Changes in the content of wheat germ agglutinin in hydrogen peroxide-treated plants

The content of wheat germ agglutinin (WGA) in hydrogen peroxide-treated seedlings was studied by indirect competitive enzyme-linked immunosorbent assay. WGA content in roots showed a transitory increase: at 10 mM hydrogen peroxide, maximum level was observed after 2 h; at 1 mM hydrogen peroxide, the maximum occurred 2 or 24 h after the treatment. Lectin induction by hydrogen peroxide is viewed as an element of a feedback mechanism limiting the operation of defense responses during pathogenetic processes.     

Peroxidase-catalyzed color removal from bleach plant effluent

Effluent from the caustic extraction stage of a bleach plant is highly colored due to the presence of dissolved products from lignin chlorination and oxidation. Color removal from the effluent by hydrogen peroxide at neutral pH was catalyzed by addition of horseradish peroxidase. The catalysis with peroxidase (20 mg/L) was observed over a wide range of peroxide concentrations (0.1mM-500mM), but the largest effect was between 1mM and 100mM. The pH optimum for catalysis was around 5.0, while the basal rate of noncatalyzed peroxide color removal simply increased with pH within the range tested (3-10). Peroxidase catalysis at pH 7.6 reached a maximum at 40 degrees C in 4 h assays with 10mM peroxide, and disappeared above 60 degrees C. Compared with mycelial color removal by Coriolus versicolor, the rate of color removal by peroxide plus peroxidase was initially faster (first 4 h), but the extent of color removal after 48 h was higher with the fungal treatment. Further addition of peroxidase to the enzyme-treated effluent did not produce additional catalysis. Thus, the peroxide/peroxidase system did not fully represent the metabolic route used by the fungus.     

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.     

Morphologic and functional correlates of plasma membrane injury during oxidant exposure

Plasma membrane injury by exposure to hydrogen peroxide was examined in a renal epithelial cell line (LLC-PK1). Morphologic and functional parameters of plasma membrane integrity were studied in an attempt to eludicate the sequence of membrane alterations during the evolution of hydrogen peroxide-mediated injury. These parameters included plasma membrane potential and permeability, plasma membrane bleb formation, cellular size, and plating efficiency. Plasma membrane potential was the earliest parameter affected by hydrogen peroxide exposure. Half maximal depolarization occurred within 15-30 min of exposure to 1 mM, after 10-15 min exposure to 100 mM and after over 150 min exposure to 10 microM hydrogen peroxide. After exposure to 1 mM hydrogen peroxide, the following sequence of events was seen; increased plasma membrane blebbing (30 min), cell swelling (90-125 min) and increased plasma membrane permeability (150-240 min). After a 30 min exposure to 1 mM hydrogen peroxide, cellular plating efficiency, measured at 24 h, was reduced by 50% (P less than .001). These changes were accelerated, although their order of appearance was unchanged, at higher concentrations of hydrogen peroxide. We conclude that functional and morphologic expressions of cellular injury in this model occur in a defined sequence with plasma membrane depolarization representing the earliest marker of membrane injury during hydrogen peroxide exposure.     

Urea hydrogen peroxide reduces the numbers of lactobacilli, nourishes yeast, and leaves no residues in the ethanol fermentation

