microM H2O2 Research Articles

The Response of Cockerel Semen Kinematic Parameters LIN, STR, WOB, ALH and BCF to Induced Oxidative Stress

Computer-assisted sperm class analyser (CASA) analysis of avian semen following cryopreservation indicates that their semen motility and viability parameters become compromised, due in part to oxidative stress. To mimic these observations we have treated cockerel semen with an oxidative stress inducing agent, namely hydrogen peroxide (H2O2) and monitored the motility, kinematic and viability parameters over time. Briefly, five healthy and fertile South African Venda cockerels were selected and their semen was collected using the abdominal massage technique. The semen was then treated with H2O2 at 0 &microM, 5 &microM, 50 &microM and 200 &microM concentrations for 0, 3, 16 and 24 hrs. The semen motility, kinematic and viability parameters were then determined using the CASA system while the viability was determined using the SYBR-14/PI staining. The Pearson’s correlation coefficient was determined to test the relationships between the levels of induced oxidative stress, period of exposure to oxidative stress inducing agent and the motility plus kinematic parameters. Our data revealed that in raw cockerel semen, there was high and positive correlations between total motility (TM), progressive motility (PM), rapid (RAP), curvilinear velocity (VCL), straight line velocity (VSL) and average path velocity (VAP) while the kinematic parameters LIN, STR, WOB, ALH and BCF had low or negative correlations with them. Furthermore, TM, PM, RAP, VCL and VSL remained highly and positively correlated with the induced oxidative stress and also, linearity (LIN), straightness (STR), wobble (WOB), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF) remained negatively correlated with the induced oxidative stress, after 3 hrs. After 24 hrs, TM, PM, RAP, VCL, VSL, VAP and ALH, became negatively correlated with the induced oxidative stress while LIN, STR, WOB and BCF became positively correlated with the induced oxidative stress. Conversely, when the H2O2 concentration used was correlated with motility and kinematic parameters over time, TM, PM, RAP, VCL, VSL, VAP became negatively correlated with oxidative stress while LIN, STR, WOB, ALH and BCF show negative or low correlations with the induced oxidative stress. This data indicates that LIN, STR, WOB, BCF and to some extend ALH, reveal the least correlations with the induced oxidative stress under persistent oxidative stress conditions in cockerel semen. In conclusion, cockerel semen, like buck semen, does not easily succumb to oxidative stress since the raw semen correlations of CASA analysed parameters are comparable to these observed after 3 hrs of H2O2 treatment. In addition, the oxidative stress levels tolerated by cockerel semen should not 5 &microM H2O2 oxidative stress levels. Lastly, lack of correlation between LIN, STR, WOB, BCF and ALH and induced oxidative stress can be used in cockerel semen to show intolerable cryopreservation conditions.

Sa1846 Metallothionein, an Emerging Danger Signal During Experimental Colitis

BACKGROUND: Danger signals have been postulated as regulators of gut mucosal immunity. During intestinal inflammation, the epithelium is compromised and signals, alerting adjacent cells of tissue damage, are released. Hence, we were interested in metallothioneins (MTs), small proteins which have been identified at inflammation sites.We explored triggers releasing MTs from colon epithelial cells and identified their role as extracellular danger signal during experimental colitis. METHODS: HT29 cells were subjected to the following treatments: 200ng/ml LPS, 200 microM H2O2, hypoxia (~1% oxygen), 100ng/ml TNF-alpha + 300ng/ ml IFN-gamma to induce apoptosis, and repeated freeze/thaw cycles to mimic necrosis. Supernatant was analysed for MT levels using western blot and for lactate dehydrogenase activity (LDH). A Boyden transwell migration assay with blood leukocytes was applied to evaluate the chemotactic potential of extracellular MT and the capacity of monoclonal antiMT antibody (100μg/ml UC1MT) to abolish this. The role of MT as chemo-attractant was further explored using dextran sulphate sodium (DSS)-induced colitis in MT knockout (MT-/-), transgenic (MT+/+) and wild type mice (WT). The therapeutical use of monoclonal therapy was tested in DSSand 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis. Inflammatory cell infiltrate was evaluated in all experiments together with standard inflammation markers. RESULTS: Necrosis and TNF-induced apoptosis resulted in detectable MT levels in supernatant of HT29 cells, which was not the case for LPS, H2O2 nor hypoxia treatment. LDH activity was not increased after stimulation with TNF, ruling out an uncontrolled release of MT from TNF-treated cells. Increased leukocyte migration towards this MT-containing supernatant was detected, whereas the addition of UC1MT was able to overcome this chemo-attraction (p,0.05). Significantly less neutrophil infiltration was observed in MT-/mice compared to MT+/+ and WT mice in DSS colitis (p,0.05). Complementary, i.p. UC1MT treatment reduced the number of F4/80-positive macrophages in DSSand TNBS-induced colitis (p,0.05). Less inflammatory infiltrate was associated with reduced histological inflammation in all three colitis experiments. CONCLUSION: We characterized metallothionein as danger signal released from HT29 cells after necrotic and TNF-induced apoptotic cell death. Inhibiting MT function by monoclonal therapy reduces leukocyte infiltration and represents a novel therapy dampening experimental colitis.

