Oxidative stress response in eukaryotes: effect of glutathione,superoxide dismutase and catalase on adaptation to peroxide and menadione stresses in Saccharomyces cerevisiae

Abstract

Aiming to clarify the mechanisms by which eukaryotes acquire tolerance to oxidative stress, adaptive and cross-protection responses to oxidants were investigated in Saccharomyces cerevisiae. Cells treated with sub-lethal concentrations of menadione (a source of superoxide anions) exhibited cross-protection against lethal doses of peroxide; however, cells treated with H₂O₂ did not acquire tolerance to a menadione stress, indicating that menadione response encompasses H₂O₂ adaptation. Although, deficiency in cytoplasmic superoxide dismutase (Sod1) had not interfered with response to superoxide, cells deficient in glutathione (GSH) synthesis were not able to acquire tolerance to H₂O₂ when pretreated with menadione. These results suggest that GSH is an inducible part of the superoxide adaptive stress response, which correlates with a decrease in the levels of intracellular oxidation. On the other hand, neither the deficiency of Sod1 nor in GSH impaired the process of acquisition of tolerance to H₂O₂ achieved by a mild pretreatment with peroxide. Using a strain deficient in the cytosolic catalase, we were able to conclude that the reduction in lipid peroxidation levels produced by the adaptive treatment with H₂O₂ was dependent on this enzyme. Corroborating these results, the pretreatment with low concentrations of H₂O₂ promoted an increase in catalase activity.     

Oxidative Stress in a Clonal Cell Line of Neuronal Origin: Effects of Antioxidant Enzyme Modulation

Abstract: The effects of intracellularly generated H₂O₂ on cell viability, morphology, and biochemical markers of injury have been investigated in a clonal cell line of neuronal origin (140-3, mouse neuroblastoma X rat glioma) as a cell culture model for the role of oxidative stress in the longterm loss of neurons in the brain. The H₂O₂ was generated from the redox cycling of menadione, or by the oxidation of serotonin catalyzed by monoamine oxidase, to simulate the effect of amine neurotransmitter turnover. Incubation with menadione at concentrations as low as 10 γM for several hours resulted in significant losses of cell viability and altered morphology. Similar effects were evident in the presence of serotonin only after incubation overnight with concentrations > 1 mM. The cytotoxicity of either agent was potentiated by preincubation with specific inhibitors of two enzymes important to cellular antioxidant defenses, 3-amino-1,2,4-trazole for catalase and 1,3-bis(chloromethyl)-1-nitrosourea for glutathione reductase. Activity of another antioxidant enzyme of particular importance to antioxidant defenses in brain, the selenoprotein glutathione peroxidase, was stimulated fourfold by growth of cultures in the presence of sodium selenite as a source of active-site Se for the enzyme. The only effect of the selenite on other functionally coupled antioxidant enzymes was a decrease in activity of superoxide dismutase at concentrations >200 nM. The selenite substantially protected cells against oxidative stress induced by combinations of menadione, 3-amino-1,2,4-trazole, and 1,3-bis(chloromethyl)-1-nitrosourea, but was only marginally effective with serotonin as a source of oxidative stress. The monoamine oxidase inhibitor pargyline increased cell survival in the presence of serotonin, demonstrating the role of this enzyme in its cytotoxicity. DNA damage (single strand breaks), but not lipid peroxidation, correlated with the cytotoxic effects of menadione.     

Modulation of Notch Signaling Pathway to Prevent H2O2/Menadione-Induced SK-N-MC Cells Death by EUK134

