyptiaca Pers. was carried out in Nepal. More than 70% of the fungal strains isolated from infected plants belonged to the genus Fusarium. Other fungi isolated were Acremonium fusidioides, Alternaria alternata, Cladosporium cladosporioides, Epicoccum nigrum, Mortierella alpina, Papulaspora sp., Phoma spp., Sordaria fimicola , Rhizoctonia sp., Trichoderma spp. and Trichothecium roseum.     

Pervanadate [peroxide(s) of vanadate] mimics insulin action in rat adipocytes via activation of the insulin receptor tyrosine kinase

Both vanadate and hydrogen peroxide (H2O2) are known to have insulin-mimetic effects. We previously reported that the mixture of vanadate plus H2O2 results in the generation of a peroxide(s) of vanadate, which strongly enhances IGF-II binding to rat adipocytes (Kadota et al., 1987b). We now report that pervanadate mimics insulin in isolated rat adipocytes to (1) stimulate lipogenesis, (2) inhibit epinephrine-stimulated lipolysis, and (3) stimulate protein synthesis. The efficacy of pervanadate is comparable to that of insulin. However, it is 10(2)-10(3) times more potent than vanadate alone. Exposure of intact rat adipocytes to pervanadate was found to activate the WGA-purified insulin receptor tyrosine kinase assayed with the exogenous substrate poly(Glu80/Tyr20) in a dose-dependent manner to a maximum of 1464% of control at 10(-3) M compared with a maximum insulin effect of 1046% at 10(-6) M. In contrast, in vitro assayed autophosphorylation of the WGA-purified extract was increased 3-fold after exposure of intact cells to insulin but not significantly increased after pervanadate. Furthermore, high concentrations of pervanadate (10(-5) M) inhibited subsequent in vitro added insulin-stimulated autophosphorylation. In vitro addition of pervanadate to WGA-purified receptors could not stimulate autophosphorylation or exogenous tyrosine kinase activity and did not inhibit insulin-stimulated autophosphorylation. Labeling of intact adipocytes with [32P]orthophosphate followed by exposure to 10(-4) M pervanadate increased insulin receptor beta-subunit phosphorylation (7.9 +/- 3.0)-fold, while 10(-7) M insulin and 10(-4) vanadate increased labeling (5.3 +/- 1.8)- and (1.1 +/- 0.2)-fold, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and physical-chemical characterization of the three hydroperoxidases from the symbiotic bacterium Sinorhizobium meliloti

Three genes encoding heme hydroperoxidases (katA, katB, and katC) have been identified in the soil bacterium Sinorhizobium meliloti. The recombinant proteins were overexpressed in Escherichia coli and purified in order to achieve a spectral and kinetic characterization. The three proteins contain heme b with high-spin Fe(III). KatB is an acidic bifunctional homodimeric catalase-peroxidase exhibiting both catalase (k(cat) = 2400 s(-1)) and peroxidase activity and having a high affinity for hydrogen peroxide (apparent K(M) = 1.6 mM). KatA and KatC are acidic monofunctional homotetrameric catalases. Although different in size (KatA is a small subunit catalase while KatC is a large subunit catalase) both enzymes exhibit the same heme type and a similar affinity for H(2)O(2) (apparent K(M) values of 160 and 150 mM). However, the turnover rate of KatA (k(cat) = 279000 s(-1)) exceeds that of KatC (k(cat) = 3100 s(-1)) significantly. The kinetic parameters are in good agreement with the physiological role of these heme proteins. KatB is the housekeeping hydroperoxidase exhibiting the highest affinity for hydrogen peroxide, while KatA has the lowest H(2)O(2) affinity but the highest k(cat)/K(M) value (1.75 x 10(6) M(-1) s(-1)), in agreement with the hydrogen peroxide inducibility of the encoding gene. Moreover, the lower catalytic efficiency of KatC (2.1 x 10(4) M(-1) s(-1)) appears to be enough for growing in the stationary phase and/or under heat or salt stress (conditions that are known to favor katC expression).     

Effect of ecological factors on spore germination and the viability of the mycelial fragments of microscopic fungi

Spore germination and the viability of the mycelial fragments of the microscopic fungi Alternaria alternata, Penicillium spinulosum, and Mucor hiemalis were studied with respect to the action of some ecological factors: sucrose concentration (0, 0.2, 2, 10, and 100 g/l), temperature (4, 20, 25, and 30 degrees C), pH (3.5, 4.0, 5.0, 6.2, and 7.0), and cadmium concentration (0, 2, 10, and 100 mg/l). The spore germination rate and the viabilities of different mycelial fragments were found to reach their maxima at different values of the ecological factors studied. This finding suggests that the vegetative and asexual types of reproduction of microscopic fungi may have different ecological optima.