P077 Emerging role of the interleukin (IL)-33/ST2 axis in gut mucosal wound healing and epithelial repair

BACKGROUND: Danger signals have been postulated as regulators of gut mucosal immunity. During intestinal inflammation, the epithelium is compromised and signals, alerting adjacent cells of tissue damage, are released. Hence, we were interested in metallothioneins (MTs), small proteins which have been identified at inflammation sites.We explored triggers releasing MTs from colon epithelial cells and identified their role as extracellular danger signal during experimental colitis. METHODS: HT29 cells were subjected to the following treatments: 200ng/ml LPS, 200 microM H2O2, hypoxia (~1% oxygen), 100ng/ml TNF-alpha + 300ng/ ml IFN-gamma to induce apoptosis, and repeated freeze/thaw cycles to mimic necrosis. Supernatant was analysed for MT levels using western blot and for lactate dehydrogenase activity (LDH). A Boyden transwell migration assay with blood leukocytes was applied to evaluate the chemotactic potential of extracellular MT and the capacity of monoclonal antiMT antibody (100μg/ml UC1MT) to abolish this. The role of MT as chemo-attractant was further explored using dextran sulphate sodium (DSS)-induced colitis in MT knockout (MT-/-), transgenic (MT+/+) and wild type mice (WT). The therapeutical use of monoclonal therapy was tested in DSSand 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis. Inflammatory cell infiltrate was evaluated in all experiments together with standard inflammation markers. RESULTS: Necrosis and TNF-induced apoptosis resulted in detectable MT levels in supernatant of HT29 cells, which was not the case for LPS, H2O2 nor hypoxia treatment. LDH activity was not increased after stimulation with TNF, ruling out an uncontrolled release of MT from TNF-treated cells. Increased leukocyte migration towards this MT-containing supernatant was detected, whereas the addition of UC1MT was able to overcome this chemo-attraction (p,0.05). Significantly less neutrophil infiltration was observed in MT-/mice compared to MT+/+ and WT mice in DSS colitis (p,0.05). Complementary, i.p. UC1MT treatment reduced the number of F4/80-positive macrophages in DSSand TNBS-induced colitis (p,0.05). Less inflammatory infiltrate was associated with reduced histological inflammation in all three colitis experiments. CONCLUSION: We characterized metallothionein as danger signal released from HT29 cells after necrotic and TNF-induced apoptotic cell death. Inhibiting MT function by monoclonal therapy reduces leukocyte infiltration and represents a novel therapy dampening experimental colitis.

Effects of chromosomal polyploidy on survival of colon cancer cells

Tetraploid cells are frequently observed in the inflamed mucosal epithelial cells of the patients with Barrett's esophagus or chronic ulcerative colitis. Polyploidy often occurs during cell fusion, abortive cell cycle, and endoreplication. Most tetraploid cells are engaged to apoptotic pathway, but some remaining stable tetraploid cells consequently cause aneuploidization and chromosomal instability. We investigated whether tetraploid cells could acquire survival advantage and hold a dominant position for natural selection. We established tetraploid cell line (HCT116GH) from parental diploid colorectal cancer cell line (HCT116) via PEG-mediated cell fusion and compared its cell viability, cell cycle response and apoptotic fractions responded to H₂O₂) with diploid HCT116 and p53 suppressed HCT116/H6 cell lines. Using MTT assay, plating efficiency and clonogenicity, we evaluated the survival of each cell line. Tetraploid cell line HCT116GH demonstrated an 83 fold greater resistance to 100 microM H₂O₂ than the parental diploid HCT116, and 6 fold greater than even the p53 negative diploid HCT116/E6. Cellular sensitivity, G2/M arrests, and apoptotic proportion were observed less in response to H₂O₂ in HCT116GH compared with HCT116 and HCT116/E6. HCT116GH expressed lower level of p53 and p21 than diploid HCT116. Stable tetraploid cell lines showed enhanced viability in comparison to parental diploid cell lines. The enhanced viability observed in tetraploidization surpassed that from downregulation of p53. Frequent appearance of tetraploid cells in stressful condition can be caused by natural selection owing to their enhanced viability and may consequently contribute to cancer cell transformation.