The brain in Alzheimer’s disease is under increased oxidative stress, and this may have a role in the pathogenesis and neural death in this disorder. It has been verified that numerous signaling pathways involved in neurodegenerative disorders are activated in response to reactive oxygen species (ROS). EUK134, a synthetic salen–manganese antioxidant complex, has been found to possess many interesting pharmacological activities awaiting exploration. The present study is to characterize the role of Notch signaling in apoptotic cell death of SK-N-MC cells. The cells were treated with hydrogen peroxide (H₂O₂) or menadione to induce oxidative stress. The free-radical scavenging capabilities of EUK134 were studied through the MTT assay, glutathione peroxidase (GPx) enzyme activity assay, and glutathione (GSH) Levels. The extents of lipid peroxidation, protein carbonyl formation, and intracellular ROS levels, as markers of oxidative stress, were also studied. Our results showed that H₂O₂/menadione reduced GSH levels and GPx activity. However, EUK134 protected cells against ROS-induced cell death by down-regulation of lipid peroxidation and protein carbonyl formation as well as restoration of antioxidant enzymes activity. ROS induced apoptosis and increased NICD and HES1 expression. Inhibition of NICD production proved that Notch signaling is involved in apoptosis through p53 activation. Moreover, H₂O₂/menadione led to Numb protein down-regulation which upon EUK134 pretreatment, its level increased and subsequently prevented Notch pathway activation. We indicated that EUK134 can be a promising candidate in designing natural-based drugs for ROS-induced neurodegenerative diseases. Collectively, ROS activated Notch signaling in SK-N-MC cells leading to cell apoptosis.     

Effects of Oxidative Stress Caused by Hyperoxia and Diquat. A Study in Isolated Hepatocytes

The effects of oxidative stress caused by hyperoxia or administration of the redox active compound diquat were studied in isolated hepatocytes, and the relative contribution of lipid peroxidation, glutathione (GSH) depletion, and NADPH oxidation to the cytotoxicity of active oxygen species was investigated.

The redox cycling of diquat occurred primarily in the microsomal fraction since diquat was found not ' to penetrate into the mitochondria. Depletion of intracellular GSH by pretreatment of the animals with diethyl maleate promoted lipid peroxidation and sensitized the cells to oxidative stress. Diquat toxicity was also greatly enhanced when glutathione reductase was inhibited by pretreatment of the cells with 1,3-bis(2-chloroethyI)-1-nitrosourea. Despite extensive lipid peroxidation, loss of cell viability was not observed, with either hyperoxia or diquat, until the GSH level had fallen below ≈ 6 nmol/10⁶ cells.

The iron chelator desferrioxamine provided complete protection against both diquat-induced lipid peroxidation and loss of cell viability. In contrast, the antioxidant a-tocopherol inhibited lipid peroxidation but provided only partial protection from toxicity. The hydroxy! radical scavenger α-keto-γ-methiol butyric acid, finally, also provided partial protection against diquat toxicity but had no effect on lipid peroxidation.

The results indicate that there is a critical GSH level above which cell death due to oxidative stress is not observed. As long as the glutathione peroxidase – glutathione reductase system is unaffected, even relatively low amounts of GSH can protect the cells by supporting glutathione peroxidase-mediated metabolism of H₂O₂ and lipid hydroperoxides.     

Tolerance of the yeast Yarrowia lipolytica to oxidative stress

The adaptive response of the yeast Yarrowia lipolytica to the oxidative stress induced by the oxidants hydrogen peroxide, menadione, and juglone has been studied. H₂O₂, menadione, and juglone completely inhibited yeast growth at concentrations higher than 120, 0.5, and 0.03 mM, respectively. The stationary-phase yeast cells were found to be more resistant to the oxidants than the exponential-phase cells. The 60-min pretreatment of logarithmic-phase cells with nonlethal concentrations of H₂O₂ (0.3 mM), menadione (0.05 mM), and juglone (0.005 mM) made the cells more resistant to high concentrations of these oxidants. The adaptation of yeast cells to H₂O₂, menadione, and juglone was associated with an increase in the activity of cellular catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and glutathione reductase, the main enzymes involved in cell defense against oxidative stress.     

Antioxidant effect of homogenetisic acid on hydrogen peroxide induced oxidative stress in human lung fibroblast cells

Homogentisic acid was found to scavenge intracellular reactive oxygen species (ROS), and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and thus prevented lipid peroxidation in human fibroblast (WI 38) cells. The radical scavenging activity of homogentisic acid was found to protect WI 38 cells against hydrogen peroxide (H₂O₂) induced oxidative stress, via the activation of extracellular signal regulated kinase (ERK) protein. Homogentisic acid increased the activity of catalase. Hence, from the present study, it is suggested that homogentisic acid protects WI 38 cells against H₂O₂ damage by enhancing the intracellular antioxidative activity.     