Urea hydrogen peroxide (UHP) at a concentration of 30 to 32 mmol/liter reduced the numbers of five Lactobacillus spp. (Lactobacillus plantarum, L. paracasei, Lactobacillus sp. strain 3, L. rhamnosus, and L. fermentum) from approximately 10(7) to approximately 10(2) CFU/ml in a 2-h preincubation at 30 degrees C of normal-gravity wheat mash at approximately 21 g of dissolved solids per ml containing normal levels of suspended grain particles. Fermentation was completed 36 h after inoculation of Saccharomyces cerevisiae in the presence of UHP, even when wheat mash was deliberately contaminated (infected) with L. paracasei at approximately 10(7) CFU/ml. There were no significant differences in the maximum ethanol produced between treatments when urea hydrogen peroxide was used to kill the bacteria and controls (in which no bacteria were added). However, the presence of L. paracasei at approximately 10(7) CFU/ml without added agent resulted in a 5.84% reduction in the maximum ethanol produced compared to the control. The bactericidal activity of UHP is greatly affected by the presence of particulate matter. In fact, only 2 mmol of urea hydrogen peroxide per liter was required for disinfection when mashes had little or no particulate matter present. No significant differences were observed in the decomposition of hydrogen peroxide in normal-gravity wheat mash at 30 degrees C whether the bactericidal agent was added as H(2)O(2) or as urea hydrogen peroxide. NADH peroxidase activity (involved in degrading H(2)O(2)) increased significantly (P = 0.05) in the presence of 0.75 mM hydrogen peroxide (sublethal level) in all five strains of lactobacilli tested but did not persist in cells regrown in the absence of H(2)O(2). H(2)O(2)-resistant mutants were not expected or found when lethal levels of H(2)O(2) or UHP were used. Contaminating lactobacilli can be effectively managed by UHP, a compound which when used at ca. 30 mmol/liter happens to provide near-optimum levels of assimilable nitrogen and oxygen that aid in vigorous fermentation performance by yeast.     

Oxidative inactivation of rhodanese by hydrogen peroxide produces states that show differential reactivation

Controlled conditions have been found that give complete reactivation and long term stabilization of rhodanese (EC 2.8.1.1) after oxidative inactivation by hydrogen peroxide. Inactivated rhodanese was completely reactivated by reductants such as thioglycolic acid (TGA) (100 mM) and dithiothreitol (DTT) (100 mM) or the substrate thiosulfate (100 mM) if these reagents were added soon after inactivation. Reactivability fell in a biphasic first order process. At pH 7.5, in the presence of DTT inactive rhodanese lost 40% of its reactivability in less than 5 min, and the remaining 60% was lost more gradually (t 1/2 = 3.5 h). TGA reactivated better than DTT, and the rapid phase was much less prominent. If excess reagents were removed by gel filtration immediately after inactivation, there was time-independent and complete reactivability with TGA for at least 24 h, and the resulting samples were stable. Reactivable enzyme was resistant to proteolysis and had a fluorescence maximum at 335 nm, just as the native protein. Oxidized rhodanese, Partially reactivated by DTT, was unstable and lost activity upon further incubation. This inactive enzyme was fully reactivated by 200 mM TGA. Also, the enzyme could be reactivated by arsenite and high concentrations of cyanide. Addition of hydrogen peroxide (40-fold molar excess) to inactive rhodanese after column chromatography initiated a time-dependent loss of reactivability. This inactivation was a single first order process (t 1/2 = 25 min). Sulfhydryl titers showed that enzyme could be fully reactivated after the loss of either one or two sulfhydryl groups. Irreversibly inactivated enzyme showed the loss of one sulfhydryl group even after extensive reduction with TGA. The results are consistent with a two-stage oxidation of rhodanese. In the first stage there can form sulfenyl and/or disulfide derivative(s) at the active site sulfhydryl that are reducible by thioglycolate. A second stage could give alternate or additional oxidation states that are not easily reducible by reagents tried to date.     

A disbalance between beta-adrenergic and muscarinic responses caused by hydrogen peroxide in rat airways in vitro

The effect of hydrogen peroxide on adrenergic and muscarinic responses of rat airway smooth muscle was studied. The trachea muscle and the lung parenchymal strip were contracted with methacholine and relaxed with (-)-isoprenaline. Recording of three (-)-isoprenaline curves on the trachea muscle and the lung parenchymal strip was followed by treatment for 30 min with hydrogen peroxide (H2O2) (1mM) after which a new dose response curve for (-)-isoprenaline was constructed. Using the trachea muscle this treatment with H2O2 resulted in a decrease of 61% of the maximum contraction by methacholine compared with the control and a complete inhibition of the relaxation by (-)-isoprenaline. In the lung parenchymal strip preparation we found, after the same treatment no reduction of the contraction by methacholine and 61% reduction of the relaxation by (-)-isoprenaline, compared with the control. The results demonstrate that the adrenergic response in rat airways is more susceptible to hydrogen peroxide than the muscarinic response.     

