Effect of carbohydrate and nitrogen on hydrogen peroxide formation by wood decay fungi in solid medium

Abstract Hydrogen peroxide (H₂O₂) has been implicated in degradation of wood by both brown-rot and white-rot fungi. This study found that low concentrations of nitrogem and carbohydrates (cellobiose, glucose, xylose and mannose) in an agar medium had little effect on H₂O₂ production by white-rot fungi. However, low concentrations of nitrogen and carbohydrates stimulated H₂O₂ production by brown-rot fungi. Use of the chromogen 2,2'-azino-di(3-ethyl benzthiazoline-6-sulphonic acid) (ABTS) with horseradish peroxidase to detect H₂O₂ by the fungi was slightly better than detection by the chromogen o -dianisidine with horseradish peroxidase. An auxiliary test to check the role of H₂O₂ in wood decay found that hydrogen peroxide-negative isolates of the white-rot fungi Pharnerochaete chrysosporium and Ganoderma applanatum were unable to decay sweetgum and southern pine.     

Temperature profile of oxygen activation and defense against oxygen toxicity in a psychrophilic member of the Bacteroidaceae

Abstract The temperature profiles have been determined for O₂ reduction by activating substrates for whole cells and cell extracts of the psychrophilic, obligately anaerobic bacterium, strain B6, belonging to the Bacteroidaceae. The profiles were similar whether the cells were grown at 15 or 1°C, and also for cells harvested in the exponential or stationary phase. The H₂O producing pyruvate oxidase displayed in cell-free extracts a considerably higher activity than the H₂O₂ producing NADH and NADPH oxidases at all temperatures in the range 30–1°C, and characteristically makes up a larger proportion of the total O₂ reduction capacity the lower the temperature. It thus seems that the O₂ scavenging property of the pyruvate oxidase, postulated to be utilized in a defense mechanism against the detrimental effects of the H₂O₂ producing pyridine nucleotide oxidases, is particularly well adapted to function at the low temperatures of the Barents Sea, from which this obligately anaerobic organism originates.     

Stress-induced activation of Nox contributes to cell survival signalling via production of hydrogen peroxide

Reactive oxygen species (ROS) have traditionally been viewed as a toxic group of molecules; however, recent publications have shown that these molecules, including H₂O₂, can also strongly promote cell survival. Even though the retina has a large capacity to produce ROS, little is known about its non-mitochondrial sources of these molecules, in particular the expression and function of NADPH oxidase (Nox) proteins which are involved in the direct generation of superoxide and indirectly H₂O₂. This study demonstrated that 661W cells, a retina-derived cell line, and mouse retinal explants express Nox2, Nox4 and certain of their well-established regulators. The roles of Nox2 and Nox4 in producing pro-survival H₂O₂ were determined using 661W cells and some of the controlling factors were identified. To ascertain if this phenomenon could have physiological relevance, the novel technique of time-lapse imaging of dichlorofluorescein fluorescence (generated upon H₂O₂ production) in retinal explants was established and it showed that explants also produce a burst of H₂O₂. The increase in H₂O₂ production was partly blocked by an inhibitor of Nox proteins. Overall, this study demonstrates a pro-survival role of Nox2 and Nox4 in retina-derived cells, elucidates some of the regulatory mechanisms and reveals that a similar phenomenon exists in retinal tissue as a whole.     

Carbohydrate oxidases in ericoid and ectomycorrhizal fungi: a possible source of Fenton radicals during the degradation of lignocellulose

Isolates of the ericoid mycorrhizal fungus Hymenoscyphus ericae (Read) Korf et Kernan, and the ectomycorrhizal fungi Suillus variegatus (Swartz ex Fr.) and Pisolithus tinctorius (Pers.) Coker & Couch, along with a Cortinarius sp. and the white rot Phanerochaete chrysosporium Burdsall were examined for the ability to oxidize carbohydrates to their corresponding lactones and to excrete the H₂O₂ produced thereby. All except Phanerochaete chrysosporium were found to express cellobiose oxidase (cellobiose dehydrogenase, EC 1.1.19.88) and glucose oxidase (β- d -glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) when grown on cellobiose and glucose respectively. Production of extracellular H₂O₂ was visualized during growth on both substrates using ABTS as the chromogen. According to the Fenton reaction, H₂O₂ will react with hydrated or chelated Fe(II) in the environment to produce hydroxyl (Fenton) radicals, HO^·. Mycelial extracts from each of the mycorrhizal fungi produced HO^· in the presence of cellobiose and Fe(II), presumably mediated by H₂O₂ produced by cellobiose oxidase activity in the extracts. Conditions favourable to HO^· production were shown to exist in Modified Melin–Norkrans medium, and the data discussed in relation to previously observed lignin degradation by mycorrhizal fungi.     