Alleviation of ultraviolet-C-induced oxidative damage through overexpression of cytosolic ascorbate peroxidase

Experiments were conducted to investigate the relationship between ultraviolet (UV) C-induced oxidative damage and the activity of ascorbate peroxidase (APX), using transgenic tobacco (Nicotiana tabacum L. cv. Petit Havana) plants overexpressing cytosolic APX gene (apx1). Transgenic plants having 2.3 fold higher total APX activity, as compared to the wild type plants, showed normal morphological characters. Exposure of 70-day-old plants to fixed intensity UV-C radiation caused an increase in the malondialdehyde (MDA) content in wild type as well as transgenic plants. However, the wild type plants showed significantly higher (p < 0.05) lipid peroxidation as compared to the transgenic plants. Higher proline accumulation was recorded in transgenic plants as compared to the wild type plants, after 24 hours of UV-C exposure. Although the ascorbate content decreased continuously with increasing exposure to UV-C radiation, yet the wild type plants exhibited higher ascorbate levels than the transgenic plants. A marked difference in H₂O₂ content, between the wild type and transgenic plants, was consistently observed up to 20 hours of UV-C exposure. A direct correlation of ascorbate, MDA and H₂O₂ levels was recorded with the extent of oxidative stress, signifying that these could be used as potential bio-marker molecules for oxidative stress. The results clearly demonstrate that overexpression of cytosolic APX can protect tobacco plants from UV-C-induced oxidative damage.     

Effect of fusaric acid on prooxidant and antioxidant properties of the potato cell suspension culture

Cell suspension cultures of potato (Solanum tuberosum, cv. Tamasha) were treated with fusaric acid (FA), a nonspecific fungal toxin produced by Fusarium species to study the effects of FA on H₂O₂ generation, lipid peroxidation, and activities of antioxidant enzymes, i.e., superoxide dismutase (SOD), catalase, and ascorbate peroxidase (APX). The toxicity of various FA doses was evaluated from viability of cultured cells of S. tuberosum. The toxic concentration of FA (10⁻³ M) reduced cell viability by 32% after 48-h incubation and induced alkalinization of the medium; the nontoxic concentration of FA (10⁻⁶ M) had no effect on cell viability and pH of the culturing medium. The treatment of cells with FA caused rapid reversible accumulation of H₂O₂ in cells, promoted lipid peroxidation, and elevated the activity of antioxidant enzymes. The toxic FA concentration elevated the intracellular H₂O₂ content by 51–59% and stimulated lipid peroxidation rate by 35–40%. The nontoxic FA concentration raised the H₂O₂ content by 84–91% and enhanced lipid peroxidation rate by 18–24%. The addition of FA induced transient biphasic induction of the antioxidant enzymes; the action of toxic and nontoxic concentrations differed in terms of the response amplitudes and dynamics. The results confirm the well-known toxic impact of high doses of FA on the cultured cells, which is determined by membrane transport disorders. In addition, the results reveal that toxic and nontoxic concentrations of FA are able to induce pro- and antioxidant systems in the cultured cells of S. tuberosum.     

Effect of Long-Term Normobaric Hyperoxia on Oxidative Stress in Mitochondria of the Guinea Pig Brain

Normobaric hyperoxia (NBO) is applied for treatment of various clinical conditions related to hypoxia, but it can potentially also induce generation of reactive oxygen species, causing cellular damage. In this study, we examined the effects of 60 h NBO treatment on lipid and protein oxidative damage and activity of superoxide dismutase (Mn-SOD) in brain mitochondria of guinea pigs. Despite significant stimulation of Mn-SOD expression and activity the NBO treatment resulted in accumulation of markers of oxidative lesions, including lipid peroxidation (conjugated dienes, thiobarbituric acid reactive substances) and protein modification (bityrosines, adducts with lipid peroxidation products, oxidized thiols). When inhaled O₂ was enriched with oxygen cation, O₂^•+, the Mn-SOD expression and activity were stimulated to similar extend, but lipid peroxidation and protein oxidation were prevented. These results suggest that long-term NBO treatment causes oxidative stress, but enrichment of inhaled oxygen by oxygen cation can protect the brain again adverse effects of hyperoxia.     