The Use of Fluorescent Probes to Assess Oxidative Processes in Isolated-Perfused Rat Heart Tissue

The formation of reactive oxygen species (ROS) in intact heart tissue has been assessed by direct ESR measurements, and indirectly by the formation of characteristic tissue products and the protective effects of various antioxidants. The development of lipid soluble esters of compounds which can be trapped intra-cellularly after hydrolysis, and which fluoresce after oxidation, has provided a new tool to investigate ROS in vitro. The utility of 2',7'-dichlorofluorescin diacetate (DCFDA) in isolated-perfused rat heart tissue was investigated in the present study. DCFDA and its deacetylated form were incubated with various levels of hydrogen peroxide or t-butylhydroperoxide (tBOOH). Conversion of the diacetate form to a fluorescent product required 4-5 h with hydrogen peroxide and up to 24 h with tBOOH. In contrast, the deacetylated form fluoresced at 80% of maximum levels 1 h after the addition of 100 mM tBOOH. DCFDA was loaded into heart tissue by infusing for lO min at a final concentration of 10,aM in Krebs-Henseleit bicarbonate buffer. After a lO min washout period, analysis of freeze-clamped heart tissue revealed that the trapped material was readily converted to a fluorescent product by tBOOH, indicating hydrolysis had occurred. Fluorescence of material trapped in heart tissue was approximately 24% of the maximum achieved after oxidation with lOOmM tBOOH. This value decreased to 18 and 13% when the loading and washout periods were from 0 to 20 or 10 to 30min of hypoxia, respectively. Similar results were obtained with the less readily oxidized dicarboxy derivative of DCFDA. Infusion of 500μM tBOOH increased the oxidation of DCFDA in heart tissue from 24 to 31%. These data demonstrate that DCFDA can be loaded into heart tissue and is capable of reflecting relative changes in the oxidative state of this organ.     

Bimodal effect of exogenous hydrogen peroxide on human neutrophils: cytotoxic effect and modulation of respiratory burst in response to an agonist]

It has been found that high concentrations of exogenous hydrogen peroxide kill human neutrophils, the range of toxic concentrations being 100 times as high as that for human endothelial cells. Whereas the H2O2 doses of 30-100 mM induce a fast massive death of neutrophils, 10 mM hydrogen peroxide induces appreciable death only within several hours after treatment. H2O2 used at 30 mM decreases superoxide anion generation by neutrophils stimulated with PMA or FMLP. This decrease is commensurate in value with cell death, thus indicating a high functional resistance of survived cells. In the dose of 10 mM hydrogen peroxide potentiates FMLP (but not PMA-)-induced generation of superoxide anions. Augmentation of superoxide anion generation by H2O2-primed neutrophils in response to FMLP amounts to 200% of the control value. Hydrogen peroxide alone is incapable of inducing superoxide anion generation. It is concluded that exogenous oxidants can alter the functional activity of leukocytes freshly recruited in inflammatory and ischemic tissues.     

The formation of 8-oxoguanine and its oxidative products in DNA in vitro at 37 degrees C

The content of 8-oxoguanine, a biomarker of DNA damage by the action of reactive oxygen species, in native and denatured DNA upon heating at 37 degrees C was studied by the enzyme-linked immunosorbent assay using monoclonal antibodies against 8-oxoguanine. It was found that the content of 8-oxoguanine changes with time in a complicated multiphase manner, the maximum changes being as great as twofold. The production of hydrogen peroxide in water and 1 mM PBS, pH 6.8, at 37 degrees C over a period of 50 h was determined by the method of enhanced chemiluminescence in a peroxide-luminol-p-iodophenol system. The generation of hydrogen peroxide also changed in a complicated multiphase manner. After heating the DNA at 80 degrees C for 24 h, guanine oxidation products were excised by 8-oxoguanine-DNA-glycosylase. The products were separated and analyzed by liquid column chromatography on Sephadex LH-20 and Toyopearl HW-40 gel. The products were identified from UV adsorption spectra. The results indicated the generation of reactive oxygen species at 37 degrees C, which leads both to the generation of 8-oxoguanine in DNA and its elimination as a result of its further oxidation. The oxidation of 8-oxoguanine was accompanied by the formation of a number of unstable products of further oxidation of 8-oxoguanine. Among these products, aminoimidazolone, spiroiminodigidantoin, and diiminoimidazole were identified from UV spectra. The appearance of the products of further oxidation of 8-oxoguanine explains the origin of G : C --> C : G transversions by the action of reactive oxygen species.     

