Necrotic cell death by hydrogen peroxide in immortal DF-1 chicken embryo fibroblast cells expressing deregulated MnSOD and catalase

The reactive oxygen species are known as endogenous toxic oxidant damaging factors in a variety of cell types, and in response, the antioxidant genes have been implicated in cell proliferation, senescence, immortalization, and tumorigenesis. The expression of manganese superoxide dismutase mRNA was shown to increase in most of the immortal chicken embryo fibroblast (CEF) cells tested, while expression of catalase mRNA appeared to be dramatically decreased in all immortal CEF cells compared to their primary counterparts. The expression of copper-zinc superoxide dismutase mRNA was shown to increase slightly in some immortal CEF cells. The glutathione peroxidase expressed relatively similar levels in both primary and immortal CEF cells. As primary and immortal DF-1 CEF cells were treated with 10-100 microM of hydrogen peroxide (concentrations known to be sublethal in human diploid fibroblasts), immortal DF-1 CEF cells were shown to be more sensitive to hydrogen peroxide, and total cell numbers were dramatically reduced when compared with primary cell counterparts. This increased sensitivity to hydrogen peroxide in immortal DF-1 cells occurred without evident changes in either antioxidant gene expression, mitochondrial membrane potential, cell cycle distribution or chromatin condensation. However, the total number of dead cells without chromatin condensation was dramatically elevated in immortal DF-1 CEFs treated with hydrogen peroxide, indicating that the inhibition of immortal DF-1 cell growth by low concentrations of hydrogen peroxide is due to increased necrotic cell death, but not apoptosis. Taken together, our observation suggests that the balanced antioxidant function might be important for cell proliferation in response to toxic oxidative damage by hydrogen peroxide.     

Nutrition- and virus-induced stress represses the expression of manganese superoxide dismutase in vitro

The relationship between oxidative stress and neuronal cell death has been suggested for many years. To understand the influence of oxidative stress on neuronal cell death, we investigated the influence of oxidative stress on DEV cells, a human glial cell line. Using enterovirus infection and/or malnutrition to induce oxidative stress, our results demonstrate that those stressors severely influence the antioxidant defense system in DEV cells. Although the expression of mitochondrial manganese superoxide dismutase (MnSOD) in DEV cells was significantly increased in acute infection with viral and nutritional stress, in persistent infection and nutritional stress, the expression of the MnSOD was drastically downregulated. We believe that this downregulation of MnSOD expression in the chronic stress model is due to repression of antioxidant defense. The downregulation of the MnSOD expression may lead to an increase of free-radical production and thus explain why the cells in the chronic stress model were more vulnerable to other oxidative stress influences. The vulnerability of DEV cells to additional stress factors resulted in progressive cell death, which may be analogous to the cell death in neurodegenerative diseases.     

Protection of insulin-producing cells against toxicity of dexamethasone by catalase overexpression

Pancreatic β cells are very sensitive to reactive oxygen species (ROS) and this might play an important role in β cell death in diabetes. Dexamethasone is a synthetic diabetogenic glucocorticoid, which impairs pancreatic β cell function. Therefore we investigated the toxicity of dexamethasone in RINm5F insulin-producing cells and its dependence on the expression level of the antioxidant enzyme catalase, which inactivates hydrogen peroxide. This was correlated with oxidative stress and cell death. An increased generation of ROS was observed in dexamethasone-treated cells together with an increase in caspase-3 activity and apoptosis rate. Interestingly, exposure to dexamethasone increased the cytosolic superoxide dismutase Cu/ZnSOD protein expression and activity, whereas the mitochondrial MnSOD isoform was not affected by the glucocorticoid. Catalase overexpression in insulin-producing cells prevented all the cytotoxic effects of dexamethasone. In conclusion, dexamethasone-induced cell death in insulin-producing cells is ROS mediated. Increased levels of expression and activity of the Cu/ZnSOD might favor the generation of hydrogen peroxide in dexamethasone-treated cells. Increased ROS scavenging capacity in insulin-producing cells, through overexpression of catalase, prevents a deleterious increase in hydrogen peroxide generation and thus prevents dexamethasone-induced apoptosis.     

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.     

