Hydrogen peroxide as an inductor of uncultivable state of Vibrio cholerae eltor in an experiment

The influence of hydrogen peroxide on the dynamics of transition into uncultivable state (UCS) and on the reversion of V. cholerae and their subcultures, resistant to hydrogen peroxide, was studied. The transition of the initial cultures in river and distilled water into UCS took place earlier than that in resistant to hydrogen peroxide variants. The capacity for reversion to hydrogen peroxide resistant subcultures preserved, on the average, 2 - 3 times longer. An increase in the level of hydrogen peroxide in uncultivable populations was found to be 2.7 - 4.4 times. Subcultures, resistant to hydrogen peroxide, in the vegetative form had lower characteristics of peroxide concentrations than in uncultivable form (UCF), but somewhat higher than in initial variants. In revertants the concentration of hydrogen peroxide was lower in UCF, but somewhat higher than in vegetative cultures. The dynamics of the formation of UCF by cholera vibrios, with different degree of stability to the action of hydrogen peroxide, the accumulation of hydrogen peroxide in uncultivable populations, the deceleration of transition into uncultivable forms, an accumulation of hydrogen peroxide and an increase in the time of the reversion of clones, resistant to hydrogen peroxide, made it possible to suggest that the accumulation of hydrogen peroxide was possible to make an essential contribution to the formation of UCF of cholera vibrios in an experiment.     

A kinetic analysis of the catalase activity of myeloperoxidase

The predominant physiological activity of myeloperoxidase is to convert hydrogen peroxide and chloride to hypochlorous acid. However, this neutrophil enzyme also degrades hydrogen peroxide to oxygen and water. We have undertaken a kinetic analysis of this reaction to clarify its mechanism. When myeloperoxidase was added to hydrogen peroxide in the absence of reducing substrates, there was an initial burst phase of hydrogen peroxide consumption followed by a slow steady state loss. The kinetics of hydrogen peroxide loss were precisely mirrored by the kinetics of oxygen production. Two mols of hydrogen peroxide gave rise to 1 mol of oxygen. With 100 microM hydrogen peroxide and 6 mM chloride, half of the hydrogen peroxide was converted to hypochlorous acid and the remainder to oxygen. Superoxide and tyrosine enhanced the steady-state loss of hydrogen peroxide in the absence of chloride. We propose that hydrogen peroxide reacts with the ferric enzyme to form compound I, which in turn reacts with another molecule of hydrogen peroxide to regenerate the native enzyme and liberate oxygen. The rate constant for the two-electron reduction of compound I by hydrogen peroxide was determined to be 2 x 10(6) M(-1) s(-1). The burst phase occurs because hydrogen peroxide and endogenous donors are able to slowly reduce compound I to compound II, which accumulates and retards the loss of hydrogen peroxide. Superoxide and tyrosine drive the catalase activity because they reduce compound II back to the native enzyme. The two-electron oxidation of hydrogen peroxide by compound I should be considered when interpreting mechanistic studies of myeloperoxidase and may influence the physiological activity of the enzyme.     

Formation of hydrogen peroxide by VUV-photolysis of water and aqueous solutions with methanol

The hydrogen peroxide production upon vacuum ultraviolet (VUV) irradiation of water is reviewed, because published results from the last 10 years lead to conflicting mechanistic interpretations. This work confirms that in pure water, hydrogen peroxide is only produced in the presence of molecular oxygen. Mechanistic schemes explain these findings and confirm earlier statements that recombination of hydroxyl radicals is kinetically disfavoured. In agreement with other recent publications, this work confirms that enhanced hydrogen peroxide production takes place upon VUV irradiation of aqueous solutions of organic compounds. For these investigations, methanol was chosen as an organic model compound. During photolyses, hydrogen peroxide, dissolved molecular oxygen, pH-value of the reaction system, methanol and its products of oxidative degradation were analyzed, and kinetic studies were undertaken to explain the evolution of the concentrations of these components.     