Drought-induced senescence is characterized by a loss of antioxidant defences in chloroplasts

The relationship between drought, oxidative stress and leaf senescence was evaluated in field‐grown sage (Salvia officinalis L.), a drought‐susceptible species that shows symptoms of senescence when exposed to stress. Despite the photoprotection conferred by the xanthophyll cycle, drought‐stressed senescing leaves showed enhanced lipid peroxidation, chlorophyll loss, reduced photosynthetic activity and strong reductions of membrane‐bound chloroplastic antioxidant defences (i.e. β‐carotene and α‐tocopherol), which is indicative of oxidative stress in chloroplasts. H₂O₂ accumulated in drought‐stressed senescing leaves. Subcellular localization studies showed that H₂O₂ accumulated first in xylem vessels and the cell wall and later in the plasma membrane of mesophyll cells, but not in chloroplasts, indicating reactive oxygen species other than H₂O₂ as direct responsible for the oxidative stress observed in the chloroplasts of drought‐stressed senescing leaves. The strong degradation of β‐carotene and α‐tocopherol suggests an enhanced formation of singlet oxygen as the putative reactive oxygen species responsible for oxidative stress to senescing chloroplasts. This study demonstrates that oxidative stress in chloroplasts mediates drought‐induced leaf senescence in sage growing in Mediterranean field conditions.     

Hydrogen peroxide is involved in the sclerotial differentiation of filamentous phytopathogenic fungi

Aims: The purpose of this study was to investigate the role of H₂O₂ and the related oxidative stress markers catalase (CAT) and lipid peroxidation in the sclerotial differentiation of the phytopathogenic filamentous fungi Sclerotium rolfsii, Sclerotinia minor, Sclerotinia sclerotiorum and Rhizoctonia solani. Methods and Results: Using the H₂O₂‐specific scopoletin fluorometric assay and the CAT‐dependent H₂O₂ consumption assays, it was found that the production rate of intra/extracellular H₂O₂ and CAT levels in the sclerotiogenic fungi were significantly higher and lower, respectively, than those of their nondifferentiating counterpart strains. They peaked in the transition between the undifferentiated and the differentiated state of the sclerotiogenic strains, suggesting both a cell proliferative and differentiative role. In addition, the indirect indicator of oxidative stress, lipid peroxidation, was substantially decreased in the nondifferentiating strains. Conclusions: These findings suggest that the differentiative role of H₂O₂ is expressed via induction of higher oxidative stress in the sclerotiogenic filamentous phytopathogenic fungi. Significance and Impact of the Study: This study shows that the direct marker of oxidative stress H₂O₂ is involved in the sclerotial differentiation of the phytopathogenic filamentous fungi S. rolfsii, S. minor, S. sclerotiorum and R. solani, which could have potential biotechnological implications in terms of developing antifungal strategies by regulating intracellular H₂O₂ levels.     

Oxidative stress perturbs cell proliferation in human K562 cells by modulating protein synthesis and cell cycle

Oxidative stress leads to perturbation of a variety of cellular processes resulting in inhibition of cell proliferation. This study has determined the effect of oxidative stress on protein synthesis in human K562 cells using a hydrophilic peroxyl radical initiator, AAPH and H₂O₂. The results indicated that oxidative stress leads to a significant decrease in the rate of protein synthesis caused due to induced activation as well as expression of the erythroid cell-specific eIF-2α kinase, called the Heme Regulated Inhibitor (HRI). Elevated levels of HRI expression and activity were accompanied by increased lipid peroxidation and decreased cell proliferation. Further, oxidative stress also caused inactivation of p34^cdc2 kinase, thereby arresting cell division leading to apoptosis. Thus, the data provides the mechanism of inhibition of protein synthesis and perturbation of a cell cycle regulatory protein leading to inhibition of cell proliferation in K562 cells during oxidative stress.     