Growth factors protect in vitro cultured embryos from the consequences of oxidative stress

The aim of the study was to evaluate the effect of insulin-like growth factors (IGF1 and IGF2), stem cell factor (SCF) and epidermal growth factor (EGF) on the development of embryos exposed to oxidative stress. C3B6F1 female mice were stimulated with 5 IU of pregnant mare serum gonadotropin and 5 IU of equine chorionic gonadotropin (eCG). Two-cell embryos were flushed out from the fallopian tubes 40 h after eCG administration and mating with DBA males. In each experiment embryos were divided into three groups and cultured in (1) control medium, (2) control medium with 0.1 mM hydrogen peroxide and (3) control medium with hydrogen peroxide and separately with IGF1, IGF2, SCF or EGF in concentrations of 1 ng/ml, 10 ng/ml and 100 ng/ml. Under phase-contrast microscopy, 8-cell and compacted embryos, and early, expanded, hatched and outgrown blastocysts were counted at 24 h. The total blastocyst (TB) and inner cell mass (ICM) cell numbers were established by differential staining. Blastocyst cell viability was examined under fluorescence microscopy. To detect apoptosis, TUNEL was performed and visualized under a laser scanning confocal microscope. Hydrogen peroxide decreased embryo growth, blastocyst rates, blastocyst cell viability as well as TB and ICM counts. The TUNEL reaction revealed significantly more apoptotic cells in oxidative stress conditions. Tested factors revealed a varying extent of protective activity against oxidative stress caused by hydrogen peroxide. In media containing hydrogen peroxide and one of the four tested factors (IGF1, IGF2, SCF or EGF) the embryos developed faster than in media with hydrogen peroxide alone. IGF1, IGF2 and EGF increased both TB and (or) ICM counts in embryos exposed to hydrogen peroxide. All tested factors reduced the number of apoptotic cells (TUNEL) in embryos exposed to hydrogen peroxide.     

Potentiation by L-cysteine of the bactericidal effect of hydrogen peroxide in Escherichia coli.

Under anaerobic conditions an exponentially growing culture of Escherichia coli K-12 was exposed to hydrogen peroxide in the presence of various compounds. Hydrogen peroxide (0.1 mM) together with 0.1 mM L-cysteine or L-cystine killed the organisms more rapidly than 10 mM hydrogen peroxide alone. The exposure of E. coli to hydrogen peroxide in the presence of L-cysteine inhibited some of the catalase. This inhibition, however, could not fully explain the 100-fold increase in hydrogen peroxide sensitivity of the organism in the presence of L-cysteine. Of other compounds tested only some thiols potentiated the bactericidal effect of hydrogen peroxide. These thiols were effective, however, only at concentrations significantly higher than 0.1 mM. The effect of L-cysteine and L-cystine could be annihilated by the metal ion chelating agent 2,2'-bipyridyl. DNA breakage in E. coli K-12 was demonstrated under conditions where the organisms were killed by hydrogen peroxide.     