The similarity and difference in the action of adriamycin and hydrogen peroxide on the myocardium

The oxidative stress in cardiomyocytes is a component of cardiotoxic action of adriamycin. To distinguish the importance of this component we have compared initial effects of hydrogen peroxide and adriamycin on the contractile function and a tone of coronary vessels of the isolated rat heart. Adriamycin in concentration 3 mcM distinctly reduced developed pressure and heart rate, but raised coronary vessel tone. The concentration 1 mcM was inefficient at usual perfusion rate, but practically prevented rise in developed pressure and rates of its rise and fall at increased perfusion rate. Hydrogen peroxide also dose-dependently reduced developed pressure, but rates of its rise and fall were reduced to a lesser degree, while the heart rate slightly raised, and coronary vessel tone was reduced. Thus, the initial actions of hydrogen peroxide and adriamycin on the heart considerably differ. It suggests that the oxidative stress is not the main component of cardiotoxic action of adriamycin, at least, in its application in concentrations close to therapeutic ones.     

Variation of the oxidation state of verdoheme in the heme oxygenase reaction

Heme oxygenase (HO) converts hemin to biliverdin, CO, and iron applying molecular oxygen and electrons. During successive HO reactions, two intermediates, α-hydroxyhemin and verdoheme, have been generated. Here, oxidation state of the verdoheme-HO complexes is controversial. To clarify this, the heme conversion by soybean and rat HO isoform-1 (GmHO-1 and rHO-1, respectively) was compared both under physiological conditions, with oxygen and NADPH coupled with ferredoxin reductase/ferredoxin for GmHO-1 or with cytochrome P450 reductase for rHO-1, and under a non-physiological condition with hydrogen peroxide. EPR measurements on the hemin-GmHO-1 reaction with oxygen detected a low-spin ferric intermediate, which was undetectable in the rHO-1 reaction, suggesting the verdoheme in the six-coordinate ferric state in GmHO-1. Optical absorption measurements on this reaction indicated that the heme degradation was extremely retarded at verdoheme though this reaction was not inhibited under high-CO concentrations, unlike the rHO-1 reaction. On the contrary, the Gm and rHO-1 reactions with hydrogen peroxide both provided ferric low-spin intermediates though their yields were different. The optical absorption spectra suggested that the ferric and ferrous verdoheme coexisted in reaction mixtures and were slowly converted to the ferric biliverdin complex. Consequently, in the physiological oxygen reactions, the verdoheme is found to be stabilized in the ferric state in GmHO-1 probably guided by protein distal residues and in the ferrous state in rHO-1, whereas in the hydrogen peroxide reactions, hydrogen peroxide or hydroxide coordination stabilizes the ferric state of verdoheme in both HOs.     

Fluctuations of pass band coefficients of water and water salt solutions in the spectral infrared region

The effect of superlow concentrations of KCl and CaCl2 solutions, obtained by diluting starting 1 M solutions of these salts 10(9)-10(15) times, on the fluctuations of pass band coefficients of water was studied. It was found that these solutions are substantially distinguished from bidistilled water by a high intensity of fluctuations of spectra in the infrared region. It was also shown that higher concentrations of solutions of these salts (at dilutions less than 10(8) times) have no strong and specific effect on fluctuations of the infrared spectrum: the intensity of fluctuations of the infrared spectra of these solutions practically coincides with that of control water. Possible reasons for this effect are discussed.     

The Tumor Suppressor Mst1 Promotes Changes in the Cellular Redox State by Phosphorylation and Inactivation of Peroxiredoxin-1 Protein

The serine/threonine protein kinases Mst1 and Mst2 can be activated by cellular stressors including hydrogen peroxide. Using two independent protein interaction screens, we show that these kinases associate, in an oxidation-dependent manner, with Prdx1, an enzyme that regulates the cellular redox state by reducing hydrogen peroxide to water and oxygen. Mst1 inactivates Prdx1 by phosphorylating it at Thr-90 and Thr-183, leading to accumulation of hydrogen peroxide in cells. These results suggest that hydrogen peroxide-stimulated Mst1 activates a positive feedback loop to sustain an oxidizing cellular state.     

