Contribution of increased glutathione content to mechanisms of oxidative stress resistance in hydrogen peroxide resistant hamster fibroblasts

An H₂O₂-resistant variant (OC14) of the HA1 Chinese hamster fibroblast cell line, which demonstrates cross resistance to 95% O₂ and a 2-fold increase in total glutathione content, was utilized to investigate mechanisms responsible for cellular resistance to H₂O₂- and O₂-toxicity. OC14 and HA1 cells were pretreated with buthionine sulfoximine (BSO) to deplete total cellular glutathione. Following BSO pretreatment, cells were either placed in 250 μM BSO to maintain the glutathione depleted condition and challenged with 95% O₂, or challenged with hydroged peroxide in the absence of BSO. Total glutathione and the activities of CuZn superoxide dismutase, Mn superoxide dismutase, catalase, glutathione peroxidase, and glutathione transferase were evaluated immediately following the BSO pretreatment as well as following 39 to 42 hr of exposure to 250 μM BSO. BSO treatment did not cause significant decreases in any cellular antioxidant tested, except total glutathione depletion resulted in significant (P < 0.05) sensitization to O₂-toxicity and H₂O₂-toxicity in both cell lines at every time point tested. However, glutathione depletion did not completely abolish the resistance to either O₂- or H₂O₂-toxicity demonstrated by OC14 cells, relative to HA1 cells. Also, glutathione depletion did not effect the ability of OC14 cells to metabolize extracellular H₂O₂. These data indicate that glutathione dependent processes significantly contribute to cellular resistance to acute H₂O₂- and O₂-toxicity, but are not the only determinants of resistance in cell lines. The contribition of aldehydes formed by lipid peroxidation in mechanisms involved with the sensitization to O₂-toxicity in glutathione depleted cells was tested by measuring the lipid peroxidation byproduct, 4-hydroxy-2-nonenal (4HNE), bound in Schiff-base linkages or in its free form in cell homogenates at 49 hr of 95% O₂-exposure. No significant increase in 4HNE was detected in glutathione depleted cells relative to glutathione competent cells, indicating that glutathione depletion does not sensitize these cells to O₂-toxicity by altering the intracellular accumulation of free or Schiff-base bound 4HNE. © 1995 Wiley-Liss Inc.     

Different protective mechanisms of human embryonic and endometrium-derived mesenchymal stem cells under oxidative stress

Oxidative stress has been shown to cause either apoptosis or stress-induced premature senescence (SIPS) in different cell types. At present, it is generally accepted that stem cells have high resistance to oxidative stress; however, data reported by various authors are disputed. In this study, we investigated stress responses of human embryonic stem cells (hESC) and human mesenchymal stem cells (hMESC) derived from desquamated endometrium to hydrogen peroxide (H₂O₂). Cell viability was evaluated by MTT assay. LD₅₀ were determined as 300–350, 370–400, and 600–700 μM for hESC, human embryonic fibroblasts, and hMESC, respectively. Thus, of the studied cell lines, hMESC exhibited the greatest resistance to increased H₂O₂ concentration. We found for the first time that a sublethal concentration of H₂O₂ induced premature senescence phenotype in hMESC, like in HEF, that was characterized by increased expression of cyclin-dependent kinase inhibitor p21^Waf1/Cip1, an irreversible cell cycle arrest, the permanent loss of proliferative potential, cell hypertrophy, and the SA-β-Gal staining. Whereas the sublethal H₂O₂ concentration (200 μM) promoted in hMESC only SIPS, higher H₂O₂ concentrations also induced apoptosis in a small part of the cell population. On the contrary, in hESC, H₂O₂, regardless of the tested concentrations (from 50 to 500 μM), triggered apoptosis, which was the only pronounced response of these cells to oxidative damage. The obtained data demonstrate that stem cells of different origins under conditions of oxidative stress use different protective mechanisms: hESC rapidly eliminate damaged cells through apoptosis, whereas hMESC are subjected to premature senescence.     

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.     