Change in antioxidant responses against oxidative damage in black chickpea following cold acclimation

Cold stress is an important factor affecting chickpea (Cicer arietinum L.) plants in winter and early spring. We evaluated the effects of cold stress by measuring lipid peroxidation, membrane permeability, and some enzyme activities involved in the ROS-scavenging system under acclimation and non-acclimation conditions in black chickpea Kaka, a popular genotype planted, and accession 4322, as a landrace genotype. Under non-acclimation conditions, the genotype 4322 prevented the H₂O₂ accumulation more efficiently, which led to a decrease in lipid peroxidation and membrane permeability compared to Kaka. Studying the activities of antioxidant enzymes showed that catalase was more effective enzyme in cell protection against H₂O₂ in 4322 plants. Such response in acclimated plants was more pronounced than in control and nonacclimated plants. In this study, the increase in guaiacol peroxidase and ascorbate peroxidase activities did not preserve cell membranes from oxidative damage in Kaka plants. It was observed that short-term acclimation can induce greater cold tolerance upon the increase of oxidative stress in chickpea plants. This was due to low levels of MDA and electrolyte leakage index, indicating the lower lipid peroxidation and higher membrane stability under the cold stress compared to non-acclimated plants.     

Phycobiliproteins from <Emphasis Type="Italic">Pseudanabaena tenuis</Emphasis> rich in c-phycoerythrin protect against HgCl<Subscript>2</Subscript>-caused oxidative stress and cellular damage in the kidney

Our objective was to study if the phycobiliproteins of the cyanobacterium Pseudanabanea tenuis rich in phycoerythrin protect renal cells against mercury-caused oxidative stress and cellular damage in the kidney. We used 40 male mice that were assigned into five groups: a control group that received phosphate buffer (PB) and saline and four treatment groups which received either PB+HgCl₂, PB+phycobiliproteins, or HgCl₂+phycobiliproteins. The kidneys of the mice were used to determine lipid peroxidation and quantification of reactive oxygen species, oxidized glutathione, and peroxidase activities (catalase and glutathione peroxidase) and were also examined histologically. Our results demonstrated that HgCl₂ causes oxidative stress and cellular damage and that all doses of phycobiliproteins prevented the increase of oxidative markers and partially protected against HgCl₂-caused cell damage. This is the first report which applied phycobiliproteins of P. tenuis rich in c-phycoerythrin, like antioxidants against mercury chloride-caused oxidative stress and renal damage.     

Hydrogen peroxide-induced oxidative damages in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

Oxidative stress causes damage to proteins, lipids and nucleic acids, and thereby compromises cell viability. Some of the oxidative stress markers in an eukaryotic model organism, fission yeast Schizosaccharomyces pombe, were evaluated in this study. Intracellular oxidation, protein carbonyls, lipid peroxidation and reduced glutathione (GSH) levels were investigated in H₂O₂-treated and non-treated control cells. It was observed that increased H₂O₂ concentration proportionally lowered the cell number and increased the intracellular oxidation, lipid peroxidation and protein carbonyl levels in S. pombe. A dose-dependent decrease in GSH level was also detected. The fission yeast S. pombe is best known for its contribution to understanding of eukaryotic cell cycle control. S. pombe displays a different physiology from Saccharomyces cerevisiae in several ways and is thus probably more closely related to higher eukaryotes. The purpose of this study was to provide some data about the effects of hydrogen peroxide on the proteins and lipids in the fission yeast. The data obtained here is expected to constitute a basis for the further studies on redox balance and related processes in yeast and mammalian cells.     

