Hydrogen peroxide suppresses U937 cell death by two different mechanisms depending on its concentration

To investigate the mechanisms of H2O2 adaptation in mammalian cells, we exposed human U937 leukemia cells to 0.05 mM H2O2. This treatment significantly suppressed cell death and DNA fragmentation induced by a subsequent challenge with 1 mM H2O2. A more dramatic protection was observed when cells were pretreated with 0.25 mM H2O2. Pretreatment with either 0.05 or 0.25 mM H2O2 also imparted cells with a survival advantage against serum withdrawal and C2-ceramide treatment. H2O2 was found to be a mediator of cell death induced by serum withdrawal, but not by the addition of C2-ceramide. Interestingly, 0.25 mM H2O2 greatly induced glutathione peroxidase, a H2O2-consuming enzyme, whereas 0.05 mM H2O2 did not. Consistent with observation, pretreatment with 0.25 mM H2O2 resulted in a great reduction of cellular oxidant levels as determined by 2'7'-dichlorofluorescein fluorescence, and it also prevented elevation of oxidant levels upon subsequent challenge with 1 mM H2O2 or with serum withdrawal. These effects were not observed in cells pretreated with 0.05 mM H2O2. The sum of the data indicated that H2O2 suppresses cell death by two different mechanisms depending on its concentration: Relatively high concentrations enhance cellular antioxidant capacity, and lower concentrations block the lethal action of H2O2.     

NaCl胁迫下交替呼吸途径对烟草悬浮细胞死亡调节作用的研究

盐分胁迫是植物在自然环境中经常遭遇的环境胁迫因素之一,会引起植物代谢紊乱乃至细胞死亡,这严重限制了植物的生长、繁育和生存。交替呼吸途径是植物较之动物独特的线粒体呼吸途径。该研究在烟草悬浮细胞中调查了交替呼吸途径对Na Cl胁迫引起的植物细胞死亡过程的调节作用及相应的内在机制,以及在200 mmol·L~(-1)Na Cl处理的烟草悬浮细胞中研究了交替呼吸途径和细胞死亡发生及H_2O_2之间的关系。结果表明:(1)随着Na Cl处理浓度的增加,烟草悬浮细胞死亡水平逐渐增加,而交替呼吸途径的容量也逐渐上升。(2)与Na Cl处理相似,外源H_2O_2的处理也能导致烟草悬浮细胞死亡水平的增加。200 mmol·L~(-1)Na Cl的胁迫导致明显的细胞死亡发生和H_2O_2产量的显著性增加;而较之200 mmol·L~(-1)Na Cl胁迫下的细胞,用水杨基氧肟酸(交替呼吸途径的抑制剂)预处理后的细胞再置于200 mmol·L~(-1)Na Cl的胁迫下导致更高水平的细胞死亡和H_2O_2的产生。综上表明,高盐胁迫诱导了烟草悬浮细胞的交替呼吸途径的增加,而交替呼吸途径则可能通过抑制活性氧的产生而起到缓解细胞死亡发生的作用。     

Cell death induced by sodium nitroprusside and hydrogen peroxide in tobacco BY-2 cell suspension

The interplay between nitric oxide (NO) and reactive oxygen species can lead to an induction of cell death in plants. The aim of our work was to find out if cyanide released from sodium nitroprusside (SNP; a donor of NO) could be involved in the cell death induction, which is triggered by SNP and H₂O₂. Cell suspension of Nicotiana tabacum L. (line BY-2) was treated with 0.5 mM SNP, 0.5 mM potassium ferricyanide (PFC; analogue of sodium nitroprusside which can not release NO) and/or by 0.5 mM glucose with 0.5 U cm⁻³ glucose oxidase (GGO; a donor system of H₂O₂). The cell death was induced only by combination of SNP and GGO. Thus cyanide released was not involved in the induction of cell death. However, SNP showed toxic effect because of decrease in activities of intracellular oxidoreductases and esterases. The cell death caused by SNP and GGO occurred within 12 h. During cell death either length or width of the cell increased. Central vacuole was formed in 20 to 40 % of cells. Most of the dead cells showed a condensed cytoplasm. Two hallmarks of programmed cell death (PCD), chromatin condensation and blebbing of nuclear periphery, were observed. However, oligonucleosomal fragmentation of DNA, another hallmark of PCD, was not detected.     