Oxygen protection of bacteriophage T1 against ionizing radiations

Bacteriophage T1 was suspended in distilled water and in phosphate buffer, saturated with oxygen, nitrogen, hydrogen, and carbon monoxide, and irradiated with gamma rays and x-rays. Under the same conditions phage was exposed to hydrogen peroxide. Oxygen acted as a protective agent against both irradiation and hydrogen peroxide inactivation. As a protective agent against irradiation, oxygen was more efficient in distilled water than in buffer. The phage was much more sensitive to irradiation in the presence of hydrogen or nitrogen than in the presence of oxygen. Survivals of phage irradiated in suspensions saturated with hydrogen and with nitrogen did not differ significantly. From this it was concluded that oxygen did not protect T1 by removing atomic hydrogen from the irradiated medium, since the hydrogen-saturated medium increased the yield of atomic hydrogen but did not increase the yield of inactivated phage. It was presumed, therefore, that phage is sensitive to OH radicals and this was confirmed by irradiating phage with UV in the presence of hydrogen peroxide and comparing this survival with the survivals obtained from hydrogen peroxide alone and from UV alone. The combined effect of hydrogen peroxide and UV acting simultaneously was greater than the effect attributable to hydrogen peroxide and UV acting separately. Evidence for sensitivity to HO₂ radicals was considered, and the effect was attributed chiefly to an oxidizing action since phage sensitivity is greater at higher hydrogen ion concentrations, which favor oxidation by HO₂ radicals. Since the OH radical is a more efficient oxidizing agent than O^-, the former being favored in an acid medium, the latter in an alkaline medium, and since the phage is more sensitive in the first situation than in the second, the present tests proved the importance of oxidation as the mechanism of inactivation. Since some inactivation was encountered when phage was exposed to reducing agents, independently of irradiation, it was concluded that phage is somewhat sensitive to reducing agents, but the inactivation attributable to ionizing radiations is due chiefly to oxidation, against which these reducing agents are very efficient protectors. Under no circumstances did hydrogen peroxide protect T1, whether produced by irradiation in the medium or added beforehand to the medium to be irradiated. The first point was investigated by irradiating T1 in the presence of hydrogen and oxygen combined; this produced a higher yield of hydrogen peroxide but a lower survival of T1. In all these tests phage survival under irradiation was directly correlated with oxygen content of the medium rather than with production of hydrogen peroxide. It is proposed that the protective effect of oxygen is due to a reaction between the phage and oxygen, and this complex confers stability upon the phage.     

Effects of rifampicin and doxycycline on the production of hydrogen peroxide by macrophages]

The influence of rifampicin and doxycycline on oxidative metabolism of macrophages was estimated in vitro by production of hydrogen peroxide. It was shown that low concentrations of rifampicin and doxycycline stimulated production of hydrogen peroxide by macrophages of guinea pigs. In concentrations of 1 to 10 micrograms/ml corresponding to the mean therapeutic ones doxycycline increased both the spontaneous and zymosan-induced production of hydrogen peroxide by the macrophages. The potentiating activity of doxycycline on the cells activated by opsonized zymosan was higher. The maximum increase in the induced production of hydrogen peroxide (by 40 per cent) was observed when the antibiotic concentration was 1 microgram/ml. Rifampicin in concentrations of 0.1 to 1 microgram/ml corresponding to the mean therapeutic ones stimulated the zymosan-induced production of hydrogen peroxide by the macrophages. The maximum increase in the production of hydrogen peroxide (by 22 per cent) was noted at the rifampicin concentration of 1 microgram/ml.     

Mechanism of dioxygen formation catalyzed by vanadium bromoperoxidase. Steady state kinetic analysis and comparison to the mechanism of bromination

The steady state kinetic mechanism of the bromide-assisted disproportionation of hydrogen peroxide, forming dioxygen, catalyzed by vanadium bromoperoxidase has been investigated and compared to the mechanism of monochlorodimedone (MCD) bromination under conditions of 0.0125-6 mM H2O2, 1-500 mM Br-, and pH 4.55-6.52. Under these conditions, 50 microM MCD was sufficient to inhibit at least 90% of the dioxygen formation during MCD bromination. The rate data is consistent with a substrate-inhibited Bi Bi Ping Pong mechanism, in which the substrate bromide, is also an inhibitor at pH 4.55 and 5.25, but not at pH 5.91 and 6.52. The kinetic parameter KmBr, KmH2O2, KisBr, and KiiBr determined for the reactions of bromide-assisted disproportionation fo hydrogen peroxide and MCD bromination are similar, indicating that the mechanisms of both reactions occur via the formation of a common intermediate, the formation of which is rate-limiting. Fluoride is a competitive inhibitor with respect to hydrogen peroxide in both reactions at pH 6.5. At high concentrations of hydrogen peroxide, the bromide-assisted disproportionation of hydrogen peroxide occurs during the bromination of MCD. The sum of the rates of MCD bromination and dioxygen formation during MCD bromination is equal to the rate of dioxygen formation in the absence of MCD. The apportionment of the reaction through the MCD bromination and dioxygen formation pathways depends on pH, with much lower hydrogen peroxide concentrations causing significant dioxygen formation at higher pH.     