Overexpression of Apg-2 increases cell proliferation and protects from oxidative damage in BaF3-BCR/ABL cells

Apg-2, a mammalian heat-shock protein belonging to the heat-shock protein 110 (Hsp110) family, was previously found to be overexpressed in BaF3-BCR/ABL cells that were treated with hydrogen peroxide (H2O2) through our comparative proteomics study. The expression of Apg-2 in chronic myelogenous leukemia (CML) cells and its role have not been investigated, forming the basis for this study. BaF3-MIGR1 and BaF3-BCR/ABL cell lines stably overexpressing Apg-2 were established and exposed to 50 microM H2O2 for 10 min. Western blot analysis of Apg-2 expression confirmed that H2O2 treatment significantly up-regulated Apg-2 expression. Apg-2 overexpression elevated BaF3-BCR/ABL cell proportions in S and G2/M phase, increased cell proliferation and colony formation in vitro. Moreover, BaF3-MIGR1 and BaF3-BCR/ABL cells were exposed to 50 microM H2O2 in the absence or presence of Apg-2 overexpression and induction of H2AX phosphorylation, the reporters of DNA damage were assessed by Western blot and immunofluorescence. Results showed that exposure to H2O2 induced H2AX phosphorylation in BaF3-MIGR1 cells, but no increase was observed in BaF3-BCR/ABL cells. Together, the data indicate that Apg-2 is overexpressed and overexpression of Apg-2 in BaF3-BCR/ABL cells increases cell proliferation and protects cells from oxidative damage, which may play an important role in CML carcinogenesis and progression.

Redox modulation of global phosphatase activity and protein phosphorylation in intact skeletal muscle

Skeletal muscles produce transient reactive oxygen species (ROS) in response to intense stimulation, disuse atrophy, heat stress, hypoxia, osmotic stress, stretch and cell receptor activation. The physiological significance is not well understood. Protein phosphatases (PPases) are known to be highly sensitive to oxidants and could contribute to many different signalling responses in muscle. We tested whether broad categories of PPases are inhibited by levels of acute oxidant exposure that do not result in loss of contractile function or gross oxidative stress. We also tested if this exposure results in elevated levels of global protein phosphorylation. Rat diaphragm muscles were treated with either 2,3-dimethoxy-1-naphthoquinone (DMNQ; 1, 10, 100 microm; a mitochondrial O(2)(.-)/H2O2 generator) or exogenous H2O2 (5, 50, 500 microm) for 30 min. Supernatants were assayed for serine/threonine PPase (Ser/Thr-PPase) or protein tyrosine PPase (PTP) activities. With the exception of 500 microm H2O2, no other oxidant exposures significantly elevated protein carbonyl formation, nor did they alter the magnitude of twitch force. DMNQ significantly decreased all categories of PPase activity at 10 and 100 microm and reduced PTP at 1 microm. Similar reductions in Ser/Thr-PPase activity were seen in response to 50 and 500 microm H2O2 and PTP at 500 microm H2O2. ROS treatments resulted a dose-dependent increase in the phosphorylation states of many proteins. The data are consistent with the concept that PPases, within intact skeletal muscles, are highly sensitive to acute changes in ROS activity and that localized ROS play a critical role in lowering the barriers for effective phosphorylation events to occur in muscle cells, thus increasing the probability for cell signalling responses to proceed.