Water restriction increases renal inner medullary manganese superoxide dismutase (MnSOD)

Oxidative stress damages cells. NaCl and urea are high in renal medullary interstitial fluid, which is necessary to concentrate urine, but which causes oxidative stress by elevating reactive oxygen species (ROS). Here, we measured the antioxidant enzyme superoxide dismutases (SODs, MnSOD, and Cu/ZnSOD) and catalase in mouse kidney that might mitigate the oxidative stress. MnSOD protein increases progressively from the cortex to the inner medulla, following the gradient of increasing NaCl and urea. MnSOD activity increases proportionately, but MnSOD mRNA does not. Water restriction, which elevates renal medullary NaCl and urea, increases MnSOD protein, accompanied by a proportionate increase in MnSOD enzymatic activity in the inner medulla, but not in the cortex or the outer medulla. In contrast, Cu/ZnSOD and TNF-α (an important regulator of MnSOD) do not vary between the regions of the kidney, and expression of catalase protein actually decreases from the cortex to the inner medulla. Water restriction increases activity of mitochondrial enzymes that catalyze production of ROS in the inner medulla, but reduces NADPH oxidase activity there. We also examined the effect of high NaCl and urea on MnSOD in Madin-Darby canine kidney (MDCK) cells. High NaCl and high urea both increase MnSOD in MDCK cells. This increase in MnSOD protein apparently depends on the elevation of ROS since it is eliminated by the antioxidant N-acetylcysteine, and it occurs without raising osmolality when ROS are elevated by antimycin A or xanthine oxidase plus xanthine. We conclude that ROS, induced by high NaCl and urea, increase MnSOD activity in the renal inner medulla, which moderates oxidative stress.     

Response of the antioxidant defense system to tert-butyl hydroperoxide and hydrogen peroxide in a human hepatoma cell line (HepG2)

The aim of this work was to investigate the response of the antioxidant defense system to two oxidative stressors, hydrogen peroxide and tert-butyl hydroperoxide, in HepG2 cells in culture. The parameters evaluated included enzyme activity and gene expression of superoxide dismutase, catalase, glutathione peroxidase, and activity of glutathione reductase. Besides, markers of the cell damage and oxidative stress evoked by the stressors such as cell viability, intracellular reactive oxygen species generation, malondialdehyde levels, and reduced glutathione concentration were evaluated. Both stressors, hydrogen peroxide and tert-butyl hydroperoxide, enhanced cell damage and reactive oxygen species generation at doses above 50 microM. The concentration of reduced glutathione decreased, and levels of malondialdehyde and activity of the antioxidant enzymes consistently increased only when HepG2 cells were treated with tert-butyl hydroperoxide but not when hydrogen peroxide was used. A slight increase in the gene expression of Cu/Zn superoxide dismutase and catalase with 500 microM tert-butyl hydroperoxide and of catalase with 200 microM hydrogen peroxide was observed. The response of the components of the antioxidant defense system evaluated in this study indicates that tert-butyl hydroperoxide evokes a consistent cellular stress in HepG2.     

Adverse effects of reduced oxygen tension on the proliferative capacity of rat kidney and insulin-secreting cell lines involve DNA damage and stress responses

Standard cell culture conditions do not reflect the physiological environment in terms of oxygen tension (20% vs 3%). The effects of lowering oxygen tension on cell proliferation in culture can be beneficial as well as detrimental depending on the cell line studied, but the molecular mechanism underlying such effects is not fully understood. We observed that the proliferative capacity of the rat cell lines NRK and INS-1 was inhibited when cultured under 3% oxygen as compared to 20% oxygen. Suppression of proliferation in NRK cells was accompanied by induction of DNA double strand breaks whereas in INS-1 cells it was accompanied by up-regulation of p53 and p27. Although Sirt1 was up-regulated in both cell lines by 3% oxygen the effects on antioxidant enzymes (MnSOD, CuZnSOD and catalase) were cell line specific. Marked up-regulation of heme oxygenase-1 (HO-1) was detected in both NRK and INS-1 cells when cultured in 3% oxygen. HO-1 expression can be readily induced by exposure to hydrogen peroxide in culture. These results suggest that reduced oxygen tension suppresses the proliferative capacity of these two cell lines through a stress response that is similar to an oxidative stress response but the molecular events that lead to the reduced cell proliferation are cell line specific.     