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.     

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.     

Correlation of resistance and H2O2 production in Ulmus pumila and Ulmus campestris cell suspension cultures inoculated with Ophiostoma novo-ulmi

The Dutch elm disease (DED) pathogen Ophiostoma novo-ulmi Buissm. elicited the production of H₂O₂ in cell suspension cultures of the resistant species Ulmus pumila L. This response was not observed in suspensions of the susceptible elm U. campestris Mill. H₂O₂ production started after a lag time of 30–40 min following inoculation, peaked between 4 and 6 h and lasted up to 24 h. Treatment of the suspensions with exogenously added H₂O₂ did not cause accumulation of the sesquiterpene phytoalexins mansonones nor of the coumarin scopoletin. Spore germination and growth of O. novo-ulmi were significantly delayed with different amounts of H₂O₂ (0.1–1 m M ). These results suggest that H₂O₂ production is an inducible defence response which may contribute to DED resistance by delaying the growth of the pathogen at the earliest stages of infection. Whether H₂O₂ is involved in other elm defence responses to the pathogen is presently unknown, but its production seems to be an independent event from phytoalexin formation.     

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.     

On the screening of hydrogen peroxide-generating lactic acid bacteria

The published plate methods for the detection of hydrogen peroxide-producing lactic acid bacteria, which employ horseradish peroxidase and a chromogen, clearly fail to detect all the organisms that produce H₂O₂. Whilst keeping the same principle, the use of a novel growth medium and of the chromogen tetramethyl-benzidine allows the detection of H₂O₂ production by strains previously classified as non-producers.     

Fusarium response to oxidative stress by H2O2 is trichothecene chemotype-dependent

The present study aims at clarifying the impact of oxidative stress on type B trichothecene production. The responses to hydrogen peroxide (H₂O₂) of an array of Fusarium graminearum and Fusarium culmorum strains were compared, both species carrying either the chemotype deoxynivalenol (DON) or nivalenol (NIV). In both cases, levels of in vitro toxin production are greatly influenced by the oxidative parameters of the medium. A 0.5 mM H₂O₂ stress induces a two- to 50-fold enhancement of DON and acetyldeoxynivalenol production, whereas the same treatment results in a 2.4- to sevenfold decrease in NIV and fusarenone X accumulation. Different effects of oxidative stress on toxin production are the result of a variation in Fusarium 's antioxidant defence responses according to the chemotype of the isolate. Compared with DON strains, NIV isolates have a higher H₂O₂-destroying capacity, which partially results from a significant enhancement of catalase activity induced by peroxide stress. A 0.5 mM H₂O₂ treatment leads to a 1.3- to 1.7-fold increase in the catalase activity of NIV isolates. Our data, which show the higher adaptation to oxidative stress developed by NIV isolates, are consistent with the higher virulence of these Fusarium strains on maize compared with DON isolates.     

Compatible and incompetent Paxillus involutus isolates for ectomycorrhiza formation in vitro with poplar (Populus x canescens) differ in H2O2 production