Ocimum sanctum extracts attenuate hydrogen peroxide induced cytotoxic ultrastructural changes in human lens epithelial cells

Hydrogen peroxide (H2O2) is the major oxidant involved in cataract formation. The present study investigated the effect of an aqueous leaf extract of Tulsi (Ocimum sanctum) against H2O2 induced cytotoxic changes in human lens epithelial cells (HLEC). Donor eyes of the age range 20-40 years were procured within 5-8 h of death. After several washings with gentamicin (50 mL/L) and betadine (10 mL/L), clear transparent lenses (n=6 in each group) were incubated in Dulbecco's modified Eagle's medium (DMEM) alone (normal) or in DMEM containing 100 microm of H2O2 (control) or in DMEM containing both H2O2 (100 microm) and 150 microg/mL of Ocimum sanctum extract (treated) for 30 min at 37 degrees C with 5% CO2 and 95% air. Following incubation, the semi-hardened epithelium of each lens was carefully removed, fixed and processed for electron microscopic studies. Thin sections (60-70 mm) were contrasted with uranyl acetate and lead citrate and viewed under a transmission electron microscope. Normal epithelial cells showed intact, euchromatic nucleus with few small vacuoles (diameter 0.58+/-0.6 microm) in well-demarcated cytoplasm. After treatment with H2O2, they showed pyknotic nuclei with clumping of chromatin and ill-defined edges. The cytoplasm was full of vacuoles (diameter 1.61+/-0.7 microm). The overall cellular morphology was typical of dying cells. Treatment of cells with Ocimum sanctum extract protected the epithelial cells from H2O2 insult and maintained their normal architecture. The mean diameter of the vacuoles was 0.66+/-0.2 microm. The results indicate that extracts of O. sanctum have an important protective role against H2O2 injury in HLEC by maintaining the normal cellular architecture. The protection could be due to its ability to reduce H2O2 through its antioxidant property and thus reinforcing the concept that the extracts can penetrate the HLEC membrane.

Effects of Hydrogen Peroxide (H2O2) on Fresh and Cryopreserved Buffalo Sperm Functions During Incubation at 37°CIn Vitro

The magnitude of damage to buffalo spermatozoa during incubation with different levels of H2O2 was assessed. A total number of 24 ejaculates from four Murrah buffalo bulls were analysed in the study. Each ejaculate was split into two parts (part I and II). Part I was extended in Tris-egg yolk-citrate extender (20% egg yolk:7% glycerol), equilibrated (4 h at 5 degrees C) and cryopreserved in 0.5-ml French straws and stored in liquid nitrogen. The other part was utilized for fresh semen studies. The sperm in fresh, equilibrated and frozen-thawed semen was separated by centrifugation (1500 g; 15 min) and were washed with sperm TALP. The sperm cells were re-suspended in incubation TALP at the rate of 10(8) sperm cells per millilitre and incubated with 0, 10, 25, and 50 microm H2O2 per ml at 37 degrees C. Sperm motility, viability and intact acrosome percentages were assessed at 15-min intervals up to 60 min of incubation. Lipid peroxidation levels of sperm were assessed at 0 and 60 min of incubation. The results of the experiment revealed that sperm motility decreased drastically during incubation with H2O2. Among the different levels of H2O2, the 50-microm H2O2-incorporated group had significantly (p<0.05) higher malonaldehyde (MDA) level than the other groups. In the 50-microm H2O2-incorporated group, the MDA levels in fresh, equilibrated and frozen-thawed semen after incubation for 60 min were 961.6+/-12.7, 991.8+/-10.3 and 1234.9+/-9.6 nm per 10(9) spermatozoa respectively. An inverse relationship was observed between sperm motility, viability, intact acrosome percentages and concentration of H2O2 and duration of incubation. The decrease in sperm functions with duration of incubation and concentration of H2O2 was significantly (p<0.05) higher in frozen-thawed than fresh and equilibrated spermatozoa.

P53 negatively regulates the transcriptional activity of FOXO3a under oxidative stress

Transcription factors P53 and FOXO are both activated in response to stresses via protein-protein interactions, leading to events such as cell survival or apoptosis. To clarify the mechanisms that regulate FOXO activity, we analyzed the intermolecular interaction of FOXO3a and P53. FOXO3a and P53 interacted in COS-7 cells, and transcriptional activity of FOXO3a was suppressed by P53, but P53 was not affected by FOXO3a. RT-PCR revealed that expression of the endogenous apoptosis-inducible genes Bim and Bcl6 was decreased markedly by co-expression of P53 with them, but expression of p27 and CyclinG2 was not. In addition, treatment with 500 microM H2O2 for 30 min to 1h to mimic oxidative stress promoted protein binding. Serum deprivation and drug treatment also affected the binding of FOXO3a and P53. These findings suggest that FOXO3a controls cellular function by changing its molecular interaction with P53 to mediate transcription factor activity under stress stimuli.