Catalase activity and hydrogen peroxide levels are inversely correlated in maize scutella during seed germination

Abstract

Temporal patterns of hydrogen peroxide (H₂O₂) levels and total catalase activity are presented for post-imbibition scutella from six maize inbred lines expressing variable catalase activity. In all lines examined, H₂O₂ levels were highest during the initial days post-imbibition (1–2 dpi) and decreased thereafter, while total catalase activity was lowest during early dpi (1–2 dpi) and reached maximal activity at 4–6 dpi. In three of the six lines tested, a simple inverse correlation between catalase activity and H₂O₂ level was significant by Spearman's rank (P <0.01). In addition to the generaldecline in H₂O₂level throughout the dpi period, a reproducible increase in H₂O₂ level was observed at 4–5 dpi in five of six lines examined. Mutant lines lacking CAT-3 activity demonstrated a temporal shift in the occurrence of this increase. The role of total catalase (and individual isozymes) in controlling H₂O₂ levels during germination and the role of H₂O₂ as a potential regulator of catalase expression during germination are discussed.     

Effects of Different Sources and Levels of Zinc on H2O2-Induced Apoptosis in IEC-6 Cells

Zinc has been shown to be an inhibitor of apoptosis for many years. The present study was designed to investigate effects of three zinc chemical forms on H₂O₂-induced cell apoptosis in IEC-6 cells via analysis of cell vitality, LDH activity, apoptosis percentage, caspase-3 activity, and Bcl-2, Bax, and caspase-3, -8, and -9 gene expression. Cells were divided into H₂O₂ and zinc sources+H₂O₂ groups, and there are three different zinc sources [zinc oxide nanoparticle (nano-ZnO), zinc oxide (ZnO), and zinc sulfate (ZnSO₄)] and three concentrations (normal = 25 μM, medium = 50 μM, and high = 100 μM) used in this article. In the present study, we found the striking cytotoxicity of H₂O₂ higher than 200 μM on cell vitality, LDH activity, and apoptosis percentage in the cells using five different concentrations (50, 100, 200, 400, and 800 μM) of H₂O₂ for 4 h. Moreover, we observed that cell vitality was increased, LDH activity and apoptotic percentage were decreased, and gene expression level of Bax and caspase-3 and -9 was markedly reduced, while gene expression level of Bcl-2 and ratio of Bcl-2/Bax were increased in normal concentration groups of nano-ZnO and ZnSO₄ compared with H₂O₂ group, but no significant difference was observed in caspase-8 gene expression. Furthermore, medium or, more intensely, high concentrations of nano-ZnO and ZnSO₄ enhanced H₂O₂-induced cell apoptosis. Compared with nano-ZnO and ZnSO₄, ZnO showed weakest protective effect on H₂O₂-induced apoptosis at normal concentration and was less toxic to cells at high level. Taken together, we proposed that preventive and protective effects of zinc on H₂O₂-induced cell apoptosis varied in IEC-6 cells with its chemical forms and concentrations, and maybe for the first time, we suggested that nano-ZnO have a protective effect on H₂O₂-induced cell apoptosis in IEC-6 cells.     

Dependence of leukemic cell proliferation and survival on H2O2 and l-arginine

The proliferation and/or survival of a variety of cells is dependent on cellular hydrogen peroxide (H₂O₂) production. We tested whether this was true of leukemic cells, using cell lines from leukemic patients (CEM, 697, Mn-60, and Tanoue). We found that addition of catalase inhibited proliferation of all cell lines and induced death in two. However, this turned out to be due to arginase contamination of the catalase. Pure arginase inhibited cell proliferation and survival, which was reversible by adding l-arginine, demonstrating the l-arginine dependency of these cells. The glutathione peroxidase mimetic ebselen killed the cells by a novel, rapid form of death, preceded by cell blebbing and prevented by N-acetylcysteine, suggesting toxicity is not due to ebselen's antioxidant activity. Addition of N-acetylcysteine to remove endogenous H₂O₂ stimulated survival and proliferation, suggesting that basal levels of H₂O₂ promoted cell death. Consistent with this, leukemic cell death was induced by adding as little as 5 μM H₂O₂. Ascorbic acid, even at 100 μM, induced death through H₂O₂ production. Thus H₂O₂ does not promote proliferation and survival, rather the opposite, and previous literature may have misinterpreted the effects of antioxidants. Arginase, H₂O₂, ascorbic acid, and ebselen might be useful in the treatment of leukemia.     

