Accelerated cell death 2 suppresses mitochondrial oxidative bursts and modulates cell death in Arabidopsis

The Arabidopsis ACCELERATED CELL DEATH 2 (ACD2) protein protects cells from programmed cell death (PCD) caused by endogenous porphyrin‐related molecules like red chlorophyll catabolite or exogenous protoporphyrin IX. We previously found that during bacterial infection, ACD2, a chlorophyll breakdown enzyme, localizes to both chloroplasts and mitochondria in leaves. Additionally, acd2 cells show mitochondrial dysfunction. In plants with acd2 and ACD2 ⁺ sectors, ACD2 functions cell autonomously, implicating a pro‐death ACD2 substrate as being cell non‐autonomous in promoting the spread of PCD. ACD2 targeted solely to mitochondria can reduce the accumulation of an ACD2 substrate that originates in chloroplasts, indicating that ACD2 substrate molecules are likely to be mobile within cells. Two different light‐dependent reactive oxygen bursts in mitochondria play prominent and causal roles in the acd2 PCD phenotype. Finally, ACD2 can complement acd2 when targeted to mitochondria or chloroplasts, respectively, as long as it is catalytically active: the ability to bind substrate is not sufficient for ACD2 to function in vitro or in vivo. Together, the data suggest that ACD2 localizes dynamically during infection to protect cells from pro‐death mobile substrate molecules, some of which may originate in chloroplasts, but have major effects on mitochondria.     

Hydrogen peroxide induces endothelial cell atypia and cytoskeleton depolymerization

Reactive oxygen intermediates induce cell injury in a variety of pathophysiological conditions. Human umbilical cord vein endothelial cell (HUVEC) cultures were exposed to 1 or 200 microM H2O2 for 15 min, and observed after 15 min, or 1, 4, 24, or 120 h. Factor VIII and the cytoskeletal proteins vimentin and tubulin were visualized immunocytochemically. Release of lactate dehydrogenase (indices of cell membrane injury) did not increase after H2O2 exposure; nor was cellular expression of factor VIII affected. 200 microM H2O2 induced cell contraction after 15 min which disappeared after 1 and 4 h, but was evident again after 24 h. Immediately after exposure, the filamentous structure of vimentin and tubulin disappeared, but normalized after 1 h. After 120 h, the cytoskeleton filaments were coarsened and disorganized, and an abundance of multinucleated giant cells were observed. Catalase (150 U/ml) abolished all effects of H2O2. One microM H2O2 did not induce any changes in HUVEC. Thus, the present concentrations of H2O2 did not induce cell necrosis or altered expression of factor VIII. Early, reversible cell contraction and depolymerization of cytoskeletal proteins were observed, followed by a delayed contraction and cell atypia after 200 microM H2O2.     

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.     

Hydrogen peroxide induces caspase activation and programmed cell death in the amitochondrial Tritrichomonas foetus

Tritrichomonas foetus is an amitochondrial parasite protist which lacks typical eukaryote organelles such as mitochondria and peroxisomes, but possesses the hydrogenosome, a double-membrane-bound organelle that produces ATP. The cell death of amitochondrial organisms is poorly studied. In the present work, the cytotoxic effects of hydrogen peroxide on T. foetus and its participation on cell death were analyzed. We took advantage of several microscopy techniques, including videomicroscopy, light microscopy immunocytochemistry for detection of caspase activation, and scanning and transmission electron microscopy. We report here that in T. foetus: (1) H₂O₂ leads to loss of motility and induces cell death, (2) the dying cells exhibit some characteristics similar to those found during the death of other organisms, and (3) a caspase-like protein seems to be activated during the death process. Thus, we propose that, although T. foetus does not present mitochondria nor any known pathways of cell death, it is likely that it bears mechanisms of cell demise. T. foetus exhibits morphological and physiological alterations in response to H₂O₂ treatment. The hydrogenosome, a unique organelle which is supposed to share a common ancestral origin with mitochondria and has an important role in oxidative responses in trichomonads, is a candidate for participating in this event.Abbreviations TUNEL Terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick-end labeling - PARP Poly (ADP-ribose) polymerase - DAPI 4,6-Diamidino-2-phenylindole dihydrochloride     

Hydrogen peroxide- and glutathione-associated mechanisms of acclimatory stress tolerance and signalling

Plants adapt to environmental stresses through specific genetic responses. The molecular mechanisms associated with signal transduction, leading to changes in gene expression early in the stress response, are largely unknown. It is clear, however, that gene expression associated with acclimatory responses is sensitive to the redox state of the cell. Of the many components which contribute to the redox balance of the cell, two factors have been shown to be crucial in mediating stress responses. Thiol/disulphide exchange reactions, particularly involving the glutathione pool and the generation of the oxidant H₂O₂, are central components of signal transduction in both environmental and biotic stresses. These molecules are multifunctional triggers, modulating metabolism and gene expression. Both are able to cross biological membranes and diffuse or be transported long distances from their sites of origin. Glutathione and H₂O₂ may act alone or in unison, in intracellular and systemic signalling systems, to achieve acclimation and tolerance to biotic and abiotic stresses.     

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.     

