Lysosomal origin of the ferric iron required for cell killing by hydrogen peroxide

The lysosomotropic amines methylamine (40 mM) and chloroquine (125 mM) prevented the killing of cultured hepatocytes by hydrogen peroxide generated in the medium by glucose oxidase. Maximum protection required several hours preincubation with either amine. Sensitivity of the hepatocytes to H2O2 was restored either by the addition of ferrous or ferric iron to the culture medium, or by incubating the cells for 4 hours in the absence of either amine prior to treatment with H2O2. Neither methylamine nor chloroquine had any effect on the cell killing by t-butyl hydroperoxide, a hepatotoxin that does not require iron. The protective effect of the lysosomotropic amines was distinguished from that of the ferric iron chelator deferoxamine in two ways: 1) deferoxamine protected hepatocytes from H2O2 toxicity but did not require a pretreatment period; and 2) in contrast to methylamine or chloroquine, deferoxamine had no effect on lysosomal pH as assessed by the fluorescent probe acridine orange. The data suggest that a lysosomal pool is the source of the ferric iron necessary for the killing of hepatocytes by H2O2.     

Prehemolytic effects of hydrogen peroxide and t-butylhydroperoxide on selected red cell properties

To provide further understanding of how oxidative damage affects red cell membrane function, the effects of low levels of two different types of oxidants on selected red cell properties have been studied. Hydrogen peroxide (H2O2), an example of a water soluble oxidant, and t-butylhydroperoxide (tBHP), a hydrophobic hydroperoxide, were compared with respect to their effects on membrane permeability, membrane mechanical properties and binding of autologous serum antibodies to the cell surface. Whereas H2O2 treatment resulted in a dose-dependent increase in membrane permeability to potassium that was evident after one hour of oxidant exposure, cells treated with tBHP at doses up to 5 mumol/ml cells showed no immediate change in cation permeability. H2O2 also caused a marked decrease in membrane deformability, whereas tBHP-treated cells showed minimal loss of deformability. However, tBHP treatment did result in a dose-dependent increase in the susceptibility of the membrane to fragmentation under high shear stress. With exclusion of treated samples that bound excess rabbit anti-spectrin antibody, indicating exposure of intracellular components, neither agent promoted the binding of autologous serum antibody in amounts comparable to that found in vivo on high density or some pathologic red cells. Taken together, the results suggest that tBHP and H2O2 cause damage to human red cells by distinct oxidative mechanisms which do not lead directly to substantive generation of binding sites for autologous serum antibodies.     

The detection of hydrogen peroxide involved in plant virus infection by fluorescence spectroscopy

The production of reactive oxygen species (ROS) forms part of the defense reaction of plants against invading pathogens. ROS have multifaceted signaling functions in mediating the establishment of multiple responses. To verify whether hydrogen peroxide (H₂O₂) contributes to plant virus infection and the development of induced symptoms, we used fluorescence to monitor the generation of H₂O₂ and confocal laser scanning microscopy (CLSM) to investigate the subcellular distribution of H₂O₂ in leaves. In this study, the M strain of Cucumber mosaic virus (M‐CMV) induced heavy chlorotic symptoms in Nicotiana tabacum cv. white burley during systemic infection. Compared with mock‐inoculated leaves, H₂O₂ accumulation in inoculated leaves increased after inoculation, then decreased after 4 days. For systemically infected leaves that showed chlorotic symptoms, H₂O₂ accumulation was always higher than in healthy leaves. Subcellular H₂O₂ localization observed using CLSM showed that H₂O₂ in inoculated leaves was generated mainly in the chloroplasts and cell wall, whereas in systemically infected leaves H₂O₂ was generated mainly in the cytosol. The levels of coat protein in inoculated and systemically infected leaves might be associated with changes in the level of H₂O₂ and symptom development. Further research is needed to elucidate the generation mechanism and the relationship between coat protein and oxidative stress during infection and symptom development. Copyright © 2016 John Wiley & Sons, Ltd.     