Application of confocal laser scanning microscopy to detect oxidative stress in human colon cells

Introduction Excess of intracellular reactive oxygen species in relation to antioxidative systems results in an oxidative environment which may modulate gene expression or damage cellular molecules. These events are expected to greatly contribute to processes of carcinogenesis. Only few studies are available on the oxidative/reductive conditions in the colon, an important tumour target tissue. It was the objective of this work to further develop methods to assess intracellular oxidative stress within human colon cells as a tool to study such associations in nutritional toxicology.

Methods We have measured H₂O₂-induced oxidative stress in different colon cell lines, in freshly isolated human colon crypts, and, for comparative purposes, in NIH3T3 mouse embryo fibroblasts. Detection was performed by loading the cells with the fluorigenic peroxide-sensitive dye 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (diacetoxymethyl ester), followed by in vitro treatment with H₂O₂ and fluorescence detection with confocal laser scanning microscopy (CLSM). Using the microgel electrophoresis (“Comet”) Assay, we also examined HT29 stem and clone 19A cells and freshly isolated primary colon cells for their relative sensitivity toward H₂O₂-induced DNA damage and for steady-state levels of endogenous oxidative DNA damage.

Results A dose-response relationship was found for the H₂O₂-induced dye decomposition in NIH3T3 cells (7.8–125 μM H₂O₂) whereas no effect occurred in the human colon tumour cell lines HT29 stem and HT29 clone 19A (62–1000 μM H₂O₂). Fluorescence was significantly increased at 62 μM H₂O₂ in the human colon adenocarcinoma cell line Caco-2. In isolated human colon crypts, the lower crypt cells (targets of colon cancer) were more sensitive towards H₂O₂ than the more differentiated upper crypt cells. In contrast to the CLSM results, oxidative DNA damage was detected in both cell lines using the Comet Assay. Endogenous oxidative DNA damage was highest in HT29 clone 19A, followed by the primary colon cells and HT29 stem cells.

Conclusions Oxidative stress in colon cells leads to damage of macromolecules which is sensitively detected in the Comet Assay. The lacking response of the CLSM-approach in colon tumour cells is probably due to intrinsic modes of protective activities of these cells. In general, however, the CLSM method is a sensitive technique to detect very low concentrations of H₂O₂-induced oxidative stress in NIH3T3 cells. Moreover, by using colon crypts it provides the unique possibility of assessing cell specific levels of oxidative stress in explanted human tissues. Our results demonstrate that the actual target cells of colon cancer induction are indeed susceptible to the oxidative activity of H₂O₂.     

Oxidative stress induces alkaloid production in Uncaria tomentosa root and cell cultures in bioreactors

The effect of oxidative stress on indole alkaloids accumulation by cell suspensions and root cultures of Uncaria tomentosa in bioreactors was investigated. Hydrogen peroxide (H₂O₂, 200 μM) added to U. tomentosa cell suspension cultures in shaken flasks induced the production of monoterpenoid oxindole alkaloids (MOA) up to 40.0 μg/L. In a stirred tank bioreactor, MOA were enhanced by exogenous H₂O₂ (200 μM) from no detection up to 59.3 μg/L. Root cultures grew linearly in shaken flasks with a μ=0.045 days^?1 and maximum biomass of 12.08±1.24 g DW/L (at day 30). Roots accumulated 3α‐dihydrocadambine (DHC) 2354.3±244.8 μg/g DW (at day 40) and MOA 348.2±32.1 μg/g DW (at day 18). Exogenous addition of H₂O₂ had a differential effect on DHC and MOA production in shaken flasks. At 200 μM H₂O₂, MOA were enhanced by 56% and DHC by 30%; while addition of 800 and 1000 μM H₂O₂, reduced by 30–40% DHC accumulation without change in MOA. Root cultures in the airlift reactor produced extracellular H₂O₂ with a characteristic biphasic profile after changing aeration. Maximum MOA was 9.06 mg/L at day 60 while at this time roots reached ca. 1 mg/L of DHC. Intracellular H₂O₂ in root cultures growing in the bioreactor was 0.87 μmol/g DW compared to 0.26 μmol/g DW of shaken flasks cultures. These results were in agreement with a higher activity of the antioxidant enzymes superoxide dismutase and peroxidase by 6‐ and 2‐times, respectively. U. tomentosa roots growing in the airlift bioreactor were exposed to an oxidative stress and their antioxidant system was active allowing them to produce oxindole alkaloids.     