Activation of murine macrophages by hydrogen peroxide

Hydrogen peroxide at concentrations from 0.1 to 20 μM enhances phagocytosis and oxidative burst of murine peritoneal macrophages. The activation of these macrophage functions is paralled by prolonged hyperpolarization and a transient increase in cytoplasmic free calcium concentration. All the effects are dose- and time-dependent. The results obtained for H₂O₂ are compared with those for a natural activator, peptide N-formyl-methionyl-leucly-phenylalanine. The data demonstrate the ability of small doses of hydrogen peroxide to stimulate macrophages through the intracellular mechanisms of ion transduction.     

Hydrogen peroxide, nitric oxide and cytosolic ascorbate peroxidase at the crossroad between defence and cell death

An increase in the production of reactive oxygen species (ROS) is a typical event occurring during different stress conditions and activating conflicting responses in plants. In order to investigate the relevance of different timing and amounts of ROS production, tobacco (Nicotiana tabacum) Bright Yellow-2 (TBY-2) cells were incubated with different amounts of glucose plus glucose oxidase, for generating H(2)O(2) during time, or directly with known amounts of H(2)O(2). Data presented here indicate that, in TBY-2 cells, a difference in H(2)O(2) level is a critical point for shifting metabolic responses towards strengthening of antioxidant defences, or their depletion with consequent cell death. Timing of ROS production is also critical because it can determine programmed cell death (PCD) or necrosis. Depending on the different kinds of activated cell death, ascorbate (ASC) and glutathione (GSH) pools are altered differently. Moreover, an H(2)O(2)-dependent activation of nitric oxide synthesis is triggered only in the conditions inducing PCD. Ascorbate peroxidase (APX) has been analysed under different conditions of H(2)O(2) generation. Under a threshold value of H(2)O(2) overproduction, a transient increase in APX occurs, whereas under conditions inducing cell necrosis, the activity of APX decreases in proportion to cell death without any evident alteration in APX gene expression. Under conditions triggering PCD, the suppression of APX involves both gene expression and alteration of the kinetic characteristics of the enzyme. The changes in ASC, GSH and APX are involved in the signalling pathway leading to PCD, probably contributing to guaranteeing the cellular redox conditions required for successful PCD.     

Turning point in apoptosis/necrosis induced by hydrogen peroxide

The turning point between apoptosis and necrosis induced by hydrogen peroxide (H₂O₂) have been investigated using human T-lymphoma Jurkat cells. Cells treated with 50 μM H₂O₂ exhibited caspase-9 and caspase-3 activation, finally leading to apoptotic cell death. Treatment with 500 μM H₂O₂ did not exhibit caspase activation and changed the mode of death to necrosis. On the other hand, the release of cytochrome c from the mitochondria was observed under both conditions. Treatment with 500 μM H₂O₂, but not with 50 μM H₂O₂, caused a marked decrease in the intracellular ATP level; this is essential for apoptosome formation. H₂O₂-reducing enzymes such as cellular glutathione peroxidase (cGPx) and catalase, which are important for the activation of caspases, were active under the 500 μM H₂O₂ condition. Prevention of intracellular ATP loss, which did not influence cytochrome c release, significantly activated caspases, changing the mode of cell death from necrosis to apoptosis. These results suggest that ATP-dependent apoptosome formation determines whether H₂O₂-induced cell death is due to apoptosis or necrosis.     