The permeability of human red blood cell membranes to hydrogen peroxide is independent of aquaporins

Hydrogen peroxide (H₂O₂) not only is an oxidant but also is an important signaling molecule in vascular biology, mediating several physiological functions. Red blood cells (RBCs) have been proposed to be the primary sink of H₂O₂ in the vasculature because they are the main cellular component of blood with a robust antioxidant defense and a high membrane permeability. However, the exact permeability of human RBC to H₂O₂ is neither known nor is it known if the mechanism of permeation involves the lipid fraction or protein channels. To gain insight into the permeability process, we measured the partition constant of H₂O₂ between water and octanol or hexadecane using a novel double-partition method. Our results indicated that there is a large thermodynamic barrier to H₂O₂ permeation. The permeability coefficient of H₂O₂ through phospholipid membranes containing cholesterol with saturated or unsaturated acyl chains was determined to be 4 × 10⁻⁴ and 5 × 10⁻³ cm s⁻¹, respectively, at 37 °C. The permeability coefficient of human RBC membranes to H₂O₂ at 37 °C, on the other hand, was 1.6 × 10⁻³ cm s⁻¹. Different aquaporin-1 and aquaporin-3 inhibitors proved to have no effect on the permeation of H₂O₂. Moreover, human RBCs devoid of either aquaporin-1 or aquaporin-3 were equally permeable to H₂O₂ as normal human RBCs. Therefore, these results indicate that H₂O₂ does not diffuse into RBCs through aquaporins but rather through the lipid fraction or a still unidentified membrane protein.     

Root growth inhibition by aluminum is probably caused by cell death due to peroxidase-mediated hydrogen peroxide production

Summary. The effect of aluminum on hydrogen peroxide production and peroxidase-catalyzed NADH oxidation was studied in barley roots germinated and grown between two layers of moistened filter paper. Guaiacol peroxidase activity significantly increased after 48h and was approximately two times higher after 72h in Al-treated roots. The oxidation of NADH was also significantly increased and, like guaiacol peroxidase activity, it was two times higher in Al-treated roots than in controls. Elevated H₂O₂ production was observed both 48 and 72h after the onset of imbibition in the presence of Al. Separation on a cation exchange column allowed the detection of two peaks with NADH peroxidase and H₂O₂ production activity. However, a difference between control and Al-treated plants was found only in one fraction, in which four times higher guaiacol peroxidase activity and five times higher NADH peroxidase activity were expressed and about three times more H₂O₂ was produced. One anionic peroxidase and three cationic peroxidases were detected in this fraction by native polyacrylamide gel electrophoresis. The anionic peroxidase was activated in the Al-treated root tips and also oxidized NADH but was detectable only after a long incubation time. Two of the cationic peroxidases were capable of oxidizing NADH and producing a significant amount of H₂O₂, but only one of these was activated by Al stress. The role of these peroxidases during Al stress in barley root tips is discussed.Correspondence and reprints: Institute of Botany, Slovak Academy of Sciences, Dúbravská cesta 14, 845 23 Bratislava, Slovakia.     

Highly sensitive determination of hydrogen peroxide and glucose by fluorescence correlation spectroscopy

Background

Because H₂O₂ is generated by various oxidase-catalyzed reactions, a highly sensitive determination method of H₂O₂ is applicable to measurements of low levels of various oxidases and their substrates such as glucose, lactate, glutamate, urate, xanthine, choline, cholesterol and NADPH. We propose herein a new, highly sensitive method for the measurement of H₂O₂ and glucose using fluorescence correlation spectroscopy (FCS).

Methodology/Principal Findings

FCS has the advantage of allowing us to determine the number of fluorescent molecules. FCS measures the fluctuations in fluorescence intensity caused by fluorescent probe movement in a small light cavity with a defined volume generated by confocal illumination. We thus developed a highly sensitive determination system of H₂O₂ by FCS, where horseradish peroxidase (HRP) catalyzes the formation of a covalent bond between fluorescent molecules and proteins in the presence of H₂O₂. Our developed system gave a linear calibration curve for H₂O₂ in the range of 28 to 300 nM with the detection limit of 8 nM. In addition, by coupling with glucose oxidase (GOD)-catalyzed reaction, the method allows to measure glucose in the range of 80 nM to 1.5 µM with detection limit of 24 nM. The method was applicable to the assay of glucose in blood plasma. The mean concentration of glucose in normal human blood plasma was determined to be 4.9 mM.

Conclusions/Significance

In comparison with commercial available methods, the detection limit and the minimum value of determination for glucose are at least 2 orders of magnitude more sensitive in our system. Such a highly sensitive method leads the fact that only a very small amount of plasma (20 nL) is needed for the determination of glucose concentration in blood plasma.     

