Hydrogen peroxide release from bacterial spores during germination

The release of free H₂O₂ from spores of Clostridium perfringens and Bacillus megaterium during germination has been demonstrated using the scopoletin fluorescence assay. Scopoletin oxidation was markedly inhibited when exogenous catalase was added, and was also influenced by the concentration of spores. H₂O₂ release into the germination medium was observed to parallel the O₂ consumption during germination, suggesting that the H₂O₂ may arise from certain O₂-dependent metabolism associated with initiation of spore germination.     

Use of superabsorbent polymer gels for surface decontamination of Bacillus anthracis spores

Aims:  This study evaluated the inactivation of Bacillus anthracis Vollum spores dried on a nonporous surface using a superabsorbent polymer (SAP) gel containing commercially available liquid decontaminants.
Methods and Results:  The first phase determining the availability of the liquid decontaminant within the SAP showed that the SAP gel containing pH-adjusted sodium hypochlorite (NaOCl) inhibited B. anthracis growth while the water control SAP gel had no affect on growth. For testing surface decontamination, B. anthracis spores were dried onto steel coupons painted with chemical agent resistant coating and exposed to SAP containing either pH-adjusted NaOCl, chlorine dioxide (ClO₂) or hydrogen peroxide/peracetic acid (H₂O₂/PA) for 5 and 30 min. At contact times of both 5 and 30 min, all of the SAP gels containing pH-adjusted NaOCl, ClO₂ or H₂O₂/PA inactivated B. anthracis spores at levels ranging from 2·2 to ≥7·6 log reductions.
Conclusions:  Incorporation of three commercially available decontaminant technologies into a SAP gel promotes inactivation of B. anthracis spores without observable physical damage to the test surface.
Significance and Impact of the Study:  This work provides preliminary data for the feasibility of using SAP in inactivating B. anthracis spores on a nonporous surface, supporting the potential use of SAP in surface decontamination.     

Activation of Endogenous Antioxidant Defenses in Neuronal Cells Prevents Free Radical-Mediated Damage

Abstract: Dopamine (DA) is oxidized to the neurotoxic prooxidant species H₂O₂, OH^•, and DA quinones. We tested whether dimethyl fumarate (DMF), an electrophile shown to induce a pleiotropic antioxidant response in nonneuronal cells, could reduce the toxicity of DA metabolites in neural cells. Treatment of the N18-RE-105 neuroblastoma-retina hybridoma cell line with 30–150 µ M dopamine led to cell death within 24 h, which increased steeply with dose, decreased with higher plating density, and was blocked by the H₂O₂-metabolizing enzyme catalase. Pretreatment with DMF (30 µ M , 24 h) significantly attenuated DA and H₂O₂ toxicity (40–60%) but not that caused by the calcium ionophore ionomycin. DMF treatment also elevated total intracellular GSH and increased activities of the antioxidant enzymes quinone reductase (QR), glutathione S -transferase (GST), glutathione reductase, and the pentose phosphate enzyme glucose-6-phosphate dehydrogenase. To assess the protective efficacy of QR and GST, a stable cell line was constructed in which these enzymes were overexpressed. Cell death in the overexpressing line was not significantly different from that in a cell line expressing normal QR and GST activities, indicating that these two enzymes alone are insufficient for protection against DA toxicity. Although the relative importance of a single antioxidant enzyme such as QR or GST may be small, antioxidant inducers such as DMF may prove valuable as agents that elicit a broad-spectrum neuroprotective response.     

Changes in Activities of Glucose Metabolising Enzymes in Germ Cells during Spermatogenesis in Rats

The rate of ¹⁴CO₂, liberation from [¹⁴C-1]glucose was identical to that from [¹⁴C-6]glucose in spermatids, but more than the latter in spermatogonia. Rotenone (1 μM) completely inhibited ¹⁴CO₂ release from [¹⁴C-1]glucose in spermatids, but decreased it only 30% in spermatogonia. The activity of glucose-6-phosphate dehydrogenase, but not 6-phosphogluconate dehydrogenase, was markedly lower in spermatocytes and spermatids than in spermatogonia. The activities of the glycolytic enzymes, glucosephosphate isomerase, fructose diphosphatase, glyceraldehyde-3-phosphate dehydrogenase and enolase, differed only slightly in spermatids and spermatogonia. It is concluded that the low glucose-6-phosphate dehydrogenase activity may contribute to the low activity of the pentose cycle in spermatocytes and spermatids.     

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.     