Enhancement of the enzymatic digestibility of sugarcane bagasse by steam pretreatment impregnated with hydrogen peroxide

Sugarcane bagasse was subjected to steam pretreatment impregnated with hydrogen peroxide. Analyses were performed using 2³ factorial designs and enzymatic hydrolysis was performed at two different solid concentrations and with washed and unwashed material to evaluate the importance of this step for obtaining high cellulose conversion. Similar cellulose conversion were obtained at different conditions of pretreatment and hydrolysis. When the cellulose was hydrolyzed using the pretreated material in the most severe conditions of the experimental design (210°C, 15 min and 1.0% hydrogen peroxide), and using 2% (w/w) water‐insoluble solids (WIS), and 15 FPU/g WIS, the cellulose conversion was 86.9%. In contrast, at a milder pretreatment condition (190°C, 15 min and 0.2% hydrogen peroxide) and industrially more realistic conditions of hydrolysis (10% WIS and 10 FPU/g WIS), the cellulose conversion reached 82.2%. The step of washing the pretreated material was very important to obtain high concentrations of fermentable sugars. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Dopamine modifies oxygen consumption and mitochondrial membrane potential in striatal mitochondria

Dopamine is a neurotransmitter that has been related to mitochondrial dysfunction. In this study, striatal intact mitochondria and submitochondrial membranes were incubated with different dopamine concentrations, and changes on mitochondrial function, hydrogen peroxide, and nitric oxide production were evaluated. A 35% decrease in state 3 oxygen uptake (active respiration state) was found after 1 mM dopamine incubation. In addition, mitochondrial respiratory control significantly decreased, indicating mitochondrial dysfunction. High dopamine concentrations induced mitochondrial depolarization. Also, evaluation of hydrogen peroxide production by intact striatal mitochondria showed a significant increase after 0.5 and 1 mM dopamine incubation. Incubation with 0.5 and 1 mM dopamine increased nitric oxide production in submitochondrial membranes by 28 and 49%, respectively, as compared with control values. This study provides evidence that high dopamine concentrations induce striatal mitochondrial dysfunction through a decrease in mitochondrial respiratory control and loss of membrane potential, probably mediated by free radical production.     

Bactericidal properties of peracetic acid and hydrogen peroxide, alone and in combination, and chlorine and formaldehyde against bacterial water strains.

The bactericidal properties of peracetic acid, hydrogen peroxide, chlorine, and formaldehyde were compared in vitro using a rapid micromethod. A combination of peracetic acid and hydrogen peroxide was also tested to assess interactions. The activities of these agents, which are widely used as disinfectants, were evaluated against water isolates and culture collection strains. Peracetic acid and chlorine exhibited an excellent antimicrobial activity, with a relatively rapid destruction of 10(5) bacteria/mL. The time-dependent bactericidal activities of hydrogen peroxide and formaldehyde were the lowest. The combination of peracetic acid and hydrogen peroxide, tested by a checkerboard micromethod, was found to be synergistic. The minimal bactericidal concentration was established in terms of time for a given mixture of peracetic acid and hydrogen peroxide. Determination of bactericidal concentrations showed that synergy was maintained with increasing contact time. Concentrations for minimal times of treatment by chemicals that provided interesting activities in vitro were tested for disinfection of ultrafiltration membranes. The bactericidal activities of peroxygen compounds were confirmed and synergism was maintained in working conditions. Chlorine showed a loss of efficacy when used on membranes.     