Interactions of nitric oxide and oxygen in cytotoxicity: Proliferation and antioxidant enzyme activities of endothelial cells in culture

Nitric oxide (NO) shows cytotoxicity, and its reaction products with reactive oxygen species, such as peroxynitrite, are potentially more toxic. To examine the role of O₂ in the NO toxicity, we have examined the proliferation of cultured human umbilical vein endothelial cells in the presence or absence of NO donor, ((Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)-amino]diazen-1-ium-1,2-diolate) (DETA-NONOate) (100–500 μM), under normoxia (air), hypoxia (< 0.04% O₂) or hyperoxia (88–94% O₂). It was found that the dose dependency on NONOate was little affected by the ambient O₂ concentration, showing no apparent synergism between the two treatments. We have also examined the effects of exogenous NO under normoxia and hyperoxia on the cellular activities of antioxidant enzymes involved in the H₂O₂ elimination, since many of them are known to be inhibited by NO or peroxynitrite in vitro. Under normoxia DETA-NONOate (500 μM) caused 25% decrease in catalase activity and 30% increases in glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities in 24 h. Under hyperoxia NO caused about 25% decreases in activities of catalase, glutathione reductase and glucose-6-phosphate dehydrogenase. The H₂O₂ removal rate by NO-treated cells was computed on the mathematical model for the enzyme system. It was concluded that the cellular antioxidant function is little affected by NO under normoxia but that it is partially impaired when the cells are exposed to NO under hyperoxia.     

Hyaluronic acid optimises therapeutic effects of hydrogen peroxide‐induced oxidative stress on breast cancer

Distinguishing the multiple effects of reactive oxygen species (ROS) on cancer cells is important to understand their role in tumour biology. On one side, ROS can be oncogenic by promoting hypoxic conditions, genomic instability and tumorigenesis. Conversely, elevated levels of ROS‐induced oxidative stress can induce cancer cell death. This is evidenced by the conflicting results of research using antioxidant therapy, which in some cases promoted tumour growth and metastasis. However, some antioxidative or ROS‐mediated oxidative therapies have also yielded beneficial effects. To better define the effects of oxidative stress, in vitro experiments were conducted on 4T1 and splenic mononuclear cells (MNCs) under hypoxic and normoxic conditions. Furthermore, hydrogen peroxide (H₂O₂; 10–1,000 μM) was used as an ROS source alone or in combination with hyaluronic acid (HA), which is frequently used as drug delivery vehicle. Our result indicated that the treatment of cancer cells with H₂O₂ + HA was significantly more effective than H₂O₂ alone. In addition, treatment with H₂O₂ + HA led to increased apoptosis, decreased proliferation, and multiphase cell cycle arrest in 4T1 cells in a dose‐dependent manner under normoxic or hypoxic conditions. As a result, migratory tendency and the messenger RNA levels of vascular endothelial growth factor, matrix metalloproteinase‐2 (MMP‐2), and MMP‐9 were significantly decreased in 4T1 cells. Of note, HA treatment combined with 100–1,000 μM H₂O₂ caused more damage to MNCs as compared to treatment with lower concentrations (10–50 μM). Based on these results, we propose to administer high‐dose H₂O₂ + HA (100–1000 μM) for intratumoural injection and low doses for systemic administration. Intratumoural route could have toxic and inhibitory effects not only on the tumour but also on residential myeloid cells defending it, whereas systemic treatment could stimulate peripheral immune responses against the tumour. More in vivo research is required to confirm this hypothesis.     

Hydrogen peroxide modulates expression of surface antigens by human umbilical vein endothelial cells in vitro

The aim of this study was to determine the immunological phenotype of human umbilical vein endothelial cells in culture and dynamics of expression of inducible CD molecules of the endothelial cell surface exposed to hydrogen peroxide (H₂O₂, 100–300 μM) in vitro. PMA was applied as a positive control. There was an insignificant increase (9–21%) of CD31 expression 3 h after the addition of H₂O₂ to the culture medium, statistically significant decrease of CD309 expression upon exposure to 200 and 300 μM H₂O₂, whereas a day after the addition of H₂O₂ we found a strongly pronounced dose-dependent increase of CD54 expression, as well as a moderate enhancement of the expression of all CD molecules.     