Synergism between Ultrasonic Waves and Hydrogen Peroxide in the Killing of Micro-organisms

The lethal effect on different micro-organisms of ultrasonic waves and hydrogen peroxide separately and in combination was examined. Ultrasonic waves were able to disintegrate Fusobacterium nucleatum within 3 min and to kill Veillonella parvula after 15 min and Streptoccus sanguis after 20 min; 20 vols H₂O₂ (6% w/v) killed V. parvula, Strep. sanguis and Staphylococcus aureus after 5 min treatment, and Clostridium sporogenes spores after 25 min. Sonication of Cl. sporogenes spores, Bacillus cereus spores and Candida albicans in 20 vols H₂O₂, using an ultrasonic probe, was lethal to the organisms after 15, 10 and 10 min, respectively. The latter 2 organisms were not killed by 30 min exposure to either agent separately. Similar results were obtained when an ultrasonic tank was used for sonication.     

Metabolism of hydrogen peroxide by the scavenging system in Chlamydomonas reinhardtii

The metabolism of hydrogen peroxide by the scavenging system was studied in Chlamydomonas grown in a selenium-lacking and a selenium-containing medium. In cells of the former, 40% of external hydrogen peroxide (H₂O₂) was scavenged by ascorbate peroxidase (AsAP; EC 1.11.1.11) and the residual H₂O₂ by catalase (EC 1.11.1.6). The enzymes involved in the ascorbate-glutathione cycle including AsAP. were localized in the chloroplast. In cells of the latter, glutathione peroxidase (GSHP; EC 1.11.1.9) functioned primarily in the removal of external H₂O₂. GSHP was located solely in the cytosol. The Chlamydomonas AsAP was relatively stable in ascorbate-depleted medium as compared with chloroplast AsAP of higher plants. No inactivation of the enzyme was found upon its incubation with hydroxyurea, an inhibitor of the chloroplast enzyme of higher plants. The enzyme showed higher specificity with pyrogallol than with ascorbate. The amino acid sequences in the N-terminal region of Chlamvdomonas AsAP showed no significant similarity to any other AsAP from higher plants and Euglena . The enzyme had a molecular mass of 34 kDa. The K_m values of the enzyme for ascorbate and H₂O₂ were 5.2±0.3 and 25±3.4 μ M , respectively. Hydrogen peroxide was generated at a rate of 6.1±0.8 μmol mg^-1 chlorophyll h^-1 in intact chloroplasts isolated from Chlamydomonas cells grown in the presence of Na-selenite, and it diffused from the organelles into the medium.     

Development of a method based on alkaline gel electrophoresis for estimation of oxidative damage to DNA in Escherichia coli

A method for estimating DNA strand breakage and subsequent repair based on alkaline gel electrophoresis was developed and tested with isogenic strains of Escherichia coli deficient in DNA repair enzymes. Samples from a cell suspension were removed at 2 min intervals following a 15 min exposure to 20 mmol l^-1 H₂O₂. Catalase was added and the cells were embedded in blocks of low-melting point agarose and lysed. After alkaline gel electrophoresis, photographs of the gels were taken and the relative lengths of the distributions of DNA fragments were measured with a scanner and computer. The lengths were correlated with survival of the cells exposed to H₂O₂ and with the importance of particular DNA repair enzymes. Alkaline gel electrophoresis appears to be a relatively simple method for analysing the level of H₂O₂-caused DNA damage and repair in E. coli.     

Interaction of α-Phenyl-N-tert-Butyl Nitrone and Alternative Electron Acceptors with Complex I Indicates a Substrate Reduction Site Upstream from the Rotenone Binding Site

Abstract: Mitochondrial complexes I, II, and III were studied in isolated brain mitochondrial preparations with the goal of determining their relative abilities to reduce O₂ to hydrogen peroxide (H₂O₂) or to reduce the alternative electron acceptors nitroblue tetrazolium (NBT) and diphenyliodonium (DPI). Complex I and II stimulation caused H₂O₂ formation and reduced NBT and DPI as indicated by dichlorodihydrofluorescein oxidation, nitroformazan precipitation, and DPI-mediated enzyme inactivation. The O₂ consumption rate was more rapid under complex II (succinate) stimulation than under complex I (NADH) stimulation. In contrast, H₂O₂ generation and NBT and DPI reduction kinetics were favored by NADH addition but were virtually unobservable during succinate-linked respiration. NADH oxidation was strongly suppressed by rotenone, but NADH-coupled H₂O₂ flux was accelerated by rotenone. α-Phenyl- N-tert -butyl nitrone (PBN), a compound documented to inhibit oxidative stress in models of stroke, sepsis, and parkinsonism, partially inhibited complex I-stimulated H₂O₂ flux and NBT reduction and also protected complex I from DPI-mediated inactivation while trapping the phenyl radical product of DPI reduction. The results suggest that complex I may be the principal source of brain mitochondrial H₂O₂ synthesis, possessing an "electron leak" site upstream from the rotenone binding site (i.e., on the NADH side of the enzyme). The inhibition of H₂O₂ production by PBN suggests a novel explanation for the broad-spectrum antioxidant and antiinflammatory activity of this nitrone spin trap.     