Runx2‐mediated bcl‐2 gene expression contributes to nitric oxide protection against hydrogen peroxide‐induced osteoblast apoptosis

Nitric oxide (NO) can regulate osteoblast activities. This study was aimed to evaluate the protective effects of pretreatment with sodium nitroprusside (SNP) as a source of NO on hydrogen peroxide‐induced osteoblast insults and its possible mechanisms. Exposure of human osteosarcoma MG63 cells to hydrogen peroxide significantly increased cellular oxidative stress, but decreased ALP activity and cell viability, inducing cell apoptosis. Pretreatment with 0.3 mM SNP significantly lowered hydrogen peroxide‐induced cell insults. Treatment of human MG63 cells with hydrogen peroxide inhibited Bcl‐2 mRNA and protein production, but pretreatment with 0.3 mM SNP significantly ameliorated such inhibition. Sequentially, hydrogen peroxide decreased the mitochondrial membrane potential, but increased the levels of cytochrome c and caspase‐3 activity. Pretreatment with 0.3 mM SNP significantly lowered such alterations. Exposure to hydrogen peroxide decreased Runx2 mRNA and protein syntheses. However, pretreatment with 0.3 mM SNP significantly lowered the suppressive effects. Runx2 knockdown using RNA interference inhibited Bcl‐2 mRNA production in human MG63 cells. Protection of pretreatment with 0.3 mM SNP against hydrogen peroxide‐induced alterations in ALP activity, caspase‐3 activity, apoptotic cells, and cell viability were also alleviated after administration of Runx2 small interference RNA. Thus, this study shows that pretreatment with 0.3 mM SNP can protect human MG63 cells from hydrogen peroxide‐induced apoptotic insults possibly via Runx2‐involved regulation of bcl‐2 gene expression. J. Cell. Biochem. 108: 1084–1093, 2009. © 2009 Wiley‐Liss, Inc.     

Time course of structure, function, and metabolic changes due to an exogenous source of oxygen metabolites in rat heart

Effects of xanthine (2 mM) and xanthine oxidase (10 U/L) perfusion on myocardial function, lipid peroxide content, high-energy phosphates and their metabolites, and ultrastructure were examined in isolated perfused rat hearts to define the time course of myocardial injury due to exogenous supply of active oxygen species. Peak-developed force and dF/dt showed a decline within 5 min and complete contractile failure was seen at 20 min. Resting tension was higher at 10 min and reached a maximum value of 400% at 40 min. These changes in contractile parameters were reduced by superoxide dismutase (1.2 x 10(5) U/L), catalase (2 and 4 X 10(4) U/L), and mannitol (10 and 20 mM). Lipid peroxide content was significantly higher at 5 min and rose continuously with xanthine-xanthine oxidase (X-XO) perfusion. A close correlation was noted (r = 0.935) between increased lipid peroxide content and a decrease in peak-developed force. Creatine phosphate and adenosine triphosphate (ATP) showed a time-dependent decrease due to X-XO perfusion. Loss of ATP also correlated (r = 0.819) with the contractile failure. Adenosine diphosphate showed an increase at 5 min followed by a decrease at 20 and 40 min. Adenosine monophosphate, adenosine, and creatine content increased with X-XO perfusion. In a semiquantitative morphometric study, significant myocardial and vascular changes became apparent only after 10 min of X-XO perfusion. When a 5-min perfusion with X-XO was followed by a control perfusion, a recovery of developed force and normal structure was noted at 40 min. These data show that X-XO induced contractile failure involves partially reduced forms of oxygen such as superoxide, hydroxyl radicals, and hydrogen peroxide. The negative inotropic effect of a vascular supply of these active oxygen species may be related to increased lipid peroxidation as well as the loss of high-energy phosphates. Structural damage to myocytes and blood vessels and a rise in resting tension were delayed events requiring a continuous and longer exposure to radical species.     

Hydrogen peroxide release from human blood platelets

The release of hydrogen peroxide from human blood platelets after stimulation with particulate membrane-perturbing agents has been determined by fluorescence using scopoletin as the detecting agent. Platelet suspensions containing less than 1 polymorphonuclear leukocyte/10⁸ platelets showed a significant release of hydrogen peroxide (6.11 nmol/10⁹ platelets per 20 min, S.D., 0.26, n=9) after addition of zymosan or latex particles, compared to unstimulated platelets. The release of hydrogen peroxide was only observed when the scopoletin was added to the platelet suspensions during the stimulation. Any attempt to determine hydrogen peroxide release in the supernatant at the end of the incubation with zymosan or latex failed. A NADH-dependent production of hydrogen peroxide was observed by measuring the difference of oxygen uptake in the presence and absence of catalase (500 units), which was not inhibited by potassium cyanide (1 mM). By this method the NADH-dependent cyanide-insensitive peroxide production and release was 6.0 nmol/10⁹ platelets per 20 min from resting platelets (S.D., 2, n=6) vs. 15 nmol/10⁹ platelets per 20 min from stimulated platelets (S.D., 2, n=6).     