Abstract: Isolates of Paxillus involutus (Batsch) Fr. collected from different hosts and environmental conditions were screened for their ability to form ectomycorrhizal symbiosis with hybrid poplar P. × canescens ( = Populus tremula L. × P. alba) in vitro. The ability to form ectomycorrhiza varied between the fungal isolates and was not correlated with the growth rate of the fungi on agar-based medium. The isolate MAJ, which was capable of mycorrhiza synthesis under axenic conditions, and the incompetent isolate NAU were characterized morphologically and anatomically. MAJ formed a typical hyphal mantle and a Hartig net, whereas NAU was not able to penetrate the host cell walls and caused thickenings of the outer cell walls of the host. MAJ, but not NAU, displayed strong H₂O₂ accumulation in the outer hyphal mantle. Increases in H₂O₂ in the outer epidermal walls and adjacent hyphae of the incompetent isolate were moderate. No increases of H₂O₂ in response to the mycobionts were found inside roots. Suggested functions of H₂O₂ production in the outer hyphal mantle of the compatible interaction are: growth regulation of the host's roots, defence against other invading microbes, or increasing plant-innate immunity. The system established here for P. × canescens compatible and incompetent fungal associations will be useful to take advantage of genomic information now available for poplar to study tree-fungal interactions at the molecular and physiological level.     

Evaluation of hydrogen peroxide-based disinfectants in a new resazurin microplate method for rapid efficacy testing of biocides

Aims:  Development of the resazurin microplate method (RMM) as a novel test system for the evaluation of the antimicrobial activity of antiseptics and disinfectants. The validated RMM was subsequently applied for the evaluation of hydrogen peroxide (H₂O₂) and stabilized H₂O₂ combination products.
Methods and Results:  The European Committee for Standardization prescribes the plate count challenge test (PCCT) for antiseptic and disinfectant efficacy testing. This protocol was adapted to a microplate-based assay, using resazurin as viability indicator. The RMM was as accurate as the PCCT, had an identical detection limit and showed high intermediate precision. Using the validated RMM, it was shown that H₂O₂ combined with silver possessed a higher bactericidal and fungicidal activity compared to native H₂O₂ with and without glycerol.
Conclusions:  Validation showed that the RMM may replace the PCCT. When applying the RMM, H₂O₂ combined with silver was clearly a more potent disinfectant compared to H₂O₂ in killing bacteria and fungi.
Significance and Impact of the Study:  The RMM is easier to use for antimicrobial efficacy testing of antiseptics and disinfectants. As the RMM is in accordance with the norms of the European Committee for Standardization, it may become a more cost-effective alternative to the more laborious PCCT reference method. H₂O₂ with silver may replace native H₂O₂ to increase overall disinfection efficiency.     

Hydrogen peroxide formation in cultured rose cells in response to UV-C radiation

Suspension-cultured rose ( Rosa damascena Mill. cv. Gloire de Guilan) cells irradiated with UV-C (254 nm. 558 J m⁻²) showed a transient production of H₂O₂ as measured by chemiluminescence of luminol in the presence of peroxidase (EC 1.1 1.1.7). The peak concentration of H₂O₂, which occurred at about 60–90 min after irradiation, was 8–9 μ M . The time course for the appearance of H₂O₂ matched that for UV–induced K⁺ efflux. Treatments that inhibited the UV-induced efflux of K⁺, including heat and overnight incubation with cycloheximide and diethylmaleate, also inhibited the appearance of H₂O₂. The converse was not always true, since catalase (EC 1.11.1.6. and salicylhydroxamic acid, which inhibited luminescence, did not stop K⁺ efflux. We conclude that H₂O₂ synthesis depends on K⁺ efflux. Because H₂.O₂ in the extracellular space is required for lignin synthesis in many plant tissues, we suggest that the UV–stimulated production of H₂O₂ is an integral part of a defensive lignin synthesis.     

Comparison of quantitative and qualitative methods of detecting hydrogen peroxide produced by human vaginal strains of lactobacilli

A quantitative method was developed for the measurement of micromolar quantities of H₂O₂ produced in Rogosa broth and peptonized milk broth by vaginal strains of lactobacilli isolated from women. The production of substantial amounts reproducibly was dependent on the growth of the organisms in acid media (pH ≤6.0) under anaerobic or micro-aerophilic conditions with continuous agitation. The addition to the media of the enzyme inhibitor, 3-amino-l,2,4-triazole, with or without catalase sometimes induced the production of H₂O₂ especially in non-agitated cultures. However, other agents such as concanavalin and o -dianisidine had no enhancing effect, and catalase or peroxidase alone completely inhibited H₂O₂ production.
The H₂O₂ produced in the acid media was stable for more than a month at 5°C but not in media at pH ≥ 7.0. Of five strains of lactobacilli tested by the quantitative method and by a chromogenic qualitative method (Rogosa-catalase or -peroxidase agar), three consistently produced H₂O₂ measurable by the former method, but none did so after growth of the organisms on Rogosa-catalase/peroxidase agar which suggested that the qualitative method was unreliable. The fact that H₂O₂ was produced in substantial quantities by some strains and not at all by others enabled H₂O₂-producers and non-producers to be distinguished easily.     