Abrupt increase in the level of hydrogen peroxide in leaves of winter wheat is caused by cold treatment

After cold treatment of seedlings of winter wheat (Triticum aestivum L.), levels of hydrogen peroxide in the leaves were measured. The concentration of hydrogen peroxide increased to about three times the control level within a few minutes, and returned to the normal level in 15 to 20 minutes. The elevated level of hydrogen peroxide was found to be equivalent to 1.5 micromoles per gram fresh weight tissues of leaves.     

Assessment of cell death studies by monitoring hydrogen peroxide in cell culture

Hydrogen peroxide (H₂O₂) has been widely used to study the oxidative stress response. However, H₂O₂ is unstable and easily decomposes into H₂O and O₂. Consequently, a wide range of exposure times and treatment concentrations has been described in the literature. In the present study, we established a ferrous oxidation-xylenol orange (FOX) assay, which was originally described for food and body liquids, as a method for the precise quantification of H₂O₂ concentrations in cell culture media. We observed that the presence of FCS and high cell densities significantly accelerate the decomposition of H₂O₂, therefore acting as a protection against cell death by accidental necrosis.     

Spermine and spermidine mediate protection against oxidative damage caused by hydrogen peroxide

Summary. The polyamines spermidine and spermine have been hypothesized to possess different functions in the protection of DNA from reactive oxygen species. The growth and survival of mouse fibroblasts unable to synthesize spermine were compared to their normal counterparts in their native and polyamine-depleted states in response to oxidative stress. The results of these studies suggest that when present at normal or supraphysiological concentrations, either spermidine or spermine can protect cells from reactive oxygen species. However, when polyamine pools are pharmacologically manipulated to produce cells with low levels of predominately spermine or spermidine, spermine appears to be more effective. Importantly, when cells are depleted of both glutathione and endogenous polyamines, they exhibit increased sensitivity to hydrogen peroxide as compared to glutathione depletion alone, suggesting that polyamines not only play a role in protecting cells from oxidative stress but this role is distinct from that played by glutathione.     

Quenching of red cell tryptophan fluorescence by mercurial compounds

Intrinsic tryptophan fluorescence in red cell ghost membranes labeled with N-ethylmaleimide (N-EM) is quenched in a dose-dependent manner by the organic mercurial p-chloromercuribenzene sulfonate (p-CMBS). Fluorescence lifetime analysis shows that quenching occurs by a static mechanism. Binding of p-CMBS occurs by a rapid (less than 5 s) biomolecular association (dissociation constant K1 = 1.8 mM) followed by a slower unimolecular transition with forward rate constant k2 = 0.015 s-1 and reverse rate constant k-2 = 0.0054 s-1. Analysis of the temperature dependence of k2 gives delta H = 6.5 kcal/mol and delta S = -21 eu. The mercurial compounds p-chloromercuribenzoic acid, p-aminophenylmercuric acetate, and mercuric chloride quench red cell tryptophan fluorescence by the same mechanism as p-CMBS does; the measured k2 value was the same for each compound, whereas K1 varied. p-CMBS also quenches the tryptophan fluorescence in vesicles reconstituted with purified band 3, the red cell anion exchange protein, in a manner similar to that in ghost membranes. These experiments define a mercurial binding site on band 3 in ghosts treated with N-EM and establish the binding mechanism to this site. The characteristics of this p-CMBS binding site on band 3 differ significantly from those of the p-CMBS binding site involved in red cell water and urea transport inhibition.     

The degradation of cytochrome c by hydrogen peroxide

Cytochrome c is degraded by a large excess of hydrogen peroxide, leading to opening of the heme porphyrin ring and loss of the Soret absorption bands. The kinetic parameters of this reaction have been determined, and it is shown that a small concentration of oxygen is liberated at the same rate as degradation. Low-level chemiluminescence and release of a hydroxylating species also accompany heme destruction. It is proposed that heme iron activates hydrogen peroxide to a more powerful oxidant, perhaps the hydroxyl radical, which remains bound to the heme iron and initiates attack on the porphyrin ring. Chemiluminescence appears to result from a side reaction involving singlet oxygen attack on the alpha-methene bridge, yielding a dioxetane. The in vivo degradation of cytochrome c by excess hydrogen peroxide may interfere with respiration, accelerate aging, and enhance the metabolism of carcinogens.     