Protective effect of salicylates against hydrogen peroxide stress in yeast

Aims:  To investigate the effects of salicylates in Saccharomyces cerevisiae exposed to oxidative stress induced by hydrogen peroxide (H₂O₂).
Methods and Results:  Saccharomyces cerevisiae was cultured through to the postlogarithmic phase of growth. Stress was induced by the addition of 1·5 mmol l⁻¹ H₂O₂ for 1 h, while N-acetyl-l-cysteine (NAC) and glutathione (GSSG) were used as control agents that affect the redox balance. Sodium salicylate, at 0·01–10 mmol l⁻¹or acetylsalicylic acid, at 0·02–2·5 mmol l⁻¹ was administered at various times before hydrogen peroxide stress. Both agents conferred resistance to a subsequent hydrogen peroxide stress, similarly to the induction of the adaptive response observed upon pretreatment with NAC and GSSG. Sodium salicylate was more potent as a short-term, but not as a long-term pretreatment agent, compared to acetylsalicylic acid.
Conclusions:  Pharmacological pretreatment with salicylates resulted in dose related increases in cell survival, indicating the induction of the protective response in yeast.
Significance and Impact of the study:  The possible role of salicylates in the modulation of the hydrogen peroxide stress response in eukaryotic cells address questions on the effects of these commonly used therapeutic agents in a number of disorders exhibiting an oxidative stress component.     

Catalase inhibition by nitric oxide potentiates hydrogen peroxide to trigger catastrophic chromosome fragmentation in Escherichia coli

Hydrogen peroxide (H₂O₂, HP) is a universal toxin that organisms deploy to kill competing or invading cells. Bactericidal action of H₂O₂ presents several questions. First, the lethal H₂O₂ concentrations in bacterial cultures are 1000x higher than, for example, those calculated for the phagosome. Second, H₂O₂-alone kills bacteria in cultures either by mode-one, via iron-mediated chromosomal damage, or by mode-two, via unknown targets, but the killing mode in phagosomes is unclear. Third, phagosomal H₂O₂ toxicity is enhanced by production of nitric oxide (NO), but in vitro studies disagree: some show NO synergy with H₂O₂ antimicrobial action, others instead report alleviation. To investigate this “NO paradox,” we treated Escherichia coli with various concentrations of H₂O₂-alone or H₂O₂+NO, measuring survival and chromosome stability. We found that all NO concentrations make sublethal H₂O₂ treatments highly lethal, via triggering catastrophic chromosome fragmentation (mode-one killing). Yet, NO-alone is not lethal, potentiating H₂O₂ toxicity by blocking H₂O₂ scavenging in cultures. Catalases represent obvious targets of NO inhibition, and catalase-deficient mutants are indeed killed equally by H₂O₂-alone or H₂O₂+NO treatments, also showing similar levels of chromosome fragmentation. Interestingly, iron chelation blocks chromosome fragmentation in catalase-deficient mutants without blocking H₂O₂-alone lethality, indicating mode-two killing. In fact, mode-two killing of WT cells by much higher H₂O₂ concentrations is transiently alleviated by NO, reproducing the “NO paradox.” We conclude that NO potentiates H₂O₂ toxicity by promoting mode-one killing (via catastrophic chromosome fragmentation) by otherwise static low H₂O₂ concentrations, while transiently suppressing mode-two killing by immediately lethal high H₂O₂ concentrations.     

Mechanistic connections between mitochondrial biology and regulated cell death

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Pyruvate secreted by human lymphoid cell lines protects cells from hydrogen peroxide mediated cell death

Reactive oxygen species (ROS) released from polymorphonuclear leukocytes and macrophages could cause DNA damage, but also induce cell death. Therefore inhibition of cell death must be an important issue for accumulation of genetic changes in lymphoid cells in inflammatory foci. Scavengers in the post culture medium of four lymphoid cell lines, lymphoblastoid cell lines (LCL), Raji, BJAB and Jurkat cells, were examined. Over 80% of cultured cells showed cell death 24 h after xanthine (X)/xanthine oxidase (XOD) treatment, which was suppressed by addition of post culture medium from four cell lines in a dose-dependent manner. H₂O₂ but not O^·-₂ produced by the X/XOD reaction was responsible for the cytotoxity, thus we used H₂O₂ as ROS stress thereafter. The H₂O₂-scavenging activity of post culture media from four cell lines increased rapidly at the first day and continued to increase in the following 2–3 days for LCL, Raji and BJAB cells. The scavenging substance was shown to be pyruvate, with various concentrations in the cultured medium among cell lines. Over 99% of total pyruvate was present in the extracellular media and less than 1% in cells. α-Cyano-4-hydroxycinnamate, a specific inhibitor of the H⁺-monocarbohydrate transporter, increased the H₂O₂-scavenging activity in the media from all four cell lines via inhibition of pyruvate re-uptake by cultured cells from the media. These findings suggest that lymphoid cells in inflammatory foci could survive even under ROS by producing pyruvate, so that accumulation of lymphoid cells with DNA damage is possible.     