NADPH oxidase is involved in H2O2-induced differentiation of human promyelocytic leukaemia HL-60 cells

The expression and activity of NADPH oxidase increase when HL‐60 cells are induced into terminally differentiated cells. However, the function of NADPH oxidase in differentiation is not well elucidated. With 150–500 μM H₂O₂ inducing differentiation of HL‐60 cells, we measured phagocytosis of latex beads and investigated cell electrophoresis. Two inhibitors of NADPH oxidase, DPI (diphenyleneiodonium) and APO (apocynin), blocked the differentiation potential of cells induced by 200 μM H₂O₂. However, H₂O₂ stimulated the generation of intracellular superoxide (O₂ ^{? ?}), which decreased in the presence of the two inhibitors. DPI also inhibited H₂O₂‐induced ERK (extracellular‐signal‐regulated kinase) activation, as detected by Western blotting. Furthermore, PD98059, the inhibitor of the ERK pathway, inhibited the differentiation of HL‐60 cells induced by H₂O₂. This shows that H₂O₂ can activate NADPH oxidase, leading to O₂ ^{? ?} production, followed by ERK activation and ultimately resulting in the differentiation of HL‐60 cells. The data indicate that NADPH oxidase is an important cell signal regulating cell differentiation.     

Vascular pro-oxidant effects secondary to the autoxidation of gallic acid in rat aorta

Gallic acid autoxidation was monitored by absorption spectroscopy and H₂O₂ production; vascular effects related to the autoxidation process were studied on intact and rubbed aortic rings from WKY rats. Gallic acid autoxidation in an oxygenated physiological salt solution (37°C, pH=7.4) mostly occurred in a 2-h time period. Superoxide anions, H₂O₂ and gallic acid quinones were produced during gallic acid autoxidation. In rings partially precontracted with phenylephrine, 0.1–3 μM gallic acid induced marked and largely endothelium-dependent contractions, 10–30 μM gallic acid induced endothelium-independent contractions and 0.1–0.3 mM gallic acid induced complete, fast-developing, endothelium-independent relaxations. Superoxide dismutase (SOD) shifted the endothelium-dependent gallic acid contractions to the right, and N^G-nitro-l-arginine abolished them. Indomethacin suppressed the endothelium-independent gallic acid contractions, and catalase abolished the endothelium-independent contractions and relaxations. Gallic acid (30 μM) inhibited the relaxant effects of acetylcholine and sodium nitroprusside. In rings maximally precontracted with KCl, 0.1–100 μM gallic acid did not modify the tone, whereas 0.3 mM induced complete, slow-developing, endothelium-independent relaxations. Moreover, 0.3 mM gallic acid induced an irreversible impairment of ring reactivity and the release of lactate dehydrogenase. Catalase and N-acetyl cysteine suppressed the deleterious effects induced by gallic acid in the rings. In conclusion: (a) gallic acid is rapidly and nonenzymatically oxidized in physiological solutions, generating superoxide anions, H₂O₂ and quinones; (b) superoxide anions (by destroying NO) and low H₂O₂ levels (by activating cyclooxygenase) both increase vascular tone; (c) moderate H₂O₂ levels decrease vascular tone; (d) high H₂O₂ and quinone levels cause irreversible relaxations due to cellular damage.     

Oxygen Uptake and Antioxidant Responses of the Free-Living Diplomonad Hexamita sp.