Physiological concentrations of butyrate favorably modulate genes of oxidative and metabolic stress in primary human colon cells

Butyrate, a metabolite of gut flora-mediated fermentation of dietary fibre, was analysed for effects on expression of genes related to oxidative stress in primary human colon cells. An induction of detoxifying, antioxidative genes is expected to contribute to dietary chemoprevention. Cells were treated with butyrate (3.125-50 mM; 0.5-8 h), and kinetics of uptake and survival were measured. Gene expression was determined with a pathway-specific cDNA array after treating colon epithelium stripes with nontoxic doses of butyrate (10 mM, 12 h). Changes of hCOX-2, hSOD2 and hCAT expression were confirmed with real-time polymerase chain reaction (PCR) and by measuring catalase-enzyme activity. Primary colon cells consumed 1.5 and 0.5 mM butyrate after 4- and 12-h treatment, respectively. Cell viability was not changed by butyrate during 0.5-2-h treatment, whereas cell yields decreased after 1 h. Metabolic activity of remaining cells was either increased (4 h, 50 mM) or retained at 97% (8 h, 50 mM). Expression of hCAT was enhanced, whereas hCOX-2 and hSOD2 were lowered according to both array and real-time PCR analysis. An enhanced catalase-enzyme activity was detected after 2 h butyrate treatment. Healthy nontransformed colon cells well tolerated butyrate (50 mM, 2 h), and lower concentrations (10 mM, 12 h) modulated cyclooxygenase 2 (COX-2) and catalase genes. This points to a dual role of chemoprotection, since less COX-2 could reduce inflammatory processes, whereas more catalase improves detoxification of hydrogen peroxide (H(2)O(2)), a compound of oxidative stress. Changes of this type could reduce damaging effects by oxidants and protect cells from initiation.     

Enhancement of cytotoxicity of hydrogen peroxide by hyperthermia in chinese hamster ovary cells: role of antioxidant defenses

Regional hyperthermia has potential for human cancer treatment, particularly in combination with systemic chemotherapy or radiotherapy. The mechanisms involved in heat-induced cell killing are currently unknown. Hyperthermia may increase oxidative stress in cells, and thus, oxidative stress could have a role in the mechanism of cell death. We use hydrogen peroxide as a model oxidant to improve understanding of interactions between heat and oxidative stress. Heat increased cytotoxicity of hydrogen peroxide in Chinese hamster ovary cells. Altered levels of cellular antioxidants should create an imbalance between prooxidant and antioxidant systems, thus modifying cytotoxic responses to heat and to oxidants. We determine the involvement of the two cellular antioxidant defenses against peroxides, catalase and the glutathione redox cycle, in cellular sensitivity to heat, to hydrogen peroxide, and to heat combined with the oxidant. Defense systems were either inhibited or increased. For inhibition studies, intracellular glutathione was diminished to less than 15% of its initial level by treatment with L-buthionine sulfoximine (1 mM, 24 h). Inhibition of catalase was achieved with 3-amino-1,2,4-triazole (20 mM, 2 h), which caused a 80% decrease in endogenous enzyme activity. To increase antioxidants, cells were pretreated with the thiol-containing reducing agents, N-acetyl-L-cysteine, 2-oxo-4-thiazolidine carboxylate, and 2-mercaptoethane sulfonate. These compounds increased intracellular glutathione levels by 30%. Catalase activity was increased by addition of exogenous enzyme to cells. We show that levels of glutathione and catalase affect cellular cytotoxic responses to heat and hydrogen peroxide, either used separately or in combination. These findings are relevant to mechanisms of cell killing at elevated temperatures and suggest the involvement of oxidative stress.