The involvement of hydrogen peroxide in plant responses to stresses

The role of reactive oxygen species, especially H₂O₂, in plant response to stresses has been the focus of much attention. Hydrogen peroxide has been postulated to play multiple functions in plant defence against pathogens. (1) H₂O₂ may possess direct microbicidal activity at the sites of pathogen invasion. (2) It is used for cell-wall reinforcing processes: lignification and oxidative cross-linking of hydroxyproline-rich proteins and other cell-wall polymers. (3) It was found to be necessary for phytoalexin synthesis. (4) H₂O₂ may trigger programmed plant cell death during the hypersensitive response that restricts the spread of infection. (5) H₂O₂ has been suggested to act as a signal in the induction of systemic acquired resistance and (6) it induces defence genes. Recently H₂O₂ has been proposed to be involved in the signal transduction pathways leading to acclimation and protection from abiotic stresses. The present review discusses new insights into the function of H₂O₂ in plant responses to biotic and abiotic stresses.     

Light-induced intracellular hydrogen peroxide generation through genetically encoded photosensitizer KillerRed-SOD1

Abstract

Hydrogen peroxide (H₂O₂) plays an important role in various biological processes in numerous organisms. Depending on the concentration and the distribution within the cell, it can act as stressor or redox signalling molecule. To analyse the effects of H₂O₂ and its diffusion within the cell we developed the new genetically encoded photosensitizer KillerRed-SOD1 which enables a light-induced spatially and temporally controlled generation of H₂O₂ in living cells. The KillerRed-SOD1 is a fusion protein of the photosensitizer KillerRed (KR) and the cytosolic superoxide dismutase isoform 1 (SOD1) connected by a helix-forming peptide linker. Light irradiation at a wavelength of 560?nm induced superoxide radical formation at the KR domain which was transformed to H₂O₂ at the SOD1 domain. H₂O₂ was specifically detected under live cell conditions using the fluorescent sensor protein HyPer. Genetically encoded photosensitizers have the advantage that appropriate tag sequences can determine the localisation of the protein within the cell. Herein, it was exemplarily shown that the peroxisomal targeting sequence 1 directed the photosensitizer KR-SOD1 to the peroxisomes and enabled H₂O₂ formation specifically in these organelles. In summary, with the photosensitizer KR-SOD1 a new valuable tool was established which allows a controlled intracellular H₂O₂ generation for the analysis of H₂O₂ effects on a subcellular level.     

Phytaspase,a relocalisable cell death promoting plant protease with caspase specificity

Caspases are cysteine‐dependent proteases and are important components of animal apoptosis. They introduce specific breaks after aspartate residues in a number of cellular proteins mediating programmed cell death (PCD). Plants encode only distant homologues of caspases, the metacaspases that are involved in PCD, but do not possess caspase‐specific proteolytic activity. Nevertheless, plants do display caspase‐like activities indicating that enzymes structurally distinct from classical caspases may operate as caspase‐like proteases. Here, we report the identification and characterisation of a novel PCD‐related subtilisin‐like protease from tobacco and rice named phytaspase (plant aspartate‐specific protease) that possesses caspase specificity distinct from that of other known caspase‐like proteases. We provide evidence that phytaspase is synthesised as a proenzyme, which is autocatalytically processed to generate the mature enzyme. Overexpression and silencing of the phytaspase gene showed that phytaspase is essential for PCD‐related responses to tobacco mosaic virus and abiotic stresses. Phytaspase is constitutively secreted into the apoplast before PCD, but unexpectedly is re‐imported into the cell during PCD providing insights into how phytaspase operates.     