ABSTRACT. The free-living anaerobic flagellate Hexamita sp. was observed to actively consume O₂ with a K_m O₂ of 13 μM. Oxygen consumption increased lineraly with O₂ tension up to a threshold level of 100 μM, above which it was inhibited. Oxygen uptake was supported by a number of substrates but probably not coupled to energy conservation as cytochromes could not be detected spectro-photometrically. In addition, inhibitors specific for respiratory chain components did not significantly affect O₂ uptake. Respiration was however, partially inhibited by flavoprotein and iron-sulfur protein inhibitors. NAD(P)H supported O₂ consumption was measured in both particulate and soluble fractions; this activity was partially inhibited by quinacrine. A chemosensory response was observed in cells exposed to air, however no response was observed in the presence of superoxide dismutase plus catalase. Catalase and nonspecific peroxidase activity could not be detected, but superoxide dismutase activity was present. Superoxide dismutase was sensitive to NaN₃ and H₂O₂ but not KCN, suggesting a Fe prosthetic group. Flow cytometric analysis revealed that thiol levels in live cells were depleted in the presence of t-butyl H₂O₂. The observed NADPH-driven glutathione reductase activity is believed to recycle oxidized thiols in order to re-establish reduced thiol levels in the cell. The corresponding thiol cycling enzyme glutathione peroxidase could not be detected. The ability to withstand high O₂ tensions (100 μM) would enable Hexamita to spend short periods in a wider range of habitats. Prologed exposure to O₂ tensions higher than 100 μM leads to irreversible damage and cell death.     

Response of Lens Epithelial Cells to Hydrogen Peroxide Stress and the Protective Effect of Caloric Restriction

Hydrogen peroxide (H₂O₂) has been reported to be present at significant levels in the lens and aqueous humor in some cataract patients and suggested as a possible source of chronically inflicted damage to lens epithelial (LE) cells. We measured H₂O₂effects on bovine and mouse LE cells and determined whether LE cells from old calorically restricted mice were more resistant to H₂O₂-induced cellular damage than those of same age ad libitum fed (AL) mice. Bovine lens epithelial cells were exposed to H₂O₂at 40 or 400 μM for 2 h and then allowed to recover from the stress. The cells were assayed for DNA damage, DNA synthesis, cell viability, cell morphology, response to growth stimuli, and proliferation potential. Hydrogen peroxide-treated cells showed an increased DNA unwinding 50% greater than that for untreated controls. These DNA strand breaks appeared to be almost completely rejoined by 30 min following removal of the cells from a 2-h exposure. The 40 μM exposure did not produce a significantly lower DNA synthesis rate than the control, it responded to growth factor stimuli, and it replicated as did the control cells after removal of H₂O₂. The 400 μM H₂O₂severely affected DNA synthesis and replication, as shown by increased cell size and by markedly reduced clonal cell growth. The cells did not respond to growth stimulation by serum or growth factors and lost irreversibly the capacity to proliferate. The responses of LE cells from old adlib diet (AL) and calorically restricted (CR) mice to H₂O₂were significantly different. Exposure of LE cells to 20, 40, or 100 μM H₂O₂for 1 h induces a significant loss of cellular proliferation in cells from old AL mice. LE cells from long-term CR mice of the same strain and age were more resistant to oxidative damage at all three concentrations of H₂O₂than those of both old and young AL mice and showed a significantly higher proliferation potential following treatment. It is concluded that CR results in superior resistance to reactive oxygen radicals in the lens epithelium.     

Salivary hydrogen peroxide produced by holding or chewing green tea in the oral cavity

Tea (Camellia sinensis) catechins have been studied for disease prevention. These compounds undergo oxidation and produce H₂O₂. We have previously shown that holding tea solution or chewing tea leaves generates high salivary catechin levels. Herein, we examined the generation of H₂O₂ in the oral cavity by green tea solution or leaves. Human volunteers holding green tea solution (0.1–0.6%) developed salivary H₂O₂ with C_max = 2.9–9.6 μM and AUC_{0 → ∞} = 8.5–285.3 μM min. Chewing 2 g green tea leaves produced higher levels of H₂O₂ (C_max = 31.2 μM, AUC_{0 → ∞} = 1290.9 μM min). Salivary H₂O₂ correlated with catechin levels and with predicted levels of H₂O₂ (C_{max(expected)} = 36 μM vs C_{max(determined)} = 31.2 μM). Salivary H₂O₂ and catechin concentrations were similar to those that are biologically active in vitro. Catechin-generated H₂O₂ may, therefore, have a role in disease prevention by green tea.     