Identification of hydrogen peroxide as a major cytotoxic component in Maillard reaction mixtures and coffee

The cytotoxic activity of Maillard reaction products and coffee was studied using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay and the neutral red uptake (NRU) assay. Equimolar mixtures of sugars and lysine were heated at 120 degrees C and used to stimulate bovine aorta endothelial cells for 24 h. The cytotoxic activity increased with increase in educt concentration and heating time. Mixtures containing ribose were most active, followed by lactose and glucose. Hydrogen peroxide, which was present in the Maillard mixtures in concentrations between 7 and 87 microM, was identified as one of their major cytotoxic components. H2O2-concentrations increased further up to 130 microM under cell culture conditions. Filter coffee, espresso, and green coffee extract reduced cell viability significantly to 10, 19, and 83% of PBS-treated control. The effect was largely attenuated by the addition of catalase. Nil, 33, and 41 microM H2O2 was measured in green coffee extract, filter coffee, and espresso, respectively, increasing to 13, 369, and 333 microM during cell culture conditions. No additional H2O2 formation was detected when coffee was incubated for up to 5 h without further treatment. In conclusion, hydrogen peroxide is a major product in Maillard mixtures and coffee inducing cell death in vitro.

Sensory Neurons and Schwann Cells Respond to Oxidative Stress by Increasing Antioxidant Defense Mechanisms

Elevated blood glucose is a key initiator of mechanisms leading to diabetic neuropathy. Increases in glucose induce acute mitochondrial oxidative stress in dorsal root ganglion (DRG) neurons, the sensory neurons normally affected in diabetic neuropathy, whereas Schwann cells are largely unaffected. We propose that activation of an antioxidant response in DRG neurons would prevent glucose-induced injury. In this study, mild oxidative stress (1 microM H2O2) leads to the activation of the transcription factor Nrf2 and expression of antioxidant (phase II) enzymes. DRG neurons are thus protected from subsequent hyperglycemia-induced injury, as determined by activation of caspase 3 and the TUNEL assay. Schwann cells display high basal antioxidant enzyme expression and respond to hyperglycemia and mild oxidative stress via further increases in these enzymes. The botanical compounds resveratrol and sulforaphane activate the antioxidant response in DRG neurons. Other drugs that protect DRG neurons and block mitochondrial superoxide, identified in a compound screen, have differential ability to activate the antioxidant response. Multiple cellular targets exist for the prevention of hyperglycemic oxidative stress in DRG neurons, and these form the basis for new therapeutic strategies against diabetic neuropathy.

Sperm quality improvement after date seed oil in vitro supplementation in spontaneous and induced oxidative stress

In vitro supplementation with date seed oil (DSO) can protect spermatozoa against hydrogen peroxide (H2O2)-mediated damage and can improve sperm function, possibly owing to antioxidant properties. We tested the antioxidant effects of DSO on human sperm motility, sperm viability, reacted acrosome and lipid peroxidation assessed in vitro after H2O2-mediated oxidative damage in spermatozoa. Sixteen patients (mean age: 35 years; range: 25-45 years) referred to the Histology-Embryology Laboratory of the Medicine Faculty of Sfax for semen analysis after 12-24 months of sexual intercourse without conception were selected. After spermiogram, sperm selection by two-interface discontinuous Sill Select gradient was performed, and selected spermatozoa were used in four experimental assays: control; incubation with 100 microm H2O2; incubation with 0.1% DSO; and co-incubation with 0.1% DSO and 100 microm H2O2. Motility and viability were determined using World Health Organization criteria. Acrosome reaction and lipid peroxidation were assessed by staining with fluorescein isothiocyanate-Pisum sativum and spectrophotometric measurement of malondialdehyde, respectively. Results showed that incubation with H2O2 alone led to a significant increase in lipid peroxidation (57.83%, P<0.05) associated with a significant decrease in sperm motility, sperm viability (after 30 min and 24 h) and percentage of reacted acrosome (P<0.05). Date seed oil improved sperm motility after 24 h of incubation (P<0.05) and protected spermatozoa against the deleterious effects of H2O2 on motility, viability, acrosome reaction and lipid peroxidation. We conclude that supplementation with DSO may have a function in antioxidant protection against male infertility.