Heat Shock Inhibits the Induced Expression of the SOS and SoxRS Regulons in Escherichia coli

Oxidative stress formed in Escherichia coli cells is known to bring about a complex induction of alternative DNA repair processes, including SOS, SoxRS, and heat-shock response (HSR). The modification by heat shock of the expression ofsfiA and soxS genes induced by oxidative agents H₂O₂, menadione and 4-nitroquinoline-1-oxide (4NQO) was studied for the first time. Quantitative parameters of gene expression were examined inE. coli strains with fused genes (promoters) sfiA::lacZ and soxS::lacZ.The expression of these genes induced by cell treatment with H₂O₂, but not menadione or 4NQO, was shown to decrease selectively after exposure to heat shock. Since genetic activity of menadione and 4NQO depends mainly on the formation of superoxide anion ,O^¯ ₂ it is assumed that the effect of selective inhibition by heat-shock of sfiA and soxS gene expression in experiments with H₂O₂ is connected with activity of DnaK heat shock protein, which, unlike other heat-shock proteins, cannot be induced by superoxide anion O^¯ ₂.     

Menadione stress in Saccharomyces cerevisiae strains deficient in the glutathione transferases

Using S. cerevisiae as a eukaryotic cell model we have analyzed the involvement of both glutathione transferase isoforms, Gtt1 and Gtt2, in constitutive resistance and adaptive response to menadione, a quinone which can exert its toxicity as redox cycling and/or electrophiles. The detoxification properties, of these enzymes, have also been analyzed by the appearance of S-conjugates in the media. Direct exposure to menadione (20 mM/60 min) showed to be lethal for cells deficient on both Gtt1 and Gtt2 isoforms. However, after pre-treatment with a low menadione concentration, cells deficient in Gtt2 displayed reduced ability to acquire tolerance when compared with the control and the Gtt1 deficient strains. Analyzing the toxic effects of menadione we observed that the gtt2 mutant showed no reduction in lipid peroxidation levels. Moreover, measuring the levels of intracellular oxidation during menadione stress we have shown that the increase of this oxidative stress parameter was due to the capacity menadione possesses in generating reactive oxygen species (ROS) and that both GSH and Gtt2 isoform were required to enhance ROS production. Furthermore, the efflux of the menadione–GSH conjugate, which is related with detoxification of xenobiotic pathways, was not detected in the gtt2 mutant. Taken together, these results suggest that acquisition of tolerance against stress generated by menadione and the process of detoxification through S-conjugates are dependent upon Gtt2 activity. This assessment was corroborated by the increase of GTT2 expression, and not of GTT1, after menadione treatment.     

Prooxidant and antioxidant effects of ascorbate on tBuOOH-induced erythrocyte membrane damage

1. t-Butylhydroperoxide (tBuOOH) a lipoperoxide analog, causes rapid and considerable sulphydryl (SH) oxidation but almost no lipid peroxidation in red blood cell membranes (ghosts) containing no detectable haemoglobin. 2. tBuOOH, in the presence of ascorbate, produces significant lipid peroxidation the level of which is proportional to the ascorbate concentration. The initiation of lipid peroxidation is thought to occur by the reactive tBuO (butoxyl) species via the reductive decomposition of tBuOOH by ascorbate. 3. Ascorbate protects ghost membranes from the tBuOOH-induced SH oxidation in a dose-dependent fashion. 4. There is no parallelism between lipid peroxidation and SH oxidation in these systems. This suggests that the two processes occur independently of each other. 5. These findings indicate that, simultaneously, ascorbate can have both a protective and a prooxidant action in different membrane components under the same oxidative stress.     

Hydrogen peroxide-induced oxidative stress to the mammalian heart-muscle cell (cardiomyocyte): Lethal peroxidative membrane injury