The role of nitric oxide in the cardiac effects of hydrogen peroxide

Oxidative stress mediated by hydrogen peroxide (H₂O₂) increases coronary flow (CF) in Langendorff-perfused rat hearts. We investigated the possible role of nitric oxide (NO) in H₂O₂-induced vasolidation. A dose-response study was conducted to find a concentration of H₂O₂ which increased CF without influencing left ventricular developed (LVDP) or end-diastolic (LVEDP) pressures. 80 (n = 10),100 (n = 7), 120 (n = 7),140 (n = 7),160 (n = 7), and 180 (n = 10) M H₂O₂ was infused for 10 min, followed by recovery for 50 min. 80 M H₂O₂ increased CF to a maximum of 143 ± 4 (mean ± S.E.M) percent of initial value after 15 min observation (p < 0.001 compared to buffer only), with no effect on LVDP or LVEDP. Another series of hearts were perfused with N-nitro-L-Arginine methylester (L-NAME, 1 M), methylene blue (MB, 50 M), or haemoglobin (Hb, 10 M), without (n = 7 in each) or with (n = 10 in each) 80 M H₂O₂ for 10 min. L-NAME, MB, and Hb alone increased CF, but attenuated the H₂O₂-induced increase of CF. LVDP was depressed when L-NAME, MB, or Hb were given in conjunction with 80 M H₂O₂. In summary, H₂O₂ concentration-dependently increased LVEDP and depressed LVDP. The H₂O₂-induced increase of CF was independent of concentration. Inhibition of NO synthesis, action, or soluble guanylate cyclase attenuated the H₂O₂-induced increase of CF, and depressed LVDP when given together with H₂O₂. H₂O₂ induces a NO-dependent vasodilation, and inhibition of NO is detrimental to left ventricular function after H₂O₂-mediated oxidative stress.     

Ethylene is required for elicitin-induced oxidative burst but not for cell death induction in tobacco cell suspension cultures

The signal compound ethylene and its relationships with oxidative burst and cell death were analyzed in cultured tobacco cells treated with the proteinaceous elicitor quercinin. Quercinin belongs to the protein family of elicitins and was isolated from the soil-born oak pathogen Phytophthora quercina. It was shown to induce a dose-dependent oxidative burst in tobacco cell culture in concentrations from 0.05 to 0.5 nM, and subsequently, cell death. The characteristics of quercinin-induced cell death included both membrane damage and DNA fragmentation in tobacco cell culture.

At higher quercinin concentrations (2 nM), H₂O₂ formation and ethylene biosynthesis were inhibited. Ethylene at low concentrations proved to be necessary for induction and maintenance of H₂O₂ production in tobacco cells treated with quercinin. It was demonstrated that external addition of inhibitors of ethylene biosynthesis such as -amino-oxy-acetic acid (AOA) and CoCl₂ also decreased or even inhibited the quercinin-induced oxidative burst, but did not influence cell death induction. These results demonstrate evidence for a requirement of the plant hormone ethylene for the onset of the quercinin-induced oxidative burst.     

昆虫细胞程序性死亡的研究进展

在昆虫发育和抵抗病原微生物的入侵过程中,细胞凋亡与自噬性死亡现象十分常见。昆虫细胞凋亡的研究已经取得了许多的成果,但是有关细胞自噬程序性死亡的研究还正在深入。昆虫细胞凋亡的信号通路至少有3条：一条类似于线虫细胞的凋亡信号通路,另一条类似于哺乳动物细胞的凋亡信号通路, 还有一条不依赖于胱天蛋白酶的凋亡信号通路。在昆虫的多种组织细胞中,细胞凋亡与自噬程序性死亡在信号通路上存在互串(cross talking),可以相互促进、抑制或替代。了解昆虫细胞程序性死亡对防治害虫具有一定的意义。     

Hypersensitive response, cell death and histochemical localisation of hydrogen peroxide in host and non-host seedlings infected with the downy mildew pathogen Sclerospora graminicola