Hydrogen peroxide detoxification in the midgut of the blood-sucking insect, Rhodnius prolixus

Here we investigated H2O2 production and detoxification in the hematophagous hemiptera, Rhodnius prolixus. Superoxide dismutase (SOD) catalyzes the dismutation of superoxide radical (O2-). This reaction produces hydrogen peroxide, which is scavenged by antioxidant enzymes such as catalase (CAT). SOD and CAT activities were found in all tissues studied, being highest in the midgut. CAT was dose-dependently inhibited in vivo by injections of 3-amino-1,2,4-triazole (AT). Insects treated with AT showed a twofold increase in H2O2 levels. Injection of DL-buthionine-[S, R]-sulfoximine (BSO), an inhibitor of glutathione synthesis, also resulted in a fourfold increase in H2O2, together with stimulation of CAT activity. Simultaneous administration of both AT and BSO had a synergistic effect on midgut H2O2 content. Taken all together, our results suggest that CAT and glutathione-dependent mechanisms cooperate to control H2O2 concentration in the midgut cell and prevent hydroxyl radical generation by Fenton reaction in this tissue.     

Induction of necrotic cell death by oxidative stress in retinal pigment epithelial cells

Age-related macular degeneration (AMD) is a degenerative disease of the retina and the leading cause of blindness in the elderly. Retinal pigment epithelial (RPE) cell death and the resultant photoreceptor apoptosis are characteristic of late-stage dry AMD, especially geographic atrophy (GA). Although oxidative stress and inflammation have been associated with GA, the nature and underlying mechanism for RPE cell death remains controversial, which hinders the development of targeted therapy for dry AMD. The purpose of this study is to systematically dissect the mechanism of RPE cell death induced by oxidative stress. Our results show that characteristic features of apoptosis, including DNA fragmentation, caspase 3 activation, chromatin condensation and apoptotic body formation, were not observed during RPE cell death induced by either hydrogen peroxide or tert-Butyl hydroperoxide. Instead, this kind of cell death can be prevented by RIP kinase inhibitors necrostatins but not caspase inhibitor z-VAD, suggesting necrotic feature of RPE cell death. Moreover, ATP depletion, receptor interacting protein kinase 3 (RIPK3) aggregation, nuclear and plasma membrane leakage and breakdown, which are the cardinal features of necrosis, were observed in RPE cells upon oxidative stress. Silencing of RIPK3, a key protein in necrosis, largely prevented oxidative stress-induced RPE death. The necrotic nature of RPE death is consistent with the release of nuclear protein high mobility group protein B1 into the cytoplasm and cell medium, which induces the expression of inflammatory gene TNFα in healthy RPE and THP-1 cells. Interestingly, features of pyroptosis or autophagy were not observed in oxidative stress-treated RPE cells. Our results unequivocally show that necrosis, but not apoptosis, is a major type of cell death in RPE cells in response to oxidative stress. This suggests that preventing oxidative stress-induced necrotic RPE death may be a viable approach for late-stage dry AMD.     

Hydrogen peroxide treatment in Atlantic salmon induces stress and detoxification response in a daily manner

Daily variation in the absorption, metabolism and excretion of toxic substances will ultimately determine the actual concentration to which the cells and tissues are exposed. In aquaculture, Atlantic salmon (Salmo salar) can be frequently exposed to hydrogen peroxide (H₂O₂) to treat topical skin and gill infections, particularly in relation to parasitic infections (e.g. sea lice Lepeophtheirus salmonis and amoebic gill disease caused by Neoparamoeba perurans). It is well accepted that the time of administration influences pharmacodynamics and pharmacokinetics of drugs which in turn affects their efficacy and toxicity. Consequently, a better understanding of drug side effects as a function of time of day exposure would help to improve treatment efficacy and fish welfare. To this end, salmon were exposed to H₂O₂ (1500 mg/L) for 20 min at six different times of the day during a 24-h cycle and we investigated the time-dependent effects of exposure on physiological stress (glucose, lactate and cortisol) and antioxidant enzyme expression (gpx1, cat, Mn-sod and hsp70) in liver and gills. In addition, at each sampling point, 8 control fish were also sampled. Our results revealed that the time of administration of H₂O₂ caused significant differences in the induction of both physiological and oxidative stress responses. Glucose and lactate were higher in the treated fish during daytime whereas cortisol levels appeared to be systematically increased (>1000 ng/mL) after H₂O₂ treatment irrespective of exposure time, although differences with control levels were higher during the day. In liver, gene expression of antioxidant enzymes displayed daily rhythmicity in both treated and control groups and showed higher mRNA expression levels in salmon treated with H₂O₂ at ZT6 (6 h after lights onset). In gills, rhythmic expression was only found for gpx1 in the control fish and for hsp70 and Mn-sod in the treated groups. However, in the treated salmon, higher gene expression levels of all the investigated enzymes were also observed at ZT6-10. Clock gene expression showed rhythmicity only in the liver in accordance with the daily rhythm of enzyme expression observed in this tissue. Altogether, this study provides first evidence of chronotoxicity in Atlantic salmon treated with H₂O₂ and suggests increased sublethal toxic effect during the first half of the day. These results have direct relevance to the salmon and broader aquaculture industry by optimising the timing of treatment administration, opening the door to chronotherapy to treat fish diseases.     