Synthesis of genistein derivatives and determination of their protective effects against vascular endothelial cell damages caused by hydrogen peroxide

A series of genistein derivatives, prepared by alkylation and difluoromethylation, were tested for their inhibitory effects on the hydrogen peroxide induced impairment in human umbilical vein endothelial (HUVE-12) cells in vitro. The HUVE-12 cells were pretreated with either the vehicle solvent (DMSO), genistein, or different amounts of the genistein derivatives for 30 min before exposed to 1 mM hydrogen peroxide for 24 h. Cell apoptosis was determined by flow cytometry with propidium iodide (PI) staining. Cellular injury was estimated by measuring the lactate dehydrogenase (LDH) release. Data suggested that the genistein derivatives possessed a protective effect on HUVE-12 cells from hydrogen peroxide induced apoptosis and reduced LDH release. Among these derivatives, 7-difluoromethyl-5,4'-dimethoxygenistein exhibited the strongest activity against hydrogen peroxide induced apoptosis of HUVE-12 cells.     

A specific fluorescence probe for hydrogen peroxide detection in peroxisomes

Hydrogen peroxide is an important mediator in cell signalling and cell death. Apart from the mitochondrion the peroxisome is the most important cellular site for the generation and scavenging of hydrogen peroxide. Peroxisomes contain various oxidases, e.g. for the metabolism of long-chain fatty acids, polyamines, and for the oxidation of urate, which form hydrogen peroxide. Widely-used chemical probes for the detection of hydrogen peroxide like dichlorofluorescein diacetate (DCFDA) often lack in specificity and the possibility of compartment-specific measurement. To overcome these disadvantages, Belousov et al. developed the novel hydrogen peroxide sensitive fluorescent protein HyPer. In the present study the HyPer protein was fused with the PTS1 tag for a specific hydrogen peroxide detection in peroxisomes. The localization of the HyPer protein in the peroxisomes was confirmed by immunofluorescence and the functionality by fluorescence microscopy and flow cytometry analyses. The presented HyPer-Peroxi fluorescent protein is a valuable tool for studying hydrogen peroxide generation within the peroxisomes.     

Inhibition of Chinese hamster ovary cell DNA synthesis by hydrogen peroxide

The DNA synthesis inhibitory effect of hydrogen peroxide has been examined under a number of experimental conditions. Results have indicated that the effect of the oxidant is more pronounced when the treatment is performed at 37 degrees C than at 4 degrees C and in low density as compared to high density cultures. In addition, similar levels of inhibition were achieved by measuring the incorporation of radiolabelled thymidine in the presence of, or following treatment with, the oxidant. Although early events seem to be responsible for the decreased rate of DNA synthesis, it would appear that hydrogen peroxide does not alter thymidine extracellularly and/or decrease the transport of the nucleoside across the plasma membrane, which may actually be slightly augmented. Thus, the previously illustrated results may represent an underestimate of the actual capacity of the oxidant to reduce DNA synthesis. This inference is further supported by the fact that the effect of hydrogen peroxide appears markedly enhanced in cells preloaded with the radiolabelled precursor. A temporal relationship seems to exist between the steady state level of DNA single strand breaks and the extent of DNA synthesis inhibition by hydrogen peroxide. The oxidant has no effect on DNA chain elongation. In conclusion, data presented in this paper suggest that early events, involving selective effects on replicon initiation, mediate the DNA synthesis inhibitory effect of hydrogen peroxide.     

The oxidation produced by hydrogen peroxide on Ca-ATP-G-actin

We report here that in vitro exposure of monomeric actin to hydrogen peroxide leads to a conversion of 6 of the 16 methionine residues to methionine sulfoxide residues. Although the initial effect of H2O2 on actin is the oxidation of Cys374, we have found that Met44, Met47, Met176, Met190, Met269, and Met355 are the other sites of the oxidative modification. Met44 and Met47 are the methionyl sites first oxidized. The methionine residues that are oxidized are not simply related to their accessibility to the external medium and are found in all four subdomains of actin. The conformations of subdomain 1, a region critical for the functional binding of different actin-binding proteins, and subdomain 2, which plays important roles in the polymerization process and stabilization of the actin filament, are changed upon oxidation. The conformational changes are deduced from the increased exposure of hydrophobic residues, which correlates with methionine sulfoxide formation, from the perturbations in tryptophan fluorescence, and from the decreased susceptibility to limited proteolysis of oxidized actin.     