Spectroscopic and quantitative analysis of the oxygenated and peroxy states of the purified cytochrome d complex of Escherichia coli

Oxygenated and peroxy states of the cytochrome d complex of Escherichia coli have been proposed as intermediates in the reaction mechanism of this ubiquinol oxidase. In this report, several stable states of the purified enzyme were examined spectroscopically at room temperature. As purified, the cytochrome d complex exists in an oxygenated state characterized by an absorbance band at 650 nm. Removal of oxygen results in loss of absorbance at this wavelength, which is restored upon the return of oxygen. The presence of one oxygen molecule in the oxygenated state was quantified by measuring oxygen released when excess hydrogen peroxide was added to the oxygenated state by passage of argon generates a "partially reduced" state with an absorbance peak at 628 nm, apparently due to reduced cytochrome d. Addition of equimolar hydrogen peroxide to the fully oxidized state produces the peroxy state. This peroxy state is also formed upon addition of excess hydrogen peroxide to the oxygenated state via a stable intermediate termed "peroxy intermediate." It is likely that 1) the oxygenated state consists of one molecule of oxygen bound to reduced heme d, and 2) there are at least two stable states that have bound peroxide at room temperature, the peroxy state and a newly discovered peroxy intermediate.     

Implication of hydrogen peroxide in the mutagenicity of coffee

A cup of instant coffee (150 ml) of normal strength (15 mg/ml) was found to contain about 500 and 750 micrograms of hydrogen peroxide soon after its preparation at 37 degrees C and 80 degrees C, respectively, but the concentration of hydrogen peroxide in the coffee increased with time for up to 24 h after its preparation. Thus coffee contains a hydrogen peroxide generating system. As extracts of green coffee beans were found to have very low capacity to generate hydrogen peroxide, this generating system is produced by roasting coffee beans. Hydrogen peroxide itself was only weakly mutagenic to Salmonella typhimurium TA100, but in the presence of methylglyoxal, which is also present as a mutagenic component in coffee, hydrogen peroxide showed strong mutagenicity. Hydrogen peroxide and methylglyoxal seem to be responsible for most of the mutagenicity of instant coffee.     

The oxidative stress response in Bacillus subtilis

Abstract Bacillus subtilis undergoes a typical bacterial stress response when exposed to low concentrations (0.1 mM) of hydrogen peroxide. Protection is thereby induced against otherwise lethal, challenge concentrations (10 mM) of this oxidant and a number of proteins are induced including the scavenging enzymes, catalase and alkyl hydroperoxide reductase, and a putative DNA binding and protecting protein. Induced protection against higher concentrations (10–30 mM) of hydrogen peroxide is eliminated in a catalase-deficient mutant. Both RecA and Spo0A influence the basal but not the induced resistance to hydrogen peroxide. A regulatory mutation has been characterized that affects the inducible phenotype and is constitutively resistant to high concentrations of hydrogen peroxide. This mutant constitutively overexpresses the proteins induced by hydrogen peroxide in the wild-type. The resistance of spores to hydrogen peroxide is partly attributable to binding of small acid soluble proteins by the spore DNA and partly to a second step which coincides with the depletion of the NADH pool, which may inhibit the generation of hydroxyl radicals from hydrogen peroxide.     

Redox Cycling of Human Methaemoglobin by H2O2 Yields Persistent Ferryl Iron and Protein Based Radicals

The formation and reactivity of ferryl haemoglobin (and myoglobin), which occurs on addition of H₂O₂, has been proposed as a mechanism contributing to oxidative stress associated with human diseases. However, relatively little is known of the reaction between hydrogen peroxide and human haemoglobin. We have studied the reaction between hydrogen peroxide and purified (catalase free) human metHbA. Addition of H₂O₂ resulted in production of both ferryl haem iron (detected by optical spectroscopy) and an associated protein radical (detected by EPR spectroscopy). Titrating metHbA with H₂O₂ showed that maximum ferryl levels could be obtained at a 1:1 stoichiometric ratio of haem to H₂O₂. No oxygen was evolved during the reaction, indicating that human metHbA does itself not possess catalatic activity. The protein radicals obtained in this reaction reached a steady state concentration, during hydrogen peroxide decomposition, but started to decay once the hydrogen peroxide had been completely exhausted. The presence of catalase, at concentrations around 10 fold lower than metHb, increased the apparent stoichiometry of the reaction to 1 mol metHb: ∼20 mol H₂O₂ and abolished the protein radical steady state. The biological implications for these results are discussed.     