Superoxide‐producing NAD(P)H oxidases in plasma membrane vesicles from elicitor responsive bean plants

Higher plants produce active oxygen species (AOS) that regulate their defence responses against pathogenic elicitation. Etiolated bean seedlings ( Phaseolus vulgaris L. cv. Limburgse vroege) were used to measure the in vivo‐induced AOS production and to search for plasma membrane bound NAD(P)H‐dependent oxidases producing AOS. Immersed bean plants showed a substantial production of H₂O₂, as determined by the peroxidase (EC 1.11.1.7)‐dependent oxidation of 3,5‐dichloro‐2‐hydroxybenzenesulfonic acid (DHBS). Addition of the elicitor polygalacturonase (PGase, EC 3.2.1.15) from Aspergillus japonicus or the phosphatase inhibitor, cantharidin, resulted in a transient increase of AOS synthesis. Plasma membrane vesicles, purified from etiolated bean seedlings, showed an NAD(P)H‐dependent superoxide (O₂⁻) production that was highly stimulated with naphthoquinones. Protein solubilisation and anion exchange chromatography resolved a basal and three naphthoquinone‐stimulated NAD(P)H‐dependent O₂⁻ oxidase fractions. The natural phenol, apigenin, was also a strong inducer of the naphthoquinone‐dependent enzymes, when it was used in the presence of peroxidase. Although, the relation of these different in vitro‐determined plasma membrane NAD(P)H‐dependent O₂⁻ oxidases to the in vivo elicitation of H ₂O₂ has not been elucidated so far.     

Effects of Fusaric Acid on Reactive Oxygen Species and Antioxidants in Tomato Cell Cultures

Generation of O₂^– and H₂O₂ as well as the activities of superoxide dismutase, catalase, ascorbate peroxidase, guaiacol peroxidase, dehydroascorbate reductase and ascorbate content were studied in tomato cell cultures in response to fusaric acid – a nonspecific toxin of phytopathogenic Fusarium species. Toxin treatment resulted in decreased cell viability which was preceded by culture medium alkalinization up to 0.65 pH unit and enhanced extracellular O₂^– production. The H₂O₂ level was not significantly affected. In toxin-treated cultures, a transient, significant increase occurred in intracellular superoxide dismutase, catalase, guaiacol peroxidase and ascorbate peroxidase activities. Fusaric acid-induced ascorbate turnover modulation led to up to a twofold increase in dehydroascorbic acid accumulation, and a decrease in the associated ascorbate redox ratio. It was concomitant with a significant decrease in dehydroascorbate reductase activity. These results support previous observations that the pro- and anti-oxidant systems are involved in response to fusaric acid treatment although differential response of H₂O₂ and its metabolism-related enzymes between the whole leaf and cell culture assays was found.     

Hydroperoxide inactivation of enzymes within spores of Bacillus megaterium ATCC19213

Abstract Hydroperoxide inactivation of the protoplast enzymes enolase, aldolase and glucose-6-phosphate dehydrogenase in intact spores of Bacillus megaterium ATCC19213 was assessed by first treating the cells with lethal levels of H₂O₂, then germinating them in the presence of chloramphenicol prior to permeabilization and enzyme assays. Glucose-6-phosphate dehydrogenase proved to be more sensitive to H₂O₂than enolase or aldolase, in agreement with findings for isolated enzymes. Average D values (time for 90% inactivation) for spores treated with 0.50% H₂O₂ were 173 min for enolase, 67 min for aldolase and 32 min for glucose-6-phosphate dehydrogenase, compared with a D value of 34 min for spore killing. H₂O₂ killing of spores was found to be conditional in that recoveries of survivors were greater on complex medium than on minimal medium. Overall, it appeared that oxidative inactivation of enzymes may be important for hydroperoxide killing of spores.