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。     

Different susceptibilities to cell death induced by t-butylhydroperoxide could depend upon cell histotype-associated growth features

The effects of the oxidizing agent t-butylhydroperoxide (t-BHP) were investigated on three human cell lines of different origin and growth features (A431 epithelial cells, ADF astrocytoma cells and U937 leukemic cells) using electron microscopy and electron paramagnetic resonance spectroscopy. The results indicate that important biophysical and ultrastructural modifications are induced in the plasma and mitochondrial membranes of these cells and that these changes can ultimately lead to cell death. In addition, the cell cytoskeleton also appears to be a target of hydroperoxide-mediated stress. In particular, all three cell types undergo cytoskeletal alterations leading to surface blebbing, a typical characteristic of cell damage. However, the timing and extent of this damage as well as that occurring at the mitochondrial and plasma membrane levels seems to be different: cells with weak (ADF) or absent (U937) cell-to-cell and cell-substrate contacts and a poorly developed cytoskeleton appear to be more susceptible than other cell types (e.g., A431) to t-BHP-mediated injury. These diverse cell susceptibilities to hydroperoxide-mediated oxidative stress could thus depend upon cell histotype-associated growth featurs.Abbreviations t-BHP tert-butylhydroperoxide - DMEM Dulbecco's Modified Eagle's Medium - EPR electron paramagnetic resonance - GSH reduced glutathione - 5-NSA nitroxystearic acid - PBS phosphate-buffered saline     

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.     

Hydrogen peroxide production by Lactobacillus johnsonii NCC 533 and its role in anti-Salmonella activity

The human intestinal isolate Lactobacillus johnsonii NCC 533 (La1) is a probiotic strain with well-documented antimicrobial properties. Previous research has identified the production of lactic acid and bacteriocins as important factors, but that other unidentified factors are also involved. We used the recently published genome sequence of L. johnsonii NCC 533 to search for novel antipathogen factors and identified three potential gene products that may catalyze the synthesis of the known antimicrobial factor hydrogen peroxide, H(2)O(2). In this work, we confirmed the ability of NCC 533 as well as eight different L. johnsonii strains and Lactobacillus gasseri to produce H(2)O(2) when resting cells were incubated in the presence of oxygen, and that culture supernatant containing NCC 533-produced H(2)O(2) was effective in killing the model pathogen Salmonella enterica serovar Typhimurium SL1344 in vitro.     

Hydrogen peroxide production by roots and its stimulation by exogenous NADH

H₂O₂ production by roots of young seedlings was monitored using a non-destructive in vivo assay at pH 5.0. A particularly high rate of H₂O₂ production was measured in the roots of soybean (Glycine max L. cv. Labrador) seedlings which were used for further investigation of the physiological and enzymological properties of apoplastic H₂O₂ production. In the soybean root H₂O₂ production can be stimulated 10-fold by exogenous NADH or NADPH. This response displays typical features of a peroxidase-catalyzed oxidase reaction using NAD(P)H as electron donor for the reduction of O₂ to H₂O₂. Comparative measurements showed that the NADH-induced H₂O₂ production of the roots resembles the H₂O₂-forming activity of horseradish peroxidase with respect to NADH and O₂ concentration requirements and sensitivity to inhibition by KCN, NaN₃, superoxide dismutase and catalase. NADH-induced H₂O₂ production can be observed with similar intensity in all regions of the root, in agreement with the distribution of apoplastic peroxidase activity. In contrast, the activity responsible for the basal H₂O₂ production in the absence of exogenous NADH was mainly confined to a short subapical zone of the root and differs from the NADH-induced reaction by insensitivity to inhibition by superoxide dismutase and a strikingly lower requirement for O₂. It is concluded that the basal H₂O₂ production of the root is mediated by an enzyme different from peroxidase, possibly a plasma membrane O₂^?-producing oxidase.     

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.     

H2O2 at physiological concentrations modulates Leydig cell function inducing oxidative stress and apoptosis

H₂O₂ is one of the active reactive oxygen species secreted by macrophages that are seen closely aligned with Leydig cells in the testicular interstitium. The present study was initiated to investigate the role of H₂O₂ on Leydig cell function in vitro at physiological concentrations. Significant decrease in both testosterone production (p < 0.05) and 3 β-hydroxysteroid dehydrogenase activity (p < 0.05) in adult Leydig cells were observed even with H₂O₂ at low concentrations (30 – 50 μM). H₂O₂ exposure increased oxidative stress in Leydig cells with the rise in lipid peroxidation and fall in the activities of the antioxidant enzymes; superoxide dismutase (SOD), catalase (CAT) & glutathione-s-transferase (GST). There was also a marginal increase (∼8%) in cell apoptosis accompanied by rise in FasL expression and caspase-3 activation. The above findings indicate that H₂O₂ as a bio-molecule modulates Leydig cell function at or below physiological concentrations through a variety of actions like decrease in steroidogenic enzyme activity and increase in oxidative stress and apoptosis.