Redox regulation of proliferation of lens epithelial cells in culture

Both oxidants and antioxidants have been shown to modulate cell proliferation. We studied the effects of hydrogen peroxide and two antioxidants on the rate of proliferation of lens epithelial cells in culture. Hydrogen peroxide at concentrations higher than 32 microM caused a significant inhibition of proliferation. However, in the concentration range of 0.01-0.5 microM, hydrogen peroxide stimulated the rate of proliferation. The effect of hydrogen peroxide was dependent on the amount of cells in an individual culture well, indicating decomposition of hydrogen peroxide by cellular enzymes. In order to eliminate the possibility of decomposition of the dose of hydrogen peroxide given as a bolus, we induced continual production of hydrogen peroxide by adding glucose oxidase to the incubation medium. We found that hydrogen peroxide, generated by 1-50 microU x ml(-1) of glucose oxidase significantly increased the rate of cell proliferation. This effect was most apparent at the beginning of the exponential phase of cellular growth. Glucose oxidase alone (100-500 microU x ml(-1)) did not produce any effect. The effects of pro-oxidative hydrogen peroxide were compared with the effects of two biologically important antioxidants, alpha-tocopherol and retinol. Both antioxidants completely inhibited proliferation at concentrations of 30 microM and higher. In contrast to retinol, the effect of alpha-tocopherol was dependent on the amount of cells, indicating cellular decomposition of alpha-tocopherol. The results document the possibility of redox regulation of cellular proliferation at physiologically relevant reactant concentrations.     

Testing the vicious cycle theory of mitochondrial ROS production: effects of H2O2 and cumene hydroperoxide treatment on heart mitochondria

Vicious cycle theories of aging and oxidative stress propose that ROS produced by the mitochondrial electron transport chain damage the mitochondria leading exponentially to more ROS production and mitochondrial damage. Although this theory is widely discussed in the field of research on aging and oxidative stress, there is little supporting data. Therefore, in order to help clarify to what extent the vicious cycle theory of aging is correct, we have exposed mitochondria in vitro to different concentrations of hydrogen peroxide or cumene-hydroperoxide (0, 30, 100 and 500 μM). We have found that 30 μM hydrogen peroxide (or higher concentrations) inhibit oxygen consumption in state 3 and increase ROS production with pyruvate/malate but not with succinate as substrate, indicating that these effects occur specifically at complex I. Similar levels of cumene-OOH inhibit state 3 respiration with both kinds of substrates, and increase ROS production in both state 4 and state 3 with pyruvate/malate and with succinate. The effects of cumene-OOH on ROS generation are due to action of the peroxide in the complex III or in the complex III plus complex I ROS generators. In all cases, the increase in ROS production occurred at a threshold level of peroxide exposure without further exponential increase in ROS generation. These results are consistent with the idea that ROS production can contribute to increase oxidative stress in old animals, but the results do not fit with a vicious cycle theory in which peroxide generation leads exponentially to more and more ROS production with age.     

Structural Analysis of Peroxide-Soaked MnSOD Crystals Reveals Side-On Binding of Peroxide to Active-Site Manganese

The superoxide dismutase (SOD) enzymes are important antioxidant agents that protect cells from reactive oxygen species. The SOD family is responsible for catalyzing the disproportionation of superoxide radical to oxygen and hydrogen peroxide. Manganese- and iron-containing SOD exhibit product inhibition whereas Cu/ZnSOD does not. Here, we report the crystal structure of Escherichia coli MnSOD with hydrogen peroxide cryotrapped in the active site. Crystallographic refinement to 1.55 Å and close inspection revealed electron density for hydrogen peroxide in three of the four active sites in the asymmetric unit. The hydrogen peroxide molecules are in the position opposite His26 that is normally assumed by water in the trigonal bipyramidal resting state of the enzyme. Hydrogen peroxide is present in active sites B, C, and D and is side-on coordinated to the active-site manganese. In chains B and D, the peroxide is oriented in the plane formed by manganese and ligands Asp167 and His26. In chain C, the peroxide is bound, making a 70° angle to the plane. Comparison of the peroxide-bound active site with the hydroxide-bound octahedral form shows a shifting of residue Tyr34 towards the active site when peroxide is bound. Comparison with peroxide-soaked Cu/ZnSOD indicates end-on binding of peroxide when the SOD does not exhibit inhibition by peroxide and side-on binding of peroxide in the product-inhibited state of MnSOD.