Enrichment of sugar content in melon fruits by hydrogen peroxide treatment

Since sweetness is one of the most important qualities of many fruits, and since sugars are translocated from leaves to fruits, the present study investigates photosynthetic activity, activity of sugar metabolizing enzymes, sugar content in leaves and fruits and endogenous levels of hydrogen peroxide in leaves of melon plants treated with various dilutions of hydrogen peroxide, a nonspecific signaling molecule in abiotic stress. For this purpose, 4-month-old melon plants were treated with various concentrations (<50mM) of hydrogen peroxide by applying 300mL per day to the soil of potted plants. The treatments resulted in increased fructose, glucose, sucrose and starch in the leaves and fruits. The most effective concentration of hydrogen peroxide was 20mM. During the day, soluble sugars in leaves were highest at 12:00h and starch at 15:00h. Furthermore, the peroxide treatment increased the photosynthetic activity and the activities of chloroplastic and cytosolic fructose-1,6-bisphosphatase, sucrose phosphate synthase and invertases. Thus, our data show that exogenous hydrogen peroxide, applied to the soil, can increase the soluble sugar content of melon fruits.     

Determination of hydrogen peroxide in exhaled breath condensate by flow injection analysis with fluorescence detection

An improved and simplified high-performance liquid chromatographic (HPLC) method at UV detection 265 nm is presented for the determination of d4T in rat plasma. The mobile phase consists of methanol-distilled water-acetic acid in the 23:77:0.2 (v/v) ratio. Quantification is achieved by the peak-area ratio method with reference to the internal standard. This paper presents linearity, accuracy, precision, limit of quantification and limit of detection, specificity-selectivity and sample stability data. Based on the intra and inter-day validation, all coefficients of variation (CV) were found less than 15%. The assay is sufficiently rapid and sensitive and was applied in a pharmacokinetic study in rats.     

The oxidation of cytochrome c oxidase by hydrogen peroxide

The reaction of H2O2 with mixed-valence and fully reduced cytochrome c oxidase was investigated by photolysis of fully reduced and mixed-valence carboxy-cytochrome c oxidase in the presence of H2O2 under anaerobic conditions. The results showed that H2O2 reacted rapidly (k = (2.5-3.1) X 10(4) M-1 X s-1) with both enzyme species. With the mixed-valence enzyme, the fully oxidised enzyme was reformed. On the time-scale of our experiments, no spectroscopically detectable intermediate was observed. This demonstrates that mixed-valence cytochrome c oxidase is able to use H2O2 as a two-electron acceptor, suggesting that cytochrome c oxidase may under suitable conditions act as a peroxidase. Upon reaction of H2O2 with the fully reduced enzyme, cytochrome a was oxidised before cytochrome a3. From this observation it was possible to estimate that the rate of electron transfer from cytochrome a to a3 is about 0.5-5 s-1.     

The production of hydrogen peroxide by high oxygen pressures

Hydrogen peroxide is formed in solutions of glutathione exposed to oxygen. This hydrogen peroxide or its precursors will decrease the viscosity of polymers like desoxyribonucleic acid and sodium alginate. Further knowledge of the mechanism of these chemical effects of oxygen might further the understanding of the biological effects of oxygen. This study deals with the rate of solution of oxygen and with the decomposition of hydrogen peroxide in chemical systems exposed to high oxygen pressures. At 6 atmospheres, the absorption coefficient for oxygen into water was about 1 cm./hour and at 143 atmospheres, it was about 2 cm./hour; the difference probably being due to the modus operandi. The addition of cobalt (II), manganese (II), nickel (II), or zinc ions in glutathione (GSH) solutions exposed to high oxygen pressure decreased the net formation of hydrogen peroxide and also the reduced glutathione remaining in the solution. Studies on hydrogen peroxide decomposition indicated that these ions act probably by accelerating the hydrogen perioxide oxidation of glutathione. The chelating agent, ethylenediaminetetraacetic acid disodium salt, inhibited the oxidation of GSH exposed to high oxygen pressure for 14 hours. However, indication that oxidation still occurred, though at a much slower rate, was found in experiments lasting 10 weeks. Thiourea decomposed hydrogen peroxide very rapidly. When GSH solutions were exposed to high oxygen pressure, there was oxidation of the GSH, which became relatively smaller with increasing concentrations of GSH.