Protection against Hydrogen Peroxide-Mediated Cytotoxicity in Friedreich's Ataxia Fibroblasts Using Novel Iron Chelators of the 2-Pyridylcarboxaldehyde Isonicotinoyl Hydrazone Class

Iron-loading diseases remain an important problem because of the toxicity of iron-catalyzed redox reactions. Iron loading occurs in the mitochondria of Friedreich's ataxia (FA) patients and may play a role in its pathogenesis. This suggests that iron chelation therapy could be useful. We developed previously the lipophilic iron chelators known as the 2-pyridylcarboxaldehyde isonicotinoyl hydrazone (PCIH) ligands and identified 2-pyridylcarboxaldehyde 2-thiophenecarboxyl hydrazone (PCTH) as the most promising analog. Hence, this study assessed the efficacy of PCTH and other PCIH analogs compared with various chelators, including deferiprone and desferrioxamine (DFO). Age- and sex-matched control and FA fibroblasts were preincubated with iron chelators and subsequently challenged with 50 microM H2O2 for up to 24 h. The current study demonstrates an interesting structure-activity relationship among the closely related PCIH series of ligands, with only PCTH being highly effective at preventing H2O2-induced cytotoxicity. PCTH increased FA fibroblast cell viability by up to 70%, whereas DFO rescued viability by 1 to 5% only. Hence, PCTH, which was well tolerated by cells was far more effective than DFO at preventing oxidative stress. It is noteworthy that kinetic studies demonstrated PCTH to rapidly penetrate cells to induce 59Fe efflux, whereas DFO, PCIH, 2-pyridylcarboxaldehyde benzoyl hydrazone, and 2-pyridylcarboxaldehyde m-bromobenzoyl hydrazone were far slower, indicating it is the rate of chelator permeation that is crucial for protection against H2O2. In addition, PCTH was found to be as effective as or more effective than conventional radical scavengers or the antioxidant idebenone (which has undergone clinical trials) at protecting cells against H2O2-mediated cytotoxicity. These findings further indicate the potential of PCTH for treatment of iron overload.

Optical Biosensor Based On Hollow Integrated Waveguides

The first absorbance biosensor based on pure silicon hollow integrated waveguides is presented in this work. With the use of horseradish peroxidase (HRP) as a model recognition element, an enzymatic sensor for the measurement of hydrogen peroxide was fabricated, numerically simulated, and experimentally characterized. Waveguides with widths ranging from 50 to 80 microm, having a depth of 50 microm and lengths up to 5 mm were easily fabricated by just one photolithographic step. These were further modified by covalent immobilization of HRP using silanization chemistry. Simulation studies of the proposed approach showed a sensor linear behavior up to 300 microM H2O2 and a sensitivity of 2.7 x 10(-3) AU/microM. Experimental results were in good agreement with the simulated ones. A linear behavior between 10 and 300 microM H2O2, a sensitivity of 3 x 10(-3) AU/microM, and a signal-to-noise ratio around 20 dB were attained. Also, kinetic studies of the activity of the immobilized enzyme on the silicon waveguide surface gave an apparent Michaelis-Menten constant of 0.44 mM. The simple technology proposed in this work enables the fabrication of cost-effective, easy-to-use, miniaturized biosensor generic platforms, these being envisioned as excellent candidates for the development of lab-on-a-chip systems.

Dihydrorhodamine 123 is superior to 2,7‐dichlorodihydrofluorescein diacetate and dihydrorhodamine 6G in detecting intracellular hydrogen peroxide in tumor cells

Dihydrorhodamine 123 (DHR 123), 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), and dihydrorhodamine 6G (DHR 6G) were evaluated as probes for detecting cellular hydrogen peroxide levels in SPC-A-1 lung adenocarcinoma cells. Imaging techniques and fluorescence-activated cell scan were used in the study of the probe responses. Obvious green fluorescence was established after a 25-min exposure. After staining with MitoTracker Orange CM-H2TMRos (a probe for mitochondria) and the abovementioned probes simultaneously, only the DHR 123 and DHR 6G groups exhibited legible green fluorescence in the mitochondrial regions. Furthermore, the DHR 6G group exhibited weaker fluorescence intensity. When 100 microM H2O2 was added to SPC-A-1 cells loaded with these probes, the intracellular fluorescence increased rapidly and significantly. Our results suggest that DHR 123 is superior for the instantaneous detection of cellular hydrogen peroxide in SPC-A-1 cells.