Hypersensitive response, cell death and release of hydrogen peroxide as measures of host and non‐host defense mechanisms upon inoculation with the downy mildew pathogen Sclerospora graminicola were studied histochemically at the light microscopy level. The materials consisted of coleoptile tissues of the highly susceptible (cv. HB3), highly resistant (cv. IP18293) and induced resistant pearl millet host seedlings and non‐host sorghum (cv. SGMN10/8) and cotyledon of french bean (cv. S9). Resistance up to 80% protection against the downy mildew pathogen was induced in the highly susceptible HB3 cultivar of pearl millet by treating the seeds with 2% aqueous leaf extract of Datura metel for 3 h. Time course study with the pathogen inoculated highly resistant pearl millet cultivar revealed the appearance of hypersensitive response in 20% of seedlings as necrotic spots as early as 2 h after inoculation. In contrast, a similar reaction was observed in the highly susceptible pearl millet cultivar only 8 h after inoculation with the pathogen. In induced resistant seedlings, appearance of hypersensitive response was recorded 4 h after inoculation. Delayed hypersensitive response was observed in both the non‐host species at 10 h after inoculation. Hypersensitive response in the seedlings of the highly resistant pearl millet cultivar 24 h after inoculation showed 100% hypersensitive response, which was not observed in susceptible and non‐host species, although the induced resistant seedlings showed 90% hypersensitive response after that period of time. Cell death in the tissues of the test seedlings was also observed to change with time. Statistical analysis revealed that the tissues of highly resistant pearl millet seedlings required 2.9 h to attain 50% cell death. Tissues of induced resistant and highly susceptible pearl millet seedlings required 4.65 and 6.50 h respectively. In non‐hosts, 50% cell death was not recorded. Quantification of hydrogen peroxide in the tissue periplasmic spaces of the test seedlings revealed 2.94 h as the time required for 50% hydrogen peroxide accumulation in the tissues of highly resistant pearl millet seedlings. Tissues of induced resistant and highly susceptible pearl millet seedlings needed 3.76 and 5.5 h respectively. Fifty percent hydrogen peroxide localisation in non‐hosts could not be recorded. These results suggested the involvement of hydrogen peroxide, cell death and hypersensitive response in pearl millet host defense against S. graminicola.     

Pyrrolidine dithiocarbamate restores gastric damages and suppressive autophagy induced by hydrogen peroxide

Abstract

It is well known that gastric barrier is very important for protecting host from various insults. Simultaneously, autophagy serving as a prominent cytoprotective and survival pathway under oxidative stress conditions is being increasingly recognized. Thus, this study was conducted for investigating the effect of pyrrolidine dithiocarbamate (PDTC) on gastric barrier function and autophagy under oxidative stress induced by intragastric administration of hydrogen peroxide (H₂O₂). The gastric tight junction proteins [zonula occludens-1 (ZO1), occludin, and claudin1], autophagic proteins [microtubule-associated protein light chain 3I(LC3I), LC3II, and beclin1], and nuclear factor kappa B (NF-κB) signaling pathway (p65 and IκB kinase α/β) were determined by Western blot. The results showed that H₂O₂ exposure disturbed gastric barrier function with decreased expression of ZO1, occludin, and claudin1, and reduced gastric autophagy with decreased conversion of LC3I into LC3II in mice. However, treatment with PDTC restored these adverse effects evidenced by increased expression of ZO1 and claudin1 and increased conversion of LC3I into LC3II. Meanwhile, H₂O₂ exposure decreased normal human gastric epithelial mucosa cell line (GES-1) viability in a concentration-dependent way. However, after being exposed to H₂O₂, GES-1 exhibited autophagic response which was inconsistent with our in vivo results in mice, while PDTC failed to decrease autophagy in GES-1 induced by H₂O₂. Simultaneously, the beneficial effect of PDTC on gastric damage and autophagy in mice might be independent of inhibition of NF-κB. In conclusion, PDTC treatment restores gastric damages and reduced autophagy induced by H₂O₂. Therefore, PDTC may serve as a potential adjuvant therapy for gastric damages.