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.     

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.     

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.     

Key enzymes of carbohydrate metabolism as targets of the 11.5-kDa Zn(2+)-binding protein (parathymosin)

The 11.5-kDa Zn(2+)-binding protein (ZnBP) was covalently linked to Sepharose. Affinity chromatography with a cytosolic subfraction from liver resulted in purification of a predominant 38-kDa protein. In comparable experiments with brain cytosol a 39-kDa protein was enriched. The ZnBP-protein interactions were zinc-specific. Both proteins were identified as fructose-1,6-bisphosphate aldolase. Experiments with crude cytosol showed zinc-specific interaction of additional enzymes involved in carbohydrate metabolism. From liver cytosol greater than 90% of the following enzymes were specifically retained: aldolase, phosphofructokinase-1, hexokinase/glucokinase, glucose-6-phosphate dehydrogenase, glycerol-3-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and fructose-1,6-bisphosphatase. Glucose-6-phosphate isomerase, phosphoglycerate kinase, enolase, lactate dehydrogenase, and most of triosephosphate isomerase remained unbound. From L-type pyruvate kinase only the phosphorylated form seems to interact with ZnBP. Using brain cytosol hexokinase, phosphofructokinase-1, and aldolase were completely bound to the affinity column, whereas glucose-6-phosphate isomerase, phosphoglycerate kinase, enolase, lactate dehydrogenase, pyruvate kinase, and most of triose-phosphate isomerase remained unbound. The behavior of glucose-6-phosphate dehydrogenase and glycerol-3-phosphate dehydrogenase from this tissue could not be followed. A possible function of ZnBP in supramolecular organization of carbohydrate metabolism is proposed.     

The effect of hydrogen peroxide on spores of Clostridium perfringens

Dithiothreitol (DTT)-treated spores of Clostridium perfringens were much more sensitive to lysis by H₂O₂ in the presence of Cu²⁺ than untreated spores. Lysis was greatly inhibited by hydroxyl radical (.OH) scavengers such as thiourea, dimethylthiourea and dimethylsulfoxide, suggesting that lysis of spores by H₂O₂ involves formation of OH by Cu²⁺-catalysed decomposition of the peroxide. DTT-treated spores took up Cu²⁺ at almost the same rate and extent as did isolated cortical fragments. Hydrogen peroxide caused both the decrease in optical density and the hexosamine solubilization of cortical fragments which bound Cu²⁺.     

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.     

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.     

Water Relations of Glucose-catabolizing Enzymes in Pseudomonas fluorescens

Examination of the catabolism of glucose via the Entner-Doudoroff pathway by standard enzyme assays showed that the activity of glucose-6-phosphate dehydrogenase, glucokinase and 2-ketoglu-conokinase plus phosphoketogluconate reductase was completely inhibited at a _w values less than 0.965, 0.98 and 0.96 respectively when NaCl was used to adjust the a _w. The other glucose-catabolizing enzymes were inhibited to a lesser degree. When sucrose was used to control a _w, glucokinase and glucose-6-phosphate dehydrogenase were inhibited at 0.92 a _w but the other enzymes remained active below 0.86 a _w. Enzymes were relatively active at reduced a _w when adjusted with glycerol and most remained active even at 0.80 a _w. When a _w was controlled by potassium glutamate, the activity of glucokinase and glucose-6-phosphate dehydrogenase was markedly less inhibited than by NaCl at similar a _w. Possible reasons for the variation in activity by glucose-catabolizing enzymes in response to a _w controlled by various solutes could be location of the enzyme in the cell, ability of the solute to penetrate the cell and ability to withstand high salt and sucrose concentrations. When the a _w of the growth medium was reduced to 0.98 by glycerol, NaCl and polyethylene glycol 400, levels of glucokinase were significantly reduced while higher levels of glucose dehydrogenase and gluconate dehydrogenase were induced. This suggests that reduction in a _w could regulate the routes of catabolism in the Entner-Doudoroff pathway. When sucrose was used to control a _w of the growth medium high levels of most enzymes were induced, suggesting catabolism of the sucrose by the organism.     

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.