Oxidative stress induced by hydrogen peroxide (H₂O₂) may contribute to the pathogenesis of ischemic-reperfusion injury in the heart. For the purpose of investigating directly the injury potential of H₂O₂ on heart muscle, a cellular model of H₂O₂-induced myocardial oxidative stress was developed. This model employed primary monolayer cultures of intact, beating neonatal-rat cardiomy-ocytes and discrete concentrations of reagent H₂O₂ in defined, supplement-free culture medium. Cardiomyocytes challenged with H₂O₂ readily metabolized it such that the culture content of H₂O₂ diminished over time, but was not depleted. The consequent H₂O₂-induced oxidative stress caused lethal sarcolemmal disruption (as measured by lactate dehydrogenase release), and cardiomyocyte integrity could be preserved by catalase. During oxidative stress, a spectrum of cellular derangements developed, including membrane phospholipid peroxidation, thiol oxidation, consumption of the major chain-breaking membrane antiperoxidant (α-tocopherol), and ATP loss. No net change in the protein or phospholipid contents of cardiomyocyte membranes accompanied H₂O₂-induced oxidative stress, but an increased turnover of these membrane constituents occurred in response to H₂O₂. Development of lethal cardiomyocyte injury during H₂O₂-induced oxidative stress did not require the presence of H₂O₂ itself; a brief “pulse” exposure of the cardiomyocytes to H₂O₂ was sufficient to incite the pathogenic mechanism leading to cell disruption. Cardiomyocyte disruption was dependent upon an intracellular source of redox-active iron and the iron-dependent transformation of internalized H₂O₂ into products (e.g., the hydroxyl radical) capable of initiating lipid peroxidation, since iron chelators and hydroxyl-radical scavengers were cytoprotective. The accelerated turnover of cardiomyocyte-membrane protein and phospholipid was inhibited by antiperoxidants, suggesting that the turnover reflected molecular repair of oxidized membrane constituents. Likewise, the consumption of α-tocopherol and the oxidation of cellular thiols appeared to be epiphenomena of peroxidation. Antiperoxidant interventions coordinately abolished both H₂O₂-induced lipid peroxidation and sarcolemmal disruption, demonstrating that an intimate pathogenic relationship exists between sarcolemmal peroxidation and lethal compromise of cardiomyocyte integrity in response to H₂O₂-induced oxidative stress. Although sarcolemmal peroxidation was causally related to cardiomyocyte disruption during H₂O₂-induced oxidative stress, a nonperoxidative route of H₂O₂ cytotoxicity was also identified, which was expressed in the complete absence of cardiomyocyte-membrane peroxidation. The latter mode of H₂O₂-induced cardiomyocyte injury involved ATP loss such that membrane peroxidation and cardiomyocyte disruption on the one hand and cellular de-energization on the other could be completely dissociated. The cellular pathophysiology of H₂O₂ as a vectorial signal for cardiomyocyte necrosis that “triggers” irreversible peroxidative disruption of the sarcolemma has implications regarding potential mechanisms of oxidative injury in the postischemic heart.     

Antioxidative defense system in pigeonpea roots under waterlogging stress

Pigeonpea [Cajanus cajan (L.) Millsp.] is a waterlogging-sensitive legume crop. We studied the effect of waterlogging stress on hydrogen peroxide (H₂O₂) content, lipid peroxidation and antioxidant enzyme activities in two pigeonpea genotypes viz., ICPL-84023 (waterlogging resistant) and MAL-18 (waterlogging susceptible). In a pot experiment, waterlogging stress was imposed for 6 days at early vegetative stage (20 days after sowing). Waterlogging treatment significantly increased hydrogen peroxide accumulation and lipid peroxidation, which indicated the extent of oxidative injury posed by stress conditions. Enzyme activities of peroxidase (POX), catalase (CAT), ascorbate peroxidase (APX), superoxide dismutase (SOD) and polyphenol oxidase (PPO) increased in pigeonpea roots as a consequence of waterlogged conditions, and all the enzyme activities were significantly higher in waterlogged ICPL-84023 than in MAL-18. POX activity was the maximum immediately after imposing stress, therefore, it was suggested to be involved in early scavenging of H₂O₂, while rest of the enzymes (CAT, APX, SOD and PPO) were more important in late responses to waterlogging. Present study revealed that H₂O₂ content is directly related to lipid peroxidation leading to oxidative damage during waterlogging in pigeonpea. Higher antioxidant potential in ICPL-84023 as evidenced by enhanced POX, CAT, APX, SOD and PPO activities increased capacity for reactive oxygen species (ROS) scavenging and indicated relationship between waterlogging resistance and antioxidant defense system in pigeonpea.