Antioxidant responses of Laeonereis acuta (Polychaeta) after exposure to hydrogen peroxide

The effects of H2O2 were evaluated in the estuarine worm Laeonereis acuta (Polychaeta, Nereididae) collected at the Patos Lagoon estuary (Southern Brazil) and maintained in the laboratory under controlled salinity (10 psu diluted seawater) and temperature (20 degrees C). The worms were exposed to H2O2 (10 and 50 microM) for 4, 7, and 10 days and the following variables were determined: oxygen consumption, catalase (CAT) and glutathione peroxidase activity in both the supernatant and pellet fractions of whole body homogenates. The concentrations of non-protein sulfhydryl and lipid peroxides (LPO) were also measured. The oxygen consumption response was biphasic, decreasing after 4 days and increasing after 7 and 10 days of exposure to 50 microM H2O2 (P < 0.05). At the same H2O2 concentration, CAT activity was lower (P < 0.05) in the pellet fraction of worms exposed for 10 days compared to control. Non-protein sulfhydryl concentration and glutathione peroxidase activity were not affected by H2O2 exposure. After 10 days, LPO levels were higher (P < 0.05) in worms exposed to 50 microM H2O2 compared to control. The reduction in the antioxidant defense was paralleled by oxidative stress as indicated by higher LPO values (441% compared to control). The reduction of CAT activity in the pellet fraction may be related to protein oxidation. These results, taken together with previous findings, suggest that the worms were not able to cope with this H2O2 concentration.

Docosahexaenoic acid‐dependent iron accumulation in oligodendroglia cells protects from hydrogen peroxide‐induced damage

Iron, a transition metal and essential nutrient, is a typical pro-oxidant forming free radicals, lipid peroxides and causing cell damage when added at high (> or = 50 microM) concentrations to oligodendroglia-like OLN-93 cells that have been enriched for 3 days with 10 microM docosahexaenoic acid (DHA, 22 : 6 n-3). At low (5 microM) iron concentrations lipid peroxides were still formed, but cells turned resistant to 250 microM H2O2, a secondary genotoxic stress. This has been attributed most likely to a time-dependent (16 h preconditioning) increase of cellular antioxidant enzyme activities i.e., glutathione peroxidase (38%) and glutathione reductase (26%). DHA but not arachidonic acid (20 : 4 n-6) supplements induced 3-fold increase in gene expression of divalent metal transporter-1, a transporter protein presumably responsible for the increase in intracellular iron. Elevated iron levels triggered a transient scrambling of membrane lipid asymmetry as evident by an accelerated ethanolamine phosphoglyceride translocation to the outer cell surface. Ethanolamine phosphoglyceride reorientation is proposed to activate certain signaling cascades leading to changes in nuclear transcription, a reaction that could represent a mechanism of preconditioning. These findings may have important implications for understanding the interactive role of iron and DHA in nutritional deficiencies, losses of polyunsaturated fatty acids in the aging brain or excessive iron accumulation in degenerative disorders.

Are So-Called Uremic Toxins Always Toxic?

Numerous substances qualify as uremic toxins by fulfilling all properties characterizing such compounds. However, their role in the development of uremic state maybe ambiguous. We followed these properties on the example of N-methyl-2-pyridone-5-carboxamide (Met2PY) from the nicotinamide end-products family. The aim of this study was to determine if these uremic compounds are toxic in all circumstances. To establish a direct toxic effect, a cytotoxicity test was performed. The influence of Met2PY on DNA damage in cultured cells was measured, using a comet assay. For in vitro experiments, Moly (L5178Y), LLCPK-1, and A549 cell lines were used. We used 250 microM H2O2 and 200 ng/mL angiotensin II (ANGII) as damaging factors. A direct cytotoxic effect of Met2PY on Moly cells was observed. In LLC-PK1 cells, co-incubation with 0.03 mM Met2PY protected cells against the DNA damage caused by ANG II. In A549 cells, the action of Met2PY was ambiguous. At lower concentrations (1 and 3 mM), it showed protective effects, although 10 mM Met2PY increased the toxic effect of 250 microM H2O2. Our results suggest that Met2PY is not always toxic or harmful. In some circumstances, it may even express beneficial and protective properties.