Mitochondrial catalase and oxidative injury

Mitochondria dysfunction induced by reactive oxygen species (ROS) is related to many human diseases and aging. In physiological conditions, the mitochondrial respiratory chain is the major source of ROS. ROS could be reduced by intracellular antioxidant enzymes including superoxide dismutase, glutathione peroxidase and catalase as well as some antioxidant molecules like glutathione and vitamin E. However, in pathological conditions, these antioxidants are often unable to deal with the large amount of ROS produced. This inefficiency of antioxidants is even more serious in mitochondria, because mitochondria in most cells lack catalase. Therefore, the excessive production of hydrogen peroxide in mitochondria will damage lipid, proteins and mDNA, which can then cause cells to die of necrosis or apoptosis. In order to study the important role of mitochondrial catalase in protecting cells from oxidative injury, a HepG2 cell line overexpressing catalase in mitochondria was developed by stable transfection of a plasmid containing catalase cDNA linked with a mitochondria leader sequence which would encode a signal peptide to lead catalase into the mitochondria. Mitochondria catalase was shown to protect cells from oxidative injury induced by hydrogen peroxide and antimycin A. However, it increased the sensitivity of cells to tumor necrosis factor-alpha-induced apoptosis by changing the redox-oxidative status in the mitochondria. Therefore, the antioxidative effectiveness of catalase when expressed in the mitochondrial compartment is dependent upon the oxidant and the locus of ROS production.     

UV-irradiation induces oxidative damage to mitochondrial DNA primarily through hydrogen peroxide: analysis of 8-oxodGuo by HPLC

Roles of reactive oxygen species (ROS) in damage to mitochondrial DNA (mtDNA) following ultraviolet (UV)-irradiation were investigated in the human hepatoma cell line SK-HEP-1. We altered the intracellular status of ROS by the overexpression of manganese superoxide dismutase (MnSOD) and/or catalase. Using HPLC, we analyzed 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), known as a marker of damage to DNA molecules. UV-irradiation resulted in the accumulation of 8-oxodGuo in these cells. The overexpression of MnSOD enhanced the accumulation of 8-oxodGuo by UV. The co-overexpression of catalase inhibited the accumulation of 8-oxodGuo by UV in MnSOD-transfectants. The overexpression of MnSOD reduced the colony forming capacity in SK-HEP-1 cells and the co-overexpression of catalase with MnSOD stimulated the capacity compared to control. UV-irradiation inhibited the colony forming capacity in these cells; no difference was observed among the capacities of control, MnSOD- and catalase-transfectants. However, the overexpression of MnSOD/catalase significantly rescued the reduction of colony forming capacity by UV-irradiation. Our results suggest that the accumulation of hydrogen peroxide plays a key role in the oxidative damage to mtDNA of UV-irradiated cells, and also that the overexpression of both MnSOD and catalase reduces the mtDNA damage and blocks the growth inhibition by UV. Our results also indicate that the increased activity of MnSOD may lead to a toxic effect on mtDNA by UV-irradiation.     

UV-irradiation induces oxidative damage to mitochondrial DNA primarily through hydrogen peroxide: Analysis of 8-oxodGuo by HPLC

Roles of reactive oxygen species (ROS) in damage to mitochondrial DNA (mtDNA) following ultraviolet (UV)-irradiation were investigated in the human hepatoma cell line SK-HEP-1. We altered the intracellular status of ROS by the overexpression of manganese superoxide dismutase (MnSOD) and/or catalase. Using HPLC, we analyzed 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo), known as a marker of damage to DNA molecules. UV-irradiation resulted in the accumulation of 8-oxodGuo in these cells. The overexpression of MnSOD enhanced the accumulation of 8-oxodGuo by UV. The co-overexpression of catalase inhibited the accumulation of 8-oxodGuo by UV in MnSOD-transfectants. The overexpression of MnSOD reduced the colony forming capacity in SK-HEP-1 cells and the co-overexpression of catalase with MnSOD stimulated the capacity compared to control. UV-irradiation inhibited the colony forming capacity in these cells; no difference was observed among the capacities of control, MnSOD- and catalase-transfectants. However, the overexpression of MnSOD/catalase significantly rescued the reduction of colony forming capacity by UV-irradiation. Our results suggest that the accumulation of hydrogen peroxide plays a key role in the oxidative damage to mtDNA of UV-irradiated cells, and also that the overexpression of both MnSOD and catalase reduces the mtDNA damage and blocks the growth inhibition by UV. Our results also indicate that the increased activity of MnSOD may lead to a toxic effect on mtDNA by UV-irradiation.     

Inhibiting catalase activity sensitizes 36B10 rat glioma cells to oxidative stress

Gliomas are extremely resistant to anticancer therapies resulting in poor patient survival, due, in part, to altered expression of antioxidant enzymes. The primary antioxidant enzyme, catalase, is elevated constitutively in gliomas compared to normal astrocytes. We hypothesized that downregulating catalase in glioma cells would sensitize these cells to oxidative stress. To test this hypothesis, we implemented two approaches. The first, a pharmacological approach, used 3-amino-1,2,4-triazole, an irreversible inhibitor that reduced catalase enzymatic activity by 75%. Pharmacological inhibition of catalase was not associated with a reduction in rat 36B10 glioma cell viability until the cells were challenged with additional oxidative stress, i.e., ionizing radiation or hydrogen peroxide (H(2)O(2)). In the second molecular approach, we generated 36B10 glioma cells stably expressing catalase shRNA; a stable cell line displayed a 75% reduction in catalase immunoreactive protein and enzymatic activity. This was accompanied by an increase in intracellular reactive oxygen species and extracellular H(2)O(2). These cells exhibited increased sensitivity to radiation and H(2)O(2), which was rescued by the antioxidant, N-acetylcysteine. These results support the hypothesis that catalase is a major participant in the defense of 36B10 glioma cells against oxidative stress mediated by anticancer agents capable of increasing steady-state levels of H(2)O(2).     

Selenium supplementation sensitizes renca cells to tert-butylhydroperoxide induced loss of viability

Mouse renal carcinoma (renca) cells growing exponentially in foetal bovine serum (1%) supplemented with selenium (1 microM, sodium selenite) were exposed to oxidative insult. It was found that glutathione peroxidase activity increased (44%), while the activities of catalase, glutathione disulfide reductase, and level of total glutathione did not change due to selenium supplementation. Selenium supplementation made renca cells susceptible to tert-butylhydroperoxide induced cell death, while it did not affect the viability when the cells were exposed to hydrogen peroxide. It suggested that the contribution of glutathione peroxidase in antioxidant defense mechanism of renca cells was possibly not crucial and the function of catalase might be important especially against hydrogen peroxide.     

Role of reactive oxygen species in cells overexpressing manganese superoxide dismutase: mechanism for induction of radioresistance

Manganese superoxide dismutase (MnSOD) catalyzes the dismutation of superoxide anions (O(2)(-)) into hydrogen peroxide (H(2)O(2)). We altered the intracellular status of reactive oxygen species by introducing human MnSOD cDNA into the human ovarian cancer cell line SK-OV-3. The overexpression of MnSOD inhibited cell growth and induced a concomitant increase in the level of H(2)O(2) in SK-OV-3 cells. The cells overexpressing MnSOD were more resistant to irradiation than parental cells. MnSOD overexpression shortened the G(2)-M duration in irradiated cells. Either inhibition of p38 mitogen-activated protein kinase (p38MAPK) or scavenging free radicals blocked the induction of radioresistance by MnSOD and also abolished the shortening of the G(2)-M duration with concomitant inhibition of p38MAPK phosphorylation. Irradiation increased the generation of H(2)O(2) even more in these transfectants. These results suggest that the accumulated H(2)O(2) potentiated the activation of p38MAPK after irradiation in cells overexpressing MnSOD, which led to the protection of cells from irradiation-mediated cell death through the G(2)-M checkpoint. SK-OV-3 cells had no constitutive expression of p53, and the overexpression of MnSOD and/or irradiation did not induce p53 or p21(WAF1), which causes cell cycle arrest. Thus, our results suggest that MnSOD alters the cell cycle progression of irradiated cells independently of p53 and p21(WAF1).     

Role of heme oxygenase-1 in the regulation of manganese superoxide dismutase gene expression in oxidatively-challenged astroglia

Manganese superoxide dismutase (MnSOD) is an antioxidant enzyme that reduces superoxide anion to hydrogen peroxide in cell mitochondria. MnSOD is overexpressed in normal aging brain and in various central nervous system disorders; however, the mechanisms mediating the upregulation of MnSOD under these conditions remain poorly understood. We previously reported that cysteamine (CSH) and other pro-oxidants rapidly induce the heme oxygenase-1 (HO-1) gene in cultured rat astroglia followed by late upregulation of MnSOD in these cells. In the present study, we demonstrate that antecedent upregulation of HO-1 is necessary and sufficient for subsequent induction of the MnSOD gene in neonatal rat astroglia challenged with CSH or dopamine, and in astroglial cultures transiently transfected with full-length human HO-1 cDNA. Treatment with potent antioxidants attenuates MnSOD expression in HO-1-transfected astroglia, strongly suggesting that intracellular oxidative stress signals MnSOD gene induction in these cells. Activation of this HO-1-MnSOD axis may play an important role in the pathogenesis of Alzheimer disease, Parkinson disease and other free radical-related neurodegenerative disorders. In these conditions, compensatory upregulation of MnSOD may protect mitochondria from oxidative damage accruing from heme-derived free iron and carbon monoxide liberated by the activity of HO-1.     

Intracellular antioxidant enzymes are not globally upregulated during hibernation in the major oxidative tissues of the 13-lined ground squirrel Spermophilus tridecemlineatus

Hibernating mammals exhibit oxidative stress resistance in brain, liver and other tissues. In many animals, cellular oxidative stress resistance is associated with enhanced expression of intracellular antioxidant enzymes. Intracellular antioxidant capacity may be upregulated during hibernation to protect against oxidative damage associated with the ischemia-reperfusion that occurs during transitions between torpor and arousal. We tested the hypothesis that the 13-lined ground squirrel (Spermophilus tridecemlineatus), upregulates intracellular antioxidant enzymes in major oxidative tissues during hibernation. The two major intracellular isoforms of superoxide dismutase (MnSOD and CuZnSOD), which catalyze the first step in superoxide detoxification, were quantified in heart, brain and liver tissue using immunodetection and an in-gel activity assay. However, no differences in SOD protein expression or activity were found between active and hibernating squirrels. Measurements of glutathione peroxidase and glutathione reductase, which catalyze hydrogen peroxide removal, were not broadly upregulated during hibernation. The activity of catalase, which catalyzes an alternative hydrogen peroxide detoxification pathway, was higher in heart and brain of torpid squirrels, but lower in liver. Taken together, these data do not support the hypothesis that hibernation is associated with enhanced oxidative stress resistance due to an upregulation of intracellular antioxidant enzymes in the major oxidative tissues.     

A novel function of poly(ADP-ribose) polymerase-1 in modulation of autophagy and necrosis under oxidative stress

Under oxidative stress, poly(ADP-ribose) polymerase-1 (PARP-1) is activated and contributes to necrotic cell death through ATP depletion. On the other hand, oxidative stress is known to stimulate autophagy, and autophagy may act as either a cell death or cell survival mechanism. This study aims to explore the regulatory role of PARP-1 in oxidative stress-mediated autophagy and necrotic cell death. Here, we first show that hydrogen peroxide (H(2)O(2)) induces necrotic cell death in Bax-/- Bak-/- mouse embryonic fibroblasts through a mechanism involving PARP-1 activation and ATP depletion. Next, we provide evidence that autophagy is activated in cells exposed to H(2)O(2). More importantly, we identify a novel autophagy signaling mechanism linking PARP-1 to the serine/threonine protein kinase LKB1-AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) pathway, leading to stimulation of autophagy. Finally, we demonstrate that autophagy plays a cytoprotective role in H(2)O(2)-induced necrotic cell death, as suppression of autophagy by knockdown of autophagy-related gene ATG5 or ATG7 greatly sensitizes H(2)O(2)-induced cell death. Taken together, these findings demonstrate a novel function of PARP-1: promotion of autophagy through the LKB1-AMPK-mTOR pathway to enhance cell survival in cells under oxidative stress.     

Prevention of hydrogen peroxide-induced oxidative stress in HDF cells by peptides derived from seaweed pipefish, Syngnathus schlegeli

Two new peptides derived from seaweed pipefish Syngnathus schlegeli, SPP-1(QLGNLGV) and SPP-2 (SVMPVVA) were assessed for their ability to prevent hydrogen peroxide induced oxidative stress in human dermal fibroblasts (HDFs). Both peptides showed a significant hydroxyl radical scavenging activity when tested by ESR technique. And also the peptides effectively suppressed the hydrogen peroxide induced ROS production and DNA damage in HDF cells. Furthermore the two peptides increase the protein expression levels of intracellular antioxidant enzymes SOD1, GSH and catalase in hydrogen peroxide stressed HDF cells. At the cellular signaling level, SPPs block the NF-κB activation which may lead to the reduction of oxidative stress mediated damage of HDF cells. These finding indicate the potential antioxidant effects of SPPs as response to H₂O₂ stimulation.