Light-dependent reduction of hydrogen peroxide by intact spinach chloroplasts

Intact spinach chloroplasts, washed four times in buffered sorbitol to decrease catalase contamination, supported O₂ evolution in the dark at very low rates (less than 2 μmol/mg Chl per h) in the presence of low concentrations of H₂O₂ (0.25 mM); H₂O₂ was not significantly metabolished under these conditions. In the light, washed chloroplasts supported H₂O₂-dependent O₂ evolution at rates of 28–46 μmol/mg Chl per h in the presence of 0.1–0.25 mM H₂O₂; the concentration of H₂O₂ supporting 0.5V_max was estimated to be 25 μM. O₂ evolution in the light was associated with H₂O₂ consumption and ceased after the production of 0.45 mol per mol H₂O₂ consumed. Both O₂ evolution and H₂O₂ consumption were abolished by 5 μM 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Washed intact chloroplasts contained endogenous pools of GSH and ascorbate estimated at 10 and 33 mM, respectively. H₂O₂-dependent O₂ evolution in the light was associated with a decrease in these levels which increased as O₂ evolution gradually ceased. The results are consistent with the hypothesis that H₂O serves as eventual electron donor for the reduction of H₂O₂ in illuminated chloroplasts and that GSH/GSSG and ascorbate/dehydroascorbate serve as intermediate electron carriers. Preincubation of chloroplasts in the dark with 0.1 mM H₂O₂ abolished O₂ evolution in the light.     

Torulene and torularhodin,protects human prostate stromal cells from hydrogen peroxide-induced oxidative stress damage through the regulation of Bcl-2/Bax mediated apoptosis

The current study was designed to elucidate the cytoprotective effects and possible mechanisms of torulene and torularhodin on hydrogen peroxide (H₂O₂)-induced oxidative stress damage in human prostate stromal cells (WPMY-1). After treated with H₂O₂, a notable decrease was appeared in cell viability, yet the decrease was attenuated when cells were pretreated with torulene and torularhodin (0.5–10?μM) as evaluated by WST-1 assay. Pretreatment with these two carotenoids significantly attenuated H₂O₂-induced apoptosis in WPMY-1 cells through the inhibition of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) overproduction, as well as the activation of the activities in catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px). Finally, pretreatment of cells with carotenoids resulted in the regulation of the mRNA and protein expression of Bcl-2 and Bax in H₂O₂-exposed prostate stromal cells. The present results indicate that both torulene and torularhodin can protect human prostate stromal cells from oxidative stress damage via Bcl-2/Bax mediated apoptosis.     

Programmed cell death is induced by hydrogen peroxide but not by excessive ionic stress of sodium chloride in the unicellular green alga Chlamydomonas reinhardtii

Eukaryotic microalgae serve as indicators of environmental change when exposed to severe seasonal fluctuations. Several environmental stress conditions are known to produce reactive oxygen species in cellular compartments, resulting in oxidative damage and apoptosis. The study of cell death in higher plants and animals has revealed the existence of an active ‘programmed cell death’ (PCD) process and similarities between such processes suggest an evolutionary origin. A study was undertaken to examine the morphological, biochemical and molecular responses of the unicellular green alga Chlamydomonas reinhardtii after exposure to oxidative (10 mM H₂O₂) and osmotic (200 mM NaCl and 360 mM sorbitol) stress. Concentrations of H₂O₂ (2–50 mM), NaCl and sorbitol (100–800 mM) were negatively correlated with growth. Biochemical analyses showed an increase in intracellular H₂O₂ production (2.2-fold with H₂O₂ and ~1.2–1.4-fold with NaCl and sorbitol) and activities of some antioxidant enzymes [super oxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX)]. Alteration of mitochondrial membrane potential (MMP) was observed upon treatment with H₂O₂ and NaCl, but not with sorbitol, indicating that the ionic stress component of NaCl altered the MMP. In addition, H₂O₂ led to the activation of a caspase-3-like protein, increase in the cleavage of a poly(ADP) ribose polymerase-1 (PARP-1)-like enzyme and formation of DNA nicks and laddering. With NaCl and sorbitol, no caspase activation, nor oligonucleosomal DNA laddering was observed, indicating non-apoptotic death. However, genomic DNA of NaCl (800 mM)-stressed cells, but not those of sorbitol-treated cells showed complete shearing. We conclude that the ionic rather than the osmotic component of NaCl leads to necrosis. These results unequivocally suggest that the vegetative cells of C. reinhardtii respond differentially to various stress agents, leading to different death types in the same organism. Moreover, unlike most other organisms, when exposed to NaCl this alga does not undergo PCD.