Peroxisome Proliferator-activated Receptor γ Up-regulates the Bcl-2 Anti-apoptotic Protein in Neurons and Induces Mitochondrial Stabilization and Protection against Oxidative Stress and Apoptosis

Peroxisome proliferator-activated receptor gamma (PPARgamma) has been proposed as a therapeutic target for neurodegenerative diseases because of its anti-inflammatory action in glial cells. However, PPARgamma agonists preventbeta-amyloid (Abeta)-induced neurodegeneration in hippocampal neurons, and PPARgamma is activated by the nerve growth factor (NGF) survival pathway, suggesting a neuroprotective anti-inflammatory independent action. Here we show that the PPARgamma agonist rosiglitazone (RGZ) protects hippocampal and dorsal root ganglion neurons against Abeta-induced mitochondrial damage and NGF deprivation-induced apoptosis, respectively, and promotes PC12 cell survival. In neurons and in PC12 cells RGZ protective effects are associated with increased expression of the Bcl-2 anti-apoptotic protein. NGF-differentiated PC12 neuronal cells constitutively overexpressing PPARgamma are resistant to Abeta-induced apoptosis and morphological changes and show functionally intact mitochondria and no increase in reactive oxygen species when challenged with up to 50 microM H2O2. Conversely, cells expressing a dominant negative mutant of PPARgamma show increased Abeta-induced apoptosis and disruption of neuronal-like morphology and are highly sensitive to oxidative stress-induced impairment of mitochondrial function. Cells overexpressing PPARgamma present a 4- to 5-fold increase in Bcl-2 protein content, whereas in dominant negative PPARgamma-expressing cells, Bcl-2 is barely detected. Bcl-2 knockdown by small interfering RNA in cells overexpressing PPARgamma results in increased sensitivity to Abeta and oxidative stress, further suggesting that Bcl-2 up-regulation mediates PPARgamma protective effects. PPARgamma prosurvival action is independent of the signal-regulated MAPK or the Akt prosurvival pathways. Altogether, these data suggest that PPARgamma supports survival in neurons in part through a mechanism involving increased expression of Bcl-2.

Cytometric assessment of DNA damage by exogenous and endogenous oxidants reports aging‐related processes

The ongoing DNA damage caused by reactive oxygen species generated during oxidative metabolism is considered a key factor contributing to cell aging as well as preconditioning cells to neoplastic transformation. We postulated before that a significant fraction of constitutive histone H2AX phosphorylation (CHP) and constitutive activation of ATM (CAA) seen in untreated normal and tumor cells occurs in response to such DNA damage. In the present study, we provide further evidence in support of this postulate. The level of ATM activation and H2AX phosphorylation, detected immunocytochemically, has been monitored in WI-38, A549, and TK6 cells treated with H2O2 as well as growing under conditions known or suspected to affect the level of endogenous oxidants. Thirty- to 60-min exposure of cells to 100 or 200 microM H2O2 led to an increase in the level of H2AX phosphorylation and ATM activation, particularly pronounced (nearly fivefold) in S-phase cells. Cell growth for 24-48 h under hypoxic conditions (3% O2) distinctly lowered the level of CHP and CAA while it had minor effect on cell cycle progression. Treatment (4 h) with 0.1 or 0.3 mM 3-bromopyruvate, an inhibitor of glycolysis and mitochondrial oxidative phosphorylation, reduced the level of CHP (up to fourfold) and also decreased the level of CAA. Growth of WI-38 cells in 2% serum concentration for 48 h led to a 25 and 30% reduction in CHP and CHA, respectively, compared with cells growing in 10% serum. The antioxidant vitamin C (2 mM) reduced CHP and CAA by 20-30% after 24 h of treatment, while the COX-2 inhibitor celecoxib (5 microM) had a minor effect on CHP and CAA, though it decreased the level of H2O2-induced H2AX phosphorylation and ATM activation. In contrast, dichloroacetate known to shift metabolism from anaerobic to oxidative glycolysis through its effect on pyruvate dehydrogenase kinase enhanced the level of CHP and CAA. Our present data and earlier observations strongly support the postulate that a large fraction of CHP and CAA occurs in response to DNA damage caused by metabolically generated oxidants. Cytometric analysis of CHP and CAA provides the means to measure the effectiveness of exogenous factors, which either through lowering aerobic metabolism or neutralizing radicals